Stacey Fynch

Liver‐specific suppressor of cytokine signaling‐3 deletion in mice enhances hepatic insulin sensitivity and lipogenesis resulting in fatty liver and obesity1

Obesity is associated with chronic inflammation and contributes to the development of insulin resistance and nonalcoholic fatty liver disease. The suppressor of cytokine signaling‐3 (SOCS3) protein is increased in inflammation and is thought to contribute to the pathogenesis of insulin resistance by inhibiting insulin and leptin signaling. Therefore, we studied the metabolic effects of liver‐specific SOCS3 deletion in vivo. We fed wild‐type (WT) and liver‐specific SOCS3 knockout (SOCS3 LKO) mice either a control diet or a high‐fat diet (HFD) for 6 weeks and examined their metabolic phenotype. We isolated hepatocytes from WT and SOCS3 LKO mice and examined the effects of tumor necrosis factor α and insulin on Akt phosphorylation and fatty acid metabolism and lipogenic gene expression. Hepatocytes from control‐fed SOCS3 LKO mice were protected from developing tumor necrosis factor α–induced insulin resistance but also had increased lipogenesis and expression of sterol response element–binding protein‐1c target genes. Lean SOCS3 LKO mice fed a control diet had enhanced hepatic insulin sensitivity; however, when fed an HFD, SOCS3 LKO mice had increased liver fat, inflammation, and whole‐body insulin resistance. SOCS3 LKO mice fed an HFD also had elevated hypothalamic SOCS3 and fatty acid synthase expression and developed greater obesity due to increased food intake and reduced energy expenditure. Conclusion: Deletion of SOCS3 in the liver increases liver insulin sensitivity in mice fed a control diet but paradoxically promotes lipogenesis, leading to the development of nonalcoholic fatty liver disease, inflammation, and obesity. (HEPATOLOGY 2010.)

Macrophage Deletion of SOCS1 Increases Sensitivity to LPS and Palmitic Acid and Results in Systemic Inflammation and Hepatic Insulin Resistance

OBJECTIVE Macrophage secretion of proinflammatory cytokines contributes to the pathogenesis of obesity-related insulin resistance. An important regulator of inflammation is the suppressor of cytokine signaling-1 (SOCS1), which inhibits the JAK-STAT and toll-like receptor-4 (TLR4) pathways. Despite the reported role of SOCS1 in inhibiting insulin signaling, it is surprising that a SOCS1 polymorphism that increases SOCS1 promoter activity is associated with enhanced insulin sensitivity despite obesity. In the current study, we investigated the physiological role of myeloid and lymphoid cell SOCS1 in regulating inflammation and insulin sensitivity. RESEARCH DESIGN AND METHODS We used mice generated by crossing SOCS1 floxed mice with mice expressing Cre recombinase under the control of the LysM-Cre promoter (SOCS1 LysM-Cre). These mice have deletion of SOCS1 in macrophages and lymphocytes. We assessed macrophage inflammation using flow cytometry and serum cytokine levels using Bioplex assays. We then measured insulin sensitivity using glucose tolerance tests and the euglycemic-hyperinsulinemic clamp. Using bone marrow–derived macrophages, we tested the effects of SOCS1 deletion in regulating responses to the TLR4 ligands: lipopolysaccharide (LPS) and palmitic acid. RESULTS SOCS1 LysM-Cre mice had increased macrophage expression of CD11c, enhanced sensitivity to LPS, and palmitic acid and increased serum concentrations of tumor necrosis factor-α, interleukin-6, and monocyte chemoattractant protein. Increased inflammation was associated with impaired glucose tolerance and hyperinsulinemia as a result of reduced hepatic but not skeletal muscle insulin sensitivity. CONCLUSIONS The expression of SOCS1 in hematopoietic cells protects mice against systemic inflammation and hepatic insulin resistance potentially by inhibiting LPS and palmitate-induced TLR4 signaling in macrophages.

Islet β-Cells Deficient in Bcl-xL Develop but Are Abnormally Sensitive to Apoptotic Stimuli

OBJECTIVE Bcl-xL is an antiapoptotic member of the Bcl-2 family of proteins and a potent regulator of cell death. We investigated the importance of Bcl-xL for β-cells by deleting the Bcl-x gene specifically in β-cells and analyzing their survival in vivo and in culture. RESEARCH DESIGN AND METHODS Islets with β-cells lacking the Bcl-x gene were assessed in vivo by histology and by treatment of mice with low-dose streptozotocin (STZ). Islets were isolated by collagenase digestion and treated in culture with the apoptosis inducers staurosporine, thapsigargin, γ-irradiation, proinflammatory cytokines, or Fas ligand. Cell death was assessed by flow cytometric analysis of subgenomic DNA. RESULTS Bcl-xL–deficient β-cells developed but were abnormally sensitive to apoptosis induced in vivo by low-dose STZ. Although a small proportion of β-cells still expressed Bcl-xL, these did not have a survival advantage over their Bcl-xL–deficient neighbors. Islets appeared normal after collagenase isolation and whole-islet culture. They were, however, abnormally sensitive in culture to a number of different apoptotic stimuli including cytotoxic drugs, proinflammatory cytokines, and Fas ligand. CONCLUSIONS Bcl-xL expression in β-cells is dispensible during islet development in the mouse. Bcl-xL is, however, an important regulator of β-cell death under conditions of synchronous stress. Bcl-xL expression at physiological levels may partially protect β-cells from apoptotic stimuli, including apoptosis because of mediators implicated in type 1 diabetes and death or degeneration of transplanted islets.

