Normal Islets Research Articles

Acalypha wilkesiana Exhibits Antihyperglycemic Potentials and Ameliorates Damages to Pancreas and Spleen of Diabetic Rat Model

Background: The present study investigated the antihyperglycemic and ameliorative effect of aqueous leaves extract of Acalypha wilkesiana on alloxan-induced diabetic male rats. The experimental rats were divided into six groups. Rats of the first group served as normal controls. Rats of the second group were negative control as they were induced with diabetes without treatment. The third group was treated with glibenclamide after induction of diabetes. The fourth, fifth and sixth groups were diabetic rats, treated with aqueous leaves extract of Acalypha wilkesiana at low dose (100mg/kg body weight), medium dose (200mg/kg body weight) and high dose (400mg/kg body weight) respectively. The blood glucose levels of the animals in each group were checked on day 5, 10 and 15 to examine the antihyperglycemic effect of the aqueous leaves extract of Acalypha wilkesiana. Also, on day 5, 10 and 15, the pancreas and spleen of experimental animals from each group were harvested for histological examination. Result: The result obtained showed that the graded doses of the extract significantly (p<0.05) reduced the blood glucose levels of the diabetic animals in a dose-dependent manner. Also, the histological examination of the pancreas and spleen of the experimental animals treated with aqueous leaves extract of Acalypha wilkesiana showed normal pancreatic islet (PI) filled with islet cells (Granular appearance) and pancreatic acini (PA), while normal spleen with white pulp containing periarteriolar lymphatic sheath (PALS) and central arteriole (CA), splenic cord (SC) and sinusoids (SS) were seen in the same animals treated with the extract. The effect of the extract was similar in action to the standard drug glibenclamide. Conclusion: The aqueous leaves extract of Acalypha wilkesiana possesses antihyperglycemic potentials and ameliorates the pancreas and spleen of diabetic albino rats.

1745-P: Voltage-Gated K+ Channel Kv7.1 Is an Important Regulator of Insulin Secretion in Normal and Hyperinsulinemic Islets

Fifty percent of infants with Beckwith-Wiedemann syndrome (BWS), an overgrowth syndrome caused by genetic / epigenetic abnormalities in imprinted genes on chromosome 11p15, have persistent hypoglycemia due to hyperinsulinism (BWS-HI). BWS-HI islets exhibit increased baseline and glucose-stimulated insulin secretion (GSIS) and are responsive to the KATP channel inhibitor glyburide, suggesting it is not caused by impaired KATP channels. KCNQ1 expression, encoding the pore-forming α subunit of voltage-gated K+ channel Kv7.1, is reduced in BWS-HI islets compared to normal islets (3-fold; p = 4.5×10−7) and KCNQ1 loss-of-function mutations in humans are associated with HI. We used genetic and pharmacologic methods to modify Kv7.1 activity and examined changes to insulin secretion. In response to glucose, Kcnq1−/− mice had lower plasma glucose and higher plasma insulin than wild-type controls. Perifusion of Kcnq1−/− pancreatic islets with increasing concentrations of glucose showed that GSIS in these islets was faster and greater than in wild-type islets. The perifusion GSIS responses in normal human, wild-type mouse and Sur1−/− mouse (KATP-HI mouse model) islets with two different Kv7.1 activators (hexachlorophene or ML277) were markedly slower and diminished. In contrast, wild-type mouse islet perifusion with the Kv7.1 inhibitor chromanol 293B strikingly fastened and increased the GSIS response compared to vehicle controls. Membrane potential (Vm) analysis of Kcnq1−/− islets demonstrated that depolarization occurs more quickly during the initiation of insulin secretion compared to wild-type controls. Treatment with Kv7.1 activators resulted in Vm changes in both wild-type mouse and normal human islets compared to controls. Our results reveal the capacity of Kv7.1 to regulate insulin secretion in pancreatic islets and that Kv7.1 is a viable target for development of therapeutics for HI. Disclosure C.Juliana: Research Support; Crinetics Pharmaceuticals, Inc., Twist Bioscience, AmideBio. D.De leon: Consultant; Zealand Pharma A/S, Crinetics Pharmaceuticals, Inc., Eiger BioPharmaceuticals, Hanmi Pharm. Co., Ltd., Research Support; Ultragenix, Crinetics Pharmaceuticals, Inc., Hanmi Pharm. Co., Ltd., Rezolute, Inc. Funding National Institutes of Health (5R01DK098517-08)

