Cordyceps inhibits ceramide biosynthesis and improves insulin resistance and hepatic steatosis.

Ectopic ceramide accumulation in insulin-responsive tissues contributes to the development of obesity and impairs insulin sensitivity. Moreover, pharmacological inhibition of serine palmitoyl transferase (SPT), the first enzyme essential for ceramide biosynthesis using myriocin in rodents reduces body weight and improves insulin sensitivity and associated metabolic indices. Myriocin was originally extracted from fruiting bodies of the fungus Isaria sinclairii and has been found abundant in a number of closely related fungal species such as the Cordyceps. Myriocin is not approved for human use but extracts from Cordyceps are routinely consumed as part of traditional Chinese medication for the treatment of numerous diseases including diabetes. Herein, we screened commercially available extracts of Cordyceps currently being consumed by humans, to identify Cordyceps containing myriocin and test the efficacy of Cordyceps extract containing myriocin in obese mice to improve energy and glucose homeostasis. We demonstrate that commercially available Cordyceps contain variable amounts of myriocin and treatment of mice with a human equivalent dose of Cordyceps extract containing myriocin, reduces ceramide accrual, increases energy expenditure, prevents diet-induced obesity, improves glucose homeostasis and resolves hepatic steatosis. Mechanistically, these beneficial effects were due to increased adipose tissue browning/beiging, improved brown adipose tissue function and hepatic insulin sensitivity as well as alterations in the abundance of gut microbes such as Clostridium and Bilophila. Collectively, our data provide proof-of-principle that myriocin containing Cordyceps extract inhibit ceramide biosynthesis and attenuate metabolic impairments associated with obesity. Moreover, these studies identify commercially available Cordyceps as a readily available supplement to treat obesity and associated metabolic diseases.

Human postnatal Mesenchymal Stem Cell Derived Islets as a Model for Diabetes Research.

The scarcity of human cadaver islets for transplantation in patients with Diabetes Mellitus (DM) has necessitated the search for alternative islet sources. With advancing islet biology research, Islet-Like Clusters (ILCs) derived from stem cells have demonstrated potential for treating DM and in novel drug discovery programs for drug and cytotoxicity testing. In vitro differentiation of ILCs from stem cells also provides an opportunity to mimic the in vivo islet developmental pathways. In vitro derived ILCs are often considered immature as they do not respond to glucose challenges efficiently. However, the in vitro and in vivo performance of ILCs can be improved by pharmacological preconditioning. In this review, we discuss how ILCs generated from human postnatal tissues can be utilized as an in-vitro model to study cytotoxicity, drug screening and enhancement of transplantation efficacy. The use of human cadaver islets is not permitted for research purposes in India. Under these restrictions, the application of ILCs in drug screening and their role in complementing, reducing, and replacing animal testing will evolve as a reliable platform for in vitro screening as well as for stem cell-based treatment in DM.

IGF-1 and somatocrinin trigger islet differentiation in human amniotic membrane derived mesenchymal stem cells.

AIM: To induce differentiation of human amniotic membrane derived mesenchymal stem cells (hAMMSCs) into insulin producing cells (IPCs) by treating with somatocrinin or growth hormone releasing hormone (GHRH) and Insulin-like growth factor-1 (IGF-1). MAIN METHOD: In this investigation, we cultivated and characterized hAMMSCs and then treated with IGF-1 and somatocrinin to find out whether this combination gives better yield of insulin producing cells. We showed that hAMMSCs can give rise to IPCs on exposure to serum-free defined media containing specific growth factors and differentiating agents in presence of IGF-1 and somatocrinin. KEY FINDING: A combination of IGF-1 and somatocrinin lead to differentiation of large number of IPCs from hAMMSCs. These IPCs were found to be positive for dithizone indicating their insulin secretory mechanism. Moreover these cells were also found to be positive for C-peptide. IPCs released insulin in response to glucose challenge. Gene expression analysis exhibited significant up-regulation of pancreatic transcription factor GLUT2 and Insulin. SIGNIFICANCE: Our data thus demonstrates for the first time that somatocrinin and IGF-1 synergistically enhance the differentiation of hAMMSCs into IPCs.

Human Umbilical Cord-Derived Stem Cells: Isolation, Characterization, Differentiation, and Application in Treating Diabetes.

