RESUMO
Congenital hyperinsulinism of infancy (CHI) can be caused by a deficiency of the ubiquitously expressed enzyme short-chain 3-hydroxyacyl-CoA dehydrogenase (SCHAD). To test the hypothesis that SCHAD-CHI arises from a specific defect in pancreatic ß-cells, we created genetically engineered ß-cell-specific (ß-SKO) or hepatocyte-specific (L-SKO) SCHAD knockout mice. While L-SKO mice were normoglycemic, plasma glucose in ß-SKO animals was significantly reduced in the random-fed state, after overnight fasting, and following refeeding. The hypoglycemic phenotype was exacerbated when the mice were fed a diet enriched in leucine, glutamine, and alanine. Intraperitoneal injection of these three amino acids led to a rapid elevation in insulin levels in ß-SKO mice compared to controls. Consistently, treating isolated ß-SKO islets with the amino acid mixture potently enhanced insulin secretion compared to controls in a low-glucose environment. RNA sequencing of ß-SKO islets revealed reduced transcription of ß-cell identity genes and upregulation of genes involved in oxidative phosphorylation, protein metabolism, and Ca2+ handling. The ß-SKO mouse offers a useful model to interrogate the intra-islet heterogeneity of amino acid sensing given the very variable expression levels of SCHAD within different hormonal cells, with high levels in ß- and δ-cells and virtually absent α-cell expression. We conclude that the lack of SCHAD protein in ß-cells results in a hypoglycemic phenotype characterized by increased sensitivity to amino acid-stimulated insulin secretion and loss of ß-cell identity.
Assuntos
3-Hidroxiacil-CoA Desidrogenase , Aminoácidos , Hiperinsulinismo Congênito , Hipoglicemia , Secreção de Insulina , Células Secretoras de Insulina , Animais , Camundongos , Aminoácidos/metabolismo , Aminoácidos/farmacologia , Hipoglicemia/enzimologia , Hipoglicemia/genética , Insulina/metabolismo , Secreção de Insulina/efeitos dos fármacos , Camundongos Knockout , 3-Hidroxiacil-CoA Desidrogenase/deficiência , 3-Hidroxiacil-CoA Desidrogenase/genética , Células Secretoras de Insulina/enzimologia , Hiperinsulinismo Congênito/genéticaRESUMO
AIM: To evaluate gastric emptying (GE) and the glycaemic response to a 75-g oral glucose load in newly diagnosed, treatment-naïve Han Chinese with type 2 diabetes (T2D) before insulin pump therapy, after 4 weeks of insulin pump therapy, and 12-15 months after insulin pump therapy. MATERIALS AND METHODS: Twenty participants with T2D (baseline glycated haemoglobin [± SD] 10.7% [± 1.2%] 93 [± 10] mmol/mol) ingested a 75-g glucose drink containing 150 mg 13C-acetate, to determine the gastric half-emptying time, and underwent assessment of plasma glucose and serum insulin, C-peptide and glucagon-like peptide-1 (GLP-1) over 180 min before and after 4 weeks of insulin pump therapy (discontinued for 48 h before re-assessment). Data were compared to those in 19 healthy participants matched for sex and age. After 12-15 months, GE was re-measured in 14 of the T2D participants. RESULTS: At baseline, participants with T2D exhibited substantially augmented fasting and post-glucose glycaemia, diminished insulin secretion, and more rapid GE (p < 0.05 each), but comparable GLP-1, compared to healthy participants. Following insulin pump therapy, insulin secretion increased, GLP-1 secretion was attenuated, fasting and post-glucose glycaemia were lower, and GE was slowed (p < 0.05 each). The slowing of GE in T2D participants was sustained over 12-15 months of follow-up. CONCLUSIONS: In newly diagnosed Han Chinese with T2D, GE is often accelerated despite poor glycaemic control and is slowed by short-term insulin pump therapy. The effect on GE is maintained for at least 12 months.
Assuntos
Glicemia , Diabetes Mellitus Tipo 2 , Esvaziamento Gástrico , Hipoglicemiantes , Sistemas de Infusão de Insulina , Insulina , Humanos , Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/sangue , Masculino , Feminino , Pessoa de Meia-Idade , Esvaziamento Gástrico/efeitos dos fármacos , Glicemia/análise , Glicemia/metabolismo , Insulina/administração & dosagem , Hipoglicemiantes/administração & dosagem , China , Hemoglobinas Glicadas/análise , Hemoglobinas Glicadas/metabolismo , Adulto , Povo Asiático , Peptídeo 1 Semelhante ao Glucagon/administração & dosagem , Peptídeo C/sangue , Secreção de Insulina/efeitos dos fármacos , Teste de Tolerância a Glucose , População do Leste AsiáticoRESUMO
AIMS: To investigate the effects of the selective serotonin reuptake inhibitors (SSRIs) sertraline and paroxetine at therapeutically relevant concentrations on beta-cell mass and function. METHODS: Viability was quantified in mouse insulinoma (MIN6) beta cells and mouse islets after 48-h exposure to sertraline (1-10 µM) or paroxetine (0.01-1 µM) using the Trypan blue exclusion test. The effects of therapeutic concentrations of these SSRIs on insulin secretion were determined by static incubation and perifusion experiments, while islet apoptosis was investigated by Caspase-Glo 3/7 assay, TUNEL staining and quantitative PCR analysis. Finally, proliferation of MIN6 and mouse islet beta cells was assessed by bromodeoxyuridine (BrdU) enzyme-linked immunosorbent assay and immunofluorescence. RESULTS: Sertraline (0.1-1 µM) and paroxetine (0.01-0.1 µM) were well tolerated by MIN6 beta cells and islets, whereas 10 µM sertraline and 1 µM paroxetine were cytotoxic. Exposure to 1 µM sertraline and 0.1 µM paroxetine significantly potentiated glucose-stimulated insulin secretion from mouse and human islets. Moreover, they showed protective effects against cytokine- and palmitate-induced apoptosis of islets, they downregulated cytokine-induced Stat1 and Traf1 mRNA expression, and they significantly increased proliferation of mouse beta cells. CONCLUSIONS: Our data demonstrate that sertraline and paroxetine act directly on beta cells to enhance glucose-stimulated insulin secretion and stimulate beta-cell mass expansion by increasing proliferation and decreasing apoptosis. These drugs are therefore likely to be appropriate for treating depression in people with type 2 diabetes.
