IL-13 improves beta-cell survival and protects against IL-1beta-induced beta-cell death.

OBJECTIVES: IL-13 is a cytokine classically produced by anti-inflammatory T-helper-2 lymphocytes; it is decreased in the circulation of type 2 diabetic patients and impacts positively on liver and skeletal muscle. Although IL-13 can exert positive effects on beta-cell lines, its impact and mode of action on primary beta-cell function and survival remain largely unexplored. METHODS: Beta-cells were cultured for 48 h in the presence of IL-13 alone or in combination with IL-1ß or cytokine cocktail (IL-1ß, IFNγ, TNFα). RESULTS: IL-13 protected human and rat beta-cells against cytokine induced death. However, IL-13 was unable to protect from IL-1ß impaired glucose stimulated insulin secretion and did not influence NFκB nuclear relocalization induced by IL-1ß. IL-13 induced phosphorylation of Akt, increased IRS2 protein expression and counteracted the IL-1ß induced regulation of several beta-cell stress response genes. CONCLUSIONS: The prosurvival effects of IL-13 thus appear to be mediated through IRS2/Akt signaling with NFκB independent regulation of gene expression. In addition to previously documented beneficial effects on insulin target tissues, these data suggest that IL-13 may be useful for treatment of type 2 diabetes by preserving beta-cell mass or slowing its rate of decline.

Circulating Follistatin Is Liver-Derived and Regulated by the Glucagon-to-Insulin Ratio.

CONTEXT: Follistatin is a plasma protein recently reported to increase under conditions with negative energy balance, such as exercise and fasting in humans. Currently, the perception is that circulating follistatin is a result of para/autocrine actions from various tissues. The large and acute increase in circulating follistatin in response to exercise suggests that it may function as an endocrine signal. OBJECTIVE: We assessed origin and regulation of circulating follistatin in humans. DESIGN/INTERVENTIONS: First, we assessed arterial-to-venous difference of follistatin over the splanchnic bed at rest and during exercise in healthy humans. To evaluate the regulation of plasma follistatin we manipulated glucagon-to-insulin ratio in humans at rest as well as in cultured hepatocytes. Finally, the impact of follistatin on human islets of Langerhans was assessed. RESULTS: We demonstrate that in humans the liver is a major contributor to circulating follistatin both at rest and during exercise. Glucagon increases and insulin inhibits follistatin secretion both in vivo and in vitro, mediated via the secondary messenger cAMP in the hepatocyte. Short-term follistatin treatment reduced glucagon secretion from islets of Langerhans, whereas long-term follistatin treatment prevented apoptosis and induced proliferation of rat ß cells. CONCLUSIONS: In conclusion, in humans, the liver secretes follistatin at rest and during exercise, and the glucagon-to-insulin ratio is a key determinant of circulating follistatin levels. Circulating follistatin may be a marker of the glucagon-to-insulin tone on the liver.

The skeleton in the closet: actin cytoskeletal remodeling in ß-cell function.

Over the last few decades, biomedical research has considered not only the function of single cells but also the importance of the physical environment within a whole tissue, including cell-cell and cell-extracellular matrix interactions. Cytoskeleton organization and focal adhesions are crucial sensors for cells that enable them to rapidly communicate with the physical extracellular environment in response to extracellular stimuli, ensuring proper function and adaptation. The involvement of the microtubular-microfilamentous cytoskeleton in secretion mechanisms was proposed almost 50 years ago, since when the evolution of ever more sensitive and sophisticated methods in microscopy and in cell and molecular biology have led us to become aware of the importance of cytoskeleton remodeling for cell shape regulation and its crucial link with signaling pathways leading to ß-cell function. Emerging evidence suggests that dysfunction of cytoskeletal components or extracellular matrix modification influences a number of disorders through potential actin cytoskeleton disruption that could be involved in the initiation of multiple cellular functions. Perturbation of ß-cell actin cytoskeleton remodeling could arise secondarily to islet inflammation and fibrosis, possibly accounting in part for impaired ß-cell function in type 2 diabetes. This review focuses on the role of actin remodeling in insulin secretion mechanisms and its close relationship with focal adhesions and myosin II.

50 years forward: beta cells.

Our understanding of beta cell development and function has increased substantially these past 50 years but much remains to be learned before this knowledge can be put to clinical use. A comprehensive business plan will be necessary to develop a detailed molecular and functional blueprint of the beta cell in health and disease based on an integrated approach involving all necessary research disciplines. This blueprint will provide a platform for the development of novel therapeutic strategies for the treatment of both major forms of diabetes, foremost among them beta cell replacement therapy. This is one of a series of commentaries under the banner '50 years forward', giving personal opinions on future perspectives in diabetes, to celebrate the 50th anniversary of Diabetologia (1965-2015).

