Marika Bogdani

β-Cell-Specific Overexpression of Glutathione Peroxidase Preserves Intranuclear MafA and Reverses Diabetes in db/db Mice

Chronic hyperglycemia causes oxidative stress, which contributes to damage in various tissues and cells, including pancreatic beta-cells. The expression levels of antioxidant enzymes in the islet are low compared with other tissues, rendering the beta-cell more susceptible to damage caused by hyperglycemia. The aim of this study was to investigate whether increasing levels of endogenous glutathione peroxidase-1 (GPx-1), specifically in beta-cells, can protect them against the adverse effects of chronic hyperglycemia and assess mechanisms that may be involved. C57BLKS/J mice overexpressing the antioxidant enzyme GPx-1 only in pancreatic beta-cells were generated. The biological effectiveness of the overexpressed GPx-1 transgene was documented when beta-cells of transgenic mice were protected from streptozotocin. The transgene was then introgressed into the beta-cells of db/db mice. Without use of hypoglycemic agents, hyperglycemia in db/db-GPx(+) mice was initially ameliorated compared with db/db-GPx(-) animals and then substantially reversed by 20 wk of age. beta-Cell volume and insulin granulation and immunostaining were greater in db/db-GPx(+) animals compared with db/db-GPx(-) animals. Importantly, the loss of intranuclear musculoaponeurotic fibrosarcoma oncogene homolog A (MafA) that was observed in nontransgenic db/db mice was prevented by GPx-1 overexpression, making this a likely mechanism for the improved glycemic control. These studies demonstrate that enhancement of intrinsic antioxidant defenses of the beta-cell protects it against deterioration during hyperglycemia.

The Role of Hyaluronan and the Extracellular Matrix in Islet Inflammation and Immune Regulation

Type 1 diabetes (T1D) is a disease that in most individuals results from autoimmune attack of a single tissue type, the pancreatic islet. A fundamental, unanswered question in T1D pathogenesis is how the islet tissue environment influences immune regulation. This crosstalk is likely to be communicated through the extracellular matrix (ECM). Here, we review what is known about the ECM in insulitis and examine how the tissue environment is synchronized with immune regulation. In particular, we focus on the role of hyaluronan (HA) and its interactions with Foxp3+ regulatory T-cells (Treg). We propose that HA is a “keystone molecule” in the inflammatory milieu and that HA, together with its associated binding proteins and receptors, is an appropriate point of entry for investigations into how ECM influences immune regulation in the islet.

Hyaluronan and Hyaluronan Binding Proteins Accumulate in both Human Type 1 Diabetic Islets and Lymphoid Tissues and Associate with Inflammatory Cells in Insulitis

Hyaluronan (HA) is an extracellular matrix glycosaminoglycan that is present in pancreatic islets, but little is known about its involvement in the development of human type 1 diabetes (T1D). We have evaluated whether pancreatic islets and lymphoid tissues of T1D and nondiabetic organ donors differ in the amount and distribution of HA and HA-binding proteins (hyaladherins), such as inter-α-inhibitor (IαI), versican, and tumor necrosis factor–stimulated gene-6 (TSG-6). HA was dramatically increased both within the islet and outside the islet endocrine cells, juxtaposed to islet microvessels in T1D. In addition, HA was prominent surrounding immune cells in areas of insulitis. IαI and versican were present in HA-rich areas of islets, and both molecules accumulated in diabetic islets and regions exhibiting insulitis. TSG-6 was observed within the islet endocrine cells and in inflammatory infiltrates. These patterns were only observed in tissues from younger donors with disease duration of <10 years. Furthermore, HA and IαI amassed in follicular germinal centers and in T-cell areas in lymph nodes and spleens in T1D patients compared with control subjects. Our observations highlight potential roles for HA and hyaladherins in the pathogenesis of diabetes.

Intrahepatic Glucose Flux as a Mechanism for Defective Intrahepatic Islet α-Cell Response to Hypoglycemia

OBJECTIVE— Glucagon responses to hypoglycemia from islets transplanted in the liver are defective. To determine whether this defect is related to intrahepatic glycogen, islets from inbred Lewis rats were transplanted into the hepatic sinus (H group), peritoneal cavity (P group), omentum (O group), and kidney capsule (K group) of recipient Lewis rats previously rendered diabetic with streptozotocin (STZ). RESEARCH DESIGN AND METHODS— Glucagon responses to hypoglycemia were obtained before and after transplantation under fed conditions and after fasting for 16 h and 48 h to deplete liver glycogen. RESULTS— Glucagon (area under the curve) responses to hypoglycemia in the H group (8,839 ± 1,988 pg/ml per 90 min) were significantly less than in normal rats (40,777 ± 8,192; P < 0.01). Fasting significantly decreased hepatic glycogen levels. Glucagon responses in the H group were significantly larger after fasting (fed 8,839 ± 1,988 vs. 16-h fasting 24,715 ± 5,210 and 48-h fasting 29,639 ± 4,550; P < 0.01). Glucagon response in the H group decreased after refeeding (48-h fasting 29,639 ± 4,550 vs. refed 10,276 ± 2,750; P < 0.01). There was no difference in glucagon response to hypoglycemia between the H and the normal control group after fasting for 48 h (H 29,639 ± 4,550 vs. control 37,632 ± 5,335; P = NS). No intragroup differences were observed in the P, O, and K groups, or normal control and STZ groups, when comparing fed or fasting states. CONCLUSIONS— These data suggest that defective glucagon responses to hypoglycemia by intrahepatic islet α-cells is due to dominance of a suppressive signal caused by increased glucose flux and glucose levels within the liver secondary to increased glycogenolysis caused by systemic hypoglycemia.

