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Hyperlipid



You need to get calories from somewhere, should it be from carbohydrate or fat?



Updated: 2018-04-23T05:13:12.270+00:00

 



AHA approved egg!

2018-04-13T07:12:13.833+00:00

One of the full size chickens miss-fired yesterday and produced this minute egg:

















I was pretty sure this would be an AHA approved egg. Zero fat. Zero cholesterol. Tiny amount of protein. You don't have to include the eggshell (unless you feel a calcium supplement is a good idea. Could treat your acid reflux at the same time!).


















Of course it should be fried in corn oil. Still hungry?

Fill up on sugar!

Ah, the decades of stupidity that have now been mostly overturned. Just the corn oil to get rid of.

Peter



Pasta for weight loss

2018-04-09T17:07:08.977+00:00

This paper hit T'internet recently and has been cited all over the place:Effect of pasta in the context of low-glycaemic index dietary patterns on body weight and markers of adiposity: a systematic review and meta-analysis of randomised controlled trials in adultsObviously the sole claim to fame for the paper is the conflict of interest statement. I've greyed it out so no-one is tempted to read it in full, the flavour is all you need:"Competing interests: All authors have completed the Unified Competing Interest form (available on request from the corresponding author) and declare: LC has worked as a clinical research coordinator at Glycaemic Index Laboratories, Toronto, Ontario, Canada. CWCK has received research support from the Advanced Food Materials Network, Agriculture and Agri-Foods Canada (AAFC), Almond Board of California, American Pistachio Growers, Barilla, California Strawberry Commission, Calorie Control Council, Canadian Institutes of Health Research (CIHR), Canola Council of Canada, International Nut and Dried Fruit Council, International Tree Nut Council Research and Education Foundation, Loblaw Brands Ltd, Pulse Canada, Saskatchewan Pulse Growers and Unilever. He has received in-kind research support from the Almond Board of California, California Walnut Council, American Peanut Council, Barilla, Unilever, Unico, Primo, Loblaw Companies, Quaker (Pepsico), Pristine Gourmet, Kellogg Canada, WhiteWave Foods. He has received travel support and/or honoraria from the American Peanut Council, American Pistachio Growers, Barilla, Bayer, California Walnut Commission, Canola Council of Canada, General Mills, International Tree Nut Council, Loblaw Brands Ltd, Nutrition Foundation of Italy, Oldways Preservation Trust, Orafti, Paramount Farms, Peanut Institute, Pulse Canada, Sabra Dipping Co., Saskatchewan Pulse Growers, Sun-Maid, Tate & Lyle, Unilever and White Wave Foods. He has served on the scientific advisory board for the International Tree Nut Council, McCormick Science Institute, Oldways Preservation Trust, Paramount Farms and Pulse Canada. He is a member of the International Carbohydrate Quality Consortium (ICQC), Executive Board Member of the Diabetes and Nutrition Study Group (DNSG) of the European Association for the Study of Diabetes (EASD), is on the Clinical Practice Guidelines Expert Committee for Nutrition Therapy of the EASD and is a Director of the Toronto 3D Knowledge Synthesis and Clinical Trials foundation. DJAJ has received research grants from Saskatchewan Pulse Growers, the Agricultural Bioproducts Innovation Program through the Pulse Research Network, the Advanced Foods and Material Network, Loblaw Companies Ltd., Unilever, Barilla, the Almond Board of California, Agriculture and Agri-food Canada, Pulse Canada, Kellogg’s Company, Canada, Quaker Oats, Canada, Procter & Gamble Technical Centre Ltd., Bayer Consumer Care, Springfield, NJ, Pepsi/Quaker, International Nut & Dried Fruit (INC), Soy Foods Association of North America, the Coca-Cola Company (investigator initiated, unrestricted grant), Solae, Haine Celestial, the Sanitarium Company, Orafti, the International Tree Nut Council Nutrition Research and Education Foundation, the Peanut Institute, the Canola and Flax Councils of Canada, the Calorie Control Council (CCC), the CIHR, the Canada Foundation for Innovation and the Ontario Research Fund. He has received in-kind supplies for trial as a research support from the Almond Board of California, Walnut Council of California, American Peanut Council, Barilla, Unilever, Unico, Primo, Loblaw Companies, Quaker (Pepsico), Kellogg Canada, and WhiteWave Foods. He has been on the speaker’s panel, served on the scientific advisory board and/or received travel support and/or honoraria from the Almond Board of California, Canadian Agriculture Policy Institute, Loblaw Companies Ltd, the Griffin Hospital (for the development of the NuVal scoring system, the Coca-Cola Company, EPICURE, Danone, Diet Quality Photo Navigation (DQPN), Better Therapeutics (FareWell[...]



Guddling in the dark for a respiratory quotient

2018-03-21T08:01:08.757+00:00

Here's a paradox: How can two groups of mice, on exactly the same chow, have different 24h averaged RQs, p less than 0.05?

















It's from here if anyone wants to peek at the methods. Two sets of animals on the same chow. It's 9F 5020, 21% of calories from fat (7% of calories from PUFA) and 55% from carbohydrate.

At all time points the SC-VIS mice have an higher RQ, ie are oxidising more glucose, than the SHAM mice. But they are all fed the same chow, which should average out at the same overall RQ.

