Hepatic Fibroblast Growth Factor 21 Is Involved in Mediating Functions of Liraglutide in Mice With Dietary Challenge.

BACKGROUND AND AIMS: Several studies have shown that expression of hepatic fibroblast growth factor 21 (FGF21) can be stimulated by glucagon-like peptide 1 (GLP-1)-based diabetes drugs. As GLP-1 receptor (GLP-1R) is unlikely to be expressed in hepatocytes, we aimed to compare such stimulation in mice and in mouse hepatocytes, determine the involvement of GLP-1R, and clarify whether FGF21 mediates certain functions of the GLP-1R agonist liraglutide. APPROACH AND RESULTS: Liver FGF21 expression was assessed in mice receiving a daily liraglutide injection for 3 days or in mouse primary hepatocytes (MPHs) undergoing direct liraglutide treatment. The effects of liraglutide on metabolic improvement and FGF21 expression were then assessed in high-fat diet (HFD)-fed mice and compared with the effects of the dipeptidyl-peptidase 4 inhibitor sitagliptin. Animal studies were also performed in Glp1r-/- mice and liver-specific FGF21-knockout (lFgf21-KO) mice. In wild-type mouse liver that underwent RNA sequencing and quantitative reverse-transcription PCR, we observed liraglutide-stimulated hepatic Fgf21 expression and a lack of Glp1r expression. In MPHs, liraglutide did not stimulate Fgf21. In mice with HFD-induced obesity, liraglutide or sitagliptin treatment reduced plasma triglyceride levels, whereas their effect on reducing body-weight gain was different. Importantly, increased hepatic FGF21 expression was observed in liraglutide-treated mice but was not observed in sitagliptin-treated mice. In HFD-fed Glp1r-/- mice, liraglutide showed no beneficial effects and could not stimulate Fgf21 expression. In lFgf21-KO mice undergoing dietary challenge, the body-weight-gain attenuation and lipid homeostatic effects of liraglutide were lost or significantly reduced. CONCLUSIONS: We suggest that liraglutide-stimulated hepatic Fgf21 expression may require GLP-1R to be expressed in extrahepatic organs. Importantly, we revealed that hepatic FGF21 is required for liraglutide to lower body weight and improve hepatic lipid homeostasis. These observations advanced our mechanistic understanding of the function of GLP-1-based drugs in NAFLD.

The developmental Wnt signaling pathway effector ß-catenin/TCF mediates hepatic functions of the sex hormone estradiol in regulating lipid metabolism.

The bipartite transcription factor ß-catenin (ß-cat)/T cell factor (TCF), formed by free ß-cat and a given TCF family member, serves as the effector of the developmental Wnt signaling cascade. ß-cat/TCFs also serve as effectors of certain peptide hormones or growth factors during adulthood. We reported that liver-specific expression of dominant-negative Transcription factor 7 like 2 (TCF7L2DN) led to impaired glucose disposal. Here we show that, in this LTCFDN transgenic mouse model, serum and hepatic lipid contents were elevated in male but not in female mice. In hepatocytes, TCF7L2DN adenovirus infection led to stimulated expression of genes that encode lipogenic transcription factors and lipogenic enzymes, while estradiol (E2) treatment attenuated the stimulation, associated with Wnt-target gene activation. Mechanistically, this E2-mediated activation can be attributed to elevated ß-cat Ser675 phosphorylation and TCF expression. In wild-type female mice, ovariectomy (OVX) plus high-fat diet (HFD) challenge impaired glucose disposal and insulin tolerance, associated with increased hepatic lipogenic transcription factor sterol regulatory element-binding protein 1-c (SREBP-1c) expression. In wild-type mice with OVX, E2 reconstitution attenuated HFD-induced metabolic defects. Some of the attenuation effects, including insulin intolerance, elevated liver-weight gain, and hepatic SREBP-1c expression, were not affected by E2 reconstitution in HFD-fed LTCFDN mice with OVX. Finally, the effects of E2 in hepatocytes on ß-cat/TCF activation can be attenuated by the G-protein-coupled estrogen receptor (GPER) antagonist G15. Our study thus expanded the scope of functions of the Wnt pathway effector ß-cat/TCF, as it can also mediate hepatic functions of E2 during adulthood. This study also enriches our mechanistic understanding of gender differences in the risk and pathophysiology of metabolic diseases.

