Revisiting the Fatty Diets & Diabetes Study ~ How to Make Mickey Fat or Fattier

High Fat Diets! 

I'm going to continue bumping up some of the hundreds of blog posts I've written on studies that support the role of dietary fat in obesity and diabetes.   As I do this I'll make some formatting changes for ease of reading, may fix a typo or awkward wording here or there, but won't alter the content meaningfully.  Instead, I'll insert Ed. Notes where I feel an update or clarification is needed.

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ORIGINAL POST 8/31/2011

In her piece over at MDA on the How Fatty Diets Cause Diabetes, Denise Minger spent a bit of time discussing the strain of mouse used in the study.  That being the not-uncute fella you see pictured here:  A C57BL/6J mouse.   Denise describes these mice as:  "uber-susceptible to obesity, high blood sugar, insulin resistance, leptin resistance, and all that other fun stuff plaguing modern humans."  This didn't really square with my memory from when I blogged on a study involving this critter.  Took me a few minutes to remember what that blog was ... Of Mice and (Wo)Men.  That post dealt with a calorie restriction study in the C57BL/6J mouse.  In looking for more info on this strain, I had come across this paper:  The High-Fat Diet–Fed Mouse.  Since I was mostly looking for info on lifespan and such at the time, the subtitle didn't "hit me", that being:  A Model for Studying Mechanisms and Treatment of Impaired Glucose Tolerance and Type 2 Diabetes.  The paper describes this mouse's propensity towards obesity (and IGT and T2 diabetes) when fed a high fat (58%) diet vs. a standard low fat (11%) chow.  However, in the calorie restriction study, these mice did not become obese on standard chow (11%F, 69%C, 20%P, Teklad Global 2016).  Therefore I think it would be more fair to say that they are susceptible to diet-induced obesity (DIO), but not obesity per se on a more appropriate diet.  I mention this as in contrast to some mice that become obese pretty much regardless of diet (though diet may impact the degree of obesity).

The C57BL/6J mouse, it thus highly susceptible to DIO and resultant diabetes, a genetic propensity highlighted by both Denise's citation, Diet-induced type II diabetes in C57BL/6J mice - Surwit et.al., and that High-Fat Diet-Fed Mouse paper.  As such, as a model it is more relevant to studies on human obesity/diabetes than a "knockout" or genetically manipulated animal model having no human equivalent. Feed these mice an appropriate diet, they don't become obese and hence they don't become diabetic. Feed them an obesity-inducing diet, they become obese and diabetic in short order. Yes, we are not rodents. Yes, we can argue until those grass-fed cows come home over the composition of the diets that make rodents fat and any real-world applicability to what makes humans fat. But this mouse is actually a pretty good model for the etiology of obesity-diabetes in humans. It is a model for how the obese state causes the diabetic state. And so, I repeat. A more appropriate headline for the study might then have been: "How Fattening Diets Cause Diabetes in Genetically Predisposed People".  The Pathways to Obesity paper identified a mechanism by which: 

Obesity 

↓ 

Elevated NEFA 

↓ 

Impaired Glucose Transport into β-cells 

= 

Impaired Sensing of Hyperglycemia 

↓ 

Impaired GSIS (Glucose Stimulated Insulin Secretion)

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ED. NOTE 8/9/2015:  This is a post from 2011 and I believe that step 2 -- Elevated NEFA → Impaired Glucose Transport = Impaired sensing  -- requires some additional fleshing out as to mechanism.   There is ample support for impaired GSIS to be the first step in β-cell dysfunction as I have blogged on in many subsequent posts.  Perhaps a place to start:  Insulin/Proinsulin/etc. in Normal, IGT and T2 Diabetics

Specific to GSIS:  Deleterious Effects of NEFA II: Pancreatic beta cell impairment

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So, let's return to the topic of the diets.  A common knock on rodent chows is that they aren't "real food".  Now this is of course true.  But how do you think you get a rodent to eat any sort of consistent diet to do any sort of nutritional study?  You have to make a homogenous food that they will eat!  Even if it were practical to feed some sort of real whole food diet, how can you ensure they eat the proper proportions of each?  With humans you can furnish foods and monitor compliance and complete consumption.  You can't do that to a mouse!  Indeed years ago I tried to put my former kitty on a diet and mixed the pellets to transition.  Wouldn't you know he ate around the diet pellets even though the only visible difference was a slight one in color.  Heck, you give CAF rats human junk foods, and they barely eat any chow.  Go figure.  So, first point:  the use of a homogenous chow is necessary.  So too is one that has some sort of reasonable shelf life, etc.

