RESUMO
AIMS: To test, for the first time in latent autoimmune diabetes in adults (LADA), the effects of autoantigen-specific immunotherapy by intralymphatic administration of aluminium-formulated recombinant human glutamic acid decarboxylase 65 (GAD-alum); specifically, to test if this treatment is safe, to test whether it induces a strong immunological response akin to a similar protocol in type 1 diabetes and to look for associations with preserved beta-cell function. MATERIALS AND METHODS: Three GAD-alum injections, 4 µg each, were administered 1 month apart into an inguinal lymph node in 14 people with newly diagnosed LADA (age 30-62 years) presenting with high levels of antibodies against glutamic acid decarboxylase (GADA). Adverse effects, immunological variables and beta-cell function were monitored, with detailed measurements at 5 and 12 months from baseline. RESULTS: Clinical adverse effects were minor and transient and measured laboratory variables were unaffected. All participants completed the study. Treatment raised levels of GADA, elicited strong effects on reactivity of peripheral blood mononuclear cells to GAD and raised cytokine/chemokine levels. Beta-cell function appeared stable preferentially in the seven participants carrying human leukocyte antigen (HLA) haplotypes DR3DQ2, as assessed by C-peptide glucagon tests (P < 0.05 vs. seven non-carriers). CONCLUSION: Intralymphatic treatment with GAD-alum in LADA is without clinical or other safety concerns over a 12-month period. As in a similar protocol used in type 1 diabetes, treatment exerts a strong immunological impact and is compatible with protection of beta-cell function preferentially in HLA-DR3DQ2 LADA patients. These findings pave the way for a randomized controlled trial in this important subgroup of LADA patients.
Assuntos
Diabetes Mellitus Tipo 1 , Intolerância à Glucose , Glutamato Descarboxilase , Diabetes Autoimune Latente em Adultos , Adulto , Humanos , Pessoa de Meia-Idade , Autoanticorpos , Diabetes Mellitus Tipo 1/terapia , Intolerância à Glucose/tratamento farmacológico , Glutamato Descarboxilase/efeitos adversos , Glutamato Descarboxilase/uso terapêutico , Injeções Intralinfáticas , Diabetes Autoimune Latente em Adultos/tratamento farmacológico , Leucócitos Mononucleares , Projetos PilotoRESUMO
BACKGROUND: Cytokines and chemokines participate in autoimmune processes at cellular targets which include insulin-producing beta cells. To which extent such participation is reflected in the circulation has not been conclusively resolved. AIM: We compared the time course of cytokines/chemokines in Latent Autoimmune Diabetes in Adults (LADA) patients heterogeneous for high or low autoimmune activity as determined by levels of antibodies against glutamic acid decarboxylase (GADA). METHODS: Serum samples to be measured were from a two-armed randomized controlled trial (RCT) in 68 LADA patients. The study encompassed 21 months with C-peptide as primary endpoint. We measured 27 immune mediators at baseline, at 9 and at 21 months (end of study). Results of measurements were analyzed by multiple linear regression. RESULTS: At baseline, a high body mass index (BMI) (>26 kg/m2) was associated with elevated levels of the interleukins (IL) IL-1 beta, IL-1ra, IL-2, IL-5, IL-6 and IL-13. Treatment during RCT (sitagliptin vs. insulin) did not affect the time course (21 months) of levels of cytokines/chemokines (by univariate analyses). However, levels of the cytokines IL-1ra and IL-1 beta decreased significantly (p < 0.04 or less) in patients with high vs. low GADA when adjusted for BMI, age, gender (male/female), treatment (insulin/sitagliptin) and study site (Norwegian/Swedish). CONCLUSIONS: In LADA, high levels of GADA, a proxy for high autoimmune activity and linked to a decline in C-peptide, was associated with a decrease of selected cytokines over time. This implies that the decline of IL-1ra and IL-1 beta in the circulation reflects autoimmune activity and beta cell demise in LADA.
