RESUMO
Type 2 diabetes (T2D) is associated with defective insulin secretion and reduced ß cell mass. Available treatments provide a temporary reprieve, but secondary failure rates are high, making insulin supplementation necessary. Reversibility of ß cell failure is a key translational question. Here, we reverse engineered and interrogated pancreatic islet-specific regulatory networks to discover T2D-specific subpopulations characterized by metabolic inflexibility and endocrine progenitor/stem cell features. Single-cell gain- and loss-of-function and glucose-induced Ca2+ flux analyses of top candidate master regulatory (MR) proteins in islet cells validated transcription factor BACH2 and associated epigenetic effectors as key drivers of T2D cell states. BACH2 knockout in T2D islets reversed cellular features of the disease, restoring a nondiabetic phenotype. BACH2-immunoreactive islet cells increased approximately 4-fold in diabetic patients, confirming the algorithmic prediction of clinically relevant subpopulations. Treatment with a BACH inhibitor lowered glycemia and increased plasma insulin levels in diabetic mice, and restored insulin secretion in diabetic mice and human islets. The findings suggest that T2D-specific populations of failing ß cells can be reversed and indicate pathways for pharmacological intervention, including via BACH2 inhibition.
Assuntos
Fatores de Transcrição de Zíper de Leucina Básica/antagonistas & inibidores , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Sinalização do Cálcio , Diabetes Mellitus Tipo 2/metabolismo , Epigênese Genética , Células Secretoras de Insulina/metabolismo , Fatores de Transcrição de Zíper de Leucina Básica/genética , Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/genética , Células HEK293 , HumanosRESUMO
Hypothalamic neurons expressing agouti-related peptide (AgRP) regulate eating and glucose metabolism. Ablation of FOXO1 in AgRP neurons of mice results in reduced food intake, leanness, improved glucose homeostasis, and increased sensitivity to insulin and leptin. We tentatively identified G-protein-coupled receptor Gpr17 as an effector of FOXO1 orexigenic signals in AgRP neurons. In this study, we generated and characterized AgRP neuron-specific Gpr17 knockout mice (Agrp-Gpr17(-/-)) to test the hypothesis that Gpr17 regulates appetite, energy expenditure, and metabolism. Agrp-Gpr17(-/-) mice show reduced food intake, increased relative energy expenditure, and increased satiety, resulting in leanness and reduced body fat. They also show increased central nervous system sensitivity to insulin and leptin and reduced plasma glucose excursions following the administration of glucose or pyruvate. In summary, AgRP neuron-specific Gpr17 knockouts phenocopy FOXO1 knockouts in the same cell type, thus supporting our original hypothesis and providing further impetus to develop Gpr17 antagonists for the treatment of obesity.
Assuntos
Proteína Relacionada com Agouti/metabolismo , Ingestão de Alimentos/fisiologia , Hipotálamo/metabolismo , Resistência à Insulina/fisiologia , Leptina/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Animais , Composição Corporal/fisiologia , Metabolismo Energético/fisiologia , Masculino , Camundongos , Camundongos Knockout , Proteínas do Tecido Nervoso/genética , Receptores Acoplados a Proteínas G/genéticaRESUMO
The central nervous system (CNS) uses glucose independent of insulin. Nonetheless, insulin receptors and insulin-responsive glucose transporters (Glut4) often colocalize in neurons (Glut4 neurons) in anatomically and functionally distinct areas of the CNS. The apparent heterogeneity of Glut4 neurons has thus far thwarted attempts to understand their function. To answer this question, we used Cre-dependent, diphtheria toxin-mediated cell ablation to selectively remove basal hypothalamic Glut4 neurons and investigate the resulting phenotypes. After Glut4 neuron ablation, mice demonstrate altered hormone and nutrient signaling in the CNS. Accordingly, they exhibit negative energy balance phenotype characterized by reduced food intake and increased energy expenditure, without locomotor deficits or gross neuronal abnormalities. Glut4 neuron ablation affects orexigenic melanin-concentrating hormone neurons but has limited effect on neuropeptide Y/agouti-related protein and proopiomelanocortin neurons. The food intake phenotype can be partially normalized by GABA administration, suggesting that it arises from defective GABAergic transmission. Glut4 neuron-ablated mice show peripheral metabolic defects, including fasting hyperglycemia and glucose intolerance, decreased insulin levels, and elevated hepatic gluconeogenic genes. We conclude that Glut4 neurons integrate hormonal and nutritional cues and mediate CNS actions of insulin on energy balance and peripheral metabolism.
