Islet Inflammation Research Articles

Huayuwendan decoction ameliorates inflammation via IL-17/NF-κB signaling pathway in diabetic rats

Ethnopharmacological relevanceHuayuwendan decoction (HYWD) is a broad used traditional Chinese medicine and therapeutic effects against type 2 diabetes mellitus (T2DM). The mechanism of HYWD on the treatment of T2DM is still unclear. Aim of the studyFor this reason, this study was performed to uncover the effects and mechanism of action of HYWD on T2DM. Materials and methodsMale Wistar rats were chosen to set up the T2DM model. This study was randomly divided into six groups: CON (control), MOD (model), HYWDL (Huayuwendan decoction Low Dose), HYWDM (Huayuwendan decoction Middle Dose), HYWDH (Huayuwendan decoction High Dose), and MET (Metformin). Body weight gains were estimated. Using H&E staining, pathological alterations was explored. The serums of fasting plasma glucose (FPG), 2-h postprandial plasma glucose (2 h PG) were detected by Roche blood glucose meter. LDL-C and HDL-C were analyzed by automatic biochemical analyzer. Network pharmacology analyzed the active ingredients, drug targets, and key pathways of HYWD in T2DM treatment. The islet function and inflammation related factors were determined by ELISA. NF-κB signaling pathway or IL-17 signaling pathway related proteins were analyzed by Western blotting. IL-17RA were determined by immunohistochemistry analyze. ResultsHYWD inhibited weight gain in T2DM rats. Histopathological results showed that HYWD inhibits liver injury. HYWD suppressed LDL-C and enhanced HDL-C in serum of T2DM rats. HYWD reduce FPG and 2 h PG, inhibit Fins, GSP and IRI, but enhance IAI in serum of T2DM rats. In addition, the network pharmacology results identified 292 chemical compounds in HYWD. 279 candidate targets were recognized, including IL-17A, IL-1β, NFкB, stats, mmp3, and cxcl2. The pathways revealed that the possible target of HYWD related with the regulation of IL-17 signaling pathway and NF-κB pathway. Then in vivo study, HYWD reduced the levels of IL-6, TNF-α, IL-17 and IL-1β in serum and inhibit the protein expression involved in IL-17/NF-κB signaling pathway. ConclusionsThe study demonstrates that HYWD may improve T2DM by repressing with the IL-17/NF-κB signaling pathway, which offer encouraging support for using alternative medicine of type 2 diabetes mellitus.

Aptabinding of tumor necrosis factor-α (TNFα) inhibits its proinflammatory effects and alleviates islet inflammation.

Pancreatic islet cell transplantation (ICT) has emerged as an effective therapy for diabetic patients lacking endogenous insulin production. However, the islet graft function is compromised by a nonspecific inflammatory and thrombotic reaction known as the instant blood-meditated inflammatory reaction (IBMIR). Here, we report the characterization of four single-stranded DNA aptamers that bind specifically to TNFα - a pivotal cytokine that causes proinflammatory signaling during the IBMIR process - using single molecule binding analysis and functional assays as a means to assess the aptamers' ability to block TNFα activity and inhibiting the downstream proinflammatory gene expression in the islets. Our single-molecule fluorescence analyses of mono- and multivalent aptamers showed that they were able to bind effectively to TNFα with monoApt2 exhibiting the strongest binding (Kd ∼0.02±0.01nM), which is ∼3 orders of magnitude smaller than the Kd of the other aptamers. Furthermore, the in vitro cell viability analysis demonstrated an optimal and safe dosage of 100μM for monoApt2 compared to 50μM for monoApt1 and significant protection from proinflammatory cytokine-mediated cell death. More interestingly, monoApt2 reversed the upregulation of IBMIR mediating genes induced by TNFα in the human islets, and this was comparable to established TNFα antagonists. Both monoaptamers showed high specificity and selectivity for TNFα. Collectively, these findings suggest the potential use of aptamers as anti-inflammatory and localized immune-modulating agents for cellular transplant therapy.

SAT071 Inhibition Of 12 Lipoxygenase In A Humanized Mouse Model Of Type 1 Diabetes Delays Progression Of Hyperglycemia

