What would happen if you fed prebiotics to genetically obese mice or mice fed an obesity-inducing high-fat diet? Would anything change? That’s the subject of today’s post, which covers such an experiment published in 2011. (1)
Two different types of mice were used in this study. The ob/ob C57BL/6 mouse and the C57BL/6J mouse. The ob/ob C57BL/6 mouse is a mutant incapable of producing leptin and is used as an animal model for type 2 diabetes.
Leptin is a hormone secreted by fat tissue and is involved in long-term weight regulation. The more fat tissue, the more leptin produced, the more leptin produced, the more reaches the arcuate nucleus of the hypothalamus. This should increase the desire to move more and reduce appetite. Or at least that is what is supposed to happen, but often times doesn’t in those battling weight gain.
Because this feedback system is absent in these mice, they have a chronic desire to overeat.
The standard C57BL/6J mouse, however, is not leptin deficient. Instead, they are quite adept at packing on the weight when fed a high-fat lab diet. They are members of the “red-line” rodent club I’ve mentioned before. I’ve known more than a few red-line people who could fully sympathize.
OK, let’s start with the ob/ob mice. One set was fed a control diet while the other was given the same diet, but with the addition of the prebiotic oligofructose.
The mice fed the prebiotic (Ob-Pre) exhibited quite dramatic changes from controls (Ob-CT) in gut flora composition. Significant shifts from predominantly Firmicutes to Bacteoidetes phylums were observed.
Also note from the table above that Bifidobacterium (bifidobacteria) was totally absent in the control mice and exclusively present in the prebiotic-fed mice. Conversely, the genus Syntrophococcus was found only in the control mice and absent in the prebiotic group.
Now I don’t read too much into these shifts between Firmicutes and Bacteoidetes because other studies have revealed opposite effects in both rodents and humans when supplemented with prebiotics. (2) What is a consistent finding in study after study, however, is the notable increase in bifidobacteria in the colons of animals or humans consuming prebiotics.
These illustrations show how well the control ob/ob (Ob-CT) and prebiotic-fed ob/ob mice (Ob-Pre) handled glucose when challenged. As you see, the prebiotic-fed rodents showed dramatic improvements in glucose control. Peak plasma glucose levels were lower in this group and returned to baseline sooner.
In chart B, viceral, epididymal and subcutaneous fat accumulations were lower in the prebiotic-fed mice, although changes in total body weight were not significant. Graph C notes statistically significant increases in lean-muscle mass in the mice fed prebiotics.
While not illustrated, cumulative food intake (Ob-CT 466.8 + 13.8, Ob-Pre 319.6 + 20.6) was much lower in the prebiotic group. I’m sure that had something to do with the increased production of gut satiety hormones I’ll highlight in a minute.
Measurements of plasma triglycerides, muscle fat and muscle triglycerides were all significantly lower in the prebiotic-fed/bifidobacteria mice. Elevated triglyceride levels are a well-known marker for metabolic syndrome and heart disease in humans.
The prebiotic-supplemented mice experienced an approximate 70% increase in muscle lipoprotein lipase (LPL). This increase in muscle LPL signals the oxidation of fatty acids to fuel muscle cells rather than storage as fat.
As you recall from my post, The Curious Case of the Gluten-Free Mice:
“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.”
In graphs H and I we see that oxidative stress, a measure of inflammation in fat cells and a common finding in the obese, was dramatically lower in the prebiotic-fed mice as were plasma levels of lipopolysaccharide (LPS).
The prebiotic-fed mice experienced improved gut-barrier function. No surprise here given the lower levels of plasma LPS. Two tight-junction proteins, small intestinal Zo-1 and occludin, were both higher in these animals. Unfortunately, no information is provided on any of the claudin tight-junction proteins.
Oxidative and inflammatory markers in the colon were greatly reduced. As inflammation is a necessary precondition for the development of cancer, including colon cancer, this is an important finding.
Here’s a fun little series of illustrations for all you science nerds out there. It appears that bifidobacteria controls the expression of L-cells as illustrated in these photos. Hmmm, L-cells? Now where have I heard about those before I wonder?
