Protocatechualdehyde restores endothelial dysfunction in streptozotocin-induced diabetic rats.

BACKGROUND: The present study was conducted with the aim of clarifying the effects of protocatechualdehyde (PCA) on the endothelial function in streptozotocin (STZ)-induced diabetic rats. METHODS: Sprague Dawley (SD) rats were intraperitoneally injected with STZ (single dose of 60 mg/kg). Diabetic model rats were given PCA (25 mg/kg/day) via gavage feeding for 6 weeks. Vascular function was studied; superoxide anion and nitrotyrosine levels were assessed; and nicotinamide adenine dinucleotide phosphate hydrogen (NADPH) oxidase as well as total superoxide dismutase (SOD) activity were detected. Protein expression of phosphorylated endothelial nitric oxide synthase (P-eNOS), total endothelial nitric oxide synthase (T-eNOS), p22phox, p47phox and Cu/Zn-SOD were measured by Western blot analysis. RESULTS: PCA treatment significantly ameliorated the impairment of acetylcholine- evoked endothelium-dependent relaxation, with no obvious effects observed on the blood glucose or body weight in the STZ-induced diabetic rats. Expression levels of aortic P-eNOS/T-eNOS and endothelial nitric oxide synthase (eNOS) activity were decreased in STZ-induced diabetic rats while they remained unchanged in PCA-treated rats. However, PCA treatment improved oxidative inactivation of nitric oxide (NO) and decreased the levels of superoxide anion and nitrotyrosine in the aorta of STZ-induced diabetic rats; these were achieved by reducing the level of nitrotyrosine and down-regulating p47phox and p22phox expression, as well as up-regulating Cu/Zn-SOD protein expression. Consistently, the effects observed were associated with a decrease in NADPH oxidase activity and an increase in total SOD activity. CONCLUSIONS: Our results indicate that the administration of PCA may be protective against oxidative stress and may restore endothelial function by improving vascular NO oxidative inactivation in diabetic condition.

Irisin Is a Positive Regulator for Ferroptosis in Pancreatic Cancer.

Regulated cell death by way of ferroptosis involves iron-dependent accumulation of cellular reactive oxygen species (ROS). Ferroptosis is attracting attention as a potential therapeutic target for cancer treatments without drug resistance. The relationship between irisin, a myokine involved in autophagy and ROS metabolism, and ferroptosis is unclear. In this study, we used erastin-induced ferroptosis in PANC-1 cells to examine potential interactions of irisin with ferroptosis. Using western blots and reverse transcriptase polymerase chain reactions, we found that irisin can further exacerbate erastin-induced upregulation in free iron, lipid ROS levels, and glutathione depletion, relative to cells treated with erastin only. Conversely, removal of irisin limited erastin effects. Furthermore, irisin modulation of ferroptosis was associated with the expression changes in molecules important for ROS metabolism, iron metabolism, and the cysteine/glutamate antiporter system (system Xc -). These study findings suggest that irisin can act as a master factor of ferroptosis, and that potential implications for harnessing irisin-mediated ferroptosis for cancer treatment are warranted.

Erastin-induced ferroptosis is a regulator for the growth and function of human pancreatic islet-like cell clusters.

Ferroptosis is a newly identified and novel form of cell death, which is characterized by an iron- and reactive oxygen species (ROS)-dependent manner. Potential utility of ferroptotic cell death has been recently proposed for cancer treatment. Meanwhile, ROS generation and apoptosis are inherently consequent to cell apoptosis and dysfunction during islet cell preparation and transplantation. Whether ferroptosis induction is a regulator for cell viability and function in human pancreatic islet-cell clusters (ICCs) derived from pancreatic progenitor cells (PPCs) remains elusive. We thus sought to induce ferroptosis in our established cell culture system of human PPCs/ICCs, examine the effects of ferroptosis on ICCs, and explore the potential regulatory pathways involved. Our results showed that ICCs were prone to the use of ferroptosis-inducing and inhibiting agents under our culture conditions. Erastin, a ferroptosis inducer, was found to trigger ferroptosis in ICCs, without the apparent detection of other types of cell death involved, such as apoptosis and autophagy. In corroboration, the use of ferroptosis inhibitor, ferrostatin-1 (Fer-1), was found to enhance the cell viability of ICCs and prevent them from ferroptosis as well as improve its function. Mechanistically, the erastin-induced ferroptosis in ICCs was probably mediated via activation of JNK/P38/MAPK pathways and upregulation of NOX4 expression. Together, our findings may provide a scientific basis of ferroptosis inhibition as a potential for the amelioration of ICC survival and functionality during islet transplantation in diabetic patients.

