Distinct alterations of gut morphology and microbiota characterize accelerated diabetes onset in nonobese diabetic mice

Abstract

The rising prevalence of type 1 diabetes (T1D) over the past decades has been linked to lifestyle changes, but the underlying mechanisms are largely unknown. Recent findings point to gut-associated mechanisms in the control of T1D pathogenesis. In nonobese diabetic (NOD) mice, a model of T1D, diabetes development accelerates after deletion of the Toll-like receptor 4 (TLR4). We hypothesized that altered intestinal functions contribute to metabolic alterations, which favor accelerated diabetes development in TLR4-deficient (TLR4−/−) NOD mice. In 70–90-day-old normoglycemic (prediabetic) female NOD TLR4+/+ and NOD TLR4−/− mice, gut morphology and microbiome composition were analyzed. Parameters of lipid metabolism, glucose homeostasis, and mitochondrial respiratory activity were measured in vivo and ex vivo. Compared with NOD TLR4+/+ mice, NOD TLR4−/− animals showed lower muscle mass of the small intestine, higher abundance of Bacteroidetes, and lower Firmicutes in the large intestine, along with lower levels of circulating short-chain fatty acids (SCFA). These changes are associated with higher body weight, hyperlipidemia, and severe insulin and glucose intolerance, all occurring before the onset of diabetes. These mice also exhibited insulin resistance–related abnormalities of energy metabolism, such as lower total respiratory exchange rates and higher hepatic oxidative capacity. Distinct alterations of gut morphology and microbiota composition associated with reduction of circulating SCFA may contribute to metabolic disorders promoting the progression of insulin-deficient diabetes/T1D development. The rising prevalence of type 1 diabetes (T1D) over the past decades has been linked to lifestyle changes, but the underlying mechanisms are largely unknown. Recent findings point to gut-associated mechanisms in the control of T1D pathogenesis. In nonobese diabetic (NOD) mice, a model of T1D, diabetes development accelerates after deletion of the Toll-like receptor 4 (TLR4). We hypothesized that altered intestinal functions contribute to metabolic alterations, which favor accelerated diabetes development in TLR4-deficient (TLR4−/−) NOD mice. In 70–90-day-old normoglycemic (prediabetic) female NOD TLR4+/+ and NOD TLR4−/− mice, gut morphology and microbiome composition were analyzed. Parameters of lipid metabolism, glucose homeostasis, and mitochondrial respiratory activity were measured in vivo and ex vivo. Compared with NOD TLR4+/+ mice, NOD TLR4−/− animals showed lower muscle mass of the small intestine, higher abundance of Bacteroidetes, and lower Firmicutes in the large intestine, along with lower levels of circulating short-chain fatty acids (SCFA). These changes are associated with higher body weight, hyperlipidemia, and severe insulin and glucose intolerance, all occurring before the onset of diabetes. These mice also exhibited insulin resistance–related abnormalities of energy metabolism, such as lower total respiratory exchange rates and higher hepatic oxidative capacity. Distinct alterations of gut morphology and microbiota composition associated with reduction of circulating SCFA may contribute to metabolic disorders promoting the progression of insulin-deficient diabetes/T1D development. Type 1 diabetes (T1D) 3The abbreviations used are T1Dtype 1 diabetesT2Dtype 2 diabetesANOVAanalysis of varianceSCFAshort-chain fatty acidNODnonobese diabeticTLR4Toll-like receptor 4LPSlipopolysaccharideFFAfree fatty acidNGSnext generation sequencingTGtriglycerideRQrespiratory exchange rateipGTTintraperitoneal glucose tolerance testipITTintraperitoneal insulin tolerance testHOMA-IRhomeostatic model assessment-insulin resistanceMMP-2matrix metalloproteinase-2. is characterized by absolute insulin deficiency resulting from progressive autoimmune destruction of pancreatic beta cells (1Atkinson M.A. Eisenbarth G.S. Michels A.W. Type 1 diabetes.Lancet. 