Pancreatic Islet Morphology Research Articles

Morphological alteration of the pancreatic islet in ovariectomized rats fed a high-fat high-fructose diet.

Diabetes and its complications are major causes of mortality worldwide. Type 2 diabetes coexists with insulin resistance and β-cell dysfunction, which are aggravated by overconsumption and estrogen-deprived conditions. However, the morphology of pancreatic islets in a combined condition of excessive caloric intake and estrogen deficiency has never been described. Herein, we examined morphological changes in the pancreatic islets of ovariectomized (OVX) rats fed a high-fat high-fructose diet (HFFD) for 12weeks. The histological changes in the size and number of pancreatic islets were assessed by hematoxylin-eosin and immunohistochemical staining. Enlarged pancreatic islets with fat deposition in OVX rats were accompanied by whole-body insulin resistance and hyperglycemia. The addition of a HFFD to OVX rats (OVX + HFFD) further aggravated insulin resistance, with a substantial increase in the density of enlarged pancreatic islets and fat accumulation. The augmented number of enlarged islets was correlated with elevated plasma glucose and insulin levels. Intriguingly, unlike the HFFD and OVX alone, the OVX + HFFD markedly expanded the area of insulin-producing β-cells and glucagon-producing α-cells. Importantly, enlarged islets, pancreatic fat deposits, and diabetic states developing in OVX + HFFD conditions were resolved by estrogen replacement. Collectively, the morphological characteristics of pancreatic islets were influenced in an insulin-resistant state caused by estrogen deficiency and HFFD consumption and were distinct from each factor alone. A combination of estrogen deficiency with HFFD consumption worsened the integrity of pancreatic islets, ultimately resulting in disease progression. These findings expand our understanding of the causal relationship between pancreatic morphology and diabetes development and suggest therapeutic strategies.

Beneficial impact of Ac3IV, an AVP analogue acting specifically at V1a and V1b receptors, on diabetes islet morphology and transdifferentiation of alpha- and beta-cells.

Ac3IV (Ac-CYIQNCPRG-NH2) is an enzymatically stable vasopressin analogue that selectively activates Avpr1a (V1a) and Avpr1b (V1b) receptors. In the current study we have employed streptozotocin (STZ) diabetic transgenic Ins1Cre/+;Rosa26-eYFP and GluCreERT2;Rosa26-eYFP mice, to evaluate the impact of sustained Ac3IV treatment on pancreatic islet cell morphology and transdifferentiation. Twice-daily administration of Ac3IV (25 nmol/kg bw) to STZ-diabetic Ins1Cre/+;Rosa26-eYFP mice for 12 days increased pancreatic insulin (p<0.01) and significantly reversed the detrimental effects of STZ on pancreatic islet morphology. Such benefits were coupled with increased (p<0.01) beta-cell proliferation and decreased (p<0.05) beta-cell apoptosis. In terms of islet cell lineage tracing, induction of diabetes increased (p<0.001) beta- to alpha-cell differentiation in Ins1Cre/+;Rosa26-eYFP mice, with Ac3IV partially reversing (p<0.05) such transition events. Comparable benefits of Ac3IV on pancreatic islet architecture were observed in STZ-diabetic GluCreERT2;ROSA26-eYFP transgenic mice. In this model, Ac3IV provoked improvements in islet morphology which were linked to increased (p<0.05-p<0.01) transition of alpha- to beta-cells. Ac3IV also increased (p<0.05-p<0.01) CK-19 co-expression with insulin in pancreatic ductal and islet cells. Blood glucose levels were unchanged by Ac3IV in both models, reflecting the severity of diabetes induced. Taken together these data indicate that activation of islet receptors for V1a and V1b positively modulates alpha- and beta-cell turnover and endocrine cell lineage transition events to preserve beta-cell identity and islet architecture.

Effects of Metformin and Sitagliptin on islet cell function in insulin-resistant prediabetic KKay mice

