Factors Influencing the Gut Microbiota, Inflammation, and Type 2 Diabetes

Abstract

The gut microbiota is a complex community of bacteria residing in the intestine. Animal models have demonstrated that several factors contribute to and can significantly alter the composition of the gut microbiota, including genetics; the mode of delivery at birth; the method of infant feeding; the use of medications, especially antibiotics; and the diet. There may exist a gut microbiota signature that promotes intestinal inflammation and subsequent systemic low-grade inflammation, which in turn promotes the development of type 2 diabetes. There are preliminary studies that suggest that the consumption of probiotic bacteria such as those found in yogurt and other fermented milk products can beneficially alter the composition of the gut microbiome, which in turn changes the host metabolism. Obesity, insulin resistance, fatty liver disease, and low-grade peripheral inflammation are more prevalent in patients with low α diversity in the gut microbiome than they are in patients with high α diversity. Fermented milk products, such as yogurt, deliver a large number of lactic acid bacteria to the gastrointestinal tract. They may modify the intestinal environment, including inhibiting lipopolysaccharide production and increasing the tight junctions of gut epithelia cells.

Introduction

The gut microbiota is a complex community of bacteria residing in the intestine. The bacterial load in the stomach is significantly lower, and the load increases exponentially through the digestive system from the stomach to the duodenum, the jejunum, and the ileum, and ultimately to the colon, which contains between 10⁹ and 10¹³ bacteria (1). Both animal and human studies have demonstrated that diet can influence the composition and function of the gut microbiome. Other factors, including genetics; the mode of delivery at birth; the method of infant feeding; and the use of medications, especially antibiotics, also contribute to the composition and function of the gut microbiome. Diet plays an important role in obesity, in addition to other factors (2–6). Yogurt, a fermented dairy product containing a variety of probiotic bacteria, is found to be associated with a reduction in inflammation markers and weight loss (7, 8). Studies found that regular yogurt consumption is involved in energy balance and/or energy homeostasis, which in turn controls body weight and reduces the risk of the development of type 2 diabetes (T2D) (9). It is well known that diet, including yogurt consumption, has a direct impact on the gut bacteria; therefore, it is possible that the effect of yogurt on energy balance and body weight control in some studies is mediated by the alteration of the gut microbiota. Despite the fact that there are certain enterotypes in the composition of the human gut microbiome, there is tremendous variability between individuals, and some of these differences are associated with chronic conditions. Dysbiosis is a state in which the homeostasis of the gut microbiome is disrupted, often leading to health problems. One of the causes of dysbiosis is diet, and studies have shown that diet may change the gut microbiota and contribute to obesity and diabetes (10, 11). Over 80% of patients with T2D in the Western world are overweight. Obesity and T2D are characterized by an altered gut microbiota, inflammation, and gut barrier disruption (12–14). Diabetes is a complex disorder that is influenced by a combination of genetic and environmental factors. However, there may exist a gut microbiota signature that promotes intestinal inflammation and subsequent systemic low-grade inflammation, which in turn promotes the development of T2D.

Composition of the Gut Microbiome

A few main phyla of bacteria exist in the gut: Actinobacteria, Bacteroidetes, Firmicutes, Proteobacteria, and Verrucomicrobia. The genes of bacteria in the gut are 150 times larger than that in the human genome, and the mass of bacteria in the body can reach 1.5 kg, or ∼2% of the weight of an average 75-kg person. Studies in germ-free animals have shown that shifts in the composition of the gut microbiome may play an important role in disease development, specifically obesity and diabetes (15–17). There is evidence demonstrating that the composition of the gut microbiota also influences metabolism and can affect energy balance (18), gut permeability (19), and inflammation (20), all of which are associated with obesity and associated disorders, including T2D (14). Gastric bypass surgery, characterized by ∼70% weight loss and an improvement in glucose metabolism, has been recommended by the American Diabetes Association as an effective treatment for obesity and T2D. The rationale of this surgical procedure was originally to cause a restriction in food intake and calorie malabsorption. Studies support the notion that gastric bypass leads to a substantial shift in the gut microbiota, which may contribute to weight loss. Samples from the duodena of bypass patients show a marked difference in the gut microbiota between patients with diabetes and those without, specifically, fewer bacterial strains exist among obese individuals with T2D (21). The change in physiology after bariatric surgery may also promote a different composition in the gut microbiota, but the microbiota alone may not be the primary driver of weight loss after bypass surgery.