2014; 383 (23890997): 69-8210.1016/S0140-6736(13)60591-7Abstract Full Text Full Text PDF PubMed Scopus (1407) Google Scholar, 2van Belle T.L. Coppieters K.T. von Herrath M.G. Type 1 diabetes: etiology, immunology, and therapeutic strategies.Physiol. Rev. 2011; 91 (21248163): 79-11810.1152/physrev.00003.2010Crossref PubMed Scopus (695) Google Scholar). Epidemiological data indicate that not only the prevalence of obesity-related type 2 diabetes (T2D) but also of T1D is continuously increasing (3Knip M. Siljander H. The role of the intestinal microbiota in type 1 diabetes mellitus.Nat. Rev. Endocrinol. 2016; 12 (26729037): 154-16710.1038/nrendo.2015.218Crossref PubMed Scopus (260) Google Scholar). In this context, accelerated childhood body weight gain (4Verbeeten K.C. Elks C.E. Daneman D. Ong K.K. Association between childhood obesity and subsequent type 1 diabetes: a systematic review and meta-analysis.Diabet. Med. 2011; 28 (21166841): 10-1810.1111/j.1464-5491.2010.03160.xCrossref PubMed Scopus (88) Google Scholar) and insulin resistance (5Xu P. Cuthbertson D. Greenbaum C. Palmer J.P. Krischer J.P. Diabetes Prevention Trial-Type 1 Study Group Role of insulin resistance in predicting progression to type 1 diabetes.Diabetes Care. 2007; 30 (17536068): 2314-232010.2337/dc06-2389Crossref PubMed Scopus (83) Google Scholar) along with impaired glucose tolerance (6Chase H.P. Cuthbertson D.D. Dolan L.M. Kaufman F. Krischer J.P. Schatz D.A. White N.H. Wilson D.M. Wolfsdorf J. First-phase insulin release during the intravenous glucose tolerance test as a risk factor for type 1 diabetes.J. Pediatr. 2001; 138 (11174623): 244-24910.1067/mpd.2001.111274Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar) associate with a higher risk for or earlier onset of T1D (7Fourlanos S. Narendran P. Byrnes G.B. Colman P.G. Harrison L.C. Insulin resistance is a risk factor for progression to type 1 diabetes.Diabetologia. 2004; 47 (15480539): 1661-166710.1007/s00125-004-1507-3Crossref PubMed Scopus (184) Google Scholar). Moreover, recent studies suggest that tissue-specific abnormalities of energy metabolism could underlie the insulin resistance not only in T2D but also in rodent models and humans with T1D (8Jelenik T. Séquaris G. Kaul K. Ouwens D.M. Phielix E. Kotzka J. Knebel B. Weiss J. Reinbeck A.L. Janke L. Nowotny P. Partke H.J. Zhang D. Shulman G.I. Szendroedi J. Roden M. Tissue-specific differences in the development of insulin resistance in a mouse model for type 1 diabetes.Diabetes. 2014; 63 (24917575): 3856-386710.2337/db13-1794Crossref PubMed Scopus (47) Google Scholar, 9Kacerovsky M. Brehm A. Chmelik M. Schmid A.I. Szendroedi J. Kacerovsky-Bielesz G. Nowotny P. Lettner A. Wolzt M. Jones J.G. Roden M. Impaired insulin stimulation of muscular ATP production in patients with type 1 diabetes.J. Intern. Med. 2011; 269 (21205021): 189-19910.1111/j.1365-2796.2010.02298.xCrossref PubMed Scopus (40) Google Scholar). type 1 diabetes type 2 diabetes analysis of variance short-chain fatty acid nonobese diabetic Toll-like receptor 4 lipopolysaccharide free fatty acid next generation sequencing triglyceride respiratory exchange rate intraperitoneal glucose tolerance test intraperitoneal insulin tolerance test homeostatic model assessment-insulin resistance matrix metalloproteinase-2. Toll-like receptor 4 (TLR4), a dominant member of the family of pattern recognition receptors (10Pasare C. Medzhitov R. Toll-like receptors: linking innate and adaptive immunity.Microbes Infect. 