Objective: To investigate the effects of metformin and sitagliptin on the function of islet β cells in insulin resistance pre-diabetic KKay mice. Methods: Thirty 6-week-old KKAy mice were randomly divided into two groups: normal diet fed group (NC group, n=10) and high-fat diet fed group (n=20). At 8 weeks, KKAy mice were randomly divided into two groups: metformin intervention group (met group, n=10) and sitagliptin intervention group (SP group, n=10), which were treated by gavage for 8 weeks. Glucose tolerance was measured by oral glucose tolerance test (OGTT), serum insulin level and plasma lipid level were measured by tail blood sampling, and HOMA-β and HOMA-IR were calculated. The mice were killed after blood collection, and the pancreas of KKAy mice was taken. The β cell volume of each group was compared by immunofluorescence staining of insulin and glucagon, respectively. The proliferation and apoptosis of β cell were analyzed by Ki67/INS. The expressions of pancreatic transcription factors PDX-1 and MafA were detected by Western blot. Results: ① The OGTT results indicated that blood glucose of KKAy mice at fast, 30, 60 and 120 min after oral administration of glucose in the Met and SP groups were decreased significantly compared with the NC group, and the area under the blood glucose time curve (AUC) was significantly reduced (P＜0.01, P＜0.01). There was no significant difference between the Met group and the SP group in blood glucose level at 30 and 60 min after oral administration of glucose. Compared with the SP group, the blood glucose of Met group at 120 min was decreased significantly (P＜0.05). There was no significant difference in AUC between the two groups. ② The results of the insulin tolerance test (ITT) indicated that, compared with NC, the fasting blood glucose and the blood glucose at 30, 60 and 90 min after insulin injection in KKAy mice in the Met and SP groups were decreased significantly, and the area under the ITT blood glucose curve (AUC) was increased significantly (P＜0.01), while there was no significant difference between the Met and SP groups. ③ In the NC group, the brightness of the areas of the islet β cells was low and the edges were scattered. After treated with metformin, the areas and brightness of the β cells were increased. After treatment with sitagliptin, the area and brightness of the β cells were increased significantly. In the NC group, the α cells were disordered in the islet distribution and the brightness was large. After the administration of metformin, the α cell area and the brightness were decreased, and distributed to the edge of the islet to a certain extent. After the administration of sitagliptin, there was a significant decrease in the area of the α cells, with a significant decrease in the brightness and distribution at the edge of the islet. ④ Compared with the NC group, the expression levels of pancreatic MafA in the Met group and SP group were increased significantly, which were 1.63 times and 1.58 times, respectively (P＜0.01, P＜0.01). There was no significant difference in the expression of pancreatic PDX-1 between the groups. Conclusion: In pre-diabetes mellitus KKAy mice with insulin resistance, metformin can maintain the function and morphology of pancreatic islets, and sitagliptin may promote the proliferation of islet βcells, improve the expression level of insulin transcription factor MafA, and prevent the occurrence and development of diabetes.

Chronic binge alcohol and ovariectomy-mediated impaired insulin responsiveness in SIV-infected female rhesus macaques.

Aging people living with HIV (PLWH), especially postmenopausal women may be at higher risk of comorbidities associated with HIV, antiretroviral therapy (ART), hypogonadism, and at-risk alcohol use. Our studies in simian immunodeficiency virus (SIV)-infected male macaques demonstrated that chronic binge alcohol (CBA) reduced acute insulin response to glucose (AIRG), and at-risk alcohol use decreased HOMA-β in PLWH. The objective of this study was to examine the impact of ovariectomy (OVX) on glucose-insulin dynamics and integrity of pancreatic endocrine function in CBA/SIV-infected female macaques. Female macaques were administered CBA (12-15 g/kg/wk) or isovolumetric water (VEH) intragastrically. Three months after initiation of CBA/VEH administration, all macaques were infected with SIVmac251, and initiated on antiretroviral therapy (ART) 2.5 mo postinfection. After 1 mo of ART, macaques were randomized to OVX or sham surgeries (n = 7 or 8/group), and euthanized 8 mo post-OVX (study endpoint). Frequently sampled intravenous glucose tolerance tests (FSIVGTT) were performed at selected time points. Pancreatic gene expression and islet morphology were determined at study endpoint. There was a main effect of CBA to decrease AIRG at Pre-SIV and study endpoint. There were no statistically significant OVX effects on AIRG (P = 0.06). CBA and OVX decreased the expression of pancreatic markers of insulin docking and release. OVX increased endoplasmic stress markers. CBA but not OVX impaired glucose-insulin expression dynamics in SIV-infected female macaques. Both CBA and OVX altered integrity of pancreatic endocrine function. These findings suggest increased vulnerability of PLWH to overt metabolic dysfunction that may be exacerbated by alcohol use and ovarian hormone loss.

High-intensity infrasound effects on glucose metabolism in rats

Recent focus has been given on the effects of high-intensity infrasound (HII) exposure, and whether it induces changes in pancreatic morphology and glucose metabolism is still unknown. As such, we have studied the impact of HII exposure on glucose tolerance, insulin sensitivity, pancreatic islet morphology, muscle GLUT4 and plasma insulin and corticosterone levels. Normal and glucose intolerant wild-type Wistar rats were randomly divided in two groups: one group not exposed to HII and the other continuously exposed to HII. Animals were sacrificed at three timepoints of exposure (1, 6 or 12 weeks). An intraperitoneal glucose tolerance test was performed, blood samples were collected and the pancreas and the quadriceps femoris muscle were excised. Circulating insulin and corticosterone levels were determined and pancreatic and muscular tissue were routinely processed for histochemistry and immunohistochemistry with an anti-GLUT4 antibody. Animals exposed to HII had higher corticosterone levels than animals not exposed. No differences were found on insulin concerning HII exposure or glucose intolerance. Glucose intolerant animals had pancreatic islet fibrosis and no differences were found in GLUT4 ratio concerning HII exposure. In conclusion, we found that continuous exposure to HII increases stress hormone levels without inducing glucose intolerance in rats.

