Tamara Saksida

Macrophage migration inhibitory factor deficiency protects pancreatic islets from palmitic acid-induced apoptosis

As a result of chronic exposure to high levels of free fatty acids, glucose and inflammatory mediators β‐cell apoptosis occurs at the end stage of obesity‐associated type 2 diabetes (T2D). One potentially deleterious molecule for β‐cell function associated with T2D and obesity in humans is macrophage migration inhibitory factor (MIF). Therefore, the aim of this study was to explore MIF expression in vivo during development of obesity and insulin resistance in high‐fat diet (HFD)‐fed C57BL/6 mice and whether MIF inhibition could affect β‐cell apoptosis and dysfunction induced by palmitic acid (PA) in vitro. Indeed, increase in systemic and locally produced MIF correlated well with the weight gain, triglyceride upregulation, glucose intolerance and insulin resistance, which developed in HFD‐fed mice. In in vitro settings PA dose‐dependently induced MIF secretion before apoptosis development in islets. Further, mif gene deletion, mRNA silencing or protein inhibition rescued β‐cells from PA‐induced apoptosis as measured by MTT assay and histone‐DNA enzyme linked immuno sorbent assay. Protection from induced apoptosis was mediated by altered activation of caspase pathway and correlated with changes in the level of Bcl‐2 family members. Further, MIF inhibition conveyed a significant resistance to PA‐induced downregulation of insulin and PDX‐1 expression and ATP content. However, β‐cell function was not entirely preserved in the absence of MIF judging by low glucose oxidation and depolarized mitochondrial membrane. In conclusion, the observed considerable preservation of β‐cells from nutrient‐induced apoptosis might implicate MIF as a potential therapeutic target in the later stage of obesity‐associated T2D.

Pharmacological application of carbon monoxide ameliorates islet-directed autoimmunity in mice via anti-inflammatory and anti-apoptotic effects

Aims/hypothesisRecent studies have identified carbon monoxide (CO) as a potential therapeutic molecule for the treatment of autoimmune diseases owing to its anti-inflammatory and anti-apoptotic properties. We explored the efficacy and the mechanisms of action of the CO-releasing molecule (CORM)-A1 in preclinical models of type 1 diabetes.MethodsThe impact of CORM-A1 on diabetes development was evaluated in models of spontaneous diabetes in NOD mice and in diabetes induced in C57BL/6 mice by multiple low-dose streptozotocin (MLDS). Ex vivo analysis was performed to determine the impact of CORM-A1 both on T helper (Th) cell and macrophage differentiation and on their production of soluble mediators in peripheral tissues and in infiltrates of pancreatic islets. The potential effect of CORM-A1 on cytokine-induced apoptosis in pancreatic islets or beta cells was evaluated in vitro.ResultsCORM-A1 conferred protection from diabetes in MLDS-induced mice and reduced diabetes incidence in NOD mice as confirmed by preserved insulin secretion and improved histological signs of the disease. In MLDS-challenged mice, CORM-A1 attenuated Th1, Th17, and M1 macrophage response and facilitated Th2 cell differentiation. In addition, CORM-A1 treatment in NOD mice upregulated the regulatory arm of the immune response (M2 macrophages and FoxP3+ regulatory T cells). Importantly, CORM-A1 interfered with in vitro cytokine-induced beta cell apoptosis through the reduction of cytochrome c and caspase 3 levels.Conclusions/interpretationThe ability of CORM-A1 to protect mice from developing type 1 diabetes provides a valuable proof of concept for the potential exploitation of controlled CO delivery in clinical settings for the treatment of autoimmune diabetes.

