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

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 using this same mouse.  In looking for info on this mouse, 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.

That said, they are highly susceptible to DIO induce diabetes.  This is a genetic propensity as 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, not 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 diobesetes(1) 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.

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 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?  I mean with humans you can instruct to comply with protocol and finish all foods.  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 difference was a slight one in color.  Heck, you give CAF rats human junk foods, and they don't eat the 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 and all that.  

Next, we get to palatability and pelletability.  Gooey pellets aren't practical so too high fat a diet is not really even possible.  Therefore there's got to be some significant carb content.  This is a drawback although ketogenic diets have been studied.  The nature of the fat has been attacked by many, and rightly so.  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.  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 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 bulletpoint the Abstract):

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

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 hyperplasia of the mesenteric fat depot - type of visceral fat. 

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.

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 {click to enlarge}

HH = High fat / High sucrose
HL = High fat / Low sucrose
LH = Low fat / High sucrose
LL = Low fat / Low sucrose

Yep ... there's the "eew" factors Denise mentions in her piece but those same things are fed to all of the mice in this study.  Casein protein is constant (at 16%) across the board, as is the soybean oil and maltodextrin (sometimes you have to have this in your primal foods)  composition 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.

So anyway, 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:  Plotted at right are A/J = red, B/6J = blue   weight vs. intake

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, but 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.  

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:

Here is the feed efficiency plot for the two strains on each diet.  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 effecient at storing fat.  

You can click on each of the two images above to enlarge.   For all plots, the top is for the mesenteric (visceral) fat, and the bottom is for inguinal (subcutaneous) fat.   The four bars on left are A/J, the four on the right are B/J6 in all plots.  In the clusters of four bars the left two are high fat, right two are high sugar, and the order goes HH, HL, LH, LL.  

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.

 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.  

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 (something I need to get back to addressing at some point!) and is considered somewhat fixed by age 18-20 or so.  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 (to the best of my knowledge not reducible w/o surgery!).  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.  

Let's revisit that CR study from a ways back.  The calorie restricted mice gained the same amount of total mass, but went into "conservation mode" and gained significantly more FAT mass.  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.

Once fully formed, what are the implications for a low carb/high fat maintenance diet for us humans?  Mmmff?  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.  

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!   

In the end, we can learn a lot from some rodent studies.  They are not perfect even in the rodent world.  They don't translate to humans in any sort of imperfect way let alone a perfect way.  But ignore 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.  

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 effective calories "in".  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:  You're a human with a family history of 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?