G. Blandino , R. Inturri , F. Lazzara , M. Di Rosa , L. Malaguarnera ∗Department of Biomedical and Biotechnological Sciences, School of Medicine, University of Catania, Catania, Italy
Received 7 January 2016; received in revised form 4 April 2016; accepted 7 April 2016
bstract
Various functions of the gut are regulated by sophisticated interactions among its functional elements, including the gut microbiota. Theseicroorganisms play a crucial role in gastrointestinal mucosa permeability. They control the fermentation and absorption of dietary polysaccharides
o produce short-chain fatty acids, which may explain their importance in the regulation of fat accumulation and the subsequent development ofbesity-related diseases, suggesting that they are a crucial mediator of obesity and its consequences. In addition, gut bacteria play a crucial role inhe host immune system, modulation of inflammatory processes, extraction of energy from the host diet and alterations of human gene expression.ietary modulation of the human colonic microbiota has been shown to confer a number of health benefits to the host. Simple therapeutic
trategies targeted at attenuating the progression of chronic low-grade inflammation and insulin resistance are urgently required to prevent or slowhe development of diabetes in susceptible individuals. The main objective of this review is to address the pathogenic association between gut
icrobiota and diabetes, and to explore any novel related therapeutic targets. New insights into the role of the gut microbiota in diabetes could
ead to the development of integrated strategies using probiotics to prevent and treat these metabolic disorders. 2016 Elsevier Masson SAS. All rights reserved.
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eywords: Diabetes mellitus; Diabetic complications; Gut microbiota; Inflamm
. Introduction
“Father of Medicine” Hippocrates’ famous statement that “allisease begins in the gut” recognized the essential role playedy the gut and diet in many of the vital homoeostatic functionsf the human body. Indeed, the role of the human gut microbiotas crucial, as it is populated by a number of different microbialroups [1]. Every one of us has our own exclusive microbiotand gut microbiome (the microorganisms’ genes). The numberf genes of the microbiota outnumbers human genes by a hun-redfold [2]. Studies examining the influence of nutrients (suchs dietary fibres and fats) and dietary habits (whether omnivores,egetarians or vegans) in different populations have allowedtratification of the human population based on three princi-
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al bacteria and their microbiome’s genetic abundance [3]. Guticrobiota from different individuals have been classified into
nterotypes, depending on their function, metabolism of dietary
omponents, and ability to tolerate and metabolise drugs [4]. healthy gut microbiome is characterized by the presence oficrobes that enhance metabolism, and are resilient to infection
nd inflammation and resistant to autoimmunity and cancer [5].Increasing evidence indicates that gut microbiota are strongly
ssociated with diabetes development [6,7]. In addition, theutoimmune mechanisms involved in the pathogenesis of type
diabetes (T1D) might also implicate peptidergic enteric neu-ons, which regulate immune-cell function and influence pro-nd anti-inflammatory cytokine production, resulting in neu-odegeneration [8]. Lymphokines produced in the pathogenicascade involved in the development of autoimmune islet-cellamage could also lead to myenteric neuropathy [9]. Notably,he gut microbiota affect the intestinal mucosa via interactionsith epithelial cells and the enteric nervous system, leading to
hanges in gut motility, sensory functions and pain perceptionmicrobiota–brain–gut axis) [10]. The present review aims torovide some mechanistic insights by highlighting the role of
gut microbiota on diabetes mellitus. Diabetes Metab (2016),
he gut microbiota in diabetes to prompt the development ofnnovative therapeutic targets for the prevention, treatment andlowing of diabetes and other metabolic-associated disorders.
smapipathogen-associated molecular patterns (PAMPs) by epithelial
G. Blandino et al. / Diabetes
. Gut microbiota
Several studies from the Human Microbiome Project and theuropean Commission’s Metagenomics of the Human Intesti-al Tract (MetaHIT) consortium have contributed to our betternowledge and understanding of the healthy composition andunctional properties of the gut microbiota [1]. These studiesndicate that host health is associated with the diversity and sta-ility of the gut microbiome [11]. Gut microbiota constitute aynamic entity that is modified by diet, lifestyle, antibiotics andenetic background [12]. Microbiota gut colonization probablytarts at birth, as no large variations appear during healthy life.nterestingly, the type of microorganisms that colonize the gutf newborns depends on the delivery procedure. Infants bornaginally display a microbiota composed of Lactobacillus, Pre-otella and Sneathia spp coming from the maternal vaginal tract.n contrast, newborns delivered by caesarean section displayredominantly Staphylococcus, Corynebacterium and Propioni-acterium spp [13]. During early childhood, Actinobacteria,nd particularly of the genus Bifidobacterium, dominate the guticrobiota of breastfed infants. Also at this time, the micro-
iota acquires a variety of new strains influenced by changesn diet, such as the introduction of solid foods, and by dis-ase, such that gradually over time, it begins to resemble thedult composition [14]. Moreover, physical exercise is ableo modulate gut microbiota, and increasing physical activityan increase the abundance of beneficial microbial species15].