297-LB: Dorzagliatin, a Glucokinase Activator, Repairs Defective Islet Function in High-Fat Diet-Induced Diabetic Mice

The DREAM study showed long-term benefits for glucose control in newly diagnosed type 2 diabetes (T2D) patients even after withdrawal of dorzagliatin treatment, a newly approved glucokinase activator for T2D treatment, who were considered to be in the remission for diabetes. The purpose of the current study is to examine the mechanisms of dorzagliatin-induced diabetes remission by evaluating the changes in islet function over time after a period of administering dorzagliatin in high-fat diet (HFD) induced obesity and diabetes mouse models. Diabetes mice were generated after 7 months of HFD, then received dorzagliatin or vehicle controls via oral gavage once a day for 19 days (8 days of 30 mg/Kg and 11 days of 15 mg/Kg). After drug withdrawal, one group of mice remained in HFD and another group changed to a normal diet. Islets were then isolated at different time points after drug withdrawal, and the serum and liver were collected for evaluation. The glucose ramp-stimulated insulin secretion (GSIS) was evaluated in islet perifusion. Gene expression was measured by qPCR in islets and by RNA-seq in the liver. Compared to normal mouse islets, HFD-induced diabetic islets in vehicle groups have impaired GSIS with about 40% reduction in maximum insulin secretion. In contrast, GSIS was fully recovered to the levels of normal islets in 10, 18 and 38 days after dorzagliatin withdrawal, but returned to defective GSIS levels after 45 days of drug discontinuation. In addition, islet GSIS can be fully recovered after 80 days of diet change. In conclusion, the defective GSIS in HFD-induced diabetes mouse islets is reversible and can be fully restored by dorzagliatin even after a long period of drug withdrawal while still remaining in HFD. The mechanisms are likely due to increased glucokinase gene expression caused by a short treatment of dorzagliatin. Diet change alone can recover islet function but requires weight reduction and a longer duration. Disclosure D. Han: Employee; Nanjing AscendRare Pharmaceutical Technology. S. Meng: Employee; Nanjing AscendRare Pharmaceutical Technology. X. Zhou: Employee; Nanjing AscendRare Pharmaceutical Technology. R. Li: Employee; Nanjing AscendRare Pharmaceutical Technology. C. Li: Employee; Hua Medicine, Nanjing AscendRare Pharmaceutical Technology.

349-OR: Selected DYRK1A Inhibitors Simultaneously Drive Both Human Beta-Cell Proliferation and Differentiation

Small molecule DYRK1A inhibitors induce adult human beta cell proliferation and expand human beta cell mass. Harmine also enhances differentiation and function in human beta cells in vitro and in vivo, increasing expression of key beta cell genes. It is unknown whether this “pro-differentiation effect” is a feature of all DYRK1A inhibitors. We compared harmine, 2-2c, 5-Iodotubericidin (5-IT), GNF4877, INDY, CC-401 and Leucettine-41 at doses that maximally drive human beta cell proliferation. As anticipated, harmine, 2-2c and 5-IT all increased expression of PDX1, MAFA, NKX6.1, SLC2A2, PCSK1, MAFB, SIX2, SLC30A8, and ENTPD3. These effects were observed in normal human islets as well as in islets from donors with T2D. Surprisingly, in simultaneous experiments, the DYRK1A inhibitors GNF4877, INDY, CC-401 and Leucettine failed to induce these beta cell differentiation markers. The pro-differentiation effect of harmine was independent of DYRK1A inhibition: silencing DYRK1A in human islets induced beta cell proliferation, but had no effect on differentiation; harmine treatment in DYRK1A-silenced islets restored beta cell pro-differentiation effects. The pro-differentiation effect was also independent of NFAT signaling and DREAM Complex effects, since NFAT overexpression, or p130, p107, E2F4 and E2F5 silencing had no effect on beta cell differentiation. Similarly, silencing other known harmine targets (DYRK2, 3, 4, the CLK and HIPK family) had no effect on either human beta cell differentiation nor proliferation. In summary, harmine, 2-2c and 5-IT are unique among the DYRK1A inhibitor family in their simultaneous ability to enhance both beta cell proliferation and differentiation. While the beta cell proliferation is mediated by DYRK1A inhibition, the pro-differentiation effects of harmine, 2-2c and 5-IT are distinct, and unexplained in mechanistic terms. These considerations have important implications for DYRK1A inhibitor drug development. Disclosure P. Wang: None. K. Kumar: None. H. Liu: None. O. Wood: None. E. Karakose: None. L. Choleva: None. L. Lambertini: None. A. Garcia-Ocana: Consultant; Sun Pharmaceutical Industries Ltd. R.J. DeVita: None. A.F. Stewart: None.

