Again via the Brent Kearney link on Stan's site.
http://phys.org/news/2012-11-scientists-key-events-early-cellular.html
discussing this paper
http://www.nature.com/nature/journal/vaop/ncurrent/full/nature11654.html
I especially like the last sentence of these two paragraphs:
These results are just the newest chapter in a "decade-old interest," according to Gottschling. He and his colleagues have made several landmark discoveries in the past 10 years, including finding that aging yeast cells exhibit the same genomic instability seen in human cancer cells and proving that mitochondrial dysfunction causes that instability. Gottschling's team also has developed innovative tools to leverage the power of yeast as a model organism, including a technique called the Mother Enrichment Program that makes experiments more efficient by enabling researchers to generate large populations of aging yeast cells.
"It's worth using yeast to study complex things like aging because a lot of person-years of research have gone into understanding the fundamentals. The genetic and cell-biology tools available for studying yeast are unparalleled," Gottschling said. "Having the proper tools is like having new glasses; you can see things you never could before, and once you start to see new things, you can dissect them to understand how they work."
The full paper is a Letter to Nature. That means it is technically very dense, but very interesting.
Caloric restriction to a yeast is, essentially, glucose restriction. In organisms with a circulation, an hormone system and a background FFA supply, caloric restriction at the cellular level (where it matters) is achieved by resisting the insulin mediated facilitation of glucose access, ie glucose restriction. Using superoxide.
Palmitate please, with just a very little glucose.
Peter
Thursday, November 22, 2012
Wednesday, November 21, 2012
Interesting times on Mars
http://boingboing.net/2012/11/20/big-news-from-mars-coming-soon.html
I picked this up through the link to Brent Kearney's blog on Stan's site. Olivine and water under a CO2 atmosphere, all present on Mars, are the precursor to biochemistry. I just wonder... Interesting times.
Peter
I picked this up through the link to Brent Kearney's blog on Stan's site. Olivine and water under a CO2 atmosphere, all present on Mars, are the precursor to biochemistry. I just wonder... Interesting times.
Peter
Tuesday, November 20, 2012
Protons: Physiological insulin resistance, addendum two
George put up the link to this paper, which allows you to tease information out about omega-3 PUFA as bulk calories vs lard as bulk calories. We are not talking about essential supplies of essential lipids here. We are looking at serious bulk calorie supply. This is quite, quite different.
Aside: The basic conclusion that FO is protective against endotoxin shock is fascinating but may be restricted to C57BL/6 mice. Pity everyone uses them. But it's interesting, and on file, never the less. End aside.
Here's the composition of the diets:
Fairly typical research diets, a little more sucrose than I would like but, well, everyone does it.
What about weight gains? Here they are:
Although body weights, at all time point, are not significantly different between groups this is just due to the initial group weights being different. If you look at weight gain rather than absolute weights, the lard group gains more weight than the fish oil group.
The insulin and glucose levels do support the idea that insulin sensitivity is controlled by the degree of unsaturation of the bulk lipid in the diet, ie PUFA diets increase insulin sensitivity. But there is no excess weight gain in the fish oil group. Why not?
C57BL/6 mice suffer an injury to their hypothalamus on exposure to a saturated fat based diet, especially if combined with sucrose. Omega-6 PUFA do not seem do this and I doubt omega-3 PUFA do either. I considered this back here.
So we are comparing obesity in an un-injured group carrying omega-3 enhanced insulin sensitive adipocytes versus a hypothalamic injured group carrying adipocytes which are partially resistant to insulin due to dietary palmitic acid and partially sensitive to insulin due to decreased sympathetic outflow from the hypothalamus to adipocytes. C57BL/6 mice are very special in their response to saturated fats.
This is a knotty problem to try and untangle. This paper helps a lot.
I just want to look at two of the control groups, both of which are C57BL/6 mice, both of which are exposed to a high palmtic acid diet and so both will have an hypothalamic injuy.
So we can have, among many, two groups of C57BL/6 mice, one fed a high fat diet to make it fat and the next fed a high fat diet to make it fat, but then add in a significant dose of omega-3 PUFA. Just to add some insult to injury. The first group gets a whammy. The second group gets a double whammy. Want to see the graph? Ok, ok, here it is:
First, strain your eyes to follow the open triangles. This is the high fat only control group. These are C57BL/6 freak mice with a brain injury triggered by palmitic acid. They have limited weight gain but, as they store palmitate without DNL, hence without desaturase activation, hence without palmitoleate generation, they develop metabolic syndrome. Visceral fat, fatty liver. Of course the group didn't measure either insulin or glucose (they are in obesity drug development), but these mice have metabolic syndrome and have lost the ability to get any fatter. They are in trouble. They don't actually weigh any more than un-injured C57BL/6 mice fed traditional crapinabag.
Now look at the open circles of ever increasing obesity. Fatties or fatties? This is what happens when you add fish oil to the diet of a palmitic acid injured C57BL/6 freak mouse. Impressive waistlines huh? Of course we don't get the insulin or glucose levels here either, but these mice do not have metabolic syndrome. They maintain the insulin sensitivity of their adipocytes, especially peripherally, and continue to become obese with sustained metabolic health. They will stay healthy until their adipocyte distension induced insulin resistance eventually over rides the insulin sensitising effects of the bulk fish oil.
We have a pair of models. Skinny-fat and obese-but-metabolically-slim. Both are explicable by looking at the basic effects of bulk lipid supply from the diet acting on the insulin signalling system within mitochondria.
Summary: These are palmitic acid injured C57BL/6 freak mice which have the added insult of having their adipocytes rendered extra insulin sensitive by the F:N ratio of a significant percentage of the fatty acids in the fish oil of their experimental diet. This postpones metabolic syndrome until they have become fat enough to develop it.
The F:N ratio concept delivers again.
Peter
BTW no one knows the omega-6 content of the fish oil is in this second study! The discussion mentions that there is zero omega-6 in the basic high fat diet, which has no added fish oil. Imagine running a Rimonabant study when you don't know the omega-6 content of the (high fat) diets. But this becomes irrelevant if you look at the basic metabolism at the molecular level. Either family of low F:N ratio PUFA will delay metabolic syndrome, while ever they assist weight gain. And you have to remember that C57BL/6 mice break by eating butter.
Aside: The basic conclusion that FO is protective against endotoxin shock is fascinating but may be restricted to C57BL/6 mice. Pity everyone uses them. But it's interesting, and on file, never the less. End aside.
Here's the composition of the diets:
Fairly typical research diets, a little more sucrose than I would like but, well, everyone does it.
What about weight gains? Here they are:
Although body weights, at all time point, are not significantly different between groups this is just due to the initial group weights being different. If you look at weight gain rather than absolute weights, the lard group gains more weight than the fish oil group.
The insulin and glucose levels do support the idea that insulin sensitivity is controlled by the degree of unsaturation of the bulk lipid in the diet, ie PUFA diets increase insulin sensitivity. But there is no excess weight gain in the fish oil group. Why not?
C57BL/6 mice suffer an injury to their hypothalamus on exposure to a saturated fat based diet, especially if combined with sucrose. Omega-6 PUFA do not seem do this and I doubt omega-3 PUFA do either. I considered this back here.
So we are comparing obesity in an un-injured group carrying omega-3 enhanced insulin sensitive adipocytes versus a hypothalamic injured group carrying adipocytes which are partially resistant to insulin due to dietary palmitic acid and partially sensitive to insulin due to decreased sympathetic outflow from the hypothalamus to adipocytes. C57BL/6 mice are very special in their response to saturated fats.
This is a knotty problem to try and untangle. This paper helps a lot.
I just want to look at two of the control groups, both of which are C57BL/6 mice, both of which are exposed to a high palmtic acid diet and so both will have an hypothalamic injuy.
So we can have, among many, two groups of C57BL/6 mice, one fed a high fat diet to make it fat and the next fed a high fat diet to make it fat, but then add in a significant dose of omega-3 PUFA. Just to add some insult to injury. The first group gets a whammy. The second group gets a double whammy. Want to see the graph? Ok, ok, here it is:
First, strain your eyes to follow the open triangles. This is the high fat only control group. These are C57BL/6 freak mice with a brain injury triggered by palmitic acid. They have limited weight gain but, as they store palmitate without DNL, hence without desaturase activation, hence without palmitoleate generation, they develop metabolic syndrome. Visceral fat, fatty liver. Of course the group didn't measure either insulin or glucose (they are in obesity drug development), but these mice have metabolic syndrome and have lost the ability to get any fatter. They are in trouble. They don't actually weigh any more than un-injured C57BL/6 mice fed traditional crapinabag.
Now look at the open circles of ever increasing obesity. Fatties or fatties? This is what happens when you add fish oil to the diet of a palmitic acid injured C57BL/6 freak mouse. Impressive waistlines huh? Of course we don't get the insulin or glucose levels here either, but these mice do not have metabolic syndrome. They maintain the insulin sensitivity of their adipocytes, especially peripherally, and continue to become obese with sustained metabolic health. They will stay healthy until their adipocyte distension induced insulin resistance eventually over rides the insulin sensitising effects of the bulk fish oil.
We have a pair of models. Skinny-fat and obese-but-metabolically-slim. Both are explicable by looking at the basic effects of bulk lipid supply from the diet acting on the insulin signalling system within mitochondria.
Summary: These are palmitic acid injured C57BL/6 freak mice which have the added insult of having their adipocytes rendered extra insulin sensitive by the F:N ratio of a significant percentage of the fatty acids in the fish oil of their experimental diet. This postpones metabolic syndrome until they have become fat enough to develop it.
The F:N ratio concept delivers again.
Peter
BTW no one knows the omega-6 content of the fish oil is in this second study! The discussion mentions that there is zero omega-6 in the basic high fat diet, which has no added fish oil. Imagine running a Rimonabant study when you don't know the omega-6 content of the (high fat) diets. But this becomes irrelevant if you look at the basic metabolism at the molecular level. Either family of low F:N ratio PUFA will delay metabolic syndrome, while ever they assist weight gain. And you have to remember that C57BL/6 mice break by eating butter.
Sunday, November 18, 2012
Protons: Physiological insulin resistance addendum
Edward sent me this paper. I think I did already have a copy on my hard drive but you can't really start to make head nor tail of what is really going on until you have a handle on F:N ratio and the physiological role for superoxide. I'd completely forgotten about the paper.
For those people who think humans are in some way different from rats, here's Fig 1 from the paper on humans eating either a saturated fat ketogenic diet or a polyunsaturated fat ketogenic diet, just for 5 days:
Look at the glucose, lowest in the PUFA group, look at the ketones, highest in the PUFA group, look at the insulin sensitivity, waaaay higher in the PUFA group. Rat or human, makes no odds. PUFA fail to generate superoxide in mitochondria. Is this good or bad?
The whole point of a ketogenic diet (epilepsy excepted) is to induce starvation-appropriate physiological insulin resistance. What is the point of setting up a ketogenic diet which does not have the ability to convert from running on glucose to running on fat?
Aside: Why might anyone want to run their metabolism on FFAs? Superoxide. I want more mitochondria to supply spare ETC capacity, to minimise the sort of levels of free radicals which wipe out mitochondria when the pressure is on. Physiological superoxide signals for mitochondrial biogenesis, without all of that tedious exercise to do the same job on a mixed diet. End aside.
Now it is just possible to argue that chronically reduced insulin may render adipocytes immune to the insulin sensitising effects of PUFA. Maybe. The obese mice of the next Protons post are on a mixed PUFA-carb diet to assist their "ballooning" experience. High insulin plus insulin hypersensitive adipocytes gives obesity. Perhaps the combination of low insulin with distendable adipocytes is OK if the insulin levels are low enough. Volunteers queue over there please.
But we are still in a situation where FFAs are high, yet glucose can still enter cells. What does this do to cellular ATP status? The whole point of insulin resistance is to avoid cellular caloric overload, full stop. If you load liver cells with PUFA and add in glucose the end result, by whatever mechanism, is cirrhosis. Perhaps other tissues fare better. Am I about to try it?
No thank you.
Peter
And for a real giggle you can see exactly what sort of utter crap made up the high PUFA diet, just read Table 1. "Imitation" if the first word of the first "food"!
For those people who think humans are in some way different from rats, here's Fig 1 from the paper on humans eating either a saturated fat ketogenic diet or a polyunsaturated fat ketogenic diet, just for 5 days:
Look at the glucose, lowest in the PUFA group, look at the ketones, highest in the PUFA group, look at the insulin sensitivity, waaaay higher in the PUFA group. Rat or human, makes no odds. PUFA fail to generate superoxide in mitochondria. Is this good or bad?
The whole point of a ketogenic diet (epilepsy excepted) is to induce starvation-appropriate physiological insulin resistance. What is the point of setting up a ketogenic diet which does not have the ability to convert from running on glucose to running on fat?
Aside: Why might anyone want to run their metabolism on FFAs? Superoxide. I want more mitochondria to supply spare ETC capacity, to minimise the sort of levels of free radicals which wipe out mitochondria when the pressure is on. Physiological superoxide signals for mitochondrial biogenesis, without all of that tedious exercise to do the same job on a mixed diet. End aside.
Now it is just possible to argue that chronically reduced insulin may render adipocytes immune to the insulin sensitising effects of PUFA. Maybe. The obese mice of the next Protons post are on a mixed PUFA-carb diet to assist their "ballooning" experience. High insulin plus insulin hypersensitive adipocytes gives obesity. Perhaps the combination of low insulin with distendable adipocytes is OK if the insulin levels are low enough. Volunteers queue over there please.
But we are still in a situation where FFAs are high, yet glucose can still enter cells. What does this do to cellular ATP status? The whole point of insulin resistance is to avoid cellular caloric overload, full stop. If you load liver cells with PUFA and add in glucose the end result, by whatever mechanism, is cirrhosis. Perhaps other tissues fare better. Am I about to try it?
No thank you.
Peter
And for a real giggle you can see exactly what sort of utter crap made up the high PUFA diet, just read Table 1. "Imitation" if the first word of the first "food"!
Protons: Physiological insulin resistance
Woody emailed me a pdf of this venerable paper. I like it. Even though it was generated in-house by Proctor and Gamble!
The core findings are that if you eat a diet with around 60% of calories from fat, then you end up lighter if that diet is based on safflower oil (omega 6 PUFA) and heavier if it's based on lard. Here's the table:
Well, there you go!
Safflower oil is more ketogenic than lard. It also allows greater fat loss during 72 hours of starvation, 10.3g of fat loss from safflower fed rats vs 3.9g from lard fed animals, here we are:
Mmmmmmmm, linoleic acid, the elixir of life and Weight Watchers' friend. And I thought it was obesogenic!
So what does the small print say? Lots!
First thing to make my ears prick up is that during the whole study the rats were only allowed to eat for 2 hours a day, in a single solid block. That means they were force fasted for 22 hours a day. We've noted from Table 3 that safflower oil allows greater weight loss under fasting conditions. So is it any surprise that the safflower rats ended up at 195g vs the 222g of the lard fed rats after eight weeks of this intermittent fasting? I'll come back to why in a moment.
In contrast most modern papers show that linoleic acid, the primary component of safflower oil, is grossly and transgenerationally obesogenic...
But that's because there is a difference between ad lib feeding and 22h per day forced fasting!