Assuntos
Diabetes Autoimune Latente em Adultos , Adulto , Autoanticorpos , Peptídeo C , Citocinas , Feminino , Glutamato Descarboxilase , Humanos , Interleucina-1beta , MasculinoRESUMO
PURPOSE: To investigate the association between fruit and vegetable intake (FVI) and the risk of developing prediabetes and type 2 diabetes (T2D) in a Swedish prospective cohort study. METHODS: Subjects were 6961 men and women aged 35-56 years old at baseline, participating in the Stockholm Diabetes Prevention Program cohort. By design, the cohort was enriched by 50% with subjects that had family history of diabetes. Anthropometric measurements, oral glucose tolerance tests and questionnaires on lifestyle and dietary factors were carried out at baseline and two follow-up occasions. Cox proportional hazard models were used to estimate hazard ratios with 95% CIs. RESULTS: During a mean follow-up time of 20 ± 4 years, 1024 subjects developed T2D and 870 prediabetes. After adjustments for confounders, the highest tertile of total FVI was associated with a lower risk of developing T2D in men (HR 0.76, 95% CI 0.60-0.96). There was also an inverse association between total fruit intake and prediabetes risk in men, with the HR for the highest tertile being 0.76 (95% CI 0.58-1.00). As for subtypes, higher intake of apples/pears was inversely associated with T2D risk in both sexes, whereas higher intakes of banana, cabbage and tomato were positively associated with T2D or prediabetes risk in either men or women. CONCLUSION: We found an inverse association between higher total FVI and T2D risk and between higher fruit intake and prediabetes risk, in men but not in women. Certain fruit and vegetable subtypes showed varying results and require further investigation.
Assuntos
Diabetes Mellitus Tipo 2 , Estado Pré-Diabético , Adulto , Estudos de Coortes , Diabetes Mellitus Tipo 2/epidemiologia , Diabetes Mellitus Tipo 2/prevenção & controle , Feminino , Frutas , Humanos , Masculino , Pessoa de Meia-Idade , Estado Pré-Diabético/epidemiologia , Estado Pré-Diabético/prevenção & controle , Estudos Prospectivos , Fatores de Risco , Inquéritos e Questionários , Suécia/epidemiologia , VerdurasRESUMO
AIMS: To compare outcomes of glucagon-stimulated C-peptide tests (GSCTs) in people with latent autoimmune diabetes in adults (LADA) after a 21-month intervention with either insulin or the dipeptidyl peptidase-4 inhibitor sitagliptin. RESEARCH DESIGN AND METHODS: We included 64 glutamic acid decarboxylase (GAD) antibody-positive individuals, who were diagnosed with diabetes <3 years before the study, aged 30 to 70 years, and without clinical need for insulin treatment. We stratified participants by age and body mass index (BMI) and evaluated ß-cell function by GSCT after a 48-hour temporary withdrawal of study medication. RESULTS: Age at randomization (mean 53 years), BMI (mean 27 kg/m2 ) and metabolic markers were similar between treatment arms. Glycated haemoglobin concentrations during intervention did not differ between arms. Fasting C-peptide concentrations after the intervention were similar, as were stimulated C-peptide levels (0.82 ± 0.63 nmol/L after insulin, 0.82 ± 0.46 nmol/L after sitagliptin; nonsignificant). Autoimmunity in the study population (estimated from GAD antibody titres and positivity/no positivity for zinc transporter 8 and islet antigen 2 antibodies) affected the evolution of the GSCT results significantly, which deteriorated in participants with high but not in those with low autoimmunity. Adjustment using analysis of covariance for the degree of autoimmunity did not alter the findings of no difference between treatment arms. CONCLUSIONS: ß-cell function after intervention was similar in patients with insulin- and sitagliptin-treated LADA, regardless of the strength of autoimmunity. Further, participants with low levels of GAD antibodies did not experience progressive deterioration of ß-cell function over a 21-month period. Taken together, these findings could be useful for clinicians' choices of treatment in people with LADA.
Assuntos
Hipoglicemiantes/uso terapêutico , Células Secretoras de Insulina , Diabetes Autoimune Latente em Adultos/tratamento farmacológico , Adulto , Idoso , Autoimunidade/fisiologia , Inibidores da Dipeptidil Peptidase IV/uso terapêutico , Feminino , Humanos , Insulina/uso terapêutico , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/fisiologia , Diabetes Autoimune Latente em Adultos/fisiopatologia , Masculino , Pessoa de Meia-Idade , Fosfato de Sitagliptina/uso terapêuticoRESUMO
Isolated pancreatic islets containing the insulin-producing beta cells are devoid of circulation. They may therefore experience hypoxia with possible negative effects on beta cell function and survival. We investigated (1) whether hyperoxia in vitro would be beneficial by counteracting putative effects of lost circulation and, further, (2) whether previous hyperoxia would attenuate the impact of subsequently induced severe hypoxia. Islets from Sprague-Dawley rats were exposed to 95% O2 for 18 h. This hyperoxic exposure diminished glucose-induced insulin secretion by 47% and inhibited oxygen consumption by 39-41%. Mitochondrial complexes I-III were decreased by 29-37%. Negative effects on insulin secretion and complexes III and IV waned after a 22 h period of normoxia following hyperoxia whereas complexes I and II were still diminished, ROS production was increased and rates of apoptosis tended to be increased (P=0.07). The effects of previous hyperoxia on susceptibility to damage by subsequent hypoxia were tested after 5.5h of 0.8% O2. Previous hyperoxia did not affect hypoxia-induced enhancement of HIF-1 alpha but modestly and significantly attenuated hypoxia-induced decreases in insulin contents. We conclude that hyperoxia exerts largely negative effects on beta cells, effects which are functional and possibly also toxic. A paradoxical positive finding (attenuation of hypoxia-induced effects) could be secondary to a protective effect of the hyperoxia-induced reduction of oxidative metabolism.