To date, around 100 large groups of bacteria, known asphyla”, have been identified, each with a different reper-oire of biochemical capabilities. In the adult gut microbiota,he majority of the microbial populations belong to the phylactinobacteria and Proteobacteria, and approximately 90% to
he Bacteroidetes and Firmicutes phyla [16]. These phyla areifferentially distributed throughout the gut and determine dif-erent microbial ecosystems [17]. In the Firmicutes phylum, thelostridium coccoides group is the dominant population in theut microbiota, with a large number of cultured and encultur-ted spp. A recent study showed that changes in the diversity ofhe C. coccoides group population in the gut microbiota corre-ates with age, and it was hypothesized that these changes couldffect the health of the host [18]. Gut microbiota fulfil struc-ural and histological functions, and play important metabolicoles for health maintenance, including amino-acid synthesisnd the absorption of dietary fats and fat-soluble vitamins, andffect the protective actions that prevent pathogenic coloniza-ion and the composition of converted bile acids (Fig. 1) [19].ile acids bind to cellular receptors, which are internalised, andhich activate distinct pathways involved in glucose homoeosta-
is and lipid energy metabolism [20–22]. Furthermore, the guticrobiota help the host to eliminate calories from indigestible
omplex carbohydrates and plant polysaccharides via enzymeshat are not encoded within the human genome [23]. Non-igestible carbohydrates are fermented by colonic microbes,
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eading to the production of short-chain fatty acids (SCFAs) suchs butyrate, which has trophic effects on intestinal epithelium24].
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tabolism xxx (2016) xxx–xxx
On the basis of the clustering patterns seen in the world’sopulation with variations in the levels of dominant micro-iota genera, three enterotypes have been proposed: Bacteroides;revotella; and Ruminococcus [25]. Differences among thesenterotypes are dependent on different combinations of micro-ial trophic chains. Bacteroides (enterotype 1) develops energyrincipally from fermentation of carbohydrates, as this genusas a very broad saccharolytic potential. Prevotella (enterotype) degrades mucin glycoproteins of the gut mucosal layer, whileuminococcus (enterotype 3) binds mucins, and transports andegrades the constituent sugars. In addition, Bacteroides andrevotella are enriched by the biosynthesis of different vitamins
25]. These enterotypes have been associated with long-termietary patterns. Bacteroides spp have been correlated with dietsominated by high levels of animal protein and saturated fats, asound in the typical Western diet, whereas Prevotella is predom-nant in people with higher consumption of carbohydrates andimple sugars, as observed in agrarian and vegetarian societies4].
In addition, microorganisms in the microbiota can regulatentestinal architecture by altering gut permeability. Intesti-al epithelium is not only responsible for the assimilation ofngested food and nutrients, but also for the prevalence ofrosstalk with the external surface of the body as well asetween gut microbes. Epithelial cells constitute a physicalarrier, impeding the translocation of the luminal contents ofhe inner tissues. The two main types of interconnecting junc-ions are the adherens junctions (AJs) and tight junctions (TJs).Js are predominantly formed by cadherins linked to the actin
ytoskeleton through a family of catenins, while TJs are theesult of interactions of occludin, claudins and junctional adhe-ion molecule (JAM)-A, linked to the actin cytoskeleton viaonula occludens proteins (ZO-1, ZO-2) and �-catenin [26].yosin phosphorylation and contraction of the actin–myosin
omplex regulate the permeability of the epithelial barrier [27].amage to intestinal permeability allows the passage of endolu-inal molecules into deeper layers which, in turn, weakens the
ntercellular connections and triggers activation of the inflam-atory response [28]. Thus, enterohaemorrhagic Escherichia
oli (EIIEC) and enteropathogenic E. coli (EPEC) have the abil-ty to adhere to intestinal epithelial cells (IECs) and disrupt thentegrity of the barrier through TJ alterations [29]. Subsequentctivation of the inflammatory response leads to increased con-entrations of proinflammatory mediators, such as interferonIFN)-� and tumour necrosis factor (TNF)-�, which can bothodulate the expression of several TJ proteins, such as ZO-1,
AM-A, occludin, claudin-1 and claudin-4 [30].Thus, the intestinal epithelium and intestinal innate immune