Assuntos
Anorexia/metabolismo , Transportador de Glucose Tipo 4/metabolismo , Glucose/metabolismo , Hipotálamo/citologia , Neurônios/metabolismo , Animais , Metabolismo Energético , Comportamento Alimentar , Regulação da Expressão Gênica/fisiologia , Genótipo , Transportador de Glucose Tipo 4/genética , Homeostase , Camundongos , Camundongos TransgênicosRESUMO
Transcription factor forkhead box O1 (FoxO1) regulates energy expenditure (EE), food intake, and hepatic glucose production. These activities have been mapped to specific hypothalamic neuronal populations using cell type-specific knockout experiments in mice. To parse out the integrated output of FoxO1-dependent transcription from different neuronal populations and multiple hypothalamic regions, we used transgenic mice expressing Cre recombinase from the Nkx2.1 promoter to ablate loxP-flanked Foxo1 alleles from a majority of hypothalamic neurons (Foxo1KO(Nkx2.1) mice). This strategy resulted in the expected inhibition of FoxO1 expression, but only produced a transient reduction of body weight as well as a decreased body length. The transient decrease of body weight in male mice was accompanied by decreased fat mass. Male Foxo1KO(Nkx2.1) mice show food intake similar to that in wild-type controls, and, although female knockout mice eat less, they do so in proportion to a reduced body size. EE is unaffected in Foxo1KO(Nkx2.1) mice, although small increases in body temperature are present. Unlike other neuron-specific Foxo1 knockout mice, Foxo1KO(Nkx2.1) mice are not protected from diet-induced obesity. These studies indicate that, unlike the metabolic effects of highly restricted neuronal subsets (proopiomelanocortin, neuropeptide Y/agouti-related peptide, and steroidogenic factor 1), those of neurons derived from the Nkx2.1 lineage either occur in a FoxO1-independent fashion or are compensated for through developmental plasticity.
Assuntos
Metabolismo Energético/fisiologia , Fatores de Transcrição Forkhead/metabolismo , Hipotálamo/metabolismo , Neurônios/metabolismo , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Proteína Relacionada com Agouti/metabolismo , Animais , Peso Corporal/fisiologia , Linhagem da Célula/fisiologia , Ingestão de Alimentos/fisiologia , Feminino , Proteína Forkhead Box O1 , Fatores de Transcrição Forkhead/genética , Masculino , Camundongos , Camundongos Transgênicos , Neuropeptídeo Y/metabolismo , Proteínas Nucleares/genética , Obesidade/genética , Obesidade/metabolismo , Pró-Opiomelanocortina/metabolismo , Regiões Promotoras Genéticas , Transdução de Sinais/fisiologia , Fator Nuclear 1 de Tireoide , Fatores de Transcrição/genéticaRESUMO
Successful development of antiobesity agents requires detailed knowledge of neural pathways controlling body weight, eating behavior, and peripheral metabolism. Genetic ablation of FoxO1 in selected hypothalamic neurons decreases food intake, increases energy expenditure, and improves glucose homeostasis, highlighting the role of this gene in insulin and leptin signaling. However, little is known about potential effects of FoxO1 in other neurons. To address this question, we executed a broad-based neuronal ablation of FoxO1 using Synapsin promoter-driven Cre to delete floxed Foxo1 alleles. Lineage-tracing experiments showed that NPY/AgRP and POMC neurons were minimally affected by the knockout. Nonetheless, Syn-Cre-Foxo1 knockouts demonstrated a catabolic energy homeostatic phenotype with a blunted refeeding response, increased sensitivity to leptin and amino acid signaling, and increased locomotor activity, likely attributable to increased melanocortinergic tone. We confirmed these data in mice lacking the three Foxo genes. The effects on locomotor activity could be reversed by direct delivery of constitutively active FoxO1 to the mediobasal hypothalamus, but not to the suprachiasmatic nucleus. The data reveal that the integrative function of FoxO1 extends beyond the arcuate nucleus, suggesting that central nervous system inhibition of FoxO1 function can be leveraged to promote hormone sensitivity and prevent a positive energy balance.
