Why We Get (Sick) Fat (and Sick Livers) - Lessons from a Cafeteria Rat

Since we're talking about fructose and the liver of late, I thought I'd bump this post.  In this study groups of rats were fed one of four diets.  The "low fat" diet is better described as a high sucrose diet as 35% of the diet was sucrose.  This replaced 35% of the fat in the 45% "high fat" diet.  While the LF and HF rats gained a little more weight than the standard (also LF at 12%) chow rats, it is clear that the high fat has rather more negative metabolic effects.  I don't think the 35% sucrose diet was beneficial, rather the contrary, but that level of sucrose consumption, every single day for 10-15 weeks (which is quite a long time for a rat) is also hardly indicative of even SAD consumption.

Original Posting:  3/8/11

It seems fairly generally accepted that whatever the cause or progression, the so-called Metabolic Syndrome, Syndrome X and Type 2 Diabetes are associated with a dysregulation of adipose tissue metabolism, and fat tissue that is infiltrated with macrophages and secretes excessive amounts of inflammatory molecules called adipokines (e.g. TNF-α, IL-6).   A term has been coined, adiposopathy, to describe this "sick fat".  

I've recently discussed the "Cafeteria Rat" study  as pertains weight gain in general.  But the other thing about this study is that it looked at the fat tissue with the different dietary interventions.  To recap, four groups of rats were fed ad libitum different diets:

1.  SC control (estimated <5% simple sugars, 12% fat)
2.  HF chow - 45% fat, lard
3.  LF - low fat (10% lard & soybean oil) matched to HF chow with sucrose substituted for fat content
4.  CAF = SC + 3 human snack foods varied daily from a list

Caloric consumption was  HF (559) < LF (660) ≈ SC (663) << CAF (908) and the weight gains were as shown below:

Of note here is that the HF rats and the LF (high sucrose) rats gained about the same amount of additional fat mass compared to the SC controls but the morphology of their fat differed considerably. Below are the slides of the eWAT (epididymal white adipose tissue).  That "eye" in slide "j" is called a crown-like structure, CLS and is a macrophage surrounded by dying adipocytes (fat cells) and/or the lipid droplet remnants of dead cells.  

The average CLS content of the eWAT was:
I found this rather striking.  It seems any degree of excess adiposity is associated with some macrophage infiltration and inflammatory state.  But it seems that adiposity induced by a high fat diet is particularly associated with the formation of this CLS-riddled "sick fat".  Despite comparable fat gains, the high fat rats had about twice the CLS as the low fat (high sugar) rats.  This is especially interesting with all the literature fingering fructose these days as "causing" insulin resistance, MetS and diabetes.  The LF rats actually consumed by far the most highly refined carbs and sugars:  more than 6.5X that of SC, over 2.5X that of HF, and about 1/3rd more than even the CAF rats.  They got as fat on average as the HF rats, but with only about half the CLS formation.  They got about half as fat as the CAF rats but only developed about 1/3rd of the CLS's of that group.  

One reasonable explanation for this might be that fat cell function as a buffer for dietary fat can easily be overwhelmed with high fat intake.  So, in this study, the high fat diet didn't make the rats fatter than a high sugar diet, but it made their fat sicker!  Perhaps the increased demand for uptake and esterification of dietary fat leads to upsetting the regulation?   Maybe ASP isn't such a good trapper of dietary fats after all leading to excess extracellular (outside the cells) fatty acid content in the fat tissue that the immune system cannot distinguish from lipids released by dying fat cells so macrophages move in?  Or does the higher lipid turnover in these cells required for high fat intake causes them to lose their sensitivity to hormonal signals regardless of size?  

Comparing the CAF to HF, we just see that at some point, excess adiposity just makes matters a whole lot worse.  If you eat a diet that causes a significant increase in ad libitum consumption (CAF ate 35% more than standard chow!) things will go awry faster.  

The CAF rats in this study were the only group that achieved a high enough and sick enough degree of adiposity to exhibit the signs of Metabolic Syndrome.    Below are the NEFA levels:
The inefficient trapping of dietary fatty acids is a possible explanation for the more elevated NEFA seen for HF v. LF given the same degree of adiposity.  Also possible that the dying adipocytes in those CLS's release fatty acids.  

Below is the liver tissue:

If you enlarge, you can see CV = central veins and PT = portal triads identified.   The 2nd column from the right is the healthy liver of the SC fed rats.  They note "macro-vessicular steatosis" in the LF rats and "micro-vessicular steatosis" in both the CAF and HF rats.  These are defined as:
  • Macrovesicular steatosis is characterized by engorgement of the hepatocyte (liver cell) by a large fat globule that displaces the nucleus.
  • Microvesicular steatosis "refers to a variant form of hepatic fat accumulation whose histologic features contrast with the much more common macrovesicular steatosis".  This is usually attributed to mitochondrial dysfunction or toxicity.
Two patterns of hepatic steatosis are recognized: (1) microvesicular steatosis: the cytoplasm is replaced by bubbles of fat that do not displace the nucleus; and (2) macrovesicular steatosis: the cytoplasm is replaced by a large bubble of fat that displaces the nucleus to the edge of the cell.
I'm not sure either form is worse or better to have, but clearly we would want to avoid this entirely.   A 35% sugar diet is NOT a good idea, but we don't really need to be told that now, do we??   But high sugar diets clearly lead to a different liver dysfunction than do high fat diets.  Is the microvessicular form one of lipotoxicity?