The Gut Microbiota, Inflammation, and Diabetes

Accumulating evidence in animal models and in humans shows that obesity and T2D are associated with dysbiosis of the gut microbiota. Given that >80% of patients with T2D are overweight in the West, and that obesity and T2D are characterized by an altered gut microbiota, inflammation, and gut barrier disruption, it is conceivable that the microbiota may play a role in the development of these conditions (14, 22).

The immune system is “educated” and matured by commensal bacteria, especially bacteria in the gut. Germ-free mice have abnormal immune systems, including the absence or underdevelopment of lymph nodes systemically and specifically in gut- associated lymphoid tissues. Homeostasis of the gut microbiota is therefore important in modulation of the host immunity and control of inflammation (23). An altered gut microbiota influenced by different factors (see “Factors that Influence Gut Bacterial Composition” below) can directly affect immune cells in the gut and indirectly affect immune cells via microbial products (24, 25) including LPS (18, 20, 26, 27), metabolites, and SCFAs (28–30), all of which can affect adipogenesis and/or insulin resistance (31). LPS is believed to cause low-grade inflammation mediated by the induction of inflammatory cytokines by immune cells and adipocytes, and acetate or butyrate can modulate immune cell function. Acetate, butyrate, and propionate are end products of the microbial fermentation of macronutrients. These SCFAs can strongly modulate gene expression of human monocytes and reduce proinflammatory cytokine and chemokine production by monocytes (32, 33). They can also promote regulatory T cell generation, thereby suppressing the function of inflammatory T cells (34). Butyrate is able to block IFNγ-inducible protein 10 (IP-10) release in human colonic subepithelial myofibroblasts (35). Thus, these immune regulatory SCFAs act not only on immune cells systemically but also on intestinal tissue cells locally.

With advanced sequencing technology, including metagenome-wide association studies, scientists have discovered significant correlations between specific intestinal bacteria, metabolic pathways, and T2D (36). Among 345 Chinese individuals, butyrate-producing Roseburia intestinalis and Faecalibacterium prausnitzii concentrations were found to be lower in subjects with T2D, whereas those of Lactobacillus gasseri and Streptococcus mutans, Proteobacteria, and certain Clostridiales were higher (36). Butyrate-producing bacteria are able to exert substantial beneficial immunometabolic effects (22), as discussed earlier. In addition, T2D is associated with increased bacterial expression of the genes involved in oxidative stress, creating a proinflammatory signature in the intestinal microbiome (36). A study cohort in European women showed that women with high glycated hemoglobin concentrations were characterized by an increase in Lactobacillales, mainly Streptococcus species, and a decrease in species belonging to Bacteroids, Eubacterium, and Clostridium (37). Although the metagenomic markers of microbiota in Chinese and European cohorts are different, it is clear that the gut microbiota closely correlates to T2D. Therefore, it has been suggested that gut microbial markers might be useful for classifying T2D. Studies also showed that the presence of Akkermansia muciniphila in the gut, which constitutes 3–5% of the gut microbiota (22) inversely correlates with body weight in rodents and humans, although the precise physiologic roles are not fully understood (14). It is also currently unclear where inflammatory processes are initiated, but a significantly altered microbiota in the gastrointestinal tract could be one of the early events in the process (22).