Assuntos
Ingestão de Alimentos , Fatores de Transcrição Forkhead/antagonistas & inibidores , Hipotálamo/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Locomoção/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Animais , Fármacos Antiobesidade/farmacologia , Desenho de Fármacos , Ingestão de Alimentos/efeitos dos fármacos , Metabolismo Energético/efeitos dos fármacos , Proteína Forkhead Box O1 , Fatores de Transcrição Forkhead/deficiência , Regulação da Expressão Gênica/efeitos dos fármacos , Genótipo , Hipotálamo/efeitos dos fármacos , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Knockout , Transdução de Sinais/efeitos dos fármacosRESUMO
Hypothalamic neurons expressing Agouti-related peptide (AgRP) are critical for initiating food intake, but druggable biochemical pathways that control this response remain elusive. Thus, genetic ablation of insulin or leptin signaling in AgRP neurons is predicted to reduce satiety but fails to do so. FoxO1 is a shared mediator of both pathways, and its inhibition is required to induce satiety. Accordingly, FoxO1 ablation in AgRP neurons of mice results in reduced food intake, leanness, improved glucose homeostasis, and increased sensitivity to insulin and leptin. Expression profiling of flow-sorted FoxO1-deficient AgRP neurons identifies G-protein-coupled receptor Gpr17 as a FoxO1 target whose expression is regulated by nutritional status. Intracerebroventricular injection of Gpr17 agonists induces food intake, whereas Gpr17 antagonist cangrelor curtails it. These effects are absent in Agrp-Foxo1 knockouts, suggesting that pharmacological modulation of this pathway has therapeutic potential to treat obesity.
Assuntos
Proteína Relacionada com Agouti/metabolismo , Ingestão de Alimentos , Fatores de Transcrição Forkhead/metabolismo , Hipotálamo/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Proteína Relacionada com Agouti/genética , Animais , Metabolismo Energético , Proteína Forkhead Box O1 , Fatores de Transcrição Forkhead/genética , Glucose/metabolismo , Leptina/metabolismo , CamundongosRESUMO
Insulin receptor (InsR) signaling through transcription factor FoxO1 is important in the development of hypothalamic neuron feeding circuits, but knowledge about underlying mechanisms is limited. To investigate the role of InsR/FoxO1 signaling in the development and maintenance of these circuits, we surveyed the pool of hypothalamic neurons expressing Pomc mRNA in different mouse models of impaired hypothalamic InsR signaling. InsR ablation in the entire hypothalamus did not affect Pomc-neuron number at birth, but resulted in a 25% increase, most notably in the middle arcuate nucleus region, in young adults. Selective restoration of InsR expression in POMC neurons in these mice partly reversed the abnormality, resulting in a 10% decrease compared to age-matched controls. To establish whether FoxO1 signaling plays a role in this process, we examined POMC neuron number in mice with POMC-specific deletion of FoxO1, and detected a 23% decrease in age-matched animals, consistent with a cell-autonomous role of InsR/FoxO1 signaling in regulating POMC neuron number, distinct from its established role to activate Pomc transcription. These changes in Pomc cells occurred in the absence of marked changes in humoral factors or hypothalamic NPY neurons.