Oh yeah, that’s right! L-cells release satiety hormones like glucagon-like peptide, peptide YY and oxyntomodulin as previously explained by your humble blogger:
“Glucagon-like peptides (GLPs) are also released by healthy L-cells of the small intestine and colon, and the alpha cells of the pancreas. These hormones influence insulin and glucagon secretion and their dysregulation may be a contributing factor in insulin resistance. These hormones reduce appetite and the rate of stomach emptying making you feel full. So again, any damage to L-cells will impede the release of these insulin-regulating and satiety-enhancing hormones.
Peptide YY…is secreted by cells in the ileum and is the focus of quite a bit of research for its appetite-suppressing effect in rodents, primates and humans. Supplementation of probiotics and prebiotics in rats has been shown to increase levels of this hormone.
Oxyntomodulin…is secreted along with GLPs and peptide YY by healthy intestinal L-cells. In both rodents and humans it reduces food intake. Like the other satiety hormones, its production will be enhanced by the presence of healthy gut flora but inhibited in the presence of gut dysbiosis.” (3)
The charts above demonstrate increases against controls in plasma glucagon-like peptide 1 (GLP-1) and proglucagon (a precursor to glucagon) along with a doubling of L-cell expression in those mice consuming prebiotics.
I suspect this effect is partly mediated by the production of short-chain fatty acid metabolites, especially butyrate, that nourish gut cells. And as always, the increase in bifidobacteria species would crowd out problematic pathogens in the colon by lowering luminal pH and out competing them for nutrition or attachment sites.
OK, enough about mutant mice. What about good old-fashioned mice who pack on the weight just by looking at food? How would these natural chubbies do if their Frankenfood, high-fat chow was supplemented with prebiotics? Glad you asked:
HF represents the control mice fed the high-fat chow and HF-Pre those fed the same diet but with prebiotics. As in the leptin-deficient mice, plasma glucose control is improved in the prebiotic group.
Weight gain is significantly less as seen in graph (B) and lean body mass is increased (graph C). This in spite of the fact that mean food intake (HF 20.9 + 0.6, HF-Pre 19.6 + 0.3) was not much affected prior to leptin treatment.
Measures of fatness or adiposity were much lower in the prebiotic group. Plasma levels of proglucagon and the satiety hormone glucagon peptide 1 were higher in the prebiotic group. This makes sense as more L-cells means more production of GLP-1.
Here we see how well these mice responded when injected with appetite-suppressing leptin. Body weight change was much greater in the prebiotic-fed mice as seen in graph G.
Illustration H shows there was no statistically significant change in food intake in the control group when administered leptin. That was not the case in the prebiotic group. The addition of prebiotics to their chow and blooming of bifidobacteria colonies in their colons made these mice less resistant to the actions of leptin suggesting that beneficial gut flora and their metabolites influences leptin sensitivity in the brain.
The last graph shows a dramatic variance in tissue aceltyl-CoA carboxylase (ACC) expression after leptin treatment. ACC is an enzyme necessary for the production of malonyl-CoA. Malonyl-CoA inhibits fatty acids from associating with carnitine thereby preventing these fats from entering the mitochondria of cells and burned for energy, not good if losing weight is your goal.
To quote the authors of this paper: “Thus, this analysis revealed that prebiotic treatment improved the anorexigenic, weight-, and lipogenesis-reducing effect of leptin compared with control obese mice.”
Translation: prebiotics, and the increase in bifidobacteria this causes, increases the effectiveness of leptin in reducing hunger and weight gain.
Imagine that! Amazing how helpful those gut critters can be.
The authors detail another interesting observation about these mice:
“Given that prebiotic treatment can reduce obesity and associated metabolic disorders, the discovery of bacteria or bacterial group(s) that is able to shape host metabolism provides an attractive mechanistic explanation. Interestingly, both 16S rRNA [gene sequencing] analyses identified significant correlations between the genus Anaerotruncus and several metabolic parameters, such as glucose intolerance, gut permeability, plasma triglyceride content, and muscle lipid content. Similarly, Clostridium lactifermentans was positively correlated with all of these parameters, except plasma triglyceride content.