The nature of the fat used in chows has been held out by many as being problematic.  Sometimes this is rightly so, but .  However, transfats aside, there is little if any evidence that high PUFA or low PUFA or anywhere in between has any impact on the development of obesity.

In the study to be discussed, yes, there are those "eew" factors (see below) that Denise mentions in her piece (e.g. soybean oil) but those same things are fed to all of the mice in this study.  Casein protein is constant (at 16%) across the board, as are the soybean oil and maltodextrin (sometimes you have to have this in your primal foods)  contents of the diets.  As to the hydrogenated coconut oil?  Well, I suppose lard could have been used.  I've tried to get a rough indication of the amounts of transfat in HCO and can't seem to find it.  But this is used for whatever reason (stability, shelf-life, rancidity resistance) and the higher melting point of HCO would seem a necessary no-brainer.  It is interesting that the increased fat is roughly 50% shorter chain fatty acids not normally associated with fat deposition, and yet induced obesity in our B/6J critters.

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ED. NOTE 8/9/2015:  In the intervening years since this post was written, this still holds with the exception of an occasional "head to head" battle between high coconut oil diets vs. other fats.  It is important to keep in mind when interpreting those studies that CO is somewhat of a unique fat.  Highly, highly saturated, but only ~50% long chain saturated fatty acids.  It is also not a majority fat source in most Western diets where obesity and diabetes are the issue.

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Also, some of these fats -- the much maligned Crisco -- have been around for a while.  Folks were eating margarine in the 50's and 60's well predating the obesity epidemic.  In any case, rats have been made fat on lard and butter based high fat diets.  Still there's that pesky inclusion of sugar in most high fat diets.  This the critics say invalidates any conclusion drawn about the fat in the diet.

Well, it just so happens that the Surwit et.al. have researched our friend the C57BL/6J further (I'll bullet point the Abstract):

Differential effects of fat and sucrose on the development of obesity and diabetes in C57BL/6J and A/J mice

Abstract

We have previously demonstrated that the C57BL/6J (B/6J) mouse will develop severe obesity, hyperglycemia, and hyperinsulinemia if weaned onto a high-fat, high-sucrose (HH) diet. ... 

... In response to such diets, B/6J mice not only become more obese generally, but show a particular increase in mesenteric (MES) fat cell number in comparison to the diabetes-resistant A/J strain.

Mesenteric fat in a mouse is a "depot [that] forms a glue-like web that supports the intestines".  Adipose tissue growth occurs by two mechanisms: 

1. Hyperplasia:  Increase in the number of cells
2. Hypertrophy:  Increase in size of the cells

Thus the B/6J mouse becomes obese compared to the A/6 strain by an increase in the number of visceral fat cells (the mesenteric fat depot).

In the present study, we compared the effects of fat and sucrose separately and in combination on diabetes- and obesity-prone B/6J and diabetes- and obesity-resistant A/J mice.   After 4 months, the feed efficiency ([FE] weight gained divided by calories consumed) did not differ across diets in A/J mice, but B/6J mice showed a significantly increased FE for fat. That is, B/6J mice gained more weight on high-fat diets without consuming more calories than A/J mice.  The increase in FE was related to adipocyte hyperplasia in B/6J mice on high-fat diets.

Well there you have it ... surely old B/6J is just more carb sensitive than A/J.  Insulin secreted even for the relatively low carb (but still considerable carb) high fat diet causes more fat cells to form in the mesenteric depot and these hoard the fat.  TWICHOO lives, CICO has been debunked!