ystem are symbiotic, and cooperate in interactions between guticrobiota and the host. This synergy arises through a mech-
nism that can destroy pathogens while equally tolerating theresence of commensals, using strategies that generate ecolog-cal niches for beneficial gut microbiota [31]. Recognition of
gut microbiota on diabetes mellitus. Diabetes Metab (2016),
ells via pathogen recognition receptors (PRRs) is important forhis equilibrium. To date, the system linking gut microbial andost signals, and the onset or progression of metabolic alterations
G. Blandino et al. / Diabetes & Metabolism xxx (2016) xxx–xxx 3
ion an
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Fig. 1. Factors influencing gut microbiota composit
ssociated with high-fat feeding, have not been fully elucidated.ecently, however, a study in a murine model demonstrated
hat inducible IEC-specific deletion of the myeloid differenti-tion primary response gene 88 (MyD88) can partially protectgainst diet-induced fat storage, inflammation and diabetes viaechanisms directly involving the gut microbiota [32]. MyD88
s a PRR at the interface of the interaction between microor-anisms and host, and one of the principal adaptor moleculesor the majority of toll-like receptors (TLRs) [33,34]. As sug-ested by Everard et al. [32], MyD88 in IECs serves as a sensor,hanging host metabolism according to diet, and influencinghe composition of gut microbiota, energy metabolism, and theevelopment of obesity and associated disorders. Nevertheless,here are no human studies showing that the host IEC MyD88ontrols gut microbiota composition and that this is associatedith metabolic disorders.Thus, further investigations of human IECs need to be per-
ormed to confirm that host intestinal epithelial MyD88 controlsut microbiota composition and is associated with metabolicisorders. Moreover, it has been proposed that IECs producenterleukin (IL)-18, which contributes to the preservation of thentestinal barrier mainly by inducing epithelial cell prolifera-ion, thereby enhancing the regeneration of damaged epithelium35,36]. Interestingly, it was also found that high-fat feed-
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ng decreases IL-18 expression in the intestine, whereas IECyD88 deletion normalizes this parameter. This result indicates
hat intestinal MyD88 contributes to the regulation of IL-18
ifv[
d its structural, protective and metabolic functions.
xpression during high-fat feeding and thus helps to improveut barrier function [32].
. Obesity and gut microbiota
Overweight and obesity are metabolic disorders that havepread all over the world. The occurrence of type 2 diabetesT2D) is mainly attributable to overweight and obesity. Thenterplay between behaviour and genetic and environmental fac-ors are the principal contributors to obesity and T2D incidence.mong ambient determinants, human overall gut bacteria appear
o be one of the crucial mediators of obesity and diabetes patho-enesis [37,38]. The microbiota possess protective functions inetabolic regulation, and play an active part in glucose and lipidetabolism [15]. A number of extrinsic and intrinsic factors can
nitiate perturbations of the gut microbiota. The consequence ofhese perturbations is a shift from normobiosis to dysbiosis, asocumented by a deficiency of microbiota compositional andunctional diversity (Fig. 3).
Obesity is linked to dysbiosis, a state characterized by alter-tions in microbiota composition, changes in bacterial metabolicctivity and/or shifts in the local distribution of bacterial com-unities. Changes in gut microbiota composition could promote
gut microbiota on diabetes mellitus. Diabetes Metab (2016),
ntestinal monosaccharide absorption and energy withdrawalrom indigestible food components (principally carbohydrates)ia SCFA production and de novo hepatic lipogenesis (Fig. 3)39]. Furthermore, this dysbiosis could intensify fatty acid
4 G. Blandino et al. / Diabetes & Metabolism xxx (2016) xxx–xxx
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ig. 2. Myeloid differentiation primary response gene 88 (MyD88) function in gnd the intestinal innate immune and neurological systems.
oading in adipocytes, limiting the fasting-induced adiposeactor in the gut which, in turn, increases lipoprotein lipasenzyme activity [40]. The main supposed mechanisms connect-ng a balanced gut microbiota composition to protection againstiet-induced obesity in germ-free mice could be the inhibi-ion of cellular energy-dependent protein kinase activation [41],ollowed by an association with SCFA signalling molecules, G-rotein-coupled receptor activation and energy storage (Fig. 3)42].