Short term exposure of beta cells to low concentrations of interleukin-1ß improves insulin secretion through focal adhesion and actin remodeling and regulation of gene expression.

Type 2 diabetes involves defective insulin secretion with islet inflammation governed in part by IL-1ß. Prolonged exposure of islets to high concentrations of IL-1ß (>24 h, 20 ng/ml) impairs beta cell function and survival. Conversely, exposure to lower concentrations of IL-1ß for >24 h improves these same parameters. The impact on insulin secretion of shorter exposure times to IL-1ß and the underlying molecular mechanisms are poorly understood and were the focus of this study. Treatment of rat primary beta cells, as well as rat or human whole islets, with 0.1 ng/ml IL-1ß for 2 h increased glucose-stimulated (but not basal) insulin secretion, whereas 20 ng/ml was without effect. Similar differential effects of IL-1ß depending on concentration were observed after 15 min of KCl stimulation but were prevented by diazoxide. Studies on sorted rat beta cells indicated that the enhancement of stimulated secretion by 0.1 ng/ml IL-1ß was mediated by the NF-κB pathway and c-JUN/JNK pathway acting in parallel to elicit focal adhesion remodeling and the phosphorylation of paxillin independently of upstream regulation by focal adhesion kinase. Because the beneficial effect of IL-1ß was dependent in part upon transcription, gene expression was analyzed by RNAseq. There were 18 genes regulated uniquely by 0.1 but not 20 ng/ml IL-1ß, which are mostly involved in transcription and apoptosis. These results indicate that 2 h of exposure of beta cells to a low but not a high concentration of IL-1ß enhances glucose-stimulated insulin secretion through focal adhesion and actin remodeling, as well as modulation of gene expression.

Fractalkine (CX3CL1), a new factor protecting ß-cells against TNFα.

OBJECTIVE: We have previously shown the existence of a muscle-pancreas intercommunication axis in which CX3CL1 (fractalkine), a CX3C chemokine produced by skeletal muscle cells, could be implicated. It has recently been shown that the fractalkine system modulates murine ß-cell function. However, the impact of CX3CL1 on human islet cells especially regarding a protective role against cytokine-induced apoptosis remains to be investigated. METHODS: Gene expression was determined using RNA sequencing in human islets, sorted ß- and non-ß-cells. Glucose-stimulated insulin secretion (GSIS) and glucagon secretion from human islets was measured following 24 h exposure to 1-50 ng/ml CX3CL1. GSIS and specific protein phosphorylation were measured in rat sorted ß-cells exposed to CX3CL1 for 48 h alone or in the presence of TNFα (20 ng/ml). Rat and human ß-cell apoptosis (TUNEL) and rat ß-cell proliferation (BrdU incorporation) were assessed after 24 h treatment with increasing concentrations of CX3CL1. RESULTS: Both CX3CL1 and its receptor CX3CR1 are expressed in human islets. However, CX3CL1 is more expressed in non-ß-cells than in ß-cells while its receptor is more expressed in ß-cells. CX3CL1 decreased human (but not rat) ß-cell apoptosis. CX3CL1 inhibited human islet glucagon secretion stimulated by low glucose but did not impact human islet and rat sorted ß-cell GSIS. However, CX3CL1 completely prevented the adverse effect of TNFα on GSIS and on molecular mechanisms involved in insulin granule trafficking by restoring the phosphorylation (Akt, AS160, paxillin) and expression (IRS2, ICAM-1, Sorcin, PCSK1) of key proteins involved in these processes. CONCLUSIONS: We demonstrate for the first time that human islets express and secrete CX3CL1 and CX3CL1 impacts them by decreasing glucagon secretion without affecting insulin secretion. Moreover, CX3CL1 decreases basal apoptosis of human ß-cells. We further demonstrate that CX3CL1 protects ß-cells from the adverse effects of TNFα on their function by restoring the expression and phosphorylation of key proteins of the insulin secretion pathway.

ß-cell failure in type 2 diabetes: postulated mechanisms and prospects for prevention and treatment.