Alcohol ingestion induces pancreatic islet dysfunction and apoptosis via mediation of FGF21 resistance.

BACKGROUND: Disruption of ß-cell insulin secretion and viability caused by excessive ethanol consumption increases type 2 diabetes mellitus (T2DM) pathogenesis risk. Fibroblast growth factor 21 (FGF21) plays a significant role in regulating lipid and glucose homeostasis. Recently, FGF21, best known for its role in lipid and glucose homeostasis regulation, and its obligate co-receptor ß-klotho have been shown to inhibit ethanol ingestion and metabolism. It remains unclear whether heavy ethanol intake modulates islet FGF21 expression and function. This study investigated the relationship between ethanol exposure, FGF21, and islet function in vivo/ex vivo islet and in vitro cell models. METHODS: Mice were gavaged with 3.5 g/kg ethanol or saline for 1-3 weeks (long-term exposure). Human MIN6 cells and isolated islets were cultured and treated with 80 mM ethanol for 24 h (short-term exposure) to mimic excessive ethanol consumption. We applied the oral glucose tolerance test (OGTT), blood glucometry, enzyme-linked immunosorbent assay (ELISAs) for insulin and FGF21, glucose stimulated insulin secretion (GSIS) testing, reverse-transcription (RT)-polymerase chain reaction (PCR), and western blot experiments. RESULTS: Long-term ethanol treatment induced FGF21 resistance in mouse pancreatic islets. Moreover, ethanol exposure damaged insulin secretory ability and glucose homeostasis. In vitro and ex vivo experiments showed that short-term ethanol treatment upregulated the expression of FGF21 signaling pathway-related genes and proteins, without affecting ß-cell survival or function. CONCLUSIONS: Long-term ethanol consumption induces FGF21 resistance-mediated pancreatic ß-cell dysfunction, and thus diabetes pathogenesis risk.

Fibroblast growth factor 21 protects against lipotoxicity-induced pancreatic ß-cell dysfunction via regulation of AMPK signaling and lipid metabolism.

Fibroblast growth factor 21 (FGF21) is known as a potent metabolic regulator but its protective mechanisms against lipotoxicity-induced ß-cell dysfunction and apoptosis remain elusive. Here, we aimed to examine the regulatory pathways whereby FGF21 mediates islet lipid metabolism in lipotoxicity-treated cells and animal models. Rat ß-cell line (INS-1E cells) and islets isolated from C57/BL6J mice were exposed to palmitic acid (PA) with/without FGF21, mimicking lipotoxic conditions. Resultant insulin secretion and intracellular signaling were analyzed with Western blotting and RNA-seq. C57/BL6J and global FGF21 knockout (KO) mice were fed with a high-fat diet (HFD) to induce lipotoxicity and given with a long-acting mimetic of FGF21. Insulin resistance and ß-cell function were then assessed using homeostasis model assessment of insulin resistance (HOMA-IR) and insulinogenic index. FGF21 ameliorated PA-induced lipid accumulation, reversed cell apoptosis, and enhanced glucose-stimulated insulin secretion (GSIS) as impaired by lipotoxicity in islet ß-cells. Mechanistically, FGF21 exerted its beneficial effects through activation of AMPK-ACC (acetyl-CoA carboxylase) pathway and peroxisome proliferation-activated receptors (PPARs) δ/γ signaling, thus increasing the levels of carnitine palmitoyltransferase-1A (CPT1A) and leading to increased fatty acid (FA) oxidation and reduced lipid deposition in ß-cells. Interestingly, FGF21 reduced PA-induced cell death via restoration of the expression of apoptosis inhibitor Birc3. In vivo studies further showed that FGF21 is critical for islet insulinogenic capacity and normal function in the context of HFD-treated animals. FGF21 down-regulates islet cell lipid accumulation, probably via activation of AMPK-ACC and PPARδ/γ signaling, and reduces cell death under lipotoxicity, indicating that FGF21 is protective against lipotoxicity-induced ß-cell dysfunction and apoptosis.

