The paradoxical fat mice (2)

This is the intra-peritoneal insulin tolerance test (ITT) result from the mice in

Caloric Restriction Paradoxically Increases Adiposity in Mice With Genetically Reduced Insulin

as mentioned a post or two ago and which needs some sort of an explanation:

The two asterisks denote that for both of the calorie restricted groups of mice there is an elevated glucose compared to the ad-lib groups in the late part of the ITT, irrespective of whether the insulin gene dose had been reduced by 50% or 75%. Obviously the effect is biologically trivial but the p value of less than 0.05 makes me think the effect is real.

I think to understand this we have to go back some time and look at the concept that metformin has no effect on blood glucose in the absence of insulin. This is the graph from here, discussed here:



My interpretation was/is that, on a background of no metformin (upper line) that insulin (given at 90 mins) generated insulin induced insulin resistance from about sixty minutes later (time 150 mins) and this become p less than 0.05 by 90 minutes after the insulin (time 180 mins), illustrated by the failure to generate insulin-induced insulin resistance in the metformin treated mice (the lower trace).

This is insulin-induced insulin resistance in type 1 diabetic mice revealed by metformin treatment.

Next we can look at type 1 diabetic mice chronically treated with long acting exogenous insulin for a few weeks before an ITT. These have pre existing insulin-induced insulin resistance before any intra-peritoneal short acting insulin is given, taken from here, previously discussed here:

In this case the mice with established insulin resistance simply developed hyperglycaemia when injected with intra-peritoneal short acting insulin. There are no p values but by eyeball the 120 minute value on the upper curve looks like it might be stastically significantly elevated compared to time zero. The message here is that insulin given to an insulin resistance patient can produce hyperglycaemia.

This is the Somogyi Effect. It is real.

In the real world insulin secretion and insulin sensitivity are carefully balanced. Anything which increases insulin secretion increases tissue exposure to insulin and down regulates insulin action. Insulin is the messenger between insulin secretion at the pancreas and insulin response/resistance at the tissues. This is in addition to the shared use of reactive oxygen species to generate both insulin secretion and insulin responsiveness.

We know that the mice with full Ins2 knockout and with or without Ins1 partial knockout are phenotypically normal and have fairly normal insulin levels in their blood.

Aside: To actually get reduced insulin levels Johnson's lab have more recently used a full Ins1 knockout with or without partial Ins2 knockout. The partial Ins2 knockouts do have lowered insulin, are slim, don't get fatty liver and live much longer than they should do. It's all in here. Nice. End aside.

So reduced insulin genotype mice should be more insulin sensitive than full insulin complement mice, though we didn't have a fully normal group in either of the papers from the Johnson lab.

Without calorie restriction (CR) Ins1 partial knockout have their insulin system in balance. With caloric restriction they are so insulin sensitive that when they have an insulogenic calorie restricted small meal they lose calories in to their adipocytes and enter torpor, ie insulin signalling is verging on pathological. So they get fat too. They are not insulin resistant.

But during an ITT they do not merely have the modest insulin levels they might produce in response to their normal small meals. They get 0.75iu/kg of insulin IP, probably more than they have ever seen before. It works. Insulin signalling drops plasma glucose for about 30 minutes. At this point the tissues realise that they are seeing more insulin than they have ever seen in their lives. Insulin-induced insulin resistance kicks in and with it the Somogyi Effect to give elevated glucose.

I think the graph at the top of this page shows an acute onset insulin-induced insulin resistance.

This insulin resistance effect appears to be releasing glucose from the liver, or failing to oppose glucagon action here. It might also have allowed a release of free fatty acids from those greedy adipocytes which precipitated daily torpor. It is just possible that the transient insulin resistant state during the brief ITT might be the only few hours of the entire life of the CR mice that they were not hungry...

Peter

Ketones in Tehran

Just a one-liner

This is a quite fascinating paper from Iran. Bear in mind that none of the authors appears to be a native english speaker and that they could really have done with some editorial assistance, but the results seem quite significant to me. Seyfried gets a thank you for input to the study design but clearly was not an author.

Feasibility, Safety, and Beneficial Effects of MCT Based Ketogenic Diet for Breast Cancer Treatment: A Randomized Controlled Trial Study

They recruited patients who were deemed to need chemotherapy before breast cancer surgery. Half got chemo alone and the other half got chemo plus 12 weeks on a calorie restricted, MCT based ketogenic diet. They all went to surgery and were followed up post-op for about 30 months.

As far as I can make out 30 people in each group completed the intervention. Each of the cross ticks ("censored") are patients lost to follow up in some way, around 10 in each group.

It looks very much like none of the available for follow up patients in the intervention group died. Forty percent (about ten people?) died in the control group, p=0.04.

This is from a 12 week ketogenic pre-surgery intervention. If a drug had produced this effect it would be a blockbuster. There was no instruction to stay ketogenic after the 12 week trial period finished, though there are hints that at least some of the women did.

Interesting, to say the least.

Peter

The paradoxical fat mice (1)

This paper is very interesting. I think I picked it up via Raphi on twitter. It comes from Jim Johnson's lab.