Assuntos
Glucose/metabolismo , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Mitocôndrias/metabolismo , Oxigênio/metabolismo , Animais , Apoptose , Separação Celular , DNA/metabolismo , Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Glucose/farmacologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Secreção de Insulina , Células Secretoras de Insulina/efeitos dos fármacos , Masculino , Fosforilação Oxidativa , Oxigênio/farmacologia , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismoRESUMO
The aim of this study was to assess the effect of four isocaloric meals with different macronutrient compositions on postprandial blood glucose, lipids, and glucagon in adults with type 1 diabetes (T1D). Seventeen subjects tested four isocaloric meals in a randomized crossover design. The meal compositions were as follows: high-carbohydrate (HC); high-carbohydrate with extra fiber (HC-fiber); low-carbohydrate high-protein (HP); and low-carbohydrate high-fat (HF). Blood glucose and lipid measurements were collected up to 4 h and glucagon up to 3 h postprandially. Mean postprandial glucose excursions were lower after the HP compared to the HC (p = 0.036) and HC-fiber meals (p = 0.002). There were no differences in mean glucose excursions after the HF meal compared to the HC and HP meals. The HF meal resulted in higher triglyceride excursions compared to the HP meal (p < 0.001) but not compared to the HC or HC-fiber meals. Glucagon excursions were higher at 180 min after the HP meal compared to the HC and HF meals. In conclusion, the low-carbohydrate HP meal showed the most favorable glycemic and metabolic effects during a 4 h postprandial period in subjects with T1D.
Assuntos
Diabetes Mellitus Tipo 1 , Adulto , Humanos , Glicemia/metabolismo , Gorduras na Dieta , Estudos Cross-Over , Glucagon , Insulina , Período Pós-Prandial , RefeiçõesRESUMO
Evidence is conflicting as to the impact of elevated levels of uncoupling protein-2 (UCP-2) on insulin-producing beta cells. Here we investigated effects of a fourfold induction of UCP-2 protein primarily on mitochondrial parameters and tested for replication of positive findings at a lower level of induction. We transfected INS-1 cells to obtain a tet-on inducible cell line. A 48 h exposure to 1 µg/ml of doxycycline (dox) induced UCP-2 fourfold (424 ± 113%, mean±SEM) and 0.1 µg/ml twofold (178 ± 29%, n=3). Fourfold induced cells displayed normal viability (MTT, apoptosis), normal cellular insulin contents and, glucose-induced insulin secretion (+27 ± 11%) as well as D-[U-(14)C]-glucose oxidation (+5 ± 9% at 11 mM glucose). Oxidation of [1-(14)C]-oleate was increased from 4088 to 5797 fmol/µg prot/2h at 3.3mM glucose, p<0.03. Oxidation of L-[(14)C(U)]-glutamine was unaffected. Induction of UCP-2 did not significantly affect measures of mitochondrial membrane potential (Rhodamine 123) or mitochondrial mass (Mitotracker Green) and did not affect ATP levels. Oligomycin-inhibited oxygen consumption (a measure of mitochondrial uncoupling) was marginally increased, the effect being significant in comparison with dox-only treated cells, p<0.05. Oxygen radicals, assessed by dichlorofluorescin diacetate, were decreased by 30%, p<0.025. Testing for the lower level of UCP-2 induction did not reproduce any of the positive findings. A fourfold induction of UCP-2 was required to exert minor metabolic effects. These findings question an impact of moderately elevated UCP-2 levels in beta cells as seen in diabetes.