2004; 6 (15596124): 1382-138710.1016/j.micinf.2004.08.018Crossref PubMed Scopus (382) Google Scholar, 11Shi H. Kokoeva M.V. Inouye K. Tzameli I. Yin H. Flier J.S. TLR4 links innate immunity and fatty acid-induced insulin resistance.J. Clin. Invest. 2006; 116 (17053832): 3015-302510.1172/JCI28898Crossref PubMed Scopus (2692) Google Scholar), is an attractive candidate to link diabetes-promoting immunologic and metabolic processes. TLR4 was initially identified as a receptor for lipopolysaccharides (LPS), which are integral components of the cell wall of Gram-negative bacteria (12Beutler B. TLR4 as the mammalian endotoxin sensor.Curr. Top. Microbiol. Immunol. 2002; 270 (12467247): 109-12010.1007/978-3-642-59430-4_7Crossref PubMed Scopus (194) Google Scholar). Based on this feature, TLR4 contributes to the activation of host defense against microorganisms (12Beutler B. TLR4 as the mammalian endotoxin sensor.Curr. Top. Microbiol. Immunol. 2002; 270 (12467247): 109-12010.1007/978-3-642-59430-4_7Crossref PubMed Scopus (194) Google Scholar). Meanwhile, however, increasing evidence suggests that TLR4 is also involved in the induction of autoimmunity, including development of T1D (13Pasare C. Medzhitov R. Toll-like receptors: balancing host resistance with immune tolerance.Curr. Opin. Immunol. 2003; 15 (14630202): 677-68210.1016/j.coi.2003.09.002Crossref PubMed Scopus (115) Google Scholar, 14Gülden E. Ihira M. Ohashi A. Reinbeck A.L. Freudenberg M.A. Kolb H. Burkart V. Toll-like receptor 4 deficiency accelerates the development of insulin-deficient diabetes in nonobese diabetic mice.PLoS ONE. 2013; 8 (24086519): e7538510.1371/journal.pone.0075385Crossref PubMed Scopus (41) Google Scholar). The presence of TLR4 on insulin target tissues, such as liver, adipose tissue, and skeletal muscle (15Jin C. Henao-Mejia J. Flavell R.A. Innate immune receptors: key regulators of metabolic disease progression.Cell Metab. 2013; 17 (23747246): 873-88210.1016/j.cmet.2013.05.011Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar), supports its relevance for diabetes-associated metabolic disorders such as obesity and insulin resistance (16Dasu M.R. Devaraj S. Park S. Jialal I. Increased toll-like receptor (TLR) activation and TLR ligands in recently diagnosed type 2 diabetic subjects.Diabetes Care. 2010; 33 (20067962): 861-86810.2337/dc09-1799Crossref PubMed Scopus (430) Google Scholar). In fact, elevated TLR4 expression correlates with increased systemic concentrations of free fatty acids (FFA) and glucose (17Dasu M.R. Jialal I. Free fatty acids in the presence of high glucose amplify monocyte inflammation via Toll-like receptors.Am. J. Physiol. Endocrinol. Metab. 2011; 300 (20959532): E145-E15410.1152/ajpendo.00490.2010Crossref PubMed Scopus (177) Google Scholar) and with the degree of insulin resistance in obesity (18Reyna S.M. Ghosh S. Tantiwong P. Meka C.S. Eagan P. Jenkinson C.P. Cersosimo E. Defronzo R.A. Coletta D.K. Sriwijitkamol A. Musi N. Elevated toll-like receptor 4 expression and signaling in muscle from insulin-resistant subjects.Diabetes. 2008; 57 (18633101): 2595-260210.2337/db08-0038Crossref PubMed Scopus (300) Google Scholar, 19Jialal I. Huet B.A. Kaur H. Chien A. Devaraj S. Increased toll-like receptor activity in patients with metabolic syndrome.Diabetes Care. 2012; 35 (22357188): 900-90410.2337/dc11-2375Crossref PubMed Scopus (134) Google Scholar). However, the mechanisms underlying TLR4-dependent modulation of the progression of insulin-deficient/T1D are not yet clear. Recent findings suggest an impact of the intestinal microbiome on the onset and progression of T1D. Patients with T1D have a less diverse and stable gut microbiome than glucose-tolerant humans (20Endesfelder D. zu Castell W. Ardissone A. Davis-Richardson A.G. Achenbach P. Hagen M. Pflueger M. Gano K.A. Fagen J.R. Drew J.C. Brown C.T. Kolaczkowski B. Atkinson M. Schatz D. Bonifacio E. et al.Compromised gut microbiota networks in children with anti-islet cell autoimmunity.Diabetes. 