Number Of Pancreatic Islets Research Articles

8786 Novel TGFbeta Ligand Regulated Genes in WT and FSTL3 KO Testis Identified by ChIP-Seq Analysis: Implications for Signalling Cross-talk

Abstract Disclosure: R. Ballesteros: None. A. Mukherjee: None. Folistatin Like 3 (FSTL3) is a known antagonist of activin and myostatin action that when deleted from the mouse genome displayed a set of phenotypic changes that are directly linked to glucose metabolism and reproductive function. Although our previous work showed that β-cell hyperplasia, increased pancreatic islet number and size and reduced visceral fat seen in FSTL3 knockout mice (FSTL3 KO) could be explained by enhanced activin and myostatin action, the molecular bases of the changes in the reproductive function seen in this mouse model remain elusive. In contrast to testicular degeneration produced by INHBA (activin A) transgenic overexpression in mice, deletion of FSTL3 results in increased testicular size, with increased Sertoli cell numbers and early meiotic entry of the first spermatogenic wave. This suggests a possibility that the phenotype in FSTL3 deleted mice might involve alteration of additional transcripts other than those induced by activin alone in the testis. To identify transcripts induced in FSTL3 deleted testis as a consequence of induced TGFβ ligand action we performed sequencing of Smad4 Chromatin immunoprecipitation (ChIP-Seq) from postnatal (3d) and post-pubertal (56d) whole testis. Our results show high activity of Smad4 signalling during postnatal stages compared to post-pubertal stages in both genotypes (Fstl3-KO vs wild-type). Interestingly, postnatal testis from FSTL3 KO contains significantly less activity from Smad4 signalling (69 peaks) compared to wild type (775 peaks). By contrast, on the whole, Smad4 signalling in post-pubertal stages is less active with 34 peaks in wild-type and 29 peaks in Fstl3-KO of which 8 genes are SMAD activated in both genotypes. To identify whether SMAD-dependent signalling produces significant differences in RNA expression, we then used the list of genes that generated peaks in the Smad4-ChIP-Seq experiment to filter our RNA-Seq data generated in a parallel experiment followed by differential gene expression analysis of GO terms by GSEA in postnatal testis from both genotypes. We found that Smad4-interacting genes in posnatal FSTL3 KO testis generate enrichment of 13 pathways including the androgen receptor signalling pathway. Taken together so far, our findings reveal a novel catalogue of SMAD-regulated genes in the testis, identifies a set of genes specifically upregulated in the testis in a SMAD-dependent manner upon FSTL3 deletion and also identify possible cross-talk between TGFβ and 13 other signalling pathways. Currently we are further analysing our data to identify whether there are SMAD regulated genes and pathways common in postnatal and postpubertal testes. We are also undertaking experiments to investigate expression pattern in FSTL3 KO testis to identify which pathways might specifically affect Sertoli cell proliferation and activity. Presentation: 6/2/2024

8786 Novel TGFbeta Ligand Regulated Genes in WT and FSTL3 KO Testis Identified by ChIP-Seq Analysis: Implications for Signalling Cross-talk

Abstract Disclosure: R. Ballesteros: None. A. Mukherjee: None. Folistatin Like 3 (FSTL3) is a known antagonist of activin and myostatin action that when deleted from the mouse genome displayed a set of phenotypic changes that are directly linked to glucose metabolism and reproductive function. Although our previous work showed that β-cell hyperplasia, increased pancreatic islet number and size and reduced visceral fat seen in FSTL3 knockout mice (FSTL3 KO) could be explained by enhanced activin and myostatin action, the molecular bases of the changes in the reproductive function seen in this mouse model remain elusive. In contrast to testicular degeneration produced by INHBA (activin A) transgenic overexpression in mice, deletion of FSTL3 results in increased testicular size, with increased Sertoli cell numbers and early meiotic entry of the first spermatogenic wave. This suggests a possibility that the phenotype in FSTL3 deleted mice might involve alteration of additional transcripts other than those induced by activin alone in the testis. To identify transcripts induced in FSTL3 deleted testis as a consequence of induced TGFβ ligand action we performed sequencing of Smad4 Chromatin immunoprecipitation (ChIP-Seq) from postnatal (3d) and post-pubertal (56d) whole testis. Our results show high activity of Smad4 signalling during postnatal stages compared to post-pubertal stages in both genotypes (Fstl3-KO vs wild-type). Interestingly, postnatal testis from FSTL3 KO contains significantly less activity from Smad4 signalling (69 peaks) compared to wild type (775 peaks). By contrast, on the whole, Smad4 signalling in post-pubertal stages is less active with 34 peaks in wild-type and 29 peaks in Fstl3-KO of which 8 genes are SMAD activated in both genotypes. To identify whether SMAD-dependent signalling produces significant differences in RNA expression, we then used the list of genes that generated peaks in the Smad4-ChIP-Seq experiment to filter our RNA-Seq data generated in a parallel experiment followed by differential gene expression analysis of GO terms by GSEA in postnatal testis from both genotypes. We found that Smad4-interacting genes in posnatal FSTL3 KO testis generate enrichment of 13 pathways including the androgen receptor signalling pathway. Taken together so far, our findings reveal a novel catalogue of SMAD-regulated genes in the testis, identifies a set of genes specifically upregulated in the testis in a SMAD-dependent manner upon FSTL3 deletion and also identify possible cross-talk between TGFβ and 13 other signalling pathways. Currently we are further analysing our data to identify whether there are SMAD regulated genes and pathways common in postnatal and postpubertal testes. We are also undertaking experiments to investigate expression pattern in FSTL3 KO testis to identify which pathways might specifically affect Sertoli cell proliferation and activity. Presentation: 6/2/2024

