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.
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
Friday, August 17, 2012
Mmmmmm eggs!
Eggs will kill you!!!!!
As a UK resident: Thank god it's not London, London but London, Ontario. Phew. Thought the goons in epidemiology at Imperial College had been at it again. Happily the shame for this has to go to Canada. Oh dear, sorry Canada.
Peter
As a UK resident: Thank god it's not London, London but London, Ontario. Phew. Thought the goons in epidemiology at Imperial College had been at it again. Happily the shame for this has to go to Canada. Oh dear, sorry Canada.
Peter
Friday, August 10, 2012
We are not alone
Obviously anyone with even a basic interest in origin of life questions will be watching the progress of Curiosity on Mars. Those of us who buy in to the serpentine and alkaline hydothermal vents concept will be interested in whether the crustal chemistry of Mars is olivine based and whether major water bodies were even present. Or equally, whether a semblance of white non-smokers might be present when acidic ground-water interacts with olivine, without needing an ocean and vents... An interesting time for testing hypotheses about whether there is life "out there", in our own back yard...
EDIT: A quick google shows olivine, serpentine and methane plumes are all present on Mars. The methane could easily be abiotic in origin, the question is whether it actually is or not...
On the more down to Earth front, if anyone thinks my basic ideas about the ratio of FADH2 based input vs NADH input to the ETC determining superoxide production are not totally incomprehensible, we are definitely not alone. I had a very nice email from Dr Speijer in Amsterdam, a fellow thinker along these lines. He has come to exactly the same conclusions and published an hypothesis paper in Bioessays back in 2011. The first section is just excellent. We may diverge in interpretation (but not FADH2:NADH ratios) very slightly late in the essay on PUFA, but it really is full of very good thinking and an excellent paper.
His ideas about peroxisomes (a very early eukaryotic invention) of course addresses that age old question of "Who's (macroscopic) fat is it anyway?", the answer being that the gut bacteria own it. On the sub cellular front, fat is primarily made in cytoplasm but at the behest of the mitochondria, only secondarily in peroxisomes and, as peroxisomes are probably a response to deal with overly long (ie excessively high FADH2 generating) fatty acids, the answer would seem to be mitochondria order fatty acid production, they own them and they have their own agenda for them. It's a sort of intracellular parallel the the fiaf series on gut bacteria and adipocytes. Very interesting concept.
If mitochondria own fatty acids I would expect them to enjoy burning fatty acids. Whatever the generation of controlled superoxide is, it's what keeps mitochondria happy. Then there is the brain to think about, its avoidance of fatty acids, it's love of ketones for an occasional fling and its very probable long term love affair with lactic acid. All based on FADH2 to NADH ratios of course.
There's a lot to post about. Back to the Protons series next (I think).
Peter
EDIT: A quick google shows olivine, serpentine and methane plumes are all present on Mars. The methane could easily be abiotic in origin, the question is whether it actually is or not...
On the more down to Earth front, if anyone thinks my basic ideas about the ratio of FADH2 based input vs NADH input to the ETC determining superoxide production are not totally incomprehensible, we are definitely not alone. I had a very nice email from Dr Speijer in Amsterdam, a fellow thinker along these lines. He has come to exactly the same conclusions and published an hypothesis paper in Bioessays back in 2011. The first section is just excellent. We may diverge in interpretation (but not FADH2:NADH ratios) very slightly late in the essay on PUFA, but it really is full of very good thinking and an excellent paper.
His ideas about peroxisomes (a very early eukaryotic invention) of course addresses that age old question of "Who's (macroscopic) fat is it anyway?", the answer being that the gut bacteria own it. On the sub cellular front, fat is primarily made in cytoplasm but at the behest of the mitochondria, only secondarily in peroxisomes and, as peroxisomes are probably a response to deal with overly long (ie excessively high FADH2 generating) fatty acids, the answer would seem to be mitochondria order fatty acid production, they own them and they have their own agenda for them. It's a sort of intracellular parallel the the fiaf series on gut bacteria and adipocytes. Very interesting concept.
If mitochondria own fatty acids I would expect them to enjoy burning fatty acids. Whatever the generation of controlled superoxide is, it's what keeps mitochondria happy. Then there is the brain to think about, its avoidance of fatty acids, it's love of ketones for an occasional fling and its very probable long term love affair with lactic acid. All based on FADH2 to NADH ratios of course.
There's a lot to post about. Back to the Protons series next (I think).
