Leonurine protects against dexamethasone-induced cytotoxicity in pancreatic ß-cells via PI3K/Akt signaling pathway.

Glucocorticoid excess induces pancreatic ß-cell apoptosis and insulin secretion impairment, which may lead to hyperglycemia and steroid diabetes. Leonurine is a natural alkaloid extracted from the Herba leonuri, which has been widely used in the treatment of obstetric and gynecological diseases. However, whether leonurine performs a protective role in pancreatic ß-cells remains unknown. In this study, we evaluated the effect of leonurine on dexamethasone -treated ß-cells. Our data showed that leonurine inhibited dexamethasone-induced INS-1 cell apoptosis and facilitated cell proliferation. Moreover, leonurine attenuated dexamethasone-impaired insulin secretion in mice islets. Leonurine ameliorated dexamethasone-induced dephosphorylation of Akt, Bad and GSK-3ß. Importantly, the protective role of leonurine on dexamethasone-induced cytotoxicity was blocked by LY294002 in INS-1 cells. Our findings revealed for the first time that leonurine could protect against dexamethasone-induced cytotoxicity in pancreatic ß-cells via PI3K/Akt signaling pathway, suggesting leonurine may be a promising therapeutic agent for steroid diabetes.

Glucose potentiates ß-cell function by inducing Tph1 expression in rat islets.

Impaired pancreatic ß-cell function is the primary defect in type 2 diabetes. Glucose is an important regulator of ß-cell growth and function; however, the mechanisms that are involved in the chronic adaptation of ß cells to hyperglycemia remain largely unknown. In the present study, global gene expression patterns revealed that tryptophan hydroxylase 1 (Tph1) was the most profound of genes that are up-regulated in rat islets exposed to high glucose. Calcium and cAMP signals synergistically mediated glucose-stimulated Tph1 transcription in ß cells by activating cAMP-responsive element-binding protein and promoting its binding with a Tph1 promoter. Similar to in vitro results, in vivo infusion of high glucose also strongly induced Tph1 expression and serotonin production in rat islets, along with enhanced islet function. Inhibition or knockdown of Tph1 markedly decreased glucose-potentiated insulin secretion. In contrast, overexpression of Tph1 augmented glucose-stimulated insulin secretion in rat islets by up-regulating the expression of genes that are related to islet function. In addition, the long-acting glucagon-like peptide 1 receptor agonist, exendin-4, stimulated Tph1 expression in a glucose-dependent manner. Knockdown of Tph1 inhibited exendin-4-potentiated insulin secretion in rat islets. These findings suggest that Tph1 mediates the compensation of islet function induced by glucose, and that promoting Tph1 expression in pancreatic ß cells will provide a new strategy for the treatment of type 2 diabetes mellitus.-Zhang, Y., Deng, R., Yang, X., Xu, W., Liu, Y., Li, F., Zhang, J., Tang, H., Ji, X., Bi, Y., Wang, X., Zhou, L., Ning, G. Glucose potentiates ß-cell function by inducing Tph1 expression in rat islets.

Glucose-derived AGEs promote migration and invasion of colorectal cancer by up-regulating Sp1 expression.

It is well established that the risk of colorectal cancer (CRC) is significantly increased in diabetic patients. As one of main forms of the advanced glycation end products (AGEs) that accumulate in vivo, glucose-derived AGEs play an important role in the pathogenesis of diabetic complications and may contribute to CRC progression. However, to date, both the contribution of glucose-derived AGEs to the course of CRC and the underlying mechanism are unclear. In the present study, the concentration of glucose-derived AGEs in the serum and tumor tissue of patients with CRC increased. A clinical data analysis demonstrated that the expression of the receptor for AGEs (RAGE), Specificity Protein 1 (Sp1), and matrix metallopeptidase -2 (MMP2) was significantly higher in cancerous tissues compared with non-tumor tissue in Chinese Han patients with CRC and that RAGE expression was closely associated with lymph node metastasis and TNM stage. Furthermore, in vivo and in vitro experiments showed that AGEs promoted invasion and migration of colorectal cancer, and the AGEs treatment increased the expression of RAGE, Sp1, and MMP2 in a dose-dependent manner. A RAGE blocking antibody and an Sp1-specific siRNA attenuated the AGE-induced effects. Moreover, the AGEs treatment increased the phosphorylation of ERK, and reducing the phosphorylation level of ERK by MEK1/2 inhibitor decreased the expression of Sp1. In conclusion, glucose-derived AGEs promote the invasion and metastasis of CRC partially through the RAGE/ERK/SP1/MMP2 cascade. These findings may provide an explanation for the poor prognoses of colorectal cancer in diabetic patients.