OBJECTIVE: This article examines the foundation of ß-cell failure in type 2 diabetes (T2D) and suggests areas for future research on the underlying mechanisms that may lead to improved prevention and treatment. RESEARCH DESIGN AND METHODS: A group of experts participated in a conference on 14-16 October 2013 cosponsored by the Endocrine Society and the American Diabetes Association. A writing group prepared this summary and recommendations. RESULTS: The writing group based this article on conference presentations, discussion, and debate. Topics covered include genetic predisposition, foundations of ß-cell failure, natural history of ß-cell failure, and impact of therapeutic interventions. CONCLUSIONS: ß-Cell failure is central to the development and progression of T2D. It antedates and predicts diabetes onset and progression, is in part genetically determined, and often can be identified with accuracy even though current tests are cumbersome and not well standardized. Multiple pathways underlie decreased ß-cell function and mass, some of which may be shared and may also be a consequence of processes that initially caused dysfunction. Goals for future research include to (1) impact the natural history of ß-cell failure; (2) identify and characterize genetic loci for T2D; (3) target ß-cell signaling, metabolic, and genetic pathways to improve function/mass; (4) develop alternative sources of ß-cells for cell-based therapy; (5) focus on metabolic environment to provide indirect benefit to ß-cells; (6) improve understanding of the physiology of responses to bypass surgery; and (7) identify circulating factors and neuronal circuits underlying the axis of communication between the brain and ß-cells.

ß-cell failure in type 2 diabetes: postulated mechanisms and prospects for prevention and treatment.

OBJECTIVE: This article examines the foundation of ß-cell failure in type 2 diabetes (T2D) and suggests areas for future research on the underlying mechanisms that may lead to improved prevention and treatment. RESEARCH DESIGN AND METHODS: A group of experts participated in a conference on 14-16 October 2013 cosponsored by the Endocrine Society and the American Diabetes Association. A writing group prepared this summary and recommendations. RESULTS: The writing group based this article on conference presentations, discussion, and debate. Topics covered include genetic predisposition, foundations of ß-cell failure, natural history of ß-cell failure, and impact of therapeutic interventions. CONCLUSIONS: ß-Cell failure is central to the development and progression of T2D. It antedates and predicts diabetes onset and progression, is in part genetically determined, and often can be identified with accuracy even though current tests are cumbersome and not well standardized. Multiple pathways underlie decreased ß-cell function and mass, some of which may be shared and may also be a consequence of processes that initially caused dysfunction. Goals for future research include to 1) impact the natural history of ß-cell failure; 2) identify and characterize genetic loci for T2D; 3) target ß-cell signaling, metabolic, and genetic pathways to improve function/mass; 4) develop alternative sources of ß-cells for cell-based therapy; 5) focus on metabolic environment to provide indirect benefit to ß-cells; 6) improve understanding of the physiology of responses to bypass surgery; and 7) identify circulating factors and neuronal circuits underlying the axis of communication between the brain and ß-cells.

Expression, phosphorylation and function of the Rab-GTPase activating protein TBC1D1 in pancreatic beta-cells.

The Rab-GTPase activating protein TBC1D1 is a paralog of AS160/TBC1D4. AS160/TBC1D4, a downstream effector of Akt, has been shown to play a central role in beta-cell function and survival. The two proteins have overlapping function in insulin signalling in muscle cells. However, the expression and the potential role of TBC1D1 in beta-cells remain unknown. Therefore, the aim of this study is to investigate whether TBC1D1 is expressed in beta-cells and whether it plays, as AS160/TBC1D4, a role in beta-cell function and survival. Using human and rat beta-cells, this study shows for the first time that TBC1D1 is expressed and phosphorylated in response to glucose in these cells. Knockdown of TBC1D1 in beta-cells resulted in increased basal and glucose-stimulated insulin release, decreased proliferation but no change in apoptosis.

Cell-type, allelic, and genetic signatures in the human pancreatic beta cell transcriptome.

Elucidating the pathophysiology and molecular attributes of common disorders as well as developing targeted and effective treatments hinges on the study of the relevant cell type and tissues. Pancreatic beta cells within the islets of Langerhans are centrally involved in the pathogenesis of both type 1 and type 2 diabetes. Describing the differentiated state of the human beta cell has been hampered so far by technical (low resolution microarrays) and biological limitations (whole islet preparations rather than isolated beta cells). We circumvent these by deep RNA sequencing of purified beta cells from 11 individuals, presenting here the first characterization of the human beta cell transcriptome. We perform the first comparison of gene expression profiles between beta cells, whole islets, and beta cell depleted islet preparations, revealing thus beta-cell-specific expression and splicing signatures. Further, we demonstrate that genes with consistent increased expression in beta cells have neuronal-like properties, a signal previously hypothesized. Finally, we find evidence for extensive allelic imbalance in expression and uncover genetic regulatory variants (eQTLs) active in beta cells. This first molecular blueprint of the human beta cell offers biological insight into its differentiated function, including expression of key genes associated with both major types of diabetes.