Clearly you can increase the RQ, even above 1.0, during de novo lipogenesis, especially when hungry mice suddenly eat carbohydrate. But there is either a payback during the sleep phase where RQ falls below the food derived RQ while that carbohydrate-derived fat is oxidised or there can be no fall in that fasting RQ if the DNL generated fat is "lost" in to adipocytes and stays there, ie under weight gain. Of course simply sequestering dietary fat in to adipocytes will generate an RQ more typical of glucose oxidation because less fat is being oxidised, full stop, during weight gain.

During on-going fat loss the extra low RQ from adipose derived fat oxidation does not have to be payed back either. "Food" of very low RQ, has been supplied from adipocytes. It's gone out of the body as CO2 and water.

But the black square mice are weight stable or actually losing adipose weight (ie should have an extra low RQ) at the time these RQs were measured, while the open diamond mice are actively gaining weight (including adipose tissue), so should have that higher RQ.

Food intakes are describes as "no significant difference" between the groups, despite the differential weight shifts.

To me this is inexplicable and should have been discussed in the paper. My feeling is the CLAMS equipment is generating a totally illogical result.

Unless I've totally missed something. I would really like to know whether I have totally missed something.

Just on general principles of substrate oxidation, never mind what they have done to the mice.......

Peter



Eating lots of meat and nothing much else

2018-03-18T11:36:36.071+00:00


Wooo has posted a couple of times about Dr Shawn Baker who eats an all meat, very high protein diet, maybe over 400g/d protein intake. His HbA1c is reported as 6.3%. Personally I have absolutely no interest in this style of eating but the underlying mechanism is obviously interesting.

How about this for a hypothetical marked protein ingestion scenario:

A person eats a lot of meat. In response to the insulinogenic amino acids present they secrete insulin. This will be amino acid specific, I’ve not looked in to how amino acids trigger insulin secretion in detail but it will NOT be through pancreatic glucokinase and subsequent glucose metabolism, as is the case for glucose triggered insulin secretion. So they secrete post prandial insulin but not using glucokinase. The insulin will be exactly in balance with the glucagon for that specific protein meal.

The expression of the gene for generating pancreatic glucokinase is controlled by the carbohydrate content of the diet. Glucose means glucokinase is required. All amino acid diet, no glucose, down-regulate glucokinase.

So, as glucose is subsequently and gradually produced from gluconeogenic amino acids and then released from the liver over several hours (in the presence of only basal insulin), there is only a mild glucose derived stimulus to trigger insulin secretion, and this slow release of glucose by the liver also provides only a minimal drive to express the gene for pancreatic glucokinase. Also hepatic glucose output shouldn’t trigger any of the gut derived insulin secretion potentiating hormones (GLP-1 and the like).

So pancreatic glucokinase is mothballed. Modest glucose release from protein metabolism won’t trigger insulin secretion without the glucose sensor. End result is low insulin with moderately elevated glucose, especially during the time protein is being processed. Which I'd guess is pretty well all of the time on greater than 300g/d. How high should glucose go? High enough to allow a slow trickle to be taken up by constitutive transporters and so deal with hepatic glucose output in this way, without insulin facilitated augmentation. Facilitated by exercise if you like that sort of thing.

How toxic is glucose in the absence of hyperinsulinaemia, given that HbA1c over 6%? Dr Baker will let us know over the next 15 years!



Of course exactly the same happens on LCHF eating, just fat does not provoke chronic glucose release from its metabolism outside of a little glycerol derived gluconeogenesis… It probably happens too in some of the weird sucrose based weight loss diets where the mice (it's mostly mice but we all know that you can do "carbosis" in humans too) are hypoinsulinaemic (otherwise they would be fat!) but glucose intolerant. A diet based on a non-insulinogenic sugar (fructose) and its palmitate derivative will mothball pancreatic glucokinase too.