Abstract Disclosure: T. Nargis: None. A. Chakraborty: None. K. Figatner: None. D. Maloney: None. M. Boxer: None. S.A. Tersey: None. R.G. Mirmira: None. Type 1 diabetes (T1D) is an autoimmune disorder characterized by islet inflammation (insulitis). We have recently shown that a key mediator of inflammatory signaling in the islet beta cell is the enzyme 12-lipoxygenase (12-LOX), which generates proinflammatory eicosanoids that augment dysfunctional insulin secretion and beta-cell visibility to the immune system. Whereas inhibition of 12-LOX offers an opportunity to modify T1D progression by altering how the beta cell responds to insulitis, a barrier to testing inhibitors in preclinical models is the specificity of next generation inhibitors toward the human enzyme. To generate a preclinical platform for the study of human-specific 12-LOX inhibitors, we developed a mouse model in which the mouse-equivalent Alox15 coding region was replaced with the human ALOX12 gene, while retaining the mouse upstream control region. These mice (hALOX12) were developed on both the C57BL/6J and NOD genetic backgrounds, then subjected to treatment with the human 12-LOX inhibitor VLX-1005 to evaluate diabetes progression. C57BL/6J-wildtype and C57BL/6J-hALOX12 mice were confirmed to express exclusively mouse Alox15 and human ALOX12, respectively, using RNA isolated from islets. To test susceptibility to toxin-induced diabetes, C57BL/6J-wildtype and C57BL/6J-hALOX12 were subjected to multiple low-dose streptozotocin (STZ) to induce diabetes. Both wildtype and humanized mice exhibited gradual and equivalent hyperglycemia (within 25 days) following STZ injection. Following intraperitoneal VLX-1005 therapy (30 mg/kg), whereas C57BL/6J-wildtype mice developed diabetes (with a delay of 10 days) following STZ, C57BL/6J-hALOX12 mice showed complete protection against STZ-induced diabetes and exhibited significantly better glucose tolerance compared to vehicle- or VLX-1005-injected wildtype controls and vehicle-injected C57BL/6J-hALOX12 mice. Next, hALOX12 mice were backcrossed onto the NOD.ShiltJ background model of autoimmune T1D. NOD-hALOX12 mice received either vehicle or VLX-1005 (30 mg/kg) orally during the prediabetic phase (6-10 weeks of age) and were followed for diabetes incidence. Mice receiving VLX-1005 showed a significant delay with only 30% of female and 7% of male mice developing diabetes over 25 weeks of age. Pancreatic sections from the mice treated with VLX-1005 showed reduced insulitis and increased beta cell mass compared to vehicle controls. In summary, the hALOX12 mouse model serves as a preclinical translational platform to interrogate effects of next generation 12-LOX inhibitors and demonstrates the potential of VLX-1005 to modify T1D disease progression. Presentation: Saturday, June 17, 2023

SAT066 Leukotriene B4 Receptor 2 Mediates Macrophage Migration To Injured β Cells

Abstract Disclosure: E. Ermis: None. T. Nargis: None. A. Kulkarni: None. S. May: None. S.A. Tersey: None. R.G. Mirmira: None. R.M. Anderson: None. Type 1 diabetes (T1D) is an autoimmune disease, characterized in part by islet inflammation and macrophage infiltration leading to β-cell injury and loss. The inflammatory mediator 12/15-lipoxygenase (12/15-LOX) and its lipid product 12-hydroxyeicosatetraenoic acid (12-HETE) have both been implicated in T1D. 12-HETE activates the low affinity receptor leukotriene B4 receptor 2 (LTB4R2, or simply BLT2), which was reported as a low-affinity receptor for 12-HETE, suggesting that BLT2 may play a proinflammatory role. Here, we used both mouse and zebrafish models to probe the roles of BLT2 in macrophage activation and/or recruitment to pancreatic islets during T1D progression. First, we analyzed Blt2 gene expression in a variety of C57BL/6J mouse tissues. We found Blt2 gene expression in bone marrow derived macrophages and peritoneal macrophages, however we found little to no expression in pancreatic islets. Although Blt2-/- mice showed a significantly reduced expression of Blt2, they exhibited overall normal growth and glucose tolerance. To uncover roles for BLT2 in macrophage function, we first interrogated its involvement in the in vitro polarization of proinflammatory macrophages to the M1-like state using LPS and IFN-γ and the M2-like state using IL-4. Under these conditions, we found no differences between Blt2-/- and wildtype macrophages, as assessed by flow cytometry analysis, suggesting that BLT2 does not play a substantial role in macrophage polarization. Next, to query roles for BLT2 in macrophage migration, we employed the transgenic zebrafish line Tg(mpeg:EGFP) in which macrophages are labeled with green fluorescent protein (GFP). Following zebrafish tailfin injury, we quantified macrophage accumulation at the injury at 6 hours post amputation. Inhibitory treatments of zebrafish larvae with either a chemical BLT2 inhibitor (LY255283) or by knockdown of the blt2a gene using an antisense morpholino, both decreased the number of GFP+ macrophages found at the injury site. Moreover, in the latter approach ectopic expression of blt2a mRNA effectively restored the wild-type phenotype. To determine if BLT2 mediates macrophage migration to injured islets, we employed a transgenic zebrafish model of β-cell injury, Tg(ins:NTR), in which metronidazole treatment rapidly induces β-cell injury. We treated the Tg(mpeg: GFP); Tg(ins: NTR) zebrafish with metronidazole and monitored macrophage migration into injured islets. We observed a significant decrease in macrophage migration towards injured β-cells in blt2a-knockdown zebrafish. Collectively, these results show that BLT2 plays a role in macrophage migration associated with tissue injury, including the islet β cell as seen in T1D. Presentation: Saturday, June 17, 2023

SAT070 The G Protein Coupled Receptor GPR31 Promotes Islet Inflammation And Diabetes