The difference is in the glucose levels. Look at these graphs from Fig 1, especially the glucose levels in the middle top graph:
Now, far be it for me to put words in to the mouth of a lab rat, but which group of rats is the hungriest? Which group becomes most hypoglycaemic perhaps? Even Dr You-are-confused-man-Guyenet seems to have, in an aberrant moment of lucidity, a glimmering of perception that hypoglycaemia makes you hungry. Or should I say drives eating behaviour? Did I ever even mention gluttony? If you are force fasted for 22h you can't overeat. You can't even eat to your metabolic needs without accessing significant amounts of stored fat.
Under full starvation a rat lives off of its fat. If linoleic acid is what is being released from the adipocytes under fasting conditions it provides significantly less FADH2 relative to NADH in to the electron transport chain of all fat burning cells than the mix of fatty acids from the adipocytes of lard fed rats. ie there is less physiological insulin resistance. This failure means you fail to keep glucose levels normal during starvation. Let's rub that in: Failure to develop physiological insulin resistance during starvation results in hypoglycaemia and hunger.
Exactly the same will happen in any soy oil fed USA citizen. The end result will still be the failure to develop the essential physiological insulin resistance which is needed to keep blood glucose normal during fasting.
When the average soy oil fattened American is asleep they HAVE to, finally, stop snacking on carbohydrate crap, which is the only way they can maintain a decent blood glucose level. At this time blood glucose falls, simply because their muscles stay insulin sensitive and glucose falls in to them. The brain will not accept hypoglycaemia. Some time, in the middle of the night, there has to be a Refrigerator Raid.
And, OMG, they eat calories! And calories count! Did I ever mention gluttony? Or, perhaps, is the Refrigerator Raid simple physiology?
The 22h daily fasted rats have a locked refrigerator. However hungry they feel due to hypoglycaemia, they are not getting any extra food. But why is there extra weight loss under starvation? Insulin was tricky to measure from a rat in the 1970s, but I know that the safflower loaded then starved rats had the lowest insulin as they are both insulin sensitive and hypoglycaemic. Low insulin = more lipolysis = more ketones and more weight loss. Logical.
Now let's go a step further. Blood glucose is low. It's low because the F:N ratio of linoleic acid is low and that's what is being released from adipocytes. This is metabolism. Individual cell by individual cell, it's a metabolic phenomenon. Picked up by the brain as hypoglycaemia.
Hypoglycaemia must drive food intake to avoid cerebral catastrophe. Glucose is not a neurotransmitter per se. The hypoglycaemia has to be converted to a neurotransmitter based message to affect behaviour. Oddly enough, a derivative of linoleic acid is a neurotransmitter. Linoleic acid is the parent molecule of the endocannabinoids. Even the cleanest nosed obesity researcher has heard of the munchies. Wouldn't it be funny if eating linoleic acid messed with your blood glucose level and this hypoglycaemia triggered the production of endocanabinoids from, you guessed, the parent molecule (of the hypoglycaemia!), linoleic acid within the brain? The same molecule which triggers the hypoglycaemia, making the hunger essential, in the first place.
You could view it as linoleic acid ingestion is a signal that hypoglycaemia is in the offing. If random evolution was looking for a substance to "choose" as a neurotransmitter to deal with hypoglycaemia, it might just be simplest to modify the basic lipid which is most prone to trigger low blood glucose throughout the body in the first place... You could then end up thinking that the endocannabinoids "just happen" to control appetite as a primary function. Of course, once the control system is in place you are all set to mess with it by demonising saturated fat and pushing corn and soy oils.
Well, I find the concept hysterical. But then I find most brain centred obesity research pretty amusing.
Peter
The core findings are that if you eat a diet with around 60% of calories from fat, then you end up lighter if that diet is based on safflower oil (omega 6 PUFA) and heavier if it's based on lard. Here's the table:
Well, there you go!
Safflower oil is more ketogenic than lard. It also allows greater fat loss during 72 hours of starvation, 10.3g of fat loss from safflower fed rats vs 3.9g from lard fed animals, here we are:
Mmmmmmmm, linoleic acid, the elixir of life and Weight Watchers' friend. And I thought it was obesogenic!
So what does the small print say? Lots!
First thing to make my ears prick up is that during the whole study the rats were only allowed to eat for 2 hours a day, in a single solid block. That means they were force fasted for 22 hours a day. We've noted from Table 3 that safflower oil allows greater weight loss under fasting conditions. So is it any surprise that the safflower rats ended up at 195g vs the 222g of the lard fed rats after eight weeks of this intermittent fasting? I'll come back to why in a moment.
In contrast most modern papers show that linoleic acid, the primary component of safflower oil, is grossly and transgenerationally obesogenic...
But that's because there is a difference between ad lib feeding and 22h per day forced fasting!
The difference is in the glucose levels. Look at these graphs from Fig 1, especially the glucose levels in the middle top graph:
Now, far be it for me to put words in to the mouth of a lab rat, but which group of rats is the hungriest? Which group becomes most hypoglycaemic perhaps? Even Dr You-are-confused-man-Guyenet seems to have, in an aberrant moment of lucidity, a glimmering of perception that hypoglycaemia makes you hungry. Or should I say drives eating behaviour? Did I ever even mention gluttony? If you are force fasted for 22h you can't overeat. You can't even eat to your metabolic needs without accessing significant amounts of stored fat.
Under full starvation a rat lives off of its fat. If linoleic acid is what is being released from the adipocytes under fasting conditions it provides significantly less FADH2 relative to NADH in to the electron transport chain of all fat burning cells than the mix of fatty acids from the adipocytes of lard fed rats. ie there is less physiological insulin resistance. This failure means you fail to keep glucose levels normal during starvation. Let's rub that in: Failure to develop physiological insulin resistance during starvation results in hypoglycaemia and hunger.
Exactly the same will happen in any soy oil fed USA citizen. The end result will still be the failure to develop the essential physiological insulin resistance which is needed to keep blood glucose normal during fasting.
When the average soy oil fattened American is asleep they HAVE to, finally, stop snacking on carbohydrate crap, which is the only way they can maintain a decent blood glucose level. At this time blood glucose falls, simply because their muscles stay insulin sensitive and glucose falls in to them. The brain will not accept hypoglycaemia. Some time, in the middle of the night, there has to be a Refrigerator Raid.
And, OMG, they eat calories! And calories count! Did I ever mention gluttony? Or, perhaps, is the Refrigerator Raid simple physiology?
The 22h daily fasted rats have a locked refrigerator. However hungry they feel due to hypoglycaemia, they are not getting any extra food. But why is there extra weight loss under starvation? Insulin was tricky to measure from a rat in the 1970s, but I know that the safflower loaded then starved rats had the lowest insulin as they are both insulin sensitive and hypoglycaemic. Low insulin = more lipolysis = more ketones and more weight loss. Logical.
Now let's go a step further. Blood glucose is low. It's low because the F:N ratio of linoleic acid is low and that's what is being released from adipocytes. This is metabolism. Individual cell by individual cell, it's a metabolic phenomenon. Picked up by the brain as hypoglycaemia.
Hypoglycaemia must drive food intake to avoid cerebral catastrophe. Glucose is not a neurotransmitter per se. The hypoglycaemia has to be converted to a neurotransmitter based message to affect behaviour. Oddly enough, a derivative of linoleic acid is a neurotransmitter. Linoleic acid is the parent molecule of the endocannabinoids. Even the cleanest nosed obesity researcher has heard of the munchies. Wouldn't it be funny if eating linoleic acid messed with your blood glucose level and this hypoglycaemia triggered the production of endocanabinoids from, you guessed, the parent molecule (of the hypoglycaemia!), linoleic acid within the brain? The same molecule which triggers the hypoglycaemia, making the hunger essential, in the first place.
You could view it as linoleic acid ingestion is a signal that hypoglycaemia is in the offing. If random evolution was looking for a substance to "choose" as a neurotransmitter to deal with hypoglycaemia, it might just be simplest to modify the basic lipid which is most prone to trigger low blood glucose throughout the body in the first place... You could then end up thinking that the endocannabinoids "just happen" to control appetite as a primary function. Of course, once the control system is in place you are all set to mess with it by demonising saturated fat and pushing corn and soy oils.
Well, I find the concept hysterical. But then I find most brain centred obesity research pretty amusing.
Peter
Thursday, November 15, 2012
Protons: SCD1 and the bomb
Our daughter has taken to watching DVDs. When we are not being tortured by Postman Pat (Special Delivery Serrrrrrviccccce, you know the tune) or the Mickey Mouse Clubhousssse we do at least get some amusement when she requests that blistering LC comedy "A Matter of Loaf and Death", by Nick Park.
Just how funny you can make a story about an obese cereal killer (no typo, the subtitles specify cereal killer, I said Park is funny!), murdering bakers as revenge for her obesity ("Are you ballooning?") has to be seen to be appreciated. It's a lot more amusing than Postman Pat.
One of the funniest scenes is where Gromit cannot get rid of Paella's bomb. It's a direct tribute to the 1960s Batman scene where "Some days you just can't get rid of a bomb".
You know, with the ducks
and the nuns. Park has kitten-enhanced the nuns
and included Yorkshire as the preferred site for bomb disposal. The Wars of the Roses are, apparently, over but not forgotten.
This post is about how physiology uses SCD1 to dispose of the metabolic bomb of hyperglycaemia in the presence elevated levels of palmitic acid.
It was pancreatic beta cells in culture which produced this picture:
I love this group because not only do they tell you in the methods section EXACTLY what glucose concentration they used in culture (5mmol/l vs 11-25mmol/l) without making you go back through three layers of references (to bury the 25mmol/l most groups use, but never discuss), but they also describe 11mmol/l as hyperglycaemia. That is, pathology.
This is Figure 4 from the same paper showing markers of apoptosis, superb:
Note the increase from palmitate to stearate. Note the complete protective effect of oleic acid and very modest toxic effect of linoleic acid. Aside: Note also the complete and total protection provided by limiting glucose to 5mmol/l, with any fatty acid, at any concentration. You have adipocytes leaking FFAs? Your best hope of keeping a functional pancreas is to limit your glucose to 5mmol/l. How? Answers on a postage stamp to...
It's also worth noting that stearic acid had to be reduced from the original 0.4mmol/l to 0.25mmol/l because at the higher concentration with high glucose they found exactly the same thing as Dave Lister did in Red Dwarf when Holly brought him out of stasis. Everyone is dead Dave. Everyone. Is. Dead. Dave. You have to U-tube the clip. Stearic acid at 0.4mmol/l with glucose at 25mmol/l, in cell culture, is utterly lethal to beta cells. If you pharmacologically block apoptosis the cells simply undergo the rather messy collapse of necrosis. This is a non survival insult.
Okay, okay, here's the clip:
The group went on to do quite intersting things with blockade of acyl-CoA synthetase and also with inhibition of fatty acid oxidation, which leads to all sorts of other threads which are, in part, where I have been wandering for the last few weeks. Far too much for this post.
So let's look at SCD1 knockout mice which have been rendered obese by also knocking out their leptin gene. Here we have rapid onset obesity due to adipocyte fat storage, free fatty acid leakage due to adipocyte insulin resistance and a complete inability to place a double bond in to palmitic acid or stearic acid. They have elevated FFAs and these are almost all saturated. This paper describes the study. It has to be noted that to obtain the FFA levels you have to reverse engineer Fig4 part A:
A ruler and calculator gives FFAs for the normal ob/ob mice as 0.32mmol/l and for the SCD1 k/o ob/ob mice as 0.56mmol/l. Any group which makes you reverse engineer in this way to get something as simple as FFA levels is, in my book, highly suspect. Does anyone think that 0.32mmol/l is quite low? Despite the greater obesity. Partly due to maintained insulin sensitivity in adipocytes (that's why they distend) while ever de novo lipogenesis produces palmitoleate using SCD1 and partly due to the higher levels of insulin production (normal beta cell mass) working on those insulin sensitive adipocytes... These mice are still in a slightly privileged position, metabolically, as they have yet to become obese enough for their SD1 equipped adipocytes to become seriously insulin resistant, they are still only six weeks old.
And here is the % of types of FFAs.
The column on the left is the one which represents about 0.32mmol/l of total FFAs and the column on the right is around 0.56mmol/l, as above. Glucose varies but fasting levels can be as high a 700mg/dl. So what happens to beta cells?
They divide up in to two types. The health ones and the dying ones.
The basic finding is that young ob/ob mice need either oleic or palmitoleic acid to maintain a functional beta cell mass. Exposure to high levels of glucose combined with palmitic and/or stearic acids induces apoptosis plus some necrosis in beta cells. Most non pancreatic tissues in the SCD1 knock out mice appear to be able to upregulate beta oxidation, especially in peroxisomes, of fatty acids which minimises both obesity and insulin resistance.
The beta cells of the pancreas do not appear to have this luxury.
They need to lower that F:N ratio with palmitoleate or oleate, otherwise they are left holding the bomb.
Peter
Just how funny you can make a story about an obese cereal killer (no typo, the subtitles specify cereal killer, I said Park is funny!), murdering bakers as revenge for her obesity ("Are you ballooning?") has to be seen to be appreciated. It's a lot more amusing than Postman Pat.
One of the funniest scenes is where Gromit cannot get rid of Paella's bomb. It's a direct tribute to the 1960s Batman scene where "Some days you just can't get rid of a bomb".
You know, with the ducks
and the nuns. Park has kitten-enhanced the nuns
and included Yorkshire as the preferred site for bomb disposal. The Wars of the Roses are, apparently, over but not forgotten.
This post is about how physiology uses SCD1 to dispose of the metabolic bomb of hyperglycaemia in the presence elevated levels of palmitic acid.
It was pancreatic beta cells in culture which produced this picture:
I love this group because not only do they tell you in the methods section EXACTLY what glucose concentration they used in culture (5mmol/l vs 11-25mmol/l) without making you go back through three layers of references (to bury the 25mmol/l most groups use, but never discuss), but they also describe 11mmol/l as hyperglycaemia. That is, pathology.
This is Figure 4 from the same paper showing markers of apoptosis, superb:
Note the increase from palmitate to stearate. Note the complete protective effect of oleic acid and very modest toxic effect of linoleic acid. Aside: Note also the complete and total protection provided by limiting glucose to 5mmol/l, with any fatty acid, at any concentration. You have adipocytes leaking FFAs? Your best hope of keeping a functional pancreas is to limit your glucose to 5mmol/l. How? Answers on a postage stamp to...
It's also worth noting that stearic acid had to be reduced from the original 0.4mmol/l to 0.25mmol/l because at the higher concentration with high glucose they found exactly the same thing as Dave Lister did in Red Dwarf when Holly brought him out of stasis. Everyone is dead Dave. Everyone. Is. Dead. Dave. You have to U-tube the clip. Stearic acid at 0.4mmol/l with glucose at 25mmol/l, in cell culture, is utterly lethal to beta cells. If you pharmacologically block apoptosis the cells simply undergo the rather messy collapse of necrosis. This is a non survival insult.
Okay, okay, here's the clip:
The group went on to do quite intersting things with blockade of acyl-CoA synthetase and also with inhibition of fatty acid oxidation, which leads to all sorts of other threads which are, in part, where I have been wandering for the last few weeks. Far too much for this post.