Benefits of Sustained Upregulated Unimolecular GLP-1 and CCK Receptor Signalling in Obesity-Diabetes

Combined activation of GLP-1 and CCK1 receptors has potential to synergistically augment the appetite-suppressive and glucose homeostatic actions of the individual parent peptides. In the current study, pancreatic beta-cell benefits of combined GLP-1 and CCK1 receptor upregulation were established, before characterising bioactivity and antidiabetic efficacy of an acylated dual-acting GLP-1/CCK hybrid peptide, namely [Lys12Pal]Ex-4/CCK. Both exendin-4 and CCK exhibited (p<0.001) proliferative and anti-apoptotic effects in BRIN BD11 beta-cells. Proliferative benefits were significantly (p<0.01) augmented by combined peptide treatment when compared to either parent peptide alone. These effects were linked to increases (p<0.001) in GLUT2 and glucokinase beta-cell gene expression, with decreased (p<0.05-p<0.001) expression of NFκB and BAX. [Lys12Pal]Ex-4/CCK exhibited prominent insulinotropic actions in vitro, coupled with beneficial (p<0.001) satiety and glucose homeostatic effects in the mice, with bioactivity evident 24 h after administration. Following twice daily injection of [Lys12Pal]Ex-4/CCK for 28 days in diabetic high fat fed (HFF) mice with streptozotocin (STZ)-induced compromised beta-cells, there were clear reductions (p<0.05-p<0.001) in energy intake and body weight. Circulating glucose was returned to lean control concentrations, with associated increases (p<0.001) in plasma and pancreatic insulin levels. Glucose tolerance and insulin secretory responsiveness were significantly (p<0.05-p<0.001) improved by hybrid peptide therapy. In keeping with this, evaluation of pancreatic histology revealed restoration of normal islet alpha- to beta-cell ratios and reduction (p<0.01) in centralised islet glucagon staining. Improvements in pancreatic islet morphology were associated with increased (p<0.05) proliferation and reduced (p<0.001) apoptosis of beta-cells. Together, these data highlight the effectiveness of sustained dual GLP-1 and CCK1 receptor activation by [Lys12Pal]Ex-4/CCK for the treatment of obesity-related diabetes.

An Insulin-Inspired Supramolecular Hydrogel for Prevention of Type 1 Diabetes.

Supramolecular peptide hydrogel has shown promising potential in vaccine development largely because of its ability to function both as antigen depot and immune adjuvant. Nap‐GdFdFdY, a tetrapeptide hydrogel that has been previously reported to exhibit adjuvant effect, is inadvertently found to contain conserved peptide sequence for insulin, proinsulin, and glutamic acid decarboxylase, 3 major autoantigens for the autoimmune type 1 diabetes (T1D). At present, despite being managed clinically with insulin replacement therapy, T1D remains a major health threat with rapidly increasing incidences, especially in children and young adults, and antigen‐specific immune tolerance induction has been proposed as a feasible approach to prevent or delay T1D progression at an early stage. Here, it is reported that innoculation of Nap‐GdFdFdY leads to complete protection of nonobese diabetic (NOD) mice from T1D development till the age of 36 weeks. Better maintenance of pancreatic islet morphology with minimal immune cell infiltration is also observed from mice exposed to Nap‐GdFdFdY. This beneficial impact is mainly due to its facilitative role on enhancing peripheral T regulatory cell (Treg) population, shown as increased splenic Treg percentage, and function, demonstrated by maintenance of circulating TGF‐β1 level. Serum cytokine microarray data further implicate a “buffering” role of Nap‐GdFdFdY on systemic inflammatory tone in NOD mice. Thus, with its versatility, applicability, and excellent potency, Nap‐GdFdFdY is posited as a novel therapeutic intervention for T1D.

A Newly Developed Synbiotic Yogurt Prevents Diabetes by Improving the Microbiome-Intestine-Pancreas Axis.