Glucose enhances rat islet function via stimulating CART expression.

Cocaine- and amphetamine-regulated transcript (CART) is an anorexigenic peptide widely expressed in the central and peripheral nervous systems, as well as in endocrine cells. CART is markedly upregulated in the ß-cells of several rodent models of type-2 diabetes. The stimulatory effect of exogenous CART peptide on insulin secretion is cAMP dependent. Glucose is the most important regulator of islet function. However, the role of CART in glucose-potentiated insulin secretion remains unclear. Here, our results showed that glucose time- and dose-dependently elicited CART mRNA expression in rat islets. Both the glucokinase agonist GKA50 and the long-acting GLP-1 analogue exendin-4 increased CART mRNA expression. The protein kinase A (PKA) inhibitor H89 and the inactivation of cAMP response element-binding protein (CREB) suppressed forskolin-stimulated CART mRNA expression. Furthermore, CART overexpression amplified insulin secretion from rat islets in response to glucose and forskolin, and ameliorated dexamethasone-impaired insulin secretion. These findings suggest that islet-derived CART is involved, at least in part, in high glucose-potentiated pancreatic ß-cell function.

Acute exposure of beta-cells to troglitazone decreases insulin hypersecretion via activating AMPK.

BACKGROUND: It has been recognized that insulin hypersecretion can lead to the development of insulin resistance and type 2 diabetes mellitus. There is substantial evidence demonstrating that thiazolidinediones are able to delay and prevent the progression of pancreatic ß-cell dysfunction. However, the mechanism underlying the protective effect of thiazolidinediones on ß-cell function remains elusive. METHODS: We synchronously detected the effects of troglitazone on insulin secretion and AMP-activated protein kinase (AMPK) activity under various conditions in isolated rat islets and MIN6 cells. RESULTS: Long-term exposure to high glucose stimulated insulin hypersecretion and inhibited AMPK activity in rat islets. Troglitazone-suppressed insulin hypersecretion was closely related to the activation of AMPK. This action was most prominent at the moderate concentration of glucose. Glucose-stimulated insulin secretion was decreased by long-term troglitazone treatment, but significantly increased after the drug withdrawal. Compound C, an AMPK inhibitor, reversed troglitazone-suppressed insulin secretion in MIN6 cells and rat islets. Knockdown of AMPKα2 showed a similar result. In MIN6 cells, troglitazone blocked high glucose-closed ATP-sensitive K(+) (KATP) channel and decreased membrane potential, along with increased voltage-dependent potassium channel currents. Troglitazone suppressed intracellular Ca(2+) response to high glucose, which was abolished by treatment with compound C. CONCLUSION: Our results suggest that troglitazone provides ß-cell "a rest" through activating AMPK and inhibiting insulin hypersecretion, and thus restores its response to glucose. GENERAL SIGNIFICANCE: These data support that AMPK activation may be an important mechanism for thiazolidinediones preserving ß-cell function.

Calycosin-7-O-ß-D-glucoside attenuates palmitate-induced lipid accumulation in hepatocytes through AMPK activation.