Stem cell therapy is one of the most promising and effective approaches for treating diabetes, due to stem cell nonimmunogenic and nonimmunosupressive properties. Mesenchymal stem cells (MSCs) derived from human umbilical cord have been widely used in cell-based therapies for clinical applications. Among their various properties, immunomodulatory and proregenerative capacities broaden their scope for treating diabetes. Owing to various problems that are involved in treating diabetes, our review focuses on modulating umbilical cord-derived MSCs (UCMSCs) without any genetic manipulation. Umbilical cord tissue contains a rich source of MSCs with intact stemness. UCMSCs have profound effects on the remodeling process, maintaining similar morphology to various organs and escalating vascularization. Because of their neonatal origin, MSCs have enormous immune properties that lead to greater therapeutic benefits, including enhanced insulin sensitivity in type 2 diabetic (T2D) animal models and treatment of complications such as diabetic ulcers and compromised wound healing. MSCs ameliorate hyperglycemia by reducing inflammation due to their anti-inflammatory nature. Furthermore, their differentiation potential enables use in T1D treatment, wherein MSCs alone or insulin-producing cells that are derived from these MSCs, when transplanted in streptozotocin, induce animals to experience reversal of hyperglycemia. In this review, we discuss methods of UCMSC isolation, characterization, differentiation potential, and various applications in diabetes treatment.

Reprogramming mouse embryo fibroblasts to functional islets without genetic manipulation.

The constant quest for generation of large number of islets aimed us to explore the differentiation potential of mouse embryo fibroblast cells. Mouse embryo fibroblast cells isolated from 12- to 14-day-old pregnant mice were characterized for their surface markers and tri-lineage differentiation potential. They were subjected to serum-free media containing a cocktail of islet differentiating reagents and analyzed for the expression of pancreatic lineage transcripts. The islet-like cell aggregates (ICAs) was confirmed for their pancreatic properties via immunofluorecence for C-peptide, glucagon, and somatostain. They were positive for CD markers-Sca1, CD44, CD73, and CD90 and negative for hematopoietic markers-CD34 and CD45 at both transcription and translational levels. The transcriptional analysis of the ICAs at different day points exhibited up-regulation of islet markers (Insulin, PDX1, HNF3, Glucagon, and Somatostatin) and down-regulation of MSC-markers (Vimentin and Nestin). They positively stained for dithizone, C-peptide, insulin, glucagon, and somatostatin indicating intact insulin producing machinery. In vitro glucose stimulation assay revealed three-fold increase in insulin secretion as compared to basal glucose with insulin content being the same in both the conditions. The preliminary in vivo data on ICA transplantation showed reversal of diabetes in streptozotocin induced diabetic mice. Our results demonstrate for the first time that mouse embryo fibroblast cells contain a population of MSC-like cells which could differentiate into insulin producing cell aggregates. Hence, our study could be extrapolated for isolation of MSC-like cells from human, medically terminated pregnancies to generate ICAs for treating type 1 diabetic patients.

Herbal pre-conditioning induces proliferation and delays senescence in Wharton's Jelly Mesenchymal Stem Cells.

BACKGROUND: Mesenchymal Stem Cells (MSCs) are multipotent stem cells which are being explored for various clinical applications. Isolation and in-vitro expansion of MSCs remain important in achieving desired cell number for the therapy. However, in-vitro proliferation of MSCs is often associated with senescence and early onset of apoptosis which limits its therapeutic ability and long term clinical use. Tinospora cordifolia and Withania somnifera are used widely in Ayurveda: the traditional Indian system of medicine and are reported to have rejuvenating and anti-aging potential. In the present study, we investigated the effect of Tinospora cordifolia and Withania somnifera on proliferation and senescence of wharton's jelly MSCs (WJMSCs) in-vitro. METHODS: WJMSCs were treated in culture medium with Tinospora cordifolia leaf and Withania somnifera root extracts to examine their effect on proliferation and senescence properties of WJMSCs. Proliferation of WJMSCs was assayed by cell count, MTT, BrdU incorporation assay, cell cycle analysis and Ki67 mRNA expression. Senescence was demonstrated using ß-galactosidase senescence assay and associated mRNA markers. RESULTS: Culture medium supplemented with Tinospora cordifolia leaf and Withania somnifera root extracts exhibited significant increase in proliferation of WJMSCs as evidenced by cell count and MTT assay. Cell cycle analysis using propidium iodide showed increase in G2/M phase and decrease in apoptotic cells. BrdU incorporation and upregulation of proliferation marker ki67 by RT PCR showed increased DNA synthesis/proliferation in Tinospora cordifolia and Withania somnifera extract treated MSCs. Delayed senescence was confirmed by ß-galactosidase senescence assay and down regulation of senescence marker p21. CONCLUSION: Our results demonstrate for the first time that Tinospora cordifolia and Withania somnifera extracts support proliferation and inhibit senescence in WJMSCs making them suitable candidates as supplements for in-vitro expansion without affecting the cell viability indicating its non-toxic nature.