Peter



On phosphorylating AKT in GHrKO Laron mice

2018-03-16T09:58:27.210+00:00

OK. I started this whole adipocyte thread because I was interested in the longevity effect in the GHrKO mouse, the Laron mouse. These posts get written because I am compelled to, I have no choice in it. I never know where they are going to end up as they start. This one has involved a lot of looking at the various types of adipocytes and how they function in normal physiology and what happened when transplanted to more unusual places. Much of it makes sense, and it does put a very different perspective on the roles of visceral and subcutaneous adipose tissue. What I was looking for was what might be special about Laron dwarf mouse derived adipocytes. You can't quite find all of the answers you want to because not all of the questions have really been asked directly, but I think you can get close. I think this is going to be the last post in the series, a relief to me, and possibly to readers too.Laron mice (GHrKO) are the longest lifespan mice ever engineered by humans. They are dwarf and obese and the obesity tends to be central. They have exquisitely low blood insulin levels and it is thought that the reduced signalling through the GH/IGF-1/insulin system is responsible for their longevity. Adding GHrKO adipocytes to the abdomen of normal mice improves their glucose tolerance significantly.The role of transplanted visceral fat from the long-lived growth hormone receptor knockout mice on insulin signalingN-S mice are normal mice with a sham implantation which adds no extra adipose tissue, N-N are normal mice receiving extra intra-abdominal adipose tissue from normal mice (these should really have had some enhanced glucose tolerance but all of these transplant models differ slightly in technique) and in this case the GTT was done at about eight days post op, ie there may well have been a lot of healing derived IL-6 visiting the liver. The N-GHrKO mice are Bl/6 mice which have received adiopcytes from GHrKO dwarves, shown as black squares:The GHrKO adipocytes are clearly a bit more effective than the normal eWAT adipocytes from the last post. Personally, I was surprised at how relatively small the enhancement of the glucose tolerance was, but then there is always that IL-6 to overcome, so perhaps they really are Super Adipocytes.GHrKO mice develop extremely elevated GH levels, probably through a total lack of IGF-1 negative feedback, but this GH does nothing. Without a receptor the GH, functionally, isn't there. The lack of GH induced lipolysis pushes the balance of adipocyte size towards the obese phenotype. It seems to affect pretty well all adipose depots fairly equally. If we then go on to look at adipose specific FaGHrKO mice, these are obese too but lack any of the insulin sensitising effects of the whole body GHrKO mice:The Role of GH in Adipose Tissue: Lessons from Adipose-Specific GH Receptor Gene-Disrupted Mice"Surprisingly, FaGHRKOs shared only a few characteristics with global GHR−/− mice. Like the GHR−/− mice, FaGHRKO mice are obese with increased total body fat and increased adipocyte size. However, FaGHRKO mice have increases in all adipose depots with no improvements in measures of glucose homeostasis".My assumption that lack it is the of growth hormone signalling in adipocytes which promotes obesity may not be the whole explanation. It is also true that these FaGHrKO adipocytes, which are possibly very insulin sensitive, are working in a mouse with normal insulin signalling outside of those KO adipocytes. This means that the mice will have normal levels of systemic insulin sensitivity/resistance. Putting calories anywhere other than their special adipocytes will have the potential to induce insulin resistance and any increase in insulin to deal with this will undoubtedly put more triglyceride in to the insulin hyper-sensitive FaGHrKO adipocytes.So much for GH.The second effect in whole body GHrKO mice is that there is essentially no IGF-1 produced either by the liver or as a local tissue h[...]



On phosphorylating AKT: the penultimate half post

2018-03-12T05:41:04.093+00:00

I'm going to use some of Konrad's data to try and understand Kahn's data and the see if it will extrapolate to growth hormone receptor knockout (GHrKO) adipoctes. That's the plan. Time will tell... I'm going to use the term eWAT for epididymal adipose tissue.There are three curves here from Konrad.The open circles are mice with extra eWAT carefully added to a mesenteric (liver draining) site only. The eWAT is inflamed, leaking IL-6 and this goes directly to the liver. This IL-6 is causing hepatic insulin resistance with glucose intolerance, as per the last post. There is no elevation of portal FFAs after a three hour fast (not surprising when you recall that you need seriously low insulin levels to access visceral fat, three hours won't hack it). So we can ignore the open circles.The black circles are the controls.The grey circles are mice with eWAT (this is normal eWAT from normal sacrificed Bl/6 mice) added to the peritoneum with all of its venous drainage going to the systemic circulation. This too is leaking IL-6 but by the time it's diluted throughout the whole systemic circulation it causes no insulin resistance. Result: adding eWAT without hitting the liver with IL-6 simply provides extra adipocytes, they accept glucose, glucose tolerance test results improve. This is a generic effect of adding extra adipose tissue, it doesn't seem to matter what the source of adipose tissue is or where you put it, so long as it isn't trickling IL-6 in to the liver, any extra fat improves glucose tolerance. Think thiazolidines, more new fat cells, they're empty, glucose tolerance improves as the cells fill up.Here is a graph from Kahn; the effect of extra fat, when it isn't dumping IL-6 directly to the liver, is always to improve insulin sensitivity, even adding eWAT to mesentery, here called VIS-VIS:Clearly only subcuticular fat transplanted to the mesenteric site reaches statistical significant (SC-VIS). But you can see the trend...This is the exact converse of the diabetes of lipodystrophy cases: in lipodydtrophy there is no adipose anywhere, nowhere to put glucose/fatty acids, all stored triglyceride is ectopic, so your end result is severe glucose intolerance.Now to look at basal lipolysis from Masternak and Bartke's group. This is an in-vitro measurement, performed on aliquots of adipose tissue in Dulbecco’s modified Eagle medium with or without 10% FBS (foetal bovine serum) where they started looking at lipolysis from GHrKO adipocytes, harvested from congenitally Laron dwarf mice. I'm guessing the 10% FBS made no difference because this is the only figure we get in the supplemental data:Now, you have to be very careful with this data. Basal lipolysis is not the same as lipolysis under fasting levels of insulin. Basal lipolysis would produce a ketoacidotic fatality because no insulin is obviously the equivalent of severe T1DM and is rapidly fatal without a very expensive trip to A and E (unless you have the NHS). Next we have the problem that this lipolysis measurement is made per gram of tissue, but in the whole animal some tissue depots are bigger than others as a % of bodyweight and all GHrKO mice are obese, so they have much more fat to provide FFAs per unit muscle etc. The approximate total fat mass of a GHrKO dwarf mouse is actually very similar to that of a normal Bl/6 mouse, it's only the fat free mass which is small. So glycerol release per gram of adipose tissue may be lower in the dwarf mice, but total lipolysis might be very similar to Bl/6 mice. Merely adding basal insulin would produce a normal, hungry mouse but we have no idea what the rates of lipolysis would be then and if they might change more in some tissues than others. So a little caution, to say the least, is needed.With that said I was going to go on to say all sorts of things about lipolysis but at this point the penny dropped, as we say in the UK. I've stopped following the trail and I think I know why GHrKO[...]