Assuntos
Fatores de Transcrição Forkhead/fisiologia , Hipotálamo/citologia , Neurônios/citologia , Pró-Opiomelanocortina/genética , Receptor de Insulina/fisiologia , Transdução de Sinais/fisiologia , Fatores Etários , Animais , Contagem de Células , Proteína Forkhead Box O1 , Camundongos , RNA Mensageiro/análise , Transcrição GênicaRESUMO
Maternal obesity can influence susceptibility to obesity and type 2 diabetes in progeny. We examined the relationship of maternal insulin resistance (IR), a metabolically important consequence of increased adiposity, to adverse consequences of obesity for fetal development. We used mice heterozygous for a null allele of the insulin receptor (Insr) to study the contributions of maternal IR to offspring phenotype without the potential confound of obesity per se, and how maternal consumption of high-fat diet (HFD) may, independently and interactively, affect progeny. In progeny fed a 60% HFD, body weight and adiposity were transiently (5-7 weeks) increased in wild-type (+/+) offspring of Insr(+/-) HFD-fed dams compared to offspring of wild-type HFD-fed dams. Offspring of HFD-fed wild-type dams had increased body weight, blood glucose, and plasma insulin concentrations compared to offspring of chow-fed wild-type dams. Quantification of proopiomelanocortin (POMC) and neuropeptide-Y (NPY) populations in the arcuate nucleus of the hypothalamus (ARH) of offspring of wild-type vs. Insr(+/-) dams was performed to determine whether maternal IR affects the formation of central feeding circuits. We found a 20% increase in the number of Pomc-expressing cells at postnatal day 9 in offspring of Insr(+/-) dams. In conclusion, maternal HFD consumption-distinct from overt obesity per se-was a major contributor to increased body weight, adiposity, IR, and liver triglyceride (TG) phenotypes in progeny. Maternal IR played a minor role in predisposing progeny to obesity and IR, though it acted synergistically with maternal HFD to exacerbate early obesity in progeny.
Assuntos
Gorduras na Dieta/efeitos adversos , Hipotálamo/metabolismo , Resistência à Insulina/fisiologia , Obesidade/etiologia , Complicações na Gravidez/metabolismo , Fenômenos Fisiológicos da Nutrição Pré-Natal , Adiposidade/efeitos dos fármacos , Animais , Glicemia/metabolismo , Feminino , Hipotálamo/citologia , Insulina/sangue , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neuropeptídeo Y/metabolismo , Obesidade/fisiopatologia , Fenótipo , Gravidez , Pró-Opiomelanocortina/metabolismo , Receptor de Insulina/genética , Triglicerídeos/metabolismo , Aumento de Peso/efeitos dos fármacosRESUMO
OBJECTIVE: To determine whether n-3 fatty acids (n-3) influence arterial cholesterol delivery and lipoprotein lipase (LpL) levels in insulin-resistant mice. METHODS AND RESULTS: Insulin resistance contributes to risk of cardiovascular disease. It was previously reported that saturated fat (SAT) diets increased, but n-3 diets decreased, arterial low-density lipoprotein (LDL) cholesterol deposition from LDL total and selective uptake; this was associated with increased or decreased arterial LpL, respectively. Insulin receptor transgenic knockout mice (L1) were fed a chow, SAT, or n-3 diet for 12 weeks. Double-fluorescent boron dipyrromethene (BODIPY)-cholesteryl ester (CE) and Alexa dye-labeled human LDL were injected to separately trace LDL-CE and LDL-apolipoprotein B whole particle uptake. In contrast to SAT, n-3 diets markedly reduced all plasma lipids, ameliorating progression of insulin resistance. As opposed to SAT, n-3 reduced arterial LDL uptake, CE deposition, and selective uptake. Disparate patterns of CE deposition between diets were comparable with arterial LpL distribution; SAT induced high LpL levels throughout aortic media; LpL was limited only to intima in n-3-fed mice. CONCLUSIONS: n-3 diets diminish arterial LDL-cholesterol deposition in mice with insulin resistance, and this is associated with changes in arterial LpL levels and distribution.