Estrogen-Wnt signaling cascade regulates expression of hepatic fibroblast growth factor 21.

We have generated the transgenic mouse line LTCFDN in which dominant negative TCF7L2 (TCF7L2DN) is specifically expressed in the liver during adulthood. Male but not female LTCFDN mice showed elevated hepatic and plasma triglyceride (TG) levels, indicating the existence of estrogen-ß-cat/TCF signaling cascade that regulates hepatic lipid homeostasis. We show here that hepatic fibroblast growth factor 21 (FGF21) expression was reduced in male but not in female LTCFDN mice. The reduction was not associated with altered hepatic expression of peroxisome proliferator-activated receptor α (PPARα). In mouse primary hepatocytes (MPH), Wnt-3a treatment increased FGF21 expression in the presence of PPARα inhibitor. Results from our luciferase-reporter assay and chromatin immunoprecipitation suggest that evolutionarily conserved TCF binding motifs (TCFBs) on Fgf21 promoter mediate Wnt-3a-induced Fgf21 transactivation. Female mice showed reduced hepatic FGF21 production and circulating FGF21 level following ovariectomy (OVX), associated with reduced hepatic TCF expression and ß-catenin S675 phosphorylation. Finally, in MPH, estradiol (E2) treatment enhanced FGF21 expression, as well as binding of TCF7L2 and ribonucleic acid (RNA) polymerase II to the Fgf21 promoter; and the enhancement can be attenuated by the G-protein-coupled estrogen receptor 1 (GPER) antagonist G15. Our observations hence indicate that hepatic FGF21 is among the effectors of the newly recognized E2-ß-cat/TCF signaling cascade.NEW & NOTEWORTHY FGF21 is mainly produced in the liver. Therapeutic effect of FGF21 analogues has been demonstrated in clinical trials on reducing hyperlipidemia. We show here that Fgf21 transcription is positively regulated by Wnt pathway effector ß-cat/TCF. Importantly, hepatic ß-cat/TCF activity can be regulated by the female hormone estradiol, involving GPER. The investigation enriched our understanding on hepatic FGF21 hormone production, and expanded our view on metabolic functions of the Wnt pathway in the liver.

Glucagon-like peptide-1 receptor mediates the beneficial effect of liraglutide in an acute lung injury mouse model involving the thioredoxin-interacting protein.

Repurposing clinically used drugs is among the important strategies in drug discovery. Glucagon-like peptide-1 (GLP-1) and its diabetes-based drugs, such as liraglutide, possess a spectrum of extra-pancreatic functions, while GLP-1 receptor (GLP-1R) is most abundantly expressed in the lung. Recent studies have suggested that GLP-1-based drugs exert beneficial effects in chronic, as well as acute, lung injury rodent models. Here, we show that liraglutide pretreatment reduced LPS induced acute lung injury in mice. It significantly reduced lung injury score, wet/dry lung weight ratio, bronchoalveolar lavage fluid immune cell count and protein concentration, and cell apoptosis in the lung, and it was associated with reduced lung inflammatory cytokine and chemokine gene expression. Importantly, these effects were virtually absent in GLP-1R-/- mice. A well-known function of GLP-1 and GLP-based drugs in pancreatic ß-cells is the attenuation of high-glucose stimulated expression of thioredoxin-interacting protein (TxNIP), a key component of inflammasome. LPS-challenged lungs showed elevated TxNIP mRNA and protein expression, which was attenuated by liraglutide treatment in a GLP-1R-dependent manner. Hence, our observations suggest that GLP-1R is essential in mediating beneficial effects of liraglutide in acute lung injury, with the inflammasome component TxNIP as a potential target.

Dietary Cyanidin-3-Glucoside Attenuates High-Fat-Diet-Induced Body-Weight Gain and Impairment of Glucose Tolerance in Mice via Effects on the Hepatic Hormone FGF21.

