Tuesday, February 20, 2018

Alcohol and weight loss

This is a paper I really like. It's about the slimming effect of alcohol:

Chronic alcohol exposure stimulates adipose tissue lipolysis in mice: role of reverse triglyceride transport in the pathogenesis of alcoholic steatosis

This is how you really examine frozen liver samples for steatosis in a real laboratory:

"Neutral lipids in the liver were detected by Oil Red O stain. Liver cryostat sections were cut at 7 μm, fixed with 10% formalin for 5 minutes, and stained with Oil Red O in 2-propynal solution for 10 minutes"

The image on the right (for Ed to enjoy, even if the approach is a little basic compared to what you can do) is from one of the alcoholic mice, lots of lovely lipid accumulation:










And if you want to know about hepatocellular damage, you measure leaked ALT in real plasma from real blood:

"... the plasma ALT level was significantly higher in alcohol-fed mice (68.8 ± 17.0 U/L) than pair-fed mice (28.4 ± 6.7 U/L)".

No suggestion of homogenising liver and measuring ALT in the supernatant!

OK, so these folks seem quite honest and to know what they are doing. That's very nice.

What did they actually do? They deuterated the fatty acids in the adipocytes of live mice, got half of the mice drunk for a few weeks and then measured how much of the deuterated triglycerides turned up in the liver.

Lots did.

They also checked out why the adipocytes released their FFAs under ethanol. The mice developed whole body insulin resistance and they particularly developed adipocyte insulin resistance. If your adipocytes resist insulin, you get thin. Vodka makes you slim, while it grossly fattens your liver and makes you (mildly) insulin resistant.

As they say in the paper:

"In conclusion, the present study demonstrated that reduction of WAT mass and adipocyte size was associated with alcoholic steatosis. Activation of ATGL and HSL due to adipose insulin resistance is likely the major cause in alcohol-induced WAT reduction"

Speculation: Combining alcohol with a carbohydrate load (Beer!) will still make you "sort-of" slim, because any lipid you manage to force in to your adipocytes, using the hyperinsulinaemia needed to achieve normoglycaemia, will be released as soon as insulin starts to fall and you end up with a central beer belly combined with skinny arms and legs peripherally... Back to the paper.

The core slimming effect of ethanol is based on the induction of insulin resistance within adipocytes... This releases FFAs and the FFAs, if not utilised, are stored in liver and visceral adipose tissue.

You really have to wonder how much of the hepatic steatosis of fructose is generated in the same manner as that of alcohol, primarily driven by reverse transport of FFAs from adipocytes to hepatic cells. It would also be interesting to know if PUFA were released from adipocytes alongside the fructose-generated palmitate we talked about in the last post. The adipocytes certainly release multiple PUFA derived FFAs for transport back to the liver under the influence of ethanol.

Peter

BTW, did anyone notice that this group, who appear to be good, didn't measure or report plasma FFAs? I'm guessing FFAs are not markedly elevated in alcoholic reverse lipid flow, so trying to work out what is happening to lipid transport (or oxidation, for that matter) from FFA levels might be somewhat fraught, in any of those studies in which FFA levels are reported in the absence of isotopic tracking... Makes things tricky when you go on to think about fructose studies.

Thursday, February 15, 2018

Systemic fructose is important

******************************************************************
TLDR: Be cautious of anyone who tells you fructose metabolism is limited to the liver.
******************************************************************

Fructose uptake by the liver is saturable. Drinking two cans of soda sweetened with high fructose corn syrup produces a peak plasma concentration 17mmol/l. Yes, 17mmol/l. On average.

Direct spectrophotometric determination of serum fructose in pancreatic cancer patients

Unfortunately the methods section makes no sense at all, so we have no idea how much fructose was actually consumed:

"In 3 of these subjects, intravenous access was obtained in an antecubital vein, and additional blood samples were taken at baseline and 15, 30, 45, 60, 90, and 120 minutes after ingestion (93 minutes) of two 75-mL cans of a proprietary soda, for determination of serum glucose and fructose concentration. Each 40-oz can of soda contained 75 g of high-fructose corn syrup, which consisted of 55% fructose and 45% glucose as constituent monosaccharides, equating to 41.25 g fructose and 33.75 g glucose, respectively".

Go figure. Two 40oz cans of soda? Some big cans there, even by USA standards!

Anyway, this is the graph they produced:




















This next group seems to have managed to write an interpretable methods section but missed peak fructose levels by only sampling at 60 and 120 minutes.