So let's look at SCD1 knockout mice which have been rendered obese by also knocking out their leptin gene. Here we have rapid onset obesity due to adipocyte fat storage, free fatty acid leakage due to adipocyte insulin resistance and a complete inability to place a double bond in to palmitic acid or stearic acid. They have elevated FFAs and these are almost all saturated. This paper describes the study. It has to be noted that to obtain the FFA levels you have to reverse engineer Fig4 part A:
A ruler and calculator gives FFAs for the normal ob/ob mice as 0.32mmol/l and for the SCD1 k/o ob/ob mice as 0.56mmol/l. Any group which makes you reverse engineer in this way to get something as simple as FFA levels is, in my book, highly suspect. Does anyone think that 0.32mmol/l is quite low? Despite the greater obesity. Partly due to maintained insulin sensitivity in adipocytes (that's why they distend) while ever de novo lipogenesis produces palmitoleate using SCD1 and partly due to the higher levels of insulin production (normal beta cell mass) working on those insulin sensitive adipocytes... These mice are still in a slightly privileged position, metabolically, as they have yet to become obese enough for their SD1 equipped adipocytes to become seriously insulin resistant, they are still only six weeks old.
And here is the % of types of FFAs.
The column on the left is the one which represents about 0.32mmol/l of total FFAs and the column on the right is around 0.56mmol/l, as above. Glucose varies but fasting levels can be as high a 700mg/dl. So what happens to beta cells?
They divide up in to two types. The health ones and the dying ones.
The basic finding is that young ob/ob mice need either oleic or palmitoleic acid to maintain a functional beta cell mass. Exposure to high levels of glucose combined with palmitic and/or stearic acids induces apoptosis plus some necrosis in beta cells. Most non pancreatic tissues in the SCD1 knock out mice appear to be able to upregulate beta oxidation, especially in peroxisomes, of fatty acids which minimises both obesity and insulin resistance.
The beta cells of the pancreas do not appear to have this luxury.
They need to lower that F:N ratio with palmitoleate or oleate, otherwise they are left holding the bomb.
Peter
Tuesday, November 06, 2012
Dalcetrapib fails as it should
Still no time to post but this one liner is another gem. Dalcetrapib, another (yawn) HDL raising drug has bombed. Who is surprised?
Funnily enough Dr Nissen (Rentaquote-you-have-a-statin-deficiency) feels dacletrapib failed because it was too weedy to do any good, only a 30% increase in HDL.
Evacetrapib (Nissen's gravy train) and anacetrapib will REALLY work because they double HDL and slash LDL.
No they won't. They will do exactly what torcetrapib did as they are as potent as torcetrapib was. Body count will rise. Dalceptrapib killed no one as it is not potent enough to do so!
Peter
Funnily enough Dr Nissen (Rentaquote-you-have-a-statin-deficiency) feels dacletrapib failed because it was too weedy to do any good, only a 30% increase in HDL.
Evacetrapib (Nissen's gravy train) and anacetrapib will REALLY work because they double HDL and slash LDL.
No they won't. They will do exactly what torcetrapib did as they are as potent as torcetrapib was. Body count will rise. Dalceptrapib killed no one as it is not potent enough to do so!
Peter
Monday, October 22, 2012
Skirting around leptin
I've been wanting to post about Wallace and Gromit, Batman and ob/ob SCD1 k/o mice for weeks now and it keeps not happening. Before we go there, just a word or two about leptin and weight gain. You can't work through anything relating to ob/ob mice (+/- SCD1 k/o) without having to, finally, sit down and read something about leptin. Or at least ob/ob mice...
To me the core question to ask is whether ob/ob mice are gaining weight because they have a brain disorder giving overeating or an adipocyte disorder storing calories. As always, there is an infinite supply of data suggesting a brain disorder, there's no denying leptin does things in the brain. The question is: Are ob/ob mice in caloric excess as they gain weight? ie Do they eat too much so gain weight or do they primarily lose calories in to their adipocytes and so have to eat more to just meet metabolic needs?
We have various rodent models of obesity which have the common feature of reducing the sympathetic nervous system drive to adipocytes, so failing to oppose insulin's lipogenic action. These animals gain fat even if you calorie restrict them. I'm thinking about hypothalamic ice-picks, various VMH neurotoxins or unprotected free radical generation. But the common thread is the loss of sympathetic nervous system driven lipolysis, facilitating insulin driven fat storage.
When confronted with the overwhelming literature on leptin it's hard to know where to start, especially when people are not asking the sorts of question which interest me, looking at the data from my very particular perspective.
I accept that ob/ob mice get fat. So too do brain injured rats and mice. Is there a common mechanism here? The smoking gun would be a period of enhanced insulin sensitivity in adipocytes, due to decreased sympathetic tone, which allows both fat gain and the preservation of insulin sensitivity in the early weeks, until adipocyte distension induced insulin resistance kicks in for the swelling adipocytes and systemic insulin resistance develops.
Of course the easy part, with absolute leptin deficiency, is asking whether leptin increases hypothalamic sympathetic nervous system outflow. That took about 30 seconds on pubmed and this was the 6th or 7th hit.
Leptin increases sympathetic drive. I think it's reasonable to conclude leptin deficiency does the converse and reduces sympathetic drive from the hypothalamus. So I'll take that as a yes. Don't forget I'm biased.
Sooooooo. Does leptin deficiency defend insulin sensitivity during rapid weight gain? As it should if the mechanism is enhanced lipid storage. And weight gain in young ob/ob mice is, well, rapid. To say the least. There will only be a very narrow window to pick up preserved insulin sensitivity before adipocyte insulin resistance and hyperinsulinaemia set in. By which time researchers have a usable model of established obesity.
I'm interested in what goes on before the model becomes "usable". We know that by rewarding volunteers to overeat we can spike their insulin levels massively within three days, probably faster. Does this happen with ob/ob mice as they over eat?
I've been working through a whole stack of papers on ob/ob mice, FFA levels, insulin levels, ketogenic diets... All the usual stuff. I ended up in a review by Lindström, giving this little gem of a quote:
"The muscle insulin resistance is not observed in very young [ob/ob] mice, but develops after 3–4 weeks [131]"
The abstract of ref 131 supports the concept of preserved insulin sensitivity, looking at muscle rather than adipocyte insulin resistance. The papers on palmitoleate as a lipokine suggest that muscle insulin resistance is controlled by adipocyte insulin resistance, via SCD1 and palmitoleate. The paper is rather nice because it is looking at ob/ob mice as ob/ob mice, not as some completely inappropriate model for hyperleptinaemic obese humans. It was, after all, 1980 when it was published.
So to summarise:
Danish volunteers who are paid to overeat spike insulin from 35pmol/l to 74pmol/l in just three days. Mice with zero leptin overeat massively, but do not show the same insulin spike. The insulin spike signifies insulin resistance, that characteristic antioxidant defence response to an excess of calories in the metabolic milieu. This does not happen with the early overeating phase of ob/ob mice. They are in metabolic caloric deficit, which they make up by eating enough to remain vaguely functional.
Absolute leptin deficiency appears to be a very harsh driver of fat storage. Losing this many calories makes you hungry. I guess some bit of the brain is involved in converting this state of actual calorie deficit in to a feeling of hunger, but that's not what interests me nearly as much as what is happening at the adipocyte level of calorie storage.
Peter
Now we can get on to Wallace and Gromit and knocking out SCD1 in ob/ob mice.
To me the core question to ask is whether ob/ob mice are gaining weight because they have a brain disorder giving overeating or an adipocyte disorder storing calories. As always, there is an infinite supply of data suggesting a brain disorder, there's no denying leptin does things in the brain. The question is: Are ob/ob mice in caloric excess as they gain weight? ie Do they eat too much so gain weight or do they primarily lose calories in to their adipocytes and so have to eat more to just meet metabolic needs?
We have various rodent models of obesity which have the common feature of reducing the sympathetic nervous system drive to adipocytes, so failing to oppose insulin's lipogenic action. These animals gain fat even if you calorie restrict them. I'm thinking about hypothalamic ice-picks, various VMH neurotoxins or unprotected free radical generation. But the common thread is the loss of sympathetic nervous system driven lipolysis, facilitating insulin driven fat storage.
When confronted with the overwhelming literature on leptin it's hard to know where to start, especially when people are not asking the sorts of question which interest me, looking at the data from my very particular perspective.
I accept that ob/ob mice get fat. So too do brain injured rats and mice. Is there a common mechanism here? The smoking gun would be a period of enhanced insulin sensitivity in adipocytes, due to decreased sympathetic tone, which allows both fat gain and the preservation of insulin sensitivity in the early weeks, until adipocyte distension induced insulin resistance kicks in for the swelling adipocytes and systemic insulin resistance develops.
Of course the easy part, with absolute leptin deficiency, is asking whether leptin increases hypothalamic sympathetic nervous system outflow. That took about 30 seconds on pubmed and this was the 6th or 7th hit.
Leptin increases sympathetic drive. I think it's reasonable to conclude leptin deficiency does the converse and reduces sympathetic drive from the hypothalamus. So I'll take that as a yes. Don't forget I'm biased.
Sooooooo. Does leptin deficiency defend insulin sensitivity during rapid weight gain? As it should if the mechanism is enhanced lipid storage. And weight gain in young ob/ob mice is, well, rapid. To say the least. There will only be a very narrow window to pick up preserved insulin sensitivity before adipocyte insulin resistance and hyperinsulinaemia set in. By which time researchers have a usable model of established obesity.
I'm interested in what goes on before the model becomes "usable". We know that by rewarding volunteers to overeat we can spike their insulin levels massively within three days, probably faster. Does this happen with ob/ob mice as they over eat?
I've been working through a whole stack of papers on ob/ob mice, FFA levels, insulin levels, ketogenic diets... All the usual stuff. I ended up in a review by Lindström, giving this little gem of a quote:
"The muscle insulin resistance is not observed in very young [ob/ob] mice, but develops after 3–4 weeks [131]"
The abstract of ref 131 supports the concept of preserved insulin sensitivity, looking at muscle rather than adipocyte insulin resistance. The papers on palmitoleate as a lipokine suggest that muscle insulin resistance is controlled by adipocyte insulin resistance, via SCD1 and palmitoleate. The paper is rather nice because it is looking at ob/ob mice as ob/ob mice, not as some completely inappropriate model for hyperleptinaemic obese humans. It was, after all, 1980 when it was published.
So to summarise:
Danish volunteers who are paid to overeat spike insulin from 35pmol/l to 74pmol/l in just three days. Mice with zero leptin overeat massively, but do not show the same insulin spike. The insulin spike signifies insulin resistance, that characteristic antioxidant defence response to an excess of calories in the metabolic milieu. This does not happen with the early overeating phase of ob/ob mice. They are in metabolic caloric deficit, which they make up by eating enough to remain vaguely functional.
Absolute leptin deficiency appears to be a very harsh driver of fat storage. Losing this many calories makes you hungry. I guess some bit of the brain is involved in converting this state of actual calorie deficit in to a feeling of hunger, but that's not what interests me nearly as much as what is happening at the adipocyte level of calorie storage.
Peter
Now we can get on to Wallace and Gromit and knocking out SCD1 in ob/ob mice.
Saturday, October 20, 2012
Protons: Love your superoxide (outside your brain)
Two off topic posts in a day! How come? I had the weirdest morning today. A three hour consulting session with only six appointments, all straight forward. Bloody hell, was I lucky for a Saturday! Can't blog at work so I had a quick browse to see what Nick Lane has been up to recently. He has a cracking article up (as a pdf) on heteroplasmy which rewards careful reading in its own right, but look at these two "throw away" quotes.
First on ROS, good old superoxide from reverse electron transport:
"ROS leak seems to optimize ATP synthesis by stimulating mitochondrial biogenesis (mtDNA copy number), an interpretation supported by the fact that antioxidants lower not only ROS leak but also mtDNA copy number and ATP synthesis. ROS leak, in effect, signals low capacity relative to demand, stimulating compensatory mitochondrial biogenesis".
How do we minimise mitochondrial biogenesis? By running metabolism on glucose of course, but don't forget the lack of superoxide generation when oxidising PUFA. But who needs mitochondria when you can lower LDL levels by swilling corn oil? Ah cardiology, you have a lot to answer for. Executive summary: Want mitochondria? Burn PALIMTATE.
And on cerebral metabolism:
"In the brain, where further mtDNA biogenesis is limited, neurons would then become compromised whenever energy demands were high, possibly causing acute cognitive and behavioral abnormalities".
The brain neurons are running on lactate under crapinabag conditions. We considered this before. No fatty acids. No glycerol 3 phosphate. No FADH2 input to the CoQ couple. No free radicals. No signal for mitochondrial biogenesis. No mitochondrial biogenesis. You could substitute ketones and maybe get a few mitochondria back if you were canny, but most medics aren't canny. What happens to the lactate supply for neurons when hyperglycaemia drops on to chronically elevated FFAs and triggers apoptosis in glial cells? I think we can attach various labels, depending on which neuronal cell types die first. Alzheimers seems a nice name for the commonest scenario.
Lovely pair of quotes. Glad I got the browse time. But don't ignore the heteroplasmy discussion at the core of the article, it's good stuff.
Peter
First on ROS, good old superoxide from reverse electron transport:
"ROS leak seems to optimize ATP synthesis by stimulating mitochondrial biogenesis (mtDNA copy number), an interpretation supported by the fact that antioxidants lower not only ROS leak but also mtDNA copy number and ATP synthesis. ROS leak, in effect, signals low capacity relative to demand, stimulating compensatory mitochondrial biogenesis".
How do we minimise mitochondrial biogenesis? By running metabolism on glucose of course, but don't forget the lack of superoxide generation when oxidising PUFA. But who needs mitochondria when you can lower LDL levels by swilling corn oil? Ah cardiology, you have a lot to answer for. Executive summary: Want mitochondria? Burn PALIMTATE.
And on cerebral metabolism:
"In the brain, where further mtDNA biogenesis is limited, neurons would then become compromised whenever energy demands were high, possibly causing acute cognitive and behavioral abnormalities".
The brain neurons are running on lactate under crapinabag conditions. We considered this before. No fatty acids. No glycerol 3 phosphate. No FADH2 input to the CoQ couple. No free radicals. No signal for mitochondrial biogenesis. No mitochondrial biogenesis. You could substitute ketones and maybe get a few mitochondria back if you were canny, but most medics aren't canny. What happens to the lactate supply for neurons when hyperglycaemia drops on to chronically elevated FFAs and triggers apoptosis in glial cells? I think we can attach various labels, depending on which neuronal cell types die first. Alzheimers seems a nice name for the commonest scenario.
Lovely pair of quotes. Glad I got the browse time. But don't ignore the heteroplasmy discussion at the core of the article, it's good stuff.
Peter
Look AHEAD trial stopped
Eat less, move more, have your heart attack on time!
With apologies for lack of any attention to the blog recently (which may be set to continue for some time) but this snippet just had to get passed on. This link from Karl:
http://www.theheart.org/article/1458351.do
More info here
http://www.nih.gov/news/health/oct2012/niddk-19.htm
And the glowing anticipation of success here from the planning stage:
https://www.lookaheadtrial.org/public/home.cfm
Pubmed gives a series of genuine success stories from the early days on all sorts of parameters. But the cardiovascular end points show how utterly useless these interventions are long term.
However the massive omission, from the quick look I've managed, is of any intention to report the all cause mortality. It seems very likely to me that more people died in the intervention group than in the usual care group, but p was > 0.05.
Call me a cynic, but I think they stopped the trial because they could see where that p number was heading. Has anyone seen a body count from anywhere in the trial?
Also, what might the outcome have been if the intervention group had been repeatedly bullied, harassed and indoctrinated to maintain a normoglycaemic, low grade ketogenic diet for 13.5 years? Say to an HbA1c of around 5%?
Ha ha ha bloody ha.
