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Review Article

Targeting gut microbiota as a possible therapy for diabetes Canxia He, Yujuan Shan⁎, Wei Song School of Food Science and Engineering, Harbin Institute of Technology, Harbin 150090, China

ARTI CLE I NFO

A BS TRACT

Article history:

The incidence of diabetes has increased rapidly across the entire world in the last 2 decades.

Received 26 August 2014

Accumulating evidence suggests that gut microbiota contribute to the pathogenesis of

Revised 26 February 2015

diabetes. Several studies have demonstrated that patients with diabetes are characterized

Accepted 9 March 2015

by a moderate degree of gut microbial dysbiosis. However, there are still substantial controversies regarding altered composition of the gut microbiota and the underlying

Keywords:

mechanisms by which gut microbiota interact with the body’s metabolism. The purpose of

Gut microbiota

this review is to define the association between gut microbiota and diabetes. In doing so an

Diabetes

electronic search of studies published in English from January 2004 to the November 2014 in

Metabolism

the National Library of Medicine, including the original studies that addressed the effects of

Inflammation

gut microbiota on diabetes, energy metabolism, inflammation, the immune system, gut

Fecal transplant

permeability and insulin resistance, was performed. Herein, we discuss the possible mechanisms by which the gut microbiota are involved in the development of diabetes, including energy metabolism, inflammation, the innate immune system, and the bowel function of the intestinal barrier. The compositional changes in the gut microbiota in type 2 and type 1 diabetes are also discussed. Moreover, we introduce the new findings of fecal transplantation, and use of probiotics and prebiotics as new treatment strategies for diabetes. Future research should be focused on defining the primary species of the gut microbiota and their exact roles in diabetes, potentially increasing the possibility of fecal transplants as a therapeutic strategy for diabetes. © 2015 Elsevier Inc. All rights reserved.

1.

Introduction

Diabetes mellitus (DM) is becoming a common problem across the entire world. According to the latest information from the International Diabetes Federation in 2013, there are 382 million people now living with diabetes. And this number will rocket to 592 million by 2035 [1]. DM had caused 5.1 million deaths and

cost 548 billion USD in healthcare expenses at the end of 2013 [1]. In 2008, a national survey in China revealed that 92.4 million adults had DM, and 148.2 million adults had pre-diabetes [2]. This high prevalence of DM in China might cause more serious diabetes-related burdens than in any other country. (See Table.) It is known that both genetic and environmental factors contribute to the pathogenesis of DM, particularly type 2

Abbreviations: Acc, acetyl-CoA carboxylase; AMPK, adenosine 5′-monophosphate (AMP)–activated protein kinase; GF, germ-free; GLP, glucagon-like peptide; GPR, G-protein coupled receptor; HbA1c, hemoglobin A1c; IL, interleukin; NOD, non-obese diabetic; SCFA, short chain fatty acid; SREBP-1c, sterol response element binding protein 1c; TLR, Toll-like receptor. ⁎ Corresponding author. Tel.: +86 451 86282021; fax: +86 451 86282906. E-mail address: [email protected] (Y. Shan). http://dx.doi.org/10.1016/j.nutres.2015.03.002 0271-5317/© 2015 Elsevier Inc. All rights reserved.

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Table – Changes in the gut microbiota associated with diabetes Disease

Models

Implicated microbiota

Function changes with DM

Reference

T2D

18 Male patients and 18 controls 345 Chinese patients and controls

Firmicutes↓ Clostridia↓

Associated with plasma glucose positively and significantly. Associated with membrane transport of sugars, branched-chain amino acid transport, methane metabolism, xenobiotics degradation and metabolism, sulphate reduction. Associated with fasting glucose and HbA1c,insulin, plasma triglycerides, adiponectin and HDL.

