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1.
Metabolism ; 111: 154339, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32777442

RESUMO

BACKGROUND: Activation of neuropeptide Y2 receptors (NPYR2) by the N-terminally truncated, dipeptidyl peptidase-4 (DPP-4) generated, Peptide YY (PYY) metabolite, namely PYY(3-36), results in satiating actions. However, PYY(3-36) is also subject to C-terminal enzymatic cleavage, which annuls anorectic effects. METHODS: Substitution of l-Arg35 with d-Arg35 in the DPP-4 stable sea lamprey PYY(1-36) peptide imparts full C-terminal stability. In the current study, we have taken this molecule and introduced DPP-4 susceptibility by Iso3 substitution. RESULTS: As expected, [Iso3]sea lamprey PYY(1-36) and [Iso3](d-Arg35)sea lamprey PYY(1-36) were N-terminally degraded to respective PYY(3-36) metabolites in plasma. Only [Iso3](d-Arg35)sea lamprey PYY(1-36) was C-terminally stable. Both peptides possessed similar insulinostatic and anti-apoptotic biological actions to native PYY(1-36) in beta-cells. Unlike native PYY(1-36) and [Iso3](d-Arg35)sea lamprey PYY(1-36), [Iso3]sea lamprey PYY(1-36) displayed some proliferative actions in Npyr1 knockout beta-cells. In addition, [Iso3]sea lamprey PYY(1-36) induced more rapid NPYR2-dependent appetite suppressive effects in mice than its C-terminally stable counterpart. Twice daily administration of either peptide to high fat fed (HFF) mice resulted in significant body weight reduction and improvements in circulating triglyceride levels. [Iso3]sea lamprey PYY(1-36) treatment also prevented elevations in glucagon. Both peptides, and especially [Iso3]sea lamprey PYY(1-36), improved glucose tolerance. The treatment interventions also partially reversed the deleterious effects of sustained high fat feeding on pancreatic islet morphology. CONCLUSION: The present study confirms that sustained NPYR2 receptor activation by [Iso3](d-Arg35)sea lamprey induced significant weight lowering actions. However, identifiable benefits of this peptide over [Iso3]sea lamprey PYY(1-36), which was not protected against C-terminal degradation, were not pronounced.


Assuntos
Peptídeo YY/metabolismo , Receptores de Neuropeptídeo Y/metabolismo , Animais , Linhagem Celular , Dipeptidil Peptidase 4/metabolismo , Glucagon/metabolismo , Humanos , Células Secretoras de Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Masculino , Camundongos
2.
Proc Natl Acad Sci U S A ; 117(36): 22204-22213, 2020 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-32848060

RESUMO

The peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) is a transcriptional coactivator that controls expression of metabolic/energetic genes, programming cellular responses to nutrient and environmental adaptations such as fasting, cold, or exercise. Unlike other coactivators, PGC-1α contains protein domains involved in RNA regulation such as serine/arginine (SR) and RNA recognition motifs (RRMs). However, the RNA targets of PGC-1α and how they pertain to metabolism are unknown. To address this, we performed enhanced ultraviolet (UV) cross-linking and immunoprecipitation followed by sequencing (eCLIP-seq) in primary hepatocytes induced with glucagon. A large fraction of RNAs bound to PGC-1α were intronic sequences of genes involved in transcriptional, signaling, or metabolic function linked to glucagon and fasting responses, but were not the canonical direct transcriptional PGC-1α targets such as OXPHOS or gluconeogenic genes. Among the top-scoring RNA sequences bound to PGC-1α were Foxo1, Camk1δ, Per1, Klf15, Pln4, Cluh, Trpc5, Gfra1, and Slc25a25 PGC-1α depletion decreased a fraction of these glucagon-induced messenger RNA (mRNA) transcript levels. Importantly, knockdown of several of these genes affected glucagon-dependent glucose production, a PGC-1α-regulated metabolic pathway. These studies show that PGC-1α binds to intronic RNA sequences, some of them controlling transcript levels associated with glucagon action.


Assuntos
Glucagon/metabolismo , Glucagon/farmacologia , Hepatócitos/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Células Cultivadas , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Gluconeogênese/fisiologia , Glucose/metabolismo , Guanosina Trifosfato/metabolismo , Fígado/metabolismo , Masculino , Metabolômica , Camundongos , Camundongos Endogâmicos C57BL , Proteínas de Transporte da Membrana Mitocondrial/genética , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Ligação Proteica , Transcriptoma
3.
Metabolism ; 111: 154324, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32712220

