mouse and the bread

I need to find me some cheese


In this post, I want to write about an interesting Brazilian study that examined the effects of feeding gluten in combination with a high-fat diet to C57BL/6 mice, the most popular strain of mice used in laboratories around the world. (1)

Would our furry friends experience any negative effects from consuming the protein found in wheat, barley and rye? Or would they merrily live their lab-shortened lives as unhealthily as any rodent fed a high-fat and sugar diet?

To find out, researchers fed one group of eight-week-old male mice with a high-fat and sugar obesity-inducing chow without gluten (the gluten-free or GF group). The second group of critters was fed this same diet but with the addition of gluten (the control group). All the boys had free access to food and water and consumed their respective diets for eight weeks.

Body weight and food intake were measured weekly. At the end of the study period, our hapless chaps were dispatched to that great rodent wheel in the sky. Their blood, visceral fat, livers, gastrocnemius muscles (leg muscles) and feces were collected for further analysis.

And what, dear reader, did our researchers discover? Well, both groups of mice gained weight, which isn’t shocking. However, the gluten-free dudes gained less weight then their gluten-eating cohorts:


gluten 1

Courtesy: Gluten-free diet reduces adiposity, inflammation and insulin resistance associated with the induction of PPAR-alpha and PPAR-gamma expression


As you can see in graph A, the gluten-free, high-fat group (represented by the black bars) gained about half the weight of the high-fat, gluten-chowing group (white bars). Epididymal fat as a percentage of body weight was much higher in the gluten-consuming group than the GF mice. Total plasma lipids or blood fats were also quite elevated in the gluten group, as were the size of fat cells.

Can these results be explained solely by the boys eating more than the gluten-free group? Apparently not:


gluten 2


Food intake was slightly higher in the gluten group, but not by any statistically significant amount. Fat excreted in feces was also lower, but again, not by much. It would seem, therefore, that the dramatic increase in weight gain in those mice fed the high-fat and gluten chow was caused by a disruption in metabolism.




These series of graphs detail glucose metabolism and levels of peroxisome proliferator-activated receptor gamma (PPAR-γ). PPAR-γ is abundant in fat tissue and is a target of anti-diabetic drugs. The more of this substance a person has, the better their insulin sensitivity and blood sugar metabolism, the lower their inflammatory markers and the smaller their fat cells.

Both fasting glucose and insulin levels were lower in the GF group. Insulin resistance, (graph C) was also lower in the gluten-free mice. Glucose levels at 15 minutes of challenge were better in the GF group as was expression of insulin and glucose receptors. Finally, PPAR-γ levels were significantly higher in the gluten-free group.




Here we see charted both gene expression and serum levels for leptin, resistin and adiponectin. Leptin levels were lower in the GF group. This isn’t surprising as leptin is produced in fat cells so the fact that this group gained less weight would explain the discrepancy.

While gene expression for resistin was higher in the GF group, serum concentrations were lower than the control group which is to be expected due to better glucose and insulin control in the gluten-free group. And both gene expression and serum concentrations of adiponectin were much higher in the GF group.

Before presenting the next series of charts, let me first define some terms.

Lipoprotein lipase (LPL) is an enzyme that breaks apart triglycerides into their constituents of three fatty acids and a glycerol molecule. An insufficiency of LPL can lead to high fat levels in the blood (hyperlipidemia) as the breakdown of triglycerides and reassembly in fat cells will not occur without it.

Hormone-sensitive lipase (HSL) is another enzyme responsible for breaking apart stored fat so it can be released into circulation for use as energy. It responds to hormones like epinephrine, but is inhibited by insulin.

HSL is also important for releasing cholesterol from chemical compounds or esters to allow the production of sex hormones in endocrine glands. Reduced levels of this enzyme, for example, can lead to low production of testosterone in men even in the presence of adequate serum cholesterol.

AcetylCoA carboxylase (ACC) is an enzyme involved in the production and regulation of fatty acids.