The prevalence of type 2 diabetes mellitus (T2D) is increasing worldwide, and there are no long-term preventive strategies to stop this growth. Emerging research shows that perturbations in the gut microbiome significantly contribute to the development of T2D, while microbiome modulators may be beneficial for T2D prevention. However, microbiome modulators that are effective, safe, affordable, and able to be administered daily are not yet available. Based on our previous pro- and prebiotic studies, we developed a novel synbiotic yogurt comprised of human-origin probiotics and plant-based prebiotics and investigated its impact on diet- and streptozotocin-induced T2D in mice. We compared the effects of our synbiotic yogurt to those of a commercially available yogurt (control yogurt). Interestingly, we found that the feeding of the synbiotic yogurt significantly reduced the development of hyperglycemia (diabetes) in response to high-fat diet feeding and streptozotocin compared to milk-fed controls. Surprisingly, the control yogurt exacerbated diabetes progression. Synbiotic yogurt beneficially modulated the gut microbiota composition compared to milk, while the control yogurt negatively modulated it by significantly increasing the abundance of detrimental bacteria such as Proteobacteria and Enterobacteriaceae. In addition, the synbiotic yogurt protected pancreatic islet morphology compared to the milk control, while the control yogurt demonstrated worse effects on islets. These results suggest that our newly developed synbiotic yogurt protects against diabetes in mice and can be used as a therapeutic to prevent diabetes progression.

Natural Protection From Type 1 Diabetes in NOD Mice Is Characterized by a Unique Pancreatic Islet Phenotype.

The NOD mouse develops spontaneous type 1 diabetes, with some features of disease that are very similar to the human disease. However, a proportion of NOD mice are naturally protected from developing diabetes, and currently, studies characterizing this cohort are very limited. Here, using both immunofluorescence and multiparameter flow cytometry, we focus on the pancreatic islet morphology and immune infiltrate observed in naturally protected NOD mice. We show that naturally protected NOD mice are characterized by an increased frequency of insulin-containing, smaller-sized, pancreatic islets. Although mice remain diabetes free, florid immune infiltrate remains. However, this immune infiltrate is skewed toward a regulatory phenotype in both T- and B-cell compartments. Pancreatic islets have an increased frequency of IL-10-producing B cells and associated cell surface markers. Resident memory CD69+CD8+ T cells show a significant shift toward reduced CD103 expression, while CD4+ T cells have increased FoxP3+CTLA4+ expression. These data indicate that naturally protected NOD mice have a unique islet signature and provide new insight into regulatory mechanisms within pancreatic islets.

Cichorium intybus attenuates Streptozotocin-induced pancreatic β-cell damage by inhibiting NF-κB activation and oxidative stress.

The aqueous extract of Cichorium intybus (CIE) leaves have shown the properties of protecting against pancreatic β-cell damage by streptozotocin (STZ), but the molecular mechanisms of its protection are not completely elucidated yet. Our current study focuses on elucidating the mechanisms of these preventive effects of CIE in MIN6 cells and an in-vivo model of Wistar rats. CIE offers protection against STZ in MIN6 cells by reducing the pro-oxidants and increasing the activity of the antioxidant enzymes. In vitro results also indicated that CIE inhibited cytotoxicity, reduced Reactive oxygen species (ROS), maintained glucose-stimulated insulin secretion and reduced NF-κB p65 translocation into the nucleus. The group administered with a 250 mg/kg dose of CIE in vivo has shown an ability to maintain blood glucose level and also to preserve the number and morphology of pancreatic islets when compared to the diabetic group treated with STZ. Probably, active compounds like quercetin, rutin, and catechin present in CIE, preserve the integrity of pancreatic islets thereby protecting β-cells from the adverse effects of STZ.

Enzymatically stable analogue of the gut-derived peptide xenin on beta-cell transdifferentiation in high fat fed and insulin-deficient Ins1Cre/+ ;Rosa26-eYFP mice.

The antidiabetic effects of the gut hormone xenin include augmenting insulin secretion and positively affecting pancreatic islet architecture. The current study has further probed pancreatic effects through sub-chronic administration of the long-acting xenin analogue, xenin-25[Lys13 PAL], in both high fat fed (HFF) and streptozotocin (STZ)-induced insulin-deficient Ins1Cre/+ ;Rosa26-eYFP transgenic mice. Parallel effects on metabolic control and pancreatic islet morphology, including islet beta-cell lineage tracing were also assessed. Xenin-25[Lys13 PAL] treatment reversed body weight loss induced by STZ, increased plasma insulin and decreased blood glucose levels. There were less obvious effects on these parameters in HFF mice, but all xenin-25[Lys13 PAL] treated mice exhibited decreased pancreatic alpha-cell areas and circulating glucagon. Xenin-25[Lys13 PAL] treatment fully, or partially, returned overall islet and beta-cell areas in STZ- and HFF mice to those of lean control animals, respectively, and was consistently associated with decreased beta-cell apoptosis. Interestingly, xenin-25[Lys13 PAL] also increased beta-cell proliferation and decreased alpha-cell apoptosis in STZ mice, with reduced alpha-cell growth noted in HFF mice. Lineage tracing studies revealed that xenin-25[Lys13 PAL] reduced the number of insulin positive pancreatic islet cells that lost their beta-cell identity, in keeping with a decreased transition of insulin positive to glucagon positive cells. These beneficial effects on islet cell differentiation were linked to maintained expression of Pdx1 within beta-cells. Xenin-25[Lys13 PAL] treatment was also associated with increased numbers of smaller sized islets in both models. Benefits of xenin-25[Lys13 PAL] on diabetes includes positive modulation of islet cell differentiation, in addition to promoting beta-cell growth and survival.