RESUMO

BACKGROUND: Clinical trials and animal studies have shown that sodium-glucose co-transporter type 2 (SGLT2) inhibitors improve pancreatic beta cell function. Our study aimed to investigate the effect of dapagliflozin on islet morphology and cell phenotype, and explore the origin and possible reason of the regenerated beta cells. METHODS: Two diabetic mouse models, db/db mice and pancreatic alpha cell lineage-tracing (glucagon-ß-gal) mice whose diabetes was induced by high fat diet combined with streptozotocin, were used. Mice were treated by daily intragastric administration of dapagliflozin (1 mg/kg) or vehicle for 6 weeks. The plasma insulin, glucagon and glucagon-like peptide-1 (GLP-1) were determined by using ELISA. The evaluation of islet morphology and cell phenotype was performed with immunofluorescence. Primary rodent islets and αTC1.9, a mouse alpha cell line, were incubated with dapagliflozin (0.25-25 µmol/L) or vehicle in the presence or absence of GLP-1 receptor antagonist for 24 h in regular or high glucose medium. The expression of specific markers and hormone levels were determined. RESULTS: Treatment with dapagliflozin significantly decreased blood glucose in the two diabetic models and upregulated plasma insulin and GLP-1 levels in db/db mice. The dapagliflozin treatment increased islet and beta cell numbers in the two diabetic mice. The beta cell proliferation as indicated by C-peptide and BrdU double-positive cells was boosted by dapagliflozin. The alpha to beta cell conversion, as evaluated by glucagon and insulin double-positive cells and confirmed by using alpha cell lineage-tracing, was facilitated by dapagliflozin. After the dapagliflozin treatment, some insulin-positive cells were located in the duct compartment or even co-localized with duct cell markers, suggestive of duct-derived beta cell neogenesis. In cultured primary rodent islets and αTC1.9 cells, dapagliflozin upregulated the expression of pancreatic endocrine progenitor and beta cell specific markers (including Pdx1) under high glucose condition. Moreover, dapagliflozin upregulated the expression of Pcsk1 (which encodes prohormone convertase 1/3, an important enzyme for processing proglucagon to GLP-1), and increased GLP-1 content and secretion in αTC1.9 cells. Importantly, the dapagliflozin-induced upregulation of Pdx1 expression was attenuated by GLP-1 receptor antagonist. CONCLUSIONS: Except for glucose-lowering effect, dapagliflozin has extra protective effects on beta cells in type 2 diabetes. Dapagliflozin enhances beta cell self-replication, induces alpha to beta cell conversion, and promotes duct-derived beta cell neogenesis. The promoting effects of dapagliflozin on beta cell regeneration may be partially mediated via GLP-1 secreted from alpha cells.


Assuntos
Compostos Benzidrílicos/farmacologia , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Células Endócrinas/efeitos dos fármacos , Células Secretoras de Glucagon/efeitos dos fármacos , Glucosídeos/farmacologia , Regeneração/efeitos dos fármacos , Animais , Glicemia/metabolismo , Peptídeo C/metabolismo , Modelos Animais de Doenças , Células Endócrinas/metabolismo , Glucagon/metabolismo , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Receptor do Peptídeo Semelhante ao Glucagon 1/metabolismo , Células Secretoras de Glucagon/metabolismo , Glucose/metabolismo , Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Masculino , Camundongos , Pró-Proteína Convertase 1/metabolismo , Inibidores do Transportador 2 de Sódio-Glicose
4.
Nat Commun ; 11(1): 2742, 2020 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-32488111

RESUMO

Next generation sequencing studies have highlighted discrepancies in ß-cells which exist between mice and men. Numerous reports have identified MAF BZIP Transcription Factor B (MAFB) to be present in human ß-cells postnatally, while its expression is restricted to embryonic and neo-natal ß-cells in mice. Using CRISPR/Cas9-mediated gene editing, coupled with endocrine cell differentiation strategies, we dissect the contribution of MAFB to ß-cell development and function specifically in humans. Here we report that MAFB knockout hPSCs have normal pancreatic differentiation capacity up to the progenitor stage, but favor somatostatin- and pancreatic polypeptide-positive cells at the expense of insulin- and glucagon-producing cells during endocrine cell development. Our results describe a requirement for MAFB late in the human pancreatic developmental program and identify it as a distinguishing transcription factor within islet cell subtype specification. We propose that hPSCs represent a powerful tool to model human pancreatic endocrine development and associated disease pathophysiology.


Assuntos
Células Secretoras de Insulina/metabolismo , Fator de Transcrição MafB/genética , Fator de Transcrição MafB/metabolismo , Células Estreladas do Pâncreas/metabolismo , Animais , Sistemas CRISPR-Cas , Diferenciação Celular , Feminino , Edição de Genes , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Glucagon/metabolismo , Células Secretoras de Glucagon/metabolismo , Humanos , Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Masculino , Camundongos , Células-Tronco , Transcriptoma
5.
Phys Rev Lett ; 124(20): 208101, 2020 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-32501061

RESUMO

Network flows often exhibit a hierarchical treelike structure that can be attributed to the minimization of dissipation. The common feature of such systems is a single source and multiple sinks (or vice versa). In contrast, here we study networks with only a single source and sink. These systems can arise from secondary purposes of the networks, such as blood sugar regulation through insulin production. Minimization of dissipation in these systems leads to vascular shunting, a single vessel connecting the inlet and outlet. We show instead how optimizing the transport time yields network topologies that match those observed in the insulin-producing pancreatic islets. These are patterns of periphery-to-center and center-to-periphery flows. The obtained flow networks are broadly independent of how the flow velocity depends on the flow flux, but continuous and discontinuous phase transitions appear at extreme flux dependencies. Lastly, we show how constraints on flows can lead to buckling of the branches of the network, a feature that is also observed in pancreatic islets.


Assuntos
Ilhotas Pancreáticas/irrigação sanguínea , Ilhotas Pancreáticas/metabolismo , Modelos Cardiovasculares , Transporte Biológico , Glucagon/sangue , Glucagon/metabolismo , Insulina/sangue , Insulina/metabolismo , Transdução de Sinais
6.
Proc Natl Acad Sci U S A ; 117(27): 15414-15422, 2020 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-32571939

RESUMO

We report on a combined activation mechanism for a class B G-protein-coupled receptor (GPCR), the glucagon receptor. By computing the conformational free-energy landscape associated with the activation of the receptor-agonist complex and comparing it with that obtained with the ternary complex (receptor-agonist-G protein) we show that the agonist stabilizes the receptor in a preactivated complex, which is then fully activated upon binding of the G protein. The proposed mechanism contrasts with the generally assumed GPCR activation mechanism, which proceeds through an opening of the intracellular region allosterically elicited by the binding of the agonist. The mechanism found here is consistent with electron cryo-microscopy structural data and might be general for class B GPCRs. It also helps us to understand the mode of action of the numerous allosteric antagonists of this important drug target.