Assuntos
Apoptose , Proliferação de Células , Secreção de Insulina , Células Secretoras de Insulina , Paroxetina , Inibidores Seletivos de Recaptação de Serotonina , Sertralina , Paroxetina/farmacologia , Sertralina/farmacologia , Animais , Inibidores Seletivos de Recaptação de Serotonina/farmacologia , Inibidores Seletivos de Recaptação de Serotonina/uso terapêutico , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Camundongos , Secreção de Insulina/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Humanos , Insulina/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Masculino , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/metabolismoRESUMO
Background this study was conducted to assess the effects of vitamin D on differentiation of bone marrow- derived mesenchymal stem cells (BM-MSCs) into insulin producing cells (IPCs). Method BM-MSCs were isolated from femur and tibia of rats and incubated in low (LG) or high glucose (HG) (5mM or 25mM), or high glucose DMEM media supplemented with vitamin D (0.2nM) (HGD) for 14 days. Cells viability was analysis by MTT assay. Differentiation of SCs was confirmed using measuring genes expression level of pdx1 and insulin, and insulin secretion, glucose stimulated insulin secretion, and insulin content by ELISA method. Results Cell viability was significantly higher in HGD than LG (p < 0.05) in day 3, also, in HG and HGD than LG (p < 0.001), and HGD vs. HG (p < 0.001) in day 7. Pdx1 and insulin level was markedly higher in HGD than LG (p < 0.05 and p < 0.01). pdx1 expression was markedly higher in HGD (p < 0.05) than LG, also insulin expression the HG (p < 0.05), and HGD (p < 0.01) groups compared to the LG group. Insulin release at 5mM glucose was notably higher in the HGD group compared to LG (p < 0.05), and at 25mM glucose, both HG and HGD showed significant increases vs. LG (p < 0.05 and p < 0.01, respectively). Insulin content was significantly higher in both 5mM and 25mM glucose for HG and HGD vs. LG (p < 0.01 and p < 0.001, respectively). In conclusion, treatment BM-MSCs with vitamin D could increase their differentiation into IPCs and it can be considered as a potential supplementary agent in enhancing differentiation SCs into insulin generating cells.
Assuntos
Células da Medula Óssea , Diferenciação Celular , Células Secretoras de Insulina , Insulina , Células-Tronco Mesenquimais , Vitamina D , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/citologia , Animais , Diferenciação Celular/efeitos dos fármacos , Vitamina D/farmacologia , Vitamina D/metabolismo , Ratos , Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/citologia , Glucose/metabolismo , Glucose/farmacologia , Proteínas de Homeodomínio/metabolismo , Proteínas de Homeodomínio/genética , Células Cultivadas , Sobrevivência Celular/efeitos dos fármacos , Masculino , Transativadores/metabolismo , Transativadores/genética , Suplementos Nutricionais , Secreção de Insulina/efeitos dos fármacosRESUMO
We aimed to determine if cheese could reduce glucose intolerance in aged rats with overt type 2 diabetes (T2D). Male Sprague-Dawley rats treated with high-fat diet (HFD) and streptozotocin (STZ) to elicit T2D were hyperglycemic. One week after STZ injection, low-fat (LOW) or regular-fat (REG) cheese was provided for 5 weeks and compared with T2D and low-fat diet reference (REF) groups. Food intake and weight gain were similar in all groups. Oral glucose tolerance tests revealed glucose intolerance in T2D rats that was partially ameliorated by LOW but not REG. Insulin secretion during the oral glucose tolerance test was impaired in T2D and REG at 10 min (p < 0.05) but the iAUC was highly variable in all groups and statistical differences were not detected (p > 0.05). ß-cell mass and pancreatic insulin content in T2D and REG were 50% lower than REF (p < 0.05), whereas LOW was not significantly different. Although isolated islets from all groups responded to glucose, the absolute amount of insulin secreted by T2D and REG was markedly reduced compared with REF, while LOW islets had relatively normal secretion. In conclusion, LOW but not REG cheese enhanced ß-cell recovery from HFD/STZ treatment that led to amelioration of glucose tolerance within 5 weeks.