Hypoxia primed placental mesenchymal stem cells for wound healing.

AIMS: To investigate how Placental Mesenchymal Stem Cells (P-MSCs) would adapt themselves and survive under hypoxic conditions which are prevalent in most injury sites. MAIN METHODS: P-MSCs were isolated from term placenta and characterised under normoxia and hypoxia (2-2.5% O2). Cells were examined for morphology and surface marker variations by flow cytometry analysis. Glucose stimulated insulin secretion was assayed by Insulin ELISA Kit. Gene expression levels were estimated using Real Time PCR for hypoxia inducible factor1 alpha, Insulin (INS), Glucose transporters (GLUT-1, GLUT-2 and GLUT-3), Adhesion Proteins- Integrins, Fibronectin1 (FN1), E-Cadherin (CDH1), and N-Cadherin (CDH2) and angiogenesis marker VEGFA. Immunofluorescence assay was done to confirm the presence of C-Peptide, GLUT 2, E-Cadherin and ITGB3. Adhesion was confirmed assessed on fibronectin binding. KEY FINDINGS: We show that insulin secretion is not hampered under hypoxia. We found an upregulation of glucose transporters under hypoxia indicating enhanced glucose uptake needed to cater to metabolic demands of proliferating cells. Up regulation of adhesion molecules was seen under hypoxia indicative of a favoured environment for retention of cells at the injury site. We also found increased level of angiogenesis of P-MSCs under hypoxia. SIGNIFICANCE: Our present study thus demonstrates for the first time that P-MSCs modulate themselves under hypoxic conditions by secreting insulin, up regulating glucose transporters and adhesion molecules and eventually exhibiting an increased angiogenic potential. We thus infer that priming P-MSCs under hypoxia, could make them more suitable for wound healing applications.

Shielding Engineered Islets With Mesenchymal Stem Cells Enhance Survival Under Hypoxia.

In the present study we focused on the improvisation of islet survival in hypoxia.The Islet like cell aggregates (ICAs) derived from wharton's jelly mesenchymal stem cells (WJ MSC) were cultured with and without WJ MSC for 48 h in hypoxia and normoxia and tested for their direct trophic effect on ß cell survival. The WJ MSCs themselves secreted insulin upon glucose challenge and expressed the pancreatic markers at both transcription and translational level (C-peptide, Insulin, Glucagon, and Glut 2). Direct contact of MSCs with ICAs facilitated highest viability under hypoxia as evidenced by fluorescein diacetate/propidium iodide and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The cytokine analysis of the co-cultured ICAs revealed amplification of anti-inflammatory cytokine like TGFß and TNFα accompanied by depletion of pro-inflammatory cytokines. The increment in VEGF and PDGFa was also seen showing their ability to vascularize upon transplantation. This was further accompanied by reduction in total reactive oxygen species, nitric oxide, and super oxide ions and down regulation of Caspase3, Caspase8, p53, and up regulation of Bcl2 confirming prevention of apoptosis in ICAs. The western blot analysis confirmed the cytoprotective effect of WJ MSC on ICAs as they enhanced the anti-apoptotic marker BCL2 and reduced the expression of apoptotic markers, Annexin 5 and Caspase 3. There was a significant reduction in the expression of p38 protein in the presence of MSCs making the ICAs responsive to glucose. Taken together our data demonstrate for the first time that the WJ MSC expressed pancreatic markers and their supplementation protected engineered islets against hypoxia and oxidative stress. J. Cell. Biochem. 118: 2672-2683, 2017. © 2017 Wiley Periodicals, Inc.