Abstract Disclosure: C. Checkcinco: None. S.A. Tersey: None. R.G. Mirmira: None. In type 1 diabetes a characteristic immune response targets insulin secreting beta cells in pancreatic islets, resulting in their destruction. This pathogenesis features inflammatory signaling cascades in both beta cells and infiltrating immune cell macrophages. 12-Lipoxygenase (12-LOX) contributes to inflammation in β cells and macrophages by producing the eicosanoid 12-HETE, which induces oxidative and ER stress resulting in cellular dysfunction. G-protein coupled receptor 31 (GPR31) was identified as a high affinity 12-HETE receptor and is increased in human islets after cytokine induced inflammation, but it remains unknown if GPR31 might mediate the downstream effects of 12-LOX. We found increased expression of GPR31 in C57Bl/6J islets, over 7 times higher relative to macrophages. To investigate the role of GPR31 during diabetic inflammation, whole body GPR31 knockout mice were generated on the C57Bl/6J background and studied for glucose homeostasis in the setting of diabetic inflammation. Islets and macrophages from these mice were studied following manipulations in vitro. Compared to wildtype (WT) littermates, Gpr31b-/- mice maintained similar body weight, beta-cell mass, and glucose homeostasis as mice aged. To mimic the setting of islet inflammation as seen in type 1 diabetes, WT and Gpr31b-/- mice were treated with the pro-diabetic toxin streptozotocin (which induces beta cell death and macrophage influx). Compared to WT, Gpr31b-/- mice showed protection from development of hyperglycemia and glucose tolerance tests showed >30% protection reduction in area-under-the-curve, suggesting that GPR31 promotes islet dysfunction and hyperglycemia. To interrogate the role of GPR31 in the beta cell, we isolated islets from WT and Gpr31b-/- mice and treated them with proinflammatory cytokines followed by RNA sequencing to interrogate gene expression pathways. Differentially expressed genes in Gpr31b-/- islets included those pertaining to oxidative stress, MAPK signaling, and cellular homeostasis by Wnt/β-catenin, a finding reminiscent of pathways that are also altered following 12-LOX inhibition. To assess how GPR31 might impact macrophage function, we induced proinflammatory M1 polarization by lipopolysaccharide and interferon gamma in bone marrow-derived macrophages. We saw no significant difference in the extent of M1 polarization between GPR31b-/- and WT macrophages by flow cytometry, as assessed by the production of iNOS. We then asked if GPR31 was involved in the mobility of macrophages using a transwell assay with chemoattractant. We saw a ∼50% reduction in migrating bone marrow-derived macrophages in GPR31b-/- compared to WT. Taken together, these data are consistent with our previously published data on the 12-LOX knockout mice, suggesting that GPR31 may be involved in mediating the proinflammatory responses of 12-HETE in both β cells and macrophages. Presentation: Saturday, June 17, 2023

Deucravacitinib, a tyrosine kinase 2 pseudokinase inhibitor, protects human EndoC-βH1 β-cells against proinflammatory insults.

Type 1 diabetes is characterized by pancreatic islet inflammation and autoimmune-driven pancreatic β-cell destruction. Interferon-α (IFNα) is a key player in early human type 1 diabetes pathogenesis. IFNα activates the tyrosine kinase 2 (TYK2)-signal transducer and activator of transcription (STAT) pathway, leading to inflammation, HLA class I overexpression, endoplasmic reticulum (ER) stress, and β-cell apoptosis (in synergy with IL-1β). As TYK2 inhibition has raised as a potential therapeutic target for the prevention or treatment of type 1 diabetes, we investigated whether the selective TYK2 inhibitor deucravacitinib could protect β-cells from the effects of IFNα and other proinflammatory cytokines (i.e., IFNγ and IL-1β). All experiments were performed in the human EndoC-βH1 β-cell line. HLA class I expression, inflammation, and ER stress were evaluated by real-time PCR, immunoblotting, and/or immunofluorescence. Apoptosis was assessed by the DNA-binding dyes Hoechst 33342 and propidium iodide or caspase 3/7 activity. The promoter activity was assessed by luciferase assay. Deucravacitinib prevented IFNα effects, such as STAT1 and STAT2 activation and MHC class I hyperexpression, in a dose-dependent manner without affecting β-cell survival and function. A comparison between deucravacitinib and two Janus kinase inhibitors, ruxolitinib and baricitinib, showed that deucravacitinib blocked IFNα- but not IFNγ-induced signaling pathway. Deucravacitinib protected β-cells from the effects of two different combinations of cytokines: IFNα + IL-1β and IFNγ + IL-1β. Moreover, this TYK2 inhibitor could partially reduce apoptosis and inflammation in cells pre-treated with IFNα + IL-1β or IFNγ + IL-1β. Our findings suggest that, by protecting β-cells against the deleterious effects of proinflammatory cytokines without affecting β-cell function and survival, deucravacitinib could be repurposed for the prevention or treatment of early type 1 diabetes.

Sodium Selenite Modulates Global Activation of Proinflammatory M1-like Macrophages, Necroinflammation and M1-like/M2-like Dichotomy at the Onset of Human Type 1 Diabetes.