Study of anti-inflammatory and immunomodulatory potential of endometrial mesenchymal stem cells-derived exosomes with micro-ultrasound (μUS) guidance in regeneration β-islets on T1D animal model

Type 1 diabetes results from the inflammatory destruction of beta cells, leading to severe insulin deficiency. MSCs have an effective role in modulating immune and inflammatory reactions and are known as a standard platform for the treatment in regenerative medicine. Despite evidence of MScs’ effectiveness in diseases, they still are associated with safety challenges. Exosomes are natural nanoparticles secreted into the extracellular space by most cells. After the isolation and characterization of human Endometrial MSC-derived exosomes (hEnEx), diabetes model was induced by destroying islets using intraperitoneal injection of streptozotocin (STZ) in male rats. The characterized EnEx and EnMSCs were injected into pancreatic areas in diabetic animals under guidance of the micro-ultrasound non-invasive method. In the current study, the animals' blood glucose levels and body weight were monitored, accompanied by increased body weight and decreased blood sugar in the treated groups. Our findings indicate specific therapeutic effects of EnEx in β-islets neogenesis, normal islets formation, and insulin secretion. Evidence that shows functional role of EnEx in regulation of inflammatory and immune responses in EnEx-treated rats. Evaluation of insulin and pdx1 proteins expression showed presence of insulin and also EnEx antidiabetics effects in β-cells through the pdx1 pathway. This study determined that exosome therapy is more effective compared to cell therapy in improving diabetes, especially regeneration of β-islets mass. Also, exosome injection with the micro-ultrasound method has more efficacy in restoring β‐cell function. Therefore, EnEx based-treatment can be a novel therapeutic agent with minimal or no unwanted side effects for diabetic patients.

Differential use of the Nkx2.2 NK2 domain in developing pancreatic islets and neurons.

The homeodomain transcription factor (TF) Nkx2.2 governs crucial cell fate decisions in several developing organs, including the central nervous system (CNS), pancreas, and intestine. How Nkx2.2 regulates unique targets in these different systems to impact their individual transcriptional programs remains unclear. In this issue of Genes & Development Abarinov and colleagues (pp. 490-504) generated and analyzed mice in which the Nkx2.2 SD is mutated and found that the SD is required for normal pancreatic islet differentiation but dispensable for most aspects of neuronal differentiation.

The nuclear localization sequence and C-terminus of parathyroid hormone-related protein regulate normal pancreatic islet development and function