Cadmium-induced pancreatic toxicity in rats: comparing vitamin C and Nigella sativa as protective agents: a histomorphometric and ultrastructural study

The study aimed to assess the toxic effect of cadmium (Cd) on the exocrine and endocrine functions of pancreas, the changes in pancreatic tissue after Cd withdrawal, and the protective effects of vitamin C (VC) and Nigella sativa (NS) against Cd-induced damage. Rats were assigned to: control, Cd-treated (0.5 mg/kg/d intraperitoneal [IP] injection), VC and Cd-treated (receiving 100 mg/kg/d VC orally and Cd concomitantly), NS and Cd-treated (receiving 20 mg/kg/d NS and Cd, simultaneously), and Cd withdrawal (receiving Cd for 30 d then living free for recovery for other 30 d). Blood samples were collected and post-sacrifice pancreatic specimens were processed for light and electron microscope study. Quantitative analyses of pancreatic collagen area%, pancreatic islet parameters, β cell density, and insulin immunoexpression were done. Fasting blood glucose was significantly increased in Cd-treated and Cd-withdrawal groups, while co-treatment with VC and NS caused significant reductions (p < 0.05). Cd-induced extensive degenerative changes in pancreatic acini and islets at light and ultrastructure levels. Obvious fibrosis and congestion of blood vessels were noticed. Significant reductions in pancreatic islet number, volume, and surface area and diminished beta cell count and insulin immunoexpression were observed. After withdrawal of Cd, the whole pancreatic tissue still showed a serious impact. Concomitant treatment with VC or NS obviously reduced these degenerative changes and significantly improved pancreatic islet parameters and insulin immunoexpression. VC showed a better amendment than NS, but this difference was statistically insignificant. Therefore, VC and NS could be used as prophylactic agents that lessen Cd consequences on the pancreas.

Marginal Zinc Deficiency Promotes Pancreatic Islet Enlargement While Zinc Supplementation Improves the Pancreatic Insulin Response in Zucker Diabetic Fatty Rats.

Zinc deficiency has been associated with the worsening of diabetes while zinc supplementation has been proposed to ameliorate diabetes. This study examined the effects of marginal zinc deficiency (MZD) and zinc supplementation (ZS) on obesity, glycemic control, pancreatic islets, hepatic steatosis and renal function of Zucker diabetic fatty (ZDF) rats. Male ZDF rats were fed an MZD, zinc control (ZC) or ZS diet (4, 30 and 300 mg Zn/kg diet, respectively), and lean Zucker rats were fed a ZC diet for 8 weeks. MZD and ZS did not alter body weight or whole-body composition in ZDF rats. MZD ZDF rats had reduced zinc concentrations in the femur and pancreas, a greater number of enlarged pancreatic islets and a diminished response to an oral glucose load based on a 1.8-fold greater incremental area-under-the-curve (AUC) for glucose compared to ZC ZDF. ZS ZDF rats had elevated serum, femur and pancreatic zinc concentrations, unchanged pancreatic parameters and a 50% reduction in the AUC for insulin compared to ZC ZDF rats, suggesting greater insulin sensitivity. Dietary zinc intake did not alter hepatic steatosis, creatinine clearance, or levels of proteins that contribute to insulin signaling, inflammation or zinc transport in epididymal fat. Potential adverse effects of ZS were suggested by reduced hepatic copper concentrations and elevated serum urea compared to ZC ZDF rats. In summary, ZS improved the pancreatic insulin response but not the glucose handling. In contrast, reduced zinc status in ZDF rats led to impaired glucose tolerance and a compensatory increase in the number and size of pancreatic islets which could lead to β-cell exhaustion.

