Episode 9 looks at a cause of obesity and diabetes: endotoxin, or lipopolysaccharide. There will be at least one more podcast on the topic, but this one looks at a number of rodent studies creating diabetes, obesity and other markers of the metabolic syndrome. I was quite surprised at the strength of data collated over dozens of studies on this topic during the initial research. Some of the experiments in animals are not done in humans but they have more descriptive power. In the next episode on the iTunes stream I’ll look at a number of studies and papers related to humans.
I’ll be uploading another podcast for patreon supporters pledging 5USD per month or more on how to mitigate endotoxin, at least the factors I came across in this research.
The referenced transcript of this podcast along with other patreon only podcasts are uploaded for patreon supporters pledging 5USD per month or more.
Biocast Episode 9 Endotoxin, Obesity and Diabetes.
In this episode I’m going to look at some of the scientific data on the effects of endotoxin relative to obesity, diabetes and markers of metabolic syndrome. The previous episode of this podcast was on endotoxin and will give you an idea of what were dealing with, the short version is that it is a toxic component of some bacteria which can cause problems when it gets into the bloodstream.
There are various factors that could increase endotoxin in the bloodstream, example dysbiosis the gut flora that increases the ratio of gram-negative to Gram-positive bacteria. Gram negative bacteria contain endotoxin so more of those means potentially more endotoxin. The other major factor is anything that would increase transport from the gastrointestinal system into the blood. There are also potentially harmful levels in some foods.
That said let’s start getting into the studies. I’m gonna start with rodent and animal studies. The first paper is called Metabolic endotoxaemia initiates obesity and insulin resistance. (1)
So the paper opens with the observation that diabetes and obesity are characterised by insulin resistance and low-grade inflammation, the authors were looking for the source of this inflammation and identified endotoxin as the cause. In this study they use the term lipopolysaccharide which usually refers to a refined portion of the endotoxin, they can be considered identical in terms of the effects.
They fed mice a high-fat carbohydrate free diet that was 72% fat and 28% protein. The fats used were corn oil and lard. They first study the effect of the high-fat diet on endotoxin levels in plasma. Normally rats have a daily rhythm of endotoxin in the plasma which peaks at the end of their dark period which is when they eat, after four weeks this pattern had been altered and the plasma endotoxin remained high all through the daily cycle. The levels recorded while increased were significantly lower than what would be needed to cause septicaemia. These levels they defined as metabolic endotoxaemia. And there in the range of 2 to 10% of what would be needed to cause septicaemia.
They also measured markers of intestinal flora, they showed that four weeks of the high-fat diet resulted in significant changes in the gut flora. One of the changes they noted was a reduction in bifidobacteria, which is something will come back to later on.
They tested oil or water tainted with endotoxin and found that only the endotoxin oil mixture increased plasma endotoxin. They also found that administration of oil allowing increased plasma endotoxin.
The then gave the rodents subcutaneous endotoxin infusion for one month, they noted that the 72% high fat diet increased endotoxaemia 2.7 fold over controls whereas a 40% fat diet increased endotoxaemia 1.4 fold over controls.
Fasting blood sugar was higher in the endotoxin infused rodent’s. Blood glucose was higher following an oral glucose challenge in the endotoxin infused rodents. The blood glucose levels were higher in the high-fat diet mice than in the endotoxin infused mice, fasting insulinemia was higher in the high fat and endotoxin mice. Glucose induced insulin was however normal in the endotoxin infused mice and was lower than control in the high-fat mice.
Increased fasting glucose and insulin levels were associated with liver insulin resistance in the endotoxin infused rodents whereas the high-fat animals had whole body but not liver insulin resistance.
Both endotoxin infusion and high-fat diet increased weight, fat, visceral and subcutaneous adipose tissue.
Liver triglycerides were higher in both groups compared to control. The animals were free feeding and the caloric intake was measured the animals on the high-fat diet had increased their energy intake when it was measured.
They measured inflammatory factors TNF alpha interleukin-1 interleukin-2 and plasminogen activator inhibitor one. All of these mediators were increased in the high fat and endotoxin infused mice over the control groups.
Then studied CD14 mutant mice. CD14 acts as one of the receptors for detecting endotoxin. So the new mice did not have this function. The CD14 deficient mice did not show any of the increases in inflammatory markers or markers of the metabolic syndrome including weight and fat gain compared with normal mice when challenged with an endotoxin infusion, further pointing towards the causal effect of endotoxin in obesity and diabetes.