Calycosin-7-O-ß-D-glucoside (CG) is the major component of Astragali Radix (AR), a traditional Chinese drug. As reported, CG could attenuate cerebral ischemia/reperfusion injury, protect blood-brain barrier integrity, and ameliorate myocardial infarction. To date, whether CG has a protective effect on metabolic diseases remains to be elucidated. In the present study, CG could attenuate palmitate-induced lipid accumulation in hepatocytes in a dose-dependent manner, with down-regulation of lipogenesis related genes expression and up-regulation of lipids ß-oxidation related genes expression. CG could decrease the triglyceride (TG) content from 0.30 mmol/g protein to 0.21 mmol/g protein and reduce the total cholesterol (TC) content from 0.39 mmol/g protein to 0.26 mmol/g protein. Moreover, CG stimulated the phosphorylation of AMP-activated protein kinase (AMPK), and the protective effect of CG on hepatocytes was partially reversed both by the inhibitor of AMPK signaling pathway and overexpression of AMPK-DN. Our findings revealed that CG could ameliorate palmitate-induced lipids accumulation in hepatocytes via AMPK activation and it may be a promising therapeutic medicine for hepatic steatosis.

Selective inhibition of CBP/p300 HAT by A-485 results in suppression of lipogenesis and hepatic gluconeogenesis.

The histone acetyltransferases CREB-binding protein (CBP) and its paralogue p300 are transcriptional coactivators which are essential for a multitude of signaling pathways and energy homeostasis. However, the role of CBP/p300 HAT domain in regulating energy balance is still unclear. Here, C57BL/6 mice fed with either normal chow diet (NCD) or high-fat diet (HFD) were administrated with A-485, a recently reported selective inhibitor of CBP/p300 HAT activity for 1 week and the metabolic change was analyzed. The white adipose tissue (WAT) weight and adipocyte size were reduced in A-485-administrated mice, with decreased expressions of lipogenic genes and transcriptional factors. In the liver of A-485-treated mice, the lipid content and lipogenic gene expressions were lowered while the binding of forkhead box O1 (FOXO1) to glucose-6-phosphatase (G6Pc) promoter was reduced, leading to decreased expression of G6Pc. In primary mouse hepatocytes, A-485 abolished cAMP-elicited mRNA expressions of key gluconeogenic enzymes and promoted FOXO1 protein degradation via increasing its ubiquitination. Thus, A-485 inhibits lipogenesis in WAT and liver as well as decreases hepatic glucose production via preventing FOXO1 acetylation, leading to its protein degradation through a proteasome-dependent pathway. The specific inhibition of CBP/p300 HAT will provide a novel therapeutic approach for metabolic diseases.

MS-275 induces hepatic FGF21 expression via H3K18ac-mediated CREBH signal.

Fibroblast growth factor 21 (FGF21) plays an important role in the regulation of lipid and glucose metabolism. MS-275, as a class I-specific histone deacetylase (HDAC) inhibitor, has also been reported to affect energy metabolism. In this current study, we investigated the effects of MS-275 on hepatic FGF21 expression in vitro and in vivo and explored whether cAMP-responsive element-binding protein H (CREBH) was involved in the action of MS-275. Our results showed that MS-275 stimulated hepatic FGF21 mRNA and protein expressions in a dose- and time-dependent manner, as well as FGF21 secretion in primary mouse hepatocytes. Serum concentration and hepatic expression of FGF21 were elevated after injection of MS-275, along with increased expressions of genes involved in fatty acid oxidation and ketogenic production (peroxisome proliferator-activated receptor gammacoactivator1α, PGC-1α; carnitine palmitoyl-transferase 1a, CPT1a; 3-hydroxy-3-methylglutaryl-CoA synthase 2, Hmgcs2) as well as improved blood lipid profile. As a proved transcription factor of FGF21, the expression of CREBH was initiated by MS-275, with increased histone H3 lysine 18 acetylation (H3K18ac) signals and hepatocyte nuclear factor 4 alpha (HNF-4α) recruitment in CREBH promoter. Adenovirus-mediated knockdown of CREBH abolished MS-275-induced hepatic FGF21 and lipid metabolism-related gene expressions. These results suggest that MS-275 induces hepatic FGF21 by H3K18ac-mediated CREBH expression.