At present, intestinal dysbiosis is crucial for understand-ng the pathophysiology of obesity and diabetes. One of the
echanisms proposed to explain the crosstalk between the guticrobiota and regulation of fat storage and development of
besity-related diseases is metabolic endotoxaemia [43]. In par-icular, abnormal gut microbiota composition may trigger a statef chronic low-grade inflammation, rendering the host suscep-ible to systemic exposure to lipopolysaccharide (LPS) [44].his large glycolipid molecule, derived from the outer mem-rane of Gram-negative bacteria, is a potent inducer of the innatemmune-system response linked to adiposity, insulin resistanceIR) and de novo synthesis of triglycerides. By binding to TLR4nd its co-receptors, LPS triggers a cascade of responses, ulti-ately resulting in the release of proinflammatory molecules
hat interfere with modulation of glucose and insulin metabolismFig. 2). Studies of gut microbiota in both humans and animalodels have helped to clarify its involvement in the pathogenesis
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f obesity. In obese humans, it was found that the relative propor-ion of Bacteroidetes was decreased in comparison to lean people45]. Moreover, a shift was demonstrated towards a higher
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crobiota immunomodulation, and the cooperation between intestinal epithelium
elative abundance of Bacteroidetes and a decreased numberf Firmicutes in mice fed high-fat diets (HFDs), but which losteight on low-calorie diets [43].Other findings have demonstrated that treatment with Bifi-
obacterium strains restored alterations in the microbiota,educing the excess numbers of Firmicutes and LPS-producingroteobacteria, as well as the production of B cells, macrophagesnd cytokines (IL-6, MCP-1, TNF-�, IL-17), thereby improvingystemic inflammation [44]. These effects were accompanied bymprovements in metabolic dysfunction, including lowered lev-ls of cholesterol, triglycerides, glucose and insulin, reducedody weight gain, and re-establishment of oral glucose toler-nce and insulin sensitivity [32]. Moreover, human oligofructoseupplementation, which increases bifidobacteria content, alsoeduced inflammatory status, and plasma and adipose tissueroinflammatory cytokines [46]. Obese adults with less bacte-ial diversity gained more weight over a 9-year follow-up period47]. Therefore, low bacterial richness appears to be associatedith more marked overall adiposity.In obese/diabetic individuals, weight loss, rapid diabetic
emission and metabolic improvement can be accomplished by type of bariatric surgery – namely, Roux-en-Y gastric bypassRYGB). In the gut microbiota of faecal samples from 30 obesendividuals before and after RYGB, it was found that the Bac-eroides/Prevotella groups were reduced in these subjects beforehe surgery, but increased 3 months after it. In addition, after
gut microbiota on diabetes mellitus. Diabetes Metab (2016),
months, E. coli spp were increased, and were inversely corre-ated with fat mass and leptin levels, independently of changesn food intake. In contrast, the decrease after 3 months in lactic
cid bacteria (LAB), including the Lactobacillus, Leuconostocnd Pediococcus groups and Bifidobacterium, correlated withmprovements in oral glucose tolerance and insulin sensitivity.his finding indicates that weight loss and changes in overall
nflammatory status can modify gut microbiota ecology [48].The gut microbiota profile may, in the near future, allow the
rediction of which obese individuals are most likely to loseeight in response to energy-restricted diets, which would be aelpful strategy for obesity treatment [49]. In a group of obeser overweight subjects who underwent a 6-week programme ofnergy restriction, followed by another 6-week period of weighttabilization, the subjects who lost the least amount of weight
and, thus, more rapidly regained it – were those who had higheractobacillus, Leuconostoc and Pediococcus numbers in theiraecal samples at baseline [49]. Given the cost of microbiotarofiling (data sequencing and analysis), diet restriction seems aealistic first option. Colonization of germ-free mice with “obeseicrobiota” resulted in a significant increase in total body fat and
R compared with colonization with “lean microbiota” [8].These results confirm that gut microbiota composition is an
dditional contributory factor to the pathophysiology of obe-ity and IR [50]. Remarkably, it has been shown that germ-freeice transplanted with the caecal microbiome of obese mice
eveloped the phenotype of the donor [51]. Similarly, after col-nizing germ-free mice with caecum-derived microbiota fromonventionalised mice, the total amount of body fat increasednd insulin sensitivity decreased, even with no changes in theiriet [40]. Taken together, these findings suggest that the guticrobiota are active players in the development of obesity
43,52].