Consumption of rapeseed honey leads to higher serum fructose levels compared with analogue glucose/fructose solutions

Ingesting 75g of neat fructose, as a solution, gives a blood concentration of 130mg/dl, ie they measured just over 7.0mmol/l in real units, at one hour post ingestion.

So fructose gets past the liver and will be taken up by any cells with GLUT3s on their surface. Whole body.

Like adipocytes.

This is a nice paper covering a lot of bases about how adipocytes deal with the fructose they are flooded with every time you down a couple of cans of soda. Or apple juice or.......

Metabolic fate of fructose in human adipocytes: a targeted 13C tracer fate association study

What do adipocytes do with fructose?

They don't oxidise much of it.

They don't convert much to lactate.

They do convert most of it to palmitate and a little to oleate.

They store the oleate.

They release the palmitate as FFAs.

You can't tell from the study how much this palmitate raises systemic FFAs because the study was being performed on "adipocyte-like" cells in cell culture. But, assuming that in most cases fructose would be co-ingested with glucose, you have here the classical situation of elevated free fatty acids, in combination with elevated glucose, in combination with elevated insulin.

This is my definition of metabolic syndrome. The hyperinsulinaemia will, until you become diabetic, eventually control the hyperglycaemia. It may well suppress the elevated FFAs. The glucose and FFAs will be pushed* in to any cell which will respond to insulin.

*Nothing is actually "pushed". Insulin facilitates diffusion (GLUT4s) and maintains a diffusion gradient by removing glucose to glycogen and FFAs to triglycerides.

The liver will be right in the frontline for accepting these FFAs, which should be in adipocytes, and experiencing sustained high levels of insulin (to control glycaemia) will make the hepatocytes hang on to those fatty acids. This is in addition to any intrahepatic trigycerides from fructose-driven DNL. Overall we end up with massively calorically overloaded liver cells. This is the prerequisite to hepatic steatosis and all that is then needed for the generation of inflammatory changes is a source of omega six PUFA. There is a desperate need for liver to say "no" to any more calories. It does by resisting insulin. Which it does by generating ROS. If those ROS meet linoleic acid, it's welcome to 13-HODE, 4-NHE and any other peroxide you care to dig up. These PUFA derivatives do cause insulin resistance per se (as well as 13-HODE stimulating cancer growth), but to me they are just an amplification system derived from what is already happening at the "front end" of the mitochondria... ROS generation by RET, essential to limit grossly excessive caloric ingress.

Peter

Wednesday, February 14, 2018

TRAK2 and HDL. Do we care?

George posted the link to this editorial in the comments of a post some considerable time ago (so it seems now). So this is another old post which has been lying around on the hard drive... Anyway the link is:

Making sense of a seemingly odd connection

It gives an overview and extension of the ideas included in a paper in the same edition of the European Heart Journal

TRAK2, a novel regulator of ABCA1 expression, cholesterol efflux and HDL biogenesis

Both papers are steeped, very deeply, in the Lipid Hypothesis. As such, the chances of them doing anything useful for anyone at all are vanishingly small. Because TRAK2 reduces HDL formation and knocking it down increases HDL, the obvious conclusion is:

"TRAK2 may therefore be an important target in the development of anti-atherosclerotic therapies"

Another target to raise HDL... Sigh, here we go again.

You have to understand that, in the 1950s, the Lipid Hypothesis was bollocks. At no point has anything ever been found to alter that situation. As such I find it very hard to worry about ApoB counts, ApoB sizes, oxidised LDL etc etc etc. including low HDL. Manipulating these numbers by sugar avoidance and saturated fat inclusion will do good. Manipulating them with drugs will bomb. We all still giggle over torcetrapib, anacetrapib, any-other-etrapib and their astronomical, yet useless, levels of HDL.

However, there is that one simple intervention which raises HDL in an effective and totally non toxic manner.

That is saturated fat. Monounsaturated fat is neutral and omega six PUFA lowers HDL. The editorial points out, very perceptively, that not only is 27-hydroxycholesterol a key messenger in HDL formation, but that HDL can be viewed as an export mechanism for free radicals.

Saturated fat raises HDL. Saturated fat drives FADH2 facilcitated RET through complex I in the mitochondria. This process is, undoubtedly, beneficial. Is it the RET which drives the HDL formation? Whether the rise in HDL itself is of benefit or whether the benefits accrue solely from the RET, generated by palmitic acid, which facilitated its formation is an interesting area to speculate in.

MUFA are less effective at RET and HDL generation than saturates. PUFA are useless at both RET and (subsequent?) HDL formation.

HDL, as a vehicle for ROS modified sterols, might be good for you per se. Raising HDL without the ROS/oxidised sterols will be useless. Forget TRAK2.

Just my two penneth.