Galectin-3 deficiency protects pancreatic islet cells from cytokine-triggered apoptosis in vitro†‡

Beta cell apoptosis is a hallmark of diabetes. Since we have previously shown that galectin‐3 deficient (LGALS3−/−) mice are relatively resistant to diabetes induction, the aim of this study was to examine whether beta cell apoptosis depends on the presence of galectin‐3 and to delineate the underlying mechanism. Deficiency of galectin‐3, either hereditary or induced through application of chemical inhibitors, β‐lactose or TD139, supported survival and function of islet beta cells compromised by TNF‐α + IFN‐γ + IL‐1β stimulus. Similarly, inhibition of galectin‐3 by β‐lactose or TD139 reduced cytokine‐triggered apoptosis of beta cells, leading to conclusion that endogenous galectin‐3 propagates beta apoptosis in the presence of an inflammatory milieu. Exploring apoptosis‐related molecules expression in primary islet cells before and after treatment with cytokines we found that galectin‐3 ablation affected the expression of major components of mitochondrial apoptotic pathway, such as BAX, caspase‐9, Apaf, SMAC, caspase‐3, and AIF. In contrast, anti‐apoptotic molecules Bcl‐2 and Bcl‐XL were up‐regulated in LGALS3−/− islet cells when compared to wild‐type (WT) counterparts (C57BL/6), resulting in increased ratio of anti‐apoptotic versus pro‐apoptotic molecules. However, Fas‐triggered apoptotic pathway as well as extracellular signal‐regulated kinase 1/2 (ERK1/2) was not influenced by LGALS‐3 deletion. All together, these results point to an important role of endogenous galectin‐3 in beta cell apoptosis in the inflammatory milieu that occurs during diabetes pathogenesis and implicates impairment of mitochondrial apoptotic pathway as a key event in protection from beta cell apoptosis in the absence of galectin‐3. J. Cell. Physiol. 228: 1568–1576, 2013.

Macrophage migration inhibitory factor deficiency protects pancreatic islets from cytokine-induced apoptosis in vitro

During pathogenesis of diabetes, pancreatic islets are exposed to high levels of cytokines and other inflammatory mediators that induce deterioration of insulin‐producing beta cells. Macrophage migration inhibitory factor (MIF) plays a key role in the onset and development of several immunoinflammatory diseases and also controls apoptotic cell death. Because the occurrence of apoptosis plays a pathogenetic role in beta cell death during type 1 diabetes development and MIF is expressed in beta cells, we explored the influence of MIF deficiency on cytokine‐induced apoptosis in pancreatic islets. The results indicated clearly that elevated MIF secretion preceded C57BL/6 pancreatic islets death induced by interferon (IFN)‐γ + tumour necrosis factor (TNF)‐α + interleukin (IL)‐1β. Consequently, MIF‐deficient [MIF‐knock‐out (KO)] pancreatic islets or islet cells showed significant resistance to cytokine‐induced death than those isolated from C57BL/6 mice. Furthermore, upon exposure to cytokines pancreatic islets from MIF‐KO mice maintained normal insulin expression and produced less cyclooxygenase‐2 (COX‐2) than those from wild‐type C57BL6 mice. The final outcome of cytokine‐induced islet apoptosis in islets from wild‐type mice was the activation of mitochondrial membrane pore‐forming protein Bcl‐2‐associated X protein and effector caspase 3. In contrast, these apoptotic mediators remained at normal levels in islets from MIF‐KO mice suggesting that MIF absence prevented initiation of the mitochondrial apoptotic pathway. Additionally, the protection from apoptosis was also mediated by up‐regulation of prosurvival kinase extracellular‐regulated kinase 1/2 in MIF‐KO islets. These data indicate that MIF is involved in the propagation of pancreatic islets apoptosis probably via nuclear factor‐κB and mitochondria‐related proteins.

Beta cell function: the role of macrophage migration inhibitory factor

During evolution, beta cells adapted to a sole aim: the production and stimulus-dependent secretion of insulin. This acquired specificity was accompanied by a loss of protection mechanisms predisposing beta cell to a high vulnerability. Among beta cell–damaging molecules, a new one has been identified recently: macrophage migration inhibitory factor (MIF). MIF was at first designated as a T-cell product that inhibits random movement of macrophages. Over the years, the number of functions attributed to this protein increased significantly, positioning MIF at the top of inflammatory cascade in the combat against infection and in immunoinflammatory and autoimmune diseases. This exceptionally versatile molecule regulates insulin secretion in physiological conditions, while in pathological states it alters beta cell function and induces their apoptosis or necrosis and affects beta cell neoplasia.