Anti-hyperglycemic effect of herbal formula of Moringa oleifera, Vernonia amygdalina and Centella asiatica extracts in streptozotocin-induced hyperglycemic mice

IntroductionMoringa oleifera, Vernonia amygdalina, and Centella asiatica are herbs used in traditional Chinese medicine and are commonly used in Southeast Asian countries to treat a variety of diseases. The experimental evidence showed that M. oleifera leaves, V. amygdalina leaves, and C. asiatica had the effect of supporting their treatment of diabetes. This study investigated the anti-hyperglycemic effect of a novel herbal formula from M. oleifera leaf, V. amygdalina leaf, and C. asiatica aerial part extracts. MethodsThe extract was examined for anti-hyperglycemic capacity in oral glucose tolerance test in normal mice. To confirm their role in a higher model, the extract was studied in the streptozotocin (STZ)-induced hyperglycemic model. Plasma glucose concentration, body weight, organ weight, malondialdehyde, glutathione in liver and kidneys, and pancreatic histology were also evaluated. Besides, in vitro properties of the extract were tested including α-amylase and α-glucosidase inhibitory, and antioxidant activities. ResultsAfter 7-day of treatment, the extract showed strong anti-hyperglycemic activity in the STZ-induced hyperglycemic mice, plasma glucose levels were reduced by 48.75 % and 54.13 % in the group administered with 200 mg/kg and 400 mg/kg of the extract, respectively, compared with the hyperglycemic control. The extract had the effect to protect the liver and kidneys against oxidation stress by enhancing glutathione levels in the liver and decreasing malondialdehyde levels in the liver and kidneys. The pancreatic β-cell protection was also observed by improving the size and number of pancreatic islets by the extract compared to the hyperglycemic control. The extract showed a significant α-amylase and α-glucosidase inhibitory activities, and antioxidant activity through DPPH and ABTS quenching and reducing power abilities. DiscussionThese results demonstrated remarkable potential of the combined extract of M. oleifera leaves, V. amygdalina leaves, and aerial part of C. asiatica in the management of diabetes.

Oleic Acid and Succinic Acid: A Potent Nutritional Supplement in Improving Hepatic Glycaemic Control in Type 2 Diabetic Sprague–Dawley Rats

Nutritional supplements are gaining traction for their effects in mitigating the impacts of various health conditions. In particular, many supplements are being proposed to reduce the impacts of type 2 diabetes (T2D), a metabolic condition that has reached global epidemic proportions. Recently, a supplement of oleic acid (OA) and succinic acid (SA; 1 : 1, w/w) was reported to improve glycaemic control in type 2 diabetic (T2D) Sprague–Dawley (S‐D) rats through ameliorating insulin release and sensitivity. Here, we investigate the effects of the supplement (OA and SA) on hepatic and pancreatic function in T2D S‐D rats. Eighteen (18) S‐D rats were rendered diabetic and were divided into three equal groups: diabetic control, diabetic treatment, and diabetic glibenclamide. Another 12 S‐D rats were obtained and served as the normal groups. The animals were treated daily with the vehicle, OA and SA (800 mg/kg body weight (bw); 1 : 1), or glibenclamide (10 mg/kg bw) which served as the positive control. The findings indicated that treatment with the supplement resulted in a 35.69 ± 4.22% reduction (p = 0.006) in blood glucose levels (BGL). Analysis of hepatic enzymes depicted that the nutritional supplement reduced the activity of the gluconeogenesis enzyme, glucose‐6‐phosphatase (G6P) while improved the activity of catabolic enzymes such as glucose‐6‐phosphate dehydrogenase (G6PD) and pyruvate kinase (PK). Furthermore, the supplement attenuated oxidative stress through restoration of catalase (CAT) and superoxide dismutase (SOD), while reducing malondialdehyde (MDA) levels. Finally, the supplement showed no liver or kidney toxicity and improved the size and number of pancreatic islets of Langerhans, indicating its potential application in treating T2D. The study highlighted that a supplement of the two organic acids may be beneficial in reducing the rate of pathogenesis of type 2 diabetes. Therefore, it may offer therapeutic value as a dietary or nutritional supplement in the approach against diabetes and its complications.

1735-P: The Dichotomic and Timely Regulation of SerpinB13 Plays a Role in Beta-Cell Neogenesis and Pancreatic Islet Expansion