Butyrate stimulates hepatic gluconeogenesis in mouse primary hepatocytes.

Butyrate is a major short-chain fatty acid (SCFA) produced by microbial fermentation of dietary fiber in the gastrointestinal tract. Butyrate is also a well-known broad-spectrum histone deacetylase (HDAC) inhibitor. Butyrate has been reported to improve energy metabolism in rodents, which is associated with its beneficial effects on skeletal muscle, brown fat tissue and pancreatic ß-cells. The present study investigated the direct effect of butyrate on hepatic gluconeogenesis in mouse primary hepatocytes and the underlying mechanism. Isolated mouse primary hepatocytes were incubated with sodium butyrate, other HDAC inhibitors and other SCFAs. Hepatic glucose production was measured and gluconeogenic gene expression was detected by polymerase chain reaction analysis. The phosphorylation of cyclic adenosine monophosphate (cAMP) response element binding protein (CREB) was assessed by western blot analysis. The results revealed that sodium butyrate dose-dependently increased hepatic glucose production and gluconeogenic gene expression in isolated mouse primary hepatocytes. Trichostatin A, a potent broad-spectrum HDAC inhibitor, had the opposite effect. Similar to sodium butyrate, propionate, which is another SCFA, promoted hepatic glucose production and gluconeogenic gene expression in the presence or absence of gluconeogenic substrates, which were further enhanced by cAMP. Furthermore, sodium butyrate also increased the accumulation of intracellular ATP and induced the phosphorylation of CREB in mouse hepatocytes. In conclusion, the present study suggested that butyrate stimulates hepatic gluconeogenesis and induces gluconeogenic gene expression as a substrate and cAMP/CREB signaling activator.

Expression profile analysis of long non-coding RNAs involved in the metformin-inhibited gluconeogenesis of primary mouse hepatocytes.

Long non-coding RNAs (lncRNAs) have been demonstrated to regulate metabolic tissue development and function, including adipogenesis, hepatic lipid metabolism, islet function and energy balance. However, the role of lncRNAs in gluconeogenesis remains completely unknown. Metformin reduces glucose output mainly via the inhibition of gluconeogenesis. In the present study, the lncRNA expression profile of primary mouse hepatocytes exposed to cyclic adenosine monophosphate (cAMP), a gluconeogenic stimulus, with or without metformin was analyzed by microarray. Among the 22,016 lncRNAs that were identified, 456 were upregulated and 409 were downregulated by cAMP (fold-change ≥2.0). Furthermore, the cAMP-induced upregulation of 189 lncRNAs and downregulation of 167 lncRNAs was attenuated by metformin. The expression levels of eight lncRNAs were validated by reverse transcription-quantitative polymerase chain reaction, and the results were consistent with those of the microarray analysis. Among them, two lncRNAs NR_027710 and ENSMUST00000138573, were identified to have an association with two protein coding genes, namely peroxisome proliferator-activated receptor-γ coactivator-1α, a critical transcriptional coactivator in gluconeogenesis, and G protein-coupled receptor 155, respectively. The two protein coding genes exhibited similar expression patterns to their associated lncRNAs. The findings of the present study suggest that lncRNAs are potentially involved in the regulation of gluconeogenesis.

Stearoyl-CoA desaturase-1 promotes colorectal cancer metastasis in response to glucose by suppressing PTEN.