SIRT1 Activation Promotes ß-Cell Regeneration by Activating Endocrine Progenitor Cells via AMPK Signaling-Mediated Fatty Acid Oxidation.

Induction of ß-cell regeneration from endogenous cells represents a highly promising strategy in stem cell-based treatment for patients with diabetes. Recently, calorie restriction has been shown to affect the regulation of tissue and cell regeneration, including ß cells, via metabolic related mechanisms. Here, we examined the potential utility of sirtuin 1 (SIRT1), a calorie restriction mimetic, for stimulating ß-cell regeneration and the underlying mechanisms of such stimulation. The present results showed that SIRT1 activation with SRT1720 promoted ß-cell regeneration in streptozotocin (STZ)-induced ß-cell-deficient neonatal rats. This beneficial effect involved enhanced activation of neurogenin3 (NGN3)-positive endocrine progenitors from pancreatic ductal cells, rather than an expansion of residual ß cells. A dynamic expression profile of SIRT1 was observed in endocrine progenitors both during ß-cell regeneration in neonatal rats and in the second transition phase of mouse pancreas development. Consistently, SRT1720 treatment upregulated endocrine progenitor differentiation in cultured pancreatic rudiments. Upregulation of NGN3 by SIRT1 activation was through stimulating AMP-activated protein kinase (AMPK) signaling-mediated fatty acid oxidation (FAO) in human pancreatic progenitor cells; AMPK inhibition abolished these effects. The present findings demonstrate a promotional effect of SIRT1 activation on ß-cell restoration and endocrine progenitor differentiation that involves regulation of AMPK signaling-mediated FAO. Stem Cells 2019;37:1416-1428.

Human Fetal Bone Marrow-Derived Mesenchymal Stem Cells Promote the Proliferation and Differentiation of Pancreatic Progenitor Cells and the Engraftment Function of Islet-Like Cell Clusters.

Pancreatic progenitor cells (PPCs) are the primary source for all pancreatic cells, including beta-cells, and thus the proliferation and differentiation of PPCs into islet-like cell clusters (ICCs) opens an avenue to providing transplantable islets for diabetic patients. Meanwhile, mesenchymal stem cells (MSCs) can enhance the development and function of different cell types of interest, but their role on PPCs remains unknown. We aimed to explore the mechanism-of-action whereby MSCs induce the in vitro and in vivo PPC/ICC development by means of our established co-culture system of human PPCs with human fetal bone marrow-derived MSCs. We examined the effect of MSC-conditioned medium on PPC proliferation and survival. Meanwhile, we studied the effect of MSC co-culture enhanced PPC/ICC function in vitro and in vivo co-/transplantation. Furthermore, we identified IGF1 as a critical factor responsible for the MSC effects on PPC differentiation and proliferation via IGF1-PI3K/Akt and IGF1-MEK/ERK1/2, respectively. In conclusion, our data indicate that MSCs stimulated the differentiation and proliferation of human PPCs via IGF1 signaling, and more importantly, promoted the in vivo engraftment function of ICCs. Taken together, our protocol may provide a mechanism-driven basis for the proliferation and differentiation of PPCs into clinically transplantable islets.

The Modulatory Action of Vitamin D on the Renin-Angiotensin System and the Determination of Hepatic Insulin Resistance.

Vitamin D deficiency or hypovitaminosis D is associated with increased risks of insulin resistance, type 2 diabetes mellitus (T2DM) and its related non-alcoholic fatty liver disease (NAFLD). Meanwhile, inappropriate over-activation of the renin-angiotensin system (RAS) in the liver leads to the hepatic dysfunction and increased risk of T2DM, such as abnormalities in lipid and glucose metabolism. Our previous findings have shown that calcitriol, an active metabolite of vitamin D, reduces hepatic triglyceride accumulation and glucose output in diabetic db/db mice and human hepatocellular cell HepG2 cells under insulin-resistant conditions. Notwithstanding the existence of this evidence, the protective action of vitamin D in the modulation of overexpressed RAS-induced metabolic abnormalities in the liver under insulin resistance remains to be elusive and investigated. Herein, we have reported the potential interaction between vitamin D and RAS; and its beneficial effects on the expression and function of the RAS components in HepG2 cells and primary hepatocytes under insulin-resistance states. Our study findings suggest that hormonal vitamin D (calcitriol) has modulatory action on the inappropriate upregulation of the hepatic RAS under insulin-resistant conditions. If confirmed, vitamin D supplementation might provide a nutraceutical potential as a cost-effective approach for the management of hepatic metabolic dysfunction as observed in T2DM and related NAFLD.