Desulfovibrionaceae (i.e., Bilophila and Desulfovibrio, both gram-negative bacteria) were less prevalent in the prebiotic-treated mice. Interestingly, two recent studies demonstrate that diet-induced obesity and diabetes are associated with a bloom of this family. Some members of Desulfovibrionaceae, shown to be involved in gut barrier disruption, are able to reduce sulfate to H2S [hydrogen sulfide is a broad-spectrum poison]. In agreement with these reports, we found a very strong correlation between gut permeability and the abundance of Streptococcus intermedius. It is important to note that this species produces a specific cytolysin (intermedilysin) [these are cell toxins] that leads to altered tight-junction architecture. Therefore, it is tempting to speculate that the lower abundance of cytolysin-producing bacteria may participate in the control of gut barrier function through these mechanisms.”
In other words, through the production of various cell toxins, the gut wall is breached and endotoxemia sets in with all the downside consequences many of you are all too familiar with. Whether these same bacteria are operative in human dysbiosis remains to be determined. Regardless, there is no way to keep gut pathogens and their toxic metabolites in check without beneficial gut bacteria.
Another mechanism that controls gut permeability is the endocannabinoid system (eCB system). (4) The eCB system is far too complex for me to cover in its entirety here, but let me briefly outline some of its functions.
If the word endocannabinoid reminds you of the word cannabis, feel free to light up a joint! Indeed, the active ingredient of marijuana, THC, acts directly on the CB1 endocannabinoid receptor to initiate what in my distant college past was a blissful haze soon followed by the intense desire to eat. Ah yes, Pink Floyd with a side of….well, whatever tasty treat was conveniently at hand.
This system is composed of a series of receptors, CB1 and CB2, that exist throughout the central and peripheral nervous system. These two receptors affect many areas of the body, including the brain and immune function.
The system is involved in memory (hey, did anyone notice where I put the bong?), appetite, energy balance and metabolism, stress response, social behavior, anxiety, pain sensation, immunity, fertility, pain control, body temperature and sleep. The expression of at least one of its receptors, CB1, can be affected by both beneficial and pathogenic bacteria.
This colorful illustration (don’t you just love the band-aid?) contrasts the obese and prebiotic state. It succinctly illustrates how “leaky gut” impacts fat accumulation via the endocannabinoid system and the translocation of lipopolysaccharides to systemic circulation.
What is not represented here, of course, are the many other downside consequences of endotoxemia I’ve covered elsewhere: activation of the hypothalamic-pituitary-adreanal axis resulting in increased cortisol production, negative impact on the conversion of thyroid T4 hormone to the more active T3 form in the liver, cytokine and immune activation, impaired production of gut-satiety hormones and increased production of 3-hydroxy-kynurenine and quinolinic acid to mention a few.
What is abundantly clear from this study is that mammalian gut flora is involved in appetite signaling, fat oxidation, lean tissue expression, regulation of plasma triglycerides, lipopolysaccharide translocation, fat-tissue inflammation, glucose control, leptin sensitivity, intestinal L-cell expression, satiety hormone secretion and altered expression of endocannabinoid receptors.
None of these observations can be explained by resorting to theories that solely revolve around food reward or food palatability or toxic-food environments or circadian rhythms or insulin spikes or sedentary lifestyles.
Please understand that I do not dismiss these theories out of hand. Far from it.
However, I do take issue with any weight-regulation hypothesis that fails to account for gut flora. Yet for many researchers and writers on obesity, the gut mircobiome is truly a forgotten organ.
How you can ignore an organ that contains 10 times more cells and 100 times more genetic material than what makes up an average human being just boggles my mind. But hey, I haven’t smoked marijuana in ages which apparently limits my ability to avoid the obvious.
Perhaps such “theorists” are partaking of the endocannabinoid system far more often than supposed. If that’s the case, I wish these folks would share some of what they’re smoking with me. I’ll happily supply my copy of The Dark Side of the Moon and some tasty gluten-free snacks for all to enjoy as we collectively bury our heads in the sand.