The researchers tallied up the number of inflammatory loci in the liver as shown below.

The pattern is similar to that seen for the fat tissue so we are offered no clues really as to progression.  The inflammation seems to correlate with NEFA release from the fat tissue as well.

In any case the inflammation in the fat tissue and liver seems to correlate with both fat content in the diet, and degree of adiposity achieved.  With modestly elevated NEFA, glucose tolerance is retained.  Dietary fat seems to cause inflammation as adiposity increases (moreso than sugar!).  Directly?  Seems more not than so.  But indirectly, perhaps as simply as by overtaxing the triglyceride/free fatty acid regulatory cycle, it sure seems so.

The high fat diets only - HF & CAF - altered the pancreatic β-cells.
LFD- and SC-fed rats displayed normal pancreatic islets with defined borders, whereas HFD-fed rats contained slightly hypertrophic and disfigured islets. Islets from CAF-fed rats were much larger in size, with dramatically distorted architecture.
FWIW, I'm not seeing much if any difference in the HF vs the LF or SC, but I have no reason to question these researchers' description of their findings.

The authors stated that only the CAF rats became prediabetic, but I see degrees/shades in the results.  One can see a progression, but only the CAF rats demonstrated a statistically significant difference - this explains the authors' statements.  The high fat and high sugar diets did appear to have a "glucose metabolism" more akin to that of CAF than the SC rats.

The first test was an intraperitoneal (IP = inject into abdominal cavity) glucose tolerance test, IPGTT.  The results are shown below:            
Note:  This test done at 7 weeks

Yes, there's no statistical significance between, but the response is similar to what we see for fat-CLS, liver-IL and NEFA.   They also conducted an IP insulin tolerance test, IPITT.  Here insulin is injected and blood glucose measured to determine insulin resistance.  The results are below:

Note:  This test done at 11 weeks

From the paper:
In line with weight gains, IP-GTT at 7 weeks on diets demonstrated that glucose intolerance and insulin resistance increased from SC < LFD < HFD < CAF (least resistant to most resistant) (Figure 3a,b), and insulin levels were significantly elevated for all diets relative to SC (Figure 3c). IP-ITT performed at 11 weeks on diets demonstrated that the insulin-mediated drop in glucose was not significantly different between diet exposures (Figure 3d,e).

I don't know about y'all, but this study is sobering to me.  I'm going to redouble my efforts to get hubby off the junk food, that's for sure!!  


Stephan Guyenet said…
I think this study basically encapsulates what's wrong with the industrial diet. It's interesting to debate the mechanism, but empirically the cause is right in front of us. Modern humans are eating a "cafeteria diet" for the first time in history, which is a broad palate of industrially refined and highly palatable foods. We're eating ourselves to death, and enjoying every bite.

I do think it's premature to blame the poorer performance of the refined HFD on the fat per se. Although HFD causes more fat gain because it's highly palatable (even though it doesn't necessarily increase energy intake in the long term, which points to a change in homeostatic mechanisms, more to follow on that eventually), adding a pretty modest amount of n-3 to the diet essentially prevents insulin resistance and adipose tissue inflammation.

Also, not autoclaving or irradiating the diet greatly attenuates its metabolic consequences:


Adding polyphenols is also protective. Polyphenols are much more abundant in unrefined diets. The point is that fat quality, as well as other properties of the diet, influence the reaction to refined HFD in rodents.
CarbSane said…
I'm really curious about the heating in that study. Tried to look up that diet and it's not on the manufacturer's site to see the type of fat. 7X higher AGE content by heating to 120C (approx 250F) for 1/2 hour?
RRX said…
Hi Sane,

I have to ask, is your husband gaining weight? How much of the effect seen here is a matter of excess energy consumption first and THEN how much is it a matter of which nutrients? Isn't a confounding variable the excess consumption? I would like to see this same study done with the rats consuming a capped amount of food, as opposed to ad lib. If you know of a study that already did that, please let me know. I'm very interested to see the results.
CarbSane said…
Hey RRX, my husband is nobody to look to on this. It peeves me to no end how hard I had to try to lose and maintain my losses, while he just gets a more active job, eats the same crap - though less of it hanging out in front of the TV - and loses weight effortlessly. Jerk! {just kidding :-) }