BACKGROUND: Diabetic patients have a higher risk factor for colorectal cancer (CRC) metastasis. Stearoyl-CoA desaturase 1 (SCD1), the main enzyme responsible for producing monounsaturated fatty acids(MUFA) from saturated fatty acids, is frequently deregulated in both diabetes and CRC. The function and mechanism of SCD1 in metastasis of CRC and its relevance to glucose remains largely unknown. METHODS: SCD1 expression levels were analyzed in human CRC tissues and the Cancer Browser database ( https://genome-cancer.ucsc.edu/ ). CRC cell lines stably transfected with SCD1 shRNAs or vector were established to investigate the role of SCD1 in modulating migration and invasion of CRC cells. A glucose concentration gradient was set to investigate regulation of SCD1 in CRC relevant to diabetic conditions. RESULTS: The clinical data analysis showed high expression of SCD1 in CRC tissues with a negative correlation with the prognosis of CRC. In vitro experiments revealed that SCD1 increased CRC progression through promoting epithelial-mesenchymal transition (EMT). Lipidomic analysis demonstrated that SCD1 increased MUFA levels and MUFA administration could rescue migration and invasion defect of CRC cells induced by SCD1 knockdown. Furthermore, SCD1-mediated progression of CRC was promoted by carbohydrate response-element binding protein (ChREBP) in response to high glucose. Mechanistically, hyperglycemia-SCD1-MUFA induced CRC cell migration and invasion by regulating PTEN. CONCLUSIONS: Our findings show that SCD1 promotes metastasis of CRC cells through MUFA production and suppressing PTEN in response to glucose, which may be a novel mechanism for diabetes-induced CRC metastasis.

Glucose-derived AGEs enhance human gastric cancer metastasis through RAGE/ERK/Sp1/MMP2 cascade.

Advanced glycation end products (AGEs) have been reported to take part in many cancer processes. Whether AGEs contribute to gastric cancer (GC) course and the underlying mechanism are still unclear. Here, glucose-derived AGEs are detected to be accumulated in tumor tissues and blood of patients with GC. As the receptor for AGEs, RAGE is highly expressed in cancer tissues, and closely associated with the depth of cancer invasion, lymph node metastasis and TNM stage. Both in vivo and in vitro treatment of AGEs accelerate the tumor invasion and metastasis, with upregualtion of RAGE, Specificity Protein 1 (Sp1), and MMP2 protein expression, as well as enhancement of MMP2 activity. Either RAGE-blocking antibody or Sp1-knockdown can partially block the AGEs-induced effects. Moreover, AGEs increased the phosphorylation of ERK, and reducing the phosphorylation level of ERK by MEK1/2 inhibitor decreased the expression of Sp1. These results indicate that accumulation of glucose-derived AGEs may act as one of potential risk factors for GC progression and promote the invasion and metastasis of gastric cancer partially through the activation of RAGE/ERK/Sp1/MMP2 pathway.

Potential role of Hsp90 in rat islet function under the condition of high glucose.

AIMS: The preservation of pancreatic ß-cell function is a key point in the treatment of type 2 diabetes mellitus. There is substantial evidence demonstrating that heat-shock protein 90 (Hsp90) is needed for the stabilization and correct folding of client proteins and plays important roles in various biological processes. Here, we revealed the important role of Hsp90 in ß-cell function. METHODS: Islets from male Sprague-Dawley rats were isolated to be used for further RT-PCR, Western blot, and insulin secretion test ex vivo in response to different stimuli. RESULTS: Our results revealed that Hsp90 expression was significantly decreased in isolated rat islets exposed to high glucose, which was involved in glucokinase activation and glucose metabolism, not calcium signaling. Two kinds of Hsp90 inhibitors 17-DMAG and CCT018159 markedly enhanced glucose-stimulated insulin secretion from rat islets, along with increased expressions of genes closely related to ß-cell function. CONCLUSIONS: These data indicate that Hsp90 may be involved in high glucose-induced islet function adaptation.

Sesamin Ameliorates Advanced Glycation End Products-Induced Pancreatic ß-Cell Dysfunction and Apoptosis.