Peter
EDIT: Have started on the SCD1 k/o ob/ob mice. The thread WILL continue.
With apologies for lack of any attention to the blog recently (which may be set to continue for some time) but this snippet just had to get passed on. This link from Karl:
http://www.theheart.org/article/1458351.do
More info here
http://www.nih.gov/news/health/oct2012/niddk-19.htm
And the glowing anticipation of success here from the planning stage:
https://www.lookaheadtrial.org/public/home.cfm
Pubmed gives a series of genuine success stories from the early days on all sorts of parameters. But the cardiovascular end points show how utterly useless these interventions are long term.
However the massive omission, from the quick look I've managed, is of any intention to report the all cause mortality. It seems very likely to me that more people died in the intervention group than in the usual care group, but p was > 0.05.
Call me a cynic, but I think they stopped the trial because they could see where that p number was heading. Has anyone seen a body count from anywhere in the trial?
Also, what might the outcome have been if the intervention group had been repeatedly bullied, harassed and indoctrinated to maintain a normoglycaemic, low grade ketogenic diet for 13.5 years? Say to an HbA1c of around 5%?
Ha ha ha bloody ha.
Peter
EDIT: Have started on the SCD1 k/o ob/ob mice. The thread WILL continue.
Wednesday, October 03, 2012
Protons: Zero fat
A bit speculative here, read with caution!
How do we lower free fatty acids? Obviously, with nicotinic acid. What does this do to insulin secretion in response to a glucose challenge? I'll just work through this figure from the same paper which gave us the insulinotropic effects of various FFAs a couple of posts ago.
Section A is very simple, it just shows that they succeeded in clamping glucose at just over 200mg/dl, about 12mmol/l, ie just in to supraphysiological levels.
Section B shows FFA levels, which they manipulated very carefully. All rats started at about 0.6mmol/l. Nicotinic acid lowered FFA levels to 0.1mmol/l. These are the black squares. Two other intervention groups were included. The white triangles had their lipolysis shut down using nicotinic acid but then had FFAs clamped back up again using a soyabean oil infusion (mostly omega 6 PUFA) and the black triangle group had an infusion of lard based lipids (a mix of lipids but with a significant palmitic acid content) to restore and hold FFAs at about 0.8mmol/l.
The nicotinic acid group, with FFAs of 0.1mmol/l, cannot secrete insulin in response to glucose. Flat line at the bottom of graph C.
The open squares are the control group. These rats show the normal response to an hyperglycaemic clamp. They reduce FFAs in response to the inhibition of lipolysis from secreted insulin, down to 0.2mmol/l. Insulin inhibits lipolysis. But the reduced FFAs also reduce insulin secretion. There is a balance struck with only a modest rise in insulin, sustained throughout the clamp. You can see this in section C, open squares.
The two lipid infused groups have clamped glucose and clamped FFAs. They secrete insulin in proportion to the amount of palmitate in the lipid infusion. A bit extra over control if you use low F:N ratio omega 6 PUFA, a ton extra when you include some palmitate. Section D is simply a summary of this.
Step by step at the mitochondrial level: The lower fatty acid supply results in decrease reduction of the CoQ couple in beta cells. This reduces the reverse electron transport and associated superoxide triggered by glucose as it feeds NADH in to complex I, so limits insulin secretion. You can virtually ablate the insulin response to glucose by eliminating beta cell fatty acid supply.
Now, nicotinic acid is one way of reducing FFAs. There have to be other, perhaps more physiological, methods. Maybe we could use insulin per se? From food perhaps? Let's try eating around 40g of carbohydrate and look at the Spanish study graph again. Insulin rises from 50pmol/l to 75pmol/l. This is enough to reduce FFAs from 0.5mmol/l to just over 0.1mmol/l. Look at the FFAs, especially the circles between 120 and 300 minutes:
Now (again, sorry!) look carefully at the insulin levels after the small carb load, bottom circles.
By 180 minutes insulin is actually lower than fasting, and FFAs are still well below fasting levels too. The rat model appears to hold in humans, not what the study was looking at, and a small effect. But I think the effect is real.
How about scaling this up to a massive dose of potato induced insulin and limiting dietary fat? Severely limiting dietary fat. And never mind pussy footing around at 40g of mixed carbs and protein. There is a limit to how low FFAs can be driven, and it seems safe to assume that a baked potato or three might just inhibit lipolysis maximally and keep it that low for rather a long time. But if you deprive beta cells of free fatty acids you blunt their ability to secrete insulin. Very, very high carbohydrate diets really ought to be able to inhibit lipolysis to the point where the knock on effect is the inhibition of insulin secretion, provided you don't supply exogenous fat. Look at the nicotinic acid treated rats...
Once you get FFA levels low enough to inhibit insulin secretion you will start to move in to the sort of territory where insulin secretion might be blunted enough to allow hyperglycaemia. But the feedback effect of reduced insulin levels is also the re commencement of lipolysis. This will restore enough FFAs to maintain functional insulin secretion and so avoid potential hyperglycaemia, which the body tries to avoid. Of course you have to throw in the increased insulin sensitivity of muscles deprived of exogenously supplied FFAs too.
So is it possible to eat an ad lib, calorie unrestricted diet based on near pure carbohydrate and lose weight? Working from the premise that lowered insulin is a pre requisite for hunger free weight loss, as I always do, the answer is possibly yes. We all remember Chris Voight on his all potato diet (plus 20ml of olive oil, low in palmitate, per day) who lost a great deal of weight over a few weeks, the rate of weight loss accelerating as the weeks progressed? I had a think about it here, well before I had any inkling as to what might be happening in the electron transport chain.
We need to know what the interaction of insulin and FFAs was during this particular n=1 self experiment, and we don't. The rats suggest to me that insulin levels were initially raised post prandially and FFAs were not then available from peripheral adipocytes. Assuming the fall in lipolysis persisted in to the post-absorptive period (the primary function of insulin, especially at low levels, is the inhibition of lipolysis rather than facilitation of glucose diffusion, we've all read Zierler and Rabinowitz) we have a method for limiting insulin secretion late post prandially using reduced free fatty acid levels.
As an aside I personally wonder it might be the ectopic lipid supplies typically found in muscle, liver and visceral adipocytes which might still be available for metabolism by the tissues when exogenous supplies are shut down. It reminds me of how metformin most likely depletes ectopic lipid to improve insulin sensitivity, despite having complex I inhibition as its primary action. You need lipid from somewhere. So reducing FFA supply by inhibiting systemic lipolysis may well be a route to lower fasting insulin levels. Especially if you are not far in to metabolic syndrome.
Once ectopic lipid becomes depleted then lipolysis would accelerate in peripheral adipocytes as systemic insulin resistance falls and fasting insulin levels too, which might be what was reported as progressively increasing weight loss by Chris Voight. Insulin levels would be low, especially during fasting, and appetite low at the same time due to hypoinsulinaemia facilitated lipolysis, much as appetite is low under LC induced hypoinsulinaemic eating. There is more than one way to skin a.... Oops let's not complete that phrase!
What would happen to a healthy person under these conditions, long term, is anyone's guess. Chis Voight gave up after a few weeks when weight loss became alarmingly rapid. But we know from the crucial study by the vegan apologist Barnard that, for diabetic people at least, that a long term, whole food, low sucrose and low fat diet is a complete disaster, once the initial weight loss ceases.
This is playing with fire (possibly near literally, at the mitochondrial level) if you are a diabetic. Please don't go there.
But the physiology of weight loss on ultra low fat diets is basically comprehensible, especially once you look at lipids and superoxide at the ETC level, and what the body needs to function effectively. Running your metabolism on pure glucose would induce, theoretically, an infinite glucose sensitivity and low fasting insulin. If we do reductio ad absurdum you would end up with no fat stores and experience death from hypoglycaemia if you ever depleted your glycogen stores. Mitochondria like (saturated) fatty acids. Fatty acids keep them in control.
I think someone in obesity research used Chris Voight's experience to support some cock and bull story about food reward and a set point of body fat. We can wait for the recant on that one, if you could care less about it. The biochemistry is, as always, the fascinating stuff.
Peter
How do we lower free fatty acids? Obviously, with nicotinic acid. What does this do to insulin secretion in response to a glucose challenge? I'll just work through this figure from the same paper which gave us the insulinotropic effects of various FFAs a couple of posts ago.
Section A is very simple, it just shows that they succeeded in clamping glucose at just over 200mg/dl, about 12mmol/l, ie just in to supraphysiological levels.
Section B shows FFA levels, which they manipulated very carefully. All rats started at about 0.6mmol/l. Nicotinic acid lowered FFA levels to 0.1mmol/l. These are the black squares. Two other intervention groups were included. The white triangles had their lipolysis shut down using nicotinic acid but then had FFAs clamped back up again using a soyabean oil infusion (mostly omega 6 PUFA) and the black triangle group had an infusion of lard based lipids (a mix of lipids but with a significant palmitic acid content) to restore and hold FFAs at about 0.8mmol/l.
The nicotinic acid group, with FFAs of 0.1mmol/l, cannot secrete insulin in response to glucose. Flat line at the bottom of graph C.
The open squares are the control group. These rats show the normal response to an hyperglycaemic clamp. They reduce FFAs in response to the inhibition of lipolysis from secreted insulin, down to 0.2mmol/l. Insulin inhibits lipolysis. But the reduced FFAs also reduce insulin secretion. There is a balance struck with only a modest rise in insulin, sustained throughout the clamp. You can see this in section C, open squares.
The two lipid infused groups have clamped glucose and clamped FFAs. They secrete insulin in proportion to the amount of palmitate in the lipid infusion. A bit extra over control if you use low F:N ratio omega 6 PUFA, a ton extra when you include some palmitate. Section D is simply a summary of this.
Step by step at the mitochondrial level: The lower fatty acid supply results in decrease reduction of the CoQ couple in beta cells. This reduces the reverse electron transport and associated superoxide triggered by glucose as it feeds NADH in to complex I, so limits insulin secretion. You can virtually ablate the insulin response to glucose by eliminating beta cell fatty acid supply.
Now, nicotinic acid is one way of reducing FFAs. There have to be other, perhaps more physiological, methods. Maybe we could use insulin per se? From food perhaps? Let's try eating around 40g of carbohydrate and look at the Spanish study graph again. Insulin rises from 50pmol/l to 75pmol/l. This is enough to reduce FFAs from 0.5mmol/l to just over 0.1mmol/l. Look at the FFAs, especially the circles between 120 and 300 minutes:
Now (again, sorry!) look carefully at the insulin levels after the small carb load, bottom circles.
By 180 minutes insulin is actually lower than fasting, and FFAs are still well below fasting levels too. The rat model appears to hold in humans, not what the study was looking at, and a small effect. But I think the effect is real.
How about scaling this up to a massive dose of potato induced insulin and limiting dietary fat? Severely limiting dietary fat. And never mind pussy footing around at 40g of mixed carbs and protein. There is a limit to how low FFAs can be driven, and it seems safe to assume that a baked potato or three might just inhibit lipolysis maximally and keep it that low for rather a long time. But if you deprive beta cells of free fatty acids you blunt their ability to secrete insulin. Very, very high carbohydrate diets really ought to be able to inhibit lipolysis to the point where the knock on effect is the inhibition of insulin secretion, provided you don't supply exogenous fat. Look at the nicotinic acid treated rats...
Once you get FFA levels low enough to inhibit insulin secretion you will start to move in to the sort of territory where insulin secretion might be blunted enough to allow hyperglycaemia. But the feedback effect of reduced insulin levels is also the re commencement of lipolysis. This will restore enough FFAs to maintain functional insulin secretion and so avoid potential hyperglycaemia, which the body tries to avoid. Of course you have to throw in the increased insulin sensitivity of muscles deprived of exogenously supplied FFAs too.
So is it possible to eat an ad lib, calorie unrestricted diet based on near pure carbohydrate and lose weight? Working from the premise that lowered insulin is a pre requisite for hunger free weight loss, as I always do, the answer is possibly yes. We all remember Chris Voight on his all potato diet (plus 20ml of olive oil, low in palmitate, per day) who lost a great deal of weight over a few weeks, the rate of weight loss accelerating as the weeks progressed? I had a think about it here, well before I had any inkling as to what might be happening in the electron transport chain.
We need to know what the interaction of insulin and FFAs was during this particular n=1 self experiment, and we don't. The rats suggest to me that insulin levels were initially raised post prandially and FFAs were not then available from peripheral adipocytes. Assuming the fall in lipolysis persisted in to the post-absorptive period (the primary function of insulin, especially at low levels, is the inhibition of lipolysis rather than facilitation of glucose diffusion, we've all read Zierler and Rabinowitz) we have a method for limiting insulin secretion late post prandially using reduced free fatty acid levels.
As an aside I personally wonder it might be the ectopic lipid supplies typically found in muscle, liver and visceral adipocytes which might still be available for metabolism by the tissues when exogenous supplies are shut down. It reminds me of how metformin most likely depletes ectopic lipid to improve insulin sensitivity, despite having complex I inhibition as its primary action. You need lipid from somewhere. So reducing FFA supply by inhibiting systemic lipolysis may well be a route to lower fasting insulin levels. Especially if you are not far in to metabolic syndrome.
Once ectopic lipid becomes depleted then lipolysis would accelerate in peripheral adipocytes as systemic insulin resistance falls and fasting insulin levels too, which might be what was reported as progressively increasing weight loss by Chris Voight. Insulin levels would be low, especially during fasting, and appetite low at the same time due to hypoinsulinaemia facilitated lipolysis, much as appetite is low under LC induced hypoinsulinaemic eating. There is more than one way to skin a.... Oops let's not complete that phrase!
What would happen to a healthy person under these conditions, long term, is anyone's guess. Chis Voight gave up after a few weeks when weight loss became alarmingly rapid. But we know from the crucial study by the vegan apologist Barnard that, for diabetic people at least, that a long term, whole food, low sucrose and low fat diet is a complete disaster, once the initial weight loss ceases.
This is playing with fire (possibly near literally, at the mitochondrial level) if you are a diabetic. Please don't go there.
But the physiology of weight loss on ultra low fat diets is basically comprehensible, especially once you look at lipids and superoxide at the ETC level, and what the body needs to function effectively. Running your metabolism on pure glucose would induce, theoretically, an infinite glucose sensitivity and low fasting insulin. If we do reductio ad absurdum you would end up with no fat stores and experience death from hypoglycaemia if you ever depleted your glycogen stores. Mitochondria like (saturated) fatty acids. Fatty acids keep them in control.
I think someone in obesity research used Chris Voight's experience to support some cock and bull story about food reward and a set point of body fat. We can wait for the recant on that one, if you could care less about it. The biochemistry is, as always, the fascinating stuff.
Peter
Thursday, September 27, 2012
Never forget, never forgive
For recreational purposes only, from here:
How toxic is palmitate at any concentration from zero to 0.4mmol/l if glucose is held at 5mmol/l? It's not.
How toxic is glucose at 25mmol/l in the presence of increasing palmitate? Glucose at this concentration becomes progressively more toxic within increasing but still physiological levels of palmitate.
Glucose at 25mmol/l is UTTERLY non physiological.
There is potentially a huge amount to discuss from this paper, one day, maybe. Even though they never delve in to the ETC, which they should do. But anyway, someone commented that papers using 5mmol/l glucose in the culture medium were rare. This one is a hen's tooth.
Back to thread next.
Peter
How toxic is palmitate at any concentration from zero to 0.4mmol/l if glucose is held at 5mmol/l? It's not.