Generation and characterisation of C-terminally stabilised PYY molecules with potential in vivo NPYR2 activity

BackgroundActivation of neuropeptide Y2 receptors (NPYR2) by the N-terminally truncated, dipeptidyl peptidase-4 (DPP-4) generated, Peptide YY (PYY) metabolite, namely PYY(3–36), results in satiating actions. However, PYY(3–36) is also subject to C-terminal enzymatic cleavage, which annuls anorectic effects. MethodsSubstitution of l-Arg35 with d-Arg35 in the DPP-4 stable sea lamprey PYY(1–36) peptide imparts full C-terminal stability. In the current study, we have taken this molecule and introduced DPP-4 susceptibility by Iso3 substitution. ResultsAs expected, [Iso3]sea lamprey PYY(1–36) and [Iso3](d-Arg35)sea lamprey PYY(1–36) were N-terminally degraded to respective PYY(3–36) metabolites in plasma. Only [Iso3](d-Arg35)sea lamprey PYY(1–36) was C-terminally stable. Both peptides possessed similar insulinostatic and anti-apoptotic biological actions to native PYY(1–36) in beta-cells. Unlike native PYY(1–36) and [Iso3](d-Arg35)sea lamprey PYY(1–36), [Iso3]sea lamprey PYY(1–36) displayed some proliferative actions in Npyr1 knockout beta-cells. In addition, [Iso3]sea lamprey PYY(1–36) induced more rapid NPYR2-dependent appetite suppressive effects in mice than its C-terminally stable counterpart. Twice daily administration of either peptide to high fat fed (HFF) mice resulted in significant body weight reduction and improvements in circulating triglyceride levels. [Iso3]sea lamprey PYY(1–36) treatment also prevented elevations in glucagon. Both peptides, and especially [Iso3]sea lamprey PYY(1–36), improved glucose tolerance. The treatment interventions also partially reversed the deleterious effects of sustained high fat feeding on pancreatic islet morphology. ConclusionThe present study confirms that sustained NPYR2 receptor activation by [Iso3](d-Arg35)sea lamprey induced significant weight lowering actions. However, identifiable benefits of this peptide over [Iso3]sea lamprey PYY(1–36), which was not protected against C-terminal degradation, were not pronounced.

Postnatal loss of the insulin receptor in osteoprogenitor cells does not impart a metabolic phenotype

The relationship between osteoblast-specific insulin signaling, osteocalcin activation and gluco-metabolic homeostasis has proven to be complex and potentially inconsistent across animal-model systems and in humans. Moreover, the impact of postnatally acquired, osteoblast-specific insulin deficiency on the pancreas-to-skeleton-to-pancreas circuit has not been studied. To explore this relationship, we created a model of postnatal elimination of insulin signaling in osteoprogenitors. Osteoprogenitor-selective ablation of the insulin receptor was induced after ~10 weeks of age in IRl°x/lox/Osx-Cre+/− genotypic male and female mice (designated postnatal-OIRKO). At ~21 weeks of age, mice were then phenotypically and metabolically characterized. Postnatal-OIRKO mice demonstrated a significant reduction in circulating concentrations of undercarboxylated osteocalcin (ucOC), in both males and females compared with control littermates. However, no differences were observed between postnatal-OIRKO and control mice in: body composition (lean or fat mass); fasting serum insulin; HbA1c; glucose dynamics during glucose tolerance testing; or in pancreatic islet area or islet morphology, demonstrating that while ucOC is impacted by insulin signaling in osteoprogenitors, there appears to be little to no relationship between osteocalcin, or its derivative (ucOC), and glucose homeostasis in this model.