Peter
Wednesday, August 08, 2012
Insulin in the brain: Hyperphagia?
Let's start with this quote from Brain insulin controls adipose tissue lipolysis and lipogenesis:
"Insulin is considered the major anti-lipolytic hormone. Its anti–lipolytic effects are thought to be exclusively mediated through insulin receptors expressed on adipocytes (Degerman et al., 2003). Cyclic–AMP (cAMP) signaling represents the major pro–lipolytic pathway in WAT, which is chiefly regulated by the sympathetic nervous system (SNS)."
and then go on to this one from the discussion:
"We draw this conclusion from the finding that denervation of WAT leads to no change in lipogenic protein expression, but completely abrogates Hsl activation leading to increased adipose depot mass (Buettner et al., 2008)".
OK, got that? Brain insulin makes you fat by damping down lipolytic neurotransmission to adipocytes. Turning off your sympathetic nervous system supply to your fat cells allows insulin to go on an obesity spree.
Then there is this quote (MBH is medial basal hypothalamus, better known as VMH, ventro medial hypothalamus):
"Our studies raise several questions. One is which neuronal subtype within the CNS and the MBH mediates the effects of insulin on the regulation of WAT metabolism".
That first one really is an interesting question, one which we can go some way towards answering. We know that the cell type is, as already noted, part of the sympathetic nervous system. In the paper they found either surgical or chemical sympathectomy of adipose tissue increases both lipogenesis and inhibits hormone sensitive lipase in that tissue. I think this is straight forward. The sympathetic nervous system is tonically opposing insulin's lipogenesis effect and insulin's inhibition of hormone sensitive lipase.
The next thing we can say is that these cells sport glutamate receptors. We can safely assume this because, if neonatal rats are injected with the excitotoxin MSG, these are some (among many) of the cells which actually die. That is, the sympathetic nervous system supply to adipose tissue dies. Lipogenesis is unrestrained. Hormone sensitive lipase shuts down. Carbohydrate easily pours in to adipocytes and stays there. Blood glucose levels are low, free fatty acid levels are low, insulin sensitivity is excellent. While ever adipocyte expansion is on going that is. As the adipocytes stretch they eventually become insulin resistant. Here's the table of metabolic parameters from pre-obese MSG injured and control rats, from a previous post:
We know you can do exactly the same by killing these cells with gold thioglucose. This neurotoxin kills those nerve cells which inhibit lipogenesis.
As the authors say: After gold thioglucose injection "systemic insulin sensitivity is preserved [actually it's increased, but these are obesity researchers, so don't quibble] during the early phase of the obesity syndrome, resulting in extensive fat production".
These hypothalamic cells don't seem to take too kindly to the application of an ice pick either:
"In this study, we have measured the expression of the insulin-sensitive glucose transporter, Glut 4 and the activities and expression of key lipogenic enzymes (fatty-acid synthase and acetyl-CoA carboxylase) in white adipose tissue, one and six weeks after the lesion. All these parameters, as well as glucose transport and metabolism determined in white adipocytes, were markedly increased one week after the lesion. They returned to control values within six weeks in VMH-lesioned rats".
All of these interventions allow calories to pour in to adipocytes and stay there. So what does the poor rat do? It's losing a sh*t load of calories in to its adipocytes but, luckily, it has access to a massive hopper of crapinabag in its cage. It simply has to eat enough calories to supply the loss in to adipocytes, plus enough to run its metabolism on. This can be described, by non comprehending people, as hyperphagia. Metabolically it is normophagia.
These rats are calorically neutral or even in mild energy deficit. They have to be running "hyperphagic" just to stand still, metabolically. They are NOT showing "voluntary" overeating. They DO NOT have an injury to any sort of "satiety" centre. They have low insulin, low FFAs and low glucose. They are NOT being paid to over eat! They will NOT be producing a ton of superoxide, despite having a hugely increased caloric intake. Until...
When does this stop? It stops when FFA leakage due to the resistance to insulin induced by adipocyte distention exactly matches the FFA releasing effect which the (now non-existent) sympathetic nervous system would have been having on non distended adipocytes. Sorry for the convoluted sentence, can't simplify it! The distension process was complete by six weeks in the ice pick rat study cited above. Obese rodents then end up with a crudely normal metabolic rate. But this injured system is a complete bodge. We are looking at the replacement of a finely tuned fuel switching system which exactly matches fuel availability to fuel needs with a system where broken adipocytes are simply leaking FFAs at a level which constantly supplements glucose use, without any semblance of fine tuning to metabolic needs. The chronic elevation of fatty acids drives, through the NADH/FADH2 ratio, superoxide production and insulin resistance. Eating glucose then becomes unacceptable because there is inappropriate whole body insulin resistance from excess and inappropriate FFAs. A large amount of insulin is need to control hyperglycaemia under these conditions. Failure to supply adequate insulin to do this, for any reason, is labelled diabetes.