The study aims to show that sodium selenite (Ss) would have an immunomodulatory effect on the functional activity of proinflammatory macrophages (Mφs) during their extended extracellular activation at the onset of human type 1 diabetes (T1D). Exacerbated activation of proinflammatory "M1" macrophages (Mφs) can promote chronic local pancreatic islet inflammation and T1D development. We investigated the ex vivo effects of Ss on the immune modulation of global/extended activation of human proinflammatory M1-like Mφs. Experiments were carried out on primary monocytes-derived Mφs (MDMs). The levels of IL-1β, TNF-α, H2O2 and intracellular free calcium ions (ifCa2+), and the ratios of IL-1β-to-IL-10 and TNF-α-to-IL-10 were markedly increased in T1D Mφs than in healthy control Mφs. Conversely, both IL-10 production and arginase 1 (ARG1) activity were downregulated in T1D Mφs. Additionally, Ss treatment induced a marked downregulation of respiratory burst, ifCa2+ levels, M1-like Mφ-associated inducible nitric oxide (NO) synthase (iNOS) activity, cell necrosis and related necroinflammation biomarkers, including IL-1β and TNF-α, CD14 expression, and the ratios of iNOS-to-ARG1, IL-1β-to-IL-10, and TNF-α-to-IL-10. Moreover, Ss upregulated anti-inflammatory "M2-like" Mφ activity as demonstrated by ARG1 activity and IL-10 production, as well as phagocytosis capacity. Ss exerts a potent immunomodulatory role on functional activities of human proinflammatory T1D M1-like Mφs subjected to extended activation, as well as on the M1-like/M2-like dichotomy. Additionally, the current study provides a novel therapeutic approach using Ss to promote the anti-inflammatory function of Mφs at the onset of T1D.

1646-P: The Effect of Glucagon-Like Peptide-1 Receptor Blockade and Acute Insulin Resistance on Islet Function and Glucose Metabolism in People without Diabetes

Intra-islet Glucagon-Like Peptide-1 (GLP-1) is increased by islet inflammation and diabetes. GLP-1 Receptor (GLP1R) blockade with exendin-9,39 impairs fasting islet function in people with and without type 2 diabetes (DM2). In addition, it decreases insulin secretion and glucagon suppression in response to hyperglycemia in people with DM2. We sought to examine if free fatty acid (FFA) elevation to induce acute insulin resistance in people without DM2 replicated the effects of GLP1R blockade seen in DM2. We studied 6 nondiabetic individuals (54 ± 4 years, 33 ± 1 kg/m2) on 4 occasions in random order. Glucose metabolism was measured after an overnight fast and during a hyperglycemic clamp (~9 mmol/l) using [3-3H] glucose. On two occasions Intralipid® and heparin were infused to raise FFA. Exendin-9,39 (300pmol/kg/min) was infused to block GLP1R in the presence or absence of FFA elevation, while on the other days saline was infused. In the absence of FFA elevation, exendin-9,39 infusion increased fasting glucose concentrations (5.0 ± 0.1 vs. 5.4 ± 0.1 mmol/l, saline vs. exendin-9,39 respectively, p = 0.01). This pattern was also observed when FFA were elevated (5.3 ± 0.1 vs. 5.7 ± 0.2 mmol/l, p = 0.02). Fasting insulin, C-peptide (without FFA: 0.9 ± 0.1 vs. 0.8 ± 0.1 nmol/l, p = 0.17; with FFA: 1.1 ± 0.1 vs. 1.0 ± 0.1 nmol/l, p = 0.08) and glucagon concentrations were unchanged, but inappropriate for the higher fasting glucose observed during exendin-9,39 infusion. Exendin-9,39 infusion did not alter islet hormone secretion during the clamp in the presence or absence of FFA elevation. The magnitude of changes induced by exendin-9,39 did not differ in the presence or absence of FFA elevation (p > 0.10). These experiments confirm the effects of GLP1R blockade on fasting α- and β-cell function in people without DM2. However, FFA elevation does not replicate the abnormalities of islet function observed in people with DM2 when exendin-9,39 is infused. Disclosure M. Zeini: None. R.A. Farahani: None. A.A. Welch: None. M.C. Laurenti: None. C. Cobelli: None. C. Dalla Man: Research Support; Sanofi-Aventis Deutschland GmbH, Becton, Dickinson and Company. A.M. Egan: None. A. Vella: Other Relationship; Novo Nordisk. Advisory Panel; Rezolute, Inc. Consultant; Crinetics Pharmaceuticals, Inc., Hanmi Pharm. Co., Ltd., Zealand Pharma A/S. Funding National Institutes of Health (DK126206)

252-LB: Dectin-2 Deficiency Promotes Proinflammatory Cytokine Release from Macrophages and Impairs Glucose-Stimulated Insulin Secretion