Advanced glycation end products (AGEs), the direct modulators of ß-cells, have been shown to cause insulin-producing ß-cell dysfunction and apoptosis through increase of intracellular reactive oxygen species (ROS) production. Sesamin has been demonstrated to possess antioxidative activity. This study was designed to investigate whether sesamin protects against AGEs-evoked ß-cell damage via its antioxidant property. The effects of sesamin were examined in C57BL/6J mice and MIN6 cell line. In in vivo studies, mice were intraperitoneally injected with AGEs (120 mg/kg) and orally treated with sesamin (160 mg/kg) for four weeks. Intraperitoneal glucose tolerance and insulin releasing tests were performed. Insulin content, ROS generation and ß-cell apoptosis in pancreatic islets were also measured. In in vitro studies, MIN6 cells were pretreated with sesamin (50 or 100 µM) and then exposed to AGEs (200 mg/L) for 24 h. Insulin secretion, ß-cell death, ROS production as well as expression and activity of NADPH oxidase were determined. Sesamin treatment obviously ameliorated AGE-induced ß-cell dysfunction and apoptosis both in vivo and in vitro. These effects were associated with decreased ROS production, down-regulated expression of p67(phox) and p22(phox), and reduced NADPH oxidase activity. These results suggest that sesamin protects ß-cells from damage caused by AGEs through suppressing NADPH oxidase-mediated oxidative stress.

Berberine Suppresses Adipocyte Differentiation via Decreasing CREB Transcriptional Activity.

Berberine, one of the major constituents of Chinese herb Rhizoma coptidis, has been demonstrated to lower blood glucose, blood lipid, and body weight in patients with type 2 diabetes mellitus. The anti-obesity effect of berberine has been attributed to its anti-adipogenic activity. However, the underlying molecular mechanism remains largely unknown. In the present study, we found that berberine significantly suppressed the expressions of CCAAT/enhancer-binding protein (C/EBP)α, peroxisome proliferators-activated receptor γ2 (PPARγ2), and other adipogenic genes in the process of adipogenesis. Berberine decreased cAMP-response element-binding protein (CREB) phosphorylation and C/EBPß expression at the early stage of 3T3-L1 preadipocyte differentiation. In addition, CREB phosphorylation and C/EBPß expression induced by 3-isobutyl-1-methylxanthine (IBMX) and forskolin were also attenuated by berberine. The binding activities of cAMP responsive element (CRE) stimulated by IBMX and forskolin were inhibited by berberine. The binding of phosphorylated CREB to the promoter of C/EBPß was abrogated by berberine after the induction of preadipocyte differentiation. These results suggest that berberine blocks adipogenesis mainly via suppressing CREB activity, which leads to a decrease in C/EBPß-triggered transcriptional cascades.

Identification of suitable reference genes for quantitative RT-PCR during 3T3-L1 adipocyte differentiation.

Quantitative reverse transcription PCR (qRT-PCR) is becoming increasingly important in the effort to gain insight into the molecular mechanisms underlying adipogenesis. However, the expression profile of a target gene may be misinterpreted due to the unstable expression of the reference genes under different experimental conditions. Therefore, in this study, we investigated the expression stability of 10 commonly used reference genes during 3T3-L1 adipocyte differentiation. The mRNA expression levels of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and transferrin receptor (TFRC) significantly increased during the course of 3T3-L1 adipocyte differentiation, which was decreased by berberine, an inhibitor of adipogenesis. Three popular algorithms, GeNorm, NormFinder and BestKeeper, identified 18 ribosomal RNA and hydroxymethylbilane synthase (HMBS) as the most stable reference genes, while GAPDH and TFRC were the least stable ones. Peptidylprolyl isomerase A [PIPA (cyclophilin A)], ribosomal protein, large, P0 (36-B4), beta-2-microglobulin (B2M), α1-tubulin, hypoxanthine-guanine phosphoribosyltransferase (HPRT) and ß-actin showed relatively stable expression levels. The choice of reference genes with various expression stabilities exerted a profound influence on the expression profiles of 2 target genes, peroxisome proliferator-activated receptor (PPAR)γ2 and C/EBPα. In addition, western blot analysis revealed that the increased protein expression of GAPDH was markedly inhibited by berberine during adipocyte differentiation. This study highlights the importance of selecting suitable reference genes for qRT-PCR studies of gene expression during the process of adipogenesis.