A study of nonobese and obese Danish individuals showed that obesity, insulin resistance, fatty liver, and low-grade inflammation (increased C-reactive protein and leptin concentrations and decreased serum adiponectin concentrations) were more prevalent in patients with low α diversity in the gut microbiome than they were in patients with high α diversity (38).

All of these studies suggest a correlation between the composition of the gut microbiome and disease. However, there are several shortcomings in human studies. The populations studied were heterogeneous in nature, they were not sex matched, and there were no data on diabetes medication that the subjects might have been taking. It is clear that more investigations are needed to better understand the mechanisms of how gut bacteria affect T2D.

Factors that Influence Gut Bacterial Composition

There are several intrinsic and extrinsic factors that can influence the composition of the gut bacteria and ultimately affect health.

Method of delivery at birth.

According to the CDC, as of 2014, 32.2% of all deliveries in the United States are performed by cesarean section (39). The composition of the gut bacterial community is different in infants delivered by cesarean section from that of infants born by vaginal delivery (40, 41). Infants born by vaginal delivery are exposed to the mother’s bacteria at birth, which influences the infant’s gut bacteria and stimulates white blood cells and other components of the immune system (42). Studies have suggested that infants born by cesarean section are at greater risk of developing obesity and/or diabetes than those born vaginally (43–47). In a recent cross-sectional study of 8900 preschool children, the authors found that the odds of overweight were 1.35 and of obesity were 1.25 in children delivered by cesarean section (48). A similar study in a small cohort also showed that the prevalence rates of overweight and obesity were 15.6% and 12.9%, respectively, in 672 preschool children who were born by cesarean section (46). However, opposite findings are also reported (49, 50). Although more studies appear to support the association, many factors can influence the outcomes of these studies, including study population, sex of the offspring, and body weight of the mother.

Infant feeding.

Infant feeding is another important factor for establishing the bacterial community in the gut, because the mother’s milk is not sterile (51). Human breast milk has been recognized as a source of commensal and potential probiotic bacteria that influence the development of infant gut bacteria (52). Human breast milk contains >700 species of bacteria (53). Although human milk bacterial communities are generally complex and vary individually, the median bacterial load is ∼10⁶ bacterial cells/mL through time (54). Thus, it has been estimated that a lactating infant consuming 800 mL breast milk/d could ingest up to 8 × 10⁸ bacterial cells daily, which is ∼100 times higher than previous estimates, and the composition changes over the course of lactation. It appears that Streptococci and Staphylococci are predominant bacterial genera in human milk (51); both of these are also predominant in the skin microbiota. Therefore, human milk may also contain some skin bacteria. However, Weissella, Leuconostoc, Staphylococcus, Streptococcus, and Lactococcus are predominant in colostrum samples of infants, whereas in milk taken at 1 and 6 mo, Veillonella, Leptotrichia, and Prevotella increased significantly (52). Evidence suggests that the transfer of microbiota from mothers to their infants affect infant growth and development (55, 56). Milk from obese mothers has been found to contain different, less-diverse bacteria than milk from normal-weight mothers. Milk from obese mothers also showed more proinflammatory properties (56). In addition, breast milk from mothers who underwent cesarean section contained bacteria that was different from milk samples from mothers who had vaginal deliveries (53). The bacteria present in breast milk, as well as those on the mother’s skin, are among the first microbes to enter the infant’s body, and they could play an important role in health (53). Breast milk is also a rich source of IgA antibodies against different pathogens (57–59). However, some infant formulas containing probiotics have been shown to beneficially affect the infant immune systems and the gut microbiota.

Genetics.

The number of specific bacteria found in the gut microbiota is influenced in part by the genetic makeup of the host in ways that affect host metabolism and ultimately can affect health (15). Family members have been found to have more similar microbiota communities than unrelated individuals, and the gut microbiota is more similar in monozygotic than in dizygotic twins (15). However, there are currently no genome-wide studies that have characterized specific genes and pathways that determine the composition of the gut microbiome (60), although certain genes in the immune system are associated with inflammatory bowel disease (61, 62).