Carnitine palmitoyl transferase-1 (CPT- 1) is an important mitochondrial enzyme responsible for fatty acid oxidation or “fat burning” by cells. Elevated CPT-1 levels increase the utilization of long-chain fatty acids for energy.

Finally, peroxisome proliferator-activated receptor alpha (PPAR-α) is an important protein involved in the expression of genes. PPARs play important roles in both glucose and lipid homeostasis. Increased expression of this protein is associated with increased insulin sensitivity and utilization (oxidation) of fat for energy.



In this series of charts, all of these lipid and glucose regulating enzymes and proteins were worse in the gluten eating group in contrast to the gluten-free cohort.

Now, does this mean that gluten is the direct cause of these observed phenomena? No, I don’t think so. I believe what these researchers witnessed is the result of increased metabolic endotoxemia in the gluten-fed rodents.

Why do I believe that?



Click to enlarge


This series of graphs tracks cytokine levels in these mice. Note the increases in tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6). Levels of anti-inflammatory interleukin 10 (IL-10) were also elevated. IL-10 is always increased in inflammatory states because it acts as a counterweight to TNF-α,  IL-6 and other inflammatory cytokines.

This increase in inflammation is a characteristic state of metabolic endotoxemia, and as I covered here, would impact metabolism for the worse. Increased cortisol levels, as occurs in endotoxemia, would be responsible for the effects on glucose and lipid metabolism witnessed in this study.

Cortisol acts inversely to PPARs, down-regulating their expression. In excess, cortisol inhibits fatty acid oxidation, promotes a lipid profile that is indicative of cardiovascular disease, decreases insulin sensitivity and promotes visceral fat accumulation. (2)

It’s a shame these researchers failed to measure plasma levels of the intestinal tight junction protein known as zonulin. Had they done so, they would have confirmed increased intestinal permeability.

Increased intestinal permeability always leads to inflammation. It causes translocation of gut pathogens and other antigens from the gut lumen first to the liver, and secondarily to systemic circulation. By activating immune cell receptors (toll-like receptors), gut pathogens provoke inflammatory immune responses.

As you recall, gluten has well-known negative impacts on intestinal tight junctions as detailed by the research work of Dr. Alessio Fasano at the University of Maryland School of Medicine. (3) Of the two molecules that compose the gluten protein, gliadin directly up-regulates zonulin production. This causes increased permeability, which stimulates inflammation and increases permeability in a continuous feed-forward manner.

Zonulin also regulates the permeability of the blood-brain barrier. Increasing its production would explain brain inflammation and give new meaning to the term “shit for brains”.

It is also quite conceivable that these gluten-fed mice developed the rodent equivalent of small intestinal bacteria overgrowth (SIBO). The combination of gluten opioid peptides and generation of the inhibitory neurotransmitter adenosine would decrease the rate of intestinal peristalsis.

Anything that inhibits small intestinal peristalsis will predispose to colonization of the distal small intestine with gram-negative gut pathogens migrating from the colon. However, whether eight-weeks of gluten feeding is enough time to observe these effects is purely speculative at this point as these researchers didn’t explore this possibility.

I need to emphasize that there is no hint that what occurred in this study had anything to do with the autoimmune disorder known as celiac disease. There is as yet no mouse model for this disease. Dr. Bana Jabri’s group at the University of Chicago is still working to develop such a strain.

No, what we had here were run-of-the-mill male lab mice displaying typical characteristics of dysregulated glucose and lipid metabolism due to increased intestinal permeability, translocating gut pathogens/antigens, along with stimulation of the hypothalamic-pituitary-adrenal (HPA) axis. In other words, good old-fashioned endotoxemia.

However, how relevant is this research to humans, and obesity in particular?

Well, not everyone eats gluten with every meal as these control mice did, but as the Western world’s favorite grain the consumption of wheat is extremely common. The breeding of wheat plants to produce higher concentrations of gluten has increased the amount of this protein consumed throughout the world.