12

T2D

T2D

145 European women

T2D

50 Japanese

T1D

4 Matched case–control in Finland 16 White children

T1D

Clostridiales sp. SS3/4↓ Eubacterium rectale↓ Faecalibacterum prausnitzii↓ Roseburia intestinalis↓ Roseburia inulinivorans↓ Bacteroides caccae↑ Clostridium↑ Akkermansia muciniphila↑ Desulfovibrio sp. 3_1_syn3↑ Lactobacillus↑ Clostridium↓

Clostridium coccoides↓ Atopobium↓ Prevotella↓ Lactobacillus↑ Firmicutes↓ Bacteroidetes↑ Clostridium↑ Bacteroides↑ Veillonella↑ Lactobacillus↓ Bifidobacterium↓ Blautia coccoides/Eubacterium rectale↓ Prevotella↓

diabetes (T2D) [3–5]. Recently, studies revealed that the gut microbiota function as an important environmental factor in the development of DM [6,7]. The human gut hosts trillions of microorganisms, including more than 1014 bacteria belonging to 1000 species [8]. The genome size of this microbial organ, collectively termed the microbiome, exceeds the size of the human nuclear genome by 100-fold and provides humans with additional biological and metabolic functions for maintaining homeostasis in the body [9]. Numerous studies have shown that the composition of the gut microbiota is altered in diabetic groups. However, there are no consistent results regarding which species are altered in diabetic patients. Moreover, the detailed mechanisms linking the gut microbiota to diabetes have not been well described. Therefore, we conducted an electronic search of English articles from 2004 to 2014 in Medline to clarify the possible relationship between gut microbiota and diabetes. The MeSH search terms used were gut microbiota, diabetes, metabolic diseases, energy metabolism, immune system, inflammation, gut permeability, and fecal transplant. In this review, we present data on the changes of the gut microbiota both in type 1 diabetes (T1D) and in T2D, and summarized the possible mechanisms through which the gut microbiota interact with diabetes. Finally, we discussed the very recent research regarding the effects of dietary modulation and fecal transplantation of intestinal microbiota as treatment strategies for diabetes. All of these compelling lines of evidence strongly suggest that gut microbiota might play a significant role in the development and treatment for diabetes.

2.

Gut microorganisms and DM

The gut microbiota in our body is part of a dynamic ecosystem, and its composition is altered at the phylum and class levels by environmental and host factors which jointly influence the gut and far removed organs. Thus, the gut microbiota is linked to several human diseases, such as obesity and diabetes [10,11]. Recently, numerous studies indicated a relationship between

13

14

15 16 Associated with the plasma glucose level and HbA1c level.

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the gut microbiota and T2D. In 2010, Larsen et al reported that the ratios of Firmicutes to Clostridia species were attenuated in T2D patients [12]. The ratio of Bacteroidetes to Firmicutes and the ratio of the Bacteroides-Prevotella group to the C coccoides-E. rectale group were positively and significantly correlated with plasma glucose concentrations [12]. Qin et al found that Chinese T2D patients exhibited a decline in butyrate-producing bacteria (eg, Clostridiales sp. SS3/4, Eubacterium rectale, Faecalibacterium prausnitzii, etc.) and an increase in several opportunistic pathogens (eg, Bacteroides caccae, Clostridium hathewayi, C ramosum, C symbiosum and others) [13]. Moreover, the mucin-degrading species Akkermansia muciniphila and sulfate-reducing species Desulfovibrio sp. 3_1_syn3 were more abundant in T2D samples [13]. Although it is important to characterize the link between gut microbiota and T2D, there are still some shortcomings that should be noted. For example, the entire gut bacteria population was not classified by age, gender, or drug treatment of subjects to minimize the sources of variation. Research was completed in Europe that examined the composition and function of gut microbiota in a well-characterized population of 70-year-old women [14]. In this T2D group, the abundances of 4 Lactobacillus species were increased, whereas the abundances of 5 Clostridium species were decreased compared to individuals without diabetes. Lactobacillus species were positively correlated with fasting glucose and glycosylated hemoglobin A1c (HbA1c). However, Clostridium species were negatively correlated to fasting glucose, HbA1c, insulin and plasma triglycerides [14]. In an cohort of Japanese T2D patients, the numbers of Clostridium coccoides, Atopobium, and Prevotella were decreased, while the quantities of total Lactobacillus were increased compared to those that were not diabetic [15]. This high Lactobacillus level might reflect the original numbers of bacteria in T2D patients because no significant differences were found between the participants who consumed yogurt and those who did not. Currently, the reason for the high counts of Lactobacillus in T2D patients remains unclear. Although these independent studies revealed an association between T2D and the gut microbiota, some other discrepancies should not be ignored. For example, neither

