RESUMEN
AIMS/HYPOTHESIS: We have previously shown that oxidative stress plays a causal role in beta cell dysfunction induced by fat. Here, we address whether the proinflammatory kinase inhibitor of (nuclear factor) κB kinase ß (IKKß), which is activated by oxidative stress, is also implicated. METHODS: Fat (oleate or olive oil) was infused intravenously in Wistar rats for 48 h with or without the IKKß inhibitor salicylate. Thereafter, beta cell function was evaluated in vivo using hyperglycaemic clamps or ex vivo in islets isolated from fat-treated rats. We also exposed rat islets to oleate in culture, with or without salicylate and 4(2'-aminoethyl)amino-1,8-dimethylimidazo(1,2-a)quinoxaline; BMS-345541 (BMS, another inhibitor of IKKß) and evaluated beta cell function in vitro. Furthermore, oleate was infused in mice treated with BMS and in beta cell-specific Ikkb-null mice. RESULTS: 48 h infusion of fat impaired beta-cell function in vivo, assessed using the disposition index (DI), in rats (saline: 1.41 ± 0.13; oleate: 0.95 ± 0.11; olive oil [OLO]: 0.87 ± 0.15; p < 0.01 for both fats vs saline) and in mice (saline: 2.51 ± 0.39; oleate: 1.20 ± 0.19; p < 0.01 vs saline) and ex vivo (i.e., insulin secretion, units are pmol insulin islet-1 h-1) in rat islets (saline: 1.51 ± 0.13; oleate: 1.03 ± 0.10; OLO: 0.91 ± 0.13; p < 0.001 for both fats vs saline) and the dysfunction was prevented by co-infusion of salicylate in rats (oleate + salicylate: 1.30 ± 0.09; OLO + salicylate: 1.33 ± 0.23) or BMS in mice (oleate + BMS: 2.25 ± 0.42) in vivo and by salicylate in rat islets ex vivo (oleate + salicylate: 1.74 ± 0.31; OLO + salicylate: 1.54 ± 0.29). In cultured islets, 48 h exposure to oleate impaired beta-cell function ([in pmol insulin islet-1 h-1] control: 0.66 ± 0.12; oleate: 0.23 ± 0.03; p < 0.01 vs saline), an effect prevented by both inhibitors (oleate + salicylate: 0.98 ± 0.08; oleate + BMS: 0.50 ± 0.02). Genetic inhibition of IKKß also prevented fat-induced beta-cell dysfunction ex vivo ([in pmol insulin islet-1 h-1] control saline: 0.16 ± 0.02; control oleate: 0.10 ± 0.02; knockout oleate: 0.17 ± 0.04; p < 0.05 control saline vs. control oleate) and in vivo (DI: control saline: 3.86 ± 0.40; control oleate: 1.95 ± 0.29; knockout oleate: 2.96 ± 0.24; p < 0.01 control saline vs control oleate). CONCLUSIONS/INTERPRETATION: Our results demonstrate a causal role for IKKß in fat-induced beta cell dysfunction in vitro, ex vivo and in vivo.
Asunto(s)
Ácidos Grasos no Esterificados/metabolismo , Quinasa I-kappa B/antagonistas & inhibidores , Células Secretoras de Insulina/efectos de los fármacos , Ácido Oléico/farmacología , Ácido Salicílico/farmacología , Animales , Femenino , Imidazoles/farmacología , Células Secretoras de Insulina/metabolismo , Ratones , Estrés Oxidativo/efectos de los fármacos , Quinoxalinas/farmacología , Ratas , Ratas WistarRESUMEN
Aggregation of islet amyloid polypeptide (IAPP) contributes to beta cell dysfunction in type 2 diabetes and islet transplantation. Like other amyloidogenic peptides, human IAPP induces macrophage IL-1ß secretion by stimulating both the synthesis and processing of proIL-1ß, a pro-inflammatory cytokine that (when chronically elevated) impairs beta cell insulin secretion. We sought to determine the specific mechanism of IAPP-induced proIL-1ß synthesis. Soluble IAPP species produced early during IAPP aggregation provided a Toll-like-receptor-2- (TLR2-) dependent stimulus for NF-κB activation in HEK 293 cells and bone marrow-derived macrophages (BMDMs). Non-amyloidogenic rodent IAPP and thioflavin-T-positive fibrillar amyloid produced by human IAPP aggregation failed to activate TLR2. Blockade of TLR6 but not TLR1 prevented hIAPP-induced TLR2 activation, consistent with stimulation of a TLR2/6 heterodimer. TLR2 and its downstream adaptor protein MyD88 were required for IAPP-induced cytokine production by BMDMs, a process that is partially dependent on autoinduction by IL-1. BMDMs treated with soluble but not fibrillar IAPP provided a TLR2-dependent priming stimulus for ATP-induced IL-1ß secretion, whereas late IAPP aggregates induced NLRP3-dependent IL-1ß secretion by LPS-primed macrophages. Moreover, inhibition of TLR2 and depletion of islet macrophages prevented up-regulation of Il1b and Tnf expression in human IAPP-expressing transgenic mouse islets. These data suggest participation by both soluble and fibrillar aggregates in IAPP-induced islet inflammation. IAPP-induced activation of TLR2 and secretion of IL-1 may be important therapeutic targets to prevent amyloid-associated beta cell dysfunction.