Fibroblast Growth Factor 21 Stimulates Pancreatic Islet Autophagy via Inhibition of AMPK-mTOR Signaling.

BACKGROUND: Islet autophagy plays a role in glucose/lipid metabolism in type 2 diabetes mellitus. Meanwhile, fibroblast growth factor 21 (FGF21) has been found to regulate insulin sensitivity and glucose homeostasis. Whether FGF21 induces islet autophagy, remains to be elucidated. This study aimed to explore the physiological roles and signaling pathways involved in FGF21-stimulated islet autophagy under glucolipotoxic conditions. METHODS: C57/BL6J mice were fed a standard diet or high-fat diet (HFD) for 12 weeks, and islets were isolated from normal and FGF21 knockout (KO) mice. Isolated islets and INS-1E cells were exposed to normal and high-concentration glucose and palmitic acid with/without FGF21 or AMPK inhibitor compound C. Real-time PCR, Western blot and immunohistochemistry/transmission electron microscopy were performed for the expression of targeted genes/proteins. RESULTS: HFD-treated mice showed increases in fasting plasma glucose, body weight and impaired glucose tolerance; islet protein expression of FGF21 was induced after HFD treatment. Protein expression levels of FGF21 and LC3-II (autophagy marker) were induced in mouse islets treated with high concentrations of palmitic acid and glucose, while phosphorylation of AMPK was reduced, compared with controls. In addition, induction of LC3-II protein expression was reduced in islets isolated from FGF21 KO mice. Furthermore, exogenous administration of FGF21 diminished phosphorylation of AMPK and stimulated protein expression of LC3-II. Consistently, compound C significantly induced increased expression of LC3-II protein. CONCLUSIONS: Our data indicate that glucolipotoxicity-induced FGF21 activation mediates islet autophagy via AMPK inhibition, and further consolidate the evidence for the FGF21/analog being a pharmacotherapeutic target for obesity and its related T2DM.

GPR120 protects lipotoxicity-induced pancreatic ß-cell dysfunction through regulation of PDX1 expression and inhibition of islet inflammation.

G-protein coupled receptor 120 (GPR120) has been shown to act as an omega-3 unsaturated fatty acid sensor and is involved in insulin secretion. However, the underlying mechanism in pancreatic ß cells remains unclear. To explore the potential link between GPR120 and ß-cell function, its agonists docosahexaenoic acid (DHA) and GSK137647A were used in palmitic acid (PA)-induced pancreatic ß-cell dysfunction, coupled with GPR120 knockdown (KD) in MIN6 cells and GPR120 knockout (KO) mice to identify the underlying signaling pathways. In vitro and ex vivo treatments of MIN6 cells and islets isolated from wild-type (WT) mice with DHA and GSK137647A restored pancreatic duodenal homeobox-1 (PDX1) expression levels and ß-cell function via inhibiting PA-induced elevation of proinflammatory chemokines and activation of nuclear factor κB, c-Jun amino (N)-terminal kinases1/2 and p38MAPK signaling pathways. On the contrary, these GPR120 agonism-mediated protective effects were abolished in GPR120 KD cells and islets isolated from GPR120 KO mice. Furthermore, GPR120 KO mice displayed glucose intolerance and insulin resistance relative to WT littermates, and ß-cell functional related genes were decreased while inflammation was exacerbated in islets with increased macrophages in pancreas from GPR120 KO mice. DHA and GSK137647A supplementation ameliorated glucose tolerance and insulin sensitivity, as well as improved Pdx1 expression and islet inflammation in diet-induced obese WT mice, but not in GPR120 KO mice. These findings indicate that GPR120 activation is protective against lipotoxicity-induced pancreatic ß-cell dysfunction, via the mediation of PDX1 expression and inhibition of islet inflammation, and that GPR120 activation may serve as a preventative and therapeutic target for obesity and diabetes.