BACKGROUND: Dietary polyphenols including anthocyanins target multiple organs. OBJECTIVE: We aimed to assess the involvement of glucagon-like peptide 1 (GLP-1), leptin, insulin and fibroblast growth factor 21 (FGF21) in mediating metabolic beneficial effects of purified anthocyanin cyanidin-3-glucoside (Cy3G). METHODS: Intestinal proglucagon gene (Gcg; encoding GLP-1) and liver Fgf21 expression were assessed in 6-wk-old male C57BL-6J mice fed a low-fat-diet (LFD; 10% of energy from fat), alone or with 1.6 mg Cy3G/L in drinking water for 3 wk [experiment (Exp.) 1; n = 5/group]. Similar mice were fed the LFD or a high-fat diet (HFD; 60% energy from fat) with or without Cy3G for 20 wk. Half of the mice administered Cy3G also received 4 broad-spectrum antibiotics (ABs) in drinking water between weeks 11 and 14, for a total of 6 groups (n = 8/group). Metabolic tolerance tests were conducted between weeks 2 and 16. Relevant hormone gene expression and plasma hormone concentrations were assessed mainly at the end of 20 wk (Exp. 2). RESULTS: In Exp. 1, Cy3G administration increased ileal but not colonic Gcg level by 2-fold (P < 0.05). In Exp. 2, Cy3G attenuated HFD-induced body-weight gain (20.3% at week 16), and improved glucose tolerance (26.5% at week 15) but not insulin tolerance. Although Cy3G had no effect on glucose tolerance in LFD mice, LFD/Cy3G/AB mice showed better glucose tolerance than LFD/Cy3G mice (23%). In contrast, HFD/Cy3G/AB mice showed worse glucose tolerance compared with HFD/Cy3G mice (15%). Beneficial effects of Cy3G in HFD mice were not associated with changes in plasma leptin, insulin or GLP-1 concentrations. However, Cy3G increased hepatic Fgf21 expression in mice in Exp. 1 by 4-fold and attenuated Fgf21 overexpression in HFD mice (Exp. 2, 22%), associated with increased expression of genes that encode FGFR1 and ß-klotho (>3-fold, P < 0.05). CONCLUSIONS: Dietary Cy3G may reduce body weight and exert metabolic homeostatic effects in mice via changes in hepatic FGF21.

Short-Term Curcumin Gavage Sensitizes Insulin Signaling in Dexamethasone-Treated C57BL/6 Mice.

BACKGROUND: Long-term dietary curcumin (>12 wk) improves metabolic homeostasis in obese mice by sensitizing insulin signaling and reducing hepatic gluconeogenesis. Whether these occur only secondary to its chronic anti-inflammatory and antioxidative functions is unknown. OBJECTIVE: In this study, we assessed the insulin sensitization effect of short-term curcumin gavage in a rapid dexamethasone-induced insulin resistance mouse model, in which the chronic anti-inflammatory function is eliminated. METHODS: Six-week-old male C57BL/6 mice received an intraperitoneal injection of dexamethasone (100 mg/kg body weight) or phosphate-buffered saline every day for 5 d, with or without simultaneous curcumin gavage (500 mg/kg body weight). On day 7, insulin tolerance tests were performed. After a booster dexamethasone injection and curcumin gavage on day 8, blood glucose and insulin concentrations were measured. Liver tissues were collected on day 10 for quantitative polymerase chain reaction and Western blotting to assess gluconeogenic gene expression, insulin signaling, and the expression of fibroblast growth factor 21 (FGF21). Primary hepatocytes from separate, untreated C57BL/6 mice were used for testing the in vitro effect of curcumin treatment. RESULTS: Dexamethasone injection impaired insulin tolerance (P < 0.05) and elevated ambient plasma insulin concentrations by ~2.7-fold (P < 0.01). Concomitant curcumin administration improved insulin sensitivity and reduced hepatic gluconeogenic gene expression. The insulin sensitization effect of curcumin was demonstrated by increased stimulation of S473 phosphorylation of protein kinase B (P < 0.01) in the dexamethasone-treated mouse liver, as well as the repression of glucose production in primary hepatocytes (P < 0.001). Finally, curcumin gavage increased FGF21 expression by 2.1-fold in the mouse liver (P < 0.05) and curcumin treatment increased FGF21 expression in primary hepatocytes. CONCLUSION: These observations suggest that the early beneficial effect of curcumin intervention in dexamethasone-treated mice is the sensitization of insulin signaling, involving the stimulation of FGF21 production, a known insulin sensitizer.

GLP-1-derived nonapeptide GLP-1(28-36)amide represses hepatic gluconeogenic gene expression and improves pyruvate tolerance in high-fat diet-fed mice.