Pancreatic islet inflammation plays an important role in the pathogenesis of diabetes mellitus. Islet macrophages, which have emerged as a key player in islet inflammation, express Dectin-2 (D2), one of the C-type lectin receptors recognizing α-mannans, while endocrine cells such as α-cells and β-cells lack the expression of D2. To elucidate the role of D2 in glucose metabolism and β-cell function, we analyzed the phenotype of D2 knockout (D2KO) mice. D2KO mice exhibited significantly impaired glucose tolerance compared to wild-type (WT) mice, although there were no significant differences in insulin sensitivity. Glucose-stimulated insulin secretion (GSIS) was significantly reduced in isolated islets from D2KO mice compared to WT mice. Comprehensive analysis of RNA-seq in isolated islets from D2KO and WT mice revealed that D2 deficiency induced immune cell infiltration and inflammation in pancreatic islets. Furthermore, RNA-seq analysis in peritoneal macrophages from D2KO and WT mice showed that macrophages from D2KO mice acquired an inflammatory phenotype and exhibited enhanced NF-κB signaling compared to those from WT mice. Next, to determine whether humoral factors secreted from macrophages affect GSIS, we examined GSIS using the culture supernatants of macrophages from D2KO and WT mice. The culture supernatants of macrophages from D2KO mice suppressed GSIS compared to WT mice. Furthermore, the multiplex cytokine assay revealed that IL-1α and IL-6 were significantly increased in the culture supernatants from macrophages of D2KO mice. Finally, we found that the stimulation of IL-1α and IL-6 reduced GSIS in β-cells. These findings suggest that macrophages from D2KO mice suppress GSIS by increasing IL-1α and IL-6 production. This study provides new insights into the mechanism by which D2 is involved in the pathogenesis of pancreatic islet inflammation. Disclosure M. Fujita: None. T. Miyazawa: None. K. Uchida: None. N. Uchida: None. T. Hatayama: None. S. Nakamura: None. Y. Takeichi: None. Y. Miyachi: None. Y. Ogawa: None.

1780-P: Assessment of Toll-Like Receptor Expression and Signaling in Islets under Metabolic and Inflammatory Conditions

Pancreatic beta cells undergoing metabolic and inflammatory stress express islet-derived cytokines or “isletokines” which can induce immune cell infiltration and cellular damage leading to loss of islet cell function. We and others have shown that toll-like receptor (TLR) 4 in islets can mediate metabolic and inflammatory response to DAMPs under diabetic conditions and in islet cell transplantation. Here we show that metabolic and inflammatory conditions result in induction and suppression of expression of multiple members of the TLR family in both MIN6 beta-cell line and mouse islets. Among upregulated TLRs, the endosomal nucleic acid-sensing TLR3 was most highly induced (up to 5 fold) in response to cytokine cocktail IL-1beta, TNF-alpha, and IFN-gamma in the presence of high glucose. Induction of TLR3 expression was blocked by PI3 kinase inhibitor wortmannin and unaffected by mTOR inhibitor rampamycin, calcineurin inhibitor FK506, and NF-κB inhibitor BMS345541. Immunoblot analyses confirmed that TLR3 but not TLR4 protein expression was wortmannin sensitive. These data indicate that TLR3 is upregulated by PI3 kinase signaling in islet beta cells in response to inflammatory conditions. As the endosomal TLR3 signaling is known to sense nucleic acids as well as influence glucose homeostasis, we propose that beta cells may sense DNA damage to induce isletokines to mediate local inflammatory responses during metabolic and inflammatory stress. Further investigations are warranted to decipher signaling pathways and molecular targets of TLR3 signaling to alleviate islet inflammation and improve beta-cell function. Disclosure J.D.Mattke: None. B.Naziruddin: None. M.C.Lawrence: None.

299-OR: A Combinatorial Approach with Monoclonal Antibodies to SerpinB13 and CD3 Is Therapeutically Superior to Single Antibody Use in Autoimmune Diabetes

A successful approach to autoimmune diabetes therapy may require simultaneous insulin-producing cell regeneration and T-cell mediated autoimmunity resolution in pancreatic islets. While CD3 antibody has been proved to reduce islet inflammation, the monoclonal antibody (mAb) to serpinB13 protease inhibitor, developed in our laboratory, was shown to cause regenerative changes in beta cells. Whether a combination of both antibodies is superior to single antibody use in the treatment of autoimmune diabetes is unclear. To address this non-obese diabetic (NOD) female mice at late pre-diabetic stage were injected with serpinB13 mAb (clone B29, 10, 40 or 200 micrograms/animal) and CD3 mAb (clone 145-2C11; at a suboptimal dose of 10 micrograms). All experimental injections were controlled with nonspecific hamster IgG and mouse IgG (clone HB-158). After the last antibody injection, random blood glucose levels were assessed for 20 weeks, and mice were declared diabetic after two measurements of glucose exceeding 300 mg/dL. We found that the combination of 40 micrograms of serpinB13 mAb with CD3 mAb offered a significant level of protection from diabetes (p=0.0027, Kaplan-Meyer with log rank test), while the combination with 10 micrograms of serpinB13 mAb had no effect. The higher dose of 200 micrograms of serpinB13 mAb showed a trend in favor of diabetes inhibition. Interestingly, at 6 to 7 weeks after completing the treatment with 40 micrograms of serpinB13 mAb alone, NOD mice showed a strong tendency to remain healthy, and separated from the double-negative control, which continued to progress to diabetes. Our results indicate that a combinatorial approach with mAbs to serpinB13 and CD3 is beneficial in autoimmune diabetes. The observations also suggest that an intermediate dose of serpinB13 mAb, and a sufficient time following administration of this mAb, is critical for its positive impact on diabetes prevention. Disclosure B.Szepietowska: None. S.V.Avdulov: None. Y.Kryvalap: None. J.Czyzyk: None. Funding JDRF (2-SRA-2022-1215-S-B)

Tracking Ca2+ Dynamics in NOD Mouse Islets During Spontaneous Diabetes Development.