2014; 63 (24608442): 2006-201410.2337/db13-1676Crossref PubMed Scopus (125) Google Scholar, 21Brown C.T. Davis-Richardson A.G. Giongo A. Gano K.A. Crabb D.B. Mukherjee N. Casella G. Drew J.C. Ilonen J. Knip M. Hyoty H. Veijola R. Simell T. Simell O. Neu J. et al.Gut microbiome metagenomics analysis suggests a functional model for the development of autoimmunity for type 1 diabetes.PLoS ONE. 2011; 6 (22043294): e2579210.1371/journal.pone.0025792Crossref PubMed Scopus (552) Google Scholar). Moreover, gut microbiome dysbiosis in early childhood associates with an increased risk of progression to T1D (22Kostic A.D. Gevers D. Siljander H. Vatanen T. Hyötyläinen T. Hämäläinen A.M. Peet A. Tillmann V. Pöhö P. Mattila I. Lähdesmäki H. Franzosa E.A. Vaarala O. de Goffau M. Harmsen H. et al.The dynamics of the human infant gut microbiome in development and in progression toward type 1 diabetes.Cell Host Microbe. 2015; 17 (25662751): 260-27310.1016/j.chom.2015.01.001Abstract Full Text Full Text PDF PubMed Scopus (695) Google Scholar, 23Harbison J.E. Roth-Schulze A.J. Giles L.C. Tran C.D. Ngui K.M. Penno M.A. Thomson R.L. Wentworth J.M. Colman P.G. Craig M.E. Morahan G. Papenfuss A.T. Barry S.C. Harrison L.C. Couper J.J. Gut microbiome dysbiosis and increased intestinal permeability in children with islet autoimmunity and type 1 diabetes: a prospective cohort study.Pediatr. Diabetes. 2019; 20 (31081243): 574-58310.1111/pedi.12865PubMed Google Scholar). Intestinal bacteria are able to modulate the host's metabolism by various mechanisms, specifically by the release of short-chain fatty acids (SCFA), a set of highly-abundant fermentation products derived from the bacterial degradation of complex macrofibrous dietary components (24Canfora E.E. Jocken J.W. Blaak E.E. Short-chain fatty acids in control of body weight and insulin sensitivity.Nat. Rev. Endocrinol. 2015; 11 (26260141): 577-59110.1038/nrendo.2015.128Crossref PubMed Scopus (1137) Google Scholar, 25Koh A. De Vadder F. Kovatcheva-Datchary P. Bäckhed F. From dietary fiber to host physiology: short-chain fatty acids as key bacterial metabolites.Cell. 2016; 165 (27259147): 1332-134510.1016/j.cell.2016.05.041Abstract Full Text Full Text PDF PubMed Scopus (2786) Google Scholar). SCFA are potent modulators of glucose and energy metabolism (26Gancheva S. Jelenik T. Álvarez-Hernandez E. Roden M. Interorgan metabolic crosstalk in human insulin resistance.Physiol. Rev. 2018; 98 (29767564): 1371-141510.1152/physrev.00015.2017Crossref PubMed Google Scholar) and thereby might affect the development of T1D (27Vatanen T. Franzosa E.A. Schwager R. Tripathi S. Arthur T.D. Vehik K. Lernmark Å. Hagopian W.A. Rewers M.J. She J.X. Toppari J. Ziegler A.G. Akolkar B. Krischer J.P. Stewart C.J. et al.The human gut microbiome in early-onset type 1 diabetes from the TEDDY study.Nature. 2018; 562 (30356183): 589-59410.1038/s41586-018-0620-2Crossref PubMed Scopus (391) Google Scholar). These findings are in line with the concept of control of the pathogenesis of insulin-deficient/T1D by TLR4 expression status via modification of morphological and functional properties of the gut. Here, we tested the hypothesis that TLR4-dependent alterations of intestinal function affect metabolic homeostasis thereby contributing to accelerated progression of insulin-deficient diabetes. To this end, we used the TLR4-deficient (TLR4−/−) nonobese diabetic (NOD) mouse strain, which shows enhanced insulitis and