On phosphorylating AKT: Interleukin-6 and a tale of two (or three) studies

2018-03-08T12:13:17.890+00:00

I've spent the last few days looking in great detail at this next paper. Mostly I'm interested in the effect of subcutaneous fat transplanted to the omentum/mesentery. It does Good Things, the title says it all. I'll probably post more about it soon:Beneficial Effects of Subcutaneous Fat Transplantation on MetabolismEven transplanting supplemetary monstrousvisceralfat to the omentum/mesentery of recipient mice improves their insulin sensitivity (admittedly ns). I like this research group. They are reporting data and don't seem to have a specific point they are trying to prove. The down side is that I think their CLAMS equipment, core to understanding certain aspects, didn't work very well. You can tell they feel the same way by the turns of phrase they use to describe some of their utterly inexplicable peripheral data like RQs. I'll call it the Kahn paper.This next paper has Konrad as senior author. He has an agenda. Just click on the author to see his other publications. He knows monstrousvisceralfat is evil. He just needs the correct model to show this. This one hit paydirt:The Portal Theory Supported by Venous Drainage–Selective Fat TransplantationOK, let's compare.Kahn's group transplanted epididymal fat in to the mesentery and omentum of recipient mice, this drains to the liver directly. By their surgical technique some of this fat will have also had systemic drainage. They waited for 12 weeks. They then ran an hyperinsulinaemic euglycaemic clamp. There was a modest (ns) improvement in insulin sensitivity. They checked the histology for macrophages, there were a few. They checked these for IL-6 production, it was minimal. Happy fat, happy mice, no hepatic insulin resistance.Konrad's group also transplanted epididymal fat in to the mesentery of recipient mice. They waited for five weeks then did a clamp and showed marked hepatic insulin resistance. There was no increase in portal FFAs or liver triglycerides. They stained the fat for macrophages. There were loads. They checked for IL-6 production. There was loads. They did it all again but with IL-6 knockout mice. Minimal macrophages in the adipose tissue, obviously no IL-6, no insulin resistance in the liver.To Konrad it's cut and dried. Visceral adipocytes cause hepatic insulin resistance using IL-6.Kahn's group check for this and found nothing of the sort. What's going on?It took me about 12 seconds on google to pull up this abstract. It's not terribly important but does bring home the functions of IL-6, there are many:Essential involvement of IL-6 in the skin wound-healing process as evidenced by delayed wound healing in IL-6-deficient miceIL-6 is deeply involved in wound healing. Obviously IL-6 does cause insulin resistance, we know from Konrad's study. And we know that all of Konrad's implants were inflamed and secreting IL-6 at the end of the study, five weeks after the surgery. Was that because visceral adipocytes are just evil and want to kill us with IL-6 or is it because the healing process after transplantation uses IL-6 and is incomplete at five weeks? What if they had waited another whole seven weeks before testing at 12weeks?In Kahn's paper the beneficial effect of adipose transplantation showed best for SC fat placed in the mesentery/omentum. The benefits started to show in bodyweight and fat percentage at eight weeks and were more obvious at 12 weeks post op. The hyperinsulinaemic clamp at 12 weeks showed insulin sensitivity was improved, p less than 0.05 in this group. The visceral to mesenteric transplants were similar but not as marked, mostly p stayed above 0.05.Kahn's paper was 2008. Konrad's was 2011. Who to believe? Who might have read whom and worked out a counter study? Perhaps the matter has now been pretty well settled by the surgeons mentioned in the last post (Oregon excepted)?I think it is quite likely that over-distended adipocytes do produce IL-6. But you're[...]



On phosphorylation of AKT in real, live humans. They're just like mice!