Asunto(s)
Inmunidad Innata , Células Secretoras de Insulina/inmunología , Polipéptido Amiloide de los Islotes Pancreáticos/inmunología , Agregación Patológica de Proteínas/inmunología , Animales , Proteínas Portadoras/genética , Proteínas Portadoras/inmunología , Células HEK293 , Humanos , Células Secretoras de Insulina/patología , Interleucina-1beta/genética , Interleucina-1beta/inmunología , Polipéptido Amiloide de los Islotes Pancreáticos/genética , Lipopolisacáridos/farmacología , Ratones , Ratones Noqueados , Proteína con Dominio Pirina 3 de la Familia NLR , Agregación Patológica de Proteínas/genética , Agregación Patológica de Proteínas/patología , Receptores Toll-Like/genética , Receptores Toll-Like/inmunología , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
BACKGROUND & AIMS: Crohn's disease (CD) is associated with a dysregulated immune response to commensal micro-organisms in the intestine. Mice deficient in inositol polyphosphate 5'-phosphatase D (INPP5D, also known as SHIP) develop intestinal inflammation resembling that of patients with CD. SHIP is a negative regulator of PI3Kp110α activity. We investigated mechanisms of intestinal inflammation in Inpp5d(-/-) mice (SHIP-null mice), and SHIP levels and activity in intestinal tissues of subjects with CD. METHODS: We collected intestines from SHIP-null mice, as well as Inpp5d(+/+) mice (controls), and measured levels of cytokines of the interleukin 1 (IL1) family (IL1α, IL1ß, IL1ra, and IL6) by enzyme-linked immunosorbent assay. Macrophages were isolated from lamina propria cells of mice, IL1ß production was measured, and mechanisms of increased IL1ß production were investigated. Macrophages were incubated with pan-phosphatidylinositol 3-kinase inhibitors or PI3Kp110α-specific inhibitors. Some mice were given an antagonist of the IL1 receptor; macrophages were depleted from ilea of mice using clodronate-containing liposomes. We obtained ileal biopsies from sites of inflammation and peripheral blood mononuclear cells (PBMCs) from treatment-naïve subjects with CD or without CD (controls), and measured SHIP levels and activity. PBMCs were incubated with lipopolysaccharide and adenosine triphosphate, and levels of IL1ß production were measured. RESULTS: Inflamed intestinal tissues and intestinal macrophages from SHIP-null mice produced higher levels of IL1B and IL18 than intestinal tissues from control mice. We found PI3Kp110α to be required for macrophage transcription of Il1b. Macrophage depletion or injection of an IL1 receptor antagonist reduced ileal inflammation in SHIP-null mice. Inflamed ileal tissues and PBMCs from patients with CD had lower levels of SHIP protein than controls (P < .0001 and P < .0002, respectively). There was an inverse correlation between levels of SHIP activity in PBMCs and induction of IL1ß production by lipopolysaccharide and adenosine triphosphate (R(2) = .88). CONCLUSIONS: Macrophages from SHIP-deficient mice have increased PI3Kp110α-mediated transcription of Il1b, which contributes to spontaneous ileal inflammation. SHIP levels and activity are lower in intestinal tissues and peripheral blood samples from patients with CD than controls. There is an inverse correlation between SHIP activity and induction of IL1ß production by lipopolysaccharide and adenosine triphosphate in PBMCs. Strategies to reduce IL1B might be developed to treat patients with CD found to have low SHIP activity.
Asunto(s)
Enfermedad de Crohn/enzimología , Ileítis/enzimología , Íleon/enzimología , Interleucina-1beta/metabolismo , Macrófagos/enzimología , Monoéster Fosfórico Hidrolasas/metabolismo , Animales , Antiinflamatorios/farmacología , Células Cultivadas , Fosfatidilinositol 3-Quinasa Clase I , Enfermedad de Crohn/diagnóstico , Enfermedad de Crohn/genética , Enfermedad de Crohn/inmunología , Modelos Animales de Enfermedad , Humanos , Ileítis/diagnóstico , Ileítis/genética , Ileítis/inmunología , Íleon/inmunología , Íleon/patología , Inositol Polifosfato 5-Fosfatasas , Interleucina-18/metabolismo , Interleucina-1beta/genética , Leucocitos Mononucleares/enzimología , Leucocitos Mononucleares/inmunología , Macrófagos/inmunología , Macrófagos/patología , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatasas , Inhibidores de las Quinasa Fosfoinosítidos-3 , Monoéster Fosfórico Hidrolasas/deficiencia , Monoéster Fosfórico Hidrolasas/genética , Inhibidores de Proteínas Quinasas/farmacología , Receptores de Interleucina-1/antagonistas & inhibidores , Receptores de Interleucina-1/metabolismo , Transcripción Genética , Regulación hacia ArribaRESUMEN
Chronic inflammation is known to promote metabolic dysregulation in obesity and type 2 diabetes. Although the precise origin of the unchecked inflammatory response in obesity is unclear, it is known that overproduction of proinflammatory cytokines by innate immune cells affects metabolism. For example, TNF-α contributes to the inability of cells to respond to insulin and to the increase in levels of insulin. Whether this hyperinsulinemia itself is part of a feedback loop that affects the progression of chronic adipose inflammation is unknown. In this article, we show that regulatory T cells (Tregs) express the insulin receptor, and that high levels of insulin impair the ability of Tregs to suppress inflammatory responses via effects on the AKT/mTOR signaling pathway. Insulin activated AKT signaling in Tregs, leading to inhibition of both IL-10 production and the ability of Tregs to suppress the production of TNF-α by macrophages in a contact-independent manner. The effect of insulin on Treg suppression was limited to IL-10 production and it did not alter the expression of other proteins associated with Treg function, including CTLA-4, CD39, and TGF-ß. In a model of diet-induced obesity, Tregs from the visceral adipose tissue of hyperinsulinemic, obese mice showed a similar specific decrease in IL-10 production, as well as a parallel increase in production of IFN-γ. These data suggest that hyperinsulinemia may contribute to the development of obesity-associated inflammation via a previously unknown effect of insulin on the IL-10-mediated function of Tregs.
