GLP-1R signaling modulates colonic energy metabolism, goblet cell number and survival in the absence of gut microbiota.

OBJECTIVES: Gut microbiota increases energy availability through fermentation of dietary fibers to short-chain fatty acids in conventionally raised mice. Energy deficiency in germ-free (GF) mice increases glucagon-like peptide-1 (GLP-1) levels, which slows intestinal transit. To further analyze the role of GLP-1-mediated signaling in this model of energy deficiency, we re-derived mice lacking GLP-1 receptor (GLP-1R KO) as GF. METHODS: GLP-1R KO mice were rederived as GF through hysterectomy and monitored for 30 weeks. Mice were subjected to rescue experiments either through feeding an energy-rich diet or colonization with a normal cecal microbiota. Histology and intestinal function were assessed at different ages. Intestinal organoids were assessed to investigate stemness. RESULTS: Unexpectedly, 25% of GF GLP-1R KO mice died before 20 weeks of age, associated with enlarged ceca, increased cecal water content, increased colonic expression of apical ion transporters, reduced number of goblet cells and loss of colonic epithelial integrity. Colonocytes from GLP-1R KO mice were energy-deprived and exhibited increased ER-stress; mitochondrial fragmentation, increased oxygen levels and loss of stemness. Restoring colonic energy levels either by feeding a Western-style diet or colonization with a normal gut microbiota normalized gut phenotypes and prevented lethality. CONCLUSIONS: Our findings reveal a heretofore unrecognized role for GLP-1R signaling in the maintenance of colonic physiology and survival during energy deprivation.

Cold Exposure and Oral Delivery of GLP-1R Agonists by an Engineered Probiotic Yeast Strain Have Antiobesity Effects in Mice.

Advanced microbiome therapeutics (AMTs) holds promise in utilizing engineered microbes such as bacteria or yeasts for innovative therapeutic applications, including the in situ delivery of therapeutic peptides. Glucagon-like peptide-1 receptor agonists, such as Exendin-4, have emerged as potential treatments for type 2 diabetes and obesity. However, current administration methods face challenges with patient adherence and low oral bioavailability. To address these limitations, researchers are exploring improved oral delivery methods for Exendin-4, including utilizing AMTs. This study engineered the probiotic yeast Saccharomyces boulardii to produce Exendin-4 (Sb-Exe4) in the gastrointestinal tract of male C57BL/6 mice to combat diet-induced obesity. The biological efficiency of Exendin-4 secreted by S. boulardii was analyzed ex vivo on isolated pancreatic islets, demonstrating induced insulin secretion. The in vivo characterization of Sb-Exe4 revealed that when combined with cold exposure (8 °C), the Sb-Exe4 yeast strain successfully suppressed appetite by 25% and promoted a 4-fold higher weight loss. This proof of concept highlights the potential of AMTs to genetically modify S. boulardii for delivering active therapeutic peptides in a precise and targeted manner. Although challenges in efficacy and regulatory approval persist, AMTs may provide a transformative platform for personalized medicine. Further research in AMTs, particularly focusing on probiotic yeasts such as S. boulardii, holds great potential for novel therapeutic possibilities and enhancing treatment outcomes in diverse metabolic disorders.

Non-Contact Face Temperature Measurement by Thermopile-Based Data Fusion.

Thermal imaging cameras and infrared (IR) temperature measurement devices act as state-of-the-art techniques for non-contact temperature determination of the skin surface. The former is cost-intensive in many cases for widespread application, and the latter requires manual alignment to the measuring point. Due to this background, this paper proposes a new method for automated, non-contact, and area-specific temperature measurement of the facial skin surface. It is based on the combined use of a low-cost thermopile sensor matrix and a 2D image sensor. The temperature values as well as the 2D image data are fused using a parametric affine transformation. Based on face recognition, this allows temperature values to be assigned to selected facial regions and used specifically to determine the skin surface temperature. The advantages of the proposed method are described. It is demonstrated by means of a participant study that the temperature absolute values, which are achieved without manual alignment in an automated manner, are comparable to a commercially available IR-based forehead thermometer.