Autoreactive Cytotoxic T Lymphocytes Acquire Higher Expression of Cytotoxic Effector Markers in the Islets of NOD Mice after Priming in Pancreatic Lymph Nodes

Cytotoxic T lymphocytes (CTLs) that cause type 1 diabetes are activated in draining lymph nodes and become concentrated as fully active CTLs in inflamed pancreatic islets. It is unclear whether CTL function is driven by signals received in the lymph node or also in the inflamed tissue. We studied whether the development of cytotoxicity requires further activation in islets. Autoreactive CTLs found in the islets of diabetes-prone NOD mice had acquired much higher expression of the cytotoxic effector markers granzyme B, interferon γ, and CD107a than had those in the pancreatic lymph node (PLN). Increased expression seemed to result from stimulation in the islet itself. T cells held up from migrating from the PLN by administration of the sphingosine-1-phosphate agonist FTY720 did not increase expression of cytotoxic molecules in the PLN. Stimulation did not require antigen presentation or cytokine secretion by the target β cells because it was not affected by the absence of class I major histocompatibility complex expression or by the overexpression of suppressor of cytokine signaling-1. Activation of CD40-expressing cells stimulated increased CTL function and β-cell destruction, suggesting that signals derived from CD40-expressing cells promote the acquisition of cytotoxicity in the islet environment. These data provide in vivo evidence that stimulation of cytotoxic effector molecule expression occurs in inflamed islets and is independent of β cells.

Intra‐islet proliferation of cytotoxic T lymphocytes contributes to insulitis progression

Infiltration of pancreatic islets by immune cells, termed insulitis, increases progressively once it begins and leads to clinical type 1 diabetes. But even after diagnosis some islets remain unaffected and infiltration is patchy rather than uniform. Traffic of autoreactive T cells into the pancreas is likely to contribute to insulitis progression but it could also depend on T‐cell proliferation within islets. This study utilizes transgenic NOD mice to assess the relative contributions of these two mechanisms. Progression of insulitis in NOD8.3 TCR transgenic mice was mildly reduced by inhibition of T‐cell migration with the drug FTY720. In FTY720‐treated mice, reduced beta cell MHC class I expression prevented progression of insulitis both within affected islets and to previously unaffected islets. CTL proliferation was significantly reduced in islets with reduced or absent beta cell expression of MHC class I protein. This indicates that intra‐islet proliferation, apparently dependent on beta cell antigen presentation, in addition to recruitment, is a significant factor in progression of insulitis.

Deficiency in Type I Interferon Signaling Prevents the Early Interferon–Induced Gene Signature in Pancreatic Islets but Not Type 1 Diabetes in NOD Mice

Type I interferons (IFNs) have been implicated in the initiation of islet autoimmunity and development of type 1 diabetes. To directly test their involvement, we generated NOD mice deficient in type I IFN receptors (NOD.IFNAR1−/−). Expression of the type I IFN-induced genes Mx1, Isg15, Ifit1, Oas1a, and Cxcr4 was detectable in NOD islets as early as 1 week of age. Of these five genes, expression of Isg15, Ifit1, Oas1a, and Mx1 peaked at 3–4 weeks of age, corresponding with an increase in Ifnα mRNA, declined at 5–6 weeks of age, and increased again at 10–14 weeks of age. Increased IFN-induced gene expression was ablated in NOD.IFNAR1−/− islets. Loss of Toll-like receptor 2 (TLR2) resulted in reduced islet expression of Mx1 at 2 weeks of age, but TLR2 or TLR9 deficiency did not change the expression of other IFN-induced genes in islets compared with wild-type NOD islets. We observed increased β-cell major histocompatibility complex class I expression with age in NOD and NOD.IFNAR1−/− mice. NOD.IFNAR1−/− mice developed insulitis and diabetes at a similar rate to NOD controls. These results indicate type I IFN is produced within islets in young mice but is not essential for the initiation and progression of diabetes in NOD mice.

The Protective Effects of CD39 Overexpression in Multiple Low-Dose Streptozotocin–Induced Diabetes in Mice

Islet allograft survival limits the long-term success of islet transplantation as a potential curative therapy for type 1 diabetes. A number of factors compromise islet survival, including recurrent diabetes. We investigated whether CD39, an ectonucleotidase that promotes the generation of extracellular adenosine, would mitigate diabetes in the T cell–mediated multiple low-dose streptozotocin (MLDS) model. Mice null for CD39 (CD39KO), wild-type mice (WT), and mice overexpressing CD39 (CD39TG) were subjected to MLDS. Adoptive transfer experiments were performed to delineate the efficacy of tissue-restricted overexpression of CD39. The role of adenosine signaling was examined using mutant mice and pharmacological inhibition. The susceptibility to MLDS-induced diabetes was influenced by the level of expression of CD39. CD39KO mice developed diabetes more rapidly and with higher frequency than WT mice. In contrast, CD39TG mice were protected. CD39 overexpression conferred protection through the activation of adenosine 2A receptor and adenosine 2B receptor. Adoptive transfer experiments indicated that tissue-restricted overexpression of CD39 conferred robust protection, suggesting that this may be a useful strategy to protect islet grafts from T cell–mediated injury.