Certain "degradation" products of GLP-1 were found to possess beneficial effects on metabolic homeostasis. Here, we investigated the function of the COOH-terminal fragment of GLP-1, the nonapeptide GLP-1(28-36)amide, in hepatic glucose metabolism. C57BL/6 mice fed a high-fat diet (HFD) for 13 wk were injected intraperitoneally with GLP-1(28-36)amide for 6 wk. A significant reduction in body weight gain in response to HFD feeding was observed in GLP-1(28-36)amide-treated mice. GLP-1(28-36)amide administration moderately improved glucose disposal during glucose tolerance test but more drastically attenuated glucose production during pyruvate tolerance test, which was associated with reduced hepatic expression of the gluconeogenic genes Pck1, G6pc, and Ppargc1a. Mice treated with GLP-1(28-36)amide exhibited increased phosphorylation of PKA targets, including cAMP response element-binding protein (CREB), ATF-1, and ß-catenin. In primary hepatocytes, GLP-1(28-36)amide reduced glucose production and expression of Pck1, G6pc, and Ppargc1a, which was associated with increased cAMP content and PKA target phosphorylation. These effects were attenuated by PKA inhibition. We suggest that GLP-1(28-36)amide represses hepatic gluconeogenesis involving the activation of components of the cAMP/PKA signaling pathway. This study further confirmed that GLP-1(28-36)amide possesses therapeutic potential for diabetes and other metabolic disorders.

GLP-1(28-36) improves ß-cell mass and glucose disposal in streptozotocin-induced diabetic mice and activates cAMP/PKA/ß-catenin signaling in ß-cells in vitro.

Recent studies have demonstrated that the COOH-terminal fragment of the incretin hormone glucagon-like peptide-1 (GLP-1), a nonapeptide GLP-1(28-36)amide, attenuates diabetes and hepatic steatosis in diet-induced obese mice. However, the effect of this nonapeptide in pancreatic ß-cells remains largely unknown. Here, we show that in a streptozotocin-induced mouse diabetes model, GLP-1(28-36)amide improved glucose disposal and increased pancreatic ß-cell mass and ß-cell proliferation. An in vitro investigation revealed that GLP-1(28-36)amide stimulates ß-catenin (ß-cat) Ser(675) phosphorylation in both the clonal INS-1 cell line and rat primary pancreatic islet cells. In INS-1 cells, the stimulation was accompanied by increased nuclear ß-cat content. GLP-1(28-36)amide was also shown to increase cellular cAMP levels, PKA enzymatic activity, and cAMP response element-binding protein (CREB) and cyclic AMP-dependent transcription factor-1 (ATF-1) phosphorylation. Furthermore, GLP-1(28-36)amide treatment enhanced islet insulin secretion and increased the growth of INS-1 cells, which was associated with increased cyclin D1 expression. Finally, PKA inhibition attenuated the effect of GLP-1(28-36)amide on ß-cat Ser(675) phosphorylation and cyclin D1 expression in the INS-1 cell line. We have thus revealed the beneficial effect of GLP-1(28-36)amide in pancreatic ß-cells in vitro and in vivo. Our observations suggest that GLP-1(28-36)amide may exert its effect through the PKA/ß-catenin signaling pathway.

Short-term semaglutide treatment improves FGF21 responsiveness in primary hepatocytes isolated from high fat diet challenged mice.

Metabolic functions of GLP-1 and its analogues have been extensively investigated. In addition to acting as an incretin and reducing body weight, we and others have suggested the existence of GLP-1/fibroblast growth factor 21 (FGF21) axis in which liver mediates certain functions of GLP-1 receptor agonists. In a more recent study, we found with surprise that four-week treatment with liraglutide but not semaglutide stimulated hepatic FGF21 expression in HFD-challenged mice. We wondered whether semaglutide can also improve FGF21 sensitivity or responsiveness and hence triggers the feedback loop in attenuating its stimulation on hepatic FGF21 expression after a long-term treatment. Here, we assessed effect of daily semaglutide treatment in HFD-fed mice for 7 days. HFD challenge attenuated effect of FGF21 treatment on its downstream events in mouse primary hepatocytes, which can be restored by 7-day semaglutide treatment. In mouse liver, 7-day semaglutide treatment stimulated FGF21 as well as genes that encode its receptor (FGFR1) and the obligatory co-receptor (KLB), and a battery of genes that are involved in lipid homeostasis. In epididymal fat tissue, expressions of a battery genes including Klb affected by HFD challenge were reversed by 7-day semaglutide treatment. We suggest that semaglutide treatment improves FGF21 sensitivity which is attenuated by HFD challenge.