Assuntos
Células Secretoras de Insulina/metabolismo , Canais Iônicos/biossíntese , Mitocôndrias/metabolismo , Proteínas Mitocondriais/biossíntese , Animais , Linhagem Celular Tumoral , Doxiciclina/farmacologia , Glucose/metabolismo , Humanos , Insulina/metabolismo , Secreção de Insulina , Canais Iônicos/genética , Potencial da Membrana Mitocondrial , Proteínas Mitocondriais/genética , Oligomicinas/farmacologia , Oxirredução , Consumo de Oxigênio/efeitos dos fármacos , Consumo de Oxigênio/fisiologia , Ratos , Transfecção , Proteína Desacopladora 2RESUMO
Background: Latent Autoimmune Diabetes in Adults (LADA) constitutes around 10% of all diabetes. Many LADA patients gradually lose their insulin secretion and progress to insulin dependency. In a recent trial BALAD (Behandling Av LADa) early insulin treatment compared with sitagliptin failed to preserve insulin secretion, which deteriorated in individuals displaying high levels of antibodies to GAD (GADA). These findings prompted us to evaluate a treatment that directly affects autoimmunity. Intra-lymphatic GAD-alum treatment has shown encouraging results in Type 1 diabetes patients. We therefore tested the feasibility of such therapy in LADA-patients (the GADinLADA pilot study). Material and Methods: Fourteen GADA-positive (>190 RU/ml), insulin-independent patients 30-70 years old, with LADA diagnosed within < 36 months were included in an open-label feasibility trial. They received an intra-nodal injection of 4 µg GAD-alum at Day 1, 30 and 60 plus oral Vitamin D 2000 U/d from screening 30 days before (Day -30) for 4 months if the vitamin D serum levels were below 100 nmol/L (40 ng/ml). Primary objective is to evaluate safety and feasibility. Mixed Meal Tolerance Test and i.v. Glucagon Stimulation Test at baseline and after 5 and 12 months are used for estimation of beta cell function. Results will be compared with those of the recent BALAD study with comparable patient population. Immunological response is followed. Results: Preliminary results show feasibility and safety, with almost stable beta cell function and metabolic control during follow-up so far (5 months). Conclusions: Intra-lymphatic GAD-alum treatment is an option to preserve beta cell function in LADA-patients. An ongoing trial in 14 LADA-patients show feasibility and safety. Clinical and immunological responses will determine how to proceed with future trials.
Assuntos
Diabetes Mellitus Tipo 1 , Intolerância à Glucose , Diabetes Autoimune Latente em Adultos , Adulto , Idoso , Compostos de Alúmen , Estudos de Viabilidade , Intolerância à Glucose/tratamento farmacológico , Glutamato Descarboxilase/uso terapêutico , Humanos , Insulina/metabolismo , Diabetes Autoimune Latente em Adultos/tratamento farmacológico , Pessoa de Meia-Idade , Projetos Piloto , Vitamina D/uso terapêuticoRESUMO
Diabetes is known to increase susceptibility to infections, partly due to impaired granulocyte function and changes in the innate immunity. Here, we investigate the effect of diabetes, and high glucose on the expression of the antimicrobial peptide, psoriasin and the putative consequences for E. coli urinary tract infection. Blood, urine, and urine exfoliated cells from patients are studied. The influence of glucose and insulin is examined during hyperglycemic clamps in individuals with prediabetes and in euglycemic hyperinsulinemic clamped patients with type 1 diabetes. Important findings are confirmed in vivo in type 2 diabetic mice and verified in human uroepithelial cell lines. High glucose concentrations induce lower psoriasin levels and impair epithelial barrier function together with altering cell membrane proteins and cytoskeletal elements, resulting in increasing bacterial burden. Estradiol treatment restores the cellular function with increasing psoriasin and bacterial killing in uroepithelial cells, confirming its importance during urinary tract infection in hyperglycemia. In conclusion, our findings present the effects and underlying mechanisms of high glucose compromising innate immunity.
Assuntos
Diabetes Mellitus Experimental , Infecções por Escherichia coli , Infecções Urinárias , Animais , Peptídeos Antimicrobianos , Escherichia coli/metabolismo , Infecções por Escherichia coli/tratamento farmacológico , Estradiol/metabolismo , Glucose/metabolismo , Humanos , Insulina/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Proteína A7 Ligante de Cálcio S100/metabolismo , Bexiga Urinária/metabolismoRESUMO
Facilitation of fatty acid uptake in beta cells could potentially affect beta cell metabolism and secretory function; however such effects have not been clearly documented. CD36 facilitates uptake of fatty acids (FA) in muscle and adipose tissue and is likely to exert a similar effect in beta cells. We investigated the impact of over-expressing CD36 on fatty acid uptake and beta cell function by a Tet-on system in INS-1 cells. Doxycycline dose-dependently increased the CD36 protein with localization mainly in the cell membrane. Over-expression increased both specific uptake and efflux of oleate whereas intracellular glycerides were only marginally increased and incorporation of 14C-oleate or -palmitate into di- or triglycerides not affected. The normal potentiation of glucose-induced insulin secretion by acute addition of FA (50-100 micromol/l oleate and palmitate) was lost and the normal inhibitory effect of high glucose both on oleate oxidation and on the activity of carnitine palmitoyltransferase I was reduced. Over-expression did not induce apoptosis. We conclude that induction of the CD36 transporter increases uptake of FA, the consequences of which are blunting of the functional interplay between glucose and FA on insulin secretion and oxidative metabolism.