Absence of gut microbiota reduces neonatal survival and exacerbates liver disease in Cyp2c70-deficient mice with a human-like bile acid composition.

Mice with deletion of Cyp2c70 have a human-like bile acid composition, display age- and sex-dependent signs of hepatobiliary disease and can be used as a model to study interactions between bile acids and the gut microbiota in cholestatic liver disease. In the present study, we rederived Cyp2c70-/- mice as germ-free (GF) and colonized them with a human or a mouse microbiota to investigate whether the presence of a microbiota can be protective in cholangiopathic liver disease associated with Cyp2c70-deficiency. GF Cyp2c70-/- mice showed reduced neonatal survival, liver fibrosis, and distinct cholangiocyte proliferation. Colonization of germ-free breeding pairs with a human or a mouse microbiota normalized neonatal survival of the offspring, and particularly colonization with mouse microbiota from a conventionally raised mouse improved the liver phenotype at 6-10 weeks of age. The improved liver phenotype in conventionalized (CD) Cyp2c70-/- mice was associated with increased levels of tauro-ursodeoxycholic acid (TUDCA) and UDCA, resulting in a more hydrophilic bile acid profile compared with GF and humanized Cyp2c70-/- mice. The hydrophobicity index of biliary bile acids of CD Cyp2c70-/- mice was associated with changes in gut microbiota, liver weight, liver transaminases, and liver fibrosis. Hence, our results indicate that neonatal survival of Cyp2c70-/- mice seems to depend on the establishment of a gut microbiota at birth, and the improved liver phenotype in CD Cyp2c70-/- mice may be mediated by a larger proportion of TUDCA/UDCA in the circulating bile acid pool and/or by the presence of specific bacteria.

Targeting the Gut in Obesity: Signals from the Inner Surface.

Obesity is caused by prolonged energy surplus. Current anti-obesity medications are mostly centralized around the energy input part of the energy balance equation by increasing satiety and reducing appetite. Our gastrointestinal tract is a key organ for regulation of food intake and supplies a tremendous number of circulating signals that modulate the activity of appetite-regulating areas of the brain by either direct interaction or through the vagus nerve. Intestinally derived messengers are manifold and include absorbed nutrients, microbial metabolites, gut hormones and other enterokines, collectively comprising a fine-tuned signalling system to the brain. After a meal, nutrients directly interact with appetite-inhibiting areas of the brain and induce satiety. However, overall feeding behaviour also depends on secretion of gut hormones produced by highly specialized and sensitive enteroendocrine cells. Moreover, circulating microbial metabolites and their interactions with enteroendocrine cells further contribute to the regulation of feeding patterns. Current therapies exploiting the appetite-regulating properties of the gut are based on chemically modified versions of the gut hormone, glucagon-like peptide-1 (GLP-1) or on inhibitors of the primary GLP-1 inactivating enzyme, dipeptidyl peptidase-4 (DPP-4). The effectiveness of these approaches shows that that the gut is a promising target for therapeutic interventions to achieve significant weigh loss. We believe that increasing understanding of the functionality of the intestinal epithelium and new delivery systems will help develop selective and safe gut-based therapeutic strategies for improved obesity treatment in the future. Here, we provide an overview of the major homeostatic appetite-regulating signals generated by the intestinal epithelial cells and how these signals may be harnessed to treat obesity by pharmacological means.

BMP4 gene therapy enhances insulin sensitivity but not adipose tissue browning in obese mice.

