Glucose-Dependent Insulinotropic Peptide Stimulates Glucagon-Like Peptide 1 Production by Pancreatic Islets via Interleukin 6, Produced by α Cells.

BACKGROUND & AIMS: Glucose-dependent insulinotropic peptide (GIP) induces production of interleukin 6 (IL6) by adipocytes. IL6 increases production of glucagon-like peptide (GLP)-1 by L cells and α cells, leading to secretion of insulin from ß cells. We investigated whether GIP regulates GLP1 and glycemia via IL6. METHODS: We obtained samples of human pancreatic islets and isolated islets from mice; human α cells and ß cells were sorted by flow cytometry and incubated with GIP. Islets were analyzed by quantitative polymerase chain reaction and immunohistochemistry. BKS.Cg-Dock7m+/+ Leprdb/J db/db mice (diabetic mice) and db/+ mice, as well as C57BL/6J IL6-knockout mice (IL6-KO) and C57BL/6J mice with the full-length Il6 gene (controls), were fed a chow or a high-fat diet; some mice were given injections of recombinant GIP, IL6, GLP, a neutralizing antibody against IL6 (anti-IL6), lipopolysaccharide, and/or IL1B. Mice were given a glucose challenge and blood samples were collected and analyzed. RESULTS: Incubation of mouse and human pancreatic α cells with GIP induced their production of IL6, leading to production of GLP1 and insulin secretion from pancreatic islets. This did not occur in islets from IL6-KO mice or in islets incubated with anti-IL6. Incubation of islets with IL1B resulted in IL6 production but directly reduced GLP1 production. Incubation of mouse islets with the sodium glucose transporter 2 inhibitor dapagliflozin induced production of GLP1 and IL6. Injection of control mice with GIP increased plasma levels of GLP1, insulin, and glucose tolerance; these effects were amplified in mice given lipopolysaccharide but reduced in IL6-KO mice or in mice given anti-IL6. Islets from diabetic mice had increased levels of IL1B and IL6, compared with db/+ mice, but injection of GIP did not lead to production of GLP1 or reduce glycemia. CONCLUSIONS: In studies of pancreatic islets from human beings and mice, we found that GIP induces production of IL6 by α cells, leading to islet production of GLP1 and insulin. This process is regulated by inflammation, via IL1B, and by sodium glucose transporter 2. In diabetic mice, increased islet levels of IL6 and IL1B might increase or reduce the production of GLP1 and affect glycemia.

TLR2/6 and TLR4-activated macrophages contribute to islet inflammation and impair beta cell insulin gene expression via IL-1 and IL-6.

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.

Large granular lymphocyte expansion after allogeneic hematopoietic stem cell transplant is associated with a cytomegalovirus reactivation and shows an indolent outcome.

Expansions of CD3+ large granular lymphocytes (LGLs) after allogeneic hematopoietic stem cell transplantation (HSCT) have been described. We sought to evaluate incidence, characteristics, and clinical significance of persistent T cell (T-)LGL after HSCT. Fourteen of 215 recipients (7%) were diagnosed with LGL expansions. Thirteen showed a CD3+/CD8+ immunophenotype, 5 of them with clonal TCR-γ rearrangement. The lymphocytes appeared at a median of 16 months (range, 3-58 months) after HSCT and lasted for a median time of 31 months (range, 2-179 months). Cytomegalovirus (CMV) reactivation (P = .001) and acute graft-versus-host disease (aGVHD) were associated with LGL expansion (P = .02). In the multivariate analysis, only CMV reactivation showed a significant association with T-LGL expansion (relative risk [RR]: 5.063; 95% confidence interval [CI]: 1.586-16.160; P = .006). The observed posttransplantation LGL expansions, even if monoclonal, showed a chronic, indolent course. Our data indicate that such expansions may be considered as an expression of chronic stimulation, triggered by CMV reactivation rather than a malignant transformation.

Publisher Correction: Loss-of-function of the long non-coding RNA A830019P07Rik in mice does not affect insulin expression and secretion.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Loss-of-function of the long non-coding RNA A830019P07Rik in mice does not affect insulin expression and secretion.

Long non-coding RNAs (lncRNAs) contribute to diverse cellular functions and the dysregulation of their expression or function can contribute to diseases, including diabetes. The contributions of lncRNAs to ß-cell development, function and survival has been extensively studied in vitro. However, very little is currently known on the in vivo roles of lncRNAs in the regulation of glucose and insulin homeostasis. Here we investigated the impact of loss-of-function in mice of the lncRNA A830019P07Rik, hereafter P07Rik, which was previously reported to be associated with reduced plasma insulin levels. Compared with wild-type littermates, male and female P07Rik mutant mice did not show any defect in glycaemia and plasma insulin levels in both fed and fasted state. Furthermore, P07Rik mutant mice displayed similar glucose and insulin levels in response to an intra-peritoneal glucose tolerance test. Ex vivo, islets from mutant P07Rik released similar amount of insulin in response to increased glucose concentration as wildtype littermates. In contrast with previous reports, our characterization of P07Rik mouse mutants revealed that loss of function of this lncRNA does not affect glucose and insulin homeostasis in mice.

Angiogenin and Osteoprotegerin are type II muscle specific myokines protecting pancreatic beta-cells against proinflammatory cytokines.