How toxic is glucose at 25mmol/l in the presence of increasing palmitate? Glucose at this concentration becomes progressively more toxic within increasing but still physiological levels of palmitate.
Glucose at 25mmol/l is UTTERLY non physiological.
There is potentially a huge amount to discuss from this paper, one day, maybe. Even though they never delve in to the ETC, which they should do. But anyway, someone commented that papers using 5mmol/l glucose in the culture medium were rare. This one is a hen's tooth.
Back to thread next.
Peter
Protons: The pancreas
We've seen the concept of superoxide being used to produce insulin resistance as a means of limiting (glucose derived) energy input in to cells which really don't want it. Superoxide appears to be the primary marker of energy excess at the cellular level.
We know from isolated mitochondrial preparations that superoxide is physiologically produced by reverse electron transport through complex I and is driven, gently, by succinic acid alone working through complex II. Far more is produced when the NADH level is high as well as having a reduced CoQ couple through FADH2 input, be that from complex II or from fatty acid oxidation products. Macroscopically fat and glucose together should produce enough superoxide to show as cellular insulin resistance, rejecting glucose from the cell, while allowing continued fatty acid oxidation. That's simple and logical.
But if you are building an energy sensor, it would be a bit dumb to restrict access to the very energy molecules which you are trying to look at to judge overall energy status, especially when energy status is high: You need to decide when to store calories...
The beta cells appear to use both fatty acids and glucose to generate superoxide, but instead of signaling beta cell insulin resistance, they signal insulin secretion. Several lines of evidence fit in with this.
You can get succinic acid itself directly in to beta cells by providing it as a methyl or ethyl ester. As a metabolic fuel source this acts as a near pure complex II substrate, pushing electrons in to the ETC through the FADH2 of succinate dehydrogenase to reduce the CoQ couple and set the scene for reverse electron transport and superoxide production, especially when NADH from glucose metabolism rises. In a commonly used model of functional beta cells, succinic acid methyl ester is a marked insulin secretion potentiator, especially at higher glucose concentrations. Glucose supplies NADH, succinate supplies FADH2, they clash at the CoQ couple and the generation of superoxide signals that there is a ton of energy available. Better store it. Better secrete insulin.
Succinic acid methyl ester drives complex II. This drives insulin secretion in response to glucose. But it's a drug. There is nothing physiological about this drug. So shall we go a little more physiological?
To recap from previous posts: Superoxide generation is directly proportional to the ratio of FADH2 generated to the amount of NADH generated for a given substrate, the F:N ratio.
Here's a nice graph of insulin secretion stimulated in response to 12.5mmol glucose on a background of assorted free fatty acids from an isolated pancreas preparation:
If you can't be bothered to work out the F:N ratios (shame on you), here they are added to the graph:
Please excuse the C8 value; as we all know, MCTs are shunted directly to the liver via the portal vein. They do not seem to feature too prominently in pancreatic superoxide generation and insulin secretion. It would take a ton of reading to see why and how they are handled differently to longer chain fatty acids. For the time being let's stay looking at C16 and longer as these make a much tidier story...
So, for the four longer fatty acids tested, the amount of insulin secreted is remarkably closely associated with the F:N ratio of the fatty acid available.
Does this work in people?
Of course it does. Remember the Spanish study? I lo0ked at it in some detail here.
In particular look at this graph:
From the top downwards we have butter, high palmitic acid seed oil, refined olive oil and a mix of fish and vegetable oils as the white triangles. It is very clear that the insulin secretion here is in direct proportion to the saturation and length of the fatty acids in the meal, in an intact group of volunteers..
Aside: Obviously, there is a glaring error in the graph. All of the curves except the control use 800kcal of total food, of which 40g is carbohydrate/protein and the bulk is fat. The graph is missing a group where 800kcal was supplied as pure carbohydrate. We can all imagine where this much bulk glucose would have put the insulin curve, needless to say there is absolutely no way it would fit on to the presented graph. We would need a much taller vertical axis, which would show the mixed meals in their true context!
But the principle, that insulin secretion at a given level of glucose is elevated in direct proportion to the F:N ratio of the background fat, holds perfectly well in this carefully contrived human study.
BTW, lucky for me they didn't include a coconut oil group!
The obvious conclusion from this finding is that to lower insulin maximally we should, taking as given that replacing carbohydrate with fat is the biggest step by far, all go for vegetable oil with some fish oil. Not butter. But in the original post on the Spanish paper I went on to discuss what appeared to be happening to the lipid from the meal. It could stay in the bloodstream and be used for metabolism, as the butter did, or it could be cleared rapidly in to adipocytes allowing metabolism to return to being glucose based. At the cost of expanding the adipocyte stores of fat.
The high PUFA meal really was rapidly stored as fat in adipocytes. The F:N based explanation is because we are supplying a low F:N ratio fat and so not generating insulin resistance phyiologically; we are allowing lipid easily in to adipocytes because the lipid does not generate adipocyte insulin resistance. We are going back to glucose metabolism as rapidly as possible. PUFA facillitates fat storage and glucose based metabolism. All is fine until you can't get any fatter. Butter limits fat storage and runs metabolism of palmitic and stearic acids. Those high PUFA-fed mice generate obesity when fed their high PUFA diets from pre-conception onwards:
In the butter group there is some excess insulin. Does this matter if no one (cellularly) is listening to it?
I next want to look at the flip side, the reduction of supply of free fatty acids to the pancreas. This was done in the same paper. You can certainly do this in intact rats (and humans if you so wish). Then we might get back to the fat mice.
I think that had better be another post as this one is getting overly long and it's light enough to let the chickens out.
Peter
We know from isolated mitochondrial preparations that superoxide is physiologically produced by reverse electron transport through complex I and is driven, gently, by succinic acid alone working through complex II. Far more is produced when the NADH level is high as well as having a reduced CoQ couple through FADH2 input, be that from complex II or from fatty acid oxidation products. Macroscopically fat and glucose together should produce enough superoxide to show as cellular insulin resistance, rejecting glucose from the cell, while allowing continued fatty acid oxidation. That's simple and logical.
But if you are building an energy sensor, it would be a bit dumb to restrict access to the very energy molecules which you are trying to look at to judge overall energy status, especially when energy status is high: You need to decide when to store calories...
The beta cells appear to use both fatty acids and glucose to generate superoxide, but instead of signaling beta cell insulin resistance, they signal insulin secretion. Several lines of evidence fit in with this.
You can get succinic acid itself directly in to beta cells by providing it as a methyl or ethyl ester. As a metabolic fuel source this acts as a near pure complex II substrate, pushing electrons in to the ETC through the FADH2 of succinate dehydrogenase to reduce the CoQ couple and set the scene for reverse electron transport and superoxide production, especially when NADH from glucose metabolism rises. In a commonly used model of functional beta cells, succinic acid methyl ester is a marked insulin secretion potentiator, especially at higher glucose concentrations. Glucose supplies NADH, succinate supplies FADH2, they clash at the CoQ couple and the generation of superoxide signals that there is a ton of energy available. Better store it. Better secrete insulin.
Succinic acid methyl ester drives complex II. This drives insulin secretion in response to glucose. But it's a drug. There is nothing physiological about this drug. So shall we go a little more physiological?
To recap from previous posts: Superoxide generation is directly proportional to the ratio of FADH2 generated to the amount of NADH generated for a given substrate, the F:N ratio.
Here's a nice graph of insulin secretion stimulated in response to 12.5mmol glucose on a background of assorted free fatty acids from an isolated pancreas preparation:
If you can't be bothered to work out the F:N ratios (shame on you), here they are added to the graph:
Please excuse the C8 value; as we all know, MCTs are shunted directly to the liver via the portal vein. They do not seem to feature too prominently in pancreatic superoxide generation and insulin secretion. It would take a ton of reading to see why and how they are handled differently to longer chain fatty acids. For the time being let's stay looking at C16 and longer as these make a much tidier story...
So, for the four longer fatty acids tested, the amount of insulin secreted is remarkably closely associated with the F:N ratio of the fatty acid available.
Does this work in people?
Of course it does. Remember the Spanish study? I lo0ked at it in some detail here.
In particular look at this graph:
From the top downwards we have butter, high palmitic acid seed oil, refined olive oil and a mix of fish and vegetable oils as the white triangles. It is very clear that the insulin secretion here is in direct proportion to the saturation and length of the fatty acids in the meal, in an intact group of volunteers..
Aside: Obviously, there is a glaring error in the graph. All of the curves except the control use 800kcal of total food, of which 40g is carbohydrate/protein and the bulk is fat. The graph is missing a group where 800kcal was supplied as pure carbohydrate. We can all imagine where this much bulk glucose would have put the insulin curve, needless to say there is absolutely no way it would fit on to the presented graph. We would need a much taller vertical axis, which would show the mixed meals in their true context!
But the principle, that insulin secretion at a given level of glucose is elevated in direct proportion to the F:N ratio of the background fat, holds perfectly well in this carefully contrived human study.
BTW, lucky for me they didn't include a coconut oil group!
The obvious conclusion from this finding is that to lower insulin maximally we should, taking as given that replacing carbohydrate with fat is the biggest step by far, all go for vegetable oil with some fish oil. Not butter. But in the original post on the Spanish paper I went on to discuss what appeared to be happening to the lipid from the meal. It could stay in the bloodstream and be used for metabolism, as the butter did, or it could be cleared rapidly in to adipocytes allowing metabolism to return to being glucose based. At the cost of expanding the adipocyte stores of fat.
The high PUFA meal really was rapidly stored as fat in adipocytes. The F:N based explanation is because we are supplying a low F:N ratio fat and so not generating insulin resistance phyiologically; we are allowing lipid easily in to adipocytes because the lipid does not generate adipocyte insulin resistance. We are going back to glucose metabolism as rapidly as possible. PUFA facillitates fat storage and glucose based metabolism. All is fine until you can't get any fatter. Butter limits fat storage and runs metabolism of palmitic and stearic acids. Those high PUFA-fed mice generate obesity when fed their high PUFA diets from pre-conception onwards:
In the butter group there is some excess insulin. Does this matter if no one (cellularly) is listening to it?
I next want to look at the flip side, the reduction of supply of free fatty acids to the pancreas. This was done in the same paper. You can certainly do this in intact rats (and humans if you so wish). Then we might get back to the fat mice.
I think that had better be another post as this one is getting overly long and it's light enough to let the chickens out.
Peter
Monday, September 17, 2012
Protons: Linoleic acid in the hypothalamus
Hi all, just getting my head above water now that we have two or three locums at work to cover some of the (rather difficult) gaps in the rota!
Before we look at the fat mouse study which wins the prize for most miserly hoarding of data, I just wanted to put up a brief post, based on that paper, about breaking your hypothalamus with a high fat diet. Just to re emphasis: This is NOT what happens to a human after 7 days on a high fat diet.
Remember Schwartz's rats? Put them on a high fat diet and this happens to food intake:
Note the very sudden and dramatic spike in the intake of food, shown by the red line which I've added to emphasise the abrupt change from baseline chow consumption. We can ignore the red oval for this post. What happens in the VMH neurons of these rats?
This is what happens:
The dark brown staining cells on the right are dying, the rats have been eating "cookie dough", which they "can't get enough of", for seven days. The nice healthy cells in the left hand photomicrograph are from rats on crapinabag. The basic idea appears to be that feeding rats a bit of fat and sugar makes them eat so much, starting in just one day, that by seven days their VMH is killed by over indulgence. You eat too much, you kill your brain. Simple. This is, of course, absolute bollocks.
At the risk of repetition, we can produce exactly the same lesions in the VMH with MSG or gold thioglucose (or an ice pick if you must be crude and don't want nice pictures). This injury results in fat gain which must be compensated for by overeating. Rats will gain weight more slowly if they are on low fat diets than on high fat diets because of the effects of increased de novo lipogenesis which I've discussed in previous posts.
Want pretty pictures from GTG injured rats? Here's some random immuno from a random paper, there's a lot of it around, only black and white though:
Gold thioglucose on the right, arrow marks the injury area. And I just noticed the same pics, also in black and white, from fat injured rats from elsewhere in the Schwartz paper (mirror imaged compared to the GTG pics, random choice of which side of brain got sectioned!) after just a week on their high fat diet:
So we can produce the pretty black stains of dying cells with gold thioglucose (or MSG if we looked at neonatal immuno) but this injury preceeds the loss of calories in to adipocytes and subsequent "hyperphagia". THE INJURY COMES FIRST.
Let's really look at the bizarre idea that non-forced "overeating" causes subsequent damages your VMH. This is how it works for over eating by a gold thioglucose injected rat, no yummie high fat diet needed: It simply decides to over eat crapinabag because this has suddenly and randomly become delicious and so it becomes obese. We all know overeating CAUSES the VMH injury in fat fed rodents. So how do GTG injured rats get the injury first and over eat secondarily? Gold thioglucose obese rodents might SEEM to have a chemical lesion causing obesity but clearly they get fat first, travel back in time (squeezing in to a time machine as obese chrononaughts) and retrospectively force the researchers to give them the injection of GTG to obtain the lesion in their VMH which they are going to produce in the future by eating too much crapinabag. Got that? You've all watched Back to the Future. I watched parts I and II but never managed part III. It's simple time travel. Ditto MSG and ice-pick (ouch!) obese rodents. Self inflicted injuries using time travel.
Or we could abandon such stupidity and say that high fat diets injure the VMH first and this injury increases fat storage by decreasing sympathetic tone to adipocytes, as it does.
And I suspect it's superoxide, generated by a high F:N ratio (classically derived from palmitic acid at an F:N ratio of 0.47) in POMC neurons, which probably does the damage. You all know POMC neurons, the ones in the VMH with both gluokinase to sense (via metablism) glucose and CD36 to monitor FFA status (via metabolism again). No lactate for the energy status sensing neurons of the VMH...
So the question is, as always, what happens to the VMH of a C57BL/6 mouse (bred to get fat on a high fat diet) when put on a high fat diet which does NOT generate superoxide in POMC neurons? You can do this.
No one has done the necessary immuno staining under these conditions to get the pretty pictures of dying (or non dying) cells, as far as I know. But it's easy to look at the weight gains, which are a reasonable surrogate for POMC injury. Schwartz again using rats:
Not the most lucid graph, but it gives the basic idea. The control weight gains on the left are comparable to the weight gains shown for day 14.
Now, here is what happens if you take a C57BL/6 mouse and put it on to 35% of calories from fat if you keep the F:N ratio of that fat well below 0.47, using omega 6 PUFA with an F:N ratio of 0.42, as the primary source of fat:
Ignore the top two lines (for now) and look at the weight gain of the mice in the bottom two lines. One group weaned on to crapinabag, the other weaned on to 35% of calories from fat, but a fat with a low F:N ratio. There is zero, zilch, nil difference in weight gain over three weeks. There is no excess weight because there is no VMH injury. No one generates significant superoxide from a low F:N ratio fat like linoleic acid. That appears to include the POMC neurons of C57BL/6 mice.
C57BL/6 mice (and Long Evans rats) are specifically bred to get fat on palmitic acid (sometimes plus fructose) based diets. They fail to deal with the absolutely normal levels of superoxide produced in POMC neurons in the VMH which are crucial to energy status sensing. They do not have the luxury of developing insulin resistance as their job is to monitor both glucose and fatty acid levels. They are not allowed to run on lactate with an F:N ratio of 0.2 the way much of the brain does. They take whatever plasma gives them and do their best to cope with it. Or, in the case of rodents bred to become fat on high fat diets, not cope with it.