CD 14 deficient mice were then tested with their high-fat diet. They found that they were able to delay but not prevent insulin resistance and increased body weight from the high-fat diet.
the paper concludes that they have demonstrated that relevant amounts of endotoxin can be transported into the blood by dietary intervention and that this increase in endotoxin in circulation was a sufficient mechanism to trigger obesity and diabetes.
The next paper is Changes in Gut Microbiota Control Metabolic Endotoxemia-Induced Inflammation in High-Fat Diet–Induced Obesity and Diabetes in Mice. (2)
This paper used antibiotic treatments to change gut microbiota in rodents in order to show that this microbiota controlled endotoxaemia, inflammation, obesity, and type II diabetes. They were also investigating some of the mechanisms.
The mice were fed a control or a high-fat non-carbohydrate diet, similar to first study. Then the treated some of the mice with antibiotics namely ampicillin and neomycin. The antibiotics removed the differences in the bacterial populations between the animals within the groups. The high-fat diet was shown to reduce some Gram-positive bacteria including lactobacillus and bifidobacterium. They showed that the high-fat diet induced metabolic endotoxaemia was mediated through increased intestinal permeability via a reduction in epithelial tight junction proteins. This was reversed with antibiotics. The antibiotics also reduced the inflammatory markers including interleukin-1 and TNF alpha. Antibiotic treatment also reduced lipid peroxidation which was increased by the high-fat diet.
The antibiotic treatment also improved all the metabolic markers of diabetes and obesity in the high fat diet mice. This included glucose tolerance blood glucose profiles, bodyweight gain, total energy intake, subcutaneous adipose weight, the only parameter was not reversed by antibiotics was the energy intake after high-fat diet which worsened during antibiotic treatment.
They then tested OB OB mice these are mice that tend to have the inflammatory markers normally seen in high-fat diet even when eating a normal rodent chow. In these animals the antibiotic treatment also reduced metabolic endotoxaemia though it did not return to control levels.
The next paper I selected is “An opportunistic pathogen isolated from the gut of an obese human causes obesity in germfree mice” (3).
So this states endotoxin is the only nonbacterial product when subcutaneously infused into mice can induce obesity and insulin resistance via inflammation. This was an experiment taking bacteria that produced endotoxin from a morbidly obese human and transplanting it into the germfree mice, that is mice with no gut flora. In the opening comments they mentioned that the volunteer donor has lost over 50 kg and recovered from hypoglycaemia and hypertension after 23 weeks on a diet of grains Chinese medicinal foods and prebiotic’s. The diet produced in the volunteer a lowering of endotoxin and inflammation.
So they inoculated the germfree mice with a strain of Enterobacter isolated from the volunteers gut, this intervention induced fully developed obesity and insulin resistance on the high-fat diet but not on a normal diet. Germfree mice on a high-fat diet did not develop any of the symptoms of metabolic disease, showing that both gut flora and high fat diet were necessary to create diabetes and obesity. The mice showed higher plasma endotoxin correlating with metabolic disorders after inoculation with the endotoxin producing bacteria.
Inoculation of germfree mice with a strain of bifidobacterium did not induce obesity showing that not any bacteria can produce metabolic disorders. Bifido are Gram-positive bacteria and do not produce endotoxin.
The next paper looks at the mechanisms in rodent models, it is titled “Propensity to high-fat diet-induced obesity in rats is associated with changes in the gut microbiota and gut inflammation” (5).
So this study was using some of the data from previous studies as its base and looking at rodents with two phenotypes, one with a tendency to be obese, and the other with a tendency to resist obesity. It then looked at whether changes in the epithelial function of the gut and the gut microbiota were altered.
They found that the obesity prone rodents had a higher expression of toll -like receptor four activation, toll -like receptor four or TLR four is it shortened to is responsible for activating the innate immune system in response to endotoxin. So higher expression of TLR4 might give a greater inflammatory response to the same level of plasma endotoxin. TLR four is associated with small intestinal inflammation and the decrease in intestinal alkaline phosphatase, this is an enzyme that detoxifies endotoxin.
Intestinal permeability and plasma endotoxin were also increased in the obesity prone rodents. The total bacterial density decreased and the relative proportion of bacteriodales and clostridiales were increased in rodents on a high-fat diet regardless of the obese or lean phenotype. They also found markers of increased appetite in rodents with obesity inducing phenotypes and fed high-fat diet.