Irisin Ameliorates Glucolipotoxicity-Associated ß-Cell Dysfunction and Apoptosis via AMPK Signaling and Anti-Inflammatory Actions.

BACKGROUND/AIMS: Islet metabolic disorder and inflammation contribute to the pathogenesis and progression of type 2 diabetes mellitus (T2DM). Irisin is a recently identified adipomyokine with protective effects on metabolic homeostasis and inflammation-suppressing effects in hepatic and vascular cells. The present study examined the effects of irisin on lipid metabolism and inflammation in ß cells under glucolipotoxic conditions. METHODS: Rat INS-1E ß cells and islets isolated from C57BL/6 mice were incubated in glucolipotoxic conditions with or without irisin. Intracellular lipid contents and lipogenic gene expression were determined by enzymatic colorimetric assays and real-time PCR, respectively. Inflammatory status was evidenced by Western blot analysis for the phosphorylation of nuclear factor-κB (NF-κB) p65 and real-time PCR analysis for the expression of pro-inflammatory genes. RESULTS: Irisin reversed glucolipotoxicity-induced intracellular non-esterified fatty acid (NEFA) and triglyceride accumulation, suppressed associated elevations in lipogenic gene expression, and phosphorylated acetyl-CoA-carboxylase (ACC) in INS-1E cells. These demonstrated effects were dependent on irisin-activated adenosine monophosphate-activated protein kinase (AMPK). Meanwhile, AMPK signaling mediated the protective effects of irisin on INS-1E cell insulin secretory ability and survival as well. Additionally, irisin inhibited phosphorylation of NF-κB p65 while decreasing the expression of pro-inflammatory genes in INS-1E cells under glucolipotoxic conditions. Moreover, irisin also improved insulin secretion, inhibited apoptosis, and restored ß-cell function-related gene expression in isolated mouse islets under glucolipotoxic conditions. CONCLUSION: Irisin attenuated excessive lipogenesis in INS-1E cells under glucolipotoxic state through activation of AMPK. Irisin also suppressed overnutrition-induced inflammation in INS-1E cells. Our findings implicate irisin as a promising therapeutic target for the treatment of islet lipid metabolic disorder and islet inflammation in T2DM.

GPR120 is an important inflammatory regulator in the development of osteoarthritis.

BACKGROUND: The aim of this study was to investigate the regulatory role of G-protein coupled receptor 120 (GPR120) in the development and progression of osteoarthritis (OA). METHODS: GPR120 knockout (KO) and wild-type (WT) mice were used to create an animal model of OA by means of anterior cruciate ligament transection (ACLT) surgery. The severity of OA was staged and evaluated by histological examination, microcomputed tomography scan and enzyme-linked immunosorbent assay (ELISA). The anti-inflammatory effects of the GPR120 agonist docosahexaenoic acid (DHA) on human chondrocytes were further evaluated by specific inflammatory markers. In addition, the healing progression of a skin defect model was determined with histological assays. RESULTS: The GPR120-KO mice displayed an accelerated development of OA after ACLT. The secondary inflammation, cartilage degeneration, and subchondral bone aberrant changes were significantly elevated in the early phase of OA in KO mice relative to those in WT mice. In addition, we found that GPR120 levels were downregulated in OA patients compared with control subjects, whereas GPR120 activation with DHA exhibited anti-inflammatory effects in primary human chondrocytes in vitro. Moreover, results from the skin defect model showed that GPR120 agonism with DHA enhanced wound repair in mice, as shown by the downregulation of the number of CD68+ cells. CONCLUSIONS: Our study suggests that GPR120 is an important inflammatory mediator during the development of OA, and that it is a potential marker for the diagnosis of high-risk patients with OA.

Na+ /H+ exchanger 3 blockade ameliorates type 2 diabetes mellitus via inhibition of sodium-glucose co-transporter 1-mediated glucose absorption in the small intestine.