Assuntos
Proteínas de Ligação ao GTP/metabolismo , Domínios Proteicos , Receptores de Glucagon/metabolismo , Regulação Alostérica , Membrana Celular/metabolismo , Cristalografia por Raios X , Desenho de Fármacos , Glucagon/análogos & derivados , Glucagon/metabolismo , Simulação de Dinâmica Molecular , Relação Estrutura-Atividade
7.
Metabolism ; 109: 154290, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32522488

RESUMO

BACKGROUND: Males absent on the first (Mof) is implicated in gene control of diverse biological processes, such as cell growth, differentiation, apoptosis and autophagy. However, the relationship between glucose regulation and Mof-mediated transcription events remains unexplored. We aimed to unravel the role of Mof in glucose regulation by using global and pancreatic α-cell-specific Mof-deficient mice in vivo and α-TC1-6 cell line in vitro. METHODS: We used tamoxifen-induced temporal Mof-deficient mice first to show Mof regulate glucose homeostasis, islet cell proportions and hormone secretion. Then we used α-cell-specific Mof-deficient mice to clarify how α-cell subsets and ß-cell mass were regulated and corresponding hormone level alterations. Ultimately, we used small interfering RNA (siRNA) to knockdown Mof in α-TC1-6 and unravel the mechanism regulating α-cell mass and glucagon secretion. RESULTS: Mof was mainly expressed in α-cells. Global Mof deficiency led to lower glucose levels, attributed by decreased α/ß-cell ratio and glucagon secretion. α-cell-specific Mof-deficient mice exhibited similar alterations, with more reduced prohormone convertase 2 (PC2)-positive α-cell mass, responsible for less glucagon, and enhanced prohormone convertase 1 (PC1/3)-positive α-cell mass, leading to more glucagon-like peptide-1 (GLP-1) secretion, thus increased ß-cell mass and insulin secretion. In vitro, increased DNA damage, dysregulated autophagy, enhanced apoptosis and altered cell fate factors expressions upon Mof knockdown were observed. Genes and pathways linked to impaired glucagon secretion were uncovered through transcriptome sequencing. CONCLUSION: Mof is a potential interventional target for glucose regulation, from the aspects of both α-cell subset mass and glucagon, intra-islet GLP-1 secretion. Upon Mof deficiency, Up-regulated PC1/3 but down-regulated PC2-positive α-cell mass, leads to more GLP-1 and insulin but less glucagon secretion, and contributed to lower glucose level.


Assuntos
Glicemia/metabolismo , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Células Secretoras de Glucagon/citologia , Glucagon/metabolismo , Histona Acetiltransferases/fisiologia , Homeostase , Animais , Linhagem Celular , Histona Acetiltransferases/deficiência , Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Camundongos , Pró-Proteína Convertase 1/metabolismo , Pró-Proteína Convertase 2/metabolismo
8.
Nat Biomed Eng ; 4(5): 507-517, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32393892

RESUMO

Treatment of patients with diabetes with insulin and pramlintide (an amylin analogue) is more effective than treatment with insulin only. However, because mixtures of insulin and pramlintide are unstable and have to be injected separately, amylin analogues are only used by 1.5% of people with diabetes needing rapid-acting insulin. Here, we show that the supramolecular modification of insulin and pramlintide with cucurbit[7]uril-conjugated polyethylene glycol improves the pharmacokinetics of the dual-hormone therapy and enhances postprandial glucagon suppression in diabetic pigs. The co-formulation is stable for over 100 h at 37 °C under continuous agitation, whereas commercial formulations of insulin analogues aggregate after 10 h under similar conditions. In diabetic rats, the administration of the stabilized co-formulation increased the area-of-overlap ratio of the pharmacokinetic curves of pramlintide and insulin from 0.4 ± 0.2 to 0.7 ± 0.1 (mean ± s.d.) for the separate administration of the hormones. The co-administration of supramolecularly stabilized insulin and pramlintide better mimics the endogenous kinetics of co-secreted insulin and amylin, and holds promise as a dual-hormone replacement therapy.


Assuntos
Diabetes Mellitus Experimental/tratamento farmacológico , Composição de Medicamentos , Glucagon/metabolismo , Insulina/uso terapêutico , Polipeptídeo Amiloide das Ilhotas Pancreáticas/uso terapêutico , Animais , Hidrocarbonetos Aromáticos com Pontes/química , Difusão , Vias de Administração de Medicamentos , Estabilidade de Medicamentos , Concentração de Íons de Hidrogênio , Imidazóis/química , Insulina/administração & dosagem , Insulina/farmacocinética , Insulina/farmacologia , Polipeptídeo Amiloide das Ilhotas Pancreáticas/administração & dosagem , Polipeptídeo Amiloide das Ilhotas Pancreáticas/farmacocinética , Polipeptídeo Amiloide das Ilhotas Pancreáticas/farmacologia , Masculino , Polietilenoglicóis/química , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Suínos
9.
Am J Physiol Endocrinol Metab ; 319(1): E133-E145, 2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32459527