The paper discusses the effect of activating toll -like receptor four, its downstream inflammatory events increasing interleukin six and TNF alpha. Interleukin-6 is a mediator of fever which is capable of crossing the blood-brain barrier and initiating the synthesis of prostaglandin E2, its type to be one of the reasons why obese individuals have higher levels of C-reactive protein. TNF alpha is a signal protein involved in systemic inflammation and regulation of immune cells also fear inducing dysregulation of TNF alpha is implicated in Alzheimer’s cancer depression psoriasis and inflammatory bowel disease.
So the study showed that the high-fat diet changes the gut microbiota and then the development of inflammation response to that change is associated with increased appetite and obesity. The increased appetite is interesting because endotoxin is also known as an anorexic agent.
They give little more information on a high-fat diet in this study and it seems only slightly different than the previous ones, the low-fat control diet was 70% carbohydrate 20% protein and 10% fat, the fat breakdown of this diet was 25.1% saturated, monounsaturated 34.7% and 40.2% polyunsaturated.
2.5% sat. 3.5% MUFA and 4% Pufa
The high-fat diet was only 45% fat, the previous experimental diets with 72% and a breakdown of the other macros in the diet were 35% carbohydrate and 20% protein. So the breakdown of the percentages of fat in the high-fat diet were 36.3% saturated, 45.3% monounsaturated and 18.5% polyunsaturated.
16% sat, 20% MUFA and 8.5% PUFA
Okay the next available capital is called “high-fat diet: bacteria interactions promote intestinal inflammation which precedes correlates with obesity and insulin resistance mouse”. (6)
So this study was looking at the idea that the high fat obesity inducing diet and gut bacteria interact to produce intestinal inflammation, which then leads to obesity and insulin resistance. They used conventional mice and germfree mice and the study looked at bodyweight adiposity TNF alpha and NFKB. NFKB is short for nuclear factor kappa light chain enhancer of activated B cells, one of the things involved in is cytokine production and responses to stress of various sources. NFKB dysregulation is correlated with cancer, inflammatory and autoimmune diseases, viral infection and improper immune development. It’s also implicated in memory as is TNF a.
The experimental diets were similar to the last study with 45% from fat and the high-fat diet and 10% in the low-fat control diet. The food given to the germfree mice was subject to radiation was not to keep that germfree . It mentioned that previous studies with conventional mice given irradiated diet gained weight similar to conventional mice on a non-irradiated diet.
Okay so they found that conventional mice on a high-fat diet significantly increased body weight relative to low-fat diet controls, however germfree mice on a high-fat diet versus low-fat diet did not differ in body weight significantly at any time and germfree mice fed either high a low-fat diet had body weights similar to conventional mice fed a low-fat diet, further there was a small increase in fat mass in germfree mice on a high-fat diet after 16 weeks but it was much less than the observed in a high-fat diet conventional mouse. So this study showed that germfree mice are very resistant to weight gain or obesity from obesity high fat diet.
With information they showed that small intestinal TNF alpha is increased with a high-fat diet but only when bacteria are present. They also showed that small intestinal TNF alpha levels are correlated with weight gain and obesity from the high-fat diet. Positive associations are shown with TNF alpha in the small intestinal and insulin resistance from high-fat diet. They tested conventional mice with high fat and low fat diets for levels of small intestinal and colonic levels of NFKB as an independent marker of intestinal inflammation and this was raised in the hi fat diet rodents.
They show that in conventional mice fed high-fat diet the increases in the inflammatory markers preceded the high-fat diet induced weight gain, and suggested the idea that the early problems are effects of the diet could serve as a trigger for subsequent systemic inflammation.
They mention a number of studies that demonstrated TNF alpha limits insulin receptor signalling and increases insulin resistance, and that rodent models with deleted TNF alpha genes were resistant to diet induced obesity and insulin resistance. The text notes that TNF a can precede an increase in NFKB and other inflammatory markers of insulin resistance and type II diabetes. The authors note some references that blocking those pathways can alleviate symptoms of insulin resistance.
Okay so the next paper looks at the permeability of the intestinal in obese mice and the fact on the liver, specifically non-alcoholic steatohepatitis which is often abbreviated to Nash NASH (7).
Nash is the most extreme form of non-alcoholic fatty liver disease and a major cause of cirrhosis. It’s physiologically characterised by excessive fat deposits known as steatosis in the liver and it is when alcohol has been ruled out as the causal factor and according to Wikipedia the cause is unknown.
The markers that the study looked at were on the effect of obesity on intestinal mucosal integrity and portal endotoxin in two strains of mice. the mice were either leptin deficient or hyperleptinemic. Both of those strains of mice have been extensively used as models of obesity due to improper leptin capacity.