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Akkermansia in European women nor Lactobacillus in Chinese people contribute to the compositions of gut microbiota in T2D patients. Altered compositions of gut microbiota have also been observed in T1D patients. In a 4-matched case–control study in Finland, the gut microbiota differed between the children who were healthy and those with autoimmune disorders. The striking differences included a reduced Firmicutes level and an increased Bacteroidetes level in the children with autoimmune disorders. Moreover, the high ratio of Firmicutes to Bacteroidetes might be an early diagnostic marker of pending autoimmunity problems such as T1D [16]. However, in another case–control study that included 16 Caucasian T1D children, the numbers of Clostridium, Bacteroides and Veillonella were all significantly increased, while the numbers of Lactobacillus, Bifidobacterium, Blautia coccoides/ Eubacterium rectale and Prevotella were all obviously decreased compared to normal subjects [17]. Interestingly, this study demonstrated that the decreased number of Bifidobacterium and Lactobacillus, as well as the decreased ratio of Firmicutes to Bacteroidetes were negatively and significantly associated with the plasma glucose level, whereas the increased numbers of Clostridium were positively and significantly linked to a higher level of HbA1c in the T1D group, which is completely contradictory to the findings in T2D patients [14,17]. These results suggest that there is some degree of gut microbial dysbiosis in diabetes, although there are differences in the altered species. These differences might be attributable to the different geographical locations, ages or gender makeup of the populations or different food habits and analysis methods used. Moreover, there are still some questions that remain unresolved regarding the association between gut microbiota and diabetes. For example, whether the alterations in the gut microbiota in diabetes are the causes or the consequences of the diabetic pathology. Hence, the interactions between diabetes and gut microbiota are more intricate than what we previously believed. Further studies are required to investigate the roles of gut microbiota in diabetes.

3. Mechanisms by which Gut microbiota are associated with DM 3.1.

The role of gut microbiota in energy metabolism

Gut microbiota hydrolyze and ferment the dietary polysaccharides to generate monosaccharides and short chain fatty acids (SCFAs) that can be absorbed and utilized for energy by the host. SCFAs, such as acetate, propionate, and butyrate, contribute approximately 5% to 10% to human energy resources [18]. The signaling actions of SCFAs are mediated by endogenous ligands of G protein-coupled receptor 41 (GPR41) and GPR43, which are particularly expressed in adipocytes and identified as receptors of fatty acids [19,20]. Acetate preferentially activates GPR43 in vitro, butyrate displays a potent effect on GPR41, and propionate is selective for both GPR41 and GPR43 [21]. Importantly, the energy-regulating effects of GPR41 and GPR43 are both microbiota-dependent. There are significant differences in body weight between Gpr41/43-deficient mice and their counterparts among conventionally raised or gut microbialcolonized mice. However, no evident differences were observed

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between germ-free (GF) conditions and following treatment with antibiotics [22,23]. Diabetes, particularly T2D, is associated with a cluster of interrelated lipid metabolism abnormalities [24]. Compared with GF mice, the levels of triglyceride in conventionalized mice were increased in adipose tissues and liver, while decreased in serum, suggesting the role of gut microbiota in lipid metabolism [25,26]. The process in which fatty acids are released from triglyceride-rich lipoproteins to the muscle and heart is mediated by fasting-induced adipose factor [26,27]. The gut microbiota also dramatically increases the synthesis of hepatic triglycerides. Carbohydrate response element binding protein (ChREBP) and sterol response element binding protein 1c (SREBP-1c) are both involved in lipogenesis due to their independent effects on increasing glucose absorption and insulin levels [19,28]. Unlike GF mice, the ChREBP and SREBP-1c mRNAs are increased in mice that have been orally gavaged with Bacteroides thetaiotaomicron strain VPI-5482 [29]. Also acetyl-CoA carboxylase and fatty acid synthase, 2 critical transcriptional targets of ChREBP and SREBP-1c are significantly elevated in the liver of conventionalized mice [29]. The increased transactivations of ChREBP, SREBP-1c, acetyl-CoA carboxylase, and fatty acid synthase are accompanied by a statistically significant increase in liver triglyceride contents [29]. Additionally, the adenosine 5′-monophosphate–activated protein kinase (AMPK) is another key factor in the regulation of lipid metabolism and insulin sensitivity [30]. In C57BL/6 J mice with deficiencies of the gut microbiota, high fat diet-induced obesity was prevented by enhancing the AMPK-mediated fatty acid oxidation in the peripheral tissues [27]. Moreover, acetylCoA carboxylase and carnitine palmitoyl transferase-1, the downstream targets of phosphor-AMPK in fatty acid oxidation, are both statistically increased [27].