Assuntos
Antígenos CD36/metabolismo , Ácidos Graxos/química , Ácidos Graxos/metabolismo , Glucose/metabolismo , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Animais , Antibacterianos/farmacologia , Apoptose/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Células Cultivadas , Doxiciclina/farmacologia , Imunofluorescência , Secreção de Insulina , Células Secretoras de Insulina/efeitos dos fármacos , Canais Iônicos/genética , Canais Iônicos/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Espécies Reativas de Oxigênio/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteína Desacopladora 2RESUMO
Diazoxide enhances glucose-induced insulin secretion from beta-cells through mechanisms that are not fully elucidated. Here, we used microarray analysis (Affymetrix) to investigate effects of diazoxide. Pancreatic islets were cultured overnight at 27, 11, or 5.5 mmol/l glucose with or without diazoxide. Inclusion of diazoxide upregulated altogether 211 genes (signal log(2) ratio > or =0.5) and downregulated 200 genes (signal log(2) ratio -0.5 or lower), and 92% of diazoxide's effects (up- and downregulation) were observed only after coculture with 11 or 27 mmol/l glucose. We found that 11 mmol/l diazoxide upregulated 97 genes and downregulated 21 genes. Increasing the glucose concentration to 27 mmol/l markedly shifted these proportions toward downregulation (101 genes upregulated and 160 genes downregulated). At 27 mmol/l glucose, most genes downregulated by diazoxide were oppositely affected by glucose (80%). Diazoxide influenced expression of several genes central to beta-cell metabolism. Diazoxide downregulated genes of fatty acid oxidation, upregulated genes of fatty acid synthesis, and downregulated uncoupling protein 2 and lactic acid dehydrogenase. Diazoxide upregulated certain genes known to support beta-cell functionality, such as NKX6.1 and PDX1. Long-term elevated glucose is permissive for most of diazoxide's effects on gene expression, the proportion of effects shifting to downregulation with increasing glucose concentration. Effects of diazoxide on gene expression could serve to enhance beta-cell functionality during continuous hyperglycemia.
Assuntos
Diazóxido/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Células Secretoras de Insulina/fisiologia , Insulina/metabolismo , Ilhotas Pancreáticas/fisiologia , Animais , Glucose/farmacologia , Secreção de Insulina , Células Secretoras de Insulina/efeitos dos fármacos , Ilhotas Pancreáticas/efeitos dos fármacos , Masculino , Análise de Sequência com Séries de Oligonucleotídeos , Ratos , Ratos Sprague-DawleyRESUMO
Pancreatic ß-cells express several ion channels of the transient receptor potential family, which play important roles in mediating the stimulus-secretion coupling. One of these channels, the TRPM4 is a Ca2+-activated monovalent cation channel. This channel is inhibited by 9-phenanthrol, which also inhibits the TMEM16a Cl- channel, and activates the Ca2+-activated K+ channel, Kca3.1. The net effects of ion-channel modulation by 9-phenantherol on the insulin secretion remain unclear. We tested the effects of 9-phenanthrol on glucose- and GLP-1-induced insulin secretion from isolated rat islets in static incubations. When applied to the islets in the presence of 3.3 mM glucose, 9-phenanthrol caused a small increase in insulin secretion (~7% of the insulin secretion stimulated by 10 mM glucose). 10 µM 9-phenanthrol did not inhibit glucose- or GLP-1-induced insulin secretion. 20 µM and 30 µM 9-phenanthrol inhibited glucose-induced insulin secretion by ~80% and ~85%, respectively. Inhibition of the GLP-1-induced insulin secretion by 20 µM and 30 µM 9-phenanthrol was 65% and 94%, respectively. Our study shows that the major effect of 9-phenanthrol on the islets is a strong inhibition of insulin secretion, and we speculate that compounds related to 9-phenanthrol may be potentially useful in treating the pancreatogenous hyperinsulinemic hypoglycemia syndromes.