There has been recent emphasis on the potential role of a functional crosstalk between the exocrine and endocrine pancreas in the development of type 1 diabetes (T1D). Our interest in this was sparked by our observations that, (i) serpinB13, a protease inhibitor of cathepsin L, is expressed in mouse pancreatic ductal cells, and (ii) an antibody response to serpinB13 is associated with better clinical outcomes in mice and humans with T1D. The exact pattern of serpinB13 expression in the human pancreas has, to date, not been examined. Similarly, the exact role of serpinB13 in beta-cell biology and diabetes remains unclear. We aimed to determine the distribution of serpinB13 expression using immunochemistry on pancreatic sections obtained from nPOD. In addition, we developed a mouse serpinB13 genetic knockout model to examine the role of this serpin in regulating pancreatic islet morphology. Visiopharm software was used for an unbiased quantitative image analysis of islet size and cellularity. We found that in healthy humans, serpinB13 expression was confined to the pancreatic ducts, mimicking the pattern we observed previously in rodents. Studies of serpinB13 genetic deficiency revealed that early inhibition of serpinB13 increased the small pancreatic islet population while with time this trend reversed, and a drop in the number of pancreatic islets was observed. Our results demonstrate that serpinB13 is expressed in the exocrine epithelial compartment of the human pancreas. In addition, quite unexpectedly, serpinB13 appears to play a dual role in regulating pancreatic islet mass. More specifically, we postulate that serpinB13 initially hampers the development of additional insulin-producing cells, but with time it promotes expansion of the already formed beta cells. Thus, we propose an approach that first impedes serpinB13, followed by later administration of this serpin, which may lead to an increase in beta-cell mass and improved resistance to T1D. Disclosure Y.Kryvalap: None. S.Z.Meng: None. R.Habte: None. J.Czyzyk: None.

The effects of trans fat diet intake on metabolic parameters and pancreatic tissue in offspring of prenatal bisphenol A exposed rats

Bisphenol A (BPA) is a plasticiser used in the manufacturing of many products and its effects on human health remain controversial. Up till now, BPA involvement in metabolic syndrome risk and development is still not fully understood. In this study, we aimed to investigate the effect of prenatal BPA exposure with postnatal trans-fat diet intake on metabolic parameters and pancreatic tissue histology. Eighteen pregnant rats were divided into control (CTL), vehicle tween 80 (VHC), and BPA (5 mg/kg/day) from gestational day (GD) 2 until GD 21, then their weaning rat’s offspring were fed with normal diet (ND) or trans-fat diet (TFD) from postnatal week (PNW) 3 until PNW 14. The rats were then sacrificed and the blood (biochemical analysis) and pancreatic tissues (histological analysis) were collected. Glucose, insulin, and lipid profile were measured. The study has shown that there was no significant difference between groups with regard to glucose, insulin, and lipid profiles (p > 0.05). All pancreatic tissues showed normal architecture with irregular islets of Langerhans in TFD intake groups compared to offspring that consumed ND. Furthermore, the pancreatic histomorphometry was also affected whereby the study findings revealed that there was a significant increase in the mean number of pancreatic islets in rats from BPA-TFD group (5.987 ± 0.3159 islets/field, p = 0.0022) compared to those fed with ND and BPA non-exposed. In addition, the results have found that prenatal BPA exposure resulted in a significant decrease in the pancreatic islets diameter of the BPA-ND group (183.3 ± 23.28 µm, p = 0.0022) compared to all other groups. In conclusion, prenatal BPA exposure with postnatal TFD in the offspring may affect glucose homeostasis and pancreatic islets in adulthood, and the effect may be more aggravated in late adulthood.

578 Impact of Acellular Fish Skin on Wound Healing in a Standardized Pre-Clinical Model

Abstract Introduction Acellular fish skin has proven to be effective in acute and chronic wound healing. Multiple human studies have to date demonstrated that products from acellular skin grafts accelerate wound healing and are efficacious, safe and cost-effective. Here we wanted to investigate the effects of acellular fish skin on wound healing in a pig model. Methods The primary aim of this study was to investigate the effects of acellular fish skin on wound healing progression in a full thickness skin defect pig model. The superiority of fish skin was tested in a standardized pig model and compared to normal wound healing (without acellular fish skin grafts) (n=6). The experiments were carried out in male landrace pigs with a mean weight of 28.8 +/- 2.8 kg at the start of the study. The experiment lasted for 21 days. Wound documentation, thermography, photo-documentation and wound perfusion were carried out 5, 9, 14 and 21 days post wounding. Results Clinical evaluation of the wounds showed that there were no major differences between the different dressings on day 5 post wounding. On the next evaluation days ( day 9, 14 and 21 post wounding) the acellular fish skin showed rapid epithelialization with minimal wound contraction, when compared to untreated control wounds, which showed significant signs of contraction (i.e. star-like wound appearance). Wound area measurements clearly showed that wound size upon treatment with the acellular fish skin and the untreated control rapidly and significantly decreased through contraction of untreated controls. However, wound perfusion seemed to be affected by the treatment with acellular fish skin. Thermography at day 9 and 14 showed higher surface temperature in treated wounds (d9 and d14; p = 0.0005), followed by a trend towards increased oxygenation and perfusion, analyzed by laser speckle and hyperspectral imaging. Conclusions Acellular fish skin seems to provide fast wound healing and counteract wound contraction. Untreated wounds showed a slight trend of faster healing, but showed significant signs of wound contraction which points towards wound healing by contraction but not by epithelialization. This contraction was not observed in wounds treated with acellular fish skin. Applicability of Research to Practice Standardized preclinical models give valuable insights to the mechanisms of wound healing. Porcine in vivo models are of special interest as the porcine skin is highly similar to human skin with regard to anatomical and physiological structures. Therefore, data generated in such models, as described here, show a high rate of translatability to the human situation.