What has this to do with the current obesity epidemic? If you are overweight I would suggest you should take the ice pick out of your brain. No ice pick? Hmmmmm, damn! Back to the drawing board on that one then.
Ah, but maybe you are a C57BL/6J mouse?
Before we can tackle such a stupid question I think we need to go back to superoxide and fatty acids, to about where we were before this digression began.
Peter
"Insulin is considered the major anti-lipolytic hormone. Its anti–lipolytic effects are thought to be exclusively mediated through insulin receptors expressed on adipocytes (Degerman et al., 2003). Cyclic–AMP (cAMP) signaling represents the major pro–lipolytic pathway in WAT, which is chiefly regulated by the sympathetic nervous system (SNS)."
and then go on to this one from the discussion:
"We draw this conclusion from the finding that denervation of WAT leads to no change in lipogenic protein expression, but completely abrogates Hsl activation leading to increased adipose depot mass (Buettner et al., 2008)".
OK, got that? Brain insulin makes you fat by damping down lipolytic neurotransmission to adipocytes. Turning off your sympathetic nervous system supply to your fat cells allows insulin to go on an obesity spree.
Then there is this quote (MBH is medial basal hypothalamus, better known as VMH, ventro medial hypothalamus):
"Our studies raise several questions. One is which neuronal subtype within the CNS and the MBH mediates the effects of insulin on the regulation of WAT metabolism".
That first one really is an interesting question, one which we can go some way towards answering. We know that the cell type is, as already noted, part of the sympathetic nervous system. In the paper they found either surgical or chemical sympathectomy of adipose tissue increases both lipogenesis and inhibits hormone sensitive lipase in that tissue. I think this is straight forward. The sympathetic nervous system is tonically opposing insulin's lipogenesis effect and insulin's inhibition of hormone sensitive lipase.
The next thing we can say is that these cells sport glutamate receptors. We can safely assume this because, if neonatal rats are injected with the excitotoxin MSG, these are some (among many) of the cells which actually die. That is, the sympathetic nervous system supply to adipose tissue dies. Lipogenesis is unrestrained. Hormone sensitive lipase shuts down. Carbohydrate easily pours in to adipocytes and stays there. Blood glucose levels are low, free fatty acid levels are low, insulin sensitivity is excellent. While ever adipocyte expansion is on going that is. As the adipocytes stretch they eventually become insulin resistant. Here's the table of metabolic parameters from pre-obese MSG injured and control rats, from a previous post:
We know you can do exactly the same by killing these cells with gold thioglucose. This neurotoxin kills those nerve cells which inhibit lipogenesis.
As the authors say: After gold thioglucose injection "systemic insulin sensitivity is preserved [actually it's increased, but these are obesity researchers, so don't quibble] during the early phase of the obesity syndrome, resulting in extensive fat production".
These hypothalamic cells don't seem to take too kindly to the application of an ice pick either:
"In this study, we have measured the expression of the insulin-sensitive glucose transporter, Glut 4 and the activities and expression of key lipogenic enzymes (fatty-acid synthase and acetyl-CoA carboxylase) in white adipose tissue, one and six weeks after the lesion. All these parameters, as well as glucose transport and metabolism determined in white adipocytes, were markedly increased one week after the lesion. They returned to control values within six weeks in VMH-lesioned rats".
All of these interventions allow calories to pour in to adipocytes and stay there. So what does the poor rat do? It's losing a sh*t load of calories in to its adipocytes but, luckily, it has access to a massive hopper of crapinabag in its cage. It simply has to eat enough calories to supply the loss in to adipocytes, plus enough to run its metabolism on. This can be described, by non comprehending people, as hyperphagia. Metabolically it is normophagia.
These rats are calorically neutral or even in mild energy deficit. They have to be running "hyperphagic" just to stand still, metabolically. They are NOT showing "voluntary" overeating. They DO NOT have an injury to any sort of "satiety" centre. They have low insulin, low FFAs and low glucose. They are NOT being paid to over eat! They will NOT be producing a ton of superoxide, despite having a hugely increased caloric intake. Until...