Assuntos
Peptídeo 1 Semelhante ao Glucagon/farmacologia , Glucose/farmacologia , Insulina/metabolismo , Ilhotas Pancreáticas/efeitos dos fármacos , Fenantrenos/farmacologia , Canais de Cátion TRPM/antagonistas & inibidores , Animais , Secreção de Insulina , Ilhotas Pancreáticas/metabolismo , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
We previously showed that hyperoxia in vitro negatively affects beta cells of the rat. Here, we tested for possible clinical significance as well as mitochondrial interactions by hyperoxia, using human islets (function and viability), INS-1 832/13 cells (mitochondrial metabolism), and mouse neonates (effects in vivo). Lastly, we assessed relevant parameters in a cohort of individuals born preterm and then exposed to hyperoxia. Human islets and INS-1 832/13 cells were exposed to 24 h of hyperoxia (90-92% oxygen). Mouse neonates were subjected to 5 days of continuous hyperoxia. Individuals born preterm were evaluated in terms of glucose homeostasis and beta cell function by HbA1c and the HOMA2 formula. In human islets, hyperoxia significantly reduced glucose-stimulated insulin secretion by 42.2 ± 5.3% and viability assessed by MTT by 22.5 ± 5.4%. Hyperoxia down-regulated mitochondrial complex II by 21 ± 5% and upregulated complex III by 26 ± 10.1% and complex IV by 37 ± 10.6%. Partly similar effects on mitochondrial complexes were found in hyperoxia-exposed INS-1 832/13 cells. Exposure to hyperoxia swiftly reduced oxygen consumption in these cells and increased mitochondrial uncoupling. Hyperoxia transiently but significantly reduced insulin release in mouse neonates. Individuals born preterm displayed higher HbA1c versus controls, as well as insulin resistance. Thus, hyperoxia exerts negative effects in vitro on human beta cells and results indicate inhibitory effects on insulin secretion in vivo in mouse neonates. Negative effects may be lessened by the demonstrated swift and profound mitochondrial adaptability. Our findings open the possibility that hyperoxia could negatively affect beta cells of preterm human neonates.
Assuntos
Hiperóxia/metabolismo , Células Secretoras de Insulina/metabolismo , Insulina/sangue , Mitocôndrias/metabolismo , Animais , Glicemia/metabolismo , Linhagem Celular , Células Cultivadas , Complexo II de Transporte de Elétrons/genética , Complexo II de Transporte de Elétrons/metabolismo , Complexo III da Cadeia de Transporte de Elétrons/genética , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Hemoglobinas/metabolismo , Humanos , Hiperóxia/sangue , Hiperóxia/patologia , Recém-Nascido , Recém-Nascido Prematuro/sangue , Camundongos , Consumo de OxigênioAssuntos
Diabetes Mellitus Tipo 1 , Resistência à Insulina , Diabetes Autoimune Latente em Adultos , Adulto , Diabetes Mellitus Tipo 1/tratamento farmacológico , Humanos , Hipoglicemiantes/efeitos adversos , Diabetes Autoimune Latente em Adultos/tratamento farmacológico , Fosfato de Sitagliptina/efeitos adversosRESUMO
We tested whether exposure of ß cells at reduced glucose leads to mitochondrial adaptions and whether such adaptions modulate effects of hypoxia. Rat islets, human islets and INS-1 832/13 cells were pre-cultured short term at half standard glucose concentrations (5.5 mM for rat islets and cells, 2.75 mM for human islets) without overtly negative effects on subsequently measured function (insulin secretion and cellular insulin contents) or on viability. Culture at half standard glucose upregulated complex I and tended to upregulate complex II in islets and INS-1 cells alike. An increased release of lactate dehydrogenase that followed exposure to hypoxia was attenuated in rat islets which had been pre-cultured at half standard glucose. In INS-1 cells exposure to half standard glucose attenuated hypoxia-induced effects on several viability parameters (MTT, cell number and incremental apoptotic DNA). Thus culture at reduced glucose of pancreatic islets and clonal ß cells leads to mitochondrial adaptions which possibly lessen the negative impact of hypoxia on ß cell viability. These findings appear relevant in the search for optimization of pre-transplant conditions in a clinical setting.