579 Histopathological Changes in the Endocrine Pancreas of Rats During the Acute Period of Burns

Abstract Introduction Severe burn victims experience a systemic inflammatory response and a hypermetabolic response. Presence of hyperglycemia, even with concurrent hyperinsulinism, is indicative of insufficient insulin secretion from beta cells to overcome the glycemia. However, the underlying molecular mechanisms of beta-cell failure in patients with acquired insulin resistance after burn trauma are not clearly understood. Our aim in this study was to describe the histopathological changes in the pancreatic islets secondary to severe burns in an experimental animal model. Methods Fourteen Wistar albino rats were randomly divided into 2 groups: the sham group and the burn group. A full-thickness burn model was designed to induce a burn of 25% total body surface area. Seven days after burn induction and sham procedure, pancreatectomy was performed. Pancreatic tissues were examined under light microscopy, and islet size and cellularity were calculated. Results The histopathologic examination was unremarkable, but the mean number of islets per pancreatic tissue was lower in the burn group than in the sham group. We observed a significant difference in the mean number of cells per one islet between the 2 groups, with the cell count higher in the burn group (P &amp;lt; .05). Conclusions During the acute phase of burn injury in rats, we observed a decrease in the number of pancreatic islets with remarkable hypercellularity. Further studies are needed to determine the histological and cellular basis of these changes. Applicability of Research to Practice Major burn injury causes severe alterations in beta-cell mass, which increases and decreases both function and mass to maintain the glycemic level within a very narrow physiological range. Patients with acute and prolonged hyperglycemia can have complications leading to impaired wound healing, increased skin graft loss, increased muscle protein catabolism, increased incidence of infections, and mortality. Therefore, a detailed understanding of the pancreatic changes under different preexisting conditions and therapeutic interventions in the context of a major burn trauma might guide treatment options.

Influence of macrophages on the insulin-synthesizing system under normal conditions and in alloxan diabetes

Insulin-synthesizing cells (ISCs) of pancreatic gland are localized both in its islets, and in exocrine portion, as single cells or cellular agglomerates. ISCs differ in their morphological and functional characteristics, depending on characteristics of the microenvironment. Resident macrophages are also involved into formation of their microenvironment. Our purpose was to assess the effect of functional macrophages upon the insulinsynthesizing system (pancreatic islets, cell agglomerates, and separately lying insulin-synthesizing cells) under normal conditions and in alloxan diabetes.Alloxan diabetes was induced in mature male Wistar rats by intraperitoneal injection of alloxan (30 mg/100 g). Functional activity of macrophages was modeled with anti-inflammatory drug aminophthalhydrazide (AMP). Contents of insulin, glucose, and glycosylated hemoglobin were measured in blood of experimental animals. The levels of IL-1α, TNFα and IFNγ were determined in pancreatic homogenate. The number of macrophages was counted in histological preparations from the insular and exocrine parts of the organ, as well as the number of pancreatic islets, agglomerates, and single ISCs. The amounts of proliferating cells (insulin+Ki-67+), apoptotic forms (TUNEL+insulin+), and insulin content of ISCs at different sites (according to their fluorescence intensity) were determined. All pancreatic islets were divided into 3 types, according to intensity of insulin fluorescence, i.e., islets with high, median and low levels of fluorescence.In healthy rats, immunomodulation reduced total level of IL-1α in pancreatic parenchyma, without changing the overall parameters of carbohydrate metabolism. In the exocrine part of pancreas, the content of single ISCs in ductal epithelium was increased. Likewise, proliferation of the ISC agglomerates became higher. The intensity of β-cell apoptosis increased in pancreatic islets. The proportion of islets with high-level insulin fluorescence was decreased, along with lower density of macrophages and proliferation rates of β-cells, and higher apoptosis rates, than in intact animals. We have also revealed there an increased ratio of cells with average insulin levels. In the islets with low insulin content, immunomodulation did not cause morphological changes. Administration of AMP in alloxan diabetes contributes to a significantly decreased concentration of IFNγ in pancreatic tissues, stabilizes IL-1α content, along with reduced apoptosis of ISCs and macrophage infiltration in all parts of the gland. In the ductal epithelium, a large number of single ISCs with high synthetic activity was observed, with retained number of agglomerates and their increased cellularity. The number of dividing β-cells is increased in pancreatic islets.Modulation of the functional activity of pancreatic macrophages under physiological conditions provides a multidirectional effect on the insulin-synthesizing cells, depending on their localization. In exocrine part of the organ, where M2 macrophages are located, we have observed activated differentiation and proliferation of ISC precursors. Meanwhile, in the islets where M1 macrophages are present, apoptosis of β-cells was enhanced. In alloxan diabetes, immunomodulation was associated with reduced destruction of insulinocytes, along with high intensity of their proliferation. Heterogenous response of ISCs to the changes in the microenvironment depends on their synthetic activity. In healthy rats, the islets with high level of insulin fluorescence, the level of apoptosis is increased, and β-cell proliferation is reduced, while the morphological and functional characteristics of islets with low-level insulin fluorescence did not change. In alloxan diabetes, apoptosis prevailed in islets with high fluorescence values, whereas β-cell proliferation predominated in the islets with low insulin contents.