Small molecules exert anti-apoptotic effect and reduce oxidative stress augmenting insulin secretion in stem cells engineered islets against hypoxia.

Transplantation of pancreatic islets is the most reliable treatment for Type 1 diabetes. However cell death mediated by hypoxia is considered as one of the main difficulties hindering success in islet transplantation. The aim of our experiment was to investigate the role of small molecules in survival of Islet like cell aggregates (ICAs) engineered from umbilical cord matrix under oxygen deprived condition (<5% O2). ICAs were analyzed for cell death via fluoroscein diacetate/propidium iodide (FDA/PI) staining, estimation of Caspase 3 and free radical release in presence and absence of small molecules. The samples were also analyzed for the presence of hypoxia inducible factor 1α (HIF1α) at both transcriptional and translational level. The addition of small molecules showed profound defensive effect on ICAs under hypoxic environment as evidenced by their viability and insulin secretion compared to untreated ICAs. The combinations of Eicosapentaenoic acid (EPA), Docosahexaenoic acid(DHA) and metformin and EPA, DHAandγ amino butyric acid (GABA) acted as anti-apoptotic agents for human ICAs when exposed to 1% O2 for 48h. The combinations of the small molecules reduced the total reactive oxygen species and malonaldehyde (MDA) levels and enhanced the production of glutathione peroxidise (GPx) enzyme under hypoxic conditions. Finally the increase in HIF1α at both protein and gene level confirmed the defensive effect of the additives in hypoxia. These results suggest that the combination of small molecules maintained the viability and functionality of the ICAs in hypoxia by up-regulating HIF1α expression and down regulating the Caspase 3 activity.

A novel GPR40 agonist, CNX-011-67, suppresses glucagon secretion in pancreatic islets under chronic glucolipotoxic conditions in vitro.

BACKGROUND: Elevated glucose concentrations lead to increased insulin secretion and suppression of glucagon secretion. In fact, insulin is a physiological inhibitor of glucagon secretion. Type 2 diabetes mellitus (T2DM) patients have defects in insulin secretion. In addition to this, lack of suppression of glucagon secretion under elevated glucose concentrations is also observed in T2DM patients. We have earlier shown that GPR40 activation by CNX-011-67 stimulates glucose stimulated insulin secretion (GSIS). Here we extended our studies to examine the impact of GPR40 activation by CNX-011-67 on glucagon secretion from intact islets under both normal and glucolipotoxic conditions. FINDINGS: Glucagon secretion from intact rat islets was suppressed under elevated glucose concentration. Activation of GPR40 by CNX-011-67 further suppressed glucagon secretion. Culturing islets under chronic glucolipotoxic (GL) conditions, we have observed increased high glucose mediated glucagon secretion and content which were reduced with GPR40 activation by CNX-011-67. Interestingly, expression of pre-proglucagon gene (GCG) remained unchanged under glucolipotoxicity in the presence or absence of GPR40 activation. CONCLUSION: Activation of GPR40 by CNX-011-67 can reduce glucagon secretion from pancreatic islets.

Activation of GPR40 attenuates chronic inflammation induced impact on pancreatic ß-cells health and function.

BACKGROUND: Chronic inflammation-mediated ß-cell apoptosis is known to decrease ß-cell mass in diabetes leading to reduced insulin secretion. Exposure to pro-inflammatory cytokines can stimulate apoptosis in pancreatic ß-cells. The G protein coupled receptor 40 (GPR40) is implicated for glucose induced insulin secretion. We hypothesized that GPR40 activation can protect ß-cells from inflammation-induced apoptosis and restore glucose stimulated insulin secretion. RESULTS: By exposing NIT1 insulinoma cells and rat islets to a cocktail of pro-inflammatory cytokines (TNFα and IL1ß), we mimicked inflammatory signaling as seen by JNK and NFκB activation and increased mRNA levels of TNFα, IL1ß and NOS2a. These changes were reversed by pharmacological activation of GPR40 by a specific, small molecule, CNX-011-67. Further, GPR40 activation reduced inflammation-mediated oxidative and endoplasmic reticulum (ER) stresses. Importantly, GPR40 activation decreased inflammation-induced apoptosis as measured by key markers. These impacts of GPR40 were mediated through activation of PLC, CaMKII, calcineurin and cAMP. Cell survival was also enhanced by GPR40 activation as seen from the increased phosphorylation of Akt/PKB and enhanced expression of BCL2 and PDX1 genes. Interestingly, GPR40 activation restored both, inflammation-mediated inhibition on insulin secretion and intracellular insulin content. CONCLUSIONS: In this study, we provide evidences that CNX-011-67, a GPR40 agonist, reduces inflammatory signaling and apoptosis in pancreatic ß-cells while promoting insulin secretion and synthesis. Activation of GPR40 leads to attenuation of ß-cell dysfunction caused by chronic inflammation and thus could be of immense clinical value to improve insulin secretion and ß-cell survival.