Peter

Collateral damage from saturophobia. People really do get hurt.

I've spent the last few posts talking about the parlous state of research in to NAFLD and the techniques for justifying saturophobia. This current post is one I wrote a few months ago but never got round to putting up. It's still fairly current, so here it is.

The president of the AHA had a heart attack at an AHA scientific conference recently. This is almost, but not quite, funny. After all, no-one got hurt (much), a little money changed hands and the president is still alive and as healthy as any other cardiologist, still able to go on promoting the ideas which led to his brief trip to the cath lab.

Not everyone is so lucky. I recently finished reading the (very depressing) biography of Tina Mokotoff, written by her husband and documenting her descent in to alcoholism and her subsequent death from alcoholic liver disease at the age of 45.

Mrs Mokotoff had an unremitting need for alcohol. It was the primary drug which allowed her to cope with the emotional scars from her childhood abuse injuries. Her husband, a interventional cardiologist, watched with palpable frustration at the failure of the gastroenterologists to manage her cirrhosis and the failure of repeated rehabs to control her need for alcohol.

WARNING: Epidemiology and rodent studies ahead.

There is significant variation in mortality between populations from alcohol related liver disease (ALD) per unit alcohol consumption. It's interesting to speculate as to why this might be and it was a recurrent thought throughout the persistently depressing account of Tina Mokotoff's journey to death. Let's start with epidemiology:

Correlations between deviations from expected cirrhosis mortality and serum uric acid and dietary protein intake

Mortality from cirrhosis in a population can range from 80% less than predicted (ie two cirrhosis deaths per 100,000 when the alcohol intake predicts 10 per 100,000) through to over 80% more deaths than predicted (ie over 18 per 100,000). That's a nine fold difference between lowest and highest risk, at the same alcohol intake. Something is real here.

In this epidemiological study, animal protein intake is associated with a markedly reduced cirrhosis death rate. The animal protein may be protective per se but I tend towards thinking of it as being a marker for saturated fat intake. But then I would.

To support this biased mindset we know that, in rodent models at least, saturated fat is either completely protective against alcoholic liver disease or shows a dose response in its protective effect up to near complete protection at 30% of calories from saturated fat, even when the other 15% of calories in the 45% calories-from-fat-diet are still PUFA. We also know that even mice fed a low carbohydrate diet derive no protection from ALD when the carbohydrate is replaced by PUFA from corn oil. I doubt anyone would argue that PUFA are good for your liver. Hepatologists have known for decades that lipid peroxides are the drivers of cirrhosis and these only come from damaged PUFA.

Through the 1990s, during his wife's descent in to cirrhosis, Dr Mokotoff worked tirelessly in the cath lab placing stents and "curing" people of occlusive coronary artery disease. His life must have been very simple. Here is a blocked artery. Here is a bit of pipework to open it. Let's put this in there and the patient is fixed. Just occasionally he might even have done some good (though far from as often as he might have thought he had done). During this period the cardiological community was deeply under the influence of the "obvious" benefits from a low fat, low saturated fat and low cholesterol diet.

Unless you are going to indulge in some weird Ornisheque low fat diet, eating a saturated fat depleted diet will will undoubtedly involve a significant intake of PUFA. This should never, under any circumstances, be combined with alcohol. Would the Morokoffs have avoided saturated fat? Dr Mokotoff was an interventional cardiologist. Just guess.

Having a cardiologist, the president of the AHA, inflict a minor injury on himself, without getting really hurt, is ironic. Reading an account of a real human being being driven to a very unpleasant death through cirrhosis is not funny. Inflicting a population wide epidemic of assorted PUFA induced diseases is, absolutely, not funny either.

Thank you, AHA.

Peter

Saturated fat and fatty liver. Payday in Colorado.

Dophamn supplied the link to another interesting study:

The role of visceral and subcutaneous adipose tissue fatty acid composition in liver pathophysiology associated with NAFLD

Here is the money shot that supports the religion of saturated fat as the devil incarnate:

"Overall, these data suggest that diets enriched in saturated fatty acids are associated with liver inflammation, ER stress and injury".

Meanwhile, in the study detail:

I would agree that the stearic acid rats stayed comparable in weight to others despite eating more calories than either Crapinabag or PUFA fed rats, as in Table 1. There is NO evidence that they developed inflammatory changes in their liver! They had a statistically significant increase in messenger RNA expression for seven genes associated with inflammatory liver disease. The question is whether these mRNA changes actually result in detectable inflammatory changes in the liver, or are they markers of the normal response to reverse electron transport though complex I derived superoxide which might also trigger life extending increases in SOD and/or catalase gene expression? ROS generation is essential to mitochondrial biogenesis. It MUST affect signalling molecules. At what level does physiological signalling degenerate in to pathology? Easy to find this out, just look at the liver histology.

