Genome-wide association studies (GWAS) have recently identified several new genetic variants associated with obesity. The majority of the variants are within introns or between genes, suggesting they affect gene expression, although it is not clear which of the nearby genes they affect. Understanding the regulation of these genes will be key to determining the role of these variants in the development of obesity and will provide support for a role of these genes in the development of obesity.

Since the development of the Edmonton protocol, islet transplantation is increasingly encouraging as a treatment for type 1 diabetes. Strategies to ameliorate problems with the intraportal site include macroencapsulating the islets in diverse biomaterials. Characterization of these biomaterials is important to optimally tune the properties to support islets and promote vascularization. In this study, we characterize the cross-linker-dependent properties of collagen-based matrices containing chondroitin-6-sulfate, chitosan, and laminin, cross-linked with 7.5, 30, or 120 mM of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide. The swelling ratio was found to be significantly negatively correlated with increasing cross-linker concentrations (p<0.0001; R2=0.718). The matrix released insulin in a reproducible logarithmic manner (R2 of 0.99 for all concentrations), demonstrating cross-linker-dependent control of drug release. The matrices with the highest cross-linker concentrations resisted degradation by collagenase for longer than the lowest concentrations (58.13%±2.22% vs. 13.69%±7.67%; p<0.05). Scanning electron microscopy images of the matrices revealed that the matrices had uniform topography and porosity, indicating efficient cross-linking and incorporation of the polymer components. Matrices were transplanted subcutaneously in naive BALB/c mice, and the number and size of vessels were quantified using von Willebrand factor staining; matrices with higher cross-linking concentrations had significantly larger capillaries at every time point up to 4 weeks after transplantation compared to the lowest cross-linker concentration group. CD31 staining visualized the capillaries at each time point. Taken together, these data show that this collagen-based matrix is reproducible with cross-linking-dependent properties that can be optimized to support vascularization and islet function.

Development of self-regulatory competencies during adolescence is partially dependent on normative brain maturation. Here, we report that adolescent rats as compared to adults exhibit impulsive and compulsive-like behavioral traits, the latter being associated with lower expression of mRNA levels of the immediate early gene zif268 in the anterior insula cortex (AIC). This suggests that underdeveloped AIC function in adolescent rats could contribute to an immature pattern of interoceptive cue integration in decision making and a compulsive phenotype. In support of this, we report that layer 5 pyramidal neurons in the adolescent rat AIC are hypoexcitable and receive fewer glutamatergic synaptic inputs compared to adults. Chemogenetic activation of the AIC attenuated compulsive traits in adolescent rats supporting the idea that in early stages of AIC maturity there exists a suboptimal integration of sensory and cognitive information that contributes to inflexible behaviors in specific conditions of reward availability.

Type 1 diabetes (T1D) is an autoimmune disease characterised by T cell mediated destruction of pancreatic beta-cells. Islet transplantation is an effective therapy, but its success is limited by islet quality and availability along with the need for immunosuppression. New approaches include use of stem cell-derived insulin-producing cells and immunomodulatory therapies, but a limitation is the paucity of reproducible animal models in which interactions between human immune cells and insulin-producing cells can be studied without the complication of xenogeneic graft- versus -host disease (xGVHD).

A positive energy balance promotes white adipose tissue (WAT) expansion which is characterized by activation of a repertoire of events including hypoxia, inflammation and extracellular matrix remodelling. The transmembrane glycoprotein CD248 has been implicated in all these processes in different malignant and inflammatory diseases but its potential impact in WAT and metabolic disease has not been explored.

The study of primary glucagon-secreting α-cells is hampered by their low abundance and scattered distribution in rodent pancreatic islets. We have designed a double-stranded adeno-associated virus containing a rat proglucagon promoter (700 bp) driving enhanced green fluorescent protein (AAV GCG-EGFP), to specifically identify α-cells. The administration of AAV GCG-EGFP by intraperitoneal or intraductal injection led to EGFP expression selectively in the α-cell population. AAV GCG-EGFP delivery to mice followed by islet isolation, dispersion and separation by FACS for EGFP resulted in an 86% pure population of α-cells. Furthermore, the administration of AAV GCG-EGFP at various doses to adult wild type mice did not significantly alter body weight, blood glucose, plasma insulin or glucagon levels, glucose tolerance or arginine tolerance. In vitro experiments in transgene positive α-cells demonstrated that EGFP expression did not alter the intracellular Ca2+ pattern in response to glucose or adrenaline. This approach may be useful for studying purified primary α-cells and for the in vivo delivery of other genes selectively to α-cells to further probe their function or to manipulate them for therapeutic purposes.

Feeding behavior is governed by homeostatic needs and motivational drive to obtain palatable foods. Here, we identify a population of glutamatergic neurons in the paraventricular thalamus of mice that express the glucose transporter Glut2 (encoded by Slc2a2) and project to the nucleus accumbens. These neurons are activated by hypoglycemia and, in freely moving mice, their activation by optogenetics or Slc2a2 inactivation increases motivated sucrose-seeking but not saccharin-seeking behavior. These neurons may control sugar overconsumption in obesity and diabetes.

Leptin signaling in the central nervous system, and particularly the arcuate hypothalamic nucleus, is important for regulating energy and glucose homeostasis. However, the roles of extra-arcuate leptin responsive neurons are less defined. In the current study, we generated mice with widespread inactivation of the long leptin receptor isoform in the central nervous system via Synapsin promoter-driven Cre (Lepr(flox/flox) Syn-cre mice). Within the hypothalamus, leptin signaling was disrupted in the lateral hypothalamic area (LHA) and ventral premammillary nucleus (PMV) but remained intact in the arcuate hypothalamic nucleus and ventromedial hypothalamic nucleus, dorsomedial hypothalamic nucleus, and nucleus of the tractus solitarius. To investigate the role of LHA/PMV neuronal leptin signaling, we examined glucose and energy homeostasis in Lepr(flox/flox) Syn-cre mice and Lepr(flox/flox) littermates under basal and diet-induced obese conditions and tested the role of LHA/PMV neurons in leptin-mediated glucose lowering in streptozotocin-induced diabetes. Lepr(flox/flox) Syn-cre mice did not have altered body weight or blood glucose levels but were hyperinsulinemic and had enhanced glucagon secretion in response to experimental hypoglycemia. Surprisingly, when placed on a high-fat diet, Lepr(flox/flox) Syn-cre mice were protected from weight gain, glucose intolerance, and diet-induced hyperinsulinemia. Peripheral leptin administration lowered blood glucose in streptozotocin-induced diabetic Lepr(flox/flox) Syn-cre mice as effectively as in Lepr(flox/flox) littermate controls. Collectively these findings suggest that leptin signaling in LHA/PMV neurons is not critical for regulating glucose levels but has an indispensable role in the regulation of insulin and glucagon levels and, may promote the development of diet-induced hyperinsulinemia and weight gain.

Alcohol use is one of the world's leading causes of death and disease, although only a small proportion of individuals develop persistent alcohol use disorder (AUD). The identification of vulnerable individuals prior to their chronic intoxication remains of highest importance. We propose here to adapt current methodologies for identifying rats at risk of losing control over alcohol intake by modeling diagnostic criteria for AUD: inability to abstain during a signaled period of reward unavailability, increased motivation assessed in a progressive effortful task and persistent alcohol intake despite aversive foot shocks. Factor analysis showed that these three addiction criteria loaded on one underlying construct indicating that they represent a latent construct of addiction trait. Further, not only vulnerable rats displayed higher ethanol consumption, and higher preference for ethanol over sweetened solutions, but they also exhibited pre-existing higher anxiety as compared to resilient rats. In conclusion, the present preclinical model confirms that development of an addiction trait not only requires prolonged exposure to alcohol, but also depends on endophenotype like anxiety that predispose a minority of individuals to lose control over alcohol consumption.

miRNAs have crucial functions in many biological processes and are candidate biomarkers of disease. Here, we show that miR-216a is a conserved, pancreas-specific miRNA with important roles in pancreatic islet and acinar cells. Deletion of miR-216a in mice leads to a reduction in islet size, β-cell mass, and insulin levels. Single-cell RNA sequencing reveals a subpopulation of β-cells with upregulated acinar cell markers under a high-fat diet. miR-216a is induced by TGF-β signaling, and inhibition of miR-216a increases apoptosis and decreases cell proliferation in pancreatic cells. Deletion of miR-216a in the pancreatic cancer-prone mouse line KrasG12D;Ptf1aCreER reduces the propensity of pancreatic cancer precursor lesions. Notably, circulating miR-216a levels are elevated in both mice and humans with pancreatic cancer. Collectively, our study gives insights into how β-cell mass and acinar cell growth are modulated by a pancreas-specific miRNA and also suggests miR-216a as a potential biomarker for diagnosis of pancreatic diseases.

Although innate immunity is linked to metabolic health, the effect of leptin signaling in cells from the innate immune system on glucose homeostasis has not been thoroughly investigated. We generated two mouse models using Cre-lox methodology to determine the effect of myeloid cell-specific leptin receptor (Lepr) reconstitution and Lepr knockdown on in vivo glucose metabolism. Male mice with myeloid cell-specific Lepr reconstitution (Lyz2Cre+LeprloxTB/loxTB) had better glycemic control as they aged compared to male mice with whole-body transcriptional blockade of Lepr (Lyz2Cre-LeprloxTB/loxTB). In contrast, Lyz2Cre+LeprloxTB/loxTB females only had a trend for diminished hyperglycemia after a prolonged fast. During glucose tolerance tests, Lyz2Cre+LeprloxTB/loxTB males had a mildly improved plasma glucose profile compared to Cre- controls while Lyz2Cre+LeprloxTB/loxTB females had a similar glucose excursion to their Cre- controls. Myeloid cell-specific Lepr knockdown (Lyz2Cre+Leprflox/flox) did not significantly alter body weight, blood glucose, insulin sensitivity, or glucose tolerance in males or females. Expression of the cytokine interleukin 10 (anti-inflammatory) tended to be higher in adipose tissue of male Lyz2Cre+LeprloxTB/loxTB mice (p = 0.0774) while interleukin 6 (pro-inflammatory) was lower in male Lyz2Cre+Leprflox/flox mice (p < 0.05) vs. their respective controls. In conclusion, reconstitution of Lepr in cells of myeloid lineage has beneficial effects on glucose metabolism in male mice.

Identifying biological markers predicting vulnerability to develop excessive alcohol consumption may lead to a real improvement of clinical care. With converging evidence suggesting that gut microbiome is capable of influencing brain and behavior, this study aimed at investigating whether changes in gut microbiome composition is associated with conditioned responses to alcohol. We trained Wistar rats to self-administer alcohol for a prolonged period before screening those exhibiting uncontrolled alcohol seeking and taking by modeling diagnostic criteria for AUD: inability to abstain during a signaled period of reward unavailability, increased motivation assessed in a progressive effortful task and persistent alcohol intake despite aversive foot shocks. Based on addiction criteria scores, rats were assigned to either Vulnerable or Resilient groups. Vulnerable rats not only displayed increased impulsive and compulsive behaviors, but also displayed increased relapse after abstinence and increased sensitivity to baclofen treatments compared to resilient animals. Then, rats underwent a 3-month wash out period before sacrifice. Dorsal striatum was collected to assess dopamine receptor mRNA expression, and 16S microbiome sequencing was performed on caecal contents. Multiple significant correlations were found between gut microbiome and impulsivity measures, as well as augmentations in striatal Dopamine 1 receptor (D1R) and reductions in D2R as vulnerability to AUD increased. Therefore, using a singular translational approach based on biobehavioral dispositions to excessive alcohol seeking without heavy intoxication, our observations suggests an association between gut microbiome composition and these specific "at risk" behavioral traits observed in our translationally relevant model.

Glucose-dependent insulinotropic polypeptide (GIP) is a gastrointestinal hormone that potentiates glucose-stimulated insulin secretion during a meal. Since GIP has also been shown to exert β-cell prosurvival and adipocyte lipogenic effects in rodents, both GIP receptor agonists and antagonists have been considered as potential therapeutics in type 2 diabetes (T2DM). In the present study, we tested the hypothesis that chronically elevating GIP levels in a transgenic (Tg) mouse model would increase adipose tissue expansion and exert beneficial effects on glucose homeostasis. In contrast, although GIP Tg mice demonstrated enhanced β-cell function, resulting in improved glucose tolerance and insulin sensitivity, they exhibited reduced diet-induced obesity. Adipose tissue macrophage infiltration and hepatic steatosis were both greatly reduced, and a number of genes involved in lipid metabolism/inflammatory signaling pathways were found to be down-regulated. Reduced adiposity in GIP Tg mice was associated with decreased energy intake, involving overexpression of hypothalamic GIP. Together, these studies suggest that, in the context of over-nutrition, transgenic GIP overexpression has the potential to improve hepatic and adipocyte function as well as glucose homeostasis.

These data suggest that insulin secretion in WSB mice is blunted specifically in vivo, either due to a reduced insulin requirement and/or due to factors that are absent or destroyed in vitro. These studies also highlight the role of post-natal growth in determining adult β-cell mass. Mice are important animal models for the study of metabolic physiology and the genetics of complex traits. Wild-derived inbred mouse strains, such as WSB/EiJ (WSB), are unrelated to the commonly studied mouse strains and are valuable tools to identify novel genes that modify disease risk. We have previously shown that in contrast to C57BL/6J (B6) mice, WSB mice fed a high fat diet do not develop hyperinsulinemia or insulin resistance, and had nearly undetectable insulin secretion in response to an intraperitoneal glucose challenge. As hyperinsulinemia may drive obesity and insulin resistance, we examined whether defects in β-cell mass or function could contribute to the low insulin levels in WSB mice. In young WSB mice, β-cell mass was similar to B6 mice. However, we found that adult WSB mice had reduced β-cell mass due to reduced pancreatic weights. Pancreatic sizes were similar between the strains when normalized to body weight, suggesting their pancreatic size is appropriate to their body size in adults, but overall post-natal pancreatic growth was reduced in WSB mice compared to B6 mice. Islet architecture was normal in WSB mice. WSB mice had markedly increased insulin secretion from isolated islets in vitro. These data suggest that insulin secretion in WSB mice is blunted specifically in vivo, either due to a reduced insulin requirement and/or due to factors that are absent or destroyed in vitro. These studies suggest that WSB mice may provide novel insight into mechanisms regulating insulin secretion and also highlight the role of post-natal growth in determining adult β-cell mass.

The relative contribution of peripheral and central leptin signalling to the regulation of metabolism and the mechanisms through which leptin affects glucose homeostasis have not been fully elucidated. We generated complementary lines of mice with either leptin receptor (Lepr) knockdown or reconstitution in adipose tissues using Cre-lox methodology. Lepr knockdown mice were modestly lighter and had lower plasma insulin concentrations following an oral glucose challenge compared to controls, despite similar insulin sensitivity. We rendered male mice diabetic using streptozotocin (STZ) and found that upon prolonged leptin therapy, Lepr knockdown mice had an accelerated decrease in blood glucose compared to controls that was associated with higher plasma concentrations of leptin and leptin receptor. Mice with transcriptional blockade of Lepr (LeprloxTB/loxTB) were obese and hyperglycemic and reconstitution of Lepr in adipose tissues of LeprloxTB/loxTB mice resulted in males reaching a higher maximal body weight. Although mice with adipose tissue Lepr reconstitution had lower blood glucose levels at several ages, their plasma insulin concentrations during an oral glucose test were elevated. Thus, attenuation or restoration of Lepr in adipocytes alters the plasma insulin profile following glucose ingestion, modifies the glucose-lowering effect of prolonged leptin therapy in insulin-deficient diabetes, and may modulate weight gain.

The prevalence of obesity has markedly increased over the past few decades. Exploration of how hunger and satiety signals influence the reward system can help us understand non-homeostatic feeding. Insulin may act in the ventral tegmental area (VTA), a critical site for reward-seeking behavior, to suppress feeding. However, the neural mechanisms underlying insulin effects in the VTA remain unknown. We demonstrate that insulin, a circulating catabolic peptide that inhibits feeding, can induce long-term depression (LTD) of mouse excitatory synapses onto VTA dopamine neurons. This effect requires endocannabinoid-mediated presynaptic inhibition of glutamate release. Furthermore, after a sweetened high-fat meal, which elevates endogenous insulin, insulin-induced LTD is occluded. Finally, insulin in the VTA reduces food anticipatory behavior in mice and conditioned place preference for food in rats. Taken together, these results suggest that insulin in the VTA suppresses excitatory synaptic transmission and reduces anticipatory activity and preference for food-related cues.

Insulin receptor (Insr) protein is present at higher levels in pancreatic β-cells than in most other tissues, but the consequences of β-cell insulin resistance remain enigmatic. Here, we use an Ins1cre knock-in allele to delete Insr specifically in β-cells of both female and male mice. We compare experimental mice to Ins1cre-containing littermate controls at multiple ages and on multiple diets. RNA-seq of purified recombined β-cells reveals transcriptomic consequences of Insr loss, which differ between female and male mice. Action potential and calcium oscillation frequencies are increased in Insr knockout β-cells from female, but not male mice, whereas only male βInsrKO islets have reduced ATP-coupled oxygen consumption rate and reduced expression of genes involved in ATP synthesis. Female βInsrKO and βInsrHET mice exhibit elevated insulin release in ex vivo perifusion experiments, during hyperglycemic clamps, and following i.p. glucose challenge. Deletion of Insr does not alter β-cell area up to 9 months of age, nor does it impair hyperglycemia-induced proliferation. Based on our data, we adapt a mathematical model to include β-cell insulin resistance, which predicts that β-cell Insr knockout improves glucose tolerance depending on the degree of whole-body insulin resistance. Indeed, glucose tolerance is significantly improved in female βInsrKO and βInsrHET mice compared to controls at 9, 21 and 39 weeks, and also in insulin-sensitive 4-week old males. We observe no improved glucose tolerance in older male mice or in high fat diet-fed mice, corroborating the prediction that global insulin resistance obscures the effects of β-cell specific insulin resistance. The propensity for hyperinsulinemia is associated with mildly reduced fasting glucose and increased body weight. We further validate our main in vivo findings using an Ins1-CreERT transgenic line and find that male mice have improved glucose tolerance 4 weeks after tamoxifen-mediated Insr deletion. Collectively, our data show that β-cell insulin resistance in the form of reduced β-cell Insr contributes to hyperinsulinemia in the context of glucose stimulation, thereby improving glucose homeostasis in otherwise insulin sensitive sex, dietary and age contexts.

Over the last few decades, there has been a progressive transition from a categorical to a dimensional approach to psychiatric disorders. Especially in the case of substance use disorders, interest in the individual vulnerability to transition from controlled to compulsive drug taking warrants the development of novel dimension-based objective stratification tools. Here we drew on a multidimensional preclinical model of addiction, namely the 3-criteria model, previously developed to identify the neurobehavioural basis of the individual's vulnerability to switch from controlled to compulsive drug taking, to test a machine-learning assisted classifier objectively to identify individual subjects as vulnerable/resistant to addiction. Datasets from our previous studies on addiction-like behaviour for cocaine or alcohol were fed into a variety of machine-learning algorithms to develop a classifier that identifies resilient and vulnerable rats with high precision and reproducibility irrespective of the cohort to which they belong. A classifier based on K-median or K-mean-clustering (for cocaine or alcohol, respectively) followed by artificial neural networks emerged as a highly reliable and accurate tool to predict if a single rat is vulnerable/resilient to addiction. Thus, each rat previously characterized as displaying 0-criterion (i.e., resilient) or 3-criteria (i.e., vulnerable) in individual cohorts was correctly labelled by this classifier. The present machine-learning-based classifier objectively labels single individuals as resilient or vulnerable to developing addiction-like behaviour in a multisymptomatic preclinical model of addiction-like behaviour in rats. This novel dimension-based classifier increases the heuristic value of these preclinical models while providing proof of principle to deploy similar tools for the future of diagnosis of psychiatric disorders.

Genetic factors affect an individual's risk of developing obesity, but in most cases each genetic variant has a small effect. Discovery of genes that regulate obesity may provide clues about its underlying biological processes and point to new ways the disease can be treated. Preclinical animal models facilitate genetic discovery in obesity because environmental factors can be better controlled compared with the human population. We studied inbred mouse strains to identify novel genes affecting obesity and glucose metabolism. BTBR T+ Itpr3tf/J (BTBR) mice are fatter and more glucose intolerant than C57BL/6J (B6) mice. Prior genetic studies of these strains identified an obesity locus on chromosome 2. Using congenic mice, we found that obesity was affected by a ∼316 kb region, with only two known genes, pyruvate dehydrogenase kinase 1 (Pdk1) and integrin α 6 (Itga6). Both genes had mutations affecting their amino acid sequence and reducing mRNA levels. Both genes have known functions that could modulate obesity, lipid metabolism, insulin secretion, and/or glucose homeostasis. We hypothesized that genetic variation in or near Pdk1 or Itga6 causing reduced Pdk1 and Itga6 expression would promote obesity and impaired glucose tolerance. We used knockout mice lacking Pdk1 or Itga6 fed an obesigenic diet to test this hypothesis. Under the conditions we studied, we were unable to detect an individual contribution of either Pdk1 or Itga6 to body weight. During our studies, with conditions outside our control, we were unable to reproduce some of our previous body weight data. However, we identified a previously unknown role for Pdk1 in cardiac cholesterol metabolism providing the basis for future investigations. The studies described in this paper highlight the importance and the challenge using physiological outcomes to study obesity genes in mice.