When does this stop? It stops when FFA leakage due to the resistance to insulin induced by adipocyte distention exactly matches the FFA releasing effect which the (now non-existent) sympathetic nervous system would have been having on non distended adipocytes. Sorry for the convoluted sentence, can't simplify it! The distension process was complete by six weeks in the ice pick rat study cited above. Obese rodents then end up with a crudely normal metabolic rate. But this injured system is a complete bodge. We are looking at the replacement of a finely tuned fuel switching system which exactly matches fuel availability to fuel needs with a system where broken adipocytes are simply leaking FFAs at a level which constantly supplements glucose use, without any semblance of fine tuning to metabolic needs. The chronic elevation of fatty acids drives, through the NADH/FADH2 ratio, superoxide production and insulin resistance. Eating glucose then becomes unacceptable because there is inappropriate whole body insulin resistance from excess and inappropriate FFAs. A large amount of insulin is need to control hyperglycaemia under these conditions. Failure to supply adequate insulin to do this, for any reason, is labelled diabetes.
What has this to do with the current obesity epidemic? If you are overweight I would suggest you should take the ice pick out of your brain. No ice pick? Hmmmmm, damn! Back to the drawing board on that one then.
Ah, but maybe you are a C57BL/6J mouse?
Before we can tackle such a stupid question I think we need to go back to superoxide and fatty acids, to about where we were before this digression began.
Peter
Tuesday, August 07, 2012
Protons: Metformin
I'll just stick this post up to get it out of the way. I was going to go on to acute uncoupling next but the link from O Numnos in the last post comments is too good not to post about. It goes some way to tying weight gain in to LACK of superoxide, so brings the thread of insulin as a "satiety" hormone and this thread on weight gain as a failure to generate superoxide in adipocytes (good and bad) together. Might take more than one post... The summary of what's coming: Is insulin a satiety hormone? Only in so far as becoming stable-obese limits your hunger. Anyway, here are a few more thoughts on superoxide first.
Metformin is generally considered to be a Good Drug.
Interestingly it is an inhibitor of complex I of the respiratory chain, which is almost certainly its primary site of action. It aborts glycolysis to lactate because pyruvate is not much use to mitochondria with blocked complex I. Acute exposure to metformin in tissue culture generates a ton of superoxide. Just what you would expect to benefit someone with T2DM!
Let's have a look at this rather nice paper.
They are using differentiated 3T3-L1 adipocytes, a strange beast if ever there was one, but "everyone does it".
They are working under room air with 25mmol/l glucose, supplemental pyruvate, glutamine and 1000pmol/l insulin. These cells are being driven, hard, generating NADH which works through complex I. Complex II will be supplying some FADH2 but there is zero beta oxidation, unless the fatty acids in the adipocyte stores are being accessed. With insulin at 1000pmol/l this is not going to be happening.
Here is the effect of metformin on oxygen consumption:
A dose dependent fall, exactly what you would expect when blocking complex I. Here is the effect of 1.0 mmol/l metformin on oxygen consumption with time:
Nice curves! And here is the effect on ECAR, a surrogate for lactate generation, over 24 hours:
Metformin is only a relatively weak inhibitor of complex I, the incidence of life threatening lactic acidosis is very low. Not so for the more effective biguanides, phenformin and buformin. Obviously the latter two are no longer used clinically, there were too many hiccups.
Now, here is the level of DHE fluoresence, a specific marker of superoxide production. It's being compared to rotenone (remember Coopers Demodectic Mange Dressing? Thought not!), a serious complex I inhibitor.
Metformin is pretty good at generating superoxide. A bit counter intuitive for a drug which is the best treatment, short of insulin, for managing T2DM, a condition essentially defined by failure to overcome insulin resistance (aka superoxide production).
Hmmmmmmmmmmm.
Now, do 3T3-L1 adipocytes like being in forced to live on ATP from glycolysis plus whatever oxphos can be squeezed through metformin inhibited complex I? Annexin V is a marker of very, very unhappy cells. This is what metformin does to the % of cells which are moribund in culture:
So what is going on? Is metformin going to kill our fat cells in vivo?
It's all back to tissue culture conditions. Glucose at 25mmol/l makes the cells utterly dependent on a combination of glycolysis and NADH oxidation at complex I, plus a little FADH2 from succinate metabolism. Our adipocytes are not in this situation.