RESUMO

Bromocriptine mesylate treatment was examined in dogs fed a high fat diet (HFD) for 8 wk. After 4 wk on HFD, daily bromocriptine (Bromo; n = 6) or vehicle (CTR; n = 5) injections were administered. Oral glucose tolerance tests were performed before beginning HFD (OGTT1), 4 wk after HFD began (Bromo only), and after 7.5 wk on HFD (OGTT3). After 8 wk on HFD, clamp studies were performed, with infusion of somatostatin and intraportal replacement of insulin (4× basal) and glucagon (basal). From 0 to 90 min (P1), glucose was infused via peripheral vein to double the hepatic glucose load; and from 90 to 180 min (P2), glucose was infused via the hepatic portal vein at 4 mg·kg-1·min-1, with the HGL maintained at 2× basal. Bromo decreased the OGTT glucose ΔAUC0-30 and ΔAUC0-120 by 62 and 27%, respectively, P < 0.05 for both) without significantly altering the insulin response. Bromo dogs exhibited enhanced net hepatic glucose uptake (NHGU) compared with CTR (~33 and 21% greater, P1 and P2, respectively, P < 0.05). Nonhepatic glucose uptake (non-HGU) was increased ~38% in Bromo in P2 (P < 0.05). Bromo vs. CTR had higher (P < 0.05) rates of glucose infusion (36 and 30%) and non-HGU (~40 and 27%) than CTR during P1 and P2, respectively. In Bromo vs. CTR, hepatic 18:0/16:0 and 16:1/16:0 ratios tended to be elevated in triglycerides and were higher (P < 0.05) in phospholipids, consistent with a beneficial effect of bromocriptine on liver fat accumulation. Thus, bromocriptine treatment improved glucose disposal in a glucose-intolerant model, enhancing both NHGU and non-HGU.


Assuntos
Glicemia/efeitos dos fármacos , Bromocriptina/farmacologia , Dieta Hiperlipídica , Agonistas de Dopamina/farmacologia , Intolerância à Glucose/metabolismo , Fígado/efeitos dos fármacos , Animais , Glicemia/metabolismo , Cães , Ácidos Graxos não Esterificados/metabolismo , Glucagon/efeitos dos fármacos , Glucagon/metabolismo , Glucose/metabolismo , Técnica Clamp de Glucose , Teste de Tolerância a Glucose , Glicogênio/metabolismo , Veias Hepáticas , Insulina/metabolismo , Ácido Láctico/metabolismo , Fígado/metabolismo , Veia Porta , Somatostatina
10.
Am J Physiol Gastrointest Liver Physiol ; 319(1): G36-G42, 2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32463335

RESUMO

After 50% proximal small bowel resection (SBR) in mice, we have demonstrated hepatic steatosis, impaired glucose metabolism without insulin resistance, and increased pancreatic islet area. We sought to determine the consequences of SBR on pancreatic ß-cell morphology, proliferation, and expression of a key regulatory hormone, glucagon-like peptide-1 (GLP-1). C57BL/6 mice underwent 50% SBR or sham operation. At 10 wk, pancreatic insulin content and secretion was measured by ELISA. Immunohistochemistry was performed to determine structural alterations in pancreatic α-and ß-cells. Western blot analysis was used to measure GLP-1R expression, and immunoassay was used to measure plasma insulin and GLP-1. Experiments were repeated by administering a GLP-1 agonist (exendin-4) to a cohort of mice following SBR. After SBR, there was pancreatic islet hypertrophy and impaired glucose tolerance. The proportion of α and ß cells was not grossly altered. Whole pancreas and pancreatic islet insulin content was not significantly different; however, SBR mice demonstrated decreased insulin secretion in both static incubation and islet perfusion experiments. The expression of pancreatic GLP-1R was decreased approximately twofold after SBR, compared with sham and serum GLP-1, was decreased. These metabolic derangements were mitigated after administration of the GLP-1 agonist. Following massive SBR, there is significant hypertrophy of pancreatic islet cells with morphologically intact α- and ß-cells. Significantly reduced pancreatic insulin release in both static and dynamic conditions demonstrate a perturbed second phase of insulin secretion. GLP-1 is a key mediator of this amplification pathway. Decreased expression of serum GLP-1 and pancreatic GLP-1R in face of no change in insulin content presents a novel pathway for enteropancreatic glucose regulation following SBR.NEW & NOTEWORTHY Metabolic changes occur following intestinal resection; however, the effects on pancreatic function are unknown. Prior studies have demonstrated that glucagon-like protein-1 (GLP-1) signaling is a crucial player in the improved insulin sensitivity after bariatric surgery. In this study, we explore the effect of massive small bowel resection on gut hormone physiology and provide novel insights into the enteropancreatic axis.


Assuntos
Peptídeo 1 Semelhante ao Glucagon/metabolismo , Intestinos/lesões , Ilhotas Pancreáticas/metabolismo , Pâncreas/metabolismo , Animais , Glucagon/metabolismo , Receptor do Peptídeo Semelhante ao Glucagon 1/metabolismo , Insulina/sangue , Células Secretoras de Insulina/metabolismo , Camundongos Endogâmicos C57BL , Pâncreas Exócrino/metabolismo
11.
Life Sci ; 253: 117651, 2020 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-32304764