Development and characterisation of a peptidergic N-and C-terminally stabilised mammalian NPY1R agonist which protects against diabetes induction

BackgroundPYY (1–36) peptides from phylogenetically ancient fish, such as sea lamprey, have previously been shown to function as specific neuropeptide Y1 receptor (NPYR1) agonists. Although, sea lamprey PYY (1–36) is N-terminally stable, we reveal in this study that the peptide is subject to endopeptidase mediated C-terminal dipeptide degradation. In an attempt to prevent this, (d-Arg35)-sea lamprey PYY (1–36) was developed. MethodsIn vitro bioassays assessed enzymatic stability, insulinostatic activity as well as beta-cell anti-apoptotic actions of (d-Arg35)-sea lamprey PYY (1–36). Follow-up studies examined the impact of twice daily administration of sea lamprey PYY (1–36) or (d-Arg35)-sea lamprey PYY (1–36) in multiple low dose STZ-induced diabetic mice. Results(d-Arg35)-sea lamprey PYY (1–36) was fully resistant to plasma enzymatic degradation. The peptide possessed similar significant insulinostatic, as well as positive anti-apoptotic biological actions, as the parent peptide. Sea lamprey PYY (1–36) and (d-Arg35)-sea lamprey PYY (1–36) delayed diabetes progression in STZ mice. Both treatment interventions induced a significant decrease in body weight, food and fluid intake as well as glucose and glucagon concentrations. In addition, glucose tolerance, plasma and pancreatic insulin were partially normalised. (d-Arg35)-sea lamprey PYY (1–36) was significantly more effective than sea lamprey PYY (1–36) in terms of enhancing glucose-stimulate insulin release. Both treatments improved pancreatic islet morphology, linked to decreased apoptosis of beta-cells. ConclusionWe present (d-Arg35)-sea lamprey PYY (1–36) as the first-in-class N- and C-terminally stable PYY (1–36) peptide analogue. General significanceEnzymatically stable, long-acting PYY (1–36) peptides highlight the therapeutic benefits of sustained activation of NPYR1's in diabetes.

Ethanol extract of Cissampelos sympodialis ameliorates lung tissue damage in streptozotocin-induced diabetic rats

Diabetes Mellitus (DM) is a metabolic syndrome characterized by hyperglycemia. Chronic complications affect a number of organs, including the lungs. Cissampelos sympodialis Eichl (Menispermaceae) is a plant used to treat respiratory diseases. The aim of this study was to evaluate the effect of Cissampelos sympodialis extract (CSE) in lungs of diabetic rats. We used 30 Wistar rats divided into three groups: control group (CG), diabetic group (DG) and diabetic Cissampelos sympodialis treatment group (DTG). Diabetes was induced by streptozotocin (40 mg/kg i.v.). The CSE (400 mg/kg, po) was administered daily, during four weeks, beginning one week after the onset of DM. The treatment with CSE was not able to reduce blood glucose levels after streptozotocin injection. However, it was able to decrease cholesterol and triglycerides and prevent damage on pancreatic islets morphology. Additionally, morphological alterations such as alveolar septa loss, inflammatory infiltrate and fibrosis were seen in lung tissue of rats with DM, and treatment with CSE apparently reversed these histopathological findings. Thus, CSE treatment reduced the lipid profile and restored the lung architecture of diabetic animals by a mechanism independent of glycemia and which might be associated with the reduction of the damage on the pancreatic islets.

Effects and mechanism of stem cells from human exfoliated deciduous teeth combined with hyperbaric oxygen therapy in type 2 diabetic rats

OBJECTIVES:Mesenchymal stem cells (MSCs) are potentially ideal for type 2 diabetes treatment, owing to their multidirectional differentiation ability and immunomodulatory properties. Here we investigated whether the stem cells from human exfoliated deciduous teeth (SHED) in combination with hyperbaric oxygen (HBO) could treat type 2 diabetic rats, and explored the underlying mechanism.METHODS:SD rats were used to generate a type 2 diabetes model, which received stem cell therapy, HBO therapy, or both together. Before and after treatment, body weight, blood glucose, and serum insulin, blood lipid, pro-inflammatory cytokines (tumor necrosis factor-alpha and interleukin-6), and urinary proteins were measured and compared. After 6 weeks, rats were sacrificed and their organs were subjected to hematoxylin and eosin staining and immunofluorescence staining for insulin and glucagon; apoptosis and proliferation were analyzed in islet cells. Structural changes in islets were observed under an electron microscope. Expression levels of Pdx1, Ngn3, and Pax4 mRNAs in the pancreas were assessed by real-time quantitative polymerase chain reaction (RT-qPCR).RESULTS:In comparison with diabetic mice, those treated with the combination or SHE therapy showed decreased blood glucose, insulin resistance, serum lipids, and pro-inflammatory cytokines and increased body weight and serum insulin. The morphology and structure of pancreatic islets improved, as evident from an increase in insulin-positive cells and a decrease in glucagon-positive cells. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining of islet cells revealed the decreased apoptosis index, while Ki67 and proliferating cell nuclear antigen staining showed increased proliferation index. Pancreatic expression of Pdx1, Ngn3, and Pax4 was upregulated.CONCLUSION:SHED combined with HBO therapy was effective for treating type 2 diabetic rats. The underlying mechanism may involve SHED-mediated increase in the proliferation and trans-differentiation of islet β-cells and decrease in pro-inflammatory cytokines and apoptosis of islets.