Now look at this graph:
This is in starvation medium. Only 2.5mmol/l glucose, no pyruvate, no glutamine. I think insulin is still supramaximal, but who cares about insulin when glucose is down at 2.5mmol/l in tissue culture? But here is the really interesting bit: They had also added 0.3mmol/l of palmitic acid to both the control cells and to the metformin cells. Compare it to the graph below, which is the same situation but with glucose and NADH drivers replacing palmitate:
So: Starvation medium plus palmitate completely reverses the fall on oxygen consumption produced by metformin. Palmitate plus starvation medium, even with metformin, actually allows more oxygen consumption that cells running flat out on glucose in the absence of metformin. It's what you would expect, the respiratory quotient is lower for fatty acids than for carbohydrate.
Metformin does not stop fatty acid oxidation. You do need some complex I activity to provide the NAD+ for beta oxidation, but no one is suggesting there is a complete block of complex I by metformin, it's not mange dressing.
So where do the free radicals come from with metformin? I would guess that the citric acid cycle still cycles, there is a build of of NADH due to complex I inhibition and complex II still reduces the CoQ couple. This could allow reverse electron transfer through whatever complex I functionality is left. There are absolutely no data on this, but I like the idea.
The group didn't look at superoxide production under starvation conditions or under starvation plus palmitate. I had a nice email reply to my query from the corresponding author along these lines, there are no data about this, as yet. I would expect the levels of superoxide to be comparable, with metformin being able to mimic palmitate based metabolism in the face of massive fat-free glucose supply, certainly for superoxide generation.
So, superoxide is insulin resistance. Adipocytes under metformin make a ton of superoxide. Are they insulin sensitive or resistant? Resistant of course.
Does an adipocyte which is insulin resistant listen to insulin's orders to store fat? Of course not. "Normal" insulin resistant adipocytes spew free fatty acids to the limit of albumin's transport provisions, with a few other moderating factors.
A metformin poisoned adipocyte is desperate for proton pumping substrate and complex I is doing bugger all to help. But electron-transfering flavoprotein dehydrogenase works perfectly well to allow an alternative electron supply...
Adipocytes under metformin have no choice but to burn fat. In vivo they have a barrel load of the stuff available as soon as they stop listening to insulin. They appear to use fatty acids for metabolism rather than dumping them as FFAs to plasma. Sounds like a recipe for treating metabolic syndrome to me.
Oh, that's what metformin is used for! Well I never...
So, do I think metformin causes adipocytes to become insulin resistant? Of course I do. Is this a Good Thing? You decide.
Peter
BTW Want an opposite to metformin? You can make adipocytes more sensitive to insulin with the thiazolidinediones. They allow insulin to become more effective on already over-distended adipocytes and generate lots of extra, nice, new, ready-to-stuff-with-fat adipoctes. They make you fatter. What would you expect?
Metformin is generally considered to be a Good Drug.
Interestingly it is an inhibitor of complex I of the respiratory chain, which is almost certainly its primary site of action. It aborts glycolysis to lactate because pyruvate is not much use to mitochondria with blocked complex I. Acute exposure to metformin in tissue culture generates a ton of superoxide. Just what you would expect to benefit someone with T2DM!
Let's have a look at this rather nice paper.
They are using differentiated 3T3-L1 adipocytes, a strange beast if ever there was one, but "everyone does it".
They are working under room air with 25mmol/l glucose, supplemental pyruvate, glutamine and 1000pmol/l insulin. These cells are being driven, hard, generating NADH which works through complex I. Complex II will be supplying some FADH2 but there is zero beta oxidation, unless the fatty acids in the adipocyte stores are being accessed. With insulin at 1000pmol/l this is not going to be happening.
Here is the effect of metformin on oxygen consumption:
A dose dependent fall, exactly what you would expect when blocking complex I. Here is the effect of 1.0 mmol/l metformin on oxygen consumption with time:
Nice curves! And here is the effect on ECAR, a surrogate for lactate generation, over 24 hours:
Metformin is only a relatively weak inhibitor of complex I, the incidence of life threatening lactic acidosis is very low. Not so for the more effective biguanides, phenformin and buformin. Obviously the latter two are no longer used clinically, there were too many hiccups.
Now, here is the level of DHE fluoresence, a specific marker of superoxide production. It's being compared to rotenone (remember Coopers Demodectic Mange Dressing? Thought not!), a serious complex I inhibitor.