RESUMO

AIMS: To investigate the combination of dimerization and PEGylation to enhance the receptor activation and in vivo stability of Oxyntomodulin (OXM). MAIN METHODS: All LDM peptides were produced by using standard method of solid phase synthesis. The in vitro effects of LDM peptides were assessed by glucagon-like peptide-1 receptor (GLP-1R) and glucagon receptor (GcgR) binding test and Proteolytic stability test. Subsequently, saline, Liraglutide and three doses of LDM-3 treated groups were subjected to the evaluation of aute and long-term efficacy. KEY FINDINGS: Five long-acting OXM conjugates, termed LDM-1 to LDM-5, were designed using cysteine (Cys)-specific modification reaction including the activated PEG, bisMal-NH2, and OXM-Cys, and all prepared with high purity. LDM-3 exhibited greater GLP-1R and GcgR activation and ameliorative serum stability. In addition, LDM-3 was identified with enhanced insulinotropic and glycemic abilities in the gene knockout mice. The prolonged glucose-lowering effects of the LDM-3 were proved by hypoglycemic duration test and multiple oral glucose tolerance tests (OGTTs) in the diet-induced obesity (DIO) mice. Furthermore, the pharmacokinetic tests in Sprague Dawley (SD) rat and cynomolgus monkey exhibited the lifespans of LDM-3 at 90 nmol·kg-1 were 101.5 h and 119.4 h, respectively. Nevertheless, consecutive 8-week administration of LDM-3 improved the cumulative body weight gain, food intake, % HbA1c, glucose tolerance and the pancreatic of the obese mice. SIGNIFICANCE: LDM-3, as a dual GLP-1R and GcgR agonist, holds potential to be developed as a promising therapeutic candidate for both diabetes and obesity.


Assuntos
Receptor do Peptídeo Semelhante ao Glucagon 1/metabolismo , Hipoglicemiantes/química , Oxintomodulina/química , Receptores de Glucagon/metabolismo , Animais , Glicemia/efeitos dos fármacos , Diabetes Mellitus/metabolismo , Dimerização , Glucagon/metabolismo , Teste de Tolerância a Glucose , Hipoglicemiantes/farmacocinética , Macaca fascicularis , Masculino , Camundongos , Camundongos Knockout , Camundongos Obesos , Obesidade/metabolismo , Oxintomodulina/farmacocinética , Polietilenoglicóis/química , Ratos Sprague-Dawley , Técnicas de Síntese em Fase Sólida , Perda de Peso/efeitos dos fármacos
12.
Am J Physiol Endocrinol Metab ; 318(6): E920-E929, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32255678

RESUMO

The aim of this study was to identify the amino acids that stimulate glucagon secretion in mice and whose metabolism depends on glucagon receptor signaling. Pancreata of female C57BL/6JRj mice were perfused with 19 individual amino acids and pyruvate (at 10 mM), and secretion of glucagon was assessed using a specific glucagon radioimmunoassay. Separately, a glucagon receptor antagonist (GRA; 25-2648, 100 mg/kg) or vehicle was administered to female C57BL/6JRj mice 3 h before an intraperitoneal injection of four different isomolar amino acid mixtures (in total 7 µmol/g body wt) as follows: mixture 1 contained alanine, arginine, cysteine, and proline; mixture 2 contained aspartate, glutamate, histidine, and lysine; mixture 3 contained citrulline, methionine, serine, and threonine; and mixture 4 contained glutamine, leucine, isoleucine, and valine. Blood glucose, plasma glucagon, amino acid, and insulin concentrations were measured using well-characterized methodologies. Alanine (P = 0.03), arginine (P < 0.0001), cysteine (P = 0.01), glycine (P = 0.02), lysine (P = 0.02), and proline (P = 0.03), but not glutamine (P = 0.9), stimulated glucagon secretion from the perfused mouse pancreas. However, when the four isomolar amino acid mixtures were administered in vivo, the four mixtures elicited similar glucagon responses (P > 0.5). Plasma concentrations of total amino acids in vivo were higher after administration of GRA when mixture 1 (P = 0.004) or mixture 3 (P = 0.04) were injected. Our data suggest that alanine, arginine, cysteine, and proline, but not glutamine, are involved in the acute regulation of the liver-α-cell axis in female mice, as they all increased glucagon secretion and their disappearance rate was altered by GRA.


Assuntos
Aminoácidos/metabolismo , Glicemia/metabolismo , Células Secretoras de Glucagon/metabolismo , Glucagon/metabolismo , Fígado/metabolismo , Alanina/metabolismo , Animais , Arginina/metabolismo , Cisteína/metabolismo , Feminino , Células Secretoras de Glucagon/efeitos dos fármacos , Glutamina/metabolismo , Técnicas In Vitro , Insulina/metabolismo , Camundongos , Prolina/metabolismo , Receptores de Glucagon/antagonistas & inibidores , Receptores de Glucagon/metabolismo
13.
Nat Commun ; 11(1): 1896, 2020 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-32312960

RESUMO

Glucagon is released from pancreatic α-cells to activate pathways that raise blood glucose. Its secretion is regulated by α-cell-intrinsic glucose sensing and paracrine control through insulin and somatostatin. To understand the inadequately high glucagon levels that contribute to hyperglycemia in type-2 diabetes (T2D), we analyzed granule behavior, exocytosis and membrane excitability in α-cells of 68 non-diabetic and 21 T2D human donors. We report that exocytosis is moderately reduced in α-cells of T2D donors, without changes in voltage-dependent ion currents or granule trafficking. Dispersed α-cells have a non-physiological V-shaped dose response to glucose, with maximal exocytosis at hyperglycemia. Within intact islets, hyperglycemia instead inhibits α-cell exocytosis, but not in T2D or when paracrine inhibition by insulin or somatostatin is blocked. Surface expression of somatostatin-receptor-2 is reduced in T2D, suggesting a mechanism for the observed somatostatin resistance. Thus, elevated glucagon in human T2D may reflect α-cell insensitivity to paracrine inhibition at hyperglycemia.