Caloric Restriction Paradoxically Increases Adiposity in Mice With Genetically Reduced Insulin

The background is in these two papers:

Phenotypic alterations in insulin-deficient mutant mice

Compensatory Responses in Mice Carrying a Null Mutation for Ins1 or Ins2

The paradoxical mice all had the Ins2 gene fully knocked out and in addition to this some mice also had one allele for the Ins1 gene knocked out (Ins1+/-). So the mice in the study had either a half or a quarter of the normal mouse insulin gene complement. Some mice were fed ad-lib, some were 40% calorie restricted (CR).

The CR, lowest insulin gene group (Ins1+/-) had significantly elevated total fat mass and a significantly elevated percentage of bodyweight as fat. That's a paradox to the insulin hypothesis of obesity and so really interesting. The Ins1+/+ group also had a (ns) increase in percentage body fat but not in absolute fat mass, so the trend is there too, but only a trend.

Metabolically, the split is between ad-lib vs CR groups.

All mice had the same maximal insulin response to a 2g/kg intraperitoneal glucose tolerance test but the CR groups had a very significantly reduced peak and AUC for glucose, ie they were much more insulin sensitive. The intra-peritoneal insulin tolerance test result might be worth a post in its own right, it's paradoxical too but there won't be space to cover it today.

So let's have a look at energy expenditure (EE) from Fig3 C.

To make things a bit clearer I've copy pasted the light period from the left half of the graph on to the end of the dark period to give more of an idea of the EE curves are really like during dark to light transition:

The red line starts horizontally with no significant difference in EE between ad-lib fed mice or CR mice. There is a modest increase during the dark (active) period when the CR mice get their three very small meals, as indicated. After the last meal a precipitous and highly significant fall in EE occurs. The mice enter torpor, a state of extreme lassitude and hypothermia. At around two hours in to the next light period the mice wake up and EE returns to just below that of the ad-lib mice and the cycle repeats. The ad-lib mice behave like normal mice.

The CR mice have a profound hypometabolic period every day. You could argue, if you are a cico-tard, that this is why they store excess fat. They eat all the food they can get but expend relatively little energy so they become fat: CICO. But I would disagree.

Here's my guess as to what is happening. Speculation warning.

We know that the CR mice are exquisitely insulin sensitive. They are that way because they have a low number of insulin genes and they never get enough food to trigger a major insulin spike anyway. The CR is the dominant factor but it needs the genetic background to get the paradox to occur. Insulin-induced insulin resistance, acute or chronic, does not occur due to lifetime low insulin exposure. The fact that all mice are capable of producing the same maximal insulin response to an IPGTT does not mean that the CR group experience an equivalent insulin exposure to the ad-lib group during their routine lives. They never get enough food to trigger a maximal insulin response.

The CR mice spent the bulk of the light period with a slightly low EE. Dark period arrives and with it food. As the food is eaten there is an upward trend in EE followed by a drop. The second small meal arrives, again an upswing followed by a drop. The third and final meal gives the same upswing but the drop in EE which follows just goes on downward. The mice enter torpor, a state of profound lassitude and hypothermia.

I think torpor happens because the mice simply have no accessible calories.

This is despite the fact that it occurs immediately after the third of their calorie restricted meals. Their problem is that the meals generate an insulin response. The mice are so insulin sensitive that calories are lost in to adipocytes (and probably hepatocytes) under the over-effective action of insulin.

They lose calories in to adipocytes. These are calories out. The adipocytes get bigger with the lost fat.

Torpor occurs BECAUSE the mice have become fatter.

This is the equivalent of the hunger which follows for a human under a euglycaemic (or even hyperglycaemic) hyperinsulinaemic clamp. There is no hypoglycaemia but fatty acids become locked in to adipocytes by the hyperisulinaemia and hunger follows due a lack of available calories. I posted about it here.

At two hours in to the light period insulin drops low enough to allow lipolysis. The mice wake up.

That's all.

Except: Why do the CR mice have paradoxically (although ns) elevated fasting insulin cf the ad lib mice? There are two reasons. Here are the blood sample times added to the EE graph. The arrows are not quite in the correct clock times, as detailed in the methods, but the times related to feeding/metabolism are approximately correct.

The green arrow is the sampling time for the ad-lib fed mice. It is about six hours in to the light period and the mice would normally have been asleep during the hours leading up to it. Light-period snacking, from the respiratory exchange ratio (RER) graph in Fig3 D, would not normally have started by this time so it's a very simple physiological fasting sample.

The blue arrow for the fasting CR mice just hits the end of torpor. I'm not sure these mice ever have a time when they wouldn't eat, given the chance, but here they are in their hypometabolic phase and have minimal access to calories. At this time insulin is actually a little (ns) higher than for the ad-lib groups (Fig1 D). Higher insulin means fat stays in adipocytes. Why is insulin high?

Calorie restriction does many things in addition to dropping metabolic rate. If you fast a hard working group of humans for 5 days they develop a post prandial increase in GIP (glucose-dependent insulinotropic hormone). This was found in the CR mice in both the fasting and fed state (Fig4 C). GIP facilitates insulin release, hence insulin is a little higher the CR mice and loss of calories in to adipocytes more severe, necessitating torpor.