Metformin is pretty good at generating superoxide. A bit counter intuitive for a drug which is the best treatment, short of insulin, for managing T2DM, a condition essentially defined by failure to overcome insulin resistance (aka superoxide production).
Hmmmmmmmmmmm.
Now, do 3T3-L1 adipocytes like being in forced to live on ATP from glycolysis plus whatever oxphos can be squeezed through metformin inhibited complex I? Annexin V is a marker of very, very unhappy cells. This is what metformin does to the % of cells which are moribund in culture:
So what is going on? Is metformin going to kill our fat cells in vivo?
It's all back to tissue culture conditions. Glucose at 25mmol/l makes the cells utterly dependent on a combination of glycolysis and NADH oxidation at complex I, plus a little FADH2 from succinate metabolism. Our adipocytes are not in this situation.
Now look at this graph:
This is in starvation medium. Only 2.5mmol/l glucose, no pyruvate, no glutamine. I think insulin is still supramaximal, but who cares about insulin when glucose is down at 2.5mmol/l in tissue culture? But here is the really interesting bit: They had also added 0.3mmol/l of palmitic acid to both the control cells and to the metformin cells. Compare it to the graph below, which is the same situation but with glucose and NADH drivers replacing palmitate:
So: Starvation medium plus palmitate completely reverses the fall on oxygen consumption produced by metformin. Palmitate plus starvation medium, even with metformin, actually allows more oxygen consumption that cells running flat out on glucose in the absence of metformin. It's what you would expect, the respiratory quotient is lower for fatty acids than for carbohydrate.
Metformin does not stop fatty acid oxidation. You do need some complex I activity to provide the NAD+ for beta oxidation, but no one is suggesting there is a complete block of complex I by metformin, it's not mange dressing.
So where do the free radicals come from with metformin? I would guess that the citric acid cycle still cycles, there is a build of of NADH due to complex I inhibition and complex II still reduces the CoQ couple. This could allow reverse electron transfer through whatever complex I functionality is left. There are absolutely no data on this, but I like the idea.
The group didn't look at superoxide production under starvation conditions or under starvation plus palmitate. I had a nice email reply to my query from the corresponding author along these lines, there are no data about this, as yet. I would expect the levels of superoxide to be comparable, with metformin being able to mimic palmitate based metabolism in the face of massive fat-free glucose supply, certainly for superoxide generation.
So, superoxide is insulin resistance. Adipocytes under metformin make a ton of superoxide. Are they insulin sensitive or resistant? Resistant of course.
Does an adipocyte which is insulin resistant listen to insulin's orders to store fat? Of course not. "Normal" insulin resistant adipocytes spew free fatty acids to the limit of albumin's transport provisions, with a few other moderating factors.
A metformin poisoned adipocyte is desperate for proton pumping substrate and complex I is doing bugger all to help. But electron-transfering flavoprotein dehydrogenase works perfectly well to allow an alternative electron supply...
Adipocytes under metformin have no choice but to burn fat. In vivo they have a barrel load of the stuff available as soon as they stop listening to insulin. They appear to use fatty acids for metabolism rather than dumping them as FFAs to plasma. Sounds like a recipe for treating metabolic syndrome to me.
Oh, that's what metformin is used for! Well I never...
So, do I think metformin causes adipocytes to become insulin resistant? Of course I do. Is this a Good Thing? You decide.
Peter
BTW Want an opposite to metformin? You can make adipocytes more sensitive to insulin with the thiazolidinediones. They allow insulin to become more effective on already over-distended adipocytes and generate lots of extra, nice, new, ready-to-stuff-with-fat adipoctes. They make you fatter. What would you expect?
Sunday, August 05, 2012
Insulin in the brain: off topic giggle
I had my septic tank emptied a fortnight ago. The contents were a load of crap, but less crappy that the paper purporting to show that insulin is a satiety hormone as quoted by some obesity researcher.
What REALLY happens when you infuse insulin in to the cerebro spinal fluid of a mouse? You know, the satiety hormone... Just in to the brain, nothing systemic, no hypoglycaemia.
Insulin = big fat adipocytes. Big fat mice. Lovely micrographs.
http://www.jci.org/articles/view/31073/figure/5 will give you the legend.
http://www.jci.org/articles/view/31073 will give you the full text. Might discuss the paper better in a few months time!