What we need to know is whether there is histological evidence of NASH development. After all, we know from the methods that they took terminal samples of liver and snap froze them in liquid nitrogen. Either sticking some in formalin at the same time or getting histology done on the frozen samples (not ideal from the histologist point of view but quite possible) would allow them to correlate their mRNAs with actual damage in the liver. They didn’t do this.

So why did they freeze liver samples at all? As they say in the methods:

“Liver tissue was homogenized in buffer (100mM Tris, pH 7.8) and alanine aminotransferase (ALT) concentration was determined from supernatant via manufacture instructions (Cayman Chemical, Ann Arbor, MI)”.

[Not my typo in the copy/paste. I can do enough of my own when I feel that way!!!]

In the results section in Figure 4 this is converted to:

Plasma alanine aminotransferase concentration was higher in SAT compared with CON and PUFA”.

[My shouting emphasis on "supernatant" and "plasma"]

Plasma???? No. The methods clearly state that it was liver homogenate supernatant! Plasma ALT is an absolutely routine, standard, everyday marker of liver damage. It is a surrogate for hepatocellular damage, i.e. a normal component of liver cytoplasm which has leaked in to plasma in response to liver injury. It’s measured every day in any patient undergoing any sort of health/illness monitoring blood work. It is a COMPLETELY normal cytoplasm component while it is contained within the liver hepatocytes. It is LEAKAGE  to the blood stream that we are interested in as a surrogate for hepatic damage. The rats all had terminal blood samples taken. The group could have measured ALT for a few pence in real plasma from this blood. They didn’t. They homogenised liver and measured ALT in the supernatant. They described this as “plasma”. All we can say from Fig 4 is that the liver of stearic acid fed rats has more of ALT within its hepatocytes. ALT is a normal enzyme used for interconverting certain components of the TCA/amino acid metabolism. Who knows why it is increased under stearate feeding, but it's not a marker of hepatocellular damage unless it is being released in to the blood stream... I think we can assume plasma ALT was completely normal. I'd be willing to bet it was measured in a pilot study and failed to pass the pay-dirt test.

The related studies cited in this paper are equally interesting and say nothing about much other than the ingenuity of the researchers. As always, my fascination is about the mindset involved.

Who decided to homogenise liver to get “plasma” ALT? Who decided to bin the histology friendly liver samples?

Why?

Peter

Monday, February 12, 2018

Saturated fat and fatty liver. Payday in Sweden.

DLS posted a link to this paper in the comments on the last post.

Overfeeding Polyunsaturated and Saturated Fat Causes Distinct Effects on Liver and Visceral Fat Accumulation in Humans

It's really fascinating. It's rather the flip side to the rodent study in the post itself. They took reasonably healthy humans and over-fed them muffins based on palm oil or sunflower oil.

The core findings, here from the conclusions:

"The fate of SFA [saturated fat] appears to be ectopic and general fat accumulation, whereas PUFA instead promotes lean tissue in healthy subjects. Given a detrimental role of liver fat and visceral fat in diabetes, the potential of early prevention of ectopic fat and hepatic steatosis by replacing some SFA with PUFA in the diet should be further investigated".

And the most important finding from the results:

"the MRI assessment showed that the SFA group gained more liver fat, total fat, and visceral fat, but less lean tissue compared with subjects in PUFA group (Table 2)".

This is pay dirt. It completely justifies saturated fat avoidance at even modest overeating. As Tom Naughton has commented recently:

Jane Brody And The American Heart Association Bravely Admit They’ve Been Right All Along

Well. I guess we can all just pack up and go home right now.

But, ultimately, you have to try to understand what is going on.

So let's have a think about it. We have two populations of adipocytes in the two study groups. Each is being provided with an excess of fatty acids to store under the influence of insulin. One population is being exposed to palmitic acid. Palmitic acid provides the maximum FADH2 of all FFAs excepting stearic acid. So it predisposes to generating insulin resistance via reverse electron transport (RET). In adipocytes this means that they are less likely to accumulate triglyceride, ie palmitic acid stops you getting fat. It does this by limiting fat storage under peak insulin. My presumption is that, under free feeding situations, this information about the state of adipocytes is transmitted to the brain, either through plasma fatty acids, hormones or via the autonomic nervous system, resulting in a cessation of eating. But there is no cessation of eating allowed in the study. If you don't gain weight you are made to eat more muffins. You have to eat. If the excess fat in the diet is not going in to the adipocytes it is going to end up somewhere else. Liver and visceral fat are good places if you have nowhere else. Sticking it in muscles might well limit the anabolic action of insulin at this site.