2018-03-11T08:20:11.571+00:00

This first paper is very neat. They enrolled study subjects who were scheduled for an elective laparotomy and persuaded them to consent to an IV bolus of insulin during surgery and to allow biopsies of SC fat and omental fat to be taken at the 6 minute mark and at the 30 minute mark after this bolus. They were given some IV glucose to keep them alive after the insulin. Omental fat is possibly the most visceral of visceral fat depots, probably similar to mesenteric fat. Here's the paper:Insulin Signaling in Human Visceral and Subcutaneous Adipose Tissue In VivoThis is what they found. The black squares are the omental fat:The x axis is non linear. Insulin signaling kicks in much faster in visceral adipocytes and is more effective at activating the whole signaling cascade (phosphorylation of AKT included) than it is in subcutaneous adipocytes. As the authors comment:"We show that visceral fat is characterized by higher expression levels of specific insulin signaling proteins and more pronounced and earlier activation of the insulin receptor, Akt, glycogen synthase kinase (GSK)-3, and ERK-1/2 in response to insulin".EDIT: Just look at those basal relative IR phosphorylation levels, visceral adipocytes have four times the insulin signaling as subcutaneous adipocytes after an overnight pre-surgical fast. I love this paper. END EDIT.I think that it is unarguable that visceral adipocytes are more insulin sensitive than subcutaneous adipocytes.Just to confirm that bias, this next paper is looking at glucose uptake in volunteers, using all sorts of clever non-invasive techniques. And some of those volunteers were obese. I think it's very clear that visceral fat, under hyperinsulinaemic euglycaemic clamp conditions, is more insulin sensitive than subcutaneous fat. Any visceral fat, any subcutaneous fat, any bodyweight of owner. From this paperGlucose uptake and perfusion in subcutaneous and visceral adipose tissue during insulin stimulation in nonobese and obese humanswe have this:First and 4th pairs of columns are SC adipose tissue, 2nd and 3rd are visceral. Even in obese subjects the glucose uptake by visceral fat, under clamp conditions, is higher than in SC adipose tissue. Lots of significant p values.Now the flip side, rather more speculative here: let's revisit this paper from several years ago, looking at healthy humans under hypoinsulinaemic states:Prolonged Fasting Identifies Skeletal Muscle Mitochondrial Dysfunction as Consequence Rather Than Cause of Human Insulin ResistanceIgnore the mindset of the researchers, just look at their data:In a normally fed Dutchman the fasting FFAs are around 0.2mmol/l at some time in the morning. This is the amount of FFAs being released under a plasma insulin of about 13.0microU/ml. At this level of insulin I very much doubt if any of these FFAs are coming from visceral adipocytes because insulin is still too high for this. This a workaday "ready for breakfast" sort of a combination of FFAs and insulin. I think it is very reasonable to consider that these FFAs are coming primarily from the subcutaneous adipose stores.By 36 hours of fasting insulin has dropped to around 7.0microU/ml and it stays there. At this level of insulin we have FFAs rising through 1.0mmol/l to 2.0mmol/l, that's very high. Insulin is now very low, low enough for visceral fat to release a lot of FFAs and the body is set up to run on fat and ketones. It could do with some hepatic insulin resistance to facilitate hepatic glucose output and the specifically portal vein draining fat depots (omental and mesenteric) are set up to do exactly this. Evolution has punished, by non survival, individuals who failed to follow this pattern.I think it is a pretty sound case that visceral fat does very little, other than hoover up a few calories, while ever insulin is above 12microU/ml. In the USA insulin probably never falls below this level. [...]



On phosphorylating AKT in visceral adipocytes under starvation

2018-03-03T12:41:55.557+00:00

I was hoping to ignore the CR mice in the paperDifferential response to caloric restriction of retroperitoneal, epididymal, and subcutaneous adipose tissue depots in ratsbut I don't think I can do it. OK, here we go.Below are the same images as in the last post showing phosphorylation of AKT as a marker of insulin signalling. The fed CR mice have an insulin level of 1787.84pg/ml, pretty much the same as the fed AL mice (1549.76pg/ml).Let's look at subcutaneous adipocytes (sWAT) first.The fed level of plasma insulin is supporting twice the level of insulin signalling in the sWAT from a starving mouse as it is in an ad lib mouse. An adipocyte from a starving mouse  is more insulin sensitive than one from a plump mouse. Not unexpected and clearly the adipocytes are small and desperate to have more fat.In the fasted state the plasma insulin is much lower in the CR mice (224.56pg/ml) vs fasting AL mice (477.25pg/ml) and this low insulin level supports the same degree of signalling in the fasted CR mice as the higher value in the plump but fasted mice. Again CR adipocytes are more insulin sensitive.Next is the situation in retroperitoneal visceral fat (rWAT). Any value of plasma insulin between the AL fasted of 477.25pg/ml and somewhere around 1500pg/ml of either AL-fed or CR-fed state gives very similar levels of insulin signalling. Maybe a little higher in the recently fed CR mice:But when we get down to the CR fasting insulin level of 224.56pg/ml we actually have significantly reduced insulin signalling in the visceral adipocytes of starving mice. A drop in insulin signalling is synonymous with increased lipolysis in adipocytes. Accessing visceral fat really does happen, but only at very low insulin levels.Does this show in fat depot size? Not really. The fall in subcutaneous fat volume is there but the fall in retroperitoneal fat is minimal. Bear in mind these guys are dissecting out very small tissue depots. If you look at the histology/computer image analysis derived values for adipocyte size you can see that the CR visceral adipocytes do really shrink and this might even achieve statistical significance. It's the two columns at the right we're looking at:Pretty much the same thing happens  in the subcutaneous adipocytes but we get no asterisks for the changes here. It's possible the visceral adipocytes might shrink more than the subcutaneous adipocytes, if you are hungry enough:So I think it is reasonable to assume that lipolysis in visceral adipocytes becomes real at plasma insulin levels somewhere between 477.25pg/ml and 224.56pg/ml. To achieve this in a mouse needs a combination of long term caloric restriction plus total fasting for about 24 hours. At this plasma insulin level it is even possible that their rate of lipolysis exceeds that of subcutaneous adipocytes but that might be stretching the data even further than I have done already. Under normal mouse husbandry conditions visceral adipose tissue is there to stay. It won't release FFAs unless the adipocytes get so large that they leak some FFAs in the presence of insulin signalling.....OK, I'll try to leave those poor starving mice alone now.Peter[...]