Assuntos
Sobrevivência Celular/fisiologia , Glucose/farmacologia , Células Secretoras de Insulina/metabolismo , Mitocôndrias/metabolismo , Animais , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Humanos , Células Secretoras de Insulina/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , RatosRESUMO
BACKGROUND: Diabetes type 1 is characterized by the failure of beta cells to produce insulin. Suppressor of cytokine signaling (SOCS) proteins are important regulators of the Janus kinase/signal transducer and activator of transcription (JAK-STAT) pathway. Previous studies have shown that GH can prevent the development of type I diabetes in mice and that SOCS2 deficiency mimics a state of increased GH sensitivity. METHODOLOGY: The elevated sensitivity of SOCS2-/- mice to GH and possibly to PRL was the rationale to analyze the effects of multiple low dose streptozotocin (MLDSTZ)-induced diabetes in SOCS2-/- mice. RESULTS: We show that 6-month-old SOCS2-/- mice, but not 2-month-old mice, were less sensitive to MLDSTZ-induced diabetes, compared to controls. MLDSTZ treatment induced glucose intolerance in both SOCS2+/+ and SOCS2-/- mice, as shown by glucose tolerance tests, with SOCS2+/+ mice showing a more marked intolerance, compared to SOCS2-/- mice. Furthermore, insulin tolerance tests showed that the SOCS2-/- mice have an improved hypoglycemic response to exogenous insulin, compared to SOCS2+/+ mice. Moreover, in isolated islets, lipotoxic effects on insulin release could partly be overcome by ligands, which bind to GH or PRL receptors. CONCLUSION: Knockdown of SOCS2 makes mice less sensitive to MLDSTZ. These results are consistent with the proposal that elimination of SOCS2 in pancreatic islets creates a state of ß-cell hypersensitivity to GH/PRL that mimics events in pregnancy, and which is protective against MLDSTZ-induced type I diabetes in mice. SOCS2-dependent control of ß-cell survival may be of relevance to islet regeneration and survival in transplantation.
Assuntos
Diabetes Mellitus Experimental/genética , Diabetes Mellitus Tipo 1/genética , Deleção de Genes , Proteínas Supressoras da Sinalização de Citocina/metabolismo , Animais , Células Cultivadas , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Glucose/metabolismo , Hormônio do Crescimento/farmacologia , Insulina/metabolismo , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Prolactina/farmacologia , Estreptozocina/toxicidade , Proteínas Supressoras da Sinalização de Citocina/genéticaRESUMO
OBJECTIVES: The use of primary human ß-cells for studying Ca signaling is limited by the scarcity of human pancreatic islets. Rodent insulinoma cell lines are widely used, but it is difficult to extrapolate results obtained from rodent cells to human. Recently, a genetically engineered human ß-cell line EndoC-BH1 has been developed. We have examined whether the EndoC-BH1 cells could be used as a model for studying Ca signaling in the ß-cells. METHODS: We used microscope-based fluorometry to measure cytoplasmic-free Ca concentration from fura-2-loaded single EndoC-BH1 cells cultured on glass cover slips. Ca responses to different agonists of insulin secretion were studied. Insulin secretion was measured by radioimmunoassay. RESULTS: EndoC-BH1 cells secreted insulin in response to glucose in a dose-dependent manner, and the secretion was enhanced by GLP-1 (glucagon-like peptide 1). Glucose, potassium chloride, carbachol, L-arginine, and tolbutamide increased cytoplasmic-free Ca concentration in the EndoC-BH1 cells. We found that GLP-1 was essential for Ca response to glucose and tolbutamide. CONCLUSIONS: We concluded that the EndoC-BH1 cells can be used as model cells to study Ca signaling and stimulus-secretion coupling in the human ß-cells.
Assuntos
Sinalização do Cálcio , Engenharia Genética , Células Secretoras de Insulina/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Corantes Fluorescentes , Fluorometria , Fura-2 , Glucose/farmacologia , Humanos , Insulina/metabolismo , Secreção de Insulina , Células Secretoras de Insulina/efeitos dos fármacos , Cinética , Potenciais da MembranaRESUMO
OBJECTIVE: To provide novel insights on mitochondrial respiration in ß-cells and the adaptive effects of hypoxia. METHODS AND DESIGN: Insulin-producing INS-1 832/13 cells were exposed to 18 hours of hypoxia followed by 20-22 hours re-oxygenation. Mitochondrial respiration was measured by high-resolution respirometry in both intact and permeabilized cells, in the latter after establishing three functional substrate-uncoupler-inhibitor titration (SUIT) protocols. Concomitant measurements included proteins of mitochondrial complexes (Western blotting), ATP and insulin secretion. RESULTS: Intact cells exhibited a high degree of intrinsic uncoupling, comprising about 50% of oxygen consumption in the basal respiratory state. Hypoxia followed by re-oxygenation increased maximal overall respiration. Exploratory experiments in peremabilized cells could not show induction of respiration by malate or pyruvate as reducing substrates, thus glutamate and succinate were used as mitochondrial substrates in SUIT protocols. Permeabilized cells displayed a high capacity for oxidative phosphorylation for both complex I- and II-linked substrates in relation to maximum capacity of electron transfer. Previous hypoxia decreased phosphorylation control of complex I-linked respiration, but not in complex II-linked respiration. Coupling control ratios showed increased coupling efficiency for both complex I- and II-linked substrates in hypoxia-exposed cells. Respiratory rates overall were increased. Also previous hypoxia increased proteins of mitochondrial complexes I and II (Western blotting) in INS-1 cells as well as in rat and human islets. Mitochondrial effects were accompanied by unchanged levels of ATP, increased basal and preserved glucose-induced insulin secretion. CONCLUSIONS: Exposure of INS-1 832/13 cells to hypoxia, followed by a re-oxygenation period increases substrate-stimulated respiratory capacity and coupling efficiency. Such effects are accompanied by up-regulation of mitochondrial complexes also in pancreatic islets, highlighting adaptive capacities of possible importance in an islet transplantation setting. Results also indicate idiosyncrasies of ß-cells that do not respire in response to a standard inclusion of malate in SUIT protocols.