So to put it another way they are looking at the permeability of the intestine, which may increase endotoxin in the blood and then the portal venous system is what directs blood from the gastrointestinal tract to the liver. And the study notes the prevalence of small intestinal bacterial overgrowth and increase in TNF a in patients with NASH as well as the fact that SIBO is improved in rat models with antibiotic treatments. Another thing that the paper notes is that endotoxin plays an important part in alcoholic liver damage.
So the results were that both the leptin deficient and hyper leptin type mice had disrupted mucosal barrier function compared to normal mice. They showed that increased permeability was strongly correlated with portal blood endotoxin. They showed again that systemic inflammation was higher in obese mice and blood glucose was significantly higher. they found much lower tight junction integrity in obese mice versus lean. The study also found that endotoxaemia initiated stronger inflammatory phenotype following exposure.
So the next study on the list looks at the effect of gut flora on the browning of adipose and obesity (9). Adipose tissue is a type of fat and there is a subtype called brown adipose tissue which is associated with promoting leanness and improving insulin sensitivity. The paper looked at the effect of depleting flora in rodent models and the changes in the colour of the adipose tissue.
In the study they used the Latin deficient mice and mice fed the high fat diet. So the study showed that microbiota depletion either by antibiotics or in germfree mice stimulated in increase in beige fat which leads to improved glucose tolerance and insulin sensitivity and decreased white fat and adipocyte size in lean mice, obese leptin deficient mice, and high-fat diet fed mice. These results were then reversed by re-colonising the antibiotic treated mice or colonising the germfree mice with microbes.
Okay so the next study I have referenced looks at the effect of the gut microbiota of human twins who have significant differences in body mass and body fat.(10)
In this experiment they selected four groups of human twins, one lean and obese in each set and transplanted the faecal microbiota from humans into previously germfree mice.
They found that their transfer of faecal microbiota to the rodents made changes in body mass and adiposity that were strongly correlated with that of the human donor. They also noted in increase in fermentation short chain fatty acids in lean rodents, increased metabolism of branched-chain amino acids and obese, and increased microbial transformation of bile acid species in lean rodents.
Okay the next study looks at the effect of the flow bacteria on high-fat diet induced diabetes in mice. (11)
The premise of this paper is that high-fat diet increases endotoxaemia and lowers bifido bacterium. Having previously shown the premises they then went on to test whether an increase in the quantity of bifido bacteria could positively affect metabolic endotoxaemia, diabetes and inflammation. It had been previously shown that bifido reduce intestinal endotoxin and increase mucosal barrier function. In order to increase the target bacteria population within the parameters of obesity and insulin resistance the mice were fed a high-fat diet with a prebiotic fibre known to increase bifido, that prebiotic was oligofructose, this is also sometimes called fructoligosaccharide. OFS or FOS
So they found that compared to normal chow fed mice the high-fat diet lowered intestinal bacteria both endotoxin and non-endotoxin producing, including bifido bacteria. The addition of the prebiotic restoring the population of bifido high-fat diet fed mice and found that that group had improved glucose tolerance, glucose induced insulin secretion and normalised inflammation as measured by endotoxaemia and adipose tissue pro-inflammatory cytokines.
Another paper look at similar metrics using the data and assumptions relating to bifido OFS and endotoxaemia (12).
There are the same measures and in the paper it is noted the correlation with liver disease and how the mechanisms may be similar and suggests that in future both metabolic disorders and NAFLD/NASH might be treated as an infectious disease.
So the next study found accumulated iron in the spleen of rodent obesity models and also found that this could be lowered by using hesperedin a flavonoid of citrus fruits (13). This is interesting because iron is implicated in compounding endotoxin damaging a number of ways and also that the flavonoid used protects against endotoxin damage in a number of other ways.
The next study investigates type I diabetes and the implication of gut flora (14).
So this was based on data that had previously been found to implicate the gastrointestinal immune system in the development of type I diabetes. The immune system does not develop properly without intestinal flora and there is evidence that administration of specific antigens early in life suppress later development of diabetes in some rodent models.
So they took a type of rat prone to diabetes known as bio breeding diabetes prone rat, examined their intestinal flora, and created a separate group were treated with antibiotics. This study showed a correlation with Bacteroides and later development of diabetes, that is those with more Bacteroides had a higher amount of diabetes. The antibiotic treatment decreased diabetes and delayed its onset when it did occur. They also found that a combination of antibiotic treatment with a hydrolysed casein diet could completely prevent type I diabetes. The antibiotics were Bactrimel – sulphamethoxazole (1.2 g/l), trimethoprim,and colistine sulphate.