AIM: To elucidate the role of Na+ /H+ exchanger 3 (NHE3) in sodium-glucose co-transporter 1 (SGLT1)-mediated small intestinal brush border membrane (BBM) glucose absorption and its functional implications in type 2 diabetes mellitus (T2DM). MATERIALS AND METHODS: Human jejunal samples were obtained from patients undergoing gastrectomy. 14 C-glucose absorption was measured by liquid scintillation counting. NHE3 expression was suppressed by siRNA-mediated knockdown or augmented in Caco2 cells. Glucose and insulin tolerance in db/db and m+/db mice was assessed with oral and intraperitoneal glucose tolerance tests, and an intraperitoneal insulin tolerance test. Insulin resistance and ß-cell function were assessed using homeostatic model assessment of insulin resistance and ß-cell function. RESULTS: NHE3 expression was upregulated in db/db mouse jejunal BBM and high-glucose-treated Caco2 cells. NHE3 blockade impaired SGLT1-mediated glucose absorption in human jejunum, m+/db and db/db mouse jejunums, and Caco2 cells, via serum/glucocorticoid-regulated kinase 1 (SGK1). NHE3 knockdown suppressed SGLT1-mediated glucose uptake and reduced mRNA and protein levels of SGK1 and SGLT1, which were conversely enhanced by NHE3 overexpression. Chronic S3226 treatment diminished postprandial glucose levels and ameliorated glucose intolerance in db/db mice. CONCLUSION: NHE3 is essential in the modulation of small intestinal BBM glucose absorption. Our findings provide a rationale for future possible clinical application of NHE3 for treatment of T2DM through reducing intestinal glucose uptake and counteracting postprandial hyperglycaemia.

Exploring brusatol as a new anti-pancreatic cancer adjuvant: biological evaluation and mechanistic studies.

Pancreatic cancer is highly resistant to chemotherapeutic agents and is known to have a poor prognosis. The development of new therapeutic entities is badly needed for this deadly malignancy. In this study, we demonstrated for the first time that brusatol, a natural quassinoid isolated from a Chinese herbal medicine named Bruceae Fructus, possessed potent cytotoxic effect against different pancreatic adenocarcinoma cell lines. Its anti-pancreatic cancer effect was comparable to that of the first-line chemotherapeutic agents such as gemcitabine and 5-fluorouracil, with a more favorable safety profile. In addition, brusatol showed a synergistic anti-proliferative effect toward PANC-1 and Capan-2 cell lines when combined with gemcitabine or 5-fluorouracil. The results of flow cytometry suggested that brusatol combination treatment with gemcitabine or 5-fluorouracil was able to cause cell cycle arrest at G2/M phase, and accentuate apoptosis in PANC-1 cells. Moreover, brusatol deactivated gemcitabine/5-fluorouracil-induced NF-κB activation. Western blot analysis and qRT-PCR results showed that brusatol significantly down-regulated the expression of vimentin and Twist, and markedly stimulated the expression of E-cadherin, the key regulatory factors of the epithelial-mesenchymal transition process. Furthermore, treatment with combination of brusatol and gemcitabine or 5-fluorouracil significantly reduced in vivo tumor growth when compared with treatment of either brusatol or gemcitabine/5-fluorouracil alone. Taken together, these results have amply demonstrated that brusatol is a potent anti-pancreatic cancer natural compound, and the synergistic anti-pancreatic cancer effects of brusatol and gemcitabine/5-fluorouracil observed both in vitro and in vivo are associated with the suppression of epithelial-mesenchymal transition process, indicating that brusatol is a promising adjunct to the current chemotherapeutic regimen.

Fibroblast growth factor 21: a regulator of metabolic disease and health span.

Fibroblast growth factor 21 (FGF21) is a potent endocrine regulator with physiological effects on glucose and lipid metabolism and thus garners much attention for its translational potential for the management of obesity and related metabolic syndromes. FGF21 is mainly expressed in several metabolically active tissue organs, such as the liver, adipose tissue, skeletal muscle, and pancreas, with profound effects and therapeutic relevance. Emerging experimental and clinical data point to the demonstrated metabolic benefits of FGF21, which include, but are not limited to, weight loss, glucose and lipid metabolism, and insulin sensitivity. In addition, FGF21 also acts directly through its coreceptor ß-klotho in the brain to alter light-dark cycle activity. In this review, we critically appraise current advances in understanding the physiological actions of FGF21 and its role as a biomarker of various metabolic diseases, especially type 2 diabetes mellitus. We also discuss the potentially exciting role of FGF21 in improving our health and prolonging our life span. This information will provide a fuller understanding for further research into FGF21, as well as providing a scientific basis for potentially establishing health care guidelines for this promising molecule.