Extracellular Matrix Components in the Pathogenesis of Type 1 Diabetes

Type 1 diabetes (T1D) results from progressive immune cell-mediated destruction of pancreatic β cells. As immune cells migrate into the islets, they pass through the extracellular matrix (ECM). This ECM is composed of different macromolecules localized to different compartments within and surrounding islets; however, the involvement of this ECM in the development of human T1D is not well understood. Here, we summarize our recent findings from human and mouse studies illustrating how specific components of the islet ECM that constitute basement membranes and interstitial matrix of the islets, and surprisingly, the intracellular composition of islet β cells themselves, are significantly altered during the pathogenesis of T1D. Our focus is on the ECM molecules laminins, collagens, heparan sulfate/heparan sulfate proteoglycans, and hyaluronan, as well as on the enzymes that degrade these ECM components. We propose that islet and lymphoid tissue ECM composition and organization are critical to promoting immune cell activation, islet invasion, and destruction of islet β cells in T1D.

Inhibition of hyaluronan synthesis restores immune tolerance during autoimmune insulitis.

We recently reported that abundant deposits of the extracellular matrix polysaccharide hyaluronan (HA) are characteristic of autoimmune insulitis in patients with type 1 diabetes (T1D), but the relevance of these deposits to disease was unclear. Here, we have demonstrated that HA is critical for the pathogenesis of autoimmune diabetes. Using the DO11.10xRIPmOVA mouse model of T1D, we determined that HA deposits are temporally and anatomically associated with the development of insulitis. Moreover, treatment with an inhibitor of HA synthesis, 4-methylumbelliferone (4-MU), halted progression to diabetes even after the onset of insulitis. Similar effects were seen in the NOD mouse model, and in these mice, 1 week of treatment was sufficient to prevent subsequent diabetes. 4-MU reduced HA accumulation, constrained effector T cells to nondestructive insulitis, and increased numbers of intraislet FOXP3+ Tregs. Consistent with the observed effects of 4-MU treatment, Treg differentiation was inhibited by HA and anti-CD44 antibodies and rescued by 4-MU in an ERK1/2-dependent manner. These data may explain how peripheral immune tolerance is impaired in tissues under autoimmune attack, including islets in T1D. We propose that 4-MU, already an approved drug used to treat biliary spasm, could be repurposed to prevent, and possibly treat, T1D in at-risk individuals.

Genetic dissection reveals diabetes loci proximal to the gimap5 lymphopenia gene

Congenic DRF.(f/f) rats are protected from type 1 diabetes (T1D) by 34 Mb of F344 DNA introgressed proximal to the gimap5 lymphopenia gene. To dissect the genetic factor(s) that confer protection from T1D in the DRF.(f/f) rat line, DRF.(f/f) rats were crossed to inbred BBDR or DR.(lyp/lyp) rats to generate congenic sublines that were genotyped and monitored for T1D, and positional candidate genes were sequenced. All (100%) DR.(lyp/lyp) rats developed T1D by 83 days of age. Reduction of the DRF.(f/f) F344 DNA fragment by 26 Mb (42.52-68.51 Mb) retained complete T1D protection. Further dissection revealed that a 2 Mb interval of F344 DNA (67.41-70.17 Mb) (region 1) resulted in 47% protection and significantly delayed onset (P < 0.001 compared with DR.(lyp/lyp)). Retaining <1 Mb of F344 DNA at the distal end (76.49-76.83 Mb) (region 2) resulted in 28% protection and also delayed onset (P < 0.001 compared with DR.(lyp/lyp)). Comparative analysis of diabetes frequency in the DRF.(f/f) congenic sublines further refined the RNO4 region 1 interval to approximately 670 kb and region 2 to the 340 kb proximal to gimap5. All congenic DRF.(f/f) sublines were prone to low-grade pancreatic mononuclear cell infiltration around ducts and vessels, but <20% of islets in nondiabetic rats showed islet infiltration. Coding sequence analysis revealed TCR Vbeta 8E, 12, and 13 as candidate genes in region 1 and znf467 and atp6v0e2 as candidate genes in region 2. Our results show that spontaneous T1D is controlled by at least two genetic loci 7 Mb apart on rat chromosome 4.