Assuntos
Queijo , Diabetes Mellitus Tipo 2 , Intolerância à Glucose , Secreção de Insulina , Células Secretoras de Insulina , Insulina , Ratos Sprague-Dawley , Animais , Masculino , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/efeitos dos fármacos , Intolerância à Glucose/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Secreção de Insulina/efeitos dos fármacos , Ratos , Insulina/metabolismo , Insulina/sangue , Teste de Tolerância a Glucose , Dieta Hiperlipídica/efeitos adversos , Dieta com Restrição de Gorduras , Modelos Animais de Doenças , Glicemia/metabolismo , Diabetes Mellitus Experimental/metabolismoRESUMO
Islet transplantation for type 1 diabetes treatment has been limited by the need for lifelong immunosuppression regimens. This challenge has prompted the development of macroencapsulation devices (MEDs) to immunoprotect the transplanted islets. While promising, conventional MEDs are faced with insufficient transport of oxygen, glucose, and insulin because of the reliance on passive diffusion. Hence, these devices are constrained to two-dimensional, wafer-like geometries with limited loading capacity to maintain cells within a distance of passive diffusion. We hypothesized that convective nutrient transport could extend the loading capacity while also promoting cell viability, rapid glucose equilibration, and the physiological levels of insulin secretion. Here, we showed that convective transport improves nutrient delivery throughout the device and affords a three-dimensional capsule geometry that encapsulates 9.7-fold-more cells than conventional MEDs. Transplantation of a convection-enhanced MED (ceMED) containing insulin-secreting ß cells into immunocompetent, hyperglycemic rats demonstrated a rapid, vascular-independent, and glucose-stimulated insulin response, resulting in early amelioration of hyperglycemia, improved glucose tolerance, and reduced fibrosis. Finally, to address potential translational barriers, we outlined future steps necessary to optimize the ceMED design for long-term efficacy and clinical utility.
Assuntos
Encapsulamento de Células/métodos , Sistemas de Liberação de Medicamentos/métodos , Células Secretoras de Insulina/metabolismo , Animais , Sobrevivência Celular/efeitos dos fármacos , Convecção , Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 1/tratamento farmacológico , Diabetes Mellitus Tipo 1/metabolismo , Sistemas de Liberação de Medicamentos/instrumentação , Insulina/metabolismo , Secreção de Insulina/efeitos dos fármacos , Secreção de Insulina/fisiologia , Células Secretoras de Insulina/efeitos dos fármacos , Ilhotas Pancreáticas/metabolismo , Transplante das Ilhotas Pancreáticas/métodos , Masculino , RatosRESUMO
Previous studies have suggested a potential role of bone morphogenetic protein 6 (BMP6) in glucose metabolism, which also seems to be regulated by serotonin (5-hydroxytryptamine, 5HT), a biogenic amine with multiple roles in the organism. In this study, we explored possible interactions between BMP6, serotonin, and glucose metabolism regulation. The effect of BMP6 or 5HT on pancreatic ß-cells has been studied in vitro using the INS-1 832/13 rat insulinoma cell line. Studies in vivo have been performed on mice with the global deletion of the Bmp6 gene (BMP6-/-) and included glucose and insulin tolerance tests, gene expression studies using RT-PCR, immunohistochemistry, and ELISA analyses. We have shown that BMP6 and 5HT treatments have the opposite effect on insulin secretion from INS-1 cells. The effect of BMP6 on the 5HT system in vivo depends on the tissue studied, with no observable systemic effect on peripheral 5HT metabolism. BMP6 deficiency does not cause diabetic changes, although a mild difference in insulin tolerance test between BMP6-/- and WT mice was observed. In conclusion, BMP6 does not directly influence glucose metabolism, but there is a possibility that its deletion causes slowly developing changes in glucose and serotonin metabolism, which would become more expressed with ageing.
Assuntos
Proteína Morfogenética Óssea 6 , Glucose , Células Secretoras de Insulina , Insulina , Serotonina , Animais , Serotonina/metabolismo , Glucose/metabolismo , Proteína Morfogenética Óssea 6/metabolismo , Proteína Morfogenética Óssea 6/genética , Camundongos , Ratos , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/efeitos dos fármacos , Insulina/metabolismo , Camundongos Knockout , Linhagem Celular Tumoral , Masculino , Secreção de Insulina/efeitos dos fármacosRESUMO
A detailed study of palmitate metabolism in pancreatic islets subject to different experimental conditions, like varying concentrations of glucose, as well as fed or starved conditions, has allowed us to explore the interaction between the two main plasma nutrients and its consequences on hormone secretion. Palmitate potentiates glucose-induced insulin secretion in a concentration-dependent manner, in a physiological range of both palmitate (0-2 mM) and glucose (6-20 mM) concentrations; at glucose concentrations lower than 6 mM, no metabolic interaction with palmitate was apparent. Starvation (48 h) increased islet palmitate oxidation two-fold, and the effect was resistant to its inhibition by glucose (6-20 mM). Consequently, labelled palmitate and glucose incorporation into complex lipids were strongly suppressed, as well as glucose-induced insulin secretion and its potentiation by palmitate. 2-bromostearate, a palmitate oxidation inhibitor, fully recovered the synthesis of complex lipids and insulin secretion. We concluded that palmitate potentiation of the insulin response to glucose is not attributable to its catabolic mitochondrial oxidation but to its anabolism to complex lipids: islet lipid biosynthesis is dependent on the uptake of plasma fatty acids and the supply of α-glycerol phosphate from glycolysis. Islet secretion of glucagon and somatostatin showed a similar dependence on palmitate anabolism as insulin. The possible mechanisms implicated in the metabolic coupling between glucose and palmitate were commented on. Moreover, possible mechanisms responsible for islet gluco- or lipotoxicity after a long-term stimulation of insulin secretion were also discussed. Our own data on the simultaneous stimulation of insulin, glucagon, and somatostatin by glucose, as well as their modification by 2-bromostearate in perifused rat islets, give support to the conclusion that increased FFA anabolism, rather than its mitochondrial oxidation, results in a potentiation of their stimulated release. Starvation, besides suppressing glucose stimulation of insulin secretion, also blocks the inhibitory effect of glucose on glucagon secretion: this suggests that glucagon inhibition might be an indirect or direct effect of insulin, but not of glucose. In summary, there seems to exist three mechanisms of glucagon secretion stimulation: 1. glucagon stimulation through the same secretion coupling mechanism as insulin, but in a different range of glucose concentrations (0 to 5 mM). 2. Direct or indirect inhibition by secreted insulin in response to glucose (5-20 mM). 3. Stimulation by increased FFA anabolism in glucose intolerance or diabetes in the context of hyperlipidemia, hyperglycemia, and hypo-insulinemia. These conclusions were discussed and compared with previous published data in the literature. Specially, we discussed the mechanism for inhibition of glucagon release by glucose, which was apparently contradictory with the secretion coupling mechanism of its stimulation.