Doxycycline Prevents Preclinical Atherosclerosis, Pancreatic Islet Loss and Improves Insulin Secretion after Glycemic Stimulation: Preclinical Study in Individuals with a High-Fat Diet

Doxycycline (Doxy) is an antibiotic, which has exhibited anti-inflammatory activity and glucose metabolism improvement. The present study was proposed to evaluate its effects on glucose metabolism and other associated processes, such as lipemia and adipogenesis, as well as, to evaluate its effects on the liver, pancreas, and aorta in subjects fed with an occidental high-fat diet (HFD). The trial followed three groups of BALB/c mice for 6 months: (1) Standard diet (SD); (2) HFD-placebo (saline solution); and (3) HFD-Doxy (10 mg/kg/day). Intrahepatic fat accumulation (steatohepatosis) and the epididymal fat pad, as well as the hepatic inflammatory infiltrate and ALT serum levels were higher in both groups with the HFD (with/without doxycycline) in comparison with the SD group. The thickness of the aorta (preclinic atherosclerosis) was significantly elevated in the HFD group with respect to the HFD + Doxy and SD group, these two being similar groups to each other. The HFD-Doxy group had pancreatic morphological parameters very similar to those of the SD group; on the contrary, the HFD group reduced the number of pancreatic islets and the number of β cells per mm2, in addition to losing large islets. The index of β cell function (∆Insulin0-30/∆Glucose0-30 ratio) was significantly higher in the HFD + Doxy group, compared to the rest of the groups.

Growth hormone treatment does not augment the anti-diabetic effects of liraglutide in UCD-T2DM rats.

The incretin hormone glucagon-like peptide-1 (GLP-1) slows gastric emptying, increases satiety and enhances insulin secretion. GLP-1 receptor agonists, such as liraglutide, are used therapeutically in humans to improve glycaemic control and delay the onset of type 2 diabetes mellitus (T2DM). In UCD-T2DM rats, a model of polygenic obesity and insulin resistance, we have previously reported that daily liraglutide administration delayed diabetes onset by >4months. Growth hormone (GH) may exert anti-diabetic effects, including increasing β-cell mass and insulin secretion, while disrupting GH signalling in mice reduces both the size and number of pancreatic islets. We therefore hypothesized that GH supplementation would augment liraglutide's anti-diabetic effects. Male UCD-T2DM rats were treated daily with GH (0.3mg/kg) and/or liraglutide (0.2mg/kg) from 2months of age. Control (vehicle) and food-restricted (with food intake matched to liraglutide-treated rats) groups were also studied. The effects of treatment on diabetes onset and weight gain were assessed, as well as measures of glucose tolerance, lipids and islet morphology. Liraglutide treatment significantly reduced food intake and body weight and improved glucose tolerance and insulin sensitivity, relative to controls. After 4.5months, none of the liraglutide-treated rats had developed T2DM (overall p=.019). Liraglutide-treated rats also displayed lower fasting triglyceride (TG) concentrations and lower hepatic TG content, compared to control rats. Islet morphology was improved in liraglutide-treated rats, with significantly increased pancreatic insulin content (p < .05 vs. controls). Although GH treatment tended to increase body weight (and gastrocnemius muscle weight), there were no obvious effects on diabetes onset or other diabetes-related outcomes. GH supplementation did not augment the anti-diabetic effects of liraglutide.

Effect of Momordica charantia on Insulin Immune-Reactive Pancreatic Beta Cells and Blood Glucose Levels in Streptozotocin-Induced Diabetic Rats.

The present study was conducted to determine the antidiabetic effect of Momordica charantia (MC) on pancreatic islets of Langerhans in streptozotocin (STZ)-induced diabetic male Wistar rats in relation to the distribution of insulin immuno-positive beta cells. MC was fed to rats at 2, 5, and 10% of the standard diet for 12 wk. After sacrification, the pancreatic tissues were obtained for histopathological and immunohistochemical observations. In addition, monitoring of fasting blood glucose was applied each month during experimental period. The results revealed that the oral doses of MC at 5% and 10% of the daily diet increased the percentage of insulin-positive pancreatic beta cells as well as the size and number of pancreatic islets. Moreover, significant (p≤0.0001) dose and time-depended reduction in fasting blood glucose levels were observed. In conclusion, these results suggest that MC induces antidiabetic effects via regeneration of insulin-positive pancreatic beta cells and increase the number of insulin secretory granules hence, blood glucose reduction.