OBJECTIVE: Bone morphogenetic protein 4 (BMP4) adeno-associated viral vectors of serotype 8 (AAV8) gene therapy targeting the liver prevents the development of obesity in initially lean mice by browning the large subcutaneous white adipose tissue (WAT) and enhancing energy expenditure. Here, we examine whether this approach could also reduce established obesity. METHODS: Dietary-induced obese C57BL6/N mice received AAV8 BMP4 gene therapy at 17-18 weeks of age. They were kept on a high-fat diet and phenotypically characterized for an additional 10-12 weeks. Following termination, the mice underwent additional characterization in vitro. RESULTS: Surprisingly, we observed no effect on body weight, browning of WAT, or energy expenditure in these obese mice, but whole-body insulin sensitivity and glucose tolerance were robustly improved. Insulin signaling and insulin-stimulated glucose uptake were increased in both adipose cells and skeletal muscle. BMP4 also decreased hepatic glucose production and reduced gluconeogenic enzymes in the liver, but not in the kidney, in addition to enhancing insulin action in the liver. CONCLUSIONS: Our findings show that BMP4 prevents, but does not reverse, established obesity in adult mice, while it improves insulin sensitivity independent of weight reduction. The BMP antagonist Noggin was increased in WAT in obesity, which may account for the lack of browning.

Erratum: EGFR signalling controls cellular fate and pancreatic organogenesis by regulating apicobasal polarity.

This corrects the article DOI: 10.1038/ncb3628.

EGFR signalling controls cellular fate and pancreatic organogenesis by regulating apicobasal polarity.

Apicobasal polarity is known to affect epithelial morphogenesis and cell differentiation, but it remains unknown how these processes are mechanistically orchestrated. We find that ligand-specific EGFR signalling via PI(3)K and Rac1 autonomously modulates apicobasal polarity to enforce the sequential control of morphogenesis and cell differentiation. Initially, EGF controls pancreatic tubulogenesis by negatively regulating apical polarity induction. Subsequently, betacellulin, working via inhibition of atypical protein kinase C (aPKC), causes apical domain constriction within neurogenin3+ endocrine progenitors, which results in reduced Notch signalling, increased neurogenin3 expression, and ß-cell differentiation. Notably, the ligand-specific EGFR output is not driven at the ligand level, but seems to have evolved in response to stage-specific epithelial influences. The EGFR-mediated control of ß-cell differentiation via apical polarity is also conserved in human neurogenin3+ cells. We provide insight into how ligand-specific EGFR signalling coordinates epithelial morphogenesis and cell differentiation via apical polarity dynamics.

Shape analysis of the cervical spinous process.

Most textbooks describe the bifid spinous process as a shape associated with the typical cervical vertebra. Somewhere later they may acknowledge that cervical vertebrae are not always bifid, and that its appearance may be asymmetric. A high incidence of bifid cervical spinous processes may be a human characteristic, but because of known racial/geographic variation it may not be a very good one. Rarely can one find a satisfactory explanation of the functional or developmental basis for this shape variation. This article explores the distinctive shape of the cervical spinous process. Analysis is based upon the spinous processes of the third through seventh cervical vertebrae from fifty individuals. Shape differences were evaluated using the techniques of geometric morphometrics. Statistical comparisons were based upon 1000 permutations of a MANOVA based analysis. Significant shape differences were identified among the cervical vertebrae. However, post hoc analysis failed to identify significant differences between the C3 and C4 and between the C4 and C5 spinous process shapes. Primary shape differences were due to the depth of the bifid separation and the length of the process. Vertebrae with shorter spinous processes tended to display a more pronounced bifid condition. Combined observations from this and several other investigations suggest that a combination of variation in the spinalis cervicis muscle and behavioral patterns associated with cervical load may provide the best explanation for the shape variation in the cervical spinous process. Clin. Anat. 30:894-900, 2017. © 2017 Wiley Periodicals, Inc.

Microbiota-induced obesity requires farnesoid X receptor.