It would be interesting to have let the HF & LF rats go long enough to reach the level of adiposity of the CAF rats as, yes, I do believe this is a confounding variable. The other option of limiting consumption of CAF rats would work too. I don't know of any, but if I come across them I'll surely post!
RRX said…
I just can't help but wonder how much of the variation is a result of increased fat levels in general. If all of the conditions had equal consumption (or access to), then would we see any differences in the results between conditions? For example, I imagine a study that has LFD 1 (increased body weight by X%), LFD 2 (weight stable from the start), HFD 1, HFD 2, SC 1, SC 2, CAF 1, and CAF 2. I think this is very relevant to humans and disease risk, as I have heard/read/had discussions about whether the skinny person that eats a CAF-style diet (represented by the weight stable conditions in this hypothetical study) is "healthy". How is their liver? How is their inflammation levels? Etc.
CarbSane said…
We do know that in part from the LF and HF diets. Even though there was a slight difference in the intake (a bit more for LF), these rats put on about the same weight. So the difference in the fat tissue and beta cells is based on "matched diets". I'm shocked, really, that the LF 35% sugar diet didn't wreak more havoc, although we see it did mess up the liver!
Stephan Guyenet said…
Hi Carbsane,

I'm pretty sure they made a mistake in the methods. D12492 is manufactured by Research Diets and the ingredients and composition are freely available online. I wrote RD a while back asking about heat treatment. D12492 is not heated much past body temp during mixing and extrusion. They sent me data for lipid peroxides, which seem to be in a reasonable range.

However, there are a few caveats. The ingredients may be heated significantly before being combined, for example spray-drying of the casein and rendering of the lard. Also, they offer an irradiation service for food going to SPF facilities, which is most rodent facilities at this point (although some may autoclave it, I don't know). So most of the food will be irradiated or autoclaved when the investigator uses it, which increases damage including lipid peroxides, AGE and ALE.

It's a pretty interesting line of investigation. There's a decent amount of human data on cooking temp and health-- it seems to be relevant, particularly in those with preexisting conditions like diabetes and kidney dysfunction. If meat consumption (esp. processed meat) really does contribute to health problems in the US, my guess is that it's because it's often cooked by high heat-- seared, grilled, roasted, etc. Processed meat especially appears to be very high in AGEs.
CarbSane said…
Thanks for that clarification.

It's a pretty interesting line of investigation. There's a decent amount of human data on cooking temp and health-- it seems to be relevant, particularly in those with preexisting conditions like diabetes and kidney dysfunction.

I agree!

I do think the obesity epidemic is explained by too many of us eating a CAF diet. I became an adult right around when the rates began to climb. It makes me feel old to talk about "back then", but today's generation can't even imagine a life without a microwave, fast food joints open 24/7/365, Starbucks and Red Bull, groceries w/o all those fancy prepped foods to pick up, gourmet frozen fare, a choice between McD/BK/Wendy's/and several more FF joints in each town, eating "out" several times a week, heck ... samples at Costco ... erm make that all such warehouse stores to begin with.
RRX said…
Being 29, I never knew a life like you describe. I can say, though, that becoming a foodie over the years since 2001 when I started losing the weight made me find out how at odds that is with most people my age and younger. Preparing and cooking my own food at home 6 nights a week, making breakfast every morning, and bringing my own prepared lunches to the lab is completely foreign to everyone I know. Hell, even the people I personally know that are older than me don't do this! I'm the "weird" one, according to all them.
CarbSane said…
Hey weirdo! ;-) It really is rather amazing isn't it? It makes a huge difference in so many ways.
eulerandothers said…
My husband ate 'my diet' when we first started dating. We ate in restaurants a lot, on our dates, and he eschewed any added oils (chicken or fist, 'broiled, dry') or butter at the table. He didn't suffer much: we still drank wine with our meals and he loaded up on .... rolls without the butter!

He lost around 20 pounds in 3 or 4 months.
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Nigel Kinbrum said…
"dying adipocytes (fat cells)". That's a good thing if you've got adipocyte hyperplasia, isn't it?
Sanjeev Sharma said…
not a slam & dunk, considering the regrowth that some liposuction vict^H^H^H^H^H patients experience

IOW, if the hyperplasia's the optimally healthy response in the given scenario killing adipocytes may be the last thing one wants.
Nigel Kinbrum said…
I meant after significant weight loss. However, I guess that macrophages won't attack shrunken adipocytes 'cos they're not leaking NEFAs. I hereby retract my initial comment.
Sanjeev Sharma said…
all hinges on whether the hyperplasia's is a healthy response

or a suboptimal response, an evolutionary tag-along, like the weakness in the abdominal wall that leads to inguinal hernias, or the "feature" that runs the urethra through the donut of the prostate.
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carbsane said…
Added fat really adds up (pardon the pun) without adding much else. For all the glories of certain fats, most are virtually devoid of nutrients, or the nutrients are in the whole food containing the fat.
carbsane said…
Yeah, after weight loss may be desired, but not in the hyperplasiac (is that a word? :-) ) state.
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