. Gut microbiota in diabetes
In diabetic humans, there is a lack of uniformity in gut micro-iota profiles. A human metagenome-wide association studyhowed significant correlations with specific gut microbes, bac-erial genes and metabolic pathways in T2D patients [38]. Theseatients displayed higher levels of Lactobacillus spp comparedith non-diabetics [53]. Lactobacillus spp correlated positivelyith fasting glucose and glycated haemoglobin (HbA1c) levels,hile Clostridium spp correlated negatively with fasting glu-
ose, HbA1c, insulin, C-peptide and plasma triglycerides, andositively with adiponectin and high-density lipoprotein (HDL)holesterol [54]. In addition, based on the onset of metagenomiclusters (MGCs) in genetically obese and diet-induced leptin-esistant mice, 26 differentially abundant clusters were foundhen comparing diabetic mice with those with normal glucose
olerance [55].Studies in different population have also shown that dia-
etic gut microbiota have lower concentrations of Roseburiantestinalis and Faecalibacterium prausnitzii (both butyrate-roducing bacteria), and higher levels of Lactobacillus gasseri,treptococcus mutans and Clostridiales members. Also, met-
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ormin administration caused an increase of Akkermansiauciniphila, a mucin-degrading Gram-negative bacterium, in
he mucous layer [56]. Several experimental studies have shownhat A. muciniphila concentrations correlate inversely with the
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tabolism xxx (2016) xxx–xxx 5
resence of obesity and diabetes [57,58], and induce improve-ents in metabolic function (weight loss), glucose tolerance
nd systemic inflammation [38–59]. Furthermore, administra-ion of a prebiotic such as oligofructose dramatically increased. muciniphila levels, with beneficial effects on metabolic con-
rol. A recent study demonstrated that giving vancomycin toon-obese diabetic (NOD) mice increased their concentra-ions of A. muciniphila and enhanced glucose homoeostasis60]. Other studies reported that the proportions of Firmicutesnd Clostridium spp were significantly reduced in diabeticsompared with controls [60]. Likewise, the ratios of Bac-eroidetes to Firmicutes and Bacteroides/Prevotella groups to. coccoides/Eubacterium rectale groups correlated positivelyith plasma glucose levels. Similarly, the Betaproteobacteria
lass was highly enriched in diabetics vs non-diabetics and wasositively correlated with plasma glucose (Fig. 3) [61]. It has alsoeen suggested that there might be an inflammation-triggeringffect of the intestinal microbiota in the development of autoim-une diabetes [62]. A pathological cascade that perturbs the
ntestinal immune system is a critical element in the developmentf autoimmune T1D. The link between the gut microbiota andhe development of autoimmune diabetes can be explained byhe shared lymphocyte-homing receptors in the gut and inflamedancreas [63].
. Gut microbiota, immunity and diabetes
The interaction between intestinal microbiota and the innatentestinal immune system has been recognized as an epige-etic factor that can modify the predisposition to T1D. It isossible that pancreatic damage originates from an initial cross-eaction of the immune system directed against a dietary antigen64]. The association with microbiota can arise because gutpithelial cells express microbe-associated molecular patternMAMP) receptors, principally TLRs, which lead to a proin-ammatory response that activates the nuclear factor (NF)-�Bathway (Fig. 4B). The activated TLRs and microbiota patternsesult in the production of cytokines, chemokines and antibac-erial products. It is known that high-fat feeding augmentslasma LPS-containing microbiota at a concentration sufficiento increase body weight, fasting glycaemia and inflammationFig. 4B) [65]. Bacterial LPS inhibits IL-1 receptor-associatedinase (IRAK) M, a modulator of IRAK1 necessary for NF-B activation. Also, the ubiquitination and degradation of I�B
s inhibited by reactive oxygen species (ROS) induced byhe microbiota, and peroxisome proliferator-activated receptorPPAR)-�, a product of TLR4 activation by LPS, shunts NF-�Brom the cell core (Fig. 4B) [66]. Subsequently and initiatedy bacterial recognition, the differentiation of effector T-helperTh) 1, Th2 and Th17 cells, the development of regulatory TTreg) cells and the production of secretory immunoglobulin AsIgA) occurs.
Many bacterial communities can induce the production of
gut microbiota on diabetes mellitus. Diabetes Metab (2016),
nflammatory T cells. In such cases, segmented filamentousacteria colonize the gut by coming into direct contact withhe epithelium, facilitated by dendritic cells (DCs; Fig. 4B).his elicits a specific effector host response, characterized by
6 G. Blandino et al. / Diabetes & Metabolism xxx (2016) xxx–xxx
l hom
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Fig. 3. Gut microbiota and how they differ in intestina
cascade of proinflammatory signals that culminates in theroduction of Th17 and Th1 cells, mediated by IL-1, IL-6nd IL-12, which can lead to autoimmunity [66]. Commensalicrobiota in the colon, such as Clostridium clusters IV andIVa, through their SCFA production stimulate the expressionf FOXP3 in CD4+ T cells and induce the differentiation of Tregells (http://www.mdpi.com/2072-6643/7/11/5461/htm–B27-utrients-07-05461) [67]. Alterations in the gut microbiotarofile, such as dysbiosis or inadequate introduction of foodsuring the first months of life, can increase susceptibility to, andenerate the development of, autoimmune diseases locally inhe gut or at a systemic level. In American and Finnish children,he fat intake from bovine milk products as well as proteinsrom fresh milk led to an increase in the risk of advanced �-cellutoimmunity and subsequent progression to T1D [68,69]. Inact, the presence of high titres of anti-�-casein at the time ofiagnosis of T1D and latent autoimmune diabetes of adultsLADA) suggests that the antibody response to this protein maye relevant to autoimmune diabetes (Fig. 4A) [70].