The PUFA group are asked to eat more too. The linoleic acid in the muffins allows easy distention of this population of adipocytes (less FADH2 per unit NADH). Insulin acts easily because peak RET is blunted and adipoctes accept more fat. Excess dietary fat ends up in adipocytes, the adipocytes don't care. At 1.6kg weight gain in a young, fit Swede there is insufficient adipocyte distention to raise FFAs in the face of insulin.  Eating surplus PUFA appears to be metabolically easier to deal with than eating palmitate beyond acute needs. With sequestration of fatty acids in adipocytes rather than in to muscle we have the possibility for the anabolic effect of insulin actually working at increasing lean muscle mass.

We know that the groups were carefully managed to reach a very tightly controlled target of weight gain. Week by week the number of muffins fed per day was adjusted to give us the desired target gain of 1.6kg in each group. It took, on average, 3.1 muffins per day in each group to achieve this over seven weeks.

What we don't know is what the pattern of weight gain was during the study. Did the PUFA group gain weight easily in the early weeks and need less and less muffins later in the study to avoid excess weight gain, with the risk of overshooting the 1.6kg target?

Did the palmitic acid group show a steady weight gain, almost all of it ending up in ectopic sites because subcutaneous adipocytes didn't want to accept more fat throughout the study?

These are interesting thoughts. It is an interesting paper!

Peter

BTW There are a whole stack more questions regarding the role of fructose in the paper but I think the basics are probably covered in the differential effects of of fatty acids on the electron transport chain.

Monday, February 05, 2018

Follow on to Tucker's post on PUFA in rats

Tucker posted an excellent discussion of this paper on his blog. Go read it:

Fat Quality Influences the Obesogenic Effect of High Fat Diets

The basic conclusion is that feeding rats a high fat diet makes them fat. If it is PUFA based, including a generous amount of omega 3 alpha linolenic acid, it will cook their liver (figuratively speaking... in actuallity it converts their liver to being full of peroxidised PUFA, en-route to cirrhosis). I have an anecdote-type post on the problems of being married to a cardiologist if you happen to be alcohol addicted somewhere. I really ought to dig it out and hit post.

So. The problems with the paper:

The rats on the PUFA diet, with the gross fatty livers, were less obese than the lard fed rats, had better lean body mass percentage and much better brown adipose tissue hypertrophy and fat oxidation.

The bottom line: If you want look slim and well muscled in your coffin then a safflower oil diet with a heavy dash of varnish might be a good choice...

How come?

The paper was not looking at insulin levels or insulin signalling so it doesn't provide the data we need to come to any conclusions but it has resonances to the comment Zoran made on the previous post.

The Protons Credo (believe if you so wish!) for the situation:

PUFA, of a carbon chain length which targets them for mitochondrial oxidation, input less FADH2 at mitochondrial electron transporting flavoprotein dehydrogenase (mtETFdh) than do saturated fats or MUFA. This lack of FADH2 input limits the ability to reduce the CoQ couple and facilitates electron flow down the electron transport chain (ETC) and so limits the generation of reverse electron transport through complex I. This damped RET limits the ROS generation (superoxide and H2O2) necessary to initiate insulin signalling under fasting and to limit excessive insulin signalling in the fed state.

So on a whole body basis PUFA maintain insulin sensitivity. Insulin acts, rather well, under PUFA compared to under saturated fat, in the fed state. It works less well in the fasted state.

A fed, insulin sensitive animal will do two things of interest on a medium carbohydrate, generous fat diet. It will utilise glucose easily in muscles to burn calories and it will continue to use glucose in adipocytes to esterify FFAs with glycolysis-derived glycerol, to store fat.

So the Protons thread expects insulin sensitivity to cause fat accumulation because of maintained insulin sensitivity in adipocytes at high levels of insulin signalling. The cost of this insulin sensitivity is obesity.

PUFA = obesity, soybean oil is the best, they used safflower here.

Slight aside: The insulin resistance associated with obesity is nothing to do with insulin per se. It is triggered by the fact that very large adipocytes leak free fatty acids irrespective of insulin levels. At elevated FFA levels more insulin is needed to translocate GLUT4s than at low FFA levels.

Back to the rats.

The lard fed rats are the most obese. The PUFA fed rats the least obese.