Assuntos
Aorta/metabolismo , Doenças da Aorta/prevenção & controle , Aterosclerose/prevenção & controle , LDL-Colesterol/sangue , Gorduras na Dieta/administração & dosagem , Óleos de Peixe/administração & dosagem , Resistência à Insulina , Lipase Lipoproteica/sangue , Animais , Antígenos CD/genética , Aorta/patologia , Doenças da Aorta/sangue , Doenças da Aorta/genética , Doenças da Aorta/patologia , Apolipoproteína B-100 , Apolipoproteínas B/sangue , Aterosclerose/sangue , Aterosclerose/genética , Aterosclerose/patologia , Peso Corporal , Óleo de Coco , Óleo de Milho/administração & dosagem , Gorduras na Dieta/metabolismo , Modelos Animais de Doenças , Ácidos Docosa-Hexaenoicos/administração & dosagem , Ácido Eicosapentaenoico/administração & dosagem , Óleos de Peixe/metabolismo , Humanos , Insulina/sangue , Resistência à Insulina/genética , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Mutação , Azeite de Oliva , Óleos de Plantas/administração & dosagem , Receptor de Insulina/genética , Óleo de Cártamo/administração & dosagem , Fatores de TempoRESUMO
Reduced food intake brings about an adaptive decrease in energy expenditure that contributes to the recidivism of obesity after weight loss. Insulin and leptin inhibit food intake through actions in the central nervous system that are partly mediated by the transcription factor FoxO1. We show that FoxO1 ablation in pro-opiomelanocortin (Pomc)-expressing neurons in mice (here called Pomc-Foxo1(-/-) mice) increases Carboxypeptidase E (Cpe) expression, resulting in selective increases of alpha-melanocyte-stimulating hormone (alpha-Msh) and carboxy-cleaved beta-endorphin, the products of Cpe-dependent processing of Pomc. This neuropeptide profile is associated with decreased food intake and normal energy expenditure in Pomc-Foxo1(-/-) mice. We show that Cpe expression is downregulated by diet-induced obesity and that FoxO1 deletion offsets the decrease, protecting against weight gain. Moreover, moderate Cpe overexpression in the arcuate nucleus phenocopies features of the FoxO1 mutation. The dissociation of food intake from energy expenditure in Pomc-Foxo1(-/-) mice represents a model for therapeutic intervention in obesity and raises the possibility of targeting Cpe to develop weight loss medications.
Assuntos
Carboxipeptidase H/genética , Ingestão de Alimentos/fisiologia , Fatores de Transcrição Forkhead/metabolismo , Neurônios/fisiologia , Obesidade/genética , Animais , Núcleo Arqueado do Hipotálamo/metabolismo , Carboxipeptidase H/metabolismo , Feminino , Proteína Forkhead Box O1 , Fatores de Transcrição Forkhead/genética , Hipotálamo/metabolismo , Masculino , Camundongos , Camundongos Knockout , Pró-Opiomelanocortina/genética , Pró-Opiomelanocortina/metabolismo , Transdução de Sinais/genética , alfa-MSH/genética , alfa-MSH/metabolismo , beta-Endorfina/genética , beta-Endorfina/metabolismoRESUMO
Partial restoration of insulin receptor Insr expression in brain, liver, and pancreatic beta cells is sufficient for rescuing Insr knockout mice from neonatal death, preventing diabetes ketoacidosis, and normalizing life span and reproductive function. However, the transgenically rescued mice (referred to as L1) have marked hyperinsulinemia, and approximately 30% develop late-onset type 2 diabetes. Analyses of protein expression indicated that L1 mice had modestly reduced Insr content but normal insulin-stimulated Akt phosphorylation in the liver. Conversely, L1 mice had a near complete ablation of Insr protein product in the arcuate and paraventricular nuclei of the hypothalamus, which was associated with a failure to undergo insulin-dependent Akt phosphorylation in the hypothalamus. To test whether reconstitution of insulin signaling in the liver is sufficient for restoring in vivo hepatic insulin action, we performed euglycemic hyperinsulinemic clamp studies in conscious L1 and WT mice. During the clamp, L1 mice required an approximately 50% lower rate of glucose infusion than did WT controls, while the rate of glucose disappearance was not significantly altered. Conversely, the rate of glucose production was increased approximately 2-fold in L1 mice. Thus, restoration of hepatic insulin signaling in Insr knockout mice fails to normalize the in vivo response to insulin.