Moreover, it has been proposed that high-gluten diets could bemong the primary drivers of gut dysbiosis associated with T1Development (Fig. 4A) [71]. Interestingly, coeliac disease (CD)s more common in patients with T1D, and is associated withoorer glycaemic control and lipid profiles [72]. This is relatedo the timing and amounts of dietary gluten fed to infants. Therogressive introduction of gluten-containing foods (in terms ofuantity) to the diet at between 3 and 7 months after birth canower the risk of T1D-associated autoimmunity [73]. Gluten has
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umerous effects on intestinal homoeostasis. Several CD studiesave reported that gluten increases gut permeability by affect-ng TJs [74,75]. As a result, long gliadin peptides can enter
pia
oeostasis vs dysbiosis. SCFA: short-chain fatty acid.
n between epithelial cells into the lamina propria. From there,Cs can then migrate to other sites, such as the pancreatic lymphodes, to activate autoreactive T cells (Fig. 4A) [73]. Pre-T1Dhildren with multiple autoantibodies and those newly diag-osed with T1D present with a T-cell response against gliadint a lower frequency and intensity than do healthy controls andatients with longer T1D durations [73]. This result supports thedea of an aberrant immune response related to the developmentf T1D.
As already mentioned, the intestinal microbiota of obeseatients have been associated with higher levels of proinflam-atory cytokines (Fig. 4B) [76]. Obese patients usually develop
hronic adipose tissue inflammation because the consumptionf an HFD in conjunction with the obese phenotype is associ-ted with changes in gut microbiota, a decrease in IFA, and anncrease in LPS and ileal inflammation. Enterocytes internalisePS from the apical surface and convey it to the Golgi complex
77], which also contains chylomicrons, lipoproteins responsibleor the transport of dietary long-chain fat through the blood andesenteric lymph. It has been observed that chylomicrons pro-ote intestinal LPS absorption. Thus, an excess of chylomicron
ormation during high-fat feeding facilitates endotoxin translo-ation via a reduction in intestinal alkaline phosphatase (IAP)ctivity, inducing the intestinal inflammation found in obesitynd insulin-resistant states.
Obesity and its metabolic complications are associated withacrophage infiltration, responsible for almost all the adipose
issue TNF-� and IL-6 expression involved in inflammatory
gut microbiota on diabetes mellitus. Diabetes Metab (2016),
athways. Moreover, it has been demonstrated that an HFDncreases plasma levels of LPS, leading to low-grade endotox-emia, and that IR is induced by LPS in differentiated adipocytes
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Fig. 4. Differences in the immunopathogenesis of type 1 (A) and type 2 (B) diabetes. LADA: latent autoimmune diabetes of adults; MCP1: monocyte chemoattractantprotein-1.
65]. The activation of macrophages is dependent on LPS/CD1478], a combination that serves as a ligand for TLR4 (Fig. 4B)79]. TLR4 inactivation reduces food intake and inflammatoryesponses, but with no significant modification of body weight79]. Thus, the LPS/CD14/TLR4 system appears to set thehreshold at which an HFD can induce IR and the onset of dia-etes and obesity. CD14 knockout mice lacking functional LPSeceptors are hypersensitive to insulin, while increased endo-oxaemia is associated with increased CD14 expression andncreased IL-6 levels after a mixed meal containing lipids inealthy humans [80]. An HFD can affect IEC integrity, leading tompaired barrier function and increased intestinal permeabilityo bacterial fragments. The endotoxins can then trigger the proin-ammatory cascade and activate TLR4 signalling [81]. In mice,hronic exposure to low-dose LPS resulted in liver steatosis,ncreased IR, dyslipidaemia, adipose tissue macrophage infiltra-ion and obesity similar to what is observed with an HFD [31].nterestingly, these effects were absent when TLR4-deficientice were fed an HFD or when ob/ob mice fed an HFD were
reated with antibiotics [33].Besides LPS, other bacterial components can interact with
nflammatory pathways [82]. Activation of innate immuneesponses by gut microbiota-derived molecular patterns isostly due to bacterial cell-wall components such as flag-
llin [83] and polysaccharide A (PSA) [84], as well asommensal genomic DNA [85]. It has been reported thatacteroides fragilis-derived PSA, a TLR2 ligand, can induce
L-10-producing CD4T cells and reciprocally suppress Th17esponses [86]. TLR5 is a flagellin-specific pattern-recognitioneceptor expressed in the gut mucosa that contributes tohe defence against infection. Interestingly, TLR5−/− miceevelop a metabolic phenotype comprising modifications ofut microbiota composition, hyperlipidaemia, hypertension, IRnd obesity. As with other studies, colonizing wild-type germ-ree mice with TLR5−/− mouse caecum microbiota transfersts metabolic phenotype to the recipient [80]. Further mecha-istic studies indicate that DNA from gut flora plays a majorole in intestinal homoeostasis through TLR9 engagement [87].herefore, malfunction of the innate intestinal immune systemay play an important role in the development of the metabolic
yndrome through modification of the gut bacterial profile. Sim-larly, TLR9-deficient mice display an elevated frequency ofOXP3+ Treg cells at intestinal effector sites, with suppressedonstitutive IL-17– and IFN-�-producing effector T cells [88].