Parathyroid hormone-related protein (PTHrP) is a pleiotropic hormone essential for morphogenesis, tissue differentiation, as well as cell regulation and function. PTHrP is expressed by pancreatic beta cells which are responsible for insulin secretion. Previous studies have reported that N-terminal PTHrP stimulated proliferation in beta cells in rodents. We have developed a knockin mouse model (PTHrP Δ/Δ) lacking the C-terminal and nuclear localization sequence (NLS) of PTHrP. These mice die at ∼day 5, are severely stunted in growth, weigh 54% less than control mice at day 1–2 and eventually fail to grow. PTHrP Δ/Δ mice are also hypoinsulinemic and hypoglycemic yet have nutrient intake proportional to size. To characterize the pancreatic islets in these mice, islets (∼10–20) were isolated from 2 to 5 day-old-mice using collagenase digestion. Islets from PTHrP Δ/Δ mice were smaller in size but secreted more insulin than littermate controls. PTHrP Δ/Δ and control mice islets were exposed to various glucose concentrations and intracellular calcium, the trigger for insulin release, was elevated for glucose concentrations of 8–20 mM. Immunofluorescence staining showed less glucagon-stained area in islets from PTHrP Δ/Δ mice (∼250 µm2) compared to islets from control mice (∼900 µm2), and ELISA confirmed there was reduced glucagon content. These data collectively demonstrate increased insulin secretion and reduced glucagon at the islet level, which may contribute to the observed hypoglycemia and early death in PTHrP Δ/Δ mice. Thus, the C-terminus and NLS of PTHrP are crucial to life, including regulation of glucose homeostasis and islet function.

Islet-1 Is Differentially Expressed Among Neuroendocrine and Non-Neuroendocrine Tumors and Its Potential Diagnostic Implication.

Islet-1 (ISL1) plays key roles in programming the epigenome and facilitating the recruitment of additional regulatory factors. Although it has been used as a marker for pancreatic neuroendocrine tumors (PanNETs), ISL1 reactivity in other tumor types are critically missing. ISL1 immunohistochemistry was performed on 147 neuroendocrine tumors (NET) originated in pancreas, gastrointestinal tract, lung, thyroid, parathyroid, pituitary, adrenal medulla, head/neck, genitourinary tract, and skin; and 110 non-neuroendocrine tumors originated in the pancreas, thymus, lung, thyroid, mesothelium, adrenal cortex, stomach, breast, head/neck, skin, and kidney. ISL1 nuclear staining was observed in normal thymic epithelium, pancreatic islets, adrenal medulla, and pituitary gland cells as well as frequently in tumors of these origins: pancreatic NET (78%), paraganglioma/pheochromocytoma (100%), thymoma (82%), and pituitary NET (50%). ISL1 was also variably expressed in certain non-pancreatic NET such as Merkel cell carcinoma (100%), medullary carcinoma of the thyroid (100%), head/neck NEC (80%), genitourinary NEC (71%), lung small cell carcinoma (46%), lung carcinoids (17%), lower intestinal tract NET (93%) but not in upper gastrointestinal tract NET nor parathyroid adenoma. For other non-NETs, focal ISL1 expression was less frequently detected in gastric adenocarcinoma (40%), mesothelioma (29%), adrenal cortical carcinoma (17%), and squamous carcinoma (24%), but not in others tested. ISL1 is not a pan-NE marker as it is consistently lacking in upper gastrointestinal NET and parathyroid adenoma. It is also differentially expressed in thymoma. ISL1 immunohistochemnistry could help to differentiate PanNET and lower intestinal NET from upper gastrointestinal NET and be used as a marker for thymoma.

Revisiting the Pathogenesis of Type 1 Diabetes: Importance of Neural Input to Pancreatic Islets and the Therapeutic Capability of Stem Cell Educator TM Therapy to Restore Their Integrity.

Type 1 diabetes (T1D) is an autoimmune disease with a shortage of islet β cells. To date, the etiology of T1D remains elusive. Increasing clinical evidence and animal studies demonstrate that autoimmune cells are directed against the nervous system of pancreatic islets, contributing to the development of T1D. Therefore, it highlights the necessity to explore novel clinical approaches to fundamentally correct the T1D autoimmunity not only focusing on islet β cells but also on protecting the islet nervous system. This allows the restoration of the integrity of islet innervation and the normal islet β-cell function. To address these issues, we developed a novel technology designated the Stem Cell Educator TM therapy, based on immune education by human cord-blood-derived multipotent stem cells (CB-SC). International amulticenter clinical trials demonstrated its clinical safety and efficacy to treat T1D and other autoimmune diseases. Stem Cell Educator TM therapy may have the potential to revolutionize the treatment of T1D, without the safety and ethical concerns associated with conventional immune and/or stem cell-based therapies.