Peptide YY (1-36) peptides from phylogenetically ancient fish targeting mammalian neuropeptide Y1 receptors demonstrate potent effects on pancreatic β-cell function, growth and survival.

To investigate the antidiabetic efficacy of enzymatically stable Peptide YY (PYY) peptides from phylogenetically ancient fish. N-terminally stabilized, PYY (1-36) sequences from Amia calva (bowfin), Oncorhynchus mykiss (trout), Petromyzon marinus (sea lamprey) and Scaphirhynchus albus (sturgeon), were synthesized, and both biological actions and antidiabetic therapeutic efficacy were assessed. All fish PYY (1-36) peptides were resistant to dipeptidyl peptidase-4 (DPP-4) degradation and inhibited glucose- and alanine-induced (P < 0.05 to P < 0.001) insulin secretion. In addition, PYY (1-36) peptides imparted significant (P < 0.05 to P < 0.001) β-cell proliferative and anti-apoptotic benefits. Proliferative effects were almost entirely absent in β cells with CRISPR-Cas9-induced knockout of Npyr1. In contrast to human PYY (1-36), the piscine-derived peptides lacked appetite-suppressive actions. Twice-daily administration of sea lamprey PYY (1-36), the superior bioactive peptide, for 21 days significantly (P < 0.05 to P < 0.001) decreased fluid intake, non-fasting glucose and glucagon in streptozotocin (STZ)-induced diabetic mice. In addition, glucose tolerance, insulin sensitivity, pancreatic insulin and glucagon content were significantly improved. Metabolic benefits were linked to positive changes in pancreatic islet morphology as a result of augmented (P < 0.001) proliferation and decreased apoptosis of β cells. Sturgeon PYY (1-36) exerted similar but less impressive effects in STZ mice. These observations reveal, for the first time, that PYY (1-36) peptide sequences from phylogenetically ancient fish replicate the pancreatic β-cell benefits of human PYY (1-36) and have clear potential for the treatment of type 2 diabetes.

Ginger extract and its compound, 6-shogaol, attenuates painful diabetic neuropathy in mice via reducing TRPV1 and NMDAR2B expressions in the spinal cord.

In silico data revealed that the active compound of ginger (Zingiber officinale Roscoe), 6-shogaol, has strong affinity toward transient receptor potential vanilloid-1 (TRPV-1). TRPV-1 is expressed in nervous tissue and pancreatic β-cells. Prolonged induction of TRPV-1 is related to the expression of N-methyl-D-aspartate receptor subunit 2B (NMDAR2B). However, there are no data on TRPV-1 and NMDAR2B expressions in nervous tissue after 6-shogaol or ginger extract treatment nor pancreatic islet morphology and insulin expression in mice model of painful diabetic neuropathy (PDN). This study aimed to investigate the mechanism of action of ginger extract and its compound, 6-shogaol, on pancreatic islets as well as on expressions of TRPV-1 and NMDAR2B in the spinal cord of streptozotocin (STZ)-induced mice model of PDN. Sixty-four 5-6 weeks old male-Balb/C mice were induced with 110 mg/kgBW STZ i.p., while eight mice were used as control group. Mice with blood glucose level ≥200mg/d, that suffered hyperalgesia and allodynia were classified as PDN mice. Hot plate and von Frey filament tests were performed once a week until termination. At day 28 after considered as PDN, ginger extracts, 6-shogaol or gabapentin as control treatment were given once daily for 21 days until day 49, except for the diabetic control group. Upon termination, mice' pancreas were fixed, processed as paraffin sections and stained with hematoxylin eosin. Total volume of pancreatic islets was estimated using Cavalieri methods. Immunohistochemistry on pancreatic sections were performed to observe insulin expression. mRNA was extracted from lumbar segments of the spinal cord, followed by cDNA preparation and quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) to measure the expressions of TRPV1 and NMDAR2B. The mean differences between groups were analyzed using one-way analysis of variance (ANOVA) with p<0.05 considered statistically significant. Ginger extracts and 6-shogaol alleviated hyperalgesia and allodynia. The groups that received ginger extract 400 mg/kgBW or 6-shogaol 15 mg/kgBW had significantly lower TRPV1 and NMDAR2B expressions in the spinal cord compared to the diabetic control group (p<0.001; p<0.05). However, no differences in volume of pancreatic islets (p>0.05) nor insulin expression were observed in all PDN groups. Ginger extracts and its compound, 6-shogaol, reduced pain symptoms in PDN via its effect on decreasing TRPV1 and NMDAR2B expressions in the spinal cord, with very limited effect on pancreatic islets.