Asunto(s)
Insulina/inmunología , Insulina/metabolismo , Interleucina-10/inmunología , Interleucina-10/metabolismo , Obesidad/inmunología , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Animales , Antígenos CD/inmunología , Antígenos CD/metabolismo , Apirasa/inmunología , Apirasa/metabolismo , Antígeno CTLA-4/inmunología , Antígeno CTLA-4/metabolismo , Células Cultivadas , Células Epiteliales/inmunología , Células Epiteliales/metabolismo , Epitelio/inmunología , Epitelio/metabolismo , Hiperinsulinismo/inmunología , Hiperinsulinismo/metabolismo , Inflamación/inmunología , Inflamación/metabolismo , Interferón gamma/inmunología , Interferón gamma/metabolismo , Interleucina-10/antagonistas & inhibidores , Grasa Intraabdominal/inmunología , Grasa Intraabdominal/metabolismo , Macrófagos/inmunología , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Obesidad/metabolismo , Proteínas Proto-Oncogénicas c-akt/inmunología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptor de Insulina/inmunología , Receptor de Insulina/metabolismo , Transducción de Señal/inmunología , Serina-Treonina Quinasas TOR/inmunología , Serina-Treonina Quinasas TOR/metabolismo , Factor de Crecimiento Transformador beta/inmunología , Factor de Crecimiento Transformador beta/metabolismo , Factor de Necrosis Tumoral alfa/inmunología , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Inflammation is a pathological feature of the pancreatic islet in type 1 and 2 diabetes, contributing to islet endocrine cell failure and the onset of hyperglycaemia in both diseases. Indeed, numerous immune targets have recently been found to be altered in type 2 diabetes, but few have yet to be translated to the clinic. Taylor-Fishwick and colleagues aimed to change this by performing proof-of-concept studies investigating the efficacy of small molecule inhibitors of 12-lipoxygenase in rodent and human beta cells exposed to proinflammatory cytokines. The results of these studies, published in this issue of Diabetologia (DOI: 10.1007/s00125-014-3452-0), build on a wealth of preclinical data that have implicated 12-lipoxygenase in rodent models of type 1 and 2 diabetes. While there remain some unanswered mechanistic questions regarding how cytokines regulate 12-lipoxygenase activation and the downstream consequences of activation, it is hoped that future studies with newly identified selective inhibitors may overcome the in vitro limitations of this study and allow for the eventual clinical translation of these highly interesting findings.
Asunto(s)
Araquidonato 12-Lipooxigenasa/metabolismo , Inhibidores Enzimáticos/farmacología , Células Secretoras de Insulina/efectos de los fármacos , Células Secretoras de Insulina/metabolismo , Animales , Humanos , MasculinoRESUMEN
AIMS/HYPOTHESIS: Aggregation of islet amyloid polypeptide (IAPP) to form amyloid contributes to beta cell dysfunction in type 2 diabetes. Human but not non-amyloidogenic rodent IAPP induces islet macrophage proIL-1ß synthesis. We evaluated the effect of IL-1 receptor antagonist (IL-1Ra) on islet inflammation and dysfunction in a mouse model of type 2 diabetes with amyloid formation. METHODS: Lean and obese male mice (A/a or A(vy)/A at the agouti locus, respectively) with or without beta cell human IAPP expression (hIAPP(Tg/0)) were treated with PBS or IL-1Ra (50 mg kg(-1) day(-1)) from 16 weeks of age. Intraperitoneal glucose and insulin tolerance tests were performed after 8 weeks. Pancreases were harvested for histology and gene expression analysis. RESULTS: Aggregation of human IAPP was associated with marked upregulation of proinflammatory gene expression in islets of obese hIAPP(Tg/0) mice, together with amyloid deposition and fasting hyperglycaemia. IL-1Ra improved glucose tolerance and reduced plasma proinsulin:insulin in both lean and obese hIAPP(Tg/0) mice with no effect on insulin sensitivity. The severity and prevalence of islet amyloid was reduced by IL-1Ra in lean hIAPP (Tg/0) mice, suggesting a feed-forward mechanism by which islet inflammation promotes islet amyloid at the early stages of disease. IL-1Ra limited Il1a, Il1b, Tnf and Ccl2 expression in islets from obese hIAPP(Tg/0) mice, suggesting an altered islet inflammatory milieu. CONCLUSIONS/INTERPRETATION: These data provide the first in vivo evidenceusing a transgenic mouse model with amyloid deposits resembling those found in human isletsthat IAPP-induced beta cell dysfunction in type 2 diabetes may be mediated by IL-1. Anti-IL-1 therapies may limit islet inflammation and dysfunction associated with amyloid formation.