Assuntos
Diabetes Mellitus Tipo 2/metabolismo , Exocitose/fisiologia , Células Secretoras de Glucagon/metabolismo , Glucagon/metabolismo , Células Secretoras de Glucagon/patologia , Glucose/metabolismo , Humanos , Hiperglicemia/metabolismo , Insulina/metabolismo , Imagem Óptica , Receptores de Somatostatina/metabolismo , Somatostatina/metabolismo
14.
Am J Physiol Gastrointest Liver Physiol ; 318(5): G912-G927, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32174131

RESUMO

Glucagon regulates the hepatic amino acid metabolism and increases ureagenesis. Ureagenesis is activated by N-acetylglutamate (NAG), formed via activation of N-acetylglutamate synthase (NAGS). With the aim to identify the steps whereby glucagon both acutely and chronically regulates ureagenesis, we investigated whether glucagon receptor-mediated activation of ureagenesis is required in a situation where NAGS activity and/or NAG levels are sufficient to activate the first step of the urea cycle in vivo. Female C57BL/6JRj mice treated with a glucagon receptor antagonist (GRA), glucagon receptor knockout (Gcgr-/-) mice, and wild-type (Gcgr+/+) littermates received an intraperitoneal injection of N-carbamoyl glutamate (Car; a stable variant of NAG), l-citrulline (Cit), Car and Cit (Car + Cit), or PBS. In separate experiments, Gcgr-/- and Gcgr+/+ mice were administered N-carbamoyl glutamate and l-citrulline (wCar + wCit) in the drinking water for 8 wk. Car, Cit, and Car + Cit significantly (P < 0.05) increased plasma urea concentrations, independently of pharmacological and genetic disruption of glucagon receptor signaling (P = 0.9). Car increased blood glucose concentrations equally in GRA- and vehicle-treated mice (P = 0.9), whereas the increase upon Car + Cit was impaired in GRA-treated mice (P = 0.008). Blood glucose concentrations remained unchanged in Gcgr-/- mice upon Car (P = 0.2) and Car + Cit (P = 0.9). Eight weeks administration of wCar + wCit did not change blood glucose (P > 0.2), plasma amino acid (P > 0.4), and urea concentrations (P > 0.3) or the area of glucagon-positive cells (P > 0.3) in Gcgr-/- and Gcgr+/+ mice. Our data suggest that glucagon-mediated activation of ureagenesis is not required when NAGS activity and/or NAG levels are sufficient to activate the first step of the urea cycle.NEW & NOTEWORTHY Hepatic ureagenesis is essential in amino acid metabolism and is importantly regulated by glucagon, but the exact mechanism is unclear. With the aim to identify the steps whereby glucagon both acutely and chronically regulates ureagenesis, we here show, contrary to our hypothesis, that glucagon receptor-mediated activation of ureagenesis is not required when N-acetylglutamate synthase activity and/or N-acetylglutamate levels are sufficient to activate the first step of the urea cycle in vivo.


Assuntos
Citrulina/administração & dosagem , Glucagon/metabolismo , Glutamatos/administração & dosagem , Fígado/efeitos dos fármacos , Receptores de Glucagon/deficiência , Receptores de Glucagon/metabolismo , Ureia/sangue , Aminoácido N-Acetiltransferase/metabolismo , Animais , Carbamoil-Fosfato Sintase (Amônia)/metabolismo , Feminino , Glutamatos/metabolismo , Antagonistas de Hormônios/administração & dosagem , Fígado/enzimologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de Glucagon/antagonistas & inibidores , Receptores de Glucagon/genética
15.
Science ; 367(6484): 1346-1352, 2020 03 20.
Artigo em Inglês | MEDLINE | ID: mdl-32193322

RESUMO

Class B G protein-coupled receptors, an important class of therapeutic targets, signal mainly through the Gs class of heterotrimeric G proteins, although they do display some promiscuity in G protein binding. Using cryo-electron microscopy, we determined the structures of the human glucagon receptor (GCGR) bound to glucagon and distinct classes of heterotrimeric G proteins, Gs or Gi1 These two structures adopt a similar open binding cavity to accommodate Gs and Gi1 The Gs binding selectivity of GCGR is explained by a larger interaction interface, but there are specific interactions that affect Gi more than Gs binding. Conformational differences in the receptor intracellular loops were found to be key selectivity determinants. These distinctions in transducer engagement were supported by mutagenesis and functional studies.


Assuntos
Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/química , Subunidades alfa Gs de Proteínas de Ligação ao GTP/química , Glucagon/química , Receptores de Glucagon/química , Sítios de Ligação , Microscopia Crioeletrônica , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/ultraestrutura , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa Gs de Proteínas de Ligação ao GTP/ultraestrutura , Glucagon/metabolismo , Humanos , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Conformação Proteica em alfa-Hélice , Receptores de Glucagon/metabolismo , Receptores de Glucagon/ultraestrutura , Transdução de Sinais
16.
Am J Physiol Endocrinol Metab ; 318(6): E956-E964, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32182123