But main conclusion:
The brain fine tunes the storage of lipid under the influence of insulin (by increasing fat storage via lipoprotein lipase and also by DNL from glucose). It uses the sympathetic nervous system outflow from the ventromedial hypothalamus to do this. Interpret with caution as these are C57BL/6, mice who may well have some very specific weakness in their ventromedial hypothalamus.
OMG did I laugh when I found this one.
Wanna loose some weight, go eat some potatoes. LMFAO!
Sorry for the crudity. Been on call too long this weekend!
Peter
What REALLY happens when you infuse insulin in to the cerebro spinal fluid of a mouse? You know, the satiety hormone... Just in to the brain, nothing systemic, no hypoglycaemia.
Insulin = big fat adipocytes. Big fat mice. Lovely micrographs.
http://www.jci.org/articles/view/31073/figure/5 will give you the legend.
http://www.jci.org/articles/view/31073 will give you the full text. Might discuss the paper better in a few months time!
But main conclusion:
The brain fine tunes the storage of lipid under the influence of insulin (by increasing fat storage via lipoprotein lipase and also by DNL from glucose). It uses the sympathetic nervous system outflow from the ventromedial hypothalamus to do this. Interpret with caution as these are C57BL/6, mice who may well have some very specific weakness in their ventromedial hypothalamus.
OMG did I laugh when I found this one.
Wanna loose some weight, go eat some potatoes. LMFAO!
Sorry for the crudity. Been on call too long this weekend!
Peter
Saturday, August 04, 2012
Protons: Fasting
OK, this is another slightly sideways look at the paper on insulin resistance as an antioxidant defence mechanism.
The basic finding is that manipulating superoxide levels as close as possible to the ETC suggests that it is THE mediator of insulin resistance. Again, I'll skip a large amount of the extreme cleverness utilised and look at the bottom line and its implications. BTW the cleverness was very, very clever. How superoxide controls responsiveness to insulin, nobody knows (though George has some interesting ideas). But it appears to be a generic finding. They looked at steroids, they looked at TNF alpha, excess insulin (good old Somogyi) and, as you might expect, palmitic acid (as in the last post, on a background of 25mmol/l glucose). All cause insulin resistance in the models used. Also bear in mind that they are looking at myotubules and rather peculiar adipocyte-like cells. But I think they are probably correct in this basic conclusion.
Superoxide is core to insulin resistance.
It is very interesting to take this concept and look at various insulin resistance syndromes over the next few weeks.
Of course these folks are in obesity research so you have to be quite cautious when looking at their models and results. You also have to be very, very wary about their conclusions. This is the last sentence of the abstract:
"These data place mitochondrial superoxide at the nexus between intracellular metabolism [tick, agree] and the control of insulin action [tick, agree] potentially defining this as a metabolic sensor of energy excess [woaaaaah, care here]."
This is a slightly tricky sentence. It's that "excess" which bugs me. Look at section L from Fig 4 in the discussion to see how they are thinking:
Here we have a schematic of inactivity and overnutrition causing increased mitochondrial superoxide production. This clearly relates to the Denmark paper where people were paid to eat to excess while deliberately reducing their exercise. Fasting insulin spiked from 35pmol/l to 74pmol/l in 3 days. You can say that overnutrition certainly generates superoxide production. But is this what is happening in weight gain outside of paying people to over eat? That is not how most obese people become obese!
Inactivity and over nutrition are macroscopic changes and superoxide generation is a sub cellular mitochondrial effect. You have to be very careful in how you link the two features together. Superoxide may always signal insulin resistance but are there other drivers of superoxide production in addition to caloric excess?
The situation which keeps coming back to me is starvation.
There is no over nutrition during starvation. There is plenty of superoxide production. Why?
Humans have a brain which is rather dependent on glucose. Using glucose for non brain purposes during starvation would be potentially fatal. All tissues which can become insulin resistant should do so under these conditions.
Superoxide is utterly essential to the survival of starvation. Insulin resistance is a complete necessity.
It looks very much as if fat oxidation (especially palmitate) is directly set up to ensure this happens. It's the reason I was blogging about beta oxidation and FADH2 here. Fat supplies only two molecules of NADH for each of FADH2 and the beta oxidation derived FADH2 enters the electron transport chain through electron-transferring flavoprotein dehydrogenase, directly to the CoQ couple. This is a good situation to generate reverse electron transport, subsequent superoxide and trigger a specific refusal to process insulin. An overnight fasted human has total FFAs of around 0.5mmol/l and they stabilise at around 1.5mmol/l by four days of starvation. They stay there until some food, especially carbohydrate, is eaten.