Human pancreatic islet microRNAs implicated in diabetes and related traits by large-scale genetic analysis

Genetic studies have identified≥240 loci associated with the risk of type 2 diabetes (T2D), yet most of these loci lie in non-coding regions, masking the underlying molecular mechanisms. Recent studies investigating mRNA expression in human pancreatic islets have yielded important insights into the molecular drivers of normal islet function and T2D pathophysiology. However, similar studies investigating microRNA (miRNA) expression remain limited. Here, we present data from 63 individuals, the largest sequencing-based analysis of miRNA expression in human islets to date. We characterized the genetic regulation of miRNA expression by decomposing the expression of highly heritable miRNAs into cis- and trans-acting genetic components and mapping cis-acting loci associated with miRNA expression [miRNA-expression quantitative trait loci (eQTLs)]. We found i) 84 heritable miRNAs, primarily regulated by trans-acting genetic effects, and ii) 5 miRNA-eQTLs. We also used several different strategies to identify T2D-associated miRNAs. First, we colocalized miRNA-eQTLs with genetic loci associated with T2D and multiple glycemic traits, identifying one miRNA, miR-1908, that shares genetic signals for blood glucose and glycated hemoglobin (HbA1c). Next, we intersected miRNA seed regions and predicted target sites with credible set SNPs associated with T2D and glycemic traits and found 32 miRNAs that may have altered binding and function due to disrupted seed regions. Finally, we performed differential expression analysis and identified 14 miRNAs associated with T2D status-including miR-187-3p, miR-21-5p, miR-668, and miR-199b-5p-and 4 miRNAs associated with a polygenic score for HbA1c levels-miR-216a, miR-25, miR-30a-3p, and miR-30a-5p.

Validating expression of beta cell maturation-associated genes in human pancreas development.

The identification of genes associated with human pancreatic beta cell maturation could stimulate a better understanding of normal human islet development and function, be informative for improving stem cell-derived islet (SC-islet) differentiation, and facilitate the sorting of more mature beta cells from a pool of differentiated cells. While several candidate factors to mark beta cell maturation have been identified, much of the data supporting these markers come from animal models or differentiated SC-islets. One such marker is Urocortin-3 (UCN3). In this study, we provide evidence that UCN3 is expressed in human fetal islets well before the acquisition of functional maturation. When SC-islets expressing significant levels of UCN3 were generated, the cells did not exhibit glucose-stimulated insulin secretion, indicating that UCN3 expression is not correlated with functional maturation in these cells. We utilized our tissue bank and SC-islet resources to test an array of other candidate maturation-associated genes, and identified CHGB, G6PC2, FAM159B, GLUT1, IAPP and ENTPD3 as markers with expression patterns that correlate developmentally with the onset of functional maturation in human beta cells. We also find that human beta cell expression of ERO1LB, HDAC9, KLF9, and ZNT8 does not change between fetal and adult stages.

Normal Pregnancy-Induced Islet Beta Cell Proliferation in Mouse Models That Are Deficient in Serotonin-Signaling.

During mouse pregnancy placental lactogens stimulate prolactin receptors on pancreatic islet beta cells to induce expression of the tryptophan hydroxylase Tph1, resulting in the synthesis and secretion of serotonin. Presently, the functional relevance of this phenomenon is unclear. One hypothesis is that serotonin-induced activation of 5-HT2B receptors on beta cells stimulates beta cell proliferation during pregnancy. We tested this hypothesis via three different mouse models: (i) total Tph1KO mice, (ii) 129P2/OlaHsd mice, which are incompetent to upregulate islet Tph1 during pregnancy, whereas Tph1 is normally expressed in the intestine, mammary glands, and placenta, and (iii) Htr2b-deficient mice. We observed normal pregnancy-induced levels of beta cell proliferation in total Tph1KO mice, 129P2/OlaHsd mice, and in Htr2b-/- mice. The three studied mouse models indicate that islet serotonin production and its signaling via 5-HT2B receptors are not required for the wave of beta cell proliferation that occurs during normal mouse pregnancy.

Abnormal Expression of an Insulin Synthesizing Enzyme in Islets of Adult Autoantibody Positive Donors.