Asunto(s)
Amiloide/metabolismo , Interleucina-1/metabolismo , Polipéptido Amiloide de los Islotes Pancreáticos/metabolismo , Animales , Humanos , Inmunohistoquímica , Inflamación/metabolismo , Interleucina-1/genética , Polipéptido Amiloide de los Islotes Pancreáticos/genética , Islotes Pancreáticos/metabolismo , Masculino , Ratones , Ratones TransgénicosRESUMEN
Pancreatic beta cell failure dictates the clinical onset of type 2 diabetes, with insulin secretion insufficient to overcome peripheral tissue insulin resistance. Over the past 5-10 years, a convincing case has emerged supporting the contribution of islet inflammation to this beta cell failure. IL-1 is central to this insult, impairing insulin secretion in preclinical and clinical studies. Further, islet-infiltrating macrophages are a major source of IL-1 and other cytokines in response to elevated levels of nutrients (glucose, saturated fatty acids), endocannabinoids and islet amyloid polypeptide (IAPP). In this issue of Diabetologia, Butcher et al have further characterised immune cell subsets present in islets from individuals with type 2 diabetes (DOI: 10.1007/s00125-013-3116-5). Increased numbers of CD45(+) leucocytes were found in these islets compared with islets from healthy controls, with an elevated proportion of CD20(+) B cells within the CD45(+) population. Their data also suggest that absolute numbers of CD3(+) T cells and CD11b(+)CD11c(+) myeloid cells may be increased in islets from individuals with type 2 diabetes. While many aspects of islet inflammation await further exploration, the study from Butcher and colleagues suggests a role for immune cell-mediated inflammation early in disease pathogenesis, and supports the concept that targeting the immune system may slow continued beta cell demise in type 2 diabetes.
Asunto(s)
Quimiocina CCL2/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Inflamación/metabolismo , Insulina/metabolismo , Islotes Pancreáticos/inmunología , Leucocitos/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Humanos , Secreción de InsulinaRESUMEN
AIMS/HYPOTHESIS: Inflammation contributes to pancreatic beta cell dysfunction in type 2 diabetes. Toll-like receptor (TLR)-2 and -4 ligands are increased systemically in recently diagnosed type 2 diabetes patients, and TLR2- and TLR4-deficient mice are protected from the metabolic consequences of a high-fat diet. Here we investigated the role of macrophages in TLR2/6- and TLR4-mediated effects on islet inflammation and beta cell function. METHODS: Genetic and pharmacological approaches were used to determine the effects of TLR2/6 and TLR4 ligands on mouse islets, human islets and purified rat beta cells. Islet macrophages were depleted and sorted by flow cytometry and the effects of TLR2/6- and TLR4-activated bone-marrow-derived macrophages (BMDMs) on beta cell function were assessed. RESULTS: Macrophages contributed to TLR2/6- and TLR4-induced islet Il1a/IL1A and Il1b/IL1B mRNA expression in mouse and human islets and IL-1ß secretion from human islets. TLR2/6 and TLR4 ligands also reduced insulin gene expression; however, this occurred in a non-beta cell autonomous manner. TLR2/6- and TLR4-activated BMDMs reduced beta cell insulin secretion partly via reducing Ins1, Ins2, and Pdx1 mRNA expression. Antagonism of the IL-1 receptor and neutralisation of IL-6 completely reversed the effects of activated macrophages on beta cell gene expression. CONCLUSIONS/INTERPRETATION: We conclude that islet macrophages are major contributors to islet IL-1ß secretion in response to TLR2/6 and TLR4 ligands. BMDMs stimulated with TLR2/6 and TLR4 ligands reduce insulin secretion from pancreatic beta cells, partly via IL-1ß- and IL-6-mediated decreased insulin gene expression.
Asunto(s)
Células Secretoras de Insulina/metabolismo , Insulina/genética , Interleucina-1/metabolismo , Interleucina-6/metabolismo , Islotes Pancreáticos/metabolismo , Macrófagos/metabolismo , Receptores Toll-Like/metabolismo , Animales , Regulación de la Expresión Génica , Humanos , Insulina/metabolismo , Ratones Noqueados , Ratas , Receptores Toll-Like/genéticaRESUMEN
The global health and economic burden of type 2 diabetes (T2D) has reached staggering proportions. Current projections estimate that 592 million people will have diabetes by 2035. T2D-which comprises 90% of cases-is a complex disease, in most cases resulting from a combination of predisposing genes and an unhealthy environment. Clinical onset of the disease occurs when pancreatic ß cells fail in the face of insulin resistance. It has long been appreciated that chronic activation of the innate immune system is associated with T2D, and many organs critical to the regulation of glucose homeostasis show signs of a chronic inflammatory process, including the pancreatic islets of Langerhans. Recent clinical trials using IL-1-targeting agents have confirmed that inflammation contributes to ß-cell failure in humans with T2D. However, little is known about the nature of the pro-inflammatory response within the islet, and there is considerable debate about the triggers for islet inflammation, which may be systemically derived and/or tissue-specific. In this review, we present evidence that Toll-like receptors 2 and 4 and the NLRP3 (Nucleotide-binding oligomerization domain, Leucine-rich Repeat and Pyrin domain containing 3) inflammasome are triggers for islet inflammation in T2D and propose that the activation of macrophages by these triggers mediates islet endocrine cell dysfunction. Therapeutically targeting these receptors may improve hyperglycemia and protect the ß cell in T2D.