OBJECTIVE: The gut microbiota has been implicated as an environmental factor that modulates obesity, and recent evidence suggests that microbiota-mediated changes in bile acid profiles and signalling through the bile acid nuclear receptor farnesoid X receptor (FXR) contribute to impaired host metabolism. Here we investigated if the gut microbiota modulates obesity and associated phenotypes through FXR. DESIGN: We fed germ-free (GF) and conventionally raised (CONV-R) wild-type and Fxr-/- mice a high-fat diet (HFD) for 10 weeks. We monitored weight gain and glucose metabolism and analysed the gut microbiota and bile acid composition, beta-cell mass, accumulation of macrophages in adipose tissue, liver steatosis, and expression of target genes in adipose tissue and liver. We also transferred the microbiota of wild-type and Fxr-deficient mice to GF wild-type mice. RESULTS: The gut microbiota promoted weight gain and hepatic steatosis in an FXR-dependent manner, and the bile acid profiles and composition of faecal microbiota differed between Fxr-/- and wild-type mice. The obese phenotype in colonised wild-type mice was associated with increased beta-cell mass, increased adipose inflammation, increased steatosis and expression of genes involved in lipid uptake. By transferring the caecal microbiota from HFD-fed Fxr-/- and wild-type mice into GF mice, we showed that the obesity phenotype was transferable. CONCLUSIONS: Our results indicate that the gut microbiota promotes diet-induced obesity and associated phenotypes through FXR, and that FXR may contribute to increased adiposity by altering the microbiota composition.

The gut microbiota contributes to a mouse model of spontaneous bile duct inflammation.

BACKGROUND & AIMS: A strong association between human inflammatory biliary diseases and gut inflammation has led to the hypothesis that gut microbes and lymphocytes activated in the intestine play a role in biliary inflammation. The NOD.c3c4 mouse model develops spontaneous biliary inflammation in extra- and intrahepatic bile ducts. We aimed to clarify the role of the gut microbiota in the biliary disease of NOD.c3c4 mice. METHODS: We sampled cecal content and mucosa from conventionally raised (CONV-R) NOD.c3c4 and NOD control mice, extracted DNA and performed 16S rRNA sequencing. NOD.c3c4 mice were rederived into a germ free (GF) facility and compared with CONV-R NOD.c3c4 mice. NOD.c3c4 mice were also co-housed with NOD mice and received antibiotics from weaning. RESULTS: The gut microbial profiles of mice with and without biliary disease were different both before and after rederivation (unweighted UniFrac-distance). GF NOD.c3c4 mice had less distended extra-hepatic bile ducts than CONV-R NOD.c3c4 mice, while antibiotic treated mice showed reduction of biliary infarcts. GF animals also showed a reduction in liver weight compared with CONV-R NOD.c3c4 mice, and this was also observed in antibiotic treated NOD.c3c4 mice. Co-housing of NOD and NOD.c3c4 mice indicated that the biliary phenotype was neither transmissible nor treatable by co-housing with healthy mice. CONCLUSIONS: NOD.c3c4 and NOD control mice show marked differences in the gut microbiota. GF NOD.c3c4 mice develop a milder biliary affection compared with conventionally raised NOD.c3c4 mice. Our findings suggest that the intestinal microbiota contributes to disease in this murine model of biliary inflammation. LAY SUMMARY: Mice with liver disease have a gut microflora (microbiota) that differs substantially from normal mice. In a normal environment, these mice spontaneously develop disease in their bile ducts. However, when these mice, are raised in an environment devoid of bacteria, the disease in the bile ducts diminishes. Overall this clearly indicates that the bacteria in the gut (the gut microbiota) influences the liver disease in these mice.

Antibiotic-mediated gut microbiome perturbation accelerates development of type 1 diabetes in mice.

The early life microbiome plays important roles in host immunological and metabolic development. Because the incidence of type 1 diabetes (T1D) has been increasing substantially in recent decades, we hypothesized that early-life antibiotic use alters gut microbiota, which predisposes to disease. Using non-obese diabetic mice that are genetically susceptible to T1D, we examined the effects of exposure to either continuous low-dose antibiotics or pulsed therapeutic antibiotics (PAT) early in life, mimicking childhood exposures. We found that in mice receiving PAT, T1D incidence was significantly higher, and microbial community composition and structure differed compared with controls. In pre-diabetic male PAT mice, the intestinal lamina propria had lower Th17 and Treg proportions and intestinal SAA expression than in controls, suggesting key roles in transducing the altered microbiota signals. PAT affected microbial lipid metabolism and host cholesterol biosynthetic gene expression. These findings show that early-life antibiotic treatments alter the gut microbiota and its metabolic capacities, intestinal gene expression and T-cell populations, accelerating T1D onset in non-obese diabetic mice.