In vitro proliferation of adult human beta-cells.

A decrease in functional beta-cell mass is a key feature of type 2 diabetes. Glucagon-like peptide 1 (GLP-1) analogues induce proliferation of rodent beta-cells. However, the proliferative capacity of human beta-cells and its modulation by GLP-1 analogues remain to be fully investigated. We therefore sought to quantify adult human beta-cell proliferation in vitro and whether this is affected by the GLP-1 analogue liraglutide.Human islets from 7 adult cadaveric organ donors were dispersed into single cells. Beta-cells were purified by FACS. Non-sorted cells and the beta-cell enriched ("beta-cells") population were plated on extracellular matrix from rat (804G) and human bladder carcinoma cells (HTB9) or bovine corneal endothelial ECM (BCEC). Cells were maintained in culture+/-liraglutide for 4 days in the presence of BrdU.Rare human beta-cell proliferation could be observed either in the purified beta-cell population (0.051±0.020%; 22 beta-cells proliferating out of 84'283 beta-cells counted) or in the non-sorted cell population (0.055±0.011%; 104 proliferating beta-cells out of 232'826 beta-cells counted), independently of the matrix or the culture conditions. Liraglutide increased human beta-cell proliferation on BCEC in the non-sorted cell population (0.082±0.034% proliferating beta-cells vs. 0.017±0.008% in control, p<0.05).These results indicate that adult human beta-cell proliferation can occur in vitro but remains an extremely rare event with these donors and particular culture conditions. Liraglutide increases beta-cell proliferation only in the non-sorted cell population and only on BCEC. However, it cannot be excluded that human beta-cells may proliferate to a greater extent in situ in response to natural stimuli.

Novel mechanistic link between focal adhesion remodeling and glucose-stimulated insulin secretion.

Actin cytoskeleton remodeling is well known to be positively involved in glucose-stimulated pancreatic ß cell insulin secretion. We have observed glucose-stimulated focal adhesion remodeling at the ß cell surface and have shown this to be crucial for glucose-stimulated insulin secretion. However, the mechanistic link between such remodeling and the insulin secretory machinery remained unknown and was the major aim of this study. MIN6B1 cells, a previously validated model of primary ß cell function, were used for all experiments. Total internal reflection fluorescence microscopy revealed the glucose-responsive co-localization of focal adhesion kinase (FAK) and paxillin with integrin ß1 at the basal cell surface after short term stimulation. In addition, blockade of the interaction between ß1 integrins and the extracellular matrix with an anti-ß1 integrin antibody (Ha2/5) inhibited short term glucose-induced phosphorylation of FAK (Tyr-397), paxillin (Tyr-118), and ERK1/2 (Thr-202/Tyr-204). Pharmacological inhibition of FAK activity blocked glucose-induced actin cytoskeleton remodeling and glucose-induced disruption of the F-actin/SNAP-25 association at the plasma membrane as well as the distribution of insulin granules to regions in close proximity to the plasma membrane. Furthermore, FAK inhibition also completely blocked short term glucose-induced activation of the Akt/AS160 signaling pathway. In conclusion, these results indicate 1) that glucose-induced activation of FAK, paxillin, and ERK1/2 is mediated by ß1 integrin intracellular signaling, 2) a mechanism whereby FAK mediates glucose-induced actin cytoskeleton remodeling, hence allowing docking and fusion of insulin granules to the plasma membrane, and 3) a possible functional role for the Akt/AS160 signaling pathway in the FAK-mediated regulation of glucose-stimulated insulin secretion.

Interleukin-6 enhances insulin secretion by increasing glucagon-like peptide-1 secretion from L cells and alpha cells.

Exercise, obesity and type 2 diabetes are associated with elevated plasma concentrations of interleukin-6 (IL-6). Glucagon-like peptide-1 (GLP-1) is a hormone that induces insulin secretion. Here we show that administration of IL-6 or elevated IL-6 concentrations in response to exercise stimulate GLP-1 secretion from intestinal L cells and pancreatic alpha cells, improving insulin secretion and glycemia. IL-6 increased GLP-1 production from alpha cells through increased proglucagon (which is encoded by GCG) and prohormone convertase 1/3 expression. In models of type 2 diabetes, the beneficial effects of IL-6 were maintained, and IL-6 neutralization resulted in further elevation of glycemia and reduced pancreatic GLP-1. Hence, IL-6 mediates crosstalk between insulin-sensitive tissues, intestinal L cells and pancreatic islets to adapt to changes in insulin demand. This previously unidentified endocrine loop implicates IL-6 in the regulation of insulin secretion and suggests that drugs modulating this loop may be useful in type 2 diabetes.