NOD mouse model of T1D also revealed that a deficiencyf the master adaptor protein MyD88 led to resistance to theevelopment of T1D through alteration of gut bacteria [89].
All these observations suggest that different innate immune-ctivating components of gut bacterial origin have a differentole in the regulation of gut immune homoeostasis, and providehe first evidence of a ‘missing link’ between gut microbiotand innate immunity in T1D development. Many findingsemonstrate that the presence/absence of specific microbes can
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odulate and programme life-long changes in immunity [90].evertheless, as T1D is an autoimmune disease, it would bef interest to determine whether dysbiosis precedes or fol-ows the development of diabetes. Also, future studies need to
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valuate more deeply how dysbiosis influences metabolic dis-ase progression.
. Microbiota and mechanisms of gut motility iniabetes
The evidence indicates that gastrointestinal (GI) symptoms,specially those related to motility disorders of the upper GIract, are more frequently seen in diabetic patients [33]. Diabetess frequently associated with a variety of GI motility abnormal-ties in which nitrergic enteric neuropathy may be the primaryysfunction, independently of vagal dysfunction [91]. A multi-ude of different, uncommon symptoms seen in diabetic patientsould be associated with the complex functions of the lowerI tract. In general, diabetics with advanced disease [3] suffer
rom watery and painless nocturnal diarrhoea alternating witheriods of constipation and intermittent normal bowel function.hanges in the brain–gut system and different parts of the cor-
ical network in diabetic patients could further modulate andontribute to the development, maintenance and subjective char-cteristics of GI symptoms in patients with diabetes [92–97].n addition, peptidergic enteric neurons, as targets of inflamma-ion, can modulate immune-cell function and therefore stimulateroinflammatory cytokine production, resulting in neurodegen-ration [94]. Thus, the pathogenic cascade, which triggers theevelopment of autoimmune diabetes through secreted lym-hokines, could also result in altered neuro-immune interactionsnd provoke myenteric neuropathy.
Of the potential environmental triggers implicated in theevelopment of diabetes-related myenteric neuropathy, thentestinal microbiome is considered a primary candidate.ccordingly, diabetes affects gut motility, thus influencing theicrobiota in the intestine and colon. Evidence indicates that the
ut microbiota communicate with the brain through the vaguserve, transmitting information from the luminal environmento the central nervous system (CNS). Microbiota can interactia the gut–brain axis through modulation of afferent sensoryerves, enhancing their excitability by inhibiting the openingf calcium-dependent potassium channels, thereby modulatingut motility and pain perception, compromising the intestinalarrier and TJs, and so interfering with all gut functions [25].urthermore, microbiota can influence enteric nervous systemENS) activity by producing molecules such as GABA, sero-onin, melatonin, histamine and acetylcholine that act as localeurotransmitters [97], and by generating biologically activeorms of catecholamines in the gut lumen [98]. Also, lactobacillise nitrate and nitrite to generate nitric oxide [99] and to produceydrogen sulphide, which modulates gut motility by interactingith the vanilloid receptor on capsaicin-sensitive nerve fibres
100]. Vagal and non-vagal muscarinic pathways influence thentestinal phase of insulin secretion, but regulation of gastricnhibitory peptide (GIP) secretion appears to be independent ofagal and muscarinic neural control mechanisms [101].
gut microbiota on diabetes mellitus. Diabetes Metab (2016),
Microbiota may interact with gut motility through severalechanisms. First, by compromising the intestinal barrier andJs, it can interfere with all gut functions. Decreased motil-
ty of the GI tract alters the gut microbial flora, which changes
AK2: Janus kinase 2; STAT1: signal transducer and activator of transcription 1a Type of cell and/or animal model used in experimental studies.
eurotransmissions by gastric afferents through the brain–gutxis [102]. In addition, the intestine is a huge and importantndocrine organ of the body. Enteroendocrine cells in the intes-
Please cite this article in press as: Blandino G, et al. Impact ofhttp://dx.doi.org/10.1016/j.diabet.2016.04.004
ine are mostly limited to deeper portions of the mucosa. Iniabetes, the most important enteroendocrine intestinal cells arehe K and L cells that produce GIP and glucagon-like peptide-1
GLP-1) incretin hormones, respectively. K cells are localized tohe duodenum and jejunum, whereas L cells are located prefer-bly in the ileum, but also throughout the entire intestinal tract.
gut microbiota on diabetes mellitus. Diabetes Metab (2016),
he effect of incretin is impaired in T2D patients with autonomiceuropathy compared with those without such neuropathy [103].he vagus nerve mediates the gastric inhibitory effects of GLP-1
nd, therefore, its effect on gastric motility may be altered in theresence of vagal neuropathy. GLP-1 displays neuroprotectiveffects in both the CNS and peripheral nervous system [104] and,hus, may have potential beneficial effects on impaired entericerves by improving their function in diabetes.