The observation that the two active forms of proprotein convertase 1/3 (PC1/3) were differentially expressed in beta cells of normal islets raised the possibility that this heterogeneity is lost during type 1 diabetes (T1D) progression. To test this hypothesis, the expression of the convertase was evaluated by confocal microscopy in sections of human pancreas of autoantibody positive (AA+) and T1D donors and compared with that of control. Islets of T1D pancreas were comprised of beta cells expressing either low or high PC1/3 levels and all islets of a pancreatic section contained only one beta cell type. Pancreata of AA+ donors contained either of these two classes of islets intermixed with normal islets comprised of beta cells with heterogeneous PC1/3 expression. This alteration affected the expression of proinsulin and insulin, which in most AA+ and T1D donors were lower than in controls. The present results indicate that the heterogeneity of PC1/3 expression is lost in all beta cells in a subset islets of AA+ donors and in all islets of T1D donors. These findings suggest that the heterogeneity of PC1/3 expression is a biomarker of human beta cell health and that its loss coincides with the initial stages of T1D.

Mice With RIP-Cre-mediated Deletion of the Long Noncoding RNA Meg3 Show Normal Pancreatic Islets and Enlarged Pituitary.

Maternally expressed gene 3 (MEG3) is a long noncoding RNA (lncRNA) that has been implicated as a tumor suppressor. The expression of MEG3 RNA is downregulated in various human tumors, including pituitary adenoma and pancreatic islet tumors due to MEG3 gene deletion or DNA hypermethylation. Mouse models with conventional germline deletion of Meg3 have shown that Meg3 is essential for perinatal or postnatal development and survival. However, a direct role of Meg3 loss in tumorigenesis has not been shown. To observe a causal relationship between Meg3 loss and tumorigenesis, we have generated a mouse model with conditional deletion of Meg3 mediated by the RIP-Cre transgene that initiated Meg3 deletion in pancreatic islet β cells and anterior pituitary. Meg3 loss did not lead to the development of islet tumors. Interestingly, RIP-Cre-mediated Meg3 loss led to the development of an enlarged pituitary. The genes in the Meg3 region are transcribed together as a 210 kb RNA that is processed into Meg3 and other transcripts. Whether these tandem transcripts play a functional role in the growth of pancreatic endocrine cells and pituitary cells remains to be determined. Our mouse model shows that Meg3 loss leads to hyperplasia in the pituitary and not in pancreatic islets, thus serving as a valuable model to study pathways associated with pituitary cell proliferation and function. Future mouse models with specific inactivation of Meg3 alone or other transcripts in the Meg3 polycistron are warranted to study tissue-specific effects on initiating neoplasia and tumor development.

Therapeutic significance of thymoquinone-loaded chitosan nanoparticles on streptozotocin/nicotinamide-induced diabetic rats: In vitro and in vivo functional analysis

To overcome the low bioavailability of lipophilic free thymoquinone (TQ), this study aims to evaluate a novel oral formula of TQ-loaded chitosan nanoparticles (TQ-CsNPs) for the effective treatment of diabetes. The XRD, FTIR, FESEM, HRTEM, and dynamic light scattering were all conducted on the prepared formula. The release pattern of TQ, cytotoxicity against MRC-5 cell line (human lung fibroblast cells), and antidiabetic activity on streptozotocin/nicotinamide (STZ/NA) rat model of diabetes were investigated. The results confirmed the formation of TQ-CsNPs with an entrapment efficiency of 75.7 ± 6.52 %, a mean Zetasizer distribution of 84.25 nm, and an average particle size of about 50 nm. After 24 h, the percentage of free TQ-cumulative release was approximately 35.8 %, whereas TQ-CsNPs showed a sustained release pattern of 78.5 %. The investigated formula was not toxic to normal lung cells, and more efficient in ameliorating the altered glycemia, dyslipidemia, inflammation, and oxidative stress induced by STZ/NA than free TQ, blank CsNPs, and metformin-HCl (as a reference drug). Additionally, TQ-CsNPs restored the normal pancreatic islets' configuration and morphometry, suggesting a potent insulinotropic action. In conclusion, the antidiabetic efficacy of TQ was improved by engaging TQ with CsNPs as an excellent nanoplatform to enhance the oral bioavailability of TQ.