On phosphorylating AKT within visceral fat

2018-03-02T13:22:41.923+00:00

I've been thinking quite a lot about the difference between subcutaneous adipocytes and visceral adipocytes. The difference appears to be much deeper than location, though that matters. The next paper is one of those terribly clever research projects where enormous amounts of information is accumulated but little integration or understanding seems to take place. Here it is:Differential response to caloric restriction of retroperitoneal, epididymal, and subcutaneous adipose tissue depots in ratsOne problem is that they are trying to do too much, so just ignore any of the bits about caloric restriction (CR) which creep in to the butchered graphs. All I'm really interested in is ad-lib (AL in the graphs) fed mice and their adipocytes. I want to know about normal insulin signalling. Here are the fed and fasted insulin levels from those mice. Fed on the left, fasted on the right, part of Table 1:The dollar signs denote statistical significance (not paydirt). Next are excerpts from Figure 5. I've cropped out the bar graphs showing the pAKT levels from fed and fasted mice. The group is using pAKT as a good marker of insulin signalling. First here's the SC (sWAT) graph:We only need to look at the AL group. There is a fed insulin of 1549.76pg/ml supporting the reference level of insulin signalling. Next to it we have the fasted level of insulin signalling, somewhere around half reference value. This is being supported by a plasma insulin level of 477.25pg/ml. Simple. More insulin, more pAKT, more insulin signalling. It seems reasonable to consider that the fed insulin level supports lipid storage in subcutaneous adipocytes and that half this level (fasting) might allow lipolysis. We can ignore the CR group.Here is the same graph from a sample of retroperitoneal adipocytes:Here we have, again, the level of insulin signalling supported by a fed state plasma insulin of 1549.76pg/ml as reference and just look at the level of insulin signalling being supported by the fasting insulin level of 477.25pg/ml. It's no different to the fed level of signalling. Hmmmmm.So, theoretically, most of the fat loss under fasting should come from the subcutaneous adipocytes. We need to go back to Table 1 for that:There we go, the tissue with the least fasting insulin signalling (sWAT) loses over four times as much lipid as the tissue where insulin signalling is maintained at the lower (fasting, 477.25pg/ml) insulin level (rWAT).This looks very much like one of the intrinsic differences between subcutaneous adipocytes and visceral adipocytes is that visceral adipocytes maintain insulin signalling at much lower levels of plasma insulin than do subcutaneous adipocytes. You have to store calories which arrive without insulin somewhere. Looks like this is the place!I'm interested in this because I want to know why VLDLs, released from the liver following lipid overload (from fructose DNL, alcohol DNL or inappropriate FFA release from adipocytes under fructose or alcohol) end up in visceral adipocytes. We know these VLDLs are only released under low insulin levels (that was a long time ago!) and this current paper tells me why the VLDL lipids end up in visceral adipocytes. It's not an address technique, it's just that visceral adipocytes are programmed to store fat under relatively low insulin levels. To get decent lipolysis from visceral fat I suspect that you need really low insulin levels, something a bit like those of ketogenic diets. Well, what do you know...What is it about the visceral adipocytes that programs this?Perhaps we should look at IGF-1 for the answer to that one.PeterEDIT We don't know the absolute level of signaling in either the sWAT or rWAT tissue. Fed is taken as reference and fasted is shown as the percentage change from the fasted state. The reference value may differ between the two dep[...]



Saturated fats vs PUFA in a 5 day human ketosis trial

2018-03-01T13:43:28.121+00:00

Just a one-liner (as if!) via mommymd commenting on an old post:

Differential Metabolic Effects of Saturated Versus Polyunsaturated Fats in Ketogenic Diets

I think I worked through this several years ago but didn't have the tools to comment on it at the time. The recall I have is of reading what the high PUFA group ate. Fake bacon, soy nuts, vegetable oil. Lovely. BUT they ended up much more insulin sensitive than the saturated fat group did over 5 days. Higher ketones, lower glucose, lower trigs, higher insulin sensitivity. I had no explanation for this.

















Once you appreciate the Protons concept this is exactly what you would expect. There is continued insulin signalling when there should be physiological insulin resistance. While ever adipocyte size is kept low, via the ketogenic nature of the diet, enhanced insulin sensitivity should persist.

The down side is that glucose metabolism will continue. If your approach to life is to stop using glucose as an energy substrate there is absolutely no need to maintain glycolysis in the face of a ketogenic diet. Simply refusing to listen to insulin is my preferred option. Someone running their metabolism of fats should have minimal need for insulin sensitivity and buying insulin sensitivity at the cost of metabolising linoleic acid, with its daughters 4-HNE and 13-HODE, is not something which appeals to me. But be aware of the study and the joy it will give to the saturophobes...