Assuntos
Glucagon , Glucose , Secreção de Insulina , Insulina , Ilhotas Pancreáticas , Glucose/metabolismo , Animais , Insulina/metabolismo , Glucagon/metabolismo , Ilhotas Pancreáticas/metabolismo , Ilhotas Pancreáticas/efeitos dos fármacos , Secreção de Insulina/efeitos dos fármacos , Ácidos Graxos/metabolismo , Ratos , Palmitatos/metabolismo , Palmitatos/farmacologia , Oxirredução/efeitos dos fármacosRESUMO
Despite the availability of different treatments for type 2 diabetes (T2D), post-diagnosis complications remain prevalent; therefore, more effective treatments are desired. Glucagon-like peptide (GLP)-1-based drugs are currently used for T2D treatment. They act as orthosteric agonists for the GLP-1 receptor (GLP-1R). In this study, we analyzed in vitro how the GLP-1R orthosteric and allosteric agonists augment glucose-stimulated insulin secretion (GSIS) and intracellular cAMP production (GSICP) in INS-1E pancreatic beta cells under healthy, diabetic, and recovered states. The findings from this study suggest that allosteric agonists have a longer duration of action than orthosteric agonists. They also suggest that the GLP-1R agonists do not deplete intracellular insulin, indicating they can be a sustainable and safe treatment option for T2D. Importantly, this study demonstrates that the GLP-1R agonists variably augment GSIS through GSICP in healthy, diabetic, and recovered INS-1E cells. Furthermore, we find that INS-1E cells respond differentially to the GLP-1R agonists depending on both glucose concentration during and before treatment and/or whether the cells have been previously exposed to these drugs. In conclusion, the findings described in this manuscript will be useful in determining in vitro how pancreatic beta cells respond to T2D drug treatments in healthy, diabetic, and recovered states.
Assuntos
Diabetes Mellitus Tipo 2 , Receptor do Peptídeo Semelhante ao Glucagon 1 , Secreção de Insulina , Células Secretoras de Insulina , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/efeitos dos fármacos , Receptor do Peptídeo Semelhante ao Glucagon 1/agonistas , Receptor do Peptídeo Semelhante ao Glucagon 1/metabolismo , Secreção de Insulina/efeitos dos fármacos , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/tratamento farmacológico , Animais , Regulação Alostérica/efeitos dos fármacos , Ratos , Humanos , Insulina/metabolismo , Glucose/metabolismo , AMP Cíclico/metabolismo , Linhagem Celular , Hipoglicemiantes/farmacologia , Peptídeo 1 Semelhante ao Glucagon/metabolismoRESUMO
Serotonin or 5-hydroxytryptamine (5-HT) is a monoamine that plays a critical role in insulin secretion, energy metabolism, and mitochondrial biogenesis. However, the action of serotonin in insulin production and secretion by pancreatic ß cells has not yet been elucidated. Here, we investigated how exogenous nanomolar serotonin concentrations regulate insulin synthesis and secretion in rat insulinoma INS-1E cells. Nanomolar serotonin concentrations (10 and 50 nM) significantly increased insulin protein expression above the constant levels in untreated control cells and decreased insulin protein levels in the media. The reductions in insulin protein levels in the media may be associated with ubiquitin-mediated protein degradation. The levels of membrane vesicle trafficking-related proteins including Rab5, Rab3A, syntaxin6, clathrin, and EEA1 proteins were significantly decreased by serotonin treatment compared to the untreated control cells, whereas the expressions of Rab27A, GOPC, and p-caveolin-1 proteins were significantly reduced by serotonin treatment. In this condition, serotonin receptors, Gαq-coupled 5-HT2b receptor (Htr2b), and ligand-gated ion channel receptor Htr3a were significantly decreased by serotonin treatment. To confirm the serotonylation of Rab3A and Rab27A during insulin secretion, we investigated the protein levels of Rab3A and Rab27A, in which transglutaminase 2 (TGase2) serotonylated Rab3A but not Rab27A. The increases in ERK phosphorylation levels were consistent with increases in the expression of p-Akt. Also, the expression level of the Bcl-2 protein was significantly increased by 50 and 100 nM serotonin treatment compared to the untreated control cells, whereas the levels of Cu/Zn-SOD and Mn-SOD proteins decreased. These results indicate that nanomolar serotonin treatment regulates the insulin protein level but decreases this level in media through membrane vesicle trafficking-related proteins (Rab5, Rab3A, syntaxin6, clathrin, and EEA1), the Akt/ERK pathway, and Htr2b/Htr3a in INS-1E cells.