Complete Diabetes Protection Despite Delayed Thymic Tolerance in NOD8.3 TCR Transgenic Mice Due to Antigen-Induced Extrathymic Deletion of T Cells

Prevention of autoimmunity requires the elimination of self-reactive T cells during their development in the thymus and maturation in the periphery. Transgenic NOD mice that overexpress islet-specific glucose 6 phosphatase catalytic subunit–related protein (IGRP) in antigen-presenting cells (NOD-IGRP mice) have no IGRP-specific T cells. To study the relative contribution of central and peripheral tolerance mechanisms to deletion of antigen-specific T cells, we crossed NOD-IGRP mice to highly diabetogenic IGRP206–214 T-cell receptor transgenic mice (NOD8.3 mice) and studied the frequency and function of IGRP-specific T cells in the thymus and periphery. Peripheral tolerance was extremely efficient and completely protected NOD-IGRP/NOD8.3 mice from diabetes. Peripheral tolerance was characterized by activation of T cells in peripheral lymphoid tissue where IGRP was expressed followed by activation-induced cell death. Thymectomy showed that thymic output of IGRP-specific transgenic T cells compensated for peripheral deletion to maintain peripheral T-cell numbers. Central tolerance was undetectable until 10 weeks and complete by 15 weeks. These in vivo data indicate that peripheral tolerance alone can protect NOD8.3 mice from autoimmune diabetes and that profound changes in T-cell repertoire can follow subtle changes in thymic antigen presentation.

Autoreactive T cells induce necrosis and not BCL-2-regulated or death receptor-mediated apoptosis or RIPK3-dependent necroptosis of transplanted islets in a mouse model of type 1 diabetes

Aims/hypothesisType 1 diabetes results from T cell-mediated destruction of pancreatic beta cells. The mechanisms of beta cell destruction in vivo, however, remain unclear. We aimed to test the relative roles of the main cell death pathways: apoptosis, necrosis and necroptosis, in beta cell death in the development of CD4+ T cell-mediated autoimmune diabetes.MethodsWe altered expression levels of critical cell death proteins in mouse islets and tested their ability to survive CD4+ T cell-mediated attack using an in vivo graft model.ResultsLoss of the B cell leukaemia/lymphoma 2 (BCL-2) homology domain 3-only proteins BIM, PUMA or BID did not protect beta cells from this death. Overexpression of the anti-apoptotic protein BCL-2 or combined deficiency of the pro-apoptotic multi-BCL2 homology domain proteins BAX and BAK also failed to prevent beta cell destruction. Furthermore, loss of function of the death receptor Fas or its essential downstream signalling molecule Fas-associated death domain (FADD) in islets was also not protective. Using electron microscopy we observed that dying beta cells showed features of necrosis. However, islets deficient in receptor-interacting serine/threonine protein kinase 3 (RIPK3), a critical initiator of necroptosis, were still normally susceptible to CD4+ T cell-mediated destruction. Remarkably, simultaneous inhibition of apoptosis and necroptosis by combining loss of RIPK3 and overexpression of BCL-2 in islets did not protect them against immune attack either.Conclusions/interpretationCollectively, our data indicate that beta cells die by necrosis in autoimmune diabetes and that the programmed cell death pathways apoptosis and necroptosis are both dispensable for this process.

Activation of the NLRP3 inflammasome complex is not required for stress-induced death of pancreatic islets.

Loss of pancreatic beta cells is a feature of type-2 diabetes. High glucose concentrations induce endoplasmic reticulum (ER) and oxidative stress-mediated apoptosis of islet cells in vitro. ER stress, oxidative stress and high glucose concentrations may also activate the NLRP3 inflammasome leading to interleukin (IL)-1β production and caspase-1 dependent pyroptosis. However, whether IL-1β or intrinsic NLRP3 inflammasome activation contributes to beta cell death is controversial. This possibility was examined in mouse islets. Exposure of islets lacking functional NLRP3 or caspase-1 to H2O2, rotenone or thapsigargin induced similar cell death as in wild-type islets. This suggests that oxidative or ER stress do not cause inflammasome-mediated cell death. Similarly, deficiency of NLRP3 inflammasome components did not provide any protection from glucose, ribose or gluco-lipotoxicity. Finally, genetic activation of NLRP3 specifically in beta cells did not increase IL-1β production or cell death, even in response to glucolipotoxicity. Overall, our results show that glucose-, ER stress- or oxidative stress-induced cell death in islet cells is not dependent on intrinsic activation of the NLRP3 inflammasome.