therefore can serve as a model of accelerated human T1D development (14Gülden E. Ihira M. Ohashi A. Reinbeck A.L. Freudenberg M.A. Kolb H. Burkart V. Toll-like receptor 4 deficiency accelerates the development of insulin-deficient diabetes in nonobese diabetic mice.PLoS ONE. 2013; 8 (24086519): e7538510.1371/journal.pone.0075385Crossref PubMed Scopus (41) Google Scholar), that allows us to examine disease-relevant metabolic processes on a genetic background predisposing to insulin-deficient diabetes (28Chaparro R.J. Dilorenzo T.P. An update on the use of NOD mice to study autoimmune (type 1) diabetes.Expert. Rev. Clin. Immunol. 2010; 6 (20979558): 939-95510.1586/eci.10.68Crossref PubMed Scopus (47) Google Scholar). Food intake, body weight development, and body composition were monitored during the prediabetic period in 70–90-day-old female normoglycemic NOD TLR4+/+ and NOD TLR4−/− mice. Despite comparable food intake (Fig. 1A), NOD TLR4−/− mice showed an accelerated development of body weight (Fig. 1B) associated with slightly greater fat mass than NOD TLR+/+ animals (Fig. 1C). Female NOD TLR4−/− mice manifested diabetes more than 7 weeks earlier (age at disease onset: 152 ± 28 days) than their NOD TLR4+/+ littermates (age at disease onset: 208 ± 40 days) (Fig. 1D). At disease onset, hyperglycemia was more pronounced in NOD TLR4−/− than in NOD TLR4+/+ mice (Fig. 1E). Detailed analyses of gut morphology showed a proportional reduction of both longitudinal and circular muscle layers of the small intestine of NOD TLR4−/− mice (Fig. 2A) resulting in lower total muscle thickness of the gut segment (Fig. 2B). The TLR4 expression status neither affected the dimensions of small intestinal villi and crypts (Fig. 2, C–E) nor the length and weight of the gut segments (Fig. 2, F and G) or weight of gut contents (Fig. 2H). Effects of TLR4 deletion on gut microbiome composition were assessed using bacterial DNA isolated from small intestine, cecum, and colon. The gut segments contained comparable microbial biomass in NOD TLR4+/+ and NOD TLR4−/− mice as estimated from the ratio of isolated bacterial DNA/pellet of gut contents (Fig. 2I). Employing next generation sequencing (NGS) revealed an increased abundance of Bacteroidetes and a decrease of Firmicutes in the cecum and colon of NOD TLR4−/− mice compared with the corresponding gut segments of NOD TLR4+/+ animals (Fig. 2J and Table S1). In line with the high quantity of bacteria in cecum and colon, these gut segments exhibited the highest α-diversity (Fig. 2K). Assessing intestinal inflammation from frequency and distribution of CD3+ lymphocytes in the gut wall revealed no differences between NOD TLR4+/+ and NOD TLR4−/− mice (Fig. S1, A and B), which resembled the distribution pattern in corresponding tissue samples of age- and sex-matched normal healthy C57BL10 mice (data not shown). NOD TLR4−/− mice had a higher activity of matrix metalloproteinase-2 (MMP-2), a marker of extracellular matrix integrity (Fig. S1C), but unchanged levels of other mediators of systemic inflammation (Fig. S1, D and E). Mucispirillum content correlated with plasma glucagon like peptide (GLP)-2 only in NOD TLR4−/− mice, again indicating impaired gut integrity of NOD TLR4−/− mice (Fig. S2). As gut wall integrity and intestinal microbiome composition strongly affect the availability of bacterial products in the host's circulation, we determined the levels of plasma components reflecting absorption of gut-derived bacterial products and lipids. When compared with NOD TLR4+/+ mice, NOD TLR4−/− mice showed higher plasma concentrations of LPS (Fig. 3A), a cell wall component of Gram-negative bacteria that can be incorporated into chylomicrons. NOD TLR4−/− mice also had higher levels