It's interesting as to why GIP might be elevated under hunger conditions. Possibly generating and saving fat becomes a priority when calories are low. The Ins1+/- CR mice certainly have the highest RER (>1.05) after their third meal, suggesting that they prioritise the conversion of glucose to fat. This DNL, should it occur in the liver, might go some way to explaining the elevated triglycerides in both of the CR groups. Maybe. Accentuated DNL in people who have undergone massive weight loss via gastric bypass surgery is routine during an OGTT. Like these people.

Anyway, I'll stop now. This post is about 1/4 the length it started out as, so if corners seem a bit cut then mea culpa.

Peter

Hyperlipid Protons ambassadors

Many people may have noticed that the blog Hyperlipid is not exactly the most user friendly of blogs. The prose clearly appears to make sense but some of the concepts are not always particularly simple unless you have the Protons idea well understood.

Last year (2018) Mike Eades made a sterling presentation which summarised the concept in a talk at Low Carb Down Under in terms that were much more accessible



and Brad Marshall now has a blog on which, throughout 2019, he has been writing around ideas which derive partly from the Protons thread on Hyperlipid. But in significantly more user friendly language, while still being on the spot.

Fire in a Bottle

is his website, a very neat name. It's good. He farms low PUFA pigs too.

Peter

*Very few things in life are quite so disappointing as finding that insulin interacts directly with the NOX4 (NADH oxidase 4) complex to generate the bulk of the initiating low levels of superoxide/H2O2 which trigger insulin signalling. Some ROS do come from the electron transport chain but NOX4 seems rather important. Sigh. Bulk ROS to terminate/blunt insulin signalling do appear to be ETC derived...

The sweet taste of DMEM

I mentioned in the last post that few papers ever specify the glucose concentration used for cell culture. This came up in comments from Alex and I think it deserves a mention in a post of its own.

The standard methods description for almost all cell culture usually specifies that DMEM was used, along with assorted ancillary chemicals and a source of growth factors, usually foetal bovine serum, but rarely the glucose concentration.

A brief trip to any commercial supplier's website gives you approximately 50 DMEM formulations. Three or four have zero glucose and are intended to allow you to specify your own glucose concentration. Another five or six use the original 1g/l of glucose giving 5mmol/l, ie a physiological glucose concentration. This is described as "low glucose".

The other forty-odd specify 4.5g/l, ie 25mmol/l.

I've never done cell culture but I gather the normal technique is to use 25mmol/l medium, let the cells use the glucose to grow and when the glucose drops toward 10mmol/l then they are re-fed with new medium at 25mmol/l of glucose. This works. For decades.

It also provides you with enormous information about the behaviour of cells under hyperglycaemic conditions.

Obviously, 25mmol/l of glucose in an intact human is pure pathology but it is well tolerated by the cells in culture because there is no other caloric source which inputs as FADH2. No free fatty acids.

At the most basic level a glucose concentration of 25mmol/l should a) never occur at all and b) if it does occur it should promptly trigger, via insulin acting on adipocytes, a fall in FFAs to 100 micromol/l or less.

This graph is from a paper on pancreatic beta cell death. From the open circles it is clear that, with glucose held at physiological concentration of 5mmol/l (G5), palmitate is harmless at up to at least 400micromol/l.



This should surprise no one because a 60 hour fast in a healthy human will provide 2000micromol/l of mixed free fatty acids with a glucose of just under 5mmol/l. And no multiple organ failure.

Compare that with the closed circles where glucose is pathologically high at 20mmol/l. Physiological fasting levels of FFAs then unmask the gross pathology of glucose at 20mmol/l. Typical of ordinary cell culture concentration.

Given the idiotic stand of the cardiologist community against saturated fat and given the apparent safety of pathologically elevated glucose in fat-free or PUFA supplemented cell culture medium it should come as no surprise that very few labs worry about the consequences of their grossly non-physiological DMEM.

Demonstrating the pathology of hyperglycaemia using (and blaming) saturated fatty acids or the converse lack of acute toxicity from PUFA (low FADH2 input) is a route to funding which would surely discourage any questioning of the techniques of cell culture which have been successfully used for decades. As folks say:

"We've always done it that way".




That's all I really wanted to say but here are a couple of odds and ends:

There are groups for whom glucose matters. Even if stuck in the incorrect complex I inhibition paradigm for metformin's action, the concentration of glucose is recognised as important by some.

This group:

Metabolic determinants of cancer cell sensitivity to glucose limitation and biguanides

have developed a system for glucose homeostasis in cell culture

Their approach is pretty well unique. It is NOT how cell culture is normally done.


The other brief aside while I'm on cell culture from an outsider viewpoint is FBS, foetal bovine serum. This contains essential growth factors. You can pretty well translate it as "insulin" or "IGF-1".

When cell culture shows that metformin works to reduce cancer cell growth, it is working in the presence of insulin signalling (usually combined with hyperglycaemia). Which metformin blocks at the glycerophosphate shuttle level, without the need for a lethal blockade of complex I.

Peter