Tissue cross-talk is emerging as a determinant way to coordinate the different organs implicated in glucose homeostasis. Among the inter-organ communication factors, muscle-secreted myokines can modulate the function and survival of pancreatic beta-cells. Using primary human myotubes from soleus, vastus lateralis and triceps brachii muscles, we report here that the impact of myokines on beta-cells depends on fiber types and their metabolic status. We show that Type I and type II primary myotubes present specific mRNA and myokine signatures as well as a different sensitivity to TNF-alpha induced insulin resistance. Finally, we show that angiogenin and osteoprotegerin are triceps specific myokines with beta-cell protective actions against proinflammatory cytokines. These results suggest that type I and type II muscles could impact insulin secretion and beta-cell mass differentially in type 2 diabetes through specific myokines secretion.

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.

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.

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.

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.

Atherosclerotic mice exhibit systemic inflammation in periadventitial and visceral adipose tissue, liver, and pancreatic islets.

OBJECTIVE: Atherosclerosis is a chronic inflammatory disease of major conduit arteries. Similarly, obesity and type 2 diabetes mellitus are associated with accumulation of macrophages in visceral white adipose tissue and pancreatic islets. Our goal was to characterize systemic inflammation in atherosclerosis with hypercholesterolemia, but without obesity. METHODS AND RESULTS: We compared 22-week-old apolipoprotein E knockout (ApoE(-/-)) with wild-type mice kept for 14 weeks on a high cholesterol (1.25%) diet (CD, n=8) and 8-week-old ApoE(-/-) with wild-type mice kept on a normal diet (ND, n=8). Hypercholesterolemic, atherosclerotic ApoE(-/-) mice on CD exhibited increased macrophages and T-cells in plaques and periadventitial adipose tissue that revealed elevated expression of MIP-1alpha, IL-1beta, IL-1 receptor, and IL-6. Mesenteric adipose tissue and pancreatic islets in ApoE(-/-) mice showed increased macrophages. Expression of IL-1beta was enhanced in mesenteric adipose tissue of ApoE(-/-) mice on CD. Furthermore, these mice exhibited steatohepatitis with macrophage and T-cell infiltrations as well as increased MIP-1alpha and IL-1 receptor expression. Blood glucose, insulin and total body weight did not differ between the groups. CONCLUSIONS: In hypercholesterolemic lean ApoE(-/-) mice, inflammation extends beyond atherosclerotic plaques to the periadventitial and visceral adipose tissue, liver, and pancreatic islets without affecting glucose homeostasis.

Low- and high-density lipoproteins modulate function, apoptosis, and proliferation of primary human and murine pancreatic beta-cells.

A low high-density lipoprotein (HDL) plasma concentration and the abundance of small dense low-density lipoproteins (LDL) are risk factors for developing type 2 diabetes. We therefore investigated whether HDL and LDL play a role in the regulation of pancreatic islet cell apoptosis, proliferation, and secretory function. Isolated mouse and human islets were exposed to plasma lipoproteins of healthy human donors. In murine and human beta-cells, LDL decreased both proliferation and maximal glucose-stimulated insulin secretion. The comparative analysis of beta-cells from wild-type and LDL receptor-deficient mice revealed that the inhibitory effect of LDL on insulin secretion but not proliferation requires the LDL receptor. HDL was found to modulate the survival of both human and murine islets by decreasing basal as well as IL-1beta and glucose-induced apoptosis. IL-1beta-induced beta-cell apoptosis was also inhibited in the presence of either the delipidated protein or the deproteinated lipid moieties of HDL, apolipoprotein A1 (the main protein component of HDL), or sphingosine-1-phosphate (a bioactive sphingolipid mostly carried by HDL). In murine beta-cells, the protective effect of HDL against IL-1beta-induced apoptosis was also observed in the absence of the HDL receptor scavenger receptor class B type 1. Our data show that both LDL and HDL affect function or survival of beta-cells and raise the question whether dyslipidemia contributes to beta-cell failure and hence the manifestation and progression of type 2 diabetes mellitus.

Involvement of 4E-BP1 in the protection induced by HDLs on pancreatic beta-cells.

High-density lipoproteins (HDLs) protect pancreatic beta-cells against apoptosis. This property might relate to the increased risk to develop diabetes in patients with low HDL blood levels. However, the mechanisms by which HDLs protect beta-cells are poorly characterized. Here we used a transcriptomic approach to identify genes differentially modulated by HDLs in beta-cells subjected to apoptotic stimuli. The transcript encoding 4E-binding protein (4E-BP)1 was up-regulated by serum starvation, and HDLs blocked this increase. 4E-BP1 inhibits cap-dependent translation in its non- or hypophosphorylated state but it loses this ability when hyperphosphorylated. At the protein level, 4E-BP1 was also up-regulated in response to starvation and IL-1beta, and this was blunted by HDLs. Whereas an ectopic increase of 4E-BP1 expression induced beta-cell death, silencing 4E-BP1 increase with short hairpin RNAs inhibited the apoptotic-inducing capacities of starvation. HDLs can therefore protect beta-cells by blocking 4E-BP1 protein expression, but this is not the sole protective mechanism activated by HDLs. Indeed, HDLs blocked apoptosis induced by endoplasmic reticulum stress with no associated decrease in total 4E-BP1 induction. Although, HDLs favored the phosphorylation, and hence the inactivation of 4E-BP1 in these conditions, this appeared not to be required for HDL protection. Our results indicate that HDLs can protect beta-cells through modulation of 4E-BP1 depending on the type of stress stimuli.