Assuntos
Secreção de Insulina , Insulina , Insulinoma , Serotonina , Animais , Serotonina/metabolismo , Serotonina/farmacologia , Ratos , Insulinoma/metabolismo , Insulinoma/patologia , Secreção de Insulina/efeitos dos fármacos , Insulina/metabolismo , Linhagem Celular Tumoral , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismoRESUMO
Epinephrine influences the function of pancreatic ß-cells, primarily through the α2A-adrenergic receptor (α2A-AR) on their plasma membrane. Previous studies indicate that epinephrine transiently suppresses insulin secretion, whereas prolonged exposure induces its compensatory secretion. Nonetheless, the impact of epinephrine-induced α2A-AR signaling on the survival and function of pancreatic ß-cells, particularly the impact of reprogramming after their removal from sustained epinephrine stimulation, remains elusive. In the present study, we applied MIN6, a murine insulinoma cell line, with 3 days of high concentration epinephrine incubation and 2 days of standard incubation, explored cell function and activity, and analyzed relevant regulatory pathways. The results showed that chronic epinephrine incubation led to the desensitization of α2A-AR and enhanced insulin secretion. An increased number of docked insulin granules and impaired Syntaxin-2 was found after chronic epinephrine exposure. Growth curve and cell cycle analyses showed the inhibition of cell proliferation. Transcriptome analysis showed the occurrence of endoplasmic reticulum stress (ER stress) and oxidative stress, such as the presence of BiP, CHOP, IRE1, ATF4, and XBP, affecting cellular endoplasmic reticulum function and survival, along with UCP2, OPA1, PINK, and PRKN, associated with mitochondrial dysfunction. Consequently, we conclude that chronic exposure to epinephrine induces α2A-AR desensitization and leads to ER and oxidative stress, impairing protein processing and mitochondrial function, leading to modified pancreatic ß-cell secretory function and cell fate.
Assuntos
Estresse do Retículo Endoplasmático , Epinefrina , Células Secretoras de Insulina , Insulina , Estresse Oxidativo , Animais , Epinefrina/farmacologia , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Camundongos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Insulina/metabolismo , Secreção de Insulina/efeitos dos fármacos , Receptores Adrenérgicos alfa 2/metabolismo , Receptores Adrenérgicos alfa 2/genética , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Mitocôndrias/metabolismo , Mitocôndrias/efeitos dos fármacosRESUMO
Filamentous actin (F-actin) cytoskeletal remodeling is critical for glucose-stimulated insulin secretion (GSIS) in pancreatic ß-cells, and its dysregulation causes type 2 diabetes. The adaptor protein APPL1 promotes first-phase GSIS by up-regulating soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein expression. However, whether APPL2 (a close homology of APPL1 with the same domain organization) plays a role in ß-cell functions is unknown. Here, we show that APPL2 enhances GSIS by promoting F-actin remodeling via the small GTPase Rac1 in pancreatic ß-cells. ß-cell specific abrogation of APPL2 impaired GSIS, leading to glucose intolerance in mice. APPL2 deficiency largely abolished glucose-induced first- and second-phase insulin secretion in pancreatic islets. Real-time live-cell imaging and phalloidin staining revealed that APPL2 deficiency abolished glucose-induced F-actin depolymerization in pancreatic islets. Likewise, knockdown of APPL2 expression impaired glucose-stimulated F-actin depolymerization and subsequent insulin secretion in INS-1E cells, which were attributable to the impairment of Ras-related C3 botulinum toxin substrate 1 (Rac1) activation. Treatment with the F-actin depolymerization chemical compounds or overexpression of gelsolin (a F-actin remodeling protein) rescued APPL2 deficiency-induced defective GSIS. In addition, APPL2 interacted with Rac GTPase activating protein 1 (RacGAP1) in a glucose-dependent manner via the bin/amphiphysin/rvs-pleckstrin homology (BAR-PH) domain of APPL2 in INS-1E cells and HEK293 cells. Concomitant knockdown of RacGAP1 expression reverted APPL2 deficiency-induced defective GSIS, F-actin remodeling, and Rac1 activation in INS-1E cells. Our data indicate that APPL2 interacts with RacGAP1 and suppresses its negative action on Rac1 activity and F-actin depolymerization thereby enhancing GSIS in pancreatic ß-cells.