In NOD mice β-cells, 8 mmol/L glucose-induced synchronized short [Ca2+]c events diminish in the early phases of islet inflammation, and long Ca2+ events became more sensitive to substimulatory 4 and 6 mmol/L glucose. In the late islet destruction phase, the synchronized short [Ca2+]c events in a subset of β-cells resumed at 8 mmol/L glucose, while the long Ca2+ events were significantly elevated at substimulatory glucose concentrations. In the α-cells, the glucose sensitivity of the [Ca2+]c events persisted throughout the course of type 1 diabetes development. α-Cell [Ca2+]c events occasionally synchronize in the islets with severe β-cell destruction.

Bi-glandular and persistent enterovirus infection and distinct changes of the pancreas in slowly progressive type 1 diabetes mellitus

In slowly progressive type 1 diabetes mellitus (SPIDDM), the pancreas shows sustained islet inflammation, pancreatitis, pancreatic acinar cell metaplasia/dysplasia (ADM), and intraepithelial neoplasia (PanIN), a precancerous lesion. The mechanisms underlying these changes remain unclear. The presence of enterovirus (EV) encoded-capsid protein 1 (VP1) and -2A protease (2Apro) and the innate immune responses of the pancreas were studied using immunohistochemistry and in situ hybridization in 12 SPIDDM and 19 non-diabetic control pancreases. VP1, 2Apro, and EV-RNA were detected in islets and the exocrine pancreas in all SPIDDM pancreases. Innate immune receptor, melanoma differentiation-associated gene 5 (MDA5), and interferon (IFN)-beta1 were intensified in the islets of SPIDDM patients with short disease duration. However, expressions of MDA5 and IFN-beta1were suppressed in those with longer disease duration. CD3+ T cell infiltration was observed in the VP1- and insulin-positive islets (insulitis) and exocrine acinar cells. CD11c+ dendritic cells (DCs) in islets were scarce in long-term SPIDDM. This study showed the consistent presence of EV, suggesting an association with inflammatory changes in the endocrine and exocrine pancreas in SPIDDM. Suppressed expressions of MDA5 and IFN-beta1, as well as decreased numbers of DCs in the host cells, may contribute to persistent EV infection and induction of ADM/PanIN lesions, which may potentially provide a scaffold for pancreatic neoplasms.

Extracellular matrix inclusion in immunoisolating alginate-based microcapsules promotes longevity, reduces fibrosis, and supports function of islet allografts in vivo.

Immunoisolation of pancreatic-islets in alginate-microcapsules is applied to treat diabetes. However, long-term islet function is limited, which might be due to damaged and lack of contact with pancreatic extracellular matrix (ECM) components. Herein we investigated the impact of collagen IV combined with laminin sequences, either RGD, LRE, or PDSGR, on graft-survival of microencapsulated bioluminescent islets in vivo. Collagen IV with RGD had the most pronounced effect. It enhanced after 8-week implantation in immune-incompetent mice the bioluminescence of allogeneic islets by 3.2-fold, oxygen consumption rate by 14.3-fold and glucose-induced insulin release by 9.6-fold. Transcriptomics demonstrated that ECM enhanced canonical pathways involving insulin-secretion and that it suppressed pathways related to inflammation and hypoxic stress. Also, 5.8-fold fewer capsules were affected by fibrosis. In a subsequent longevity study in immune-competent mice, microencapsulated allografts containing collagen IV and RGD had a 2.4-fold higher functionality in the first week after implantation and remained at least 2.1-fold higher during the study. Islets in microcapsules containing collagen IV and RGD survived 211 ± 24.1 days while controls survived 125 ± 19.7 days. Our findings provide in vivo evidence for the efficacy of supplementing immunoisolating devices with specific ECM components to enhance functionality and longevity of islet-grafts in vivo. STATEMENT OF SIGNIFICANCE: Limitations in duration of survival of immunoisolated pancreatic islet grafts is a major obstacle for application of the technology to treat diabetes. Accumulating evidence supports that incorporation of extracellular matrix (ECM) molecules in the capsules enhances longevity of pancreatic islets. After selection of the most efficacious laminin sequence in vitro, we show in vivo that inclusion of collagen IV and RGD in alginate-based microcapsules enhances survival, insulin secretion function, and mitochondrial function. It also suppresses fibrosis by lowering proinflammatory cytokines secretion. Moreover, transcriptomic analysis shows that ECM-inclusion promotes insulin-secretion related pathways and attenuates inflammation and hypoxic stress related pathways in islets. We show that inclusion of ECM in immunoisolating devices is a promising strategy to promote long-term survival of islet-grafts.

Long-term exercise preserves pancreatic islet structure and β-cell mass through attenuation of islet inflammation and fibrosis.