3.2. Metabolic endotoxemia involved in the low-grade inflammation of DM Current views suggest that low-grade chronic systemic inflammation contributes to the development of insulin resistance, diabetes, and obesity [31,32]. The increased circulating concentration of plasma lipopolysaccharide (LPS), which is defined as metabolic endotoxemia, is a trigger factor for the maintenance of a low-tone continuous inflammatory state in the host responding to high-fat diets [33,34]. The decreased cecal contents of Bifidobacterium spp upon high-fat diets are significantly and negatively correlated with high portal plasma levels of LPS [35]. The results from a population-based cohort demonstrated that LPS is significantly associated with increased risks for the incidence and prevalence of diabetes [36]. Moreover, a broad-spectrum antibiotic treatment (ampicillin and neomycin) dramatically reduces the metabolic endotoxemia and cecal contents of LPS in ob/ob mice, as well as the glucose intolerance, inflammation and body weight [33]. Additionally, antibiotic treatment significantly reduces the numbers of Lactobacillus spp, Bifidobacterium spp, and Bacteroides-Prevotella spp in ob/ob mice [34]. Accumulating in vitro and in vivo evidence suggests that these inflammatory responses initiated by LPS in the host are mediated through Toll-like receptor (TLR)-2/-4–related pathways [37–39]. The Toll-like receptor-4/cluster of differentiation-14

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(CD14)/myeloid differentiation-2 pathway is induced by LPS in response to high-fat diets and mediates the activation of transcription factor nuclear factor κB, which ultimately results in the secretion of pro-inflammatory cytokines such as tumor necrosis factor-α, interleukin-1 (IL-1) and IL-6 [33,40]. Moreover, TLR4 gene silencing with siRNA or pharmacologic blockade suppresses the inflammation and insulin resistance triggered by LPS [37,41]. TLR-2 recognizes the components of bacterial cell walls and lipid-containing molecules and then transduces inflammatory signaling by activating nuclear factor κB and producing pro-inflammatory cytokines in cells [42]. Mice lacking TLR-2 exhibit an increased insulin sensitivity and a faster clearance of glucose that are accompanied by attenuated expression of inflammatory cytokines [42–44].

3.3. Gut microbiota and gut permeability: a novel insight into diabetes Recent studies suggested that an altered bowel function of the intestinal barrier contributes to the pathogenesis of metabolic diseases such as diabetes [45,46]. The disruption of the intestinal barrier in genetically obese mice enhances intestinal mucosa permeability, leads to a profound and consistent leakage of LPS into the portal blood circulation, which in turn increases metabolic endotoxemia and inflammatory cytokine levels [47]. After feeding mice with high-fat diets, the expressions of the tight junction proteins zonula occludens-1 and occludin are significantly reduced, resulting in a obvious increase in intestinal permeability and metabolic endotoxemia, inflammation and metabolic disorders [34]. A further study showed that the greater production of endogenous glucagonlike peptide-2 (GLP-2) is associated with improved tight junctions. GLP-2 is an intestinal meal-stimulated peptide hormone that mediates the cleavage of proglucagon in the intestinal endocrine L cells [48]. Prebiotic or GLP-2 pharmacological treatment induces GLP-2 production and improves tight junctions, which ultimately decreases LPS levels in the plasma and blunts the inflammatory state of ob/ob mice [49].

3.4.