Before we go looking at the linoleic acid paper a bit more carefully I think it's worth trying to look at energy sensing rather more peripherally than the POMC neurons of the VMH. Then we can come back to the fat mice and try to think about what's going on using the meagre data available. Because it's quite interesting.
Peter
Before we look at the fat mouse study which wins the prize for most miserly hoarding of data, I just wanted to put up a brief post, based on that paper, about breaking your hypothalamus with a high fat diet. Just to re emphasis: This is NOT what happens to a human after 7 days on a high fat diet.
Remember Schwartz's rats? Put them on a high fat diet and this happens to food intake:
Note the very sudden and dramatic spike in the intake of food, shown by the red line which I've added to emphasise the abrupt change from baseline chow consumption. We can ignore the red oval for this post. What happens in the VMH neurons of these rats?
This is what happens:
The dark brown staining cells on the right are dying, the rats have been eating "cookie dough", which they "can't get enough of", for seven days. The nice healthy cells in the left hand photomicrograph are from rats on crapinabag. The basic idea appears to be that feeding rats a bit of fat and sugar makes them eat so much, starting in just one day, that by seven days their VMH is killed by over indulgence. You eat too much, you kill your brain. Simple. This is, of course, absolute bollocks.
At the risk of repetition, we can produce exactly the same lesions in the VMH with MSG or gold thioglucose (or an ice pick if you must be crude and don't want nice pictures). This injury results in fat gain which must be compensated for by overeating. Rats will gain weight more slowly if they are on low fat diets than on high fat diets because of the effects of increased de novo lipogenesis which I've discussed in previous posts.
Want pretty pictures from GTG injured rats? Here's some random immuno from a random paper, there's a lot of it around, only black and white though:
Gold thioglucose on the right, arrow marks the injury area. And I just noticed the same pics, also in black and white, from fat injured rats from elsewhere in the Schwartz paper (mirror imaged compared to the GTG pics, random choice of which side of brain got sectioned!) after just a week on their high fat diet:
So we can produce the pretty black stains of dying cells with gold thioglucose (or MSG if we looked at neonatal immuno) but this injury preceeds the loss of calories in to adipocytes and subsequent "hyperphagia". THE INJURY COMES FIRST.
Let's really look at the bizarre idea that non-forced "overeating" causes subsequent damages your VMH. This is how it works for over eating by a gold thioglucose injected rat, no yummie high fat diet needed: It simply decides to over eat crapinabag because this has suddenly and randomly become delicious and so it becomes obese. We all know overeating CAUSES the VMH injury in fat fed rodents. So how do GTG injured rats get the injury first and over eat secondarily? Gold thioglucose obese rodents might SEEM to have a chemical lesion causing obesity but clearly they get fat first, travel back in time (squeezing in to a time machine as obese chrononaughts) and retrospectively force the researchers to give them the injection of GTG to obtain the lesion in their VMH which they are going to produce in the future by eating too much crapinabag. Got that? You've all watched Back to the Future. I watched parts I and II but never managed part III. It's simple time travel. Ditto MSG and ice-pick (ouch!) obese rodents. Self inflicted injuries using time travel.
Or we could abandon such stupidity and say that high fat diets injure the VMH first and this injury increases fat storage by decreasing sympathetic tone to adipocytes, as it does.
And I suspect it's superoxide, generated by a high F:N ratio (classically derived from palmitic acid at an F:N ratio of 0.47) in POMC neurons, which probably does the damage. You all know POMC neurons, the ones in the VMH with both gluokinase to sense (via metablism) glucose and CD36 to monitor FFA status (via metabolism again). No lactate for the energy status sensing neurons of the VMH...
So the question is, as always, what happens to the VMH of a C57BL/6 mouse (bred to get fat on a high fat diet) when put on a high fat diet which does NOT generate superoxide in POMC neurons? You can do this.
No one has done the necessary immuno staining under these conditions to get the pretty pictures of dying (or non dying) cells, as far as I know. But it's easy to look at the weight gains, which are a reasonable surrogate for POMC injury. Schwartz again using rats:
Not the most lucid graph, but it gives the basic idea. The control weight gains on the left are comparable to the weight gains shown for day 14.
Now, here is what happens if you take a C57BL/6 mouse and put it on to 35% of calories from fat if you keep the F:N ratio of that fat well below 0.47, using omega 6 PUFA with an F:N ratio of 0.42, as the primary source of fat:
Ignore the top two lines (for now) and look at the weight gain of the mice in the bottom two lines. One group weaned on to crapinabag, the other weaned on to 35% of calories from fat, but a fat with a low F:N ratio. There is zero, zilch, nil difference in weight gain over three weeks. There is no excess weight because there is no VMH injury. No one generates significant superoxide from a low F:N ratio fat like linoleic acid. That appears to include the POMC neurons of C57BL/6 mice.
C57BL/6 mice (and Long Evans rats) are specifically bred to get fat on palmitic acid (sometimes plus fructose) based diets. They fail to deal with the absolutely normal levels of superoxide produced in POMC neurons in the VMH which are crucial to energy status sensing. They do not have the luxury of developing insulin resistance as their job is to monitor both glucose and fatty acid levels. They are not allowed to run on lactate with an F:N ratio of 0.2 the way much of the brain does. They take whatever plasma gives them and do their best to cope with it. Or, in the case of rodents bred to become fat on high fat diets, not cope with it.
Before we go looking at the linoleic acid paper a bit more carefully I think it's worth trying to look at energy sensing rather more peripherally than the POMC neurons of the VMH. Then we can come back to the fat mice and try to think about what's going on using the meagre data available. Because it's quite interesting.
Peter
Thursday, August 30, 2012
Guess the weight of the mouse competition
Here's the Brownie points quiz.
What is the weight of either mouse?
If you find the answer, in this study, please let we know!
Peter
BTW you are allowed/obliged to scour results, discussion and all supplementary data. Please.
Saturday, August 25, 2012
Protons: SCD1 knockout mice
Let's peep inside an adipocyte belonging to a mouse which has had its stearoyl-CoA desaturase gene deleted.
It's busy making lipid, being an adipocyte. Two carbons, four carbons, six, eight, ten, twelve, fourteen and hey, there's the sixteen for palmitic acid. Now, how much glucose and insulin is there around? Ah, lots. Need to signal this with palmitoleate. In goes the double bond to prove it... Oops. No SCD1. Hmmmm. We now have a ton of palmitic acid and no chance to convert any of it to palmitoleic acid. Tricky.
Is the adipocyte going to become insulin resistant? Unless it runs on glucose and never uses any lipid this seems likely. Will the adipocyte stay small? It should do, it's insulin resistant, so won't store fat. Should it export saturated fat as FFAs? Yes. Should we have a slim but insulin resistant mouse? On chow it should become hyperinsulinaemic. Well, you might expect so.
But that's not what happens. The mice stay slim alright, but have excellent insulin sensitivity. Like really, really good insulin sensitivity. You can even feed them on toffee fudge cheesecake and they stay fairly slim and very insulin sensitive.
The SCD1 deleted mice also eat more despite being slimmer than WT mice when on chow, ie they are in CICO-denial:
"On average, the SCD1−/− mice consumed 25% more food than wild-type mice (4.1 g/day vs. 5.6 g/day; n = 9, P < 0.05). Nonetheless, they were leaner and accumulated less fat in their adipose tissue"
Huh. Bloody gym sneaks again. Even while they are asleep:
"The SCD1−/− mice exhibited consistently higher rates of oxygen consumption (had higher metabolic rates) than their wild-type littermates throughout the day and night (Fig. 3A). After adjusting for allometric scaling and gender, the effect of the knockout allele was highly significant (P = 0.00019, multiple ANOVA, Fig. 3B)."
These animals have a hugely increased metabolic rate. The brown adipose tissue "looks normal". That's not the answer.
They are also ketogenic during fasting (daytime is sleep time but they don't really go to the gym while they are asleep). Fasting BHB was 4.4mg/dl. For those watching their ketone meter at home, eat your heart out. They do this even when living on toffee fudge cheesecake.
OK. Utter basics:
What is the F:N ratio within the mitochondria of these mice? Is it:
a) <0.45
b) <0.45
c) <0.45
d) <0.45
e) <0.45
f) Huh????
g) >0.48 (trick answer, don't choose this one!)
The mice are insulin sensitive. They do not have undiluted palmitic acid oxidation going on in their mitochondria. This would produce a ton of superoxide and severe insulin resistance. We know that their mitochondrial F:N ratio must be low. Their metabolism is ketogenic. What fats produce ketones on a high carbohydrate diet? Those MCTs from coconuts and breast milk do. Where do you get C8 caprylic acid from if you are an SCD1 knockout mouse on a low fat diet?
From your peroxisiomes.
Mice with palmitic acid on tap and no ability to lower the F:N ratio by desaturation simply oxidise it in peroxisiomes, FADH2 free, to C8 which is ketogenic, has a low F:N ratio and they produce a lot of heat in the process.
In the words of the paper:
"Northern blot analysis also supports changes in fatty acid oxidation and lipid biosynthesis. Probes for acyl–CoA oxidase (ACO), very long chain acyl–CoA dehydrogenase (VLCAD), and carnitine palmitoyltransferase-1 (CPT-1) indicate increases in β-oxidation"
My emphasis. VLCAD is the main one in peroxisomes, as well as being present in mitochondria. The authors do not come up with any comprehensive explanation of what is going on. The F:N ratio delivers.
I think I mentioned some time ago the explanatory ability of the F:N ratio is awesome. It just goes on.
I was going to leave it there, back to work next week so blogging will diminish, but here is some idle rambling which followed on from this post.
Now here's the question. If some guy like me set out to maintain the lowest practical insulin level (which will minimise SCD1 activation) and bases his diet on the very longest chain, most fully saturated fat practical, would you expect me to activate my peroxisomes? Might the result be that I might stay slim and be cold tolerant?
When we moved in to our current house I unpacked the scales after they had spent nearly a year in a box provided by Pickfords. I was 63.8kg after a year of not checking anything, down by about a kilo from Glasgow. But I was getting a great deal of hill walking in Scotland and probably had more muscle. I forgot about the scales for another year but dug them out recently. Down to 62.8kgs. I eat a huge amount of palmitic acid. I generate enough superoxide to maintain the needed physiological insulin resistance to eat LCHF and I suspect I might have quite active peroxisomes.
I still run a dawn phenomemon FBG of around 5.5mmol/l, if I get up early enough to check it beforehand it's about 4.3mmol/l, once 3.9mmol/l. Random BG through the day vary from 3.3mmol/l after a half day of walking to and from the beach while the car was being MOTed to 6ishmmol/l post prandial if I had parsnip chips (yum) with my high fat beef burgers. Yes, I pour the cooking fat over the chips. A big carb load will get me above 7.0mmol/l easily but only for a couple of hours. I try not to do this too often.
Posting-wise I have no idea what time will allow next week but beta cell failure in SCD1 knockout ob/ob-ve mice tells us interesting things about cells which have minimal antioxidant defences and are deprived of palmitoleic and oleic acids.
Peter
It's busy making lipid, being an adipocyte. Two carbons, four carbons, six, eight, ten, twelve, fourteen and hey, there's the sixteen for palmitic acid. Now, how much glucose and insulin is there around? Ah, lots. Need to signal this with palmitoleate. In goes the double bond to prove it... Oops. No SCD1. Hmmmm. We now have a ton of palmitic acid and no chance to convert any of it to palmitoleic acid. Tricky.
Is the adipocyte going to become insulin resistant? Unless it runs on glucose and never uses any lipid this seems likely. Will the adipocyte stay small? It should do, it's insulin resistant, so won't store fat. Should it export saturated fat as FFAs? Yes. Should we have a slim but insulin resistant mouse? On chow it should become hyperinsulinaemic. Well, you might expect so.
But that's not what happens. The mice stay slim alright, but have excellent insulin sensitivity. Like really, really good insulin sensitivity. You can even feed them on toffee fudge cheesecake and they stay fairly slim and very insulin sensitive.
The SCD1 deleted mice also eat more despite being slimmer than WT mice when on chow, ie they are in CICO-denial:
"On average, the SCD1−/− mice consumed 25% more food than wild-type mice (4.1 g/day vs. 5.6 g/day; n = 9, P < 0.05). Nonetheless, they were leaner and accumulated less fat in their adipose tissue"
Huh. Bloody gym sneaks again. Even while they are asleep:
"The SCD1−/− mice exhibited consistently higher rates of oxygen consumption (had higher metabolic rates) than their wild-type littermates throughout the day and night (Fig. 3A). After adjusting for allometric scaling and gender, the effect of the knockout allele was highly significant (P = 0.00019, multiple ANOVA, Fig. 3B)."
These animals have a hugely increased metabolic rate. The brown adipose tissue "looks normal". That's not the answer.
They are also ketogenic during fasting (daytime is sleep time but they don't really go to the gym while they are asleep). Fasting BHB was 4.4mg/dl. For those watching their ketone meter at home, eat your heart out. They do this even when living on toffee fudge cheesecake.
OK. Utter basics:
What is the F:N ratio within the mitochondria of these mice? Is it:
a) <0.45
b) <0.45
c) <0.45
d) <0.45
e) <0.45
f) Huh????
g) >0.48 (trick answer, don't choose this one!)
The mice are insulin sensitive. They do not have undiluted palmitic acid oxidation going on in their mitochondria. This would produce a ton of superoxide and severe insulin resistance. We know that their mitochondrial F:N ratio must be low. Their metabolism is ketogenic. What fats produce ketones on a high carbohydrate diet? Those MCTs from coconuts and breast milk do. Where do you get C8 caprylic acid from if you are an SCD1 knockout mouse on a low fat diet?
From your peroxisiomes.
Mice with palmitic acid on tap and no ability to lower the F:N ratio by desaturation simply oxidise it in peroxisiomes, FADH2 free, to C8 which is ketogenic, has a low F:N ratio and they produce a lot of heat in the process.
In the words of the paper:
"Northern blot analysis also supports changes in fatty acid oxidation and lipid biosynthesis. Probes for acyl–CoA oxidase (ACO), very long chain acyl–CoA dehydrogenase (VLCAD), and carnitine palmitoyltransferase-1 (CPT-1) indicate increases in β-oxidation"
My emphasis. VLCAD is the main one in peroxisomes, as well as being present in mitochondria. The authors do not come up with any comprehensive explanation of what is going on. The F:N ratio delivers.
I think I mentioned some time ago the explanatory ability of the F:N ratio is awesome. It just goes on.
I was going to leave it there, back to work next week so blogging will diminish, but here is some idle rambling which followed on from this post.
Now here's the question. If some guy like me set out to maintain the lowest practical insulin level (which will minimise SCD1 activation) and bases his diet on the very longest chain, most fully saturated fat practical, would you expect me to activate my peroxisomes? Might the result be that I might stay slim and be cold tolerant?
When we moved in to our current house I unpacked the scales after they had spent nearly a year in a box provided by Pickfords. I was 63.8kg after a year of not checking anything, down by about a kilo from Glasgow. But I was getting a great deal of hill walking in Scotland and probably had more muscle. I forgot about the scales for another year but dug them out recently. Down to 62.8kgs. I eat a huge amount of palmitic acid. I generate enough superoxide to maintain the needed physiological insulin resistance to eat LCHF and I suspect I might have quite active peroxisomes.