Assuntos
Actinas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/farmacologia , Glucose/metabolismo , Secreção de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Citoesqueleto de Actina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Diabetes Mellitus Tipo 2/metabolismo , Feminino , Proteínas Ativadoras de GTPase/metabolismo , Técnicas de Silenciamento de Genes , Intolerância à Glucose , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Knockout , Neuropeptídeos/genética , Neuropeptídeos/metabolismo , Proteínas SNARE/metabolismo , Transcriptoma , Proteínas rac1 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
Voltage-gated calcium 3.1 (CaV3.1) channels are absent in healthy mouse ß cells and mediate minor T-type Ca2+ currents in healthy rat and human ß cells but become evident under diabetic conditions. Whether more active CaV3.1 channels affect insulin secretion and glucose homeostasis remains enigmatic. We addressed this question by enhancing de novo expression of ß cell CaV3.1 channels and exploring the consequent impacts on dynamic insulin secretion and glucose homeostasis as well as underlying molecular mechanisms with a series of in vitro and in vivo approaches. We now demonstrate that a recombinant adenovirus encoding enhanced green fluorescent protein-CaV3.1 subunit (Ad-EGFP-CaV3.1) efficiently transduced rat and human islets as well as dispersed islet cells. The resulting CaV3.1 channels conducted typical T-type Ca2+ currents, leading to an enhanced basal cytosolic-free Ca2+ concentration ([Ca2+]i). Ad-EGFP-CaV3.1-transduced islets released significantly less insulin under both the basal and first phases following glucose stimulation and could no longer normalize hyperglycemia in recipient rats rendered diabetic by streptozotocin treatment. Furthermore, Ad-EGFP-CaV3.1 transduction reduced phosphorylated FoxO1 in the cytoplasm of INS-1E cells, elevated FoxO1 nuclear retention, and decreased syntaxin 1A, SNAP-25, and synaptotagmin III. These effects were prevented by inhibiting CaV3.1 channels or the Ca2+-dependent phosphatase calcineurin. Enhanced expression of ß cell CaV3.1 channels therefore impairs insulin release and glucose homeostasis by means of initial excessive Ca2+ influx, subsequent activation of calcineurin, consequent dephosphorylation and nuclear retention of FoxO1, and eventual FoxO1-mediated down-regulation of ß cell exocytotic proteins. The present work thus suggests an elevated expression of CaV3.1 channels plays a significant role in diabetes pathogenesis.
Assuntos
Canais de Cálcio Tipo T/metabolismo , Diabetes Mellitus Experimental/metabolismo , Secreção de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Adolescente , Adulto , Animais , Células COS , Bloqueadores dos Canais de Cálcio/farmacologia , Bloqueadores dos Canais de Cálcio/uso terapêutico , Canais de Cálcio Tipo T/genética , Núcleo Celular/metabolismo , Chlorocebus aethiops , Citosol/metabolismo , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/tratamento farmacológico , Exocitose/efeitos dos fármacos , Estudos de Viabilidade , Feminino , Proteínas de Fluorescência Verde/genética , Células HEK293 , Humanos , Insulina/metabolismo , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/transplante , Masculino , Pessoa de Meia-Idade , Fosforilação , Cultura Primária de Células , Ratos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Estreptozocina/toxicidade , Proteínas de Transporte Vesicular/metabolismo , Adulto JovemRESUMO
The inhibitory G protein alpha-subunit (Gαz) is an important modulator of beta-cell function. Full-body Gαz-null mice are protected from hyperglycemia and glucose intolerance after long-term high-fat diet (HFD) feeding. In this study, at a time point in the feeding regimen where WT mice are only mildly glucose intolerant, transcriptomics analyses reveal islets from HFD-fed Gαz KO mice have a dramatically altered gene expression pattern as compared with WT HFD-fed mice, with entire gene pathways not only being more strongly upregulated or downregulated versus control-diet fed groups but actually reversed in direction. Genes involved in the "pancreatic secretion" pathway are the most strongly differentially regulated: a finding that correlates with enhanced islet insulin secretion and decreased glucagon secretion at the study end. The protection of Gαz-null mice from HFD-induced diabetes is beta-cell autonomous, as beta cell-specific Gαz-null mice phenocopy the full-body KOs. The glucose-stimulated and incretin-potentiated insulin secretion response of islets from HFD-fed beta cell-specific Gαz-null mice is significantly improved as compared with islets from HFD-fed WT controls, which, along with no impact of Gαz loss or HFD feeding on beta-cell proliferation or surrogates of beta-cell mass, supports a secretion-specific mechanism. Gαz is coupled to the prostaglandin EP3 receptor in pancreatic beta cells. We confirm the EP3γ splice variant has both constitutive and agonist-sensitive activity to inhibit cAMP production and downstream beta-cell function, with both activities being dependent on the presence of beta-cell Gαz.
Assuntos
Diabetes Mellitus Tipo 2/patologia , Dieta Hiperlipídica , Subunidades alfa de Proteínas de Ligação ao GTP/metabolismo , Células Secretoras de Insulina/patologia , Obesidade/complicações , Animais , Glicemia/metabolismo , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/etiologia , Modelos Animais de Doenças , Subunidades alfa de Proteínas de Ligação ao GTP/genética , Secreção de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
Glucose-mediated signaling regulates the expression of a limited number of genes in human pancreatic ß-cells at the transcriptional level. However, it is unclear whether glucose plays a role in posttranscriptional RNA processing or translational control of gene expression. Here, we asked whether glucose affects posttranscriptional steps and regulates protein synthesis in human ß-cell lines. We first showed the involvement of the mTOR pathway in glucose-related signaling. We also used the surface sensing of translation technique, based on puromycin incorporation into newly translated proteins, to demonstrate that glucose treatment increased protein translation. Among the list of glucose-induced proteins, we identified the proconvertase PCSK1, an enzyme involved in the proteolytic conversion of proinsulin to insulin, whose translation was induced within minutes following glucose treatment. We finally performed global proteomic analysis by mass spectrometry to characterize newly translated proteins upon glucose treatment. We found enrichment in proteins involved in translation, glycolysis, TCA metabolism, and insulin secretion. Taken together, our study demonstrates that, although glucose minorly affects gene transcription in human ß-cells, it plays a major role at the translational level.