Not so fast :-)  First let's get through the remainder of the abstract summary of results:

Fat-induced obesity in B/6J mice was unrelated to adrenal cortical activity.  In the absence of fat, sucrose produced a decreased in FE in both strains. Animals fed a low-fat, high-sucrose (LH) diet were actually leaner than animals fed a high—complex-carbohydrate diet.  Fat was also found to be the critical stimulus for hyperglycemia and hyperinsulinemia in B/6J mice. In the absence of fat, sucrose had no effect on plasma glucose or insulin.   These data clearly show that across these two strains of mice, genetic differences in the metabolic response to fat are more important in the development of obesity and diabetes than the increased caloric content of a high-fat diet.

Methods

The study involved feeding rats, housed 5 per cage, ad libitum one of four diets.  There were 30 rats for each of the low fat diets and only 20 rats for each of the high fat diets.  (Perhaps anticipating that the LF mice would be leaner, there were more of them as samples from two mice were pooled to provide sufficient sample for some analyses).  All diets were iso-protein and the composition is shown in the table 

HH = High fat / High sucrose 

HL = High fat / Low sucrose

LH = Low fat / High sucrose

LL = Low fat / Low sucrose

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ED. NOTE 8/9/2015:    The low fat high sucrose diet is 60% sugar!   This is one of those "accidental evidences" I talk about in my upcoming book, Restriction Addiction, where even given absurdly high amounts of this so-called addictive substance, rodents fail to demonstrate any sort of physiological dependence on the substance.  

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At 4 weeks of age, the mice were weaned onto one of the four diets and maintained for 4 months.    Intake was assessed as follows:

To avoid the stress of individual housing, food intake was measured on a per-cage basis for 24 hours once per week. Caloric content of food intake was determined based on 5.55 kcal/g for high-fat diets and 4.07 kcal/g for lowfat diets. The feed efficiency (FE) [(weight gained/kcal consumed) x 100] was determined after 16 weeks on the respective diets for each of 40 cages.

Measured during study: Fasting BG & insulin levels at monthly intervals. Plasma corticosterone levels were determined in the morning under basal conditions and after a brief stress (shaking) at month 4 in 10 animals per strain per diet. 

Measured at end of study (off site): fat pad weight, fat cell size, fat cell number, lipoprotein lipase activity (LPL), and basal and norepinephrine-stimulated lipolysis.

Results

It is clear from either the table or the plot that on low fat diets (the left cluster of four) there's not much difference in weight, while on the high fat diets, there's a clear difference in bodyweight.  Within each strain, there's a rough positive correlation between intake and weight, but the slope of such is rather steeper for the more obesity-prone B/6J.  I think this is important to keep in mind when comparing between the strains.  (This is why I chose both color and shape for the data points to help distinguish.)

It is abundantly clear, however, that in the obesity prone strain, which eventually develops a "deranged carbohydrate metabolism", it is fat intake that does it, not carbohydrate intake.  Within each strain, it is also clearly higher caloric intake on the high fat diets that is responsible for increased weight gain.

OK now for some graphics from the study:

Feed Efficiency: This was weight gained/calories consumed. Also note: weight, not fat mass. The A/J mice don't seem to have much difference. The B/6J's on the other hand are highly efficient at storing fat. 

Fat Cell Weight & Number: This was determined for two fat depots, the mesenteric and inguinal. To repeat, mesenteric is representative of visceral fat while inguinal is subcutaneous fat.

CELL WEIGHT                                               CELL NUMBER

Total Fat Pad Weight   

A few generalizations/comments:

• The relative distribution between mesenteric (VAT) and inguinal depots (SAT) of total fat mass appears to be relatively constant for both strains across all diet compositions
• Increased SAT mass in the A/J mice fed HF diets is due to slight increases in cell number, but mostly to increases in cell size, while in B/6J mice, this is due to marked increase in both size and number.
• Increased VAT in the A/J mice fed HF diets is almost exclusively attributable to cell size with no considerable difference in cell number across all diets.  This is in stark contrast with the observations in B/6J mice where HF diets result in increases in both size and number of cells.

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ED. NOTE 8/9/2015: Looking at these data again, I feel I should stress that whether we are concerned about the number or the size of cells, it is the high fat diets that increase these parameters -- differently in different fat depots, differently in different strains -- consistently when a dietary effect is seen.  All mice gained more body fat on the high fat diets.  