Infections.

Although the gut microbiota affects viral and bacterial infections, the reverse is also true (63–68). One study investigated the effect of an enteropathogenic infection with Citrobacter rodentium on the microbiota of mice and found that certain bacterial groups in the gut are altered in response to C. rodentium infection, including a reduction in the relative abundance of Lactobacillus (69). A human study of Clostridium difficile patients and asymptomatic carriers with the use of 16S ribosomal RNA gene pyrosequencing found that both had reduced microbial richness and diversity compared with healthy subjects (70). C. difficile infection is a typical result of severe dysbiosis in the gut microbiota (71, 72). Interestingly, transplantation of the gut microbiome from healthy donors to infected patients increased microbial richness and diversity, and it is currently applied clinically (73–76). These studies demonstrated that the characterization and diversity of the gut microbiota are altered with bacterial infections. While using a mouse model of hepatitis B virus infection, Chou et al. (63) showed that the clearance of hepatitis B virus infection requires the establishment of the gut microbiota. It is evident that the shift in the host gut microbiota affects both pathogenesis and clearance of bacterial and viral infections.

Medications.

Increasing evidence suggests that many nonantibiotic drugs have an impact on the gut microbiota (77–79), including the drugs used to treat T2D (80). Likewise, the gut microbiota also affects the efficacy of drugs (81, 82). Antibiotics are commonly prescribed drugs that have saved millions of lives from infections; antibiotics also have a profound effect on the normal gut microbiota. The effect is rapid and sometimes persistent. Broad-spectrum antibiotics reduce bacterial diversity while increasing the abundance of some bacteria that can be used by opportunistic pathogens and decreasing the number of beneficial bacteria (83). The use of broad-spectrum antibiotics, such as clindamycin, in infants and young children has been found to have the longest-lasting effects on the composition of the gut microbiota (84–86). Early antibiotic exposure in neonates can lead to microbial dysbiosis, which may be a predisposing factor to inflammatory bowel disease (87). There also appears to be an interaction between antibiotic administration and diet. Studies in both mice and humans have found that the use of antibiotics early in life could promote obesity later in life, mediated by the alteration of the gut microbiota (88–90). However, there are limitations in those studies. The retrospective nature, the diversity of diet, and the heterogeneous (or not heterogeneous) population are the inevitable limitations in human studies. Most mouse studies on obesity induced by a high-fat diet with or without antibiotic treatment used only male mice because they gain more weight than female mice, whereas there is no obvious sex bias in human obesity. A recent study showed that antibiotics altered the host’s gut microbiota without changing the metabolism of the hosts (91, 92). Other studies demonstrated that antibiotics reduce body weight and increase insulin sensitivity (93, 94). Berberine, the main component of a Chinese herb extract used to treat bacterial diarrhea, also has an antidiabetic effect by modulating the gut microbiota and lowering glucose and insulin resistance (95, 96). Thus, it is not clear whether antibiotics are to be blamed for the sharp rise in obesity, especially childhood obesity. It is clear, however, that the use of antibiotics has a profound effect on the alteration in the gut microbiota.

Metformin is routinely used to help with control of hyperglycemia in T2D. The drug increases the insulin sensitivity of body cells, especially fat cells, muscle cells, and hepatocytes. Metformin also prevents the overproduction of glucose by hepatocytes. Furthermore, metformin delays glucose absorption during digestion after a meal. Interestingly, recent studies have found that the administration of metformin alters the composition of the microbiota (97–99). In a study in which obesity was induced in mice by feeding a high-fat diet, the authors found that metformin led to a greater abundance of the mucin-degrading bacterium Akkermansia in the obese mice than in their obese counterparts not given metformin (98). A recent human study confirmed the effect of metformin on the gut microbiota (80). It is possible that an altered gut microbiota may be the cause of the drug’s common side effect in the digestive system, and the altered gut microbiota is also likely to influence the drug’s antidiabetic efficacy.