The lard fed rats are on about 10% of their calories as PUFA in their diet. They are probably almost as fat as a 10% PUFA diet would like them to be, ie their adipocytes are almost as distended as a 10% PUFA diet dictates. The rats are almost as fat as they need to be. They are doing this on 380kJ per day. Because the rats are only allowed a total of 380kJ of energy per day. Did you pick that up in the methods?

The PUFA fed rats want to be truely, grossly obese, much more so than the lard fed rats do, because they are on somewhere between 50% and 60% of their energy intake as PUFA. But there, in the hopper, is that same old 380kJ per rat per day. It doesn't matter how much your adipocytes are crying out for more fat, how empty they feel, how hungry they tell your brain to feel. There, in the hopper, is 380kJ.

These rats are intensely insulin sensitive because their adipocytes are "empty" compared to how thery would like to be. They are "starving" compared to how they would like to be. Their muscles respond to insulin's anabolic effect and I'd be willing to bet their growth hormone levels are through the roof and IGF-1 through the floor (another post there, GH, IGF-1 and starvation). Insulin is going to be low because any glucose released from the liver is easily utilised in the fed state. In the fasting state insulin fails to act effectively so that, while FFAs may be the same as in the lard fed rats, we know (from Figure 2) that lipids are being oxidised much more rapidly on a 24h basis.

PUFA sensitise adipocytes to insulin. Given the choice the animal will eat until obese and become insulin resistant due to adipocyte distension. Combine PUFA with starvation and insulin sensitivity will be maintained. Or enhanced.

Just the Protons view. Any other explanations welcome.

Peter

Of course people should ask how the action of PUFA compares to the action of metformin. They are superficially similar. That might need more doodles I'm afraid!

Saturday, January 20, 2018

Metformin (07) glargine 50iu/kg causes diabetes

The group which demonstrated that exogenous insulin induces insulin resistance in T1DM NOD mice went on to demonstrate that exogenous insulin induces T2DM in normal* mice on a normal chow diet.

*If you can describe Bl/6 mice as normal, with their failure to assemble mitochondrial super complexes and all of the potential implications that has on all sorts of metabolic effects. But I digress, as always.

Exposure to excess insulin (glargine) induces type 2 diabetes mellitus in mice fed on a chow diet

So you can imagine that I quite like this group. But they are naughty and the naughtiness is very annoying.

If you read about insulin administration in the current paper you will be presented with this bollocks:

"The dose of glargine was determined according to our previous experiments (Liu et al. 2009a )..."

What they did in Liu et al 2009a was to titrate the dose of insulin determir upwards to just achieve normoglycaemia. Different doses were needed in individual mice because that's what diabetes is like in NOD mice. That is NOT what they did in this current paper to the Bl/6 mice. Here they used glargine and they went straight in at a massive supraphysiological dose:

"C57BL/6 (B6) mice (male, 6–9-week old) from Charles River were treated with either saline or a long- and slow-acting insulin reagent, glargine (50 Unit/kg body weight, s.c. injection, once a day), for 8 weeks".

Even allowing for metabolic scaling from humans down to mice this is a massive dose of glargine. It's not remotely what they did with the NOD mice.

EDIT: Going back through the first paper the NOD T1 mice did actually end up with the average group dose of detemir being 25iu/kg twice daily. The difference in protocol is that the group is assuming that glargine 50iu/kg once daily is equivalent to detemir 25iu/kg twice daily. A big assumption. And that this would be fine for all mice. And that going in at the full dose rate on day one rather than titrating up over two weeks would also be equivalent. But the dose rate is more reasonable than I expected. END EDIT.

So there are fibs in the methods. Can you really trust these folks? Not much choice really...

None of the mice died on the glargine 50iu/kg dose, so I think we can assume that they developed a marked and rapid onset insulin-induced insulin resistance.

When you wade through the IRS1 serine phosphorylation, IRS1 tyrosine phosphorylation, Akt phosphorylation, total Akt etc etc etc in the results section the end conclusion is that the liver became insulin resistant but the gastrocnemius muscle (representing what I called "systemic" tissues) did not. Bummer for my nice, plausible and apparently incorrect ideas.

So to ease my cognitative dissonance I went back to our initial paper and waded through the IRS1 serine phosphorylation, IRS1 tyrosine phosphorylation, Akt phosphorylation, total Akt etc etc etc and discoved that, lo and behold, that under a clinical protocol the gastrocnemius did become insulin resistant. So did the liver but I can live with that, after all the liver does have a (bloody enormous) arterial blood supply in addition to receiving flow from the portal vein.

Phew, biases in-tact.

It's interesting to see in the paper that exogenous glargine actually destroys the pancreas. Beta cell mass falls, their mitochondria undergo marked oxidative stress and this allows the failure to deal with the hepatic insulin resistance induced by glargine 50iu/kg, hence the induction of diabetes.