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This table below has the metabolic measurements of fasting glucose, insulin, and LPL activity

The hyperinsulinemia of the genetically predisposed B/6J mice is "brought out" by the high fat diet.  On the low fat diet?  These mice don't become obese or diabetic.  I'm going to shout this out in bold colors here!

• In both strains of mice, the HF diets led to higher caloric consumption and higher body weight and fat mass.
• The genetically predisposed strain accumulated more fat in response to a high fat diet
• The fat accumulation and its distribution causes the hyperinsulinemia and hyperglycemia in the B/6J mice, NOT the other way around.
• The fat accumulation was triggered in the genetical predisposed mice by non-insulinogenic dietary fat consumption
• All mice fared better on higher carb lower fat diets. 

Did sugar make them fat or diabetic? NO.  The sugar junkie mice were leaner in both strains.  

I would also note that all diets were ad libitum.  Across the board the mice ate more calories of the high fat diet.  

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ED. NOTE 8/9/2015:  In light of BabyGate (most posts with that label should contain links to relevant other posts in the preface), where Professor Tim Noakes is advocating for weaning children onto #LCHF diets.  In addition to the high protein content of those diets, the high fat content proposed -- on the order seen in this study which is considerably higher than the 40-50% fat in most "high fat diets" used to induce obesity in rodents -- needs to be addressed.   I am going to leave my initial discussion below intact and do a stand-alone BabyGate in the near future.

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A couple of caveats and thoughts for those of you with kids.  Yes ... back of mind ALWAYS ... these were mice.  But, there are parallels between what we see in rodents and humans especially in growth phases.  Since the mice in this study were weaned onto the diets during their "formative" months (I would say akin to childhood/adolescents), we see that those genetically predisposed to obesity fare far worse on a high fat diet. The number and distribution of our fat cells seems to be established towards the end of our gestation, and is considered somewhat fixed by age 18-20 or so.

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ED. NOTE 8/9/2015:  I need to address this in the future to clarify.  A lot of new research indicates that these generalizations are overly broad.   It is important to know that new fat cells can be created in adulthood, and there is considerable fat cell turnover.

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Diabetes in these mice -- remember, not genetic "knockout" mutants, but just a strain that seems predisposed -- is associated with something about a high fat diet that favors energy partitioning into fat tissue by increasing both visceral fat cell number and size.  They didn't measure energy expenditure but I'm willing to bet that, for whatever reason, like in the calorie restriction study, these mice are prone to serious metabolic adaptation to ... carbohydrate restriction.

The Calorie Restriction Study

Let's revisit that CR study from a ways back.

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Ed Note 8/9/2015:  I thought I'd add some more information on this study as it appears to even be missing from the original blog post on it.

There were two separate protocols that I'll summarize briefly below.  All mice received the same standard (low fat) chow.

Experiment 1:  Eight week old mice were individually housed for 6 weeks and fed ad libitum chow.  After that, based on prior intake, they were assigned to either 4 weeks at 5% calorie restriction or another 4 weeks of ad libitum intake.

Experiment 2:  Ten week old mice were individually housed for 3 weeks in a rodent metabolic chamber and fed ad libitum.  After that they were assigned to either 3 weeks at 5% CR or ad libitum.

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The calorie restricted mice and ad libitum mice statistically remained weight stable, but gained small amounts of weight, with no differences between groups.  The CRs went into "conservation mode" and gained significantly more FAT mass at the expense of lean mass (lost).  This has been seen with truly ketogenic diets in rats.   True starvation in childhood often leads to adulthood obesity.  Are the mechanisms the same?  Do you want to chance it?  In any case, it is rather interesting that a strain of mouse prone to obesity would also preferentially sequester energy to fat during growth under mild intake restriction.

The Diabetic Rats

Once fully formed, what are the implications for a low carb/high fat maintenance diet for us humans?  Perhaps whatever damage in terms of fat distribution and whatnot has been done.  Perhaps nothing you can do with diet changes that.  Or maybe hedge your bets.  Here I find myself back at all the studies I've read on the etiology of diabetes.  That it is the NEFA that "get ya" if you've lost the genetic lottery.