Assuntos
Metabolismo Energético/genética , Glucose/farmacologia , Secreção de Insulina/genética , Células Secretoras de Insulina/metabolismo , Biossíntese de Proteínas/genética , Linhagem Celular , Subunidade RIIalfa da Proteína Quinase Dependente de AMP Cíclico/metabolismo , Metabolismo Energético/efeitos dos fármacos , Humanos , Secreção de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/efeitos dos fármacos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Pró-Proteína Convertase 1/metabolismo , Biossíntese de Proteínas/efeitos dos fármacos , Puromicina/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismoRESUMO
Mitochondrial homeostasis is crucial for the function of pancreatic ß-cells. ATP synthase inhibitory factor subunit 1 (IF1) is a mitochondrial protein interacting with ATP synthase to inhibit its enzyme activity. IF1 may also play a role in maintaining ATP synthase oligomerization and mitochondrial inner membrane formation. A recent study confirmed IF1 expresses in ß-cells. IF1 knockdown in cultured INS-1E ß-cells enhances glucose-induced insulin release. However, the role of IF1 in islet ß-cells remains little known. The present study investigates islets freshly isolated from mouse lines with global IF1 knockout (IF1-/-) and overexpression (OE). The glucose-stimulated insulin secretion was increased in islets from IF1-/- mice but decreased in islets from IF1 OE mice. Transmitted Electronic Microscopic assessment of isolated islets revealed that the number of matured insulin granules (with dense core) was relatively higher in IF1-/-, but fewer in IF1 OE islets than those of controlled islets. The mitochondrial ultrastructure within ß-cells of IF1 overexpressed islets was comparable with those of wild-type mice, whereas those in IF1-/- ß-cells showed increased mitochondrial mass. Mitochondrial network analysis in cultured INS-1 ß-cells showed a similar pattern with an increased mitochondrial network in IF1 knockdown cells. IF1 overexpressed INS-1 ß-cells showed a compromised rate of mitochondrial oxidative phosphorylation with attenuated cellular ATP content. In contrast, INS-1 cells with IF1 knockdown showed markedly increased cellular respiration with improved ATP production. These results support that IF1 is a negative regulator of insulin production and secretion via inhibiting mitochondrial mass and respiration in ß-cells. Therefore, inhibiting IF1 to improve ß-cell function in patients can be a novel therapeutic strategy to treat diabetes.
Assuntos
Homeostase , Células Secretoras de Insulina/metabolismo , Mitocôndrias/metabolismo , Proteínas/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Linhagem Celular Tumoral , Glucose/farmacologia , Secreção de Insulina/efeitos dos fármacos , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/metabolismo , Ilhotas Pancreáticas/ultraestrutura , Potencial da Membrana Mitocondrial , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Microscopia Eletrônica de Transmissão , Mitocôndrias/ultraestrutura , Fosforilação Oxidativa , Proteínas/genética , Ratos , Espécies Reativas de Oxigênio/metabolismo , Proteína Inibidora de ATPaseRESUMO
Low serum bicarbonate is closely related to type 2 diabetes mellitus. However, the precise role of bicarbonate on glucose homeostasis and insulin secretion remains unknown. In this study, we investigated the effects of bicarbonate concentration on pancreatic ß-cells. It was observed that the high bicarbonate concentration of the cell culture medium significantly increased the glucose-induced insulin secretion (GSIS) levels in mouse islets, MIN6, and the INS-1E ß cells. MIN6 cells presented an impaired GSIS; the cells produced a lower bicarbonate concentration when co-cultured with Capan-1 than when with CFPAC-1. NBCe1, a major bicarbonate transporter was observed to block the increasing insulin secretions, which were promoted by a high concentration of bicarbonate. In addition, higher extracellular bicarbonate concentration significantly increased the intracellular cAMP level, pHi, and calcium concentration with a 16.7 mM of glucose stimulation. Further study demonstrated that a low concentration of extracellular bicarbonate significantly impaired the functioning of pancreatic ß cells by reducing coupling Ca2+ influx, whose process may be modulated by NBCe1. Taken together, our results conclude that bicarbonate may serve as a novel target in diabetes prevention-related research.
Assuntos
Bicarbonatos/farmacologia , Glucose/farmacologia , Secreção de Insulina , Células Secretoras de Insulina/metabolismo , Animais , Cálcio/metabolismo , Linhagem Celular , AMP Cíclico/metabolismo , Concentração de Íons de Hidrogênio , Secreção de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/efeitos dos fármacos , Camundongos , Simportadores de Sódio-Bicarbonato/metabolismoRESUMO
AIMS: Metformin, rosiglitazone and sulfonylureas enhance either insulin action or secretion and thus have been used extensively as early stage anti-diabetic medication, independently of the aetiology of the disease. When administered to newly diagnosed diabetes patients, these drugs produce variable results. Here, we examined the effects of the three early stage oral hypoglycaemic agents in mice with diabetes induced by multiple low doses of streptozotocin, focusing specifically on the developmental biology of pancreatic islets. METHODS: Streptozotocin-treated diabetic mice expressing a fluorescent reporter specifically in pancreatic islet α-cells were administered the biguanide metformin (100 mg/kg), thiazolidinedione rosiglitazone (10 mg/kg), or sulfonylurea tolbutamide (20 mg/kg) for 10 days. We assessed the impact of the treatment on metabolic status of the animals as well as on the morphology, proliferative potential and transdifferentiation of pancreatic islet cells, using immunofluorescence. RESULTS: The effect of the therapy on the islet cells varied depending on the drug and included enhanced pancreatic islet ß-cell proliferation, in case of metformin and rosiglitazone; de-differentiation of α-cells and ß-cell apoptosis with tolbutamide; increased relative number of ß-cells and bi-hormonal insulin + glucagon + cells with metformin. These effects were accompanied by normalisation of food and fluid intake with only minor effects on glycaemia at the low doses of the agents employed. CONCLUSIONS: Our data suggest that metformin and rosiglitazone attenuate the depletion of the ß-cell pool in the streptozotocin-induced diabetes, whereas tolbutamide exacerbates the ß-cell apoptosis, but is likely to protect ß-cells from chronic hyperglycaemia by directly elevating insulin secretion.