2197-P: SEL1L-HRD1 Endoplasmic Reticulum-Associated Degradation Is Required for Maintaining Glucagon Production in Pancreatic Alpha Cells

Endoplasmic reticulum-associated degradation (ERAD) is a key component of cellular protein quality control, an essential process for pancreatic islet endocrine cells, which synthesize peptide prohormones in high quantities. Two highly conserved ER membrane proteins comprise the core ERAD machinery: HRD1, a ubiquitin ligase that retrotranslocates and ubiquitinates misfolded proteins for proteasomal degradation, and SEL1L, an adaptor protein that identifies ERAD target proteins and is required for HRD1 stability and function. Pancreatic alpha cells, intestinal L-cells, and certain neurons synthesize and differentially cleave proglucagon into tissue-specific hormones, including glucagon and glucagon-like peptide 1 (GLP-1). To investigate the role of ERAD in these cells, we targeted SEL1L deletion with the Cre-lox system, using mice expressing Cre recombinase under the proglucagon promoter. We found that Sel1LGcg mice showed normal growth and glucose homeostasis in vivo. Oral glucose tolerance was equally impaired by administration of the GLP-1 receptor antagonist exendin 9-39 in both Sel1LGcg and control mice, suggesting intact GLP-1 signaling in Sel1LGcg mice. Glucagon secretion in vivo was not significantly different following a 24 hour fast or insulin-induced hypoglycemia. Interestingly, pancreatic glucagon content was reduced by half in Sel1LGcg mice, despite unchanged pancreatic weight. By immunofluorescence, pancreatic islet morphology in Sel1LGcg mice was normal, though α-cells demonstrated increased expression of the ER chaperone BiP and ERAD-associated proteins OS9 and XTP3-B. By transmission electron microscopy, a large subset of α-cells showed significant ER dilation and fewer, smaller glucagon granules. Our data demonstrate that SEL1L-HRD1 ERAD is required for maintaining normal glucagon expression and ER morphology in pancreatic α-cells but does not entirely disrupt proglucagon signaling in vivo. Disclosure R.B. Reinert: None. N. Shrestha: None. Y. Ji: None. L. Qi: None. Funding National Institutes of Health (5T32DK007245)

Amorphous solid dispersion of Berberine mitigates apoptosis via iPLA2β/Cardiolipin/Opa1 pathway in db/db mice and in Palmitate-treated MIN6 β-cells.

Aims: Berberine (BBR) improves beta-cell function in Type 2 diabetes (T2D) because of its anti-apoptotic activity, and our laboratory developed a new preparation named Huang-Gui Solid Dispersion (HGSD) to improve the oral bioavailability of BBR. However, the mechanism by which BBR inhibits beta-cell apoptosis is unclear. We hypothesized that the Group VIA Ca2+-Independent Phospholipase A2 (iPLA2β)/Cardiolipin(CL)/Opa1 signaling pathway could exert a protective role in T2D by regulating beta-cell apoptosis and that HGSD could inhibit β-cell apoptosis through iPLA2β/CL/Opa1 upregulation.Methods: We examined how iPLA2β and BBR regulated apoptosis and insulin secretion through CL/Opa1 in vivo and in vitro. In in vitro studies, we developed Palmitate(PA)-induced apoptotic cell death model in mouse insulinoma cells (MIN6). iPLA2β overexpression and silencing technology were used to examine how the iPLA2β/CL/Opa1 interaction may play an important role in BBR treatment. In in vivo studies, db/db mice were used as a diabetic animal model. The pancreatic islet function and morphology, beta-cell apoptosis and mitochondrial injury were examined to explore the effects of HGSD. The expression of iPLA2β/CL/Opa1 was measured to explore whether the signaling pathway was damaged in T2D and was involved in HGSD treatment.Results: The overexpression of iPLA2β and BBR treatment significantly attenuated Palmitate- induced mitochondrial injury and apoptotic death compared with Palmitate-treated MIN6 cell. In addition, iPLA2β silencing could simultaneously partly abolish the anti-apoptotic effect of BBR and decrease CL/Opa1 signaling in MIN6 cells. Moreover, HGSD treatment significantly decreased beta-cell apoptosis and resulted in the upregulation of iPLA2β/CL/Opa1 compared to those of the db/db mice.Conclusion: The results indicated that the regulation of iPLA2β/CL/Opa1 by HGSD may prevent beta-cell apoptosis and may improve islet beta-cell function in Type 2 diabetic mice and in palmitate-treated MIN6 cells.