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ED. NOTE 8/9/2015:  NEFA, or non-esterified aka "free" fatty acids, are by all means not the only issue here.  However, metabolically, they are the universal culprit.  This is something that requires careful detail and discussion in context.  One thing I have tried to do, despite some overarching premise to the blog, is mention this when it is seen, regardless of whether the study addressed or intended to address the subject.  There are many studies where I look at reported NEFA and they are not addressed in any way (either to acknowledge or dismiss) and I wonder ... why not?   This study that follows is not one of those.  The researchers posit a role for excessive NEFA in the process.

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I was mocked pretty well by a very high fat blogger over this post:  Diabetes progresses on LC/HF Diet.  Yeah ... genetically predisposed ... fed a crappy diet ... apparently morally repugnant researchers ... but, it all seems to come back to those NEFA.  Just look at them!   

These rats, predisposed to "spontaneously develop" diabetes, fared better on the LFHC than the HFLC diet.  

Concluding ...

In the end, we can learn a lot from some rodent studies.  They are not perfect even in the rodent world.  They don't always translate to humans in any sort of imperfect way let alone a perfect way.  But ignore them at your own peril.  We don't really have any human cultures eating the version of VLC/VHF diets to look too.  Not the Masai and not the Inuit (and at least some Inuit diets may not have been as high fat as some think).  Aside from them (Masai had more carb) we've got bupkiss.

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ED. NOTE 8/9/2015:  I smile when I read those last sentences when I realize how much more I know that supports them, and how often the lies have been repeated by IHC "experts" in the interim.

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Last some thoughts on rodents, starch and fructose and the results.  Rodents are hindgut fermenters ... they are thus more efficient at converting fat to fat but do extract more calories from their carbs than we do through fermentation.  Sugar is easily metabolized, but I'm beginning to wonder if fructose doesn't offer somewhat of a  metabolic advantage in excess of fructose per se (as opposed to contributing to an excess of total caloric intake).  I've seen reports of considerable malabsorption.  Unpleasant digestive upsets aside, malabsorption = calories that pass on through.  Calories not absorbed are not ultimately considered calories "in" in the energy balance though they will be included in gross intake.  At the 60% level of sucrose = 30% fructose, an analysis of the stool might have demonstrated some significantly different caloric content.  Further, since fructose must be metabolized by the liver, I suspect the TEF for fructose is higher than for starch/glucose.  In my Nutrient Fates post I relay that I was surprised by the TEF of alcohol -- 20% -- more than double that of generic "carb".  It is possible that fructose metabolism has some sort of hierarchy preference and increase in TEF.

In Conclusion:  Let's say you're a human with a family history of  obesity and/or T2 diabetes, that's kinda like being our B/6J mouse.  You don't want to get fat and diabetic.  What seems to be the best dietary approach to hedge your bets on?

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Bonus!!   Today's FeDiabesity Question -- aka Doing Due Diligence Debunking Your Own Hypothesis -- aka CarbSane Pre-Debunked Long Ago 

The ONLY commentary I will accept on this issue is to answer this question.  I'd prefer you took this rhetorically.  I'm inviting you to think on why the information in this post -- specifically the primary study  discussed --  counters any sort of significant role of iron in the diet on the development of obesity or diabetes.  I'll help.  All mice received highly controlled diets.  They all received the same amount of iron.   Fructose increases iron absorption.  Fat inhibits iron absorption.

How is dietary iron a factor in developing obesity and diabetes?

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Bonus Two!!   Today's BlaineBrain Challenge -- aka Now's Your Chance to Discuss Something CarbSane Wrote!

The ONLY commentary I will accept on this issue is to answer these questions only.  I'd prefer you take these rhetorically, but do invite relevant discussion.  I'm inviting you to think on why the information in this post -- specifically the primary study  discussed --  counters the idea that refined sugar is in any way uniquely obesogenic, causes diabetes or is addictive causing overeating.  Remember, the LH diet was low fat and 60% carbohydrate as sugar.

1. Why didn't mice overeat the LH diet?
2. Why are the LH mice the leanest 
3. Why are the LH mice metabolically healthy?

Cheers!