Methanolic extract of Origanum vulgare ameliorates type 1 diabetes through antioxidant, anti-inflammatory and anti-apoptotic activity

Type 1 diabetes (T1D), an autoimmune inflammatory disorder, develops as a consequence of pancreatic β-cell destruction and results in hyperglycaemia. Since current T1D therapy mainly involves insulin replacement, the aim of the present study was to evaluate the therapeutic potential of Origanum vulgare L. ssp. hirtum (Greek oregano) leaf extract rich in biophenols for the treatment of T1D. The phytochemical profile of methanolic oregano extract (MOE) and aqueous oregano extract (AOE) was determined by liquid chromatography/electrospray ion-trap tandem MS (LC/DAD/ESI-MSn), while their main compounds were quantified by HPLC with diode array detection. After establishing their potent in vitro antioxidant activity, the extracts were administered to C57BL/6 mice treated with multiple low doses of streptozotocin for diabetes induction. While prophylactic AOE therapy had no impact on diabetes induction, MOE reduced diabetes incidence and preserved normal insulin secretion. In addition, MOE scavenged reactive oxygen and nitrogen species and, therefore, alleviated the need for the up-regulation of antioxidant enzymes. MOE treatment specifically attenuated the pro-inflammatory response mediated by T helper 17 cells and enhanced anti-inflammatory T helper 2 and T regulatory cells through the impact on specific signalling pathways and transcription factors. Importantly, MOE preserved β-cells from in vitro apoptosis via blockade of caspase 3. Finally, rosmarinic acid, a predominant compound in MOE, exhibited only partial protection from diabetes induction. In conclusion, acting as an antioxidant, immunomodulator and in an anti-apoptotic manner, MOE protected mice from diabetes development. Seemingly, there is more than one compound responsible for the beneficial effect of MOE.

Carbon Monoxide–Releasing Molecule‐A1 Inhibits Th1/Th17 and Stimulates Th2 Differentiation In vitro

Carbon monoxide (CO) is endogenously produced by haeme oxygenase‐1 and has profound effects on intracellular signalling processes, generating anti‐inflammatory, antiproliferative and antiapoptotic effects. A boron‐containing compound CORM‐A1 is capable of releasing CO in such a way to mimic physiological functions of haeme oxygenase‐1. Considering the importance of Th1/Th17 versus Th2 balance in the final outcome of immune and inflammatory responses in this study we focused on immune‐modulatory effects of CORM‐A1 on murine lymph node–derived T cells in vitro and its influence on T‐cell proliferation, activation and differentiation. Anti‐CD3/CD28 antibody‐triggered lymph node cells proliferation remained unaffected after 24‐hour CORM‐A1 treatment, as well as the expression of the early activation marker CD25. However, CORM‐A1 successfully reduced the secretion of the two representative pro‐inflammatory cytokines, IFN‐γ and IL‐17, while the secretion of anti‐inflammatory cytokine IL‐4 remained unchanged. Furthermore, CORM‐A1 efficiently reduced the percentage of CD4+IFN‐γ+ and CD4+IL‐17+ cells, whereas CD4+IL‐4+ cell population increased after treatment. Also, CORM‐A1 significantly reduced expression of transcription factor RORγT, necessary for Th17 development, but the expression of Th1‐related and Th2‐related transcription factors (T‐bet and GATA‐3, respectively) remained unchanged. In conclusion, our findings indicate that CO has anti‐inflammatory role through the regulation of balance between pro‐inflammatory Th1/Th17 and anti‐inflammatory Th2 cells. Observed immunomodulatory effects of CORM‐A1 could be useful for developing novel therapeutic approaches in managing Th1/Th17‐mediated immune disorders.

The role of endogenous glucocorticoids in glucose metabolism and immune status of MIF-deficient mice.