Islet fibrosis is associated with the disruption of islet structure and contributes to β-cell dysfunction, playing an essential role in the pathogenesis of type 2 diabetes. Physical exercise has been shown to attenuate fibrosis in various organs; however, the effect of exercise on islet fibrosis has not been defined. Male Sprague-Dawley rats were divided into four groups: normal diet sedentary [N-Sed], normal diet + exercise [N-Ex], high-fat diet sedentary [H-Sed], and high-fat diet + exercise [H-Ex]. After 60 weeks of exercise, 4452 islets from Masson-stained slides were analyzed. Exercise led to a 68% and 45% reduction in islet fibrosis in the normal and high-fat diet groups and was correlated with a lower serum blood glucose. Fibrotic islets were characterized by irregular shapes and substantial loss of β-cell mass, which were significantly reduced in the exercise groups. Remarkably, the islets from exercised rats at week 60 were morphologically comparable to those of sedentary rats at 26 weeks. In addition, the protein and RNA levels of collagen and fibronectin, and the protein levels of hydroxyproline in the islets were also attenuated by exercise. This was accompanied by a significant reduction in inflammatory markers in the circulation Interleukin-1 beta (IL-1β)] and pancreas [IL-1β, Tumor Necrosis Factor-alpha, Transforming Growth Factor-β, and Phosphorylated Nuclear Factor Kappa-B p65subunit], lower macrophage infiltration, and stellate cell activation in the islets of exercised rats. In conclusion, we have demonstrated that long-term exercise preserves pancreatic islet structure and β-cell mass through anti-inflammatory and anti-fibrotic actions, suggesting additional rationales for the success of exercise training in the prevention and treatment of type 2 diabetes that should be further explored.

Extracellular Vesicles Derived from Three-Dimensional-Cultured Human Umbilical Cord Blood Mesenchymal Stem Cells Prevent Inflammation and Dedifferentiation in Pancreatic Islets.

It is unclear whether extracellular vesicles (EVs) from mesenchymal stem cells (MSCs) have a direct protective effect on pancreatic islets. In addition, whether culturing MSCs in three dimensions (3D) instead of a monolayer (2D) can induce changes in the cargo of EVs that facilitate the polarization of macrophages into an M2 phenotype has not been investigated. We sought to determine whether EVs from MSCs cultured in 3D can prevent inflammation and dedifferentiation in pancreatic islets and, if so, whether the protective effect is superior to that of EVs from 2D MSCs. Human umbilical cord blood- (hUCB-) MSCs cultured in 3D were optimized according to cell density, exposure to hypoxia, and cytokine treatment based on the ability of the hUCB-MSC-derived EVs to induce the M2 polarization of macrophages. Islets isolated from human islet amyloid polypeptide (hIAPP) heterozygote transgenic mice were cultured in serum-deprived conditions with hUCB-MSC-derived EVs. EVs derived from 3D hUCB-MSCs had more abundant microRNAs involved in M2 polarization of macrophages and had an enhanced M2 polarization ability on macrophages, which was optimized when the 3D culture condition was 2.5 × 104 cells per spheroid without preconditioning with hypoxia and cytokine exposure. When islets isolated from hIAPP heterozygote transgenic mice were cultured in serum-deprived conditions with hUCB-MSC-derived EVs, the EVs derived from 3D hUCB-MSCs suppressed the expression of proinflammatory cytokines and caspase-1 in pancreatic islets and increased the proportion of M2-polarized islet-resident macrophages. They improved glucose-stimulated insulin secretion, reduced the expression of Oct4 and NGN3, and induced the expression of Pdx1 and FoxO1. The greater suppression of IL-1β, NLRP3 inflammasome, caspase-1, and Oct4 and induction of Pdx1 and FoxO1 were found in islets cultured with the EVs derived from 3D hUCB-MSCs. In conclusion, EVs derived from 3D hUCB-MSCs optimized for M2 polarization attenuated nonspecific inflammation and preserved β-cell identity of pancreatic islets.

Therapeutic efficacy of liraglutide versus metformin in modulating the gut microbiota for treating type 2 diabetes mellitus complicated with nonalcoholic fatty liver disease.

Metformin and liraglutide are used in the treatment of type 2 diabetes mellitus (T2DM) complicated with nonalcoholic fatty liver disease (NAFLD). Although these drugs can alter the intestinal microbiome, clinical data are required to explore their mechanisms of action. Using 16S sequencing technology, we analyzed and compared the intestinal bacterial community structure and function between patients before and after treatment (12 weeks) with the two drugs (metformin or liraglutide, n = 15) and healthy controls (n = 15). Moreover, combined with 19 clinical indices, the potential therapeutic mechanisms of the two drugs were compared. The studied clinical indices included those associated with islet β-cell function (FPG, FINS, HbA1c, and HOMA-IR), inflammation (TNF-α, IL-6, and APN), lipid metabolism (TC, TG, and LDL-C), and liver function (ALT, AST, and GGT); the values of all indices changed significantly after treatment (p < 0.01). In addition, the effect of the two drugs on the intestinal bacterial community varied. Liraglutide treatment significantly increased the diversity and richness of the intestinal bacterial community (p < 0.05); it significantly increased the relative abundances of Bacteroidetes, Proteobacteria, and Bacilli, whereas metformin treatment significantly increased the relative abundance of Fusobacteria and Actinobacteria (p < 0.05). Metformin treatment increased the complexity and stability of the intestinal bacterial network. However, liraglutide treatment had a weaker effect on the intestinal bacterial network, and the network after treatment was similar to that in healthy controls. Correlation matrix analysis between dominant genera and clinical indicators showed that the correlation between the bacterial community and islet β-cell function was stronger after liraglutide treatment, whereas the correlation between the bacterial community and inflammation-related factors was stronger after metformin treatment. Functional prediction showed that liraglutide could significantly affect the abundance of functional genes related to T2DM and NAFLD (p < 0.05), but the effect of metformin was not significant. This study is the first to report the changes in the intestinal bacterial community in patients treated with metformin or liraglutide and the differences between the mechanisms of action of metformin and liraglutide. Metformin or liraglutide has a therapeutic value in T2DM complicated with NAFLD, with liraglutide having a weaker effect on the intestinal bacterial community but a better therapeutic efficacy.