Assuntos
Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Secreção de Insulina/efeitos dos fármacos , Ilhotas Pancreáticas , Metformina/farmacologia , Rosiglitazona/farmacologia , Animais , Glicemia/metabolismo , Diferenciação Celular/efeitos dos fármacos , Transdiferenciação Celular/efeitos dos fármacos , Diabetes Mellitus Experimental/tratamento farmacológico , Hipoglicemiantes/farmacologia , Células Secretoras de Insulina/efeitos dos fármacos , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/metabolismo , CamundongosRESUMO
Given the global epidemic in type 2 diabetes, novel antidiabetic drugs with increased efficacy and reduced side effects are urgently needed. Previous work has shown that M3 muscarinic acetylcholine (ACh) receptors (M3Rs) expressed by pancreatic ß cells play key roles in stimulating insulin secretion and maintaining physiological blood glucose levels. In the present study, we tested the hypothesis that a positive allosteric modulator (PAM) of M3R function can improve glucose homeostasis in mice by promoting insulin release. One major advantage of this approach is that allosteric agents respect the ACh-dependent spatiotemporal control of M3R activity. In this study, we first demonstrated that VU0119498, a drug known to act as a PAM at M3Rs, significantly augmented ACh-induced insulin release from cultured ß cells and mouse and human pancreatic islets. This stimulatory effect was absent in islets prepared from mice lacking M3Rs, indicative of the involvement of M3Rs. VU0119498 treatment of wild-type mice caused a significant increase in plasma insulin levels, accompanied by a striking improvement in glucose tolerance. These effects were mediated by ß-cell M3Rs, since they were absent in mutant mice selectively lacking M3Rs in ß cells. Moreover, acute VU0119498 treatment of obese, glucose-intolerant mice triggered enhanced insulin release and restored normal glucose tolerance. Interestingly, doses of VU0119498 that led to pronounced improvements in glucose homeostasis did not cause any significant side effects due to activation of M3Rs expressed by other peripheral cell types. Taken together, the data from this proof-of-concept study strongly suggest that M3R PAMs may become clinically useful as novel antidiabetic agents.
Assuntos
Diabetes Mellitus Tipo 2/tratamento farmacológico , Hipoglicemiantes/farmacologia , Ilhotas Pancreáticas/efeitos dos fármacos , Agonistas Muscarínicos/farmacologia , Receptor Muscarínico M3/efeitos dos fármacos , Acetilcolina/metabolismo , Adulto , Regulação Alostérica/efeitos dos fármacos , Animais , Glicemia/análise , Glicemia/metabolismo , Linhagem Celular Tumoral , Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/metabolismo , Modelos Animais de Doenças , Feminino , Intolerância à Glucose/sangue , Intolerância à Glucose/tratamento farmacológico , Intolerância à Glucose/metabolismo , Humanos , Hipoglicemiantes/uso terapêutico , Secreção de Insulina/efeitos dos fármacos , Ilhotas Pancreáticas/metabolismo , Masculino , Camundongos , Camundongos Obesos , Camundongos Transgênicos , Pessoa de Meia-Idade , Agonistas Muscarínicos/uso terapêutico , Obesidade/sangue , Obesidade/tratamento farmacológico , Obesidade/metabolismo , Cultura Primária de Células , Estudo de Prova de Conceito , Receptor Muscarínico M3/genética , Receptor Muscarínico M3/metabolismo , Adulto JovemRESUMO
Prevalence of type 2 diabetes increased from 2.5% of the US population in 1990 to 10.5% in 2018. This creates a major public health problem, due to increases in long-term complications of diabetes, including neuropathy, retinopathy, nephropathy, skin ulcers, amputations, and atherosclerotic cardiovascular disease. In this review, we evaluated the scientific basis that supports the use of physiologic insulin resensitization. Insulin resistance is the primary cause of type 2 diabetes. Insulin resistance leads to increasing insulin secretion, leading to beta-cell exhaustion or burnout. This triggers a cascade leading to islet cell destruction and the long-term complications of type 2 diabetes. Concurrent with insulin resistance, the regular bursts of insulin from the pancreas become irregular. This has been treated by the precise administration of insulin more physiologically. There is consistent evidence that this treatment modality can reverse the diabetes-associated complications of neuropathy, diabetic ulcers, nephropathy, and retinopathy, and that it lowers HbA1c. In conclusion, physiologic insulin resensitization has a persuasive scientific basis, significant treatment potential, and likely cost benefits.