Resveratrol intervention attenuates chylomicron secretion via repressing intestinal FXR-induced expression of scavenger receptor SR-B1.

Two common features of dietary polyphenols have hampered our mechanistic understanding of their beneficial effects for decades: targeting multiple organs and extremely low bioavailability. We show here that resveratrol intervention (REV-I) in high-fat diet (HFD)-challenged male mice inhibits chylomicron secretion, associated with reduced expression of jejunal but not hepatic scavenger receptor class B type 1 (SR-B1). Intestinal mucosa-specific SR-B1-/- mice on HFD-challenge exhibit improved lipid homeostasis but show virtually no further response to REV-I. SR-B1 expression in Caco-2 cells cannot be repressed by pure resveratrol compound while fecal-microbiota transplantation from mice on REV-I suppresses jejunal SR-B1 in recipient mice. REV-I reduces fecal levels of bile acids and activity of fecal bile-salt hydrolase. In Caco-2 cells, chenodeoxycholic acid treatment stimulates both FXR and SR-B1. We conclude that gut microbiome is the primary target of REV-I, and REV-I improves lipid homeostasis at least partially via attenuating FXR-stimulated gut SR-B1 elevation.

The Wnt signaling pathway effector TCF7L2 is upregulated by insulin and represses hepatic gluconeogenesis.

Certain single nucleotide polymorphisms (SNPs) in transcription factor 7-like 2 (TCF7L2) are strongly associated with the risk of type 2 diabetes. TCF7L2 and ß-catenin (ß-cat) form the bipartite transcription factor cat/TCF in stimulating Wnt target gene expression. cat/TCF may also mediate the effect of other signaling cascades, including that of cAMP and insulin in cell-type specific manners. As carriers of TCF7L2 type 2 diabetes risk SNPs demonstrated increased hepatic glucose production, we aimed to determine whether TCF7L2 expression is regulated by nutrient availability and whether TCF7L2 and Wnt regulate hepatic gluconeogenesis. We examined hepatic Wnt activity in the TOPGAL transgenic mouse, assessed hepatic TCF7L2 expression in mice upon feeding, determined the effect of insulin on TCF7L2 expression and ß-cat Ser675 phosphorylation, and investigated the effect of Wnt activation and TCF7L2 knockdown on gluconeogenic gene expression and glucose production in hepatocytes. Wnt activity was observed in pericentral hepatocytes in the TOPGAL mouse, whereas TCF7L2 expression was detected in human and mouse hepatocytes. Insulin and feeding stimulated hepatic TCF7L2 expression in vitro and in vivo, respectively. In addition, insulin activated ß-cat Ser675 phosphorylation. Wnt activation by intraperitoneal lithium injection repressed hepatic gluconeogenic gene expression in vivo, whereas lithium or Wnt-3a reduced gluconeogenic gene expression and glucose production in hepatic cells in vitro. Small interfering RNA-mediated TCF7L2 knockdown increased glucose production and gluconeogenic gene expression in cultured hepatocytes. These observations suggest that Wnt signaling and TCF7L2 are negative regulators of hepatic gluconeogenesis, and TCF7L2 is among the downstream effectors of insulin in hepatocytes.

Hepatic hormone FGF21 and its analogues in clinical trials.

Fibroblast growth factor 21 (FGF21) is a fasting or stress inducible metabolic hormone produced mainly in the liver. It plays important roles in regulating both glucose and lipid homeostasis via interacting with a heterodimeric receptor complex comprising FGF receptor 1 (FGFR1) and ß-klotho (KLB). For the past decade, great effort has been made on developing FGF21 derivatives or specific FGF21 receptor agonists into therapeutic agents for various metabolic disorders including type 2 diabetes (T2D), obesity, and more importantly, nonalcoholic fatty liver disease (NAFLD). Here we have reviewed FGF21 gene and protein structures, its expression pattern, cellular signaling cascades that mediate FGF21 production and function. We have then summarized the six clinical trials utilizing four FGF21 analogues. Finally, two recent literatures on the development of GLP-1 and FGF21 dual agonists were presented briefly.

Comparison of Beneficial Metabolic Effects of Liraglutide and Semaglutide in Male C57BL/6J Mice.

OBJECTIVES: Semaglutide and liraglutide are glucagon-like peptide-1 (GLