RESUMO
We report that astrocytic insulin signaling co-regulates hypothalamic glucose sensing and systemic glucose metabolism. Postnatal ablation of insulin receptors (IRs) in glial fibrillary acidic protein (GFAP)-expressing cells affects hypothalamic astrocyte morphology, mitochondrial function, and circuit connectivity. Accordingly, astrocytic IR ablation reduces glucose-induced activation of hypothalamic pro-opio-melanocortin (POMC) neurons and impairs physiological responses to changes in glucose availability. Hypothalamus-specific knockout of astrocytic IRs, as well as postnatal ablation by targeting glutamate aspartate transporter (GLAST)-expressing cells, replicates such alterations. A normal response to altering directly CNS glucose levels in mice lacking astrocytic IRs indicates a role in glucose transport across the blood-brain barrier (BBB). This was confirmed in vivo in GFAP-IR KO mice by using positron emission tomography and glucose monitoring in cerebral spinal fluid. We conclude that insulin signaling in hypothalamic astrocytes co-controls CNS glucose sensing and systemic glucose metabolism via regulation of glucose uptake across the BBB.
Assuntos
Astrócitos/metabolismo , Glucose/metabolismo , Hipotálamo/metabolismo , Insulina/metabolismo , Transdução de Sinais , Sistema X-AG de Transporte de Aminoácidos/genética , Sistema X-AG de Transporte de Aminoácidos/metabolismo , Animais , Barreira Hematoencefálica , Retículo Endoplasmático/metabolismo , Proteína Glial Fibrilar Ácida/genética , Proteína Glial Fibrilar Ácida/metabolismo , Homeostase , Camundongos , Mitocôndrias/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Pró-Opiomelanocortina/metabolismo , Receptor de Insulina/genética , Receptor de Insulina/metabolismoRESUMO
Prader-Willi Syndrome (PWS) is a rare neurodevelopmental disorder of genetic etiology, characterized by paternal deletion of genes located at chromosome 15 in 70% of cases. Two distinct genetic subtypes of PWS deletions are characterized, where type I (PWS T1) carries four extra haploinsufficient genes compared to type II (PWS T2). PWS T1 individuals display more pronounced physiological and cognitive abnormalities than PWS T2, yet the exact neuropathological mechanisms behind these differences remain unclear. Our study employed postmortem hypothalamic tissues from PWS T1 and T2 individuals, conducting transcriptomic analyses and cell-specific protein profiling in white matter, neurons, and glial cells to unravel the cellular and molecular basis of phenotypic severity in PWS sub-genotypes. In PWS T1, key pathways for cell structure, integrity, and neuronal communication are notably diminished, while glymphatic system activity is heightened compared to PWS T2. The microglial defect in PWS T1 appears to stem from gene haploinsufficiency, as global and myeloid-specific Cyfip1 haploinsufficiency in murine models demonstrated. Our findings emphasize microglial phagolysosome dysfunction and altered neural communication as crucial contributors to the severity of PWS T1's phenotype.
Assuntos
Síndrome de Prader-Willi , Humanos , Camundongos , Animais , Síndrome de Prader-Willi/genética , Síndrome de Prader-Willi/psicologia , Microglia , Proteínas de Transporte/genética , Fenótipo , Fagossomos , Proteínas Adaptadoras de Transdução de Sinal/genéticaRESUMO
BACKGROUND: Obesity, metabolic disease and some psychiatric conditions are associated with changes to relative abundance of bacterial species and specific genes in the faecal microbiome. Little is known about the impact of pharmacologically induced weight loss on distinct microbiome species and their respective gene programs in obese individuals. METHODOLOGY: Using shotgun metagenomics, the composition of the microbiome was obtained for two cohorts of obese female Wistar rats (n = 10-12, total of 82) maintained on a high fat diet before and after a 42-day treatment with a panel of four investigatory or approved anti-obesity drugs (tacrolimus/FK506, bupropion, naltrexone and sibutramine), alone or in combination. RESULTS: Only sibutramine treatment induced consistent weight loss and improved glycaemic control in the obese rats. Weight loss was associated with reduced food intake and changes to the faecal microbiome in multiple microbial taxa, genes, and pathways. These include increased ß-diversity, increased relative abundance of multiple Bacteroides species, increased Bacteroides/Firmicutes ratio and changes to abundance of genes and species associated with obesity-induced inflammation, particularly those encoding components of the flagellum and its assembly. CONCLUSIONS: Sibutramine-induced weight loss in obese rats is associated with improved metabolic health, and changes to the faecal microbiome consistent with a reduction in obesity-induced bacterially-driven inflammation.
Assuntos
Microbioma Gastrointestinal , Animais , Bacteroides , Feminino , Inflamação , Obesidade/microbiologia , Ratos , Ratos Wistar , Redução de PesoRESUMO
The mechanisms underlying the transport of leptin into the brain are still largely unclear. While the leptin receptor has been implicated in the transport process, recent evidence has suggested an additional role of LRP2 (megalin). To evaluate the function of LRP2 for leptin transport across the blood-brain barrier (BBB), we developed a novel leptin-luciferase fusion protein (pLG), which stimulated leptin signaling and was transported in an in vitro BBB model based on porcine endothelial cells. The LRP inhibitor RAP did not affect leptin transport, arguing against a role of LRP2. In line with this, the selective deletion of LRP2 in brain endothelial cells and epithelial cells of the choroid plexus did not influence bodyweight, body composition, food intake, or energy expenditure of mice. These findings suggest that LRP2 at the BBB is not involved in the transport of leptin into the brain, nor in the development of obesity as has previously been described.
Assuntos
Barreira Hematoencefálica/metabolismo , Leptina/metabolismo , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Obesidade/metabolismo , Obesidade/patologia , Animais , Sítios de Ligação , Composição Corporal , Peso Corporal , Células CHO , Plexo Corióideo/metabolismo , Cricetulus , Células Endoteliais/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Luciferases/metabolismo , Masculino , Modelos Biológicos , Fosforilação , Transporte Proteico , Receptores para Leptina/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , SuínosRESUMO
The sirtuins are a family of highly conserved NAD(+)-dependent deacetylases that act as cellular sensors to detect energy availability and modulate metabolic processes. Two sirtuins that are central to the control of metabolic processes are mammalian sirtuin 1 (SIRT1) and sirtuin 3 (SIRT3), which are localized to the nucleus and mitochondria, respectively. Both are activated by high NAD(+) levels, a condition caused by low cellular energy status. By deacetylating a variety of proteins that induce catabolic processes while inhibiting anabolic processes, SIRT1 and SIRT3 coordinately increase cellular energy stores and ultimately maintain cellular energy homeostasis. Defects in the pathways controlled by SIRT1 and SIRT3 are known to result in various metabolic disorders. Consequently, activation of sirtuins by genetic or pharmacological means can elicit multiple metabolic benefits that protect mice from diet-induced obesity, type 2 diabetes, and nonalcoholic fatty liver disease.
Assuntos
Metabolismo Energético , Transdução de Sinais , Sirtuína 1/metabolismo , Sirtuína 3/metabolismo , Animais , Ritmo Circadiano , Metabolismo Energético/efeitos dos fármacos , Metabolismo Energético/genética , Ativação Enzimática , Ativadores de Enzimas/farmacologia , Predisposição Genética para Doença , Homeostase , Humanos , Doenças Metabólicas/tratamento farmacológico , Doenças Metabólicas/genética , Doenças Metabólicas/metabolismo , Modelos Moleculares , Fenótipo , Polimorfismo Genético , Conformação Proteica , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Sirtuína 1/genética , Sirtuína 3/genética , Relação Estrutura-AtividadeRESUMO
BACKGROUND/OBJECTIVES: Individuals carrying loss-of-function gene mutations for the adipocyte hormone leptin are morbidly obese, but respond favorably to replacement therapy. Recombinant leptin is however largely ineffective for the vast majority of obese individuals due to leptin resistance. One theory underlying leptin resistance is impaired leptin transport across the blood-brain-barrier (BBB). Here, we aim to gain new insights into the mechanisms of leptin BBB transport, and its role in leptin resistance. METHODS: We developed a novel tool for visualizing leptin transport using infrared fluorescently labeled leptin, combined with tissue clearing and light-sheet fluorescence microscopy. We corroborated these data using western blotting. RESULTS: Using 3D whole brain imaging, we display comparable leptin accumulation in circumventricular organs of lean and obese mice, predominantly in the choroid plexus (CP). Protein quantification revealed comparable leptin levels in microdissected mediobasal hypothalami (MBH) of lean and obese mice (p = 0.99). We further found increased leptin receptor expression in the CP (p = 0.025, p = 0.0002) and a trend toward elevated leptin protein levels in the MBH (p = 0.17, p = 0.078) of obese mice undergoing weight loss interventions by calorie restriction or exendin-4 treatment. CONCLUSIONS: Overall, our findings suggest a crucial role for the CP in controlling the transport of leptin into the cerebrospinal fluid and from there to target areas such as the MBH, potentially mediated via the leptin receptor. Similar leptin levels in circumventricular organs and the MBH of lean and obese mice further suggest intact leptin BBB transport in leptin resistant mice.
Assuntos
Barreira Hematoencefálica/metabolismo , Encéfalo/metabolismo , Leptina/metabolismo , Camundongos Obesos/metabolismo , Obesidade Mórbida/metabolismo , Animais , Transporte Biológico , Barreira Hematoencefálica/diagnóstico por imagem , Western Blotting , Encéfalo/diagnóstico por imagem , Modelos Animais de Doenças , Fluorescência , Células HEK293 , Humanos , Imageamento Tridimensional , Camundongos , Imagem MolecularRESUMO
á : Astrocytosis is a reactive process involving cellular, molecular, and functional changes to facilitate neuronal survival, myelin preservation, blood brain barrier function and protective glial scar formation upon brain insult. The overall pro- or anti-inflammatory impact of reactive astrocytes appears to be driven in a context- and disease-driven manner by modulation of astrocytic Ca2+ homeostasis and activation of Ca2+/calmodulin-activated serine/threonine phosphatase calcineurin. Here, we aimed to assess whether calcineurin is dispensable for astrocytosis in the hypothalamus driven by prolonged high fat diet (HFD) feeding. Global deletion of calcineurin A beta (gene name: Ppp3cb) led to a decrease of glial fibrillary acidic protein (GFAP)-positive cells in the ventromedial hypothalamus (VMH), dorsomedial hypothalamus (DMH), and arcuate nucleus (ARC) of mice exposed chronically to HFD. The concomitant decrease in Iba1-positive microglia in the VMH further suggests a modest impact of Ppp3cb deletion on microgliosis. Pharmacological inhibition of calcineurin activity by Fk506 had no impact on IBA1-positive microglia in hypothalami of mice acutely exposed to HFD for 1 week. However, Fk506-treated mice displayed a decrease in GFAP levels in the ARC. In vivo effects could not be replicated in cell culture, where calcineurin inhibition by Fk506 had no effect on astrocytic morphology, astrocytic cell death, GFAP, and vimentin protein levels or microglia numbers in primary hypothalamic astrocytes and microglia co-cultures. Further, adenoviral overexpression of calcineurin subunit Ppp3r1 in primary glia culture did not lead to an increase in GFAP fluorescence intensity. Overall, our results point to a prominent role of calcineurin in mediating hypothalamic astrocytosis as response to acute and chronic HFD exposure. Moreover, discrepant findings in vivo and in cell culture indicate the necessity of studying astrocytes in their "natural" environment, i.e., preserving an intact hypothalamic microenvironment with neurons and non-neuronal cells in close proximity.
Assuntos
Calcineurina/deficiência , Dieta Hiperlipídica/efeitos adversos , Gliose/metabolismo , Gliose/prevenção & controle , Hipotálamo/metabolismo , Animais , Astrócitos/metabolismo , Sobrevivência Celular/fisiologia , Células Cultivadas , Gliose/patologia , Hipotálamo/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
Monoamines, acting as hormones and neurotransmitters, play a critical role in multiple physiological processes ranging from cognitive function and mood to sympathetic nervous system activity, fight-or-flight response and glucose homeostasis. In addition to brain and blood, monoamines are abundant in several tissues, and dysfunction in their synthesis or signaling is associated with various pathological conditions. It was our goal to develop a method to detect these compounds in peripheral murine tissues. In this study, we employed a high-performance liquid chromatography method using electrochemical detection that allows not only detection of catecholamines but also a detailed analysis of nine monoamines and metabolites in murine tissues. Simple tissue extraction procedures were optimized for muscle (gastrocnemius, extensor digitorum longus and soleus), liver, pancreas and white adipose tissue in the range of weight 10-200 mg. The system allowed a limit of detection between 0.625 and 2.5 pg µL-1 for monoamine analytes and their metabolites, including dopamine, 3,4-dihydroxyphenylacetic acid, 3-methoxytyramine, homovanillic acid, norepinephrine, epinephrine, 3-methoxy-4-hydroxyphenylglycol, serotonin and 5-hydroxyindoleacetic acid. Typical concentrations for different monoamines and their metabolization products in these tissues are presented for C57Bl/6 J mice fed a high-fat diet.
Assuntos
Monoaminas Biogênicas/análise , Monoaminas Biogênicas/metabolismo , Cromatografia Líquida de Alta Pressão/métodos , Técnicas Eletroquímicas/métodos , Tecido Adiposo Branco/química , Animais , Monoaminas Biogênicas/química , Dieta Hiperlipídica , Sistema Digestório/química , Hipotálamo/química , Limite de Detecção , Modelos Lineares , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Músculo Esquelético/química , Especificidade de Órgãos , Reprodutibilidade dos TestesRESUMO
AIMS/HYPOTHESIS: Ketogenic diets (KDs) have increasingly gained attention as effective means for weight loss and potential adjunctive treatment of cancer. The metabolic benefits of KDs are regularly ascribed to enhanced hepatic secretion of fibroblast growth factor 21 (FGF21) and its systemic effects on fatty-acid oxidation, energy expenditure (EE) and body weight. Ambiguous data from Fgf21-knockout animal strains and low FGF21 concentrations reported in humans with ketosis have nevertheless cast doubt regarding the endogenous function of FGF21. We here aimed to elucidate the causal role of FGF21 in mediating the therapeutic benefits of KDs on metabolism and cancer. METHODS: We established a dietary model of increased vs decreased FGF21 by feeding C57BL/6J mice with KDs, either depleted of protein or enriched with protein. We furthermore used wild-type and Fgf21-knockout mice that were subjected to the respective diets, and monitored energy and glucose homeostasis as well as tumour growth after transplantation of Lewis lung carcinoma cells. RESULTS: Hepatic and circulating, but not adipose tissue, FGF21 levels were profoundly increased by protein starvation, independent of the state of ketosis. We demonstrate that endogenous FGF21 is not essential for the maintenance of normoglycaemia upon protein and carbohydrate starvation and is therefore not needed for the effects of KDs on EE. Furthermore, the tumour-suppressing effects of KDs were independent of FGF21 and, rather, driven by concomitant protein and carbohydrate starvation. CONCLUSIONS/INTERPRETATION: Our data indicate that the multiple systemic effects of KD exposure in mice, previously ascribed to increased FGF21 secretion, are rather a consequence of protein malnutrition.
Assuntos
Dieta Cetogênica , Fatores de Crescimento de Fibroblastos/genética , Glucose/metabolismo , Homeostase/genética , Cetose/genética , Neoplasias/genética , Deficiência de Proteína/genética , Tecido Adiposo/metabolismo , Animais , Fatores de Crescimento de Fibroblastos/metabolismo , Cetose/metabolismo , Fígado/metabolismo , Camundongos , Camundongos Knockout , Neoplasias/dietoterapia , Neoplasias/metabolismo , Deficiência de Proteína/metabolismoRESUMO
OBJECTIVE: Surgical interventions that prevent nutrient exposure to the duodenum are among the most successful treatments for obesity and diabetes. However, these interventions are highly invasive, irreversible and often carry significant risk. The duodenal-endoluminal sleeve (DES) is a flexible tube that acts as a barrier to nutrient-tissue interaction along the duodenum. We implanted this device in Zucker Diabetic Fatty (ZDF) rats to gain greater understanding of duodenal nutrient exclusion on glucose homeostasis. DESIGN: ZDF rats were randomised to four groups: Naive, sham ad libitum, sham pair-fed, and DES implanted. Food intake, body weight (BW) and body composition were measured for 28â days postoperatively. Glucose, lipid and bile acid metabolism were evaluated, as well as histological assessment of the upper intestine. RESULTS: DES implantation induced a sustained decrease in BW throughout the study that was matched by pair-fed sham animals. Decreased BW resulted from loss of fat, but not lean mass. DES rats were also found to be more glucose tolerant than either ad libitum-fed or pair-fed sham controls, suggesting fat mass independent metabolic benefits. DES also reduced circulating triglyceride and glycerol levels while increasing circulating bile acids. Interestingly, DES stimulated a considerable increase in villus length throughout the upper intestine, which may contribute to metabolic improvements. CONCLUSIONS: Our preclinical results validate DES as a promising therapeutic approach to diabetes and obesity, which offers reversibility, low risk, low invasiveness and triple benefits including fat mass loss, glucose and lipid metabolism improvement which mechanistically may involve increased villus growth in the upper gut.
Assuntos
Glicemia/metabolismo , Duodeno/fisiologia , Absorção Intestinal , Síndrome Metabólica/terapia , Próteses e Implantes , Animais , Ácidos e Sais Biliares/sangue , Composição Corporal , Peso Corporal , Diabetes Mellitus Experimental/terapia , Duodeno/patologia , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Teste de Tolerância a Glucose , Glicerol/sangue , Homeostase , Íleo/patologia , Jejuno/patologia , Masculino , Obesidade/terapia , Distribuição Aleatória , Ratos , Ratos Zucker , Triglicerídeos/sangueRESUMO
AIMS/HYPOTHESIS: Genome-wide association studies have revealed an association of the transcription factor ETS variant gene 5 (ETV5) with human obesity. However, its role in glucose homeostasis and energy balance is unknown. METHODS: Etv5 knockout (KO) mice were monitored weekly for body weight (BW) and food intake. Body composition was measured at 8 and 16 weeks of age. Glucose metabolism was studied, and glucose-stimulated insulin secretion was measured in vivo and in vitro. RESULTS: Etv5 KO mice are smaller and leaner, and have a reduced BW and lower fat mass than their wild-type controls on a chow diet. When exposed to a high-fat diet, KO mice are resistant to diet-induced BW gain. Despite a greater insulin sensitivity, KO mice have profoundly impaired glucose tolerance associated with impaired insulin secretion. Morphometric analysis revealed smaller islets and a reduced beta cell size in the pancreatic islets of Etv5 KO mice. Knockdown of ETV5 in an insulin-secreting cell line or beta cells from human donors revealed intact mitochondrial and Ca(2+) channel activity, but reduced insulin exocytosis. CONCLUSION/INTERPRETATION: This work reveals a critical role for ETV5 in specifically regulating insulin secretion both in vitro and in vivo.
Assuntos
Peptídeo C/metabolismo , Proteínas de Ligação a DNA/metabolismo , Exocitose/fisiologia , Glucose/metabolismo , Homeostase/fisiologia , Insulina/metabolismo , Fatores de Transcrição/metabolismo , Animais , Composição Corporal , Peso Corporal , Dieta Hiperlipídica , Ingestão de Alimentos , Estudo de Associação Genômica Ampla , Teste de Tolerância a Glucose , Resistência à Insulina , Secreção de Insulina , Camundongos , Camundongos KnockoutRESUMO
BACKGROUND: Abnormal glucose metabolism is a central feature of disorders with increased rates of cardiovascular disease. Low levels of high-density lipoprotein (HDL) are a key predictor for cardiovascular disease. We used genetic mouse models with increased HDL levels (apolipoprotein A-I transgenic [apoA-I tg]) and reduced HDL levels (apoA-I-deficient [apoA-I ko]) to investigate whether HDL modulates mitochondrial bioenergetics in skeletal muscle. METHODS AND RESULTS: ApoA-I ko mice exhibited fasting hyperglycemia and impaired glucose tolerance test compared with wild-type mice. Mitochondria isolated from gastrocnemius muscle of apoA-I ko mice displayed markedly blunted ATP synthesis. Endurance capacity during exercise exhaustion test was impaired in apoA-I ko mice. HDL directly enhanced glucose oxidation by increasing glycolysis and mitochondrial respiration rate in C2C12 muscle cells. ApoA-I tg mice exhibited lower fasting glucose levels, improved glucose tolerance test, increased lactate levels, reduced fat mass, associated with protection against age-induced decline of endurance capacity compared with wild-type mice. Circulating levels of fibroblast growth factor 21, a novel biomarker for mitochondrial respiratory chain deficiencies and inhibitor of white adipose lipolysis, were significantly reduced in apoA-I tg mice. Consistent with an increase in glucose utilization of skeletal muscle, genetically increased HDL and apoA-I levels in mice prevented high-fat diet-induced impairment of glucose homeostasis. CONCLUSIONS: In view of impaired mitochondrial function and decreased HDL levels in type 2 diabetes mellitus, our findings indicate that HDL-raising therapies may preserve muscle mitochondrial function and address key aspects of type 2 diabetes mellitus beyond cardiovascular disease.
Assuntos
Glicemia/metabolismo , Intolerância à Glucose/metabolismo , Hiperglicemia/metabolismo , Lipoproteínas HDL/metabolismo , Músculo Esquelético/metabolismo , Animais , Apolipoproteína A-I/genética , Respiração Celular/fisiologia , Células Cultivadas , Diabetes Mellitus Tipo 2/metabolismo , Modelos Animais de Doenças , Ácidos Graxos não Esterificados/sangue , Fatores de Crescimento de Fibroblastos/sangue , Homeostase/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias Musculares/metabolismo , Resistência Física/fisiologiaRESUMO
Reductions in levels of the hunger-stimulating hormone ghrelin have been proposed to mediate part of the effects of vertical sleeve gastrectomy (VSG) and Roux-en-Y gastric bypass surgeries for obesity. We studied circulating levels of acyl and desacyl ghrelin in rats after these surgeries. We found that blood levels of ghrelin were reduced after VSG, but not after Roux-en-Y gastric bypass, based on enzyme-linked immunosorbent assay and mass-spectrometry analyses. We compared the effects of VSG in ghrelin-deficient mice and wild-type mice on food intake, body weight, dietary fat preference, and glucose tolerance. We found that VSG produced comparable outcomes in each strain. Reduced ghrelin signaling therefore does not appear to be required for these effects of VSG.
Assuntos
Ingestão de Alimentos , Comportamento Alimentar , Gastrectomia , Grelina/sangue , Animais , Peso Corporal , Gorduras na Dieta , Genótipo , Grelina/deficiência , Grelina/genética , Teste de Tolerância a Glucose , Masculino , Camundongos , Camundongos Knockout , Ratos , Ratos Long-Evans , Transdução de SinaisRESUMO
We present a consolidated view of the complexity and challenges of designing studies for measurement of energy metabolism in mouse models, including a practical guide to the assessment of energy expenditure, energy intake and body composition and statistical analysis thereof. We hope this guide will facilitate comparisons across studies and minimize spurious interpretations of data. We recommend that division of energy expenditure data by either body weight or lean body weight and that presentation of group effects as histograms should be replaced by plotting individual data and analyzing both group and body-composition effects using analysis of covariance (ANCOVA).
Assuntos
Ingestão de Energia , Metabolismo Energético , Camundongos/fisiologia , Animais , Composição Corporal , Meio Ambiente , Abrigo para Animais , Camundongos Mutantes/genética , Obesidade/etiologia , FenótipoRESUMO
Background: The development of obesity-associated comorbidities such as type 2 diabetes (T2D) and hepatic steatosis has been linked to selected microRNAs in individual studies; however, an unbiased genome-wide approach to map T2D induced changes in the miRNAs landscape in human liver samples, and a subsequent robust identification and validation of target genes are still missing. Methods: Liver biopsies from age- and gender-matched obese individuals with (n=20) or without (n=20) T2D were used for microRNA microarray analysis. The candidate microRNA and target genes were validated in 85 human liver samples, and subsequently mechanistically characterized in hepatic cells as well as by dietary interventions and hepatic overexpression in mice. Results: Here, we present the human hepatic microRNA transcriptome of type 2 diabetes in liver biopsies and use a novel seed prediction tool to robustly identify microRNA target genes, which were then validated in a unique cohort of 85 human livers. Subsequent mouse studies identified a distinct signature of T2D-associated miRNAs, partly conserved in both species. Of those, human-murine miR-182-5 p was the most associated with whole-body glucose homeostasis and hepatic lipid metabolism. Its target gene LRP6 was consistently lower expressed in livers of obese T2D humans and mice as well as under conditions of miR-182-5 p overexpression. Weight loss in obese mice decreased hepatic miR-182-5 p and restored Lrp6 expression and other miR-182-5 p target genes. Hepatic overexpression of miR-182-5 p in mice rapidly decreased LRP6 protein levels and increased liver triglycerides and fasting insulin under obesogenic conditions after only seven days. Conclusions: By mapping the hepatic miRNA-transcriptome of type 2 diabetic obese subjects, validating conserved miRNAs in diet-induced mice, and establishing a novel miRNA prediction tool, we provide a robust and unique resource that will pave the way for future studies in the field. As proof of concept, we revealed that the repression of LRP6 by miR-182-5 p, which promotes lipogenesis and impairs glucose homeostasis, provides a novel mechanistic link between T2D and non-alcoholic fatty liver disease, and demonstrate in vivo that miR-182-5 p can serve as a future drug target for the treatment of obesity-driven hepatic steatosis. Funding: This work was supported by research funding from the Deutsche Forschungsgemeinschaft (KI 1887/2-1, KI 1887/2-2, KI 1887/3-1 and CRC-TR296), the European Research Council (ERC, CoG Yoyo LepReSens no. 101002247; PTP), the Helmholtz Association (Initiative and Networking Fund International Helmholtz Research School for Diabetes; MB) and the German Center for Diabetes Research (DZD Next Grant 82DZD09D1G).
Assuntos
Diabetes Mellitus Tipo 2 , Fígado , MicroRNAs , Obesidade , Transcriptoma , MicroRNAs/metabolismo , MicroRNAs/genética , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Animais , Humanos , Obesidade/genética , Obesidade/metabolismo , Fígado/metabolismo , Camundongos , Masculino , Fígado Gorduroso/genética , Fígado Gorduroso/metabolismo , Feminino , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Perfilação da Expressão GênicaRESUMO
Hypothalamic gonadotropin-releasing hormone (GnRH) neurons regulate fertility and integrate hormonal status with environmental cues to ensure reproductive success. Here we show that GnRH neurons in the olfactory bulb (GnRHOB) of adult mice can mediate social recognition. Specifically, we show that GnRHOB neurons extend neurites into the vomeronasal organ and olfactory epithelium and project to the median eminence. GnRHOB neurons in males express vomeronasal and olfactory receptors, are activated by female odors and mediate gonadotropin release in response to female urine. Male preference for female odors required the presence and activation of GnRHOB neurons, was impaired after genetic inhibition or ablation of these cells and relied on GnRH signaling in the posterodorsal medial amygdala. GnRH receptor expression in amygdala kisspeptin neurons appear to be required for GnRHOB neurons' actions on male mounting behavior. Taken together, these results establish GnRHOB neurons as regulating fertility, sex recognition and mating in male mice.
Assuntos
Hormônio Liberador de Gonadotropina , Neurônios , Odorantes , Bulbo Olfatório , Comportamento Sexual Animal , Órgão Vomeronasal , Animais , Masculino , Hormônio Liberador de Gonadotropina/metabolismo , Bulbo Olfatório/fisiologia , Bulbo Olfatório/metabolismo , Camundongos , Neurônios/metabolismo , Neurônios/fisiologia , Comportamento Sexual Animal/fisiologia , Feminino , Órgão Vomeronasal/fisiologia , Órgão Vomeronasal/metabolismo , Camundongos Endogâmicos C57BL , Olfato/fisiologia , Tonsila do Cerebelo/metabolismo , Tonsila do Cerebelo/fisiologiaRESUMO
Thyroid hormone (TH) effects are mediated through TH receptors (TRs), TRα1, TRß1, and TRß2. The TRs bind to the DNA and regulate expression of TH target genes (canonical signaling). In addition, they mediate activation of signaling pathways (noncanonical signaling). Whether noncanonical TR action contributes to the spectrum of TH effects is largely unknown. The aim of this study was to attribute physiological effects to the TR isoforms and their canonical and noncanonical signaling. We conducted multiparameter phenotyping in male and female TR knockout mice (TRαKO, TRßKO), mice with disrupted canonical signaling due to mutations in the TR DNA binding domain (TRαGS, TRßGS), and their wild-type littermates. Perturbations in senses, especially hearing (mainly TRß with a lesser impact of TRα), visual acuity, retinal thickness (TRα and TRß), and in muscle metabolism (TRα) highlighted the role of canonical TR action. Strikingly, selective abrogation of canonical TR action often had little phenotypic consequence, suggesting that noncanonical TR action sufficed to maintain the wild-type phenotype for specific effects. For instance, macrocytic anemia, reduced retinal vascularization, or increased anxiety-related behavior were only observed in TRαKO but not TRαGS mice. Noncanonical TRα action improved energy utilization and prevented hyperphagia observed in female TRαKO mice. In summary, by examining the phenotypes of TRα and TRß knockout models alongside their DNA binding-deficient mutants and wild-type counterparts, we could establish that the noncanonical actions of TRα and TRß play a crucial role in modulating sensory, behavioral, and metabolic functions and, thus, contribute to the spectrum of physiological TH effects.
Assuntos
Camundongos Knockout , Fenótipo , Receptores alfa dos Hormônios Tireóideos , Receptores beta dos Hormônios Tireóideos , Animais , Feminino , Masculino , Receptores alfa dos Hormônios Tireóideos/genética , Receptores alfa dos Hormônios Tireóideos/metabolismo , Receptores beta dos Hormônios Tireóideos/genética , Receptores beta dos Hormônios Tireóideos/metabolismo , Camundongos , Transdução de Sinais/genética , Hormônios Tireóideos/metabolismo , Camundongos Endogâmicos C57BLRESUMO
OBJECTIVE: The glucose-dependent insulinotropic polypeptide (GIP) decreases body weight via central GIP receptor (GIPR) signaling, but the underlying mechanisms remain largely unknown. Here, we assessed whether GIP regulates body weight and glucose control via GIPR signaling in cells that express the leptin receptor (Lepr). METHODS: Hypothalamic, hindbrain, and pancreatic co-expression of Gipr and Lepr was assessed using single cell RNAseq analysis. Mice with deletion of Gipr in Lepr cells were generated and metabolically characterized for alterations in diet-induced obesity (DIO), glucose control and leptin sensitivity. Long-acting single- and dual-agonists at GIPR and GLP-1R were further used to assess drug effects on energy and glucose metabolism in DIO wildtype (WT) and Lepr-Gipr knock-out (KO) mice. RESULTS: Gipr and Lepr show strong co-expression in the pancreas, but not in the hypothalamus and hindbrain. DIO Lepr-Gipr KO mice are indistinguishable from WT controls related to body weight, food intake and diet-induced leptin resistance. Acyl-GIP and the GIPR:GLP-1R co-agonist MAR709 remain fully efficacious to decrease body weight and food intake in DIO Lepr-Gipr KO mice. Consistent with the demonstration that Gipr and Lepr highly co-localize in the endocrine pancreas, including the ß-cells, we find the superior glycemic effect of GIPR:GLP-1R co-agonism over single GLP-1R agonism to vanish in Lepr-Gipr KO mice. CONCLUSIONS: GIPR signaling in cells/neurons that express the leptin receptor is not implicated in the control of body weight or food intake, but is of crucial importance for the superior glycemic effects of GIPR:GLP-1R co-agonism relative to single GLP-1R agonism.
Assuntos
Peso Corporal , Ingestão de Alimentos , Polipeptídeo Inibidor Gástrico , Camundongos Knockout , Obesidade , Receptores dos Hormônios Gastrointestinais , Receptores para Leptina , Animais , Masculino , Camundongos , Polipeptídeo Inibidor Gástrico/metabolismo , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Receptor do Peptídeo Semelhante ao Glucagon 1/metabolismo , Receptor do Peptídeo Semelhante ao Glucagon 1/genética , Glucose/metabolismo , Leptina/metabolismo , Camundongos Endogâmicos C57BL , Obesidade/metabolismo , Receptores dos Hormônios Gastrointestinais/metabolismo , Receptores dos Hormônios Gastrointestinais/genética , Receptores para Leptina/metabolismo , Receptores para Leptina/genética , Transdução de SinaisRESUMO
The neuronal circuits involved in the regulation of feeding behavior and energy expenditure are soft-wired, reflecting the relative activity of the postsynaptic neuronal system, including the anorexigenic proopiomelanocortin (POMC)-expressing cells of the arcuate nucleus. We analyzed the synaptic input organization of the melanocortin system in lean rats that were vulnerable (DIO) or resistant (DR) to diet-induced obesity. We found a distinct difference in the quantitative and qualitative synaptology of POMC cells between DIO and DR animals, with a significantly greater number of inhibitory inputs in the POMC neurons in DIO rats compared with DR rats. When exposed to a high-fat diet (HFD), the POMC cells of DIO animals lost synapses, whereas those of DR rats recruited connections. In both DIO rats and mice, the HFD-triggered loss of synapses on POMC neurons was associated with increased glial ensheathment of the POMC perikarya. The altered synaptic organization of HFD-fed animals promoted increased POMC tone and a decrease in the stimulatory connections onto the neighboring neuropeptide Y (NPY) cells. Exposure to HFD was associated with reactive gliosis, and this affected the structure of the blood-brain barrier such that the POMC and NPY cell bodies and dendrites became less accessible to blood vessels. Taken together, these data suggest that consumption of an HFD has a major impact on the cytoarchitecture of the arcuate nucleus in vulnerable subjects, with changes that might be irreversible due to reactive gliosis.
Assuntos
Dieta , Gliose/metabolismo , Melanocortinas/metabolismo , Obesidade/metabolismo , Sinapses/metabolismo , Animais , Núcleo Arqueado do Hipotálamo/metabolismo , Núcleo Arqueado do Hipotálamo/patologia , Núcleo Arqueado do Hipotálamo/fisiopatologia , Gorduras na Dieta/efeitos adversos , Feminino , Gliose/etiologia , Hipotálamo/metabolismo , Hipotálamo/patologia , Hipotálamo/fisiopatologia , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia Eletrônica , Neurônios/metabolismo , Neurônios/ultraestrutura , Neuropeptídeo Y/metabolismo , Obesidade/etiologia , Pró-Opiomelanocortina/metabolismo , Ratos , Ratos Sprague-Dawley , Transmissão Sináptica/fisiologiaRESUMO
Until menopause, women have a lower propensity to develop metabolic diseases than men, suggestive of a protective role for sex hormones. Although a functional synergy between central actions of estrogens and leptin has been demonstrated to protect against metabolic disturbances, the underlying cellular and molecular mechanisms mediating this crosstalk have remained elusive. By using a series of embryonic, adult-onset, and tissue/cell-specific loss-of-function mouse models, we document an unprecedented role of hypothalamic Cbp/P300-interacting transactivator with Glu/Asp-rich carboxy-terminal domain 1 (Cited1) in mediating estradiol (E2)-dependent leptin actions that control feeding specifically in pro-opiomelanocortin (Pomc) neurons. We reveal that within arcuate Pomc neurons, Cited1 drives leptin's anorectic effects by acting as a co-factor converging E2 and leptin signaling via direct Cited1-ERα-Stat3 interactions. Together, these results provide new insights on how melanocortin neurons integrate endocrine inputs from gonadal and adipose axes via Cited1, thereby contributing to the sexual dimorphism in diet-induced obesity.