Microbially produced glucagon-like peptide 1 improves glucose tolerance in mice.

OBJECTIVE: The enteroendocrine hormone glucagon-like peptide 1 (GLP-1) is an attractive anti-diabetic therapy. Here, we generated a recombinant Lactococcus lactis strain genetically modified to produce GLP-1 and investigated its ability to improve glucose tolerance in mice on chow or high-fat diet (HFD). METHODS: We transformed L. lactis FI5876 with either empty vector (pUK200) or murine GLP-1 expression vector to generate LL-UK200 and LL-GLP1, respectively, and determined their potential to induce insulin secretion by incubating primary islets from wild-type (WT) and GLP-1 receptor knockout (GLP1R-KO) mice with culture supernatant of these strains. In addition, we administered these strains to mice on chow or HFD. At the end of the study period, we measured plasma GLP-1 levels, performed intraperitoneal glucose tolerance and insulin tolerance tests, and determined hepatic expression of the gluconeogenic genes G6pc and Pepck. RESULTS: Insulin release from primary islets of WT but not GLP1R-KO mice was higher following incubation with culture supernatant from LL-GLP1 compared with LL-UK200. In mice on chow, supplementation with LL-GLP1 versus LL-UK200 promoted increased vena porta levels of GLP-1 in both WT and GLP1R-KO mice; however, LL-GLP1 promoted improved glucose tolerance in WT but not in GLP1R-KO mice, indicating a requirement for the GLP-1 receptor. In mice on HFD and thus with impaired glucose tolerance, supplementation with LL-GLP1 versus LL-UK200 promoted a pronounced improvement in glucose tolerance together with increased insulin levels. Supplementation with LL-GLP1 versus LL-UK200 did not affect insulin tolerance but resulted in reduced expression of G6pc in both chow and HFD-fed mice. CONCLUSIONS: The L. lactis strain genetically modified to produce GLP-1 is capable of stimulating insulin secretion from islets and improving glucose tolerance in mice.

Microbial regulation of GLP-1 and L-cell biology.

BACKGROUND: The gut microbiota is associated with several of metabolic diseases, including obesity and type 2 diabetes and affects host physiology through distinct mechanisms. The microbiota produces a vast array of metabolites that signal to host cells in the intestine as well as in more distal organs. SCOPE OF REVIEW: Enteroendocrine cells acts as 'chemo sensors' of the intestinal milieu by expressing a large number of receptors, which respond to different metabolites and nutrients, and signal to host by a wide variety of hormones. However, enteroendocrine cells differ along the length of the gut in terms of hormones expressed and receptor repertoire. Also, the microbial ecology and dietary substrates differ along the length of the gut, providing further evidence for unique functions of specific subpopulations among enteroendocrine cells. Here we will review how the gut microbiota interacts with L-cells in the small and large intestine and the resulting effects on the host. MAJOR CONCLUSIONS: Microbial metabolites can be sensed differently by specific subpopulations of enteroendocrine cells. Furthermore, hormones such as GLP-1 can have different functions when originating from the small intestine or colon. This article is part of a special issue on microbiota.

The gut microbiota modulates glycaemic control and serum metabolite profiles in non-obese diabetic mice.

Islet autoimmunity in children who later progress to type 1 diabetes is preceded by dysregulated serum metabolite profiles, but the origin of these metabolic changes is unknown. The gut microbiota affects host metabolism and changes in its composition contribute to several immune-mediated diseases; however, it is not known whether the gut microbiota is involved in the early metabolic disturbances in progression to type 1 diabetes. We rederived non-obese diabetic (NOD) mice as germ free to explore the potential role of the gut microbiota in the development of diabetic autoimmunity and to directly investigate whether the metabolic profiles associated with the development of type 1 diabetes can be modulated by the gut microbiota. The absence of a gut microbiota in NOD mice did not affect the overall diabetes incidence but resulted in increased insulitis and levels of interferon gamma and interleukin 12; these changes were counterbalanced by improved peripheral glucose metabolism. Furthermore, we observed a markedly increased variation in blood glucose levels in the absence of a microbiota in NOD mice that did not progress to diabetes. Additionally, germ-free NOD mice had a metabolite profile similar to that of pre-diabetic children. Our data suggest that germ-free NOD mice have reduced glycaemic control and dysregulated immunologic and metabolic responses.