Peter



More on drinking varnish

2018-02-28T11:54:08.130+00:00

This paper is a gem.Reducing the Dietary Omega-6:Omega-3 Utilizing α-Linolenic Acid; Not a Sufficient Therapy for Attenuating High-Fat-Diet-Induced Obesity Development Nor Related Detrimental Metabolic and Adipose Tissue Inflammatory OutcomesWhat did they do? They fed rats chow or they fed them on one of four other diets enriched in PUFA. The extra PUFA were based around various mixtures of linoleic acid with alpha-linolenic acid, some  were mostly corn oil, some were slanted towards varnish (flax/linseed oil). Total 18-C PUFA made up 9.4% of calories, ie was obesogenic, and this was identical for all of the high fat diets. Overall macros were identical in all of the high fat diets too. There was no sucrose. The rats were fed ad lib.Here is the link to Table 1 which lists the compositions, it's too big for putting it up as a jpeg. Just look at how utterly fair the composition of the high fat diets were. Even if the absolute amount of linoleic acid in the lard is not accurate, there will be a consistent error across the diets and the results stay plausible. My only complaint is that there was no group where the omega-3 lipids predominated in the diet PUFA, a 50:50 mix was the maximum. Whereas the maximum omega-6 fed group got essentially all of their PUFA from omega-6 PUFA.The second excellent feature is that the rats were neither semi-starved nor forcibly overfed. Rats are not people. They cannot be verbally asked to overeat to maintain a stable bodyweight nor to calorie restrict to lose weight. They will simply eat until they are no longer feeling hungry. If that happens while they are svelte or not until they are morbidly obese, the rats don't care.What happened?Almost nothing. The chow fed rats, with around 3.5% of calories as PUFA, stayed at a reasonable weight. The obesogenic high fat diets (ie nearly 10% of total calories as PUFA) each caused almost exactly the same progression of obesity:Why almost?Can you see that the open squares group gain weight slightly more slowly than the other PUFA diet groups? This shows between week six and week 17. The two hashtags mark out a couple of time points where this achieved statistical significance. This slightly less obese group of rats is the group which ate the least alpha-linolenic acid, the most linoleic acid. This suggests that omega-6 PUFA are less fattening than omega-3 PUFA. I like that. Protons likes that.The effect was fairly small and only shows as an early facilitation of weight gain. By the end of the study the rats and their adipocytes were all about as fat as they were going to get on 9.4% of calories from any family of PUFA.You can easily hide this effect by under feeding (pair feeding to the same calories as a chow fed group or arbitrarily reducing overall caloric availability) or overfeeding (paid humans or intragastric cannula over-fed rats). If you are an omega-3 lover this can be necessary. But, given a decent study, it shows.Consuming the 18-C omega-3 rich linseed oil/flax oil/varnish may not make you terribly much fatter than corn oil will eventually make you, but it should get you there quicker. The situation for EPA and DHA is different. Oxidising these will increase the cytoplasmic NADH:NAD+ ratio via peroxisomal oxidation (bad) and give reasonable mitochondrial function from oxidising the residual saturated caprylic acid C-8 (good), which is the normal fate of very long chain fatty acids of any ilk.Executive summary: Omega-3 18-C fatty acids are more obesogenic than omega-6 18-C fatty acids. The effect is small but real, it might show better if all of the PUFA were alpha-linolenic acid rather than to 50:50 mixture used. It still makes me happy.PeterThe Protons view (skip this if you're fed up with hearing it over and over again)...I [...]



Alcohol steatosis and NASH

2018-02-28T11:02:46.986+00:00

This came to me via George in the comments to an earlier post:Supplementation of Saturated Long-Chain Fatty Acids Maintains Intestinal Eubiosis and Reduces Ethanol-induced Liver Injury in MiceAgain, it is a pro-saturated fat paper, always nice to read. They did some odd things such as using fully hydrogenated soya oil, mostly stearic and palmitic acids, versus corn oil and their feeding protocol used controlled intragastric feeding, with or without ethanol. Obviously there is no fructose in any of the feeds.But they generated some lovely micrographs. These next ones are Oil Red O stained. There is essentially no lipid accumulation in either of the control groups:And here it is in numerical form:USF diet supplies 35% of calories from corn oil (which is roughly 60% linoleic acid, ie 20% of total calories as PUFA) and there is no lipid accumulation at all without ethanol. PUFA alone to not appear to cause fatty liver. Adding ethanol produces spectacular steatosis (top right).The SF diet also included 5% of calories as corn oil which, combined with ethanol, does produce some steatosis (bottom right).The other images of great interest are the 4-hydroxynonenal stains looking at lipid peroxidation, obviously derived from linoleic acid. These use an immunohistochemical stain and so this will be come up as brown, that's what we're looking at on the top right image. Obviously 4-hydroxynonenal is a marker of the process leading to cirrhosis and eventually to hepatocellular carcinoma. Another gift from your cardiologist:If you'd like it in more numerical form they measured TBARS too:My feeling is that fructose is going to behave in exactly the same way as alcohol, through a very similar process. If that is correct then saturated fat will protect your liver from peroxidation. I'd not suggest that fructose won't cause problems, it might even generate steatosis and hepatic insulin resistance, just conversion of that steatosis to NASH seems very unlikely without the PUFA.Peter[...]