RESUMO

Enhanced meal-related enteroendocrine secretion, particularly of glucagon-like peptide-1 (GLP-1), contributes to weight-loss and improved glycemia after Roux-en-Y gastric bypass (RYGB). Dietary glucose drives GLP-1 and glucose-dependent insulinotropic polypeptide (GIP) secretion postoperatively. Understanding how glucose triggers incretin secretion following RYGB could lead to new treatments of diabetes and obesity. In vitro, incretin release depends on glucose absorption via sodium-glucose cotransporter 1 (SGLT1). We investigated the importance of SGLT1/SGLT2 for enteropancreatic hormone concentrations and glucose metabolism after RYGB in a randomized, controlled, crossover study. Ten RYGB-operated patients ingested 50 g of oral glucose with and without acute pretreatment with 600 mg of the SGLT1/SGLT2-inhibitor canagliflozin. Paracetamol and 3-O-methyl-d-glucopyranose (3-OMG) were added to the glucose drink to evaluate rates of intestinal entry and absorption of glucose, respectively. Blood samples were collected for 4 h. The primary outcome was 4-h plasma GLP-1 (incremental area-under the curve, iAUC). Secondary outcomes included glucose, GIP, insulin, and glucagon. Canagliflozin delayed glucose absorption (time-to-peak 3-OMG: 50 vs. 132 min, P < 0.01) but did not reduce iAUC GLP-1 (6,067 vs. 7,273·min·pmol-1·L-1, P = 0.23), although peak GLP-1 concentrations were lowered (-28%, P = 0.03). Canagliflozin reduced GIP (iAUC -28%, P = 0.01; peak concentrations -57%, P < 0.01), insulin, and glucose excursions, whereas plasma glucagon (AUC 3,216 vs. 4,160 min·pmol·L-1, P = 0.02) and amino acids were increased. In conclusion, acute SGLT1/SGLT2-inhibition during glucose ingestion did not reduce 4-h plasma GLP-1 responses in RYGB-patients but attenuated the early rise in GLP-1, GIP, and insulin, whereas late glucagon concentrations were increased. The results suggest that SGLT1-mediated glucose absorption contributes to incretin hormone secretion after RYGB.


Assuntos
Canagliflozina/farmacologia , Derivação Gástrica , Polipeptídeo Inibidor Gástrico/metabolismo , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Transportador 1 de Glucose-Sódio/metabolismo , Inibidores do Transportador 2 de Sódio-Glicose/farmacologia , Transportador 2 de Glucose-Sódio/metabolismo , Glicemia/efeitos dos fármacos , Glicemia/metabolismo , Peptídeo C/efeitos dos fármacos , Peptídeo C/metabolismo , Estudos Cross-Over , Polipeptídeo Inibidor Gástrico/efeitos dos fármacos , Glucagon/efeitos dos fármacos , Glucagon/metabolismo , Peptídeo 1 Semelhante ao Glucagon/efeitos dos fármacos , Teste de Tolerância a Glucose , Humanos , Incretinas/metabolismo , Insulina/metabolismo , Pessoa de Meia-Idade , Polipeptídeo Pancreático/efeitos dos fármacos , Polipeptídeo Pancreático/metabolismo , Transportador 1 de Glucose-Sódio/antagonistas & inibidores
17.
Endocrinology ; 161(4)2020 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-32065829

RESUMO

ß-Cell dysfunction in type 2 diabetes (T2D) is associated with loss of cellular identity and mis-expression of alternative islet hormones, including glucagon. The molecular basis for these cellular changes has been attributed to dysregulation of core ß-cell transcription factors, which regulate ß-cell identity through activating and repressive mechanisms. The TLE1 gene lies near a T2D susceptibility locus and, recently, the glucagon repressive actions of this transcriptional coregulator have been demonstrated in vitro. We investigated whether TLE1 expression is disrupted in human T2D, and whether this is associated with increased islet glucagon-expressing cells. Automated image analysis following immunofluorescence in donors with (n = 7) and without (n = 7) T2D revealed that T2D was associated with higher islet α/ß cell ratio (Control: 0.7 ± 0.1 vs T2D: 1.6 ± 0.4; P < .05) and an increased frequency of bihormonal (insulin+/glucagon+) cells (Control: 0.8 ± 0.2% vs T2D: 2.0 ± 0.4%, P < .05). In nondiabetic donors, the majority of TLE1-positive cells were mono-hormonal ß-cells (insulin+/glucagon-: 98.2 ± 0.5%; insulin+/glucagon+: 0.7 ± 0.2%; insulin-/glucagon+: 1.1 ± 0.4%; P < .001). TLE1 expression was reduced in T2D (Control: 36 ± 2.9% vs T2D: 24 ± 2.6%; P < .05). Reduced islet TLE1 expression was inversely correlated with α/ß cell ratio (r = -0.55; P < .05). TLE1 knockdown in EndoC-ßH1 cells was associated with a 2.5-fold increase in glucagon gene mRNA and mis-expression of glucagon in insulin-positive cells. These data support TLE1 as a putative regulator of human ß-cell identity, with dysregulated expression in T2D associated with increased glucagon expression potentially reflecting ß- to α-cell conversion.


Assuntos
Proteínas Correpressoras/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Células Secretoras de Glucagon/metabolismo , Glucagon/metabolismo , Adulto , Idoso , Diabetes Mellitus Tipo 2/patologia , Feminino , Células Secretoras de Glucagon/patologia , Humanos , Secreção de Insulina/fisiologia , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/patologia , Masculino , Pessoa de Meia-Idade
18.
Diabetes ; 69(4): 647-660, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32005707

RESUMO

Aging-dependent changes in tissue function are associated with the development of metabolic diseases. However, the molecular connections linking aging, obesity, and diabetes remain unclear. Lamin A, lamin C, and progerin, products of the Lmna gene, have antagonistic functions on energy metabolism and life span. Lamin C, albeit promoting obesity, increases life span, suggesting that this isoform is crucial for maintaining healthy conditions under metabolic stresses. Because ß-cell loss during obesity or aging leads to diabetes, we investigated the contribution of lamin C to ß-cell function in physiopathological conditions. We demonstrate that aged lamin C only-expressing mice (Lmna LCS/LCS ) become obese but remain glucose tolerant due to adaptive mechanisms including increased ß-cell mass and insulin secretion. Triggering diabetes in young mice revealed that Lmna LCS/LCS animals normalize their fasting glycemia by both increasing insulin secretion and regenerating ß-cells. Genome-wide analyses combined to functional analyses revealed an increase of mitochondrial biogenesis and global translational rate in Lmna LCS/LCS islets, two major processes involved in insulin secretion. Altogether, our results demonstrate for the first time that the sole expression of lamin C protects from glucose intolerance through a ß-cell-adaptive transcriptional program during metabolic stresses, highlighting Lmna gene processing as a new therapeutic target for diabetes treatment.