Asunto(s)
Proteínas Portadoras/metabolismo , Diabetes Mellitus Tipo 2/inmunología , Diabetes Mellitus Tipo 2/patología , Inflamación/inmunología , Inflamación/patología , Islotes Pancreáticos/patología , Receptores Toll-Like/metabolismo , Animales , Diabetes Mellitus Tipo 2/terapia , Humanos , Islotes Pancreáticos/inmunología , Macrófagos/patologíaRESUMEN
Islets from patients with type 2 diabetes exhibit ß cell dysfunction, amyloid deposition, macrophage infiltration, and increased expression of proinflammatory cytokines and chemokines. We sought to determine whether human islet amyloid polypeptide (hIAPP), the main component of islet amyloid, might contribute to islet inflammation by recruiting and activating macrophages. Early aggregates of hIAPP, but not nonamyloidogenic rodent islet amyloid polypeptide, caused release of CCL2 and CXCL1 by islets and induced secretion of TNF-α, IL-1α, IL-1ß, CCL2, CCL3, CXCL1, CXCL2, and CXCL10 by C57BL/6 bone marrow-derived macrophages. hIAPP-induced TNF-α secretion was markedly diminished in MyD88-, but not TLR2- or TLR4-deficient macrophages, and in cells treated with the IL-1R antagonist (IL-1Ra) anakinra. To determine the significance of IL-1 signaling in hIAPP-induced pancreatic islet dysfunction, islets from wild-type or hIAPP-expressing transgenic mice were transplanted into diabetic NOD/SCID recipients implanted with mini-osmotic pumps containing IL-1Ra (50 mg/kg/d) or saline. IL-1Ra significantly improved the impairment in glucose tolerance observed in recipients of transgenic grafts 8 wk following transplantation. Islet grafts expressing hIAPP contained amyloid deposits in close association with F4/80-expressing macrophages. Transgenic grafts contained 50% more macrophages than wild-type grafts, an effect that was inhibited by IL-1Ra. Our results suggest that hIAPP-induced islet chemokine secretion promotes macrophage recruitment and that IL-1R/MyD88, but not TLR2 or TLR4 signaling is required for maximal macrophage responsiveness to prefibrillar hIAPP. These data raise the possibility that islet amyloid-induced inflammation contributes to ß cell dysfunction in type 2 diabetes and islet transplantation.
Asunto(s)
Citocinas/metabolismo , Interleucina-1/antagonistas & inhibidores , Polipéptido Amiloide de los Islotes Pancreáticos/inmunología , Trasplante de Islotes Pancreáticos/inmunología , Islotes Pancreáticos/metabolismo , Transducción de Señal/inmunología , Animales , Diabetes Mellitus Tipo 2/inmunología , Diabetes Mellitus Tipo 2/metabolismo , Humanos , Inmunohistoquímica , Inflamación/inmunología , Inflamación/metabolismo , Polipéptido Amiloide de los Islotes Pancreáticos/metabolismo , Islotes Pancreáticos/inmunología , Macrófagos/inmunología , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones Transgénicos , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Diabetes mellitus is associated with increased risk for cardiovascular disorders, which are major causes of mortality in this disease. Hyperhomocysteinemia, defined by high plasma homocysteine levels, is an independent risk factor for the development of cardiovascular diseases. Type 2 diabetic patients have higher circulating homocysteine levels than healthy subjects and these levels are even higher in plasma of obese than nonobese diabetic patients. Homocysteine metabolism that has been studied in 2 animal models of type 2 diabetes with obesity led to conflicting data. The aim of the present study was to analyze homocysteine metabolism in a spontaneous nonobese model of type 2 diabetes, the Goto-Kakizaki rats at various successive and well characterized stages of the disease: during early postnatal normoglycemia, at the onset of hyperglycemia (around weaning), and during chronic mild hyperglycemia with progressive insulin resistance. Compared to age-matched Wistar controls, Goto-Kakizaki rats showed lower plasma levels of homocysteine and a falling trend in its major byproduct antioxidant, glutathione, from the prediabetic stage onwards. Concomitantly, Goto-Kakizaki rats exhibited increased liver activity of cystathionine beta synthase, which catalyzes the condensation of homocysteine with serine in the first step of the transsulfuration pathway. These results emphasize a strong association between homocysteine metabolism and insulin via the first step of the hepatic transsulfuration pathway in Goto-Kakizaki rats.
Asunto(s)
Diabetes Mellitus Tipo 2/sangre , Homocisteína/sangre , Factores de Edad , Animales , Peso Corporal , Modelos Animales de Enfermedad , Glutatión/metabolismo , Resistencia a la Insulina , Masculino , Ratas , Ratas WistarRESUMEN
Interleukin-6 (IL-6) is systemically elevated in obesity and is a predictive factor to develop type 2 diabetes. Pancreatic islet pathology in type 2 diabetes is characterized by reduced beta-cell function and mass, an increased proportion of alpha-cells relative to beta-cells, and alpha-cell dysfunction. Here we show that the alpha cell is a primary target of IL-6 actions. Beginning with investigating the tissue-specific expression pattern of the IL-6 receptor (IL-6R) in both mice and rats, we find the highest expression of the IL-6R in the endocrine pancreas, with highest expression on the alpha-cell. The islet IL-6R is functional, and IL-6 acutely regulates both pro-glucagon mRNA and glucagon secretion in mouse and human islets, with no acute effect on insulin secretion. Furthermore, IL-6 stimulates alpha-cell proliferation, prevents apoptosis due to metabolic stress, and regulates alpha-cell mass in vivo. Using IL-6 KO mice fed a high-fat diet, we find that IL-6 is necessary for high-fat diet-induced increased alpha-cell mass, an effect that occurs early in response to diet change. Further, after high-fat diet feeding, IL-6 KO mice without expansion of alpha-cell mass display decreased fasting glucagon levels. However, despite these alpha-cell effects, high-fat feeding of IL-6 KO mice results in increased fed glycemia due to impaired insulin secretion, with unchanged insulin sensitivity and similar body weights. Thus, we conclude that IL-6 is necessary for the expansion of pancreatic alpha-cell mass in response to high-fat diet feeding, and we suggest that this expansion may be needed for functional beta-cell compensation to increased metabolic demand.