Assuntos
Respiração Celular , Células Secretoras de Insulina/metabolismo , Mitocôndrias/metabolismo , Adaptação Biológica , Trifosfato de Adenosina/metabolismo , Animais , Morte Celular , Linhagem Celular , Proliferação de Células , Complexo I de Transporte de Elétrons/metabolismo , Complexo II de Transporte de Elétrons/metabolismo , Humanos , Hipóxia/metabolismo , Insulina/metabolismo , Malatos/metabolismo , Consumo de Oxigênio , Permeabilidade , Ratos , Especificidade por SubstratoRESUMO
Toll-like receptors (TLRs) have been implicated in the pathogenesis of type 2 diabetes. We examined the function of TLR3 in glucose metabolism and type 2 diabetes-related phenotypes in animals and humans. TLR3 is highly expressed in the pancreas, suggesting that it can influence metabolism. Using a diet-induced obesity model, we show that TLR3-deficient mice had enhanced glycemic control, facilitated by elevated insulin secretion. Despite having high insulin levels, Tlr3(-/-) mice did not experience disturbances in whole-body insulin sensitivity, suggesting that they have a robust metabolic system that manages increased insulin secretion. Increase in insulin secretion was associated with upregulation of islet glucose phosphorylation as well as exocytotic protein VAMP-2 in Tlr3(-/-) islets. TLR3 deficiency also modified the plasma lipid profile, decreasing VLDL levels due to decreased triglyceride biosynthesis. Moreover, a meta-analysis of two healthy human populations showed that a missense single nucleotide polymorphism in TLR3 (encoding L412F) was linked to elevated insulin levels, consistent with our experimental findings. In conclusion, our results increase the understanding of the function of innate receptors in metabolic disorders and implicate TLR3 as a key control system in metabolic regulation.
Assuntos
Glucose/metabolismo , Insulina/metabolismo , Receptor 3 Toll-Like/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Área Sob a Curva , Glicemia/metabolismo , VLDL-Colesterol/sangue , Gorduras na Dieta/efeitos adversos , Gorduras na Dieta/farmacologia , Glucose/farmacocinética , Teste de Tolerância a Glucose , Homeostase , Humanos , Insulina/sangue , Secreção de Insulina , Células Secretoras de Insulina/metabolismo , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Polimorfismo Genético , Receptor 3 Toll-Like/genéticaRESUMO
OBJECTIVE: Several studies have reported that the transient receptor potential melastatin-like subtype 5 (TRPM5) channel, a Ca(2+)-activated monovalent cation channel, is involved in the stimulus-secretion coupling in the mouse pancreatic ß-cells. We have studied the role of the TRPM5 channel in regulating insulin secretion and cytoplasmic free Ca(2+) concentration ([Ca(2+)](i)) in the rat ß-cells by using triphenylphosphine oxide, a selective inhibitor of the channel. METHODS: Insulin secretion from islets from Sprague-Dawley rats was measured in batch incubations. Cytoplasmic free Ca(2+) concentration was measured from single ß-cells by fura-2-based microfluorometry. RESULTS: Triphenylphosphine oxide did not alter insulin secretion and [Ca(2+)](i) response triggered by KCl or fructose. It inhibited insulin secretion in response to glucose, L-arginine, and glucagon-like peptide 1. It also inhibited glucose-induced insulin secretion by mechanisms that are independent of the adenosine triphosphate-sensitive potassium channels and [Ca(2+)](i) increase. CONCLUSIONS: Our results suggest that in the rat islets, TRPM5 is involved in mediating insulin secretion by glucose and l-arginine and in potentiating the glucose-induced insulin secretion by glucagon-like peptide 1.