Kynurenine-3-monooxygenase expression is activated in the pancreatic endocrine cells by diabetes and its blockade improves glucose-stimulated insulin secretion

Type 2 diabetes is associated with an inflammatory phenotype in the pancreatic islets. We previously demonstrated that proinflammatory cytokines potently activate the tryptophan/kynurenine pathway (TKP) in INS-1 cells and in normal rat islets. Here we examined: (1) the TKP enzymes expression in the diabetic GK islets; (2) the TKP enzymes expression profiles in the GK islets before and after the onset of diabetes; (3) The glucose-stimulated insulin secretion (GSIS) in vitro in GK islets after KMO knockdown using specific morpholino-oligonucleotides against KMO or KMO blockade using the specific inhibitor Ro618048; (4) The glucose tolerance and GSIS after acute in vivo exposure to Ro618048 in GK rats. We report a remarkable induction of the kmo gene in GK islets and in human islets exposed to proinflammatory conditions. It occurred prominently in beta cells. The increased expression and activity of KMO reflected an acquired adaptation. Both KMO knockdown and specific inhibitor Ro618048 enhanced GSIS in vitro in GK islets. Moreover, acute administration of Ro618048 in vivo improved glucose tolerance, GSIS and basal blood glucose levels in GK rats. These results demonstrate that targeting islet TKP is able to correct defective GSIS. KMO inhibition could represent a potential therapeutic strategy for type 2 diabetes.

1461-P: Quantifying Proinsulin Misfolding in the Endoplasmic Reticulum

We recently reported that normal pancreatic islets or beta cell lines contain a subset of proinsulin molecules that have not achieved the three intramolecular disulfide bonds required for native insulin structure. Indeed, we found that non-native proinsulin monomers can form intermolecular disulfide-linked complexes, which are detectable by immunoblotting after nonreducing SDS-PAGE. However, we have recognized difficulties in the quantitative interpretation of these analyses, as we frequently identified conditions in which little or no proinsulin monomer was detected, and non-native disulfide-linked complexes amounted to greater cumulative proinsulin band intensity than the entire population of proinsulin molecules detected by immunoblotting upon reducing SDS-PAGE. Here we have developed several critical modifications of our methodology to rectify these discrepancies to undertake a more accurate determination of the pool of misfolded proinsulin molecules in pancreatic beta cells under conditions of health and disease. Using these modifications, we find that the size of the proinsulin monomer pool has previously been underestimated and the fraction of disulfide-linked complexes overestimated — and this can be readily explained by proinsulin antibody affinity in the immunoblotting protocol. Additionally by immunoblotting, we now resolve native and non-native proinsulin monomers, highlighting the inefficiency of non-native proinsulin transport through the secretory pathway. Disclosure A.Arunagiri: None. M.Alam: None. L.Haataja: None. P.J.Samy: None. S.Soleimanpour: None. L.S.Satin: None. P.Itkin-ansari: None. M.Liu: None. P.Arvan: n/a. Funding National Institutes of Health (R01-DK48280) , National Institutes of Health (U01-DK127747) , and National Institutes of Health (R24 DK110973)

Pairing 3D-Printing with Nanotechnology to Manage Metabolic Syndrome.