I still run a dawn phenomemon FBG of around 5.5mmol/l, if I get up early enough to check it beforehand it's about 4.3mmol/l, once 3.9mmol/l. Random BG through the day vary from 3.3mmol/l after a half day of walking to and from the beach while the car was being MOTed to 6ishmmol/l post prandial if I had parsnip chips (yum) with my high fat beef burgers. Yes, I pour the cooking fat over the chips. A big carb load will get me above 7.0mmol/l easily but only for a couple of hours. I try not to do this too often.
Posting-wise I have no idea what time will allow next week but beta cell failure in SCD1 knockout ob/ob-ve mice tells us interesting things about cells which have minimal antioxidant defences and are deprived of palmitoleic and oleic acids.
Peter
Thursday, August 23, 2012
Protons: de novo lipogenesis
Okay, time to think about whole body insulin sensitivity, adipocytes and insulin.
First the core process; adipocytes which are listening to insulin will post GLUT4s on their surface, accept glucose and do enough de novo lipogenesis to both store and release palmitoleate. The palmitoleate is a signal that there is plenty of glucose around, let's use it.
Adipocytes are accepting glucose for signalling purposes and DNL lipid formation, plus they are sequestering away whatever lipid is available from the diet. The core function of insulin is the storage of DIETARY fat under the influence of carbohydrate. Boy, that is an old post! But the fact that there is plenty of glucose around means that the body should maintain insulin sensitivity, to make use of that glucose. But DNL in adipocytes which are insulin sensitive makes you, err, fat. As Cao et al point out in their lipokine paper:
"Additionally, genetic or pharmacological manipulations that boost de novo lipogenesis in adipose tissue (even though this sometimes leads to expansion of the fat depot) are associated with improved metabolic homeostasis (Kuriyama et al., 2005; Waki et al., 2007)."
I think this is a long winded paraphrase of the Hyperlipid concept "Getting fat is bad when you stop".
Increased insulin sensitivity in adipocytes makes you fat. That's as you would expect.
Back to the ice pick rats with their acute onset insulin hypersensitivity in adipocytes. During rapidly increasing bodyweight (on a low fat diet) there is a marked increase in obesity with excellent insulin sensitivity. Ended by six weeks.
Ditto MSG rats, but for in 4 weeks rather than 6 weeks. Can't tell from the gold thioglucose abstract, but at least a few weeks. Probably depends on all sorts of minutiae.
While ever these rodent models are gaining weight they maintain insulin sensitivity because they are doing DNL to get fat. On a low fat diet increasing obesity means DNL, palmitoleate and the ability to run metabolism on glucose. Logical.
Only once a brain-damaged rat becomes obese enough does hyperinsulinaemia set in, with attendant glucose intolerance. By this stage adipocytes are insulin resistant so have reduced ability to respond to insulin, reduced GLUT4 expression and, presumably, reduced palmitoleate synthesis. From the adipocyte's point of view there is not a lot of insulin around, whatever the blood concentration might be.
Lets look at the converse to obesity:
What does a lack of insulin signify? No food (or no carbohydrate, pax protein). Starvation requires insulin resistance as an obligate state for survival. How much good is palmitoleate going to do you under starvation or ketogenic dieting? Not a lot, unless you enjoy dropping precious glucose in to muscles until you brain falls to pieces.
An adipocyte which sees no insulin will not generate palmitoleate. If it generates anything at all (doubtful) it will be palmitate. Releasing some residual palmitoleate from adipocytes is fine for a few days, as long as there is glycogen hanging around. By three days this will be gone and so too should the palmitoleate. You are now in to hard core survival driven insulin resistance.
That's where I live.
Adipocyte distension induced insulin resistance is completely different. Here the adipocytes see low insulin when there is a ton of it around. There is a ton of glucose around too. But an adipocyte acts as per starvation and does what would be absolutely the correct thing under starvation circumstances. It releases palmitic acid and stops generating palmitoleate. Doing this while the macroscopic organism is eating bagels and french fries is bad. It's bound to generate massive hyperinsulinaemia to normalise glucose in the face of a ton of palmitic acid.
I'm just wondering whether there is time to look at the C57BL/6 mice. Just briefly as there is a lot of Mickey to be extracted on this subject when we get to idiots in detail. Briefly:
By an utter quirk of metabolism the VMH of C57BL/6 mice breaks under high dietary fat levels. So they have access to ample dietary fat when their VMH is injured, by definition. They store this fat because sympathetic tone to adipocytes is acutely lost and adipocytes become exquisitely insulin sensitive. Fat falls in to adipocytes as soon as the injury occurs, probably within hours of eating some butter, almost any amount of butter, however small.
But the fat they store is dietary fat. No DNL. They do not need to gain fat by DNL as it is there in the hopper. Dietary fat falls in to adipocytes. Palmitoleate synthesis? When fat is distending adipocytes so fast they are leaking FFAs despite losing lipolytic sympathetic tone? There is a ton of dietary fat dropping in to adipocytes, this is what gets released as they distend. By day three C57BL/6 mice are systemically insulin resistant. Their palmitoleate levels will be low and palmitic acid levels high. They are just a modification of the ice-pick/MSG/gold thioglucose family, but the process happens at warp speed due to the availability of DNL-free bulk fat.
Even on high fat plus high sucrose diets humans do not injure their VMH, at least not immediately. But C57BL/6 mice do and they have taught me a great deal over the years, made me think a great deal too. But they are still just a model, as explicable as the rest of the models, from the insulocentric point of view.
Once you have enough data.
To summarise: Palmitoleate is released by adipocytes when glucose and insulin are plentiful. Palmitate is released when glucose is sparse and insulin is low.
The sh!t hits the fan when glucose and insulin are plentiful but adipocytes are so distended that they THINK glucose and insulin are low. When both insulin and glucose are high you want palmitoleate. If your adipocytes give you palmitate under these circumstances you had better have a pancreas of steel or diabetes here you come.
I think we might go to PUFA and SCD1 in adipocytes before hepatic DNL in this series.
BTW It's nice to see people in comments being a post or two ahead! At least this isn't complete gobbledegook to everyone!
Peter
First the core process; adipocytes which are listening to insulin will post GLUT4s on their surface, accept glucose and do enough de novo lipogenesis to both store and release palmitoleate. The palmitoleate is a signal that there is plenty of glucose around, let's use it.
Adipocytes are accepting glucose for signalling purposes and DNL lipid formation, plus they are sequestering away whatever lipid is available from the diet. The core function of insulin is the storage of DIETARY fat under the influence of carbohydrate. Boy, that is an old post! But the fact that there is plenty of glucose around means that the body should maintain insulin sensitivity, to make use of that glucose. But DNL in adipocytes which are insulin sensitive makes you, err, fat. As Cao et al point out in their lipokine paper:
"Additionally, genetic or pharmacological manipulations that boost de novo lipogenesis in adipose tissue (even though this sometimes leads to expansion of the fat depot) are associated with improved metabolic homeostasis (Kuriyama et al., 2005; Waki et al., 2007)."
I think this is a long winded paraphrase of the Hyperlipid concept "Getting fat is bad when you stop".
Increased insulin sensitivity in adipocytes makes you fat. That's as you would expect.
Back to the ice pick rats with their acute onset insulin hypersensitivity in adipocytes. During rapidly increasing bodyweight (on a low fat diet) there is a marked increase in obesity with excellent insulin sensitivity. Ended by six weeks.
Ditto MSG rats, but for in 4 weeks rather than 6 weeks. Can't tell from the gold thioglucose abstract, but at least a few weeks. Probably depends on all sorts of minutiae.
While ever these rodent models are gaining weight they maintain insulin sensitivity because they are doing DNL to get fat. On a low fat diet increasing obesity means DNL, palmitoleate and the ability to run metabolism on glucose. Logical.
Only once a brain-damaged rat becomes obese enough does hyperinsulinaemia set in, with attendant glucose intolerance. By this stage adipocytes are insulin resistant so have reduced ability to respond to insulin, reduced GLUT4 expression and, presumably, reduced palmitoleate synthesis. From the adipocyte's point of view there is not a lot of insulin around, whatever the blood concentration might be.
Lets look at the converse to obesity:
What does a lack of insulin signify? No food (or no carbohydrate, pax protein). Starvation requires insulin resistance as an obligate state for survival. How much good is palmitoleate going to do you under starvation or ketogenic dieting? Not a lot, unless you enjoy dropping precious glucose in to muscles until you brain falls to pieces.
An adipocyte which sees no insulin will not generate palmitoleate. If it generates anything at all (doubtful) it will be palmitate. Releasing some residual palmitoleate from adipocytes is fine for a few days, as long as there is glycogen hanging around. By three days this will be gone and so too should the palmitoleate. You are now in to hard core survival driven insulin resistance.
That's where I live.
Adipocyte distension induced insulin resistance is completely different. Here the adipocytes see low insulin when there is a ton of it around. There is a ton of glucose around too. But an adipocyte acts as per starvation and does what would be absolutely the correct thing under starvation circumstances. It releases palmitic acid and stops generating palmitoleate. Doing this while the macroscopic organism is eating bagels and french fries is bad. It's bound to generate massive hyperinsulinaemia to normalise glucose in the face of a ton of palmitic acid.
I'm just wondering whether there is time to look at the C57BL/6 mice. Just briefly as there is a lot of Mickey to be extracted on this subject when we get to idiots in detail. Briefly:
By an utter quirk of metabolism the VMH of C57BL/6 mice breaks under high dietary fat levels. So they have access to ample dietary fat when their VMH is injured, by definition. They store this fat because sympathetic tone to adipocytes is acutely lost and adipocytes become exquisitely insulin sensitive. Fat falls in to adipocytes as soon as the injury occurs, probably within hours of eating some butter, almost any amount of butter, however small.
But the fat they store is dietary fat. No DNL. They do not need to gain fat by DNL as it is there in the hopper. Dietary fat falls in to adipocytes. Palmitoleate synthesis? When fat is distending adipocytes so fast they are leaking FFAs despite losing lipolytic sympathetic tone? There is a ton of dietary fat dropping in to adipocytes, this is what gets released as they distend. By day three C57BL/6 mice are systemically insulin resistant. Their palmitoleate levels will be low and palmitic acid levels high. They are just a modification of the ice-pick/MSG/gold thioglucose family, but the process happens at warp speed due to the availability of DNL-free bulk fat.
Even on high fat plus high sucrose diets humans do not injure their VMH, at least not immediately. But C57BL/6 mice do and they have taught me a great deal over the years, made me think a great deal too. But they are still just a model, as explicable as the rest of the models, from the insulocentric point of view.
Once you have enough data.
To summarise: Palmitoleate is released by adipocytes when glucose and insulin are plentiful. Palmitate is released when glucose is sparse and insulin is low.
The sh!t hits the fan when glucose and insulin are plentiful but adipocytes are so distended that they THINK glucose and insulin are low. When both insulin and glucose are high you want palmitoleate. If your adipocytes give you palmitate under these circumstances you had better have a pancreas of steel or diabetes here you come.
I think we might go to PUFA and SCD1 in adipocytes before hepatic DNL in this series.
BTW It's nice to see people in comments being a post or two ahead! At least this isn't complete gobbledegook to everyone!
Peter
Tuesday, August 21, 2012
Protons: Palmitoleate
I think we have to start with the results section of Cao et al's very interesting (and free to study if you want all the detail) paper:
Identification of a Lipokine, a Lipid Hormone Linking Adipose Tissue to Systemic Metabolism
As always, the paper is a superb piece of detective work featuring a superabundance of genetically engineered mice from the C57BL/6J background fed an high fat diet, the nature of which doesn't make it in to the methods, but we can just assume that it's all the usual fare. They started from the protective effect of knocking out certain fatty acid receptors in the mice, which prevented the development of metabolic syndrome, and ran with this concept for the massive project detailed in the paper. It's big. It ended up with them doing the following to confirm that they got it correct. From the very end of the results:
"To define the effects of individual fatty acids on metabolic regulation, we prepared Intralipid with triglycerides composed of a single fatty acid, either TG-palmitoleate or TG-palmitate. Infusion of either lipid resulted in a two-fold increase in total plasma FFA levels with similar dynamics (Figure S13). While TG-palmitate suppressed the entire proximal insulin-signaling pathway including activation of insulin receptor and phosphorylation of insulin receptor substrate 1, 2 and AKT in liver, TG-palmitoleate strongly potentiated these insulin actions (Figure 7A). We observed similar effects of both lipids on muscle tissue where palmitoleate enhanced and palmitate impaired insulin signaling (Figure 7B)."
It's a switch, at the crude level of Intralipid infusions. Viewed macroscopically:
Palmitoleate = insulin sensitive
Palmitate = insulin resistant
I may have mentioned this before!
If you take a light switch apart, under the plastic there are some metal parts. The metal provides a sea of probability through which electrons can flow, provided the metal is continuous from light bulb to the powerstation (pax transformers). Or not flow, if we replace a few mm of metal with a few mm of room air.
If we accept that superoxide from complex I reverse electron transport is insulin resistance, then fatty acid binding proteins are a macroscopic overlay over this process, they are part of the plastic of the switch.
Superoxide never leaves the mitochondria, it probably converts to H2O2 to talk to the nucleus or acts locally to activate transcription factors which then talk to the nucleus. Adipocytes don't talk to muscles using superoxide either. The intermediary they use appears to be palmitoleate, probably the ratio of palmitoleate to palmitic acids, once you get away from bulk Intralipid infusions.
Why is it arranged this way? The body has to know what substrates are available. Ignoring protein, carbohydrate talks to the body through insulin, and through insulin transporting glucose in to adipocytes. That's the next post.
There: Not a mention of FADH2 or NADH. Even if I'm thinking about them, as per the last post...
Peter
BTW, Charles commented on the depressing amount of superoxide associated with a high fat, low carb diet. True, but about as scary as going for a walk at the brisk-but-not-excessive pace which is reputed to burn fat best. Burning fat is what LCHF eating is all about. Useful if you don't have the hours a day to walk for health purposes. Walking seems to be quite good for you!
Identification of a Lipokine, a Lipid Hormone Linking Adipose Tissue to Systemic Metabolism
As always, the paper is a superb piece of detective work featuring a superabundance of genetically engineered mice from the C57BL/6J background fed an high fat diet, the nature of which doesn't make it in to the methods, but we can just assume that it's all the usual fare. They started from the protective effect of knocking out certain fatty acid receptors in the mice, which prevented the development of metabolic syndrome, and ran with this concept for the massive project detailed in the paper. It's big. It ended up with them doing the following to confirm that they got it correct. From the very end of the results:
"To define the effects of individual fatty acids on metabolic regulation, we prepared Intralipid with triglycerides composed of a single fatty acid, either TG-palmitoleate or TG-palmitate. Infusion of either lipid resulted in a two-fold increase in total plasma FFA levels with similar dynamics (Figure S13). While TG-palmitate suppressed the entire proximal insulin-signaling pathway including activation of insulin receptor and phosphorylation of insulin receptor substrate 1, 2 and AKT in liver, TG-palmitoleate strongly potentiated these insulin actions (Figure 7A). We observed similar effects of both lipids on muscle tissue where palmitoleate enhanced and palmitate impaired insulin signaling (Figure 7B)."
It's a switch, at the crude level of Intralipid infusions. Viewed macroscopically:
Palmitoleate = insulin sensitive
Palmitate = insulin resistant
I may have mentioned this before!