Modulation of diabetes in NOD mice by GAD65-specific monoclonal antibodies is epitope specific and accompanied by anti-idiotypic antibodies

Type 1 diabetes is caused by the autoimmune destruction of pancreatic beta cells. Here we show that administration of a human monoclonal antibody (b96·11) specific to the 65‐kDa isoform of glutamate decarboxylase (GAD65) to prediabetic non‐obese diabetic (NOD) mice significantly delays the onset of autoimmune diabetes. We found this effect to be epitope‐specific, as only b96·11 showed this therapeutic property, while a GAD65‐specific human monoclonal control antibody (b78) derived from the same patient, but specific to a different determinant of GAD65, had no significant effect on the progression of disease. Administration of b96·11 or b78 to NOD mice was accompanied by the generation of anti‐idiotypic antibodies. Importantly, the induced anti‐idiotypic antibodies were specific for the immunizing antibody and blocked the binding of GAD65 by the respective antibody. These findings suggest a potential role for the internal image of the GAD65 determinant recognized by b96·11 in the anti‐idiotypic antibody, supporting an immunomodulatory role for GAD65‐specific autoantibodies, as originally postulated by Jerne.

Hyaluronan A Mediator of Islet Dysfunction and Destruction in Diabetes

Hyaluronan (HA) is an extracellular matrix (ECM) component that is present in mouse and human islet ECM. HA is localized in peri-islet and intra-islet regions adjacent to microvessels. HA normally exists in a high molecular weight form, which is anti-inflammatory. However, under inflammatory conditions, HA is degraded into fragments that are proinflammatory. HA accumulates in islets of human subjects with recent onset type 1 diabetes (T1D), and is associated with myeloid and lymphocytic islet infiltration, suggesting a possible role for HA in insulitis. A similar accumulation of HA, in amount and location, occurs in non-obese diabetic (NOD) and DORmO mouse models of T1D. Furthermore, HA accumulates in follicular germinal centers and in T-cell areas in lymph nodes and spleen in both human and mouse models of T1D, as compared with control tissues. Whether HA accumulates in islets in type 2 diabetes (T2D) or models thereof has not been previously described. Here we show evidence that HA accumulates in a mouse model of islet amyloid deposition, a well-known component of islet pathology in T2D. In summary, islet HA accumulation is a feature of both T1D and a model of T2D, and may represent a novel inflammatory mediator of islet pathology.Hyaluronan (HA) is an extracellular matrix (ECM) component that is present in mouse and human islet ECM. HA is localized in peri-islet and intra-islet regions adjacent to microvessels. HA normally exists in a high molecular weight form, which is anti-inflammatory. However, under inflammatory conditions, HA is degraded into fragments that are proinflammatory. HA accumulates in islets of human subjects with recent onset type 1 diabetes (T1D), and is associated with myeloid and lymphocytic islet infiltration, suggesting a possible role for HA in insulitis. A similar accumulation of HA, in amount and location, occurs in non-obese diabetic (NOD) and DORmO mouse models of T1D. Furthermore, HA accumulates in follicular germinal centers and in T-cell areas in lymph nodes and spleen in both human and mouse models of T1D, as compared with control tissues. Whether HA accumulates in islets in type 2 diabetes (T2D) or models thereof has not been previously described. Here we show evidence that HA accumulates in a mouse model of islet amyloid deposition, a well-known component of islet pathology in T2D. In summary, islet HA accumulation is a feature of both T1D and a model of T2D, and may represent a novel inflammatory mediator of islet pathology.

Thinking Outside the Cell: A Key Role for Hyaluronan in the Pathogenesis of Human Type 1 Diabetes

The important thing in science is not so much to obtain new facts as to discover new ways of thinking about them.—William Lawrence Bragg For more than 50 years, chronic immunological processes have been considered central to type 1 diabetes pathogenesis. Studies in pancreata from patients with type 1 diabetes have revealed the presence of insulitis, identified histologically as immune cell infiltrates around and within the islets. Finding the insulitis lesion in a portion of patients with recent-onset type 1 diabetes indicates heterogeneity of the pathogenic process, while the uneven occurrence of the lesion within diabetic pancreata supports the view that the process of islet damage does not take place in all the islets concomitantly. The intermittent pattern of insulitis and the differential recruitment of islets into the pathological process despite the continuous presence of β-cell autoreactive immune cells in circulation suggest the islet pathological process may not be solely dependent on the presence of these cells. Changes in islet tissue-specific structural characteristics and in the local microenvironment may take place in the course of islet inflammation, which predisposes for islet invasion by the immune cells. Local tissue extracellular matrix (ECM) constituents are active participants in the regulation of in situ inflammatory processes during which the functional and structural properties of the local tissue components and of the immune cells themselves are continuously modulated. Recent studies in human diabetic pancreata have indicated the presence of greatly altered hyaluronan (HA), a major ECM component, in the diabetic islets, which is associated with the extent of invasive insulitis and β-cell loss. These novel observations led to the hypothesis that HA guides immune cell migration into the islets and regulates the immune cell phenotype and that alterations in islet HA contribute to the increased vulnerability of the β-cells to inflammatory insult. This …