Desmoglein-2 is important for islet function and β-cell survival

Type 1 diabetes is a complex disease characterized by the lack of endogenous insulin secreted from the pancreatic β-cells. Although β-cell targeted autoimmune processes and β-cell dysfunction are known to occur in type 1 diabetes, a complete understanding of the cell-to-cell interactions that support pancreatic function is still lacking. To characterize the pancreatic endocrine compartment, we studied pancreata from healthy adult donors and investigated a single cell surface adhesion molecule, desmoglein-2 (DSG2). Genetically-modified mice lacking Dsg2 were examined for islet cell mass, insulin production, responses to glucose, susceptibility to a streptozotocin-induced mouse model of hyperglycaemia, and ability to cure diabetes in a syngeneic transplantation model. Herein, we have identified DSG2 as a previously unrecognized adhesion molecule that supports β-cells. Furthermore, we reveal that DSG2 is within the top 10 percent of all genes expressed by human pancreatic islets and is expressed by the insulin-producing β-cells but not the somatostatin-producing δ-cells. In a Dsg2 loss-of-function mice (Dsg2lo/lo), we observed a significant reduction in the number of pancreatic islets and islet size, and consequently, there was less total insulin content per islet cluster. Dsg2lo/lo mice also exhibited a reduction in blood vessel barrier integrity, an increased incidence of streptozotocin-induced diabetes, and islets isolated from Dsg2lo/lo mice were more susceptible to cytokine-induced β-cell apoptosis. Following transplantation into diabetic mice, islets isolated from Dsg2lo/lo mice were less effective than their wildtype counterparts at curing diabetes. In vitro assays using the Beta-TC-6 murine β-cell line suggest that DSG2 supports the actin cytoskeleton as well as the release of cytokines and chemokines. Taken together, our study suggests that DSG2 is an under-appreciated regulator of β-cell function in pancreatic islets and that a better understanding of this adhesion molecule may provide new opportunities to combat type 1 diabetes.

1450-P: Identifying Hidden Sources of Tissue Plasticity in Pancreatic Islets with a Novel Multicolor Flow Cytometry Approach

Identifying pancreatic islet cells that acquire a beta-cell like phenotype is important for developing new diabetes therapies. Our studies have revealed a novel serpinB13 protease inhibitor antibody, that augments cathepsin L protease activity, resulting in Notch1 receptor cleavage and mobilization of endocrine progenitor cells to become beta cells in the embryonic pancreas. It is unclear, however, whether a similar induction of additional beta cells occurs with exposure to serpinB13 monoclonal antibody (mAb) in later life. To explore the exact source (s) of new beta cells in response to serpinB13 mAb after birth we developed a novel, multicolor flow cytometry analysis approach to study pancreatic islets. Five-week old normal Balb/c mice were injected with serpinB13 mAb (or control IgG) , and sacrificed at 8 weeks for islet isolation. Islet cell suspensions were stained with a viability dye, and a panel of antibodies to CD31, CD45, cytokeratin 19, EpCAM, insulin (Ins) , glucagon (Glu) , somatostatin (SS) , c-peptide, and four transcription factors, Maf-A, Nkx6.1, NeuroD1, and Pdx-1. Exposure to the serpinB13 mAb caused a significant increase in the number of hormonal-negative (e.g., Ins-Glu-SS-c-peptide-) cells (p=0.0011) , and hormonal negative, but c-peptide positive, cells (p=0.002) . At least one of four transcription factors examined were positive in 5-10% and 10-30% of these populations, respectively. At the same time, there was a trend in the drop of Glu+ and SS+ cells. The average number of pancreatic islets, and the total islet cellularity per mouse, was augmented in animals receiving serpinB13 mAb. Although additional studies are necessary to characterize the hormonal negative cells and assess whether Glu+ and SS+ cells transdifferentiate to beta cells, these findings demonstrate that enhanced tissue plasticity and beta-cell output can be achieved in the pancreas following treatment with antibodies to protease inhibitors. Disclosure Y.Kryvalap: None. S.Z.Meng: None. J.Czyzyk: None.

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.