Diet.

The role that food-ingested bacteria play in the gut microbiome had been underestimated in the past, possibly because of methodologic limitations that have been overcome in recent years (100). Numerous studies, both in research mice and in humans, have shown that high-calorie diets contribute to obesity and T2D (101–105). However, increasing evidence suggests that the link between diet and obesity lies in the gut microbiota (106–112). Understanding that diet is an important contributing factor to the composition of the gut microbiome makes it the most logical target to manipulate. Interventional studies show that dietary changes result in substantial and rapid changes in the make-up of the gut microbiome (10, 113). Studies in mice have demonstrated that a high-fat diet (60% fat) decreases the number of bacterial species (α diversity) in the gut microbiome, and the composition of the gut microbiome between mice given a high-fat diet (unpurified) and those given a regular unpurified diet is very different (β diversity). One study in mice found that the abundance of A. muciniphila decreased in obese mice and those with type 2 diabetes and that prebiotic feeding of A. muciniphila normalized its abundance and improved metabolic profiles (14). Treatment with A. muciniphila also reduced fat mass, inflammation, and insulin resistance induced by a high-fat diet (14). A fiber-rich diet has been shown to be beneficial to health because it modulates the gut microbiome (114).

Studies in humans by 16S ribosomal RNA sequencing have characterized the human gut microbiota into different enterotypes distinguished by the types of bacteria present (115). Enterotypes were strongly associated with long-term diets, particularly those with protein and animal fat. Wu et al. (10) showed that protein and animal fat were associated with Bacteroides, whereas carbohydrates were associated with Prevotella. In that study, the authors also investigated controlled feeding in 10 subjects and found that the microbiome composition changed within 24 h of initiating a high-fat and low-fiber or low-fat and high-fiber diet, and remained stable during the 10-d study (10). The results suggest that diet is particularly strongly associated with enterotype partitioning. The issue then becomes whether the effects of a Western-type diet on gut microbiota composition is associated with a higher or lower incidence of disease and whether long-term dietary interventions can create stable alterations in the bacterial enterotype.

In another diet and enterotype study that involved 6 male and 4 female volunteers between the ages of 21 and 33 y with a BMI (in kg/m²) ranging from 19 to 32, the study subjects consumed either a plant-based diet rich in grains, legumes, fruits, and vegetables, or an animal-based diet composed of meat, eggs, and cheese. Each diet was consumed ad libitum for 5 consecutive days, and the subects’ fecal samples were cultured or directly analyzed by 16S ribosomal RNA gene sequencing (113). The authors showed that microbiota changes in the animal-based diet, which was high in fat, were hypothetically linked to altered fecal bile acid profiles and the growth of microorganisms capable of triggering inflammatory bowel disease (113). The results showed that a high-fat diet can alter gut bacteria and lead to dysbiosis and ultimately disease.

Yogurt and the gut microbiome.