I think the take home message is that taking glargine 50iu/kg won't help your own clinical diabetes, should you be so affected. It's also too high a dose if you are attempting to do harm!

Anyhow. Growth hormone and insulin resistance next, probably.

Peter

Friday, January 19, 2018

Metformin (06) Insulin-induced insulin resistance is real

When I started reading about insulin-induced insulin resistance I began with this paper:

Insulin Is a Stronger Inducer of Insulin Resistance than Hyperglycemia in Mice with Type 1 Diabetes Mellitus (T1DM)

It's a nice paper. They took NOD mice which had developed their NOD mouse version of T1DM and either treated them with insulin detemir, or didn't. They had a third group which never developed T1DM so were never treated and these served as a control group.

The treated diabetic NOD mice were gradually stabilised over a two week period then kept normoglycaemic for a further two days. They were assessed for insulin sensitivity using an insulin tolerance test, where a dose of neutral insulin is injected then you track what happens to the blood glucose concentration. The more insulin sensitive the animal, the more the glucose level drops:















It's pretty obvious that the detemir treated mice (top line) have absolutely no response to neutral insulin and that both non-treated diabetic mice and never-diabetic mice drop their blood glucose levels by about 50% on this particular dose of neutral insulin.

I could stop this post here. Exogenous insulin induces insulin resistance in T1DM mice, as it does in people. This is fact.

But of course you should not just accept this. The question is: Why?

Why does "enough" insulin as secreted by the pancreas to produce normoglycaemia (in the never-diabetic control group of mice) cause no insulin resistance whereas insulin detemir given to produce the same level of normoglycaemia induces striking insulin resistance in those treated NOD mice?

Recall that the hyperglycaemia in T1DM has little to do with the lack of insulin per se. The hyperglycaemia is caused by an excess of glucagon from the alpha cells of the pancreas. Insulin starts its control of hyperglycaemia by the suppression of pancreatic glucagon secretion, it's a local action within the islets. How high this concentration of insulin is under normal physiological conditions is quite hard to determine but it is likely to be a lot higher than the diluted insulin concentration in the portal vein, heading towards the liver.

The diluted insulin within the portal vein arrives at the liver where its next job is to suppress hepatic glucose output, again in antagonism to glucagon.

Finally, if glucose from the liver continues to enter the systemic circulation, the function of insulin here is to push that glucose in to any cells that will take it. Muscle and adipose tissue being two major targets.

So under normal physiology there is a gradient of insulin concentrations from very high within the Islets of Langerhans, to significantly lower at the hepatocytes, down to much lower in the systemic circulation.

Exogenous insulin produces no such gradient. It drains from its injection site into the systemic veins and is then redistributed, at a single concentration, throughout the body.

This will never effectively suppress alpha cell glucagon secretion and will only do a modestly effective job of suppressing hepatic glucose output. So glucose will be continuously secreted in to the systemic circulation. The dose of detemir used has to be enough to mop up this excess glucose supply, and it can only put it in to cells sensitive to insulin throughout the body. Muscle cells. Adipocytes.

Now look at it from recipient cell's point of view. Glucagon is high, hepatic glucose output is high and this continuous supply of glucose is being allowed in to systemic cells by the exogenous insulin. To these cells the glucose supply looks like a meal being digested (high glucose, high insulin). The cells rapidly realise that they have enough calories (High NADH levels, high mitochondrial delta psi, reduced electron transport chain). They don't want any more. Their solution: insulin-induced insulin resistance (ie reverse electron transport to generate H2O2 at complex I and so inactivate insulin signalling).

That's what happens.

So the very effective control of blood glucose in these NOD mice is at the cost of continuous exposure to supraphysiological insulin levels coupled with a supraphysiological glucose supply, because the systemic cells are "covering for" the failure to replicate the normal gradient from islet to liver to systemic circulation.

Exogenous insulin can never be physiological.

Aside: Except, of course, under deeply ketogenic eating where only minimal insulin is ever secreted, very little is metabolised, the gradients between alpha cells, hepatocytes and adipocytes flattens out and the correct physiology is for glucagon to be elevated with minimal insulin. I've posted this before. T1DM patients have no choice, ketosis is the only physiological state which can be fairly well mimicked using very low doses of exogenous insulin. End aside.

I would never suggest that exogenous insulin has no effect on pancreatic glucagon secretion or elicits no suppression of hepatic glucose output. It will always have some effect, but there will always be an abnormal emphasis of its effect on systemic tissues.