of triglycerides (TG) and FFA than NOD TLR4+/+ mice in plasma (Fig. 3, B and C). We next measured circulating levels of fetuin A in serum, an abundant liver-derived glycoprotein, acting as an adaptor to enable the interaction of FFA with TLR4 (29Pal D. Dasgupta S. Kundu R. Maitra S. Das G. Mukhopadhyay S. Ray S. Majumdar S.S. Bhattacharya S. Fetuin-A acts as an endogenous ligand of TLR4 to promote lipid-induced insulin resistance.Nat. Med. 2012; 18 (22842477): 1279-128510.1038/nm.2851Crossref PubMed Scopus (634) Google Scholar). However, both NOD TLR4+/+ and NOD TLR4−/− mice showed high but comparable fetuin A concentrations (Fig. 3D). As intestinal bacteria-derived SCFA are potent modulators of host energy metabolism, we determined their concentrations in the lumen of small intestine, cecum, and colon using GC-MS. Highest SCFA concentrations were found in the two distal gut segments with acetic acid, propionic acid, and butyric acid as the most abundant species (Fig. 4). No differences were observed in SCFA concentrations in gut segments from TLR4+/+ and NOD TLR4−/− mice. In the plasma of both NOD TLR4+/+ and NOD TLR4−/− mice, acetic acid, propionic acid, and butyric acid were also identified as the most abundant SCFA. However, NOD TLR4−/− mice had 12, 43, and 68% lower concentrations of acetic acid, propionic acid, and butyric acid, respectively (Fig. 5A). Only low concentrations of isobutyric acid, valeric and isovaleric acid, isocaproic acid, and hexanoic acid were detectable. The total concentration of peripheral SCFA was markedly lower in NOD TLR4−/− than in NOD TLR4+/+ mice (p < 0.001) (Fig. 5B). Metabolic phenotyping during three dark and two light phases revealed lower energy expenditure during light phases, occurring independently of the TLR4 expression status (Fig. 6A). Physical activity followed a similar pattern, showing comparable low levels during the light phase and 3–4-fold increases in the dark phase (Fig. 6B). Interestingly, NOD TLR4−/− mice had greater maximum physical activity than NOD TLR4+/+ mice during the dark phase. All mice also exhibited the typical pattern of circadian rhythmicity of the respiratory exchange rate (RQ) with maximum values in the dark phases (Fig. 6, C and D). NOD TLR4−/− mice had consistently lower RQ values than NOD TLR4+/+ mice (Fig. 6D), suggesting increased utilization of dietary fat as energy source. Intraperitoneal glucose tolerance tests (ipGTT) revealed severe glucose intolerance with elevation of both glucose peak (Fig. 6E) and overall glucose appearance, as measured from the area under the glucose concentration curve, in NOD TLR4−/− mice (Fig. 6F). Of note, peak glucose levels occurred 25 min later, pointing to delayed glucose absorption in the TLR4-deficient mice. Serum insulin concentration, measured at the end of the ipGTT, was not appropriately increased in NOD TLR4−/−, indicating impaired glucose-dependent insulin secretion (Fig. 6G). To assess insulin sensitivity in more detail, the animals underwent an intraperitoneal insulin tolerance test (ipITT), which showed marked whole-body insulin resistance in NOD TLR4−/− mice (Fig. 6H). In addition, in line with hepatic insulin resistance these mice had higher HOMA-IR values (Fig. 6I). To examine glucose-responsive insulin secretion, we isolated islets of Langerhans from pancreata of the mice. These studies revealed comparable dose-dependent glucose-stimulated insulin release in NOD TLR4+/+ and NOD TLR4−/− mice (Fig. 6J). Reduced RQ and glucose tolerance along with higher plasma lipids in NOD TLR4−/− mice indicate abnormal energy metabolism. For the direct measurement of substrate-dependent oxidation, we employed high-resolution respirometry on samples from soleus muscle and liver to