Asunto(s)
Células Secretoras de Glucagón/citología , Interleucina-6/metabolismo , Animales , Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Grasas de la Dieta/farmacología , Conducta Alimentaria/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Glucagón/genética , Glucagón/metabolismo , Células Secretoras de Glucagón/efectos de los fármacos , Prueba de Tolerancia a la Glucosa , Humanos , Células Secretoras de Insulina/citología , Células Secretoras de Insulina/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fosforilación/efectos de los fármacos , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Receptores de Interleucina-6/genética , Receptores de Interleucina-6/metabolismo , Factor de Transcripción STAT3/metabolismoRESUMEN
Onset of Type 2 diabetes occurs when the pancreatic beta-cell fails to adapt to the increased insulin demand caused by insulin resistance. Morphological and therapeutic intervention studies have uncovered an inflammatory process in islets of patients with Type 2 diabetes characterized by the presence of cytokines, immune cells, beta-cell apoptosis, amyloid deposits, and fibrosis. This insulitis is due to a pathological activation of the innate immune system by metabolic stress and governed by IL-1 signaling. We propose that this insulitis contributes to the decrease in beta-cell mass and the impaired insulin secretion observed in patients with Type 2 diabetes.
Asunto(s)
Diabetes Mellitus Tipo 2/patología , Células Secretoras de Insulina/patología , Animales , Citocinas/inmunología , Diabetes Mellitus Tipo 2/inmunología , Humanos , Inflamación/inmunología , Inflamación/patología , Resistencia a la Insulina/inmunología , Células Secretoras de Insulina/inmunologíaRESUMEN
BACKGROUND: The expression of interleukin-1-receptor antagonist is reduced in pancreatic islets of patients with type 2 diabetes mellitus, and high glucose concentrations induce the production of interleukin-1beta in human pancreatic beta cells, leading to impaired insulin secretion, decreased cell proliferation, and apoptosis. METHODS: In this double-blind, parallel-group trial involving 70 patients with type 2 diabetes, we randomly assigned 34 patients to receive 100 mg of anakinra (a recombinant human interleukin-1-receptor antagonist) subcutaneously once daily for 13 weeks and 36 patients to receive placebo. At baseline and at 13 weeks, all patients underwent an oral glucose-tolerance test, followed by an intravenous bolus of 0.3 g of glucose per kilogram of body weight, 0.5 mg of glucagon, and 5 g of arginine. In addition, 35 patients underwent a hyperinsulinemic-euglycemic clamp study. The primary end point was a change in the level of glycated hemoglobin, and secondary end points were changes in beta-cell function, insulin sensitivity, and inflammatory markers. RESULTS: At 13 weeks, in the anakinra group, the glycated hemoglobin level was 0.46 percentage point lower than in the placebo group (P=0.03); C-peptide secretion was enhanced (P=0.05), and there were reductions in the ratio of proinsulin to insulin (P=0.005) and in levels of interleukin-6 (P<0.001) and C-reactive protein (P=0.002). Insulin resistance, insulin-regulated gene expression in skeletal muscle, serum adipokine levels, and the body-mass index were similar in the two study groups. Symptomatic hypoglycemia was not observed, and there were no apparent drug-related serious adverse events. CONCLUSIONS: The blockade of interleukin-1 with anakinra improved glycemia and beta-cell secretory function and reduced markers of systemic inflammation. (ClinicalTrials.gov number, NCT00303394 [ClinicalTrials.gov].).
Asunto(s)
Diabetes Mellitus Tipo 2/tratamiento farmacológico , Proteína Antagonista del Receptor de Interleucina 1/uso terapéutico , Receptores de Interleucina-1/antagonistas & inhibidores , Glucemia/metabolismo , Índice de Masa Corporal , Proteína C-Reactiva/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Método Doble Ciego , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Prueba de Tolerancia a la Glucosa , Transportador de Glucosa de Tipo 4/genética , Transportador de Glucosa de Tipo 4/metabolismo , Hemoglobina Glucada/metabolismo , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Humanos , Resistencia a la Insulina/fisiología , Células Secretoras de Insulina/efectos de los fármacos , Células Secretoras de Insulina/metabolismo , Proteína Antagonista del Receptor de Interleucina 1/efectos adversos , Proteína Antagonista del Receptor de Interleucina 1/farmacología , Interleucina-6/sangre , Masculino , Persona de Mediana Edad , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma , ARN Mensajero/metabolismo , Proteínas Recombinantes/farmacología , Proteínas Recombinantes/uso terapéutico , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Macrophages play a dynamic role in tissue repair following injury. Here we found that following streptozotocin (STZ)-induced beta-cell death, mouse islet macrophages had increased Igf1 expression, decreased proinflammatory cytokine expression, and transcriptome changes consistent with macrophages undergoing efferocytosis and having an enhanced state of metabolism. Macrophages were the major, if not sole, contributors to islet insulin-like growth factor-1 (IGF-1) production. Adoptive transfer experiments showed that macrophages can maintain insulin secretion in vivo following beta-cell death with no effects on islet cell turnover. IGF-1 neutralization during STZ treatment decreased insulin secretion without affecting islet cell apoptosis or proliferation. Interestingly, high-fat diet (HFD) combined with STZ further skewed islet macrophages to a reparative state. Finally, islet macrophages from db/db mice also expressed decreased proinflammatory cytokines and increased Igf1 mRNA. These data have important implications for islet biology and pathology and show that islet macrophages preserve their reparative state following beta-cell death even during HFD feeding and severe hyperglycemia.