AGE FEATURES OF THE STRUCTURE OF THE BLOOD VESSELS OF THE SOME DIGESTIVE GLANDS AND ITS RESTRUCTURING IN THE INITIAL STAGES OF EXPERIMENTAL DIABETES MELLITUS

The aim: The purpose of the work was to study the peculiarities of blood supply the pancreatic islet of the 24-month-old rat, and its restructuring during the initial periods of experimental diabetes mellitus. Materials and methods: The work was performed on 20 white outbred rats - males weighing 340-420g. 24 months of age, kept in standard vivarium conditions in compliance with all accepted ethical rules. Experimental streptozotocin diabetes mellitus was simulated in 16 animals. The material was taken on the 14th and 28th day of the experiment. Results: Reorganization of the endocrine part of the pancreas in the early stages of experimental diabetes is characterized by a decrease in the number and area of pancreatic islets, a decrease in the diameter of the lumen of arterioles, precapillaries, postcapillaries compared to the control group of animals by 7% and 5%. The diameter of the capillaries decreases by 16% and reaches 3.8 ± 0.62 μm2, and the diameter of the venules increases by 12%. In some blood vessels there are phenomena of desolation and edema of perivascular connective tissue, which is manifested by a decrease in optical density and stratification of collagen fibers. Conclusions: Thus, the reorganization of the circulatory system of the endocrine part of the pancreas of 24-month-old rats in the early stages of experimental diabetes mellitus is characterized by a decrease in the number and area of pancreatic islets.

Effect of Momordica charantia on Insulin Immune-reactive Pancreatic Beta Cells and Blood Glucose Levels in Streptozotocin-induced Diabetic Rats

The present study was conducted to determine the antidiabetic effect of Momordica charantia on pancreatic islets of Langerhans in streptozotocin-induced diabetic male Wistar rats in relation to the distribution of insulin immuno-positive beta cells. Momordica charantia was fed to rats at 2 %, 5 % and 10 % of the standard diet for 12 w. After sacrifice, the pancreatic tissues were obtained for histopathological and immunohistochemical observations. In addition, monitoring of fasting blood glucose was applied each month during experimental period. The results revealed that the oral doses of Momordica charantia at 5 % and 10 % of the daily diet increased the percentage of insulin-positive pancreatic beta cells as well as the size and number of pancreatic islets. Moreover, significant (p≤0.01) dose and time-dependent reduction in fasting blood glucose levels were observed. In conclusion, these results suggest that Momordica charantia induces antidiabetic effects via regeneration of insulin-positive pancreatic beta cells and increase the number of insulin secretory granules; hence, blood glucose reduction.

Abrus precatorius Leaf Extract Reverses Alloxan/Nicotinamide-Induced Diabetes Mellitus in Rats through Hormonal (Insulin, GLP-1, and Glucagon) and Enzymatic (α-Amylase/α-Glucosidase) Modulation.

Background Abrus precatorius is used in folk medicine across Afro-Asian regions of the world. Earlier, glucose lowering and pancreato-protective effects of Abrus precatorius leaf extract (APLE) was confirmed experimentally in STZ/nicotinamide-induced diabetic rats; however, the underlying mechanism of antidiabetic effect and pancreato-protection remained unknown. Objective This study elucidated antidiabetic mechanisms and pancreato-protective effects of APLE in diabetic rats. Materials and Methods APLE was prepared by ethanol/Soxhlet extraction method. Total phenols and flavonoids were quantified calorimetrically after initial phytochemical screening. Diabetes mellitus (DM) was established in adult Sprague-Dawley rats (weighing 120–180 g) of both sexes by daily sequential injection of nicotinamide (48 mg/kg; ip) and Alloxan (120 mg/kg; ip) over a period of 7 days. Except control rats which had fasting blood glucose (FBG) of 4.60 mmol/L, rats having stable FBG (16–21 mmol/L) 7 days post-nicotinamide/Alloxan injection were considered diabetic and were randomly reassigned to one of the following groups (model, APLE (100, 200, and 400 mg/kg, respectively; po) and metformin (300 mg/kg; po)) and treated daily for 18 days. Bodyweight and FBG were measured every 72 hours for 18 days. On day 18, rats were sacrificed under deep anesthesia; organs (kidney, liver, pancreas, and spleen) were isolated and weighed. Blood was collected for estimation of serum insulin, glucagon, and GLP-1 using a rat-specific ELISA kit. The pancreas was processed, sectioned, and H&E-stained for histological examination. Effect of APLE on enzymatic activity of alpha (α)-amylase and α-glucosidase was assessed. Antioxidant and free radical scavenging properties of APLE were assessed using standard methods. Results APLE dose-dependently decreased the initial FBG by 68.67%, 31.07%, and 4.39% compared to model (4.34%) and metformin (43.63%). APLE (100 mg/kg) treatment restored weight loss relative to model. APLE increased serum insulin and GLP-1 but decreased serum glucagon relative to model. APLE increased both the number and median crosssectional area (×106μm2) of pancreatic islets compared to that of model. APLE produced concentration-dependent inhibition of α-amylase and α-glucosidase relative to acarbose. APLE concentration dependently scavenged DPPH and nitric oxide (NO) radicals and demonstrated increased ferric reducing antioxidant capacity (FRAC) relative to standards. Conclusion Antidiabetic effect of APLE is mediated through modulation of insulin and GLP-1 inversely with glucagon, noncompetitive inhibition of α-amylase and α-glucosidase, free radical scavenging, and recovery of damaged/necro-apoptosized pancreatic β-cells.