DIAMAP: a road map for diabetes research in Europe.

The DIAMAP Project, which has drawn up a road map for diabetes research in Europe, has now concluded, and the results are available in the form of a report and searchable databases. The DIAMAP road maps provide strategic guidance for diabetes research activity and investment in Europe, with the person with diabetes and a broad approach to research being integral to the process.

Bimodal effect on pancreatic ß-cells of secretory products from normal or insulin-resistant human skeletal muscle.

OBJECTIVE: Type 2 diabetes is characterized by insulin resistance with a relative deficiency in insulin secretion. This study explored the potential communication between insulin-resistant human skeletal muscle and primary (human and rat) ß-cells. RESEARCH DESIGN AND METHODS: Human skeletal muscle cells were cultured for up to 24 h with tumor necrosis factor (TNF)-α to induce insulin resistance, and mRNA expression for cytokines was analyzed and compared with controls (without TNF-α). Conditioned media were collected and candidate cytokines were measured by antibody array. Human and rat primary ß-cells were used to explore the impact of exposure to conditioned media for 24 h on apoptosis, proliferation, short-term insulin secretion, and key signaling protein phosphorylation and expression. RESULTS: Human myotubes express and release a different panel of myokines depending on their insulin sensitivity, with each panel exerting differential effects on ß-cells. Conditioned medium from control myotubes increased proliferation and glucose-stimulated insulin secretion (GSIS) from primary ß-cells, whereas conditioned medium from TNF-α-treated insulin-resistant myotubes (TMs) exerted detrimental effects that were either independent (increased apoptosis and decreased proliferation) or dependent on the presence of TNF-α in TM (blunted GSIS). Knockdown of ß-cell mitogen-activated protein 4 kinase 4 prevented these effects. Glucagon-like peptide 1 protected ß-cells against decreased proliferation and apoptosis evoked by TMs, while interleukin-1 receptor antagonist only prevented the latter. CONCLUSIONS: Taken together, these data suggest a possible new route of communication between skeletal muscle and ß-cells that is modulated by insulin resistance and could contribute to normal ß-cell functional mass in healthy subjects, as well as the decrease seen in type 2 diabetes.

Focal adhesion remodeling is crucial for glucose-stimulated insulin secretion and involves activation of focal adhesion kinase and paxillin.

OBJECTIVE: Actin cytoskeleton remodeling is known to be involved in glucose-stimulated insulin secretion (GSIS). We have observed glucose-stimulated changes at the ß-cell basal membrane similar to focal adhesion remodeling in cell migration. This led us to study the role of two key focal adhesion proteins, focal adhesion kinase (FAK) and paxillin, in GSIS. RESEARCH DESIGN AND METHODS: All studies were performed using rat primary ß-cells or isolated islets. Protein phosphorylation and subcellular localization were determined by Western blotting and confocal immunofluorescence, respectively. Insulin was measured by radioimmunoassay. Both siRNA and pharmacological approaches were used to assess the role of FAK and paxillin in glucose-stimulated focal adhesion remodeling and insulin secretion. RESULTS: Glucose stimulation of ß-cells in monolayer significantly increased phosphorylation of FAK and paxillin as well as cell surface area. This coincided with the appearance at the basal membrane of numerous shorter actin filopodial extensions, containing not only phosphorylated paxillin, FAK, and extracellular signal-related kinase 1/2 but also two SNARE proteins, synaptosomal-associated protein 25 and syntaxin 1, indicating involvement in exocytosis. SR7037 completely inhibited this sequence of events, indicating the requirement of increased cytosolic Ca²(+). Furthermore, knockdown of paxillin significantly decreased GSIS, as did inhibition of glucose-induced FAK phosphorylation by compound Y15. Key findings were confirmed in ß-cells within the natural setting of islets. CONCLUSIONS: Glucose-stimulated remodeling of focal adhesions and phosphorylation of FAK and paxillin are involved in full development of GSIS, indicating a previously unknown role for focal adhesion remodeling in pancreatic ß-cell function.