Kinematics of primate midfoot flexibility.

This study describes a unique assessment of primate intrinsic foot joint kinematics based upon bone pin rigid cluster tracking. It challenges the assumption that human evolution resulted in a reduction of midfoot flexibility, which has been identified in other primates as the "midtarsal break." Rigid cluster pins were inserted into the foot bones of human, chimpanzee, baboon, and macaque cadavers. The positions of these bone pins were monitored during a plantarflexion-dorsiflexion movement cycle. Analysis resolved flexion-extension movement patterns and the associated orientation of rotational axes for the talonavicular, calcaneocuboid, and lateral cubometatarsal joints. Results show that midfoot flexibility occurs primarily at the talonavicular and cubometatarsal joints. The rotational magnitudes are roughly similar between humans and chimps. There is also a similarity among evaluated primates in the observed rotations of the lateral cubometatarsal joint, but there was much greater rotation observed for the talonavicular joint, which may serve to differentiate monkeys from the hominines. It appears that the capability for a midtarsal break is present within the human foot. A consideration of the joint axes shows that the medial and lateral joints have opposing orientations, which has been associated with a rigid locking mechanism in the human foot. However, the potential for this same mechanism also appears in the chimpanzee foot. These findings demonstrate a functional similarity within the midfoot of the hominines. Therefore, the kinematic capabilities and restrictions for the skeletal linkages of the human foot may not be as unique as has been previously suggested.

Cdc42/N-WASP signaling links actin dynamics to pancreatic ß cell delamination and differentiation.

Delamination plays a pivotal role during normal development and cancer. Previous work has demonstrated that delamination and epithelial cell movement within the plane of an epithelium are associated with a change in cellular phenotype. However, how this positional change is linked to differentiation remains unknown. Using the developing mouse pancreas as a model system, we show that ß cell delamination and differentiation are two independent events, which are controlled by Cdc42/N-WASP signaling. Specifically, we show that expression of constitutively active Cdc42 in ß cells inhibits ß cell delamination and differentiation. These processes are normally associated with junctional actin and cell-cell junction disassembly and the expression of fate-determining transcription factors, such as Isl1 and MafA. Mechanistically, we demonstrate that genetic ablation of N-WASP in ß cells expressing constitutively active Cdc42 partially restores both delamination and ß cell differentiation. These findings elucidate how junctional actin dynamics via Cdc42/N-WASP signaling cell-autonomously control not only epithelial delamination but also cell differentiation during mammalian organogenesis.

Microbial modulation of energy availability in the colon regulates intestinal transit.

Gut microbiota contribute to host metabolic efficiency by increasing energy availability through the fermentation of dietary fiber and production of short-chain fatty acids (SCFAs) in the colon. SCFAs are proposed to stimulate secretion of the proglucagon (Gcg)-derived incretin hormone GLP-1, which stimulates insulin secretion (incretin response) and inhibits gastric emptying. We find that germ-free (GF) and antibiotic-treated mice, which have severely reduced SCFA levels, have increased basal GLP-1 levels in the plasma and increased Gcg expression in the colon. Increasing energy supply, either through colonization with polysaccharide-fermenting bacteria or through diet, suppressed colonic Gcg expression in GF mice. Increased GLP-1 levels in GF mice did not improve the incretin response but instead slowed intestinal transit. Thus, microbiota regulate the basal levels of GLP-1, and increasing these levels may be an adaptive response to insufficient energy availability in the colon that slows intestinal transit and allows for greater nutrient absorption.