Assuntos
Envelhecimento/metabolismo , Diabetes Mellitus/metabolismo , Intolerância à Glucose/metabolismo , Células Secretoras de Insulina/metabolismo , Lamina Tipo A/metabolismo , Obesidade/metabolismo , Envelhecimento/genética , Animais , Glicemia/metabolismo , Diabetes Mellitus/genética , Metabolismo Energético/fisiologia , Glucagon/metabolismo , Intolerância à Glucose/genética , Insulina/metabolismo , Lamina Tipo A/genética , Camundongos , Camundongos Transgênicos , Obesidade/genética , Pâncreas/metabolismo
19.
Am J Physiol Gastrointest Liver Physiol ; 318(4): G661-G672, 2020 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-32068442

RESUMO

Postprandial gut hormone responses change after Roux-en-Y gastric bypass (RYGB), and we investigated the impact of glucose, protein, and fat (with and without pancreas lipase inhibition) on plasma responses of gut and pancreas hormones, bile acids, and fibroblast growth factor 21 (FGF-21) after RYGB and in nonoperated control subjects. In a randomized, crossover study 10 RYGB operated and 8 healthy weight-matched control subjects were administered 4 different 4-h isocaloric (200 kcal) liquid meal tests containing >90 energy (E)% of either glucose, protein (whey protein), or fat (butter with and without orlistat). The primary outcome was glucagon-like peptide-1 (GLP-1) secretion (area under the curve above baseline). Secondary outcomes included responses of peptide YY (PYY), glucose-dependent insulinotropic polypeptide (GIP), cholecystokinin (CCK), glicentin, neurotensin, ghrelin, insulin, glucagon, bile acids, and FGF-21. In the RYGB group the responses of GLP-1, GIP, glicentin, FGF-21, and C-peptide were increased after glucose compared with the other meals. The neurotensin and bile acids responses were greater after fat, while the glucagon and CCK responses were greater after protein ingestion. Furthermore, compared with control subjects, RYGB subjects had greater responses of total PYY after glucose, glucagon after glucose and fat, glicentin after glucose and protein, and GLP-1 and neurotensin after all meals, while GIP and CCK responses were lower after fat. Ghrelin responses did not differ between meals or between groups. Orlistat reduced all hormone responses to fat ingestion, except for ghrelin in the RYGB group. In conclusion, after RYGB glucose is a more potent stimulator of most gut hormones, especially for the marked increased secretion of GLP-1 compared with fat and protein.NEW & NOTEWORTHY We investigated the impact of glucose, protein, and fat meals on intestinal and pancreatic hormones, bile acid, and fibroblast growth factor 21 (FGF-21) secretion in gastric bypass-operated patients compared with matched nonoperated individuals. The fat meal was administered with and without a pancreas lipase inhibitor. We found that the impact of the different meals on gut hormones, bile, and FGF 21 secretion differ and was different from the responses observed in nonoperated control subjects.


Assuntos
Ácidos e Sais Biliares/metabolismo , Fatores de Crescimento de Fibroblastos/metabolismo , Derivação Gástrica , Trato Gastrointestinal/metabolismo , Glucose/administração & dosagem , Pâncreas/metabolismo , Acetaminofen/administração & dosagem , Acetaminofen/sangue , Acetaminofen/farmacocinética , Adolescente , Adulto , Analgésicos não Entorpecentes/administração & dosagem , Analgésicos não Entorpecentes/sangue , Analgésicos não Entorpecentes/farmacocinética , Glicemia , Colecistocinina/metabolismo , Gorduras na Dieta , Proteínas na Dieta/administração & dosagem , Feminino , Polipeptídeo Inibidor Gástrico/metabolismo , Grelina/metabolismo , Glicentina/metabolismo , Glucagon/metabolismo , Glucose/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Neurotensina/metabolismo , Adulto Jovem
20.
Diabetes ; 69(5): 882-892, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32005706

RESUMO

Glucagon is classically described as a counterregulatory hormone that plays an essential role in the protection against hypoglycemia. In addition to its role in the regulation of glucose metabolism, glucagon has been described to promote ketosis in the fasted state. Sodium-glucose cotransporter 2 inhibitors (SGLT2i) are a new class of glucose-lowering drugs that act primarily in the kidney, but some reports have described direct effects of SGLT2i on α-cells to stimulate glucagon secretion. Interestingly, SGLT2 inhibition also results in increased endogenous glucose production and ketone production, features common to glucagon action. Here, we directly test the ketogenic role of glucagon in mice, demonstrating that neither fasting- nor SGLT2i-induced ketosis is altered by interruption of glucagon signaling. Moreover, any effect of glucagon to stimulate ketogenesis is severely limited by its insulinotropic actions. Collectively, our data suggest that fasting-associated ketosis and the ketogenic effects of SGLT2 inhibitors occur almost entirely independent of glucagon.


Assuntos
Compostos Benzidrílicos/farmacologia , Privação de Alimentos , Glucagon/metabolismo , Glucosídeos/farmacologia , Insulina/sangue , Transportador 2 de Glucose-Sódio/metabolismo , Animais , Glicemia , Epinefrina/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Insulina/metabolismo , Lipólise/efeitos dos fármacos , Camundongos , Transportador 2 de Glucose-Sódio/genética , Inibidores do Transportador 2 de Sódio-Glicose/farmacologia
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