quantify the mitochondrial oxygen fluxes via complex I and complexes I and II and to assess maximum respiratory capacity (Fig. 7). In soleus muscle, TLR4 expression status did not affect oxygen fluxes (Fig. 7A). In contrast, livers from NOD TLR4−/− mice showed 60–86% higher rates of oxygen flux in complex I and complexes I and II as well as maximal respiratory capacity compared with those obtained from NOD TLR4+/+ mice (Fig. 7B). This indicates hepatic mitochondrial adaptation to greater glucose and lipid availability as reported previously in insulin-resistant rodent models and humans (8Jelenik T. Séquaris G. Kaul K. Ouwens D.M. Phielix E. Kotzka J. Knebel B. Weiss J. Reinbeck A.L. Janke L. Nowotny P. Partke H.J. Zhang D. Shulman G.I. Szendroedi J. Roden M. Tissue-specific differences in the development of insulin resistance in a mouse model for type 1 diabetes.Diabetes. 2014; 63 (24917575): 3856-386710.2337/db13-1794Crossref PubMed Scopus (47) Google Scholar, 30Koliaki C. Szendroedi J. Kaul K. Jelenik T. Nowotny P. Jankowiak F. Herder C. Carstensen M. Krausch M. Knoefel W.T. Schlensak M. Roden M. Adaptation of hepatic mitochondrial function in humans with non-alcoholic fatty liver is lost in steatohepatitis.Cell Metab. 2015; 21 (25955209): 739-74610.1016/j.cmet.2015.04.004Abstract Full Text Full Text PDF PubMed Scopus (549) Google Scholar). These data show that profound alterations of gut morphology and microbiota and tissue-specific abnormalities of energy metabolism associate with altered availability of SCFA and precede accelerated diabetes progression in a model of T1D. This study also suggests a complex relationship between TLR4-dependent intestinal abnormalities and altered inter-organ communication by an imbalance between SCFA and FFA resulting in disease-promoting conditions in the prediabetic state. The earlier onset of diabetes and higher blood glucose levels at disease onset in NOD TLR4−/− mice confirmed the diabetes-accelerating effect of TLR4 deficiency in this NOD mouse model (14Gülden E. Ihira M. Ohashi A. Reinbeck A.L. Freudenberg M.A. Kolb H. Burkart V. Toll-like receptor 4 deficiency accelerates the development of insulin-deficient diabetes in nonobese diabetic mice.PLoS ONE. 2013; 8 (24086519): e7538510.1371/journal.pone.0075385Crossref PubMed Scopus (41) Google Scholar). This study provides evidence that TLR4 deficiency promotes the development of diabetes under conditions of early alterations of gut microbiota composition and gut morphology before disease onset. The TLR4 expression status determines important structural and functional properties of the intestinal system. By controlling the proliferation of smooth muscle cells, this receptor is involved in the regulation of muscle tissue expansion (31Yin Q. Jiang D. Li L. Yang Y. Wu P. Luo Y. Yang R. Li D. LPS promotes vascular smooth muscle cells proliferation through the TLR4/Rac1/Akt signalling pathway.Cell. Physiol. Biochem. 2017; 44 (29298445): 2189-220010.1159/000486024Crossref PubMed Scopus (19) Google Scholar). As intestinal smooth muscle cells express functional TLR4 (32Rumio C. Besusso D. Arnaboldi F. Palazzo M. Selleri S. Gariboldi S. Akira S. Uematsu S. Bignami P. Ceriani V. Ménard S. Balsari A. Activation of smooth muscle and myenteric plexus cells of jejunum via Toll-like receptor 4.J. Cell. Physiol. 2006; 208 (16523497): 47-5410.1002/jcp.20632Crossref PubMed Scopus (57) Google Scholar), TLR4 deficiency may foster decreasing the thickness of the intestinal muscle layer as observed in the NOD TLR4−/− mice in this study. Reduced intestinal muscle thickness is known to impair gut motility (33Uranga J.A. Garcia-Martinez J.M. Garcia-Jimenez C. Vera G. Martin-Fontelles M.I. Abalo R. Alterations in the small intestinal wall and motor function after repeated cisplatin in rat.Neurogastroenterol. Motil. 