A body of evidence suggests that the gut microbiota plays an important role in obesity and chronic inflammatory disorders. Obesity and/or T2D have been considered to be chronic inflammatory disorders. As discussed earlier, the Western diet is an important contributor to obesity and T2D. Different from consumption of a high-fat diet, yogurt consumption is believed to be beneficial to health, and the mechanism of that is possibly mediated by altering the host gut microbiota in addition to other nutritional factors. Elie Metchnikoff theorized over a century ago that health could be improved by manipulating the intestinal microbiome with bacteria found in yogurt (116). Few studies had been conducted to examine the effect of yogurt consumption and the composition of the gut microbiota for decades. However, with advanced technology, especially high-throughput sequencing technology and adequate bioinformatical computation, there has been a sharp rise in the number of studies in the last 8 y or so, including clinical trials, that have investigated the effect of yogurt consumption on the change in the gut microbiota and its benefits on health (117–120). Many fermented foods, and yogurt in particular, contain up to 10⁹ CFU live bacteria/g yogurt (100). Yogurt is fermented with a combination of Lactobacillus delbrueckii subspecies bulgaricus and Streptococcus salivarius subspecies thermophiles. Other bacteria, mainly Lactobacilli or Bifidobacteria, are often added to yogurt as well. In a phase-I clinical trial, Merenstein et al. (117) showed that the consumption of yogurt containing Bifidobacterium lactis activated an array of immune genes in the immune cells from the peripheral blood of study subjects. It is interesting that both anti-inflammatory and inflammatory genes were activated, including genes that regulate interferons and T cell differentiation. It is also interesting that there were no major changes in the composition of the gut microbiota in the subjects in the 10-d trial period (117). However, the consumption of yogurt that contained Lactobacilli (6 × 10⁷ – 2.4 × 10⁸/g yogurt) in a Japanese study showed a significant effect on alteration in the gut microbiota (120). Fermented milk products, such as yogurt, deliver a large number of lactic acid bacteria to the gastrointestinal tract. Lactic acid bacteria may modify the intestinal environment by increasing tight junctions in the gut epithelium (121) and by decreasing potentially harmful enzymes produced by the resident bacteria (122). Olivares et al. (123) reported that dietary deprivation of fermented foods caused a fall in innate immune response, including decreased phagocytosis by leukocytes and decreased immune response against infections, whereas lactic acid bacteria can reverse the impaired immune response. Childhood obesity has been a critical public health issue for at least the past 20 y. Marette and Picard-Deland (124) recently reviewed yogurt consumption and its impact on childhood obesity and cardiometabolic disease risk based on the outcome of different clinical trials. The authors concluded that yogurt not only provided important nutrients, but was also beneficial to the regulation of body weight and cardiovascular health in the children and adolescents studied. The beneficial effect was in part contributed by the change in the gut microbiota. Studies have shown that patients with obesity and/or T2D have elevated endotoxin, such as LPS, in the circulation, which in turn activates immune cells and promotes inflammation. The increase in LPS is likely due to the change in the gut microbiota in obesity. Odamaki et al. (119) studied 420 healthy subjects and found that the consumption of yogurt containing Bifidobacterium longum BB536 significantly decreases enterotoxigenic Bacteroides fragilis in the gut microbiota. Although this study was not designed for investigating obesity and inflammation, the results support the notion that the consumption of certain types of yogurt suppresses endotoxin-producing gut bacteria, which may ameliorate endotoxinemia and inflammation. The consumption of yogurt containing Bifidobacterium animalis subspecies lactis BB-12 also led to a reduction in inflammatory cytokines and the suppression of Toll-like receptor 2 (TLR2) expression in immune cells (125). However, not all yogurt consumption has beneficial effects. Some yogurts have no effect on the gut microbiota and/or weight (126). Moreover, long-term yogurt consumption in immune-deficient patients can cause fatal Lactobacillus rhamnosus septicemia (127). Although yogurt consumption, in most cases, has beneficial effects on the health of hosts, we need to be careful in applying this popular biotherapy in different clinical settings.

Conclusion

Jean Anthelme Brillat-Savarin, a French lawyer and politician from the 18th century, is credited with saying, “Tell me what you eat, and I will tell you what you are” (128). Today, this statement has been contracted to, “You are what you eat.” The statement is particularly applicable to the gut microbiome. Numerous studies suggest that a high-fat diet can lead to gut microbiota dysbiosis, which contributes to increased gut permeability and metabolic endotoxemia. This in turn contributes to low-grade inflammation and insulin resistance and, ultimately, obesity, diabetes, and other metabolic disorders. Although not conclusive, research strongly suggests that the consumption of probiotic bacteria such as those found in yogurt and other fermented milk products can beneficially alter the composition of the gut microbiome. The unanswered question is whether a gut signature exists that promotes intestinal inflammation, low-grade systemic inflammation, and T2D, and what the most effective methods would be, including dietary changes, to alter the gut microbiome to one that is conducive to disease prevention.