Macrophage migration inhibitory factor (MIF)-deficient mice develop glucose intolerance and hyperglycemia, but remain entirely responsive to exogenous insulin in adult age. Furthermore, as a consequence of MIF deficiency, the immune response in these mice is predominantly anti-inflammatory. Since MIF is a natural counter-regulator of glucocorticoid action, and it is known that excessive concentration of glucocorticoids contribute both to beta cell dysfunction and immunosuppression, we hypothesized that MIF absence enables elevation of glucocorticoids which in turn caused the observed condition. Our results confirm that MIF-knockout (MIF-KO) mice possess higher levels of circulating corticosterone, but lower expression of glucocorticoid receptor in pancreatic islets, liver and adipose tissue to the one observed in wild type (WT) mice. A significant up-regulation of glucocorticoid receptor expression was however noticed in MIF-deficient lymph node cells. The inhibition of glucocorticoid receptor by RU486 improved tolerance to glucose in MIF-KO mice and restored euglycemia. Although RU486 treatment did not alter the level of glucose receptor GLUT2, it enhanced insulin secretion and up-regulated insulin-triggered Akt phosphorylation within hepatic tissue. Finally, inhibition of glucocorticoid receptor changed anti-inflammatory phenotype of MIF-KO lymphocytes toward a physiological profile. Our results indicate that deregulated glucocorticoid secretion and glucocorticoid receptor expression in the absence of MIF possibly contributes to the development of glucose intolerance and immunosuppression in MIF-KO mice. However, since MIF-KO mice respond normally to insulin and their beta cell function is within physiological range, additional cause for glucose intolerance could be sought in the possible malfunction of their insulin.

The critical role of macrophage migration inhibitory factor in insulin activity

Macrophage migration inhibitory factor (MIF) is a molecule with plethora of functions such as regulation of immune response, hormone-like, enzymatic and chaperone-like activity. Further, MIF is a major participant in glucose homeostasis since it is an autocrine stimulator of insulin secretion. MIF absence in male knockout mice (MIF-KO) results in development of glucose intolerance, while sensitivity to insulin is fully preserved. Since our results confirm that beta cells from MIF-KO mice express, produce and secrete insulin similarly to beta cells of their wild type (WT) counterparts C57BL/6 mice, we hypothesize that MIF-KO-derived insulin is less active. Indeed, insulin from MIF-KO islets is unable to significantly induce glucose uptake into hepatocytes and to efficiently promote insulin-triggered Akt phosphorylation determined by immunoblot. However, MIFs tautomerase function is not crucial for insulin biosynthesis since MIF inhibitors had no impact on WT insulin activity. Importantly, MIF recognition by anti-MIF antibody (ELISA) after in vitro co-incubation with purified insulin was significantly lower suggesting that insulin covers MIF immunodominant epitope. In addition, MIF binds insulin within beta cell as confirmed by co-immunoprecipitation. WT and MIF-KO-derived insulin exhibited different cleavage patterns suggesting different protein conformations. Finally, pre-incubation of recombinant MIF with insulin promotes formation of insulin hexamers. These results imply that MIF probably enables proper insulin folding what results in insulin full activity. This newly discovered feature of the cytokine MIF could be potentially important for commercially produced insulin, for increasing its stability and/or bioavailability.

Macrophage migration inhibitory factor (MIF) enhances palmitic acid- and glucose-induced murine beta cell dysfunction and destruction in vitro.

Although several reports suggest a potentially deleterious role of macrophage migration inhibitory factor (MIF) in type 2 diabetes (T2D) pathology, it is still unclear how this pro-inflammatory cytokine acts on pancreatic beta cells. The aim of the present study was to evaluate MIF effects on murine beta cells in the in vitro settings mimicking T2D-associated conditions. Results indicate that recombinant MIF further increased apoptosis of pancreatic islets or MIN6 cells upon exposure to palmitic acid or glucose. This was accompanied by upregulation of several pro-apoptotic molecules. Furthermore, MIF potentiated nutrient-induced islet cell dysfunction, as revealed by lower glucose oxidation rate, ATP content, and depolarized mitochondrial membrane. The final outcome was potentiation of mitochondrial apoptotic pathway. The observed upregulation of nutrient-induced islet cell dysfunction and apoptosis by MIF implicates that silencing MIF may be beneficial for maintaining integrity of endocrine pancreas in obesity-associated T2D.