2017; 29 (28261911)10.1111/nmo.13047Crossref Scopus (15) Google Scholar). Moreover, the contractile function of intestinal smooth muscle cells also depends on the appropriate expression of TLR4 (34Forcén R. Latorre E. Pardo J. Alcalde A.I. Murillo M.D. Grasa L. Toll-like receptors 2 and 4 modulate the contractile response induced by serotonin in mouse ileum: analysis of the serotonin receptors involved.Neurogastroenterol. Motil. 2015; 27 (26053401): 1258-126610.1111/nmo.12619Crossref PubMed Scopus (21) Google Scholar). Thus, TLR4 deficiency likely promotes prolongation of the intestinal transit time of gut contents (35Anitha M. Vijay-Kumar M. Sitaraman S.V. Gewirtz A.T. Srinivasan S. Gut microbial products regulate murine gastrointestinal motility via Toll-like receptor 4 signaling.Gastroenterology. 2012; 143 (22732731): 1006-1016.e410.1053/j.gastro.2012.06.034Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar) as reported recently in metabolically healthy C57BL6 mice carrying a homozygous TLR4 defect (36Caputi V. Marsilio I. Cerantola S. Roozfarakh M. Lante I. Galuppini F. Rugge M. Napoli E. Giulivi C. Orso G. Giron M.C. Toll-like receptor 4 modulates small intestine neuromuscular function through nitrergic and purinergic pathways.Front. Pharmacol. 2017; 8 (28642706): 35010.3389/fphar.2017.00350Crossref PubMed Scopus (33) Google Scholar). Consequently, reduced gut motility and prolonged intestinal transit time cause alterations of the digestive process and the accumulation of absorbable food components (37Kasubuchi M. Hasegawa S. Hiramatsu T. Ichimura A. Kimura I. Dietary gut microbial metabolites, short-chain fatty acids, and host metabolic regulation.Nutrients. 2015; 7 (25875123): 2839-284910.3390/nu7042839Crossref PubMed Scopus (521) Google Scholar), thereby modulating the survival conditions for distinct bacterial communities. A recent study demonstrated such an association between delayed intestinal transit and alterations of gut microbiome composition (38Liu Y. Jin Y. Li J. Zhao L. Li Z. Xu J. Zhao F. Feng J. Chen H. Fang C. Shilpakar R. Wei Y. Small bowel transit and altered gut microbiota in patients with liver cirrhosis.Front. Physiol. 2018; 9 (29780327): 47010.3389/fphys.2018.00470Crossref PubMed Scopus (16) Google Scholar). In this study, NOD TLR4−/− mice showed an imbalance of the two most abundant bacterial phyla when compared with NOD TLR4+/+ mice. Bacteroidetes were increased while Firmicutes were decreased in the distal gut segments of normoglycemic TLR4-deficient mice at an age of 70–90 days, i.e. before the age of diabetes-onset. This finding corresponds to observations in patients with T1D and in children at increased risk for T1D showing a predominance of distinct Bacteroidetes species in their gut microbiome prior to the clinical onset of auto-immunity (21Brown C.T. Davis-Richardson A.G. Giongo A. Gano K.A. Crabb D.B. Mukherjee N. Casella G. Drew J.C. Ilonen J. Knip M. Hyoty H. Veijola R. Simell T. Simell O. Neu J. et al.Gut microbiome metagenomics analysis suggests a functional model for the development of autoimmunity for type 1 diabetes.PLoS ONE. 2011; 6 (22043294): e2579210.1371/journal.pone.0025792Crossref PubMed Scopus (552) Google Scholar). Of note, some previous studies suggested an involvement of gut microbiota in the development of insulin-deficient diabetes (3Knip M. Siljander H. The role of the intestinal microbiota in type 1 diabetes mellitus.Nat. Rev. Endocrinol. 2016; 12 (26729037): 154-16710.1038/nrendo.2015.218Crossref PubMed Scopus (260) Google Scholar, 39Wen L. Ley R.E. Volchkov P.Y. Stranges P.B. Avanesyan L. Stonebraker A.C. Hu C. Wong F.S. Szot G.L. Bluestone