CNX-011-67, a novel GPR40 agonist, enhances glucose responsiveness, insulin secretion and islet insulin content in n-STZ rats and in islets from type 2 diabetic patients.

BACKGROUND: GPR40 is a G-protein coupled receptor regulating free fatty acid induced and also glucose induced insulin secretion. We generated neonatally-streptozotocin-treated female rats (n-STZ) and treated them with CNX-011-67, a GPR40 agonist to examine the role of GPR40 in modulation of glucose metabolism, insulin secretion and content. METHODS: Female n-STZ animals were orally administered with CNX-011-67 (15 mg/kg body weight, twice daily) or with vehicle for 8 weeks (n = 8 per group). Glucose tolerance in treated animals and insulin secretion, islet insulin content and gene expression in isolated islets were determined. Islets from type 2 diabetic mellitus (T2DM) patients were treated with different concentrations of glucose in presence or absence of CNX-011-67 and insulin secretion was measured. RESULTS: Treatment of n-STZ rats with GPR40 agonist CNX-011-67 enhanced insulin secretion in response to oral glucose load on day 0 and this response persisted during the treatment period. The treatment also produced a 'memory effect' during which insulin secretion in response to oral glucose load remained enhanced, for a week, even in absence of the agonist. Activation of GPR40 enhanced responsiveness of islets to glucose and increased glucose induced insulin secretion and islet insulin content. An increase in islet mRNA expression of GCK, PDX1, insulin and PC was also observed. Acute treatment of islets from n-STZ rats with GPR40 agonist enhanced cellular ATP content. Activation of GPR40 enhanced mitochondrial calcium level in NIT-1 insulinoma cells. CNX-011-67 increased insulin secretion in islets from T2DM patients which were non-responsive to increased glucose concentration CONCLUSIONS: Our data provide evidence that activation of GPR40 with CNX-011-67 stimulates glucose metabolism, enhances glucose responsiveness, increases insulin secretion and content and that pharmacological activation of GPR40 will prove beneficial for treatment of T2DM.

High Recovery of Functional Islets Stored at Low and Ultralow Temperatures.

BACKGROUND: Poor recovery of islets upon cryopreservation is the main hurdle in islet banking. Pancreatic islets have a poor antioxidative defense mechanism, and exposure of islets to low temperature leads to oxidative stress. AIM: We aimed to investigate whether known compounds such as metformin, γ aminobutyric acid (GABA), docosahexanoic acid (DHA), or eicosapentaenoic acid (EPA) alone or in combination are capable of reducing oxidative stress for better islet recovery upon storage at suboptimal temperatures. METHODS: Islets isolated from mouse pancreas were stored at low temperature (4°C) for 15 days and at ultralow temperature (-196°C) for 30 days with or without additives. After revival from cold storage, islets were assessed by using three methods: (1) specificity by dithizone (DTZ), (2) viability by fluorescein diacetate/propidium iodide (FDA/PI) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra-zolium bromide (MTT) assay, and (3) functionality by glucose-stimulated insulin secretion (GSIS). The oxidative status of the islets stored at suboptimal temperatures was determined by both intracellular free radical release (fluorometric analysis) and lipid peroxidation (enzymatic determination). RESULTS: Supplementation with additives led to an improvement in islet survival upon storage at suboptimal temperatures, without depletion of insulin secretory activity, which was comparable to that of controls. The additives acted as cryoprotectants and antioxidants as revealed by high recovery of viable islets and reduction in total reactive oxygen species (ROS) and malonidealdehyde (MDA), respectively. CONCLUSIONS: Our results demonstrate for the first time that supplementation with EPA, DHA, and metformin may lead to higher islet recovery from -196°C storage, enabling proper islet banking.