RESUMEN
CONTEXT: Elevated glucose levels impair islet function and survival, and it has been proposed that intraislet expression of IL-1beta contributes to glucotoxicity. OBJECTIVE: The objective was to investigate IL-1beta mRNA expression in near-pure beta-cells of patients with type 2 diabetes (T2DM) and study the regulation of IL-1beta by glucose in isolated human islets. METHODS: Laser capture microdissection was performed to isolate beta-cells from pancreas sections of 10 type 2 diabetic donors and nine controls, and IL-1beta mRNA expression was analyzed using gene arrays and PCR. Cultured human islets and fluorescence-activated cell sorter-purified human beta-cells were used to study the regulation of IL-1beta expression by glucose and IL-1beta. RESULTS: Gene array analysis of RNA from beta-cells of individuals with T2DM revealed increased expression of IL-1beta mRNA. Real-time PCR confirmed increased IL-1beta expression in six of 10 T2DM samples, with minimal or no expression in nine control samples. In cultured human islets, IL-1beta mRNA and protein expression was induced by high glucose and IL-1beta autostimulation and decreased by the IL-1 receptor antagonist IL-1Ra. The glucose response was negatively correlated with basal IL-1beta expression levels. Autostimulation was transient and nuclear factor-kappaB dependent. Glucose-induced IL-1beta was biologically active and stimulated IL-8 release. Low picogram per milliliter concentrations of IL-1beta up-regulated inflammatory factors IL-8 and IL-6. CONCLUSION: Evidence that IL-1beta mRNA expression is up-regulated in beta-cells of patients with T2DM is presented, and glucose-promoted IL-1beta autostimulation may be a possible contributor.
Asunto(s)
Comunicación Autocrina/fisiología , Diabetes Mellitus Tipo 2/genética , Glucosa/farmacología , Células Secretoras de Insulina/metabolismo , Interleucina-1beta/genética , Islotes Pancreáticos/efectos de los fármacos , Comunicación Autocrina/efectos de los fármacos , Comunicación Autocrina/genética , Células Cultivadas , Diabetes Mellitus Tipo 2/metabolismo , Perfilación de la Expresión Génica , Humanos , Interleucina-1beta/metabolismo , Interleucina-1beta/farmacología , Interleucina-6/genética , Interleucina-8/genética , Islotes Pancreáticos/metabolismo , FN-kappa B/fisiología , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Mensajero/metabolismo , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Gastric emptying is a critical regulator of postprandial glucose and delayed gastric emptying is an important mechanism of improved glycemic control achieved by short-acting glucagon-like peptide-1 (GLP-1) analogs in clinical practice. Here we report on a novel regulatory mechanism of gastric emptying in humans. We show that increasing interleukin (IL)-6 concentrations delays gastric emptying leading to reduced postprandial glycemia. IL-6 furthermore reduces insulin secretion in a GLP-1-dependent manner while effects on gastric emptying are GLP-1 independent. Inhibitory effects of IL-6 on gastric emptying were confirmed following exercise-induced increases in IL-6. Importantly, gastric- and insulin-reducing effects were maintained in individuals with type 2 diabetes. These data have clinical implications with respect to the use of IL-6 inhibition in autoimmune/inflammatory disease, and identify a novel target that could be exploited pharmacologically to delay gastric emptying and spare insulin, which may be beneficial for the beta cell in type 2 diabetes.