IntroductionThis work was aimed to develop a Curcuma oil-based self-nanoemulsifying drug delivery system (SNEDDS) 3D-printed polypills containing glimepiride (GMD) and rosuvastatin (RSV) for treatment of dyslipidemia in patients with diabetes as a model for metabolic syndrome (MS).MethodsCompartmentalized 3D printed polypills were prepared and studied in streptozotocin/poloxamer induced diabetic/dyslipidemic rats. The pharmacokinetic parameters of GMD and RSV in the prepared polypills were evaluated. Blood glucose level, lipid profile, antioxidant, and biochemical markers activities were investigated. Also, histopathological examination of the liver and pancreas was carried out. The atherosclerotic index, the area of islets of Langerhans, and liver steatosis lesion scores were calculated.ResultsThe developed SNEDDS-loaded GMD/RSV polypills showed acceptable quality control characteristics with a high relative bioavailability of 217.16% and 224.28% for GMD and RSV, respectively, when compared with the corresponding non-SNEDDS pills. The prepared polypills showed dramatic lowering in blood glucose levels and substantial improvement in lipid profile and hepatic serum biomarkers as well as remarkable decrease in serum antioxidants in response to Poloxamer 407 intoxication. The prepared polypills decreased the risk of atherosclerosis and coronary disease by boosting the level of high-density lipoprotein and lowering both triglyceride and low-density lipoprotein. Microscopic examination showed normal hepatic sinusoids and high protection level with less detectable steatosis in the examined hepatocytes. Normal size pancreatic islets with apparently normal exocrine acini and pancreatic duct were also noticed.ConclusionThis formulation strategy clearly shows the potential of the developed polypills in personalized medicine for treatment of patients with MS.

Notch-mediated Ephrin signaling disrupts islet architecture and β cell function.

Altered islet architecture is associated with β cell dysfunction and type 2 diabetes (T2D) progression, but molecular effectors of islet spatial organization remain mostly unknown. Although Notch signaling is known to regulate pancreatic development, we observed “reactivated” β cell Notch activity in obese mouse models. To test the repercussions and reversibility of Notch effects, we generated doxycycline-dependent, β cell–specific Notch gain-of-function mice. As predicted, we found that Notch activation in postnatal β cells impaired glucose-stimulated insulin secretion and glucose intolerance, but we observed a surprising remnant glucose intolerance after doxycycline withdrawal and cessation of Notch activity, associated with a marked disruption of normal islet architecture. Transcriptomic screening of Notch-active islets revealed increased Ephrin signaling. Commensurately, exposure to Ephrin ligands increased β cell repulsion and impaired murine and human pseudoislet formation. Consistent with our mouse data, Notch and Ephrin signaling were increased in metabolically inflexible β cells in patients with T2D. These studies suggest that β cell Notch/Ephrin signaling can permanently alter islet architecture during a morphogenetic window in early life.

Effects of Moringa oliefera Lam. Aqueous Root Extract on the Histology of Pancreas in Alloxan-Induced Diabetic Rats

This study was to determine the effects of aqueous root extract of Moringa oleifera on antioxidant activities and on the histology of pancreas in Alloxan-induced-diabetic rats. Twenty Albino Wistar rats of both sexes weighing between100 and 194g were used for the study. The animals were divided in to four groups (Groups I, II, II and IV) of five rats each. Group I was the control group and were administered distilled water per body weight. Group II was induced with diabetes by injecting the rats with 150mg/kg Alloxan single dose and not treated. group III was induced with diabetes and treated with 50mg/kg Moringa for 28 days and group IV were not induced with diabetes but were treated with the aqueous extract of Moringa oleifera root for a period of twenty-eight days at 50mg/kg orally. The antioxidant activity of Moringa oliefera aqueous root extract was studied by assaying serum marker substances of Catalase, Glutathione peroxidase (GPx), Superoxide Dismutase (SOD) and Malondialdehyde (MDA) to determine the extent of antioxidant activity of the extract on the pancreas. At the end of the experiment, the animals were sacrificed and the Pancreas processed for routing light microscopic analyses. The results of this study showed that antioxidant activities of Malondialdehyde (MDA) decreased significantly in diabetic not treated and diabetic treated Moringa groups and Glutathione peroxidase (GPx), activity increased significantly in diabetic non-treated group and non-diabetic treated group (p<0.05). Glutathione Superoxide Dismutase (SOD) and Catalase changes were not significant. The results of this study also showed that photomicrograph of pancreas of diabetic non treated rats showed highly lobulated pancreatic tissue, glands exhibits loose collagenous capsule which can be seen extending as delicate septae, chronic inflammation and degeneration of islets of Langerhans. Diabetic treated group also showed septea between the lobule similar to the control group and glandular acini undergoing regeneration. Non-diabetic but treated group showed diffused micro and macro acini and normal islets of Langerhans similar to control.