A small molecule improves diabetes in mice expressing human islet amyloid polypeptide

ABSTRACT In recent years, the number of studies on islet and beta cell autophagy have substantially increased due to growing interest in the role of autophagy in maintaining cellular homeostasis in diabetes. In type 2 diabetes, human islet amyloid polypeptide (hIAPP) aggregates to form higher structure oligomers and fibrils that are toxic to beta cells and induce islet inflammation. The primary mechanism of oligomer and fibril clearance in beta cells is through the autophagic pathway, a process that is impaired in type 2 diabetes. Thus, toxic oligomeric and fibrillar forms of hIAPP accumulate in type 2 diabetic islets. Recently, Kim et al. characterized the ability of a small molecule autophagy enhancer, MSL-7, to clear hIAPP oligomers in mice expressing hIAPP. Herein, we outline the primary findings of the study, limitations, and future directions to further investigate the therapeutic potential of autophagy enhancers to treat diabetes.

High Dietary Iron in Western Diet-Fed Male Rats Causes Pancreatic Islet Injury and Acute Pancreatitis

BackgroundHigh dietary iron has been linked to an increased type 2 diabetes risk. We have previously shown that intrauterine growth restriction (IUGR) and feeding a Western diet (WD) to male Sprague-Dawley rats independently, as well as together, cause pancreatic islet inflammation, fibrosis, and hemosiderosis. ObjectivesTo investigate whether iron has a role in the pathogenesis of this inflammatory islet injury caused by IUGR and WD intake. MethodsMale Sprague-Dawley offspring of bilateral uterine artery ligated (IUGR) and sham-operated (Sham) dams, fostered to nonoperated dams, were fed a WD [45% sucrose, 19.4% protein and 23% fat (w/w)] containing low iron (LI, 20 mg/kg) or high iron (HI, 500 mg/kg) from weaning. Four groups were studied: Sham-LI, Sham-HI, IUGR-LI, and IUGR-HI. Serial measurements of rat body weight, blood glucose, lipids and insulin, an intraperitoneal glucose tolerance test (age 13 wk), and histological analysis of pancreas and liver (age 14 wk) were recorded. The effects of iron, IUGR, and their interaction, on these measurements have been analyzed. ResultsWD with HI compared with LI caused an 11% greater weight gain by age 14 wk (P < 0.001), impaired glucose tolerance [AUC for glucose (G-AUC) 17% higher; P < 0.001), acute pancreatitis (17/18, HI; 6/17, LI; P < 0.001), pancreas-associated fat necrosis and saponification (7/18, HI; 0/17 LI; P < 0.01), and a trend to islet fibrotic injury (7/18, HI; 1/17 LI; P = 0.051). Although pancreatic and hepatic steatosis was evident in almost all WD-fed rats, pancreatic and hepatic iron accumulation was prevalent only in HI-fed rats (P < 0.0001 for both), being only mild in the livers. IUGR, independent of dietary iron, also caused impairment in glucose tolerance (G-AUC: 17% higher; P < 0.05). ConclusionsA postweaning WD containing HI, independent of IUGR, causes acute pancreatitis and islet injury in Sprague-Dawley rats suggesting a role of dietary iron in the development of steatopancreatitis.

Sustained high glucose intake accelerates type 1 diabetes in NOD mice.

Epidemiological studies have suggested that dietary factors, especially high consumption of high glycaemic index carbohydrates and sugars, may trigger or exacerbate the progression of type 1 diabetes. We aimed to provide experimental evidence to confirm this relevance and to explore the underlying mechanisms. NOD mice were given sustained high-glucose drinking or glucose-free water and observed for the incidence of type 1 diabetes and islet inflammation. RNAseq was performed to detect the transcriptome changes of the NOD islet beta cell line NIT-1 after high glucose treatment, and mass spectrometry was performed to detect the proteome changes of NIT-1-cells-derived sEVs. Sustained high glucose drinking significantly aggravates islet inflammation and accelerates the onset of type 1 diabetes in NOD mice. Mechanistically, high glucose treatment induces aberrant ER stress and up-regulates the expression of autoantigens in islet beta cell. Moreover, high glucose treatment alters the proteome of beta-cells-derived sEVs, and significantly enhances the ability of sEVs to promote DC maturation and stimulate immune inflammatory response. This study provides evidence for negative effect of high glucose intake as a dietary factor on the pathogenesis of type 1 diabetes in genetically predisposed individuals. Therefore, avoiding high sugar intake may be an effective disease prevention strategy for children or adults susceptible to type 1 diabetes.