This is the situation in NOD mice and T1DM people. They become insulin resistant by simply using exogenous insulin to ensure normoglycaemia.

I guess the next question, which was asked by the same group whose paper we've just been looking at, is whether simply injecting exogenous insulin in to normal mice induces insulin resistance. That's the next paper, in the next post.

Spoiler: Of course it does.

Peter

Sunday, January 14, 2018

A wander off in to dietary protein calories

There is prize for developing the longest-lived mouse in the world. It was set up in 2003 and the first award went to Dr Bartke.

"On June 8th, 2003, the inaugural Methuselah Prize was awarded to Dr. Andrzej Bartke for the "Methuselah Mouse" that lived the equivalent of 180 human years".

You can read a bit more about growth hormone receptor knockout mice and other forms of dwarf mice in Dr Bartke's review, written soon after winning the prize:

Life extension in the dwarf mouse.

It's now 2018 and no one appears to have improved on the Laron mouse model which won that initial prize. Over the last 15 years there has been a lot of interesting research but no numerical progress. I think it is worth noting that Laron mice are not GH deficient, they have tons of the stuff. They simply do not have the receptor to do anything with it. Which, in particular, means they cannot generate IGF-1.

How do Laron humans fare? The best studied group live in Colombia. They're of very short stature. They have no recorded cases of diabetes and only one recorded cancer, which was non lethal*. Their every biochemical parameter is exemplary, especially insulin level and HOMA score. Do they all live to be centenarians? Apparently not. Being a dwarf in Columbia requires alcohol in large amounts to render life tolerable, plus accidental trauma is another huge problem. Quite what would happen if these people lived under similar conditions to the Laron mice in Dr Bartke's laboratory is a question which is unlikely ever to be answered! Longevity in the real world vs what works under ideal conditions...

*Dr Laron has reported two cases of Laron Syndrome people developing diabetes (and there are others), including the complications such as atherosclerosis, renal disease and diabetic retinopathy. This is an interesting observation and might be worth a post on its own some time.

There are tantalising suggestions from other GH modifying mutations in humans. One of the better studied of these is carried by the "Little people" of Krk in Croatia. More from Dr Laron:

Do deficiencies in growth hormone and insulin-like growth factor-1 (IGF-1) shorten or prolong longevity?

Longevity of the hypopituitary patients from the island Krk: a follow-up study

They have a mutation which causes multiple pituitary hormone deficits, ACTH secretion excepted. There are too few documented people with this genetic problem to say a great deal about longevity but ages of 68, 77, 83 and 91 years have been recorded in the four individuals to have died since detailed observations began. The equivalent syndrome in mice under lab conditions promotes longevity.

One of the nicer studies looking at human height (viewing this as a GH/IGF-1 signalling surrogate) and longevity is this one:

Shorter Men Live Longer: Association of Height with Longevity and FOXO3 Genotype in American Men of Japanese Ancestry

It found, as you might expect, an inverse relationship between height at enrolment and longevity. They also tied the relationship, observationally, to a down-regulating SNP of the FOXO3 gene, FOXO genes being major controllers of the insulin/IGF-1 signalling system.

Which genes you have is not under your control. What you do with then might well be...

Let's finish this post with the LoBAG diet. It's modest (20% of calories) in carbohydrate, has 30% of calories from protein and the rest as fat. It's being compared to a diet with similar carbohydrate content, 15% of calories from protein, with the rest as fat. Lots of details in here:

The metabolic response to a high-protein, low-carbohydrate diet in men with type 2 diabetes mellitus

As they say in the discussion:

"The present data indicate that the increase in IGF-1 is the result of the increase in protein content. The further decrease in carbohydrate did not result in a further increase in IGF-1. In fact, the increase was approximately the same (138% and 136%, respectively)".























What interested me initially (and had made me chase the paper) was the effect on GH itself. The LoBAG diet actually drops GH levels, admittedly by a ns amount. What turns out to be a much more interesting incidental finding is that, despite the downward trend in GH, IGF-1 rises by a statistically significantly and possibly by a biologically significant amount. Especially when you consider a whole slew of cancers sprout IGF-1 receptors on their surface.

Brief aside. You have to be very careful with GH and IGF-1 levels in papers like this one as both hormones come with a whole load of plasma binding proteins which very few people, including the LoBAG folks, ever measure. These may well alter the effective concentration of the hormone either upwards or downwards. Caution is needed with simple measurements like those in this paper. End aside.

So. Folks should eat whatever they feel comfortable with, protein-wise. I probably eat a little more protein than I would prefer, but then I'm no perfectionist. What I wouldn't do is to add protein gratuitously to any meal...

But that's just me I guess.

Peter