If you take a light switch apart, under the plastic there are some metal parts. The metal provides a sea of probability through which electrons can flow, provided the metal is continuous from light bulb to the powerstation (pax transformers). Or not flow, if we replace a few mm of metal with a few mm of room air.
If we accept that superoxide from complex I reverse electron transport is insulin resistance, then fatty acid binding proteins are a macroscopic overlay over this process, they are part of the plastic of the switch.
Superoxide never leaves the mitochondria, it probably converts to H2O2 to talk to the nucleus or acts locally to activate transcription factors which then talk to the nucleus. Adipocytes don't talk to muscles using superoxide either. The intermediary they use appears to be palmitoleate, probably the ratio of palmitoleate to palmitic acids, once you get away from bulk Intralipid infusions.
Why is it arranged this way? The body has to know what substrates are available. Ignoring protein, carbohydrate talks to the body through insulin, and through insulin transporting glucose in to adipocytes. That's the next post.
There: Not a mention of FADH2 or NADH. Even if I'm thinking about them, as per the last post...
Peter
BTW, Charles commented on the depressing amount of superoxide associated with a high fat, low carb diet. True, but about as scary as going for a walk at the brisk-but-not-excessive pace which is reputed to burn fat best. Burning fat is what LCHF eating is all about. Useful if you don't have the hours a day to walk for health purposes. Walking seems to be quite good for you!
Monday, August 20, 2012
Protons: FADH2:NADH ratios and MUFA
A few more thoughts building on F:N ratios of differing metabolic substrates:
Each cycle of beta oxidation (assuming an even numbered carbon chain fully saturated fatty acid) produces one FADH2, one NADH and one acetyl-CoA. This gives a total of 2FADH2 inputs and 4 NADHs per cycle of beta oxidation. But the very last pair of carbon atoms in a saturated fat do not need to go through beta oxidation as they already comprise acetate attached to CoA, so they can simply enter the TCA as acetyl-CoA. This last step only produces 1 FADH2 and 3 NADHs, with no extras.
So the shorter the fatty acid, the less FADH2 per unit NADH it produces. Short chain fatty acids like C4 butyric acid have an F:N ratio of 0.43 while very long chain fatty acids, up at 26 carbons, have an F:N ratio of about 0.49.
As Dr Speijer points out, differing length fatty acids are dealt with differently. Very short chain fatty acids head straight for the liver and get metabolised by hepatic mitochondria immediately. Any excess acetyl-CoA gets off-loaded as ketones.
Very long chain fatty acids end up in peroxisomes for shortening, usually to C8, which is then shunted to mitochondria for routine beta oxidation. Of course peroxisomal beta oxidation generates zero FADH2, except that from acetyl-CoA, because peroxisomal FADH2 is reacted directly with oxygen to give H2O2. And heat, of course.
Bear in mind that the ratio of F:N generated by a metabolic fuel sets the ability to generate reverse electron flow through complex I and subsequent superoxide production, macroscopically described as insulin resistance.
So fatty acids up to C8 are cool, dump them to the liver and make a few ketones. Very long chain fatty acids over C18, shorten to C8 in peroxisomes, shift them to mitochondria and make some ketones if needs must. The F:N ratio of C8 is about 0.47, a value chosen by metabolism as the end product of peroxisomal shortening. The number is important. Actually the number is even lower as peroxisomal beta oxidation generates the NADHs of beta oxidation, just not the FADH2s, but why allow facts like this to spoil a great argument. C8 from breast milk and/or coconuts seems fine and has that F:N ratio of 0.47.
Now the area of interest is, of course, C16, palmitic acid. This has an F:N ratio of about 0.48, almost as superoxide generating as a C26 fatty acid up at 0.49. And palmitic acid does, without any shadow of a doubt, produce macroscopic insulin resistance. That's 15 FADH2s and 31 NADHs.
So an F:N of 0.47 is not a serious generator of superoxide and an F:N of 0.48 is.
What happens when we drop a double bond in to palmitic acid? Mitochondrial beta oxidation generates FADH2 as it drops a double bond in to the saturated fat chain. If the double bond is already there, hey, no FADH2!
Palmitoleate has one double bond. This of course gives 14 FADH2s and 31 NADHs, an F:N ratio of 0.45.
Palmitate 0.48
C8 caprylic 0.47, chosen by peroxisomes to hand to mitochondria
Palmitoleic 0.45
Adding a single double bond to palmitic acid drops its F:N ratio from significantly superoxide generating to minimally superoxide generating. It looks like a switch to me.
I just love the way the numbers pan out. Of course we can now go on to what these number signify and what determines unsaturation. And uncoupling too, I guess. We are then back to insulin and stearoyl-CoA desaturase and also de novo lipogenesis. It might be worth an aside to PUFA and how these behave too, especially in adipocytes.
Peter
Each cycle of beta oxidation (assuming an even numbered carbon chain fully saturated fatty acid) produces one FADH2, one NADH and one acetyl-CoA. This gives a total of 2FADH2 inputs and 4 NADHs per cycle of beta oxidation. But the very last pair of carbon atoms in a saturated fat do not need to go through beta oxidation as they already comprise acetate attached to CoA, so they can simply enter the TCA as acetyl-CoA. This last step only produces 1 FADH2 and 3 NADHs, with no extras.
So the shorter the fatty acid, the less FADH2 per unit NADH it produces. Short chain fatty acids like C4 butyric acid have an F:N ratio of 0.43 while very long chain fatty acids, up at 26 carbons, have an F:N ratio of about 0.49.
As Dr Speijer points out, differing length fatty acids are dealt with differently. Very short chain fatty acids head straight for the liver and get metabolised by hepatic mitochondria immediately. Any excess acetyl-CoA gets off-loaded as ketones.
Very long chain fatty acids end up in peroxisomes for shortening, usually to C8, which is then shunted to mitochondria for routine beta oxidation. Of course peroxisomal beta oxidation generates zero FADH2, except that from acetyl-CoA, because peroxisomal FADH2 is reacted directly with oxygen to give H2O2. And heat, of course.
Bear in mind that the ratio of F:N generated by a metabolic fuel sets the ability to generate reverse electron flow through complex I and subsequent superoxide production, macroscopically described as insulin resistance.
So fatty acids up to C8 are cool, dump them to the liver and make a few ketones. Very long chain fatty acids over C18, shorten to C8 in peroxisomes, shift them to mitochondria and make some ketones if needs must. The F:N ratio of C8 is about 0.47, a value chosen by metabolism as the end product of peroxisomal shortening. The number is important. Actually the number is even lower as peroxisomal beta oxidation generates the NADHs of beta oxidation, just not the FADH2s, but why allow facts like this to spoil a great argument. C8 from breast milk and/or coconuts seems fine and has that F:N ratio of 0.47.
Now the area of interest is, of course, C16, palmitic acid. This has an F:N ratio of about 0.48, almost as superoxide generating as a C26 fatty acid up at 0.49. And palmitic acid does, without any shadow of a doubt, produce macroscopic insulin resistance. That's 15 FADH2s and 31 NADHs.
So an F:N of 0.47 is not a serious generator of superoxide and an F:N of 0.48 is.
What happens when we drop a double bond in to palmitic acid? Mitochondrial beta oxidation generates FADH2 as it drops a double bond in to the saturated fat chain. If the double bond is already there, hey, no FADH2!
Palmitoleate has one double bond. This of course gives 14 FADH2s and 31 NADHs, an F:N ratio of 0.45.
Palmitate 0.48
C8 caprylic 0.47, chosen by peroxisomes to hand to mitochondria
Palmitoleic 0.45
Adding a single double bond to palmitic acid drops its F:N ratio from significantly superoxide generating to minimally superoxide generating. It looks like a switch to me.
I just love the way the numbers pan out. Of course we can now go on to what these number signify and what determines unsaturation. And uncoupling too, I guess. We are then back to insulin and stearoyl-CoA desaturase and also de novo lipogenesis. It might be worth an aside to PUFA and how these behave too, especially in adipocytes.
Peter
Saturday, August 18, 2012
Protons: Lactate
I've been aware for some time that there is a reasonable idea that the brain runs on lactate. Dr Speijer emailed me a link to a very recent paper which supports this concept at the cutting edge of modern research, without having to go back to that old stuff from over five years ago which no one ever reads because it has no lovely photomicrographs and no ultracool transgenic mice.
The editorial has this nice diagram which sums up what might be going on:
Let's get back to electron donors. The brain hates superoxide. It hates fatty acids. It's a bit ambivalent about glucose (gasp). I don't think I would say it rejects glucose, just there are better fuels.
Is there anything the brain does like? Well, ketone bodies seem to be okay, but what the brain really seems to like is lactate. Perhaps I should rephrase all of this and say that the neurons of the brain love lactate. The rest of the brain seems fine on glucose and will even dabble with fatty acids at a pinch. But glucose is fed to neurons, pre digested by the glial cells, as lactate. The FFAs are fed as ketones, yes the glial cells in the brain are ketogenic, it's not just the liver that does this. I suppose the neurons might use glucose directly, but they become quite sick if you knock out lactate supply by eliminating MCT1 (mono carboxylic acid transporter 1).
Neurons are irreplaceable, more or less. They aim for zero superoxide production. This means behaving like a mitochondrial preparation which is being fed on glutamate, a provider of NADH only. Near zero free radical production is the closest you can come to having no mitochondria at all, yet still have the powerhouse of the electron transport chain at your command. When thinking about apoptosis that is. Apoptosis is not a good idea in non-replaceable cells...
This means minimising FADH2 utilisation. Fatty acids, with their beta oxidation derived FADH2, are out. No way in neurons.
Glucose is not ideal either. Why not? Well glucose supplies the best possible neuronal FADH2:NADH (F/N) ratio of 0.2, ie it gives one FADH2 for 5 NADHs. Usually. This is superb for minimising superoxide production (and maintaining insulin sensitivity). But not always. What about glycerol-phosphate dehydrogenase or glycerol-phosphate oxidase? Both of these, in much the same manner as the FADH2 moiety within electron-transporting flavoprotein dehydrogenase, can reduce the CoQ couple and promote superoxide production. That's without thinking about simply over driving the TCA with pathological hyperglycaemia. There is absolutely no doubt that hyperglycaemia generates superoxide production. Unfortunately most of the people discussing this on pubmed have no real concept of F:N ratios or what exactly goes on in the respiratory chain to generate superoxide. There is no nice neat diagram to copy paste. My own assumption is that massive enough inputs of glucose drive huge amounts of NADH production which cannot be accommodated once FADH2 from succinate dehydrogenase reaches a critical level or is supplemented by glycerol-phosphate dehydrogenase based FADH2. At this point a cell says no to glucose calories, ie it makes superoxide and becomes insulin resistant. As has been observed, insulin resistance is an antioxidant defence mechanism, you need it. If pushed hard enough to overcome insulin resistance a cell will take one step closer to apoptosis.
Not so with lactate. Lactate supplies acetyl-CoA (which itself has an F:N ratio of 0.25) along side a couple of extra NADH molecules (one each from lactate dehydrogenase and pyruvate dehydrogenase) which reduce the overall F:N ratio to 0.2, the same as glucose). Yet pre-prepared lactate does not need any glycolysis to take place in the neurons themselves. It has no possibility of supplying ANY FADH2-like input to the CoQ couple, outside of succinate dehydrogenase (complex II) activation in the turning of the TCA. It's the purest of complex I inputs available to any intact organism. No wonder the brain loves lactate. Lactate usage appears to be the best way of postponing apoptosis, short of abandoning mitochondria altogether. Glucose comes second.
I had always though of lactate in the brain as a sort of direct mitochondrial fuel injection system. Lactate dehydrogenase then mitochondrial uptake of pyruvate. Just a fast response time. But looking at FADH2 to NADH ratios gives a much deep insight in to what is going on.
What about fat????? Not for the brain.
But for the rest of the body? What makes mitochondria happy? Hint: It's not glucose.
Peter
The editorial has this nice diagram which sums up what might be going on:
Let's get back to electron donors. The brain hates superoxide. It hates fatty acids. It's a bit ambivalent about glucose (gasp). I don't think I would say it rejects glucose, just there are better fuels.
Is there anything the brain does like? Well, ketone bodies seem to be okay, but what the brain really seems to like is lactate. Perhaps I should rephrase all of this and say that the neurons of the brain love lactate. The rest of the brain seems fine on glucose and will even dabble with fatty acids at a pinch. But glucose is fed to neurons, pre digested by the glial cells, as lactate. The FFAs are fed as ketones, yes the glial cells in the brain are ketogenic, it's not just the liver that does this. I suppose the neurons might use glucose directly, but they become quite sick if you knock out lactate supply by eliminating MCT1 (mono carboxylic acid transporter 1).
Neurons are irreplaceable, more or less. They aim for zero superoxide production. This means behaving like a mitochondrial preparation which is being fed on glutamate, a provider of NADH only. Near zero free radical production is the closest you can come to having no mitochondria at all, yet still have the powerhouse of the electron transport chain at your command. When thinking about apoptosis that is. Apoptosis is not a good idea in non-replaceable cells...
This means minimising FADH2 utilisation. Fatty acids, with their beta oxidation derived FADH2, are out. No way in neurons.
Glucose is not ideal either. Why not? Well glucose supplies the best possible neuronal FADH2:NADH (F/N) ratio of 0.2, ie it gives one FADH2 for 5 NADHs. Usually. This is superb for minimising superoxide production (and maintaining insulin sensitivity). But not always. What about glycerol-phosphate dehydrogenase or glycerol-phosphate oxidase? Both of these, in much the same manner as the FADH2 moiety within electron-transporting flavoprotein dehydrogenase, can reduce the CoQ couple and promote superoxide production. That's without thinking about simply over driving the TCA with pathological hyperglycaemia. There is absolutely no doubt that hyperglycaemia generates superoxide production. Unfortunately most of the people discussing this on pubmed have no real concept of F:N ratios or what exactly goes on in the respiratory chain to generate superoxide. There is no nice neat diagram to copy paste. My own assumption is that massive enough inputs of glucose drive huge amounts of NADH production which cannot be accommodated once FADH2 from succinate dehydrogenase reaches a critical level or is supplemented by glycerol-phosphate dehydrogenase based FADH2. At this point a cell says no to glucose calories, ie it makes superoxide and becomes insulin resistant. As has been observed, insulin resistance is an antioxidant defence mechanism, you need it. If pushed hard enough to overcome insulin resistance a cell will take one step closer to apoptosis.
Not so with lactate. Lactate supplies acetyl-CoA (which itself has an F:N ratio of 0.25) along side a couple of extra NADH molecules (one each from lactate dehydrogenase and pyruvate dehydrogenase) which reduce the overall F:N ratio to 0.2, the same as glucose). Yet pre-prepared lactate does not need any glycolysis to take place in the neurons themselves. It has no possibility of supplying ANY FADH2-like input to the CoQ couple, outside of succinate dehydrogenase (complex II) activation in the turning of the TCA. It's the purest of complex I inputs available to any intact organism. No wonder the brain loves lactate. Lactate usage appears to be the best way of postponing apoptosis, short of abandoning mitochondria altogether. Glucose comes second.
I had always though of lactate in the brain as a sort of direct mitochondrial fuel injection system. Lactate dehydrogenase then mitochondrial uptake of pyruvate. Just a fast response time. But looking at FADH2 to NADH ratios gives a much deep insight in to what is going on.
What about fat????? Not for the brain.
But for the rest of the body? What makes mitochondria happy? Hint: It's not glucose.
Peter
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