Can anyone imagine the typical American diet (hamburgers, hotdogs, pizza, apple pie, cake, cookies, crackers, cereal, pancakes, waffles, sandwiches, mac and cheese, etc.) without it? Those of us who have given up gluten grains are very aware of how difficult it can be to avoid gluten-containing foods when shopping at the local grocery store or dining out.

So while the effect of gluten on intestinal permeability is transitory in non-celiacs, exposing small intestinal cells to gluten multiple times a day is not in the least bit unusual for many. Nor is it unlikely that a good amount of wheat germ agglutinin is also ingested with these foods. (4)

A year ago, my close friend and her family visited us for a week. During the time they were with us, every breakfast, lunch, snack or dinner they all ate contained some component of wheat. Everyone.

As for explaining obesity in humans, I’m less than convinced. Yes, it is definitely part of the problem, but gluten is not the only substance in food that causes disruption to gut health and all that flows from that. Let’s not forget binge drinking and excess omega-6 fat and fructose intake.

And there is more to endotoxemia than just the food you eat. As I’ve written, psychological stress can play a key role. It should not be surprising to learn that poverty and low social economic status are associated with negative health outcomes, including obesity.

That said, it is quite possible to suffer from metabolic endotoxemia, whether induced by gluten or not, yet still remain thin or normal-weight as I covered here. Much depends on genetics and gut flora composition.


Courtesy: Genetic Control of Obesity and Gut Microbiota Composition in Response to High-Fat, High-Sucrose Diet in Mice

Courtesy: Genetic Control of Obesity and Gut Microbiota Composition in Response to High-Fat, High-Sucrose Diet in Mice


This chart tracks how much weight is gained when you feed 100 different mouse strains the same high-fat and high-sugar or “cafeteria” diet.(5)

Some mouse strains see increases of over 300% in body-fat accumulation as represented by the red lines. Other mouse strains, represented by the black lines, see no increase whatsoever. Others fall somewhere in the middle.

I would imagine we would arrive at similar results if we added gluten to the chow of these different breeds of mice. There is obviously a genetic and gut flora component to weight gain that explains this wide divergence to the same diet.

However, does this mean that the mice represented by the black lines are not experiencing endotoxemia when ingesting the “cafeteria” diet? Perhaps. They may have gut flora or genes that protect them.

Or perhaps, like some of their normal-weight human counterparts who can seemingly eat anything they want, yet never pack on the pounds, they may be genetically unlucky.


Ray, how could it possibly be unlucky to eat a diet that promotes obesity in most but not in these normal-weight people?

Because the ability to put on weight appears to be protective against the consequences of endotoxemia and a chronically activated HPA axis as I’ve explained when discussing the obesity paradox. Why this is so still remains a mystery, but that is clearly what the evidence shows.

So while these people are “lucky” in never having to face weight discrimination or ridicule from friends, family members, strangers or comedians, they may be genetically unlucky because the weight gain that would provide some level of protection from endotoxemia-induced inflammation is non-existent.

These are the people who when confronted with the bad news that they’ve developed type-2 diabetes, or an autoimmune disorder, or cardiovascular disease, or cancer, or Alzheimer’s sputter in absolute disbelief to their doctor “But, but, but, I’ve never been overweight my entire life.”

True, but irrelevant. As I’ve said before, weight is an imperfect predictor of health, longevity and the state of your intestinal gut barrier.

So while this study is intriguing for its findings on weight gain in this particular mouse strain, I’m far more interested in its findings of increased inflammation and dysregulated glucose/lipid metabolism in the gluten-fed mice. That, and not weight gain, are the real gems of this study.

Let’s hope this research continues so we can better identify those foods and dietary practices most inimical to gut health and well being.

OK, it’s time for me to start cooking my gluten-free pasta for tonight’s dinner.


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