The immune system and diabetes

Recent studies have suggested that there is an interaction between the intestinal microbiota and the immune system in diabetes. T1D, a well-known autoimmune disease that is characterized by lymphocytic infiltration or inflammation in pancreatic islets, that is usually associated with infiltration of innate immune cells. The cytokines produced by innate immune cells in the gut could destroy β-cells through promoting β-cells apoptosis and islet-specific T cells infiltration, suggesting the linkage between intestinal microbiota and immune system in T1D [50,51]. After treatment with a cocktail of antibiotics (metronidazole, neomycin and polymyxin), non-obese diabetic (NOD) pregnant mice exhibit significant alterations in the peripheral composition of the T cell compartment and alterations in their offspring including a higher frequency of cluster of differentiation (CD3+CD8+) T cells in the mesenteric lymph nodes. Additionally, lymphocytic infiltration into the pancreatic islets is slightly lower in the offspring of NOD mice, and there are changes in the clusters of gut microbiota [52]. Another result revealed that treatment with vancomycin

protects NOD mice from T1D in the early postnatal period and increases the cluster of differentiation CD 4+ T cells. Moreover, most of the gram-positive and -negative microbes are depleted with the exception of Akkermansia muciniphila [53]. The increased population of Akkermansia muciniphila induces Foxp3 regulatory T cells in visceral adipose tissues and significantly enhances glucose tolerance, and these effects suggest that the modulation of the immune system by Akkermansia muciniphila might be a potential treatment for diabetes [54]. T2D has been traditionally known as a solely metabolic disease, however, recent studies have suggested the association between immune system and the pathogenesis of T2D. The obesity-related chronic inflammation and β-cells stress induced by gluco-and lipotoxicity could activate both innate and adaptive immunity in T2D [55]. The local or generalized immune responses stimulate TLRs and nucleotide-binding oligomerization domain (NOD) receptors, and promote the production of cytokines such as IL-1β which destroy β-cells [55–57]. But more detailed effects of gut microbiota and the immune system on diabetes warrant further studies. The innate immune system could be activated by TLR, which induces dendritic cell maturation and inflammatory cytokine secretion, also generates favorable conditions for the activation of naïve T-cells [58]. TLR-5, a component of innate immune system that was expressed in intestinal mucosa, plays an important role in the development of metabolic syndromes [58]. TLR-5–deficient mice exhibit hyperphagia and develop metabolic diseases such as hyperlipidemia, hypertension, insulin resistance and obesity. After transplantation of the gut microbiota from TLR5-deficient mice to their wide-type GF counterparts, the increased levels of proinflammatory cytokines and features of metabolic diseases, such as insulin resistance and obesity, have been observed in the recipients [59]. The elevated inflammatory mediators in diabetes induce oxidative and endoplasmic reticulum stress in pancreatic islet β-cells, which then influences insulin sensitivity and glucose homeostasis [60]. These data suggest that the interaction between the innate immune system and the intestinal microbiota collectively contributes to the development of metabolic syndrome. However, the underlying mechanisms remain poorly understood and no consistent results have been concluded. For example, 2 recent studies revealed that deficiency of TLR5 in mice has no relationship with changes in the composition of the gut microbiota and metabolic syndromes such as obesity and intestinal inflammation [61,62].

4. Novel therapeutic strategies targeting gut microbiota for diabetes Experimental and clinical studies have shown that targeting gut microbiota might be an effective strategy to prevent and manage diabetes [63,64]. Diet is one of the main factors that influences the composition of gut microbiota. Prebiotic, defined as a nonviable food component that confers a health benefit to modulate the composition of gut microbiota in the host, are mainly inulin, fructo-oligosaccharides and galactooligosaccharides and lactulose [65]. The therapeutic effects of prebiotics on metabolic diseases have been confirmed by a