Asunto(s)
Glucemia/efectos de los fármacos , Diabetes Mellitus Tipo 2/metabolismo , Vaciamiento Gástrico/efectos de los fármacos , Péptido 1 Similar al Glucagón/metabolismo , Hipoglucemia/metabolismo , Secreción de Insulina/efectos de los fármacos , Interleucina-6/farmacología , Proteínas Recombinantes/farmacología , Anciano , Estudios de Casos y Controles , Método Doble Ciego , Ejercicio Físico , Humanos , Insulina/metabolismo , Células Secretoras de Insulina/efectos de los fármacos , Interleucina-6/administración & dosificación , Masculino , Proteínas Recombinantes/administración & dosificación , Adulto JovenRESUMEN
As we showed previously, the extracellular matrix (ECM) derived from rat bladder carcinoma cells (804G-ECM) has positive effects on rat primary beta-cell function and survival in vitro. The aim of this study was to define beta-cell genes induced by this ECM with a specific focus on cytokines. Analysis of differential gene expression by oligonucleotide microarrays, RT-PCR, and in situ hybridization was performed to identify cytokine mRNA induced by this matrix. Four cytokines were overexpressed on 804G-ECM compared with poly-L-lysine: C-X-C motif ligand 1 (CXCL1), CXCL2, interferon-inducible protein-10, and IL-1beta. A time-course experiment indicated that maximal induction by 804G-ECM of CXCL1/2 and interferon-inducible protein-10 occurred at 4 h. Stimulation of CXCL1 release by beta-cells on 804G-ECM was confirmed at the protein level. Moreover, secreted CXCL1 was shown to be functionally active by attracting rat granulocytes. Preventing the interaction of beta1 integrins and laminin-5 (a major component of 804G-ECM) with specific antibodies resulted in a 40-50% inhibition of CXCL1 expression. Using the nuclear factor-kappaB pathway inhibitor Bay 11-7082 it is demonstrated that CXCL1 expression and secretion are dependent on nuclear factor-kappaB activation. IL-1 secreted by beta-cells plated on 804G-ECM was found to be a key soluble mediator because treatment of cells with the IL-1 receptor antagonist significantly reduced both CXCL1 gene expression and secretion. It is concluded that ECM induces expression of cytokines including CXCL1 with amplification by IL-1 acting via a positive autocrine feedback loop.
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Comunicación Autocrina/efectos de los fármacos , Quimiocina CXCL1/genética , Matriz Extracelular/fisiología , Células Secretoras de Insulina/efectos de los fármacos , Interleucina-1/farmacología , Animales , Movimiento Celular/efectos de los fármacos , Células Cultivadas , Quimiocina CXCL1/metabolismo , Quimiocina CXCL1/farmacología , Medios de Cultivo Condicionados/farmacología , Citocinas/genética , Regulación de la Expresión Génica/efectos de los fármacos , Granulocitos/citología , Granulocitos/efectos de los fármacos , Células Secretoras de Insulina/metabolismo , Masculino , Modelos Biológicos , Ratas , Ratas WistarRESUMEN
A decrease in the number of functional insulin-producing beta-cells contributes to the pathophysiology of type 2 diabetes. Opinions diverge regarding the relative contribution of a decrease in beta-cell mass versus an intrinsic defect in the secretory machinery. Here we review the evidence that glucose, dyslipidemia, cytokines, leptin, autoimmunity, and some sulfonylureas may contribute to the maladaptation of beta-cells. With respect to these causal factors, we focus on Fas, the ATP-sensitive K+ channel, insulin receptor substrate 2, oxidative stress, nuclear factor-kappaB, endoplasmic reticulum stress, and mitochondrial dysfunction as their respective mechanisms of action. Interestingly, most of these factors are involved in inflammatory processes in addition to playing a role in both the regulation of beta-cell secretory function and cell turnover. Thus, the mechanisms regulating beta-cell proliferation, apoptosis, and function are inseparable processes.
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Muerte Celular , Diabetes Mellitus Tipo 2/patología , Células Secretoras de Insulina/patología , Animales , Diabetes Mellitus Tipo 2/inmunología , Modelos Animales de Enfermedad , Dislipidemias/fisiopatología , Humanos , Inmunidad Innata , Inflamación/fisiopatología , Leptina/fisiología , Factores de RiesgoRESUMEN
Recent studies into the physiology of the incretins glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) have added stimulation of beta-cell growth, differentiation, and cell survival to well-documented, potent insulinotropic effects. Unfortunately, the therapeutic potential of these hormones is limited by their rapid enzymatic inactivation in vivo by dipeptidyl peptidase IV (DP IV). Inhibition of DP IV, so as to enhance circulating incretin levels, has proved effective in the treatment of type 2 diabetes both in humans and in animal models, stimulating improvements in glucose tolerance, insulin sensitivity, and beta-cell function. We hypothesized that enhancement of the cytoprotective and beta-cell regenerative effects of GIP and GLP-1 might extend the therapeutic potential of DP IV inhibitors to include type 1 diabetes. For testing this hypothesis, male Wistar rats, exposed to a single dose of streptozotocin (STZ; 50 mg/kg), were treated twice daily with the DP IV inhibitor P32/98 for 7 weeks. Relative to STZ-injected controls, P32/98-treated animals displayed increased weight gain (230%) and nutrient intake, decreased fed blood glucose ( approximately 26 vs. approximately 20 mmol/l, respectively), and a return of plasma insulin values toward normal (0.07 vs. 0.12 nmol/l, respectively). Marked improvements in oral glucose tolerance, suggesting enhanced insulin secretory capacity, were corroborated by pancreas perfusion and insulin content measurements that revealed two- to eightfold increases in both secretory function and insulin content after 7 weeks of treatment. Immunohistochemical analyses of pancreatic sections showed marked increases in the number of small islets (+35%) and total beta-cells (+120%) and in the islet beta-cell fraction (12% control vs. 24% treated) in the treated animals, suggesting that DP IV inhibitor treatment enhanced islet neogenesis, beta-cell survival, and insulin biosynthesis. In vitro studies using a beta-(INS-1) cell line showed a dose-dependent prevention of STZ-induced apoptotic cell-death by both GIP and GLP-1, supporting a role for the incretins in eliciting the in vivo results. These novel findings provide evidence to support the potential utility of DP IV inhibitors in the treatment of type 1 and possibly late-stage type 2 diabetes.