This level (1.5mmol/l) should, by necessity, develop enough insulin resistance to stop GLUT4 dependent tissues from using glucose, to spare it for brain tissue.
Survival during starvation does not just necessitate using stored fat for energy. It necessitates the near complete abrogation of glucose usage for anything other than brain function. Not after that mere 14 hour fast before an oral glucose tolerance test, but certainly by four days without food. This abrogation cannot be reversed in a couple of hours during an OGTT. This is the "diabetes of starvation".
Superoxide is not always a marker of excess, though this is certainly one way of generating it. It is more accurately a marker of any situation in which insulin resistance is beneficial to survival.
Peter
And I really will get to emails some time soon (mea culpa!)
The basic finding is that manipulating superoxide levels as close as possible to the ETC suggests that it is THE mediator of insulin resistance. Again, I'll skip a large amount of the extreme cleverness utilised and look at the bottom line and its implications. BTW the cleverness was very, very clever. How superoxide controls responsiveness to insulin, nobody knows (though George has some interesting ideas). But it appears to be a generic finding. They looked at steroids, they looked at TNF alpha, excess insulin (good old Somogyi) and, as you might expect, palmitic acid (as in the last post, on a background of 25mmol/l glucose). All cause insulin resistance in the models used. Also bear in mind that they are looking at myotubules and rather peculiar adipocyte-like cells. But I think they are probably correct in this basic conclusion.
Superoxide is core to insulin resistance.
It is very interesting to take this concept and look at various insulin resistance syndromes over the next few weeks.
Of course these folks are in obesity research so you have to be quite cautious when looking at their models and results. You also have to be very, very wary about their conclusions. This is the last sentence of the abstract:
"These data place mitochondrial superoxide at the nexus between intracellular metabolism [tick, agree] and the control of insulin action [tick, agree] potentially defining this as a metabolic sensor of energy excess [woaaaaah, care here]."
This is a slightly tricky sentence. It's that "excess" which bugs me. Look at section L from Fig 4 in the discussion to see how they are thinking:
Here we have a schematic of inactivity and overnutrition causing increased mitochondrial superoxide production. This clearly relates to the Denmark paper where people were paid to eat to excess while deliberately reducing their exercise. Fasting insulin spiked from 35pmol/l to 74pmol/l in 3 days. You can say that overnutrition certainly generates superoxide production. But is this what is happening in weight gain outside of paying people to over eat? That is not how most obese people become obese!
Inactivity and over nutrition are macroscopic changes and superoxide generation is a sub cellular mitochondrial effect. You have to be very careful in how you link the two features together. Superoxide may always signal insulin resistance but are there other drivers of superoxide production in addition to caloric excess?
The situation which keeps coming back to me is starvation.
There is no over nutrition during starvation. There is plenty of superoxide production. Why?
Humans have a brain which is rather dependent on glucose. Using glucose for non brain purposes during starvation would be potentially fatal. All tissues which can become insulin resistant should do so under these conditions.
Superoxide is utterly essential to the survival of starvation. Insulin resistance is a complete necessity.
It looks very much as if fat oxidation (especially palmitate) is directly set up to ensure this happens. It's the reason I was blogging about beta oxidation and FADH2 here. Fat supplies only two molecules of NADH for each of FADH2 and the beta oxidation derived FADH2 enters the electron transport chain through electron-transferring flavoprotein dehydrogenase, directly to the CoQ couple. This is a good situation to generate reverse electron transport, subsequent superoxide and trigger a specific refusal to process insulin. An overnight fasted human has total FFAs of around 0.5mmol/l and they stabilise at around 1.5mmol/l by four days of starvation. They stay there until some food, especially carbohydrate, is eaten.
This level (1.5mmol/l) should, by necessity, develop enough insulin resistance to stop GLUT4 dependent tissues from using glucose, to spare it for brain tissue.
Survival during starvation does not just necessitate using stored fat for energy. It necessitates the near complete abrogation of glucose usage for anything other than brain function. Not after that mere 14 hour fast before an oral glucose tolerance test, but certainly by four days without food. This abrogation cannot be reversed in a couple of hours during an OGTT. This is the "diabetes of starvation".
Superoxide is not always a marker of excess, though this is certainly one way of generating it. It is more accurately a marker of any situation in which insulin resistance is beneficial to survival.
Peter
And I really will get to emails some time soon (mea culpa!)