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clinical trial. Six obese volunteers with T2D and/or hypertension were fed on a strict vegetarian diet for one month, the metabolic parameters such as body weight, the levels of triglycerides and HbA1c were significantly reduced, and the levels of fasting glucose and postprandial glucose were also improved. Such a strict vegetarian diet led to compositional changes of the gut microbiota, for example, a reduced ratio of Firmicutes to Bacteroidetes and an increase in the species of Bacteroides fragilis and Clostridium, which decreased intestinal inflammation and SCFA levels [66]. Another area of therapeutic interest is probiotics, which are the live microorganisms either in the form of food or supplement. After consuming probiotic yogurt (Lactobacillus acidophilus La5 and Bifidobacterium lactis Bb12) for 6 weeks at the dose of 300 g/d, fasting blood glucose and HbA1c were significantly decreased in T2D patients. The antioxidant levels such as the serum malondialdehyde were also significantly decreased. In mice, the antidiabetic effect of probiotics was demonstrated by feeding with probiotics containing Lactobacillus acidophilus and Lactobacillus casei [67]. Recently, fecal transplant as a therapeutic strategy has attracted more attention. Vrieze et al investigated the effects of transferring the intestinal microbiota from lean subjects to male recipients with metabolic syndrome [68]. Six weeks after the infusion of the gut microbiota, the insulin sensitivity of the recipients and the levels of butyrate-producing intestinal microbiota were both significantly increased [68]. The effects of the transfer of gut microbiota on diabetes in mice were subsequently further discussed. After the transfer of bacteria

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from MyD88-deficient NOD mice, insulitis and the onset of diabetes were significantly improved. Moreover, after the oral transfer of fecal bacteria over 3 weeks, the composition of the gut microbiota were stably altered, mainly in terms of increases in Lachnospiraceae and Clostridiaceae and decreases in Lactobacillaceae [69]. These results indicate that the oral administration of gut microbiota represents a possible therapeutic intervention for improving insulin sensitivity in diabetes. However, the safety of fecal microbiota transplantation should be noted because potential adverse effects have also been reported. For example, 2 patients died in a study involving 18 patients who underwent bacterial transplantations to treat recurrent Clostridium. difficile colitis [70]. More clinical data and critical discussions of fecal microbiota transplantation protocols are required to prove whether this approach is beneficial for diabetic patients overall. Moreover, much more attention should be paid to the safety and composition of donor microbiota.

5.

Summary and future research

The human gut hosts trillions of microorganisms, which are collectively termed the “microbiome” and provide us with genetic and metabolic attributes pertinent to the maintenance of our homeostasis. Recent studies have revealed an important role of gut microbiota in the development of diabetes. However, the available data in this field remain limited, and the relevant scientific work has only just begun. Moreover, it is worth noting

Figure – Possible pathways linking gut microbiota and diabetes. The variety of independent mechanisms through which the gut microbiota influence the development of diabetes are summarized. Energy metabolism, inflammation, gut permeability and the immune system have been considered to be the key mechanisms through which the gut microbiota regulate the development of diabetes. The gut microbiota regulates energy metabolism through various pathways including the SCFA-GPR41/43, ChREBP/ SREBP-1c, AMPK, and fasting-induced adipose factor pathways. Additionally, high-fat food promotes the plasma levels of LPS and enhances intestinal mucosa permeability, which are followed by an inflammatory response in the host that contributes to the development of insulin resistance and diabetes. The innate immune system has been linked to diabetes through T lymphocytes and TLR-5 in the intestinal mucosa, which have also been linked to inflammation, insulin sensitivity and glucose homeostasis.

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that the available data are heterogeneous in nature and include examinations of different populations or rodent models and various types of food. In addition, similar to all systematic reviews, the findings in this review depend on other factors, such as the nature of the search strategy. Therefore, the limited availability and complexity of the relevant research in this area are limitations of this review. In summary, we conclude that the gut microbiota influence the development of diabetes through a variety of independent mechanisms (Figure). Although compelling evidence support the concept that gut microbiota are potentially a new therapy target for diabetes, more research is needed to elucidate the associations between the microbiota and diabetes. Future research should address the following issues: (1) a thorough characterization of the main subspecies of gut microbiota that contribute to the incidence or development of diabetes, and the exact role of the individual’s gut microorganisms in the pathogenesis of diabetes. (2) Detailed interactions between the host and the gut bacteria is needed. (3) Additional well-controlled clinical studies with the gut microbiota should be done to confirm safety in the patient.

Acknowledgment This work was supported by the Fundamental Research Funds for the Central Universities (Grant No. HIT. IBRSEM.201335).

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