The next is titled bacterial endotoxin stimulates adipose lipolysis via toll -like receptor 4 and extracellular signal-regulated kinase pathway (15).
So this study tells us that the inflammatory response to endotoxin increases circulating levels of free fatty acids and impairs insulin sensitivity, that free fatty acid elevation may be due to the disregulating effect of endotoxin on lipid disposal. They look at the effect of endotoxin on lipolysis and found that endotoxin increases lipolysis in adipose tissues, that it elevates circulating free fatty acid levels, and create insulin resistance.
I want to mention the randle cycle as it may be relevant here. The Randall cycle also known as the glucose fatty acid cycle is the observation of the competition between glucose and fatty acids for uses energy substrate. This has been theorised to play a role in explaining type II diabetes and insulin resistance, elevated free fatty acids are linked with the onset of insulin resistance and increased free fatty acids in circulation will decrease glucose oxidation (5).
The paper points out that in toll -like receptor 4 deficient mice increase plasma endotoxin does not increase lipolysis and does not liberate free fatty acids. It also notes the causal role of free fatty acids in inducing dyslipidemia and insulin resistance.
The next looks at portal endotoxaemia liver inflammation and pancreatic beta cell dysfunction in rodents (16).
Portal endotoxaemia is an increase in endotoxin in the blood from the digestive system to the liver, pancreatic beta cells store and release insulin. They created two groups of rats and gave them either portal saline or endotoxin infusions for four weeks before examining the liver and the pancreatic insulin secretions.
They found an increase in white blood cell count after endotoxin infusion, these are the cells of the immune system that protect against infectious disease. They found an increase in superoxide ,TNF a and interleukin-6 in both the liver and the pancreas. The levels of C-reactive protein did not differ in either group, insulin secretions were significantly decreased in the endotoxin treated rats. Markers of reactive oxygen substrate were significantly increased in the endotoxin infused rats in both organs showing that portal endotoxaemia may be causal in liver inflammation and pancreatic beta cell dysfunction.
The next looks at the effects of high-fat diet again on insulin resistance type II diabetes and low-grade inflammation (17).
in this study they showed that before the onset of diabetes and obesity from the high-fat diet in rodents there was increased mucosal adherents of the bacteria and translocation of the bacteria into the adipose tissue. This pre-seeded diabetes and they showed that the mechanism requires CD14 which I mentioned earlier and NOD1. Both of these are immune system receptors involved in recognising endotoxin.
Note refs are out of sequence and some original references were removed due to repetition.
Metabolic endotoxaemia initiates obesity and insulin resistance
Changes in Gut Microbiota Control Metabolic Endotoxemia-Induced Inflammation in High-Fat Diet–Induced Obesity and Diabetes in Mice
An opportunistic pathogen isolated from the gut of an obese human causes obesity in germfree mice
Propensity to high-fat diet-induced obesity in rats is associated with changes in the gut microbiota and gut inflammation
High-Fat Diet: Bacteria Interactions Promote Intestinal Inflammation Which Precedes and Correlates with Obesity and Insulin Resistance in Mouse
Increased intestinal permeability in obese mice: new evidence in the pathogenesis of nonalcoholic steatohepatitis
Microbiota depletion promotes browning of white adipose tissue and reduces obesity
Gut Microbiota from Twins Discordant for Obesity Modulate Metabolism in Mice
Mice study twins bacteria
Selective increases of bifidobacteria in gut microflora improve high-fat-diet-induced diabetes in mice through a mechanism associated with endotoxaemia
The gut–liver-axis: Endotoxemia, inflammation, insulin resistance and NASH
Mice study liver bacteria bifido
Involvement of splenic iron accumulation in the development of nonalcoholic steatohepatitis in Tsumura Suzuki Obese Diabetes mice
Antibiotic treatment partially protects against type 1 diabetes in the bio-breeding diabetes-prone rat: is the gut flora involved in the development of type 1 diabetes?
Bacterial endotoxin stimulates adipose lipolysis via toll-like receptor 4 and extracellular signal-regulated kinase pathway.
Free fatty acids in obesity and type 2 diabetes: defining their role in the development of insulin resistance and beta-cell dysfunction.
Mild portal endotoxaemia induces subacute hepatic inflammation and pancreatic beta-cell dysfunction in rats.
Intestinal mucosal adherence and translocation of commensal bacteria at the early onset of type 2 diabetes: molecular mechanisms and probiotic treatment