. Probiotics and their therapeutic potential in diabetes
In general, the above-mentioned findings, which confirm atrong link between the GI system and diabetes, have focusedonsiderable attention on the use of biotherapy to regulatentestinal microbiota function. Probiotics are live microorgan-sms that, when administered in sufficient quantities, provideealth benefits to the host [105,106]. However, the idea of
‘universal strain’ that offers all of the beneficial improve-ents simultaneously is unlikely. In fact, the positive effects
btained with probiotics are probably strain-specific; thus, dif-erent strains of the same species may exert distinct effects106]. The probiotic components associated with positive effectsnclude a variety of cell constituents, such as peptidoglycan,eichoic acids, polysaccharides, fimbrial/pili constituents andacteriocins [107]. Within the gut, probiotics are in competitionor nutrients, metabolites and antimicrobial proteins, therebyodulating gut microbiota population diversity in various ways
9–14].Probiotic administration can stimulate the immune response,
mprove lactose tolerance, avoid diarrhoea, restore obesity-inked gut dysbiosis and exert anti-inflammatory effects108–110]. In particular, the beneficial effects of probiotictrains include the prevention and treatment of obesity andnflammation, as well as of associated metabolic disorders suchs diabetes. These positive effects arise through a direct influ-nce on the mucosal barrier and, in particular, on the surroundingells, which can hamper chronic inflammation [109]. In theontext of obesity and metabolic disorders, probiotic supple-entation can help to reduce hyperphagia, so improving control
f weight gain, fat mass loss and glucose tolerance. More-ver, these positive effects may be obtained with no modulationf caloric intakes [111]. The majority of the probiotic strainshowing positive effects on glucose metabolism in humanselong to the Lactobacillus genus (Firmicutes phylum) and,o a lesser extent, the Bifidobacterium genus (Actinobacteriahylum) [33,60–111]. To demonstrate the benefits of probio-ics for improving metabolic disorders, researchers now haveccess to a variety of assays for plasma and liver cholesterol, freeatty acids, alanine and aspartate transaminases (hepatotoxicityarkers), and gene and protein expression (involved in inflam-atory and metabolic pathways). Two recent reviews [112,113]
emonstrated the beneficial effects of probiotics (mostly Lac-obacillus spp) in the prevention and management of T1D and2D. A summary of all clinical and experimental studies of dia-etes involving popular probiotic strains is presented in Table 1114–123].
Please cite this article in press as: Blandino G, et al. Impact ofhttp://dx.doi.org/10.1016/j.diabet.2016.04.004
Propionibacterium freudenreichii, a promising well-known,on-LAB species, produces 1,4-dihydroxy-2-naphthoic acidDHNA), which can reduce inflammation in IL-10-deficientice by suppressing proinflammatory cytokines [124].
tabolism xxx (2016) xxx–xxx
oreover, P. freudenreichii ssp shermanii JS, in combina-ion with Lactobacillus rhamnosus GG, has anti-inflammatoryffects on HFD-induced inflammation in ApoE*3-Leiden micey decreasing intestinal mast-cell numbers [125]. More impor-ant, it would be of interest to investigate other “beneficial”icroorganisms that are decreased in the gut microbiota of dia-
etic patients. Of the ones that might potentially be used inhe treatment of T2D, A. muciniphila is of particular interest56–61]. The administration of A. muciniphila MucT (ATTCAA-835) to a diet-induced mouse model of T2D has beenroved to exert beneficial effects on glucose metabolism bylleviating glucose intolerance in HFD-induced diabetic mice126], suggesting that A. muciniphila could prevent the delete-ious increase of gluconeogenesis in diabetic mice. In additiono A. muciniphila, other bacteria might be beneficial in the treat-ent of diabetes. For instance, F. prausnitzii plays an important
ole in preserving the gut barrier and controlling inflamma-ion and diabetes progression [58,127]. A traditional Chineseerberine-containing herbal formula given to T2D patients [128]hanged the gut microbiota by increasing F. prausnitzii, whichas negatively correlated with fasting blood glucose, HbA1c andostprandial blood glucose levels, and positively correlated withomoeostasis model assessment of beta-cell function (HOMA-). As F. prausnitzii is the least abundant spp found in T2Datients [62,129], it may be of particular interest.
Although clinical and experimental studies have revealed themportant potential of these probiotic strains in the managementf diabetes, further investigations are still required to elucidatehe molecular mechanisms involved in order to develop moreffective strategies against diabetes and its complications.
isclosure of interest
The authors declare that they have no competing interest.
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