Fructose and lipolysis

2018-02-28T11:49:59.897+00:00

You have to be very, very careful with fructose feeding papers. It is very easy to slant your methods to give strange and conflicting results. Some really weird stuff happens when you give a sugar which fails to trigger insulin secretion and itself rapidly turns in to fat. The combination of low insulin secretion and high fat production can end up looking very much like a genuine high fat diet! There are papers out there where this can be pushed to the point where fructose fed rats are slim, exquisitely insulin sensitive and apparently very health. Increasing starch/glucose alongside the fructose seems, generally, to produce more obesity. PUFA add a whole new dimension. So be careful...Onwards.Another confirmationally biasing paper:Adipose tissue remodeling in rats exhibiting fructose-induced obesityHere are the dietsNot too bad. Some changes between sucrose and starch but most of the other variables are pretty well held constant. The study ran for eight weeks. Here are the body compositions at the end:The fructose fed rats carried 18g of extra fat, just over 12g of which were in mesentery and the epididymal fat pads. Visceral fat. The fructose fed subcutaneous adipocytes had an average volume of 25,200μm3 vs 40,950μm3 in the controls. The situation is reversed in the visceral adipocytes, fructose fed are 28,540μm3 vs 19,870μm3 in the controls.So, are FFAs being released from adipocytes under the influence of fructose, being picked up by the liver, repackaged in VLDLs and stored in visceral adipocytes long term? Well, as far as I can find, no one has done the tracer studies to check this. We do have these measurements in this paper relating to lipids:Those elevated FFAs along with elevated fasting triglycerides are both suggesting routes in to and out from the liver respectively. I also rather like the elevated lipid peroxidation, this is not happening to palmitate!So it's all very suggestive that fructose might be working on subcutaneous adipocytes much the way that alcohol does. I suppose it could be acting on all adipocytes, subcutaneous and visceral, just the repackaged FFAs are targeted to visceral adipocytes, hence the overall shift in size differential. Just as neat vodka makes you thin so a very high fructose diet should do the same. Adding in more starch and/or glucose should go more towards the beer belly look. Of course you could just argue that fructose or ethanol simply generated lipid in the liver which was shipped out destined for visceral adipocytes. Until you look at the alcohol tracer study and realise that it is certainly not that simple for ethanol. I just have to wish that someone had done a similar tracer study on fructose feeding. Can't have everything I guess.Peter[...]



Registered Dietitian Health Educators: how fat do you want to get?

2018-02-25T09:38:38.876+00:00

I guess everyone has seen this:

Effect of Low-Fat vs Low-Carbohydrate Diet on 12-Month Weight Loss in Overweight Adults and the Association With Genotype Pattern or Insulin Secretion The DIETFITS Randomized Clinical Trial

How do you sum up the trial (apart from tedious)? These quotes do it up for me:

"The intervention involved 22 instructional sessions held over 12 months in diet-specific groups of approximately 17 participants per class. Sessions were held weekly for 8 weeks, then every 2 weeks for 2 months, then every 3 weeks until the sixth month, and monthly thereafter. Classes were led by 5 registered dietitian health educators who each taught 1 healthy low-fat class and 1 healthy low-carbohydrate class per cohort"

"...an emphasis on high-quality foods and beverages"

"...focus on whole foods that were minimally processed, nutrient dense, and prepared at home whenever possible."

This should be a good intervention.

Except decision making was then handed to the participants:

"Then individuals slowly added fats or carbohydrates back to their diets in increments of 5 to 15 g/d per week until they reached the lowest level of intake they believed could be maintained indefinitely"

End result of this is that 10% of the participants weighed more at the end of 12 months of closely supervised healthy eating by a Registered Dietitian Health Educator than they did at the start. Cracking intervention for these poor folks.

And in both the low fat and the low carb groups just under 5% (LF 4.3%, LC 3.6%) of participants developed metabolic syndrome, who had been relatively healthy before the start of the intervention. How can you manage this with "healthy" food and 22 meetings with a Registered Dietitian Health Educator? I'm impressed. If a Registered Dietitian Health Educator ever comes your way, RUN. Especially if they use the words "healthy" and "diet" in the same sentence.

Bottom line: Low carb diets only work when you limit the amount of carbs you eat. However "healthy" those carbs you add back in might be, depending on the opinion of a Registered Dietitian Health Educator, it's no longer a low carb diet. You'll get fat again.

Of course the same applies to low fat diets, especially if they are sugar restricted at the same time. Ultimately if you follow a low fat diet with as much added fat as you feel comfortable with, you're going to be disappointed with the results too. Adding back sugar will be even more disastrous. Sad but true.

Peter

Addendum: Gardner did essentially the same study in 2007 but made the mistake of publishing the weights alongside the carb intakes at each assessment interval. I wrote all over his graphs here. He didn't repeat the mistake. No one should imagine he's stupid. Or honest.



Alcohol and weight loss

2018-02-20T06:01:50.558+00:00

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 steatosisThis 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.PeterBTW, 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.[...]



Systemic fructose is important

2018-02-15T14:59:57.543+00:00

******************************************************************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 patientsUnfortunately 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 solutionsIngesting 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 studyWhat 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"[...]



TRAK2 and HDL. Do we care?

2018-02-15T05:42:25.011+00:00

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.

2018-02-14T14:12:55.615+00:00

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 intakeMortality 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 chol[...]



Saturated fat and fatty liver. Payday in Colorado.

2018-02-14T13:26:37.784+00:00

Dophamn supplied the link to another interesting study:The role of visceral and subcutaneous adipose tissue fatty acid composition in liver pathophysiology associated with NAFLDHere 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 s[...]



Saturated fat and fatty liver. Payday in Sweden.

2018-02-12T10:25:27.243+00:00

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 HumansIt'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 AlongWell. 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 [...]



Follow on to Tucker's post on PUFA in rats

2018-02-05T21:30:10.567+00:00

Tucker posted an excellent discussion of this paper on his blog. Go read it:Fat Quality Influences the Obesogenic Effect of High Fat DietsThe 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, beca[...]



Metformin (07) glargine 50iu/kg causes diabetes

2018-01-22T11:17:53.969+00:00

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 dietSo 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 t[...]



Metformin (06) Insulin-induced insulin resistance is real

2018-01-19T13:01:35.854+00:00

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 cell[...]



A wander off in to dietary protein calories

2018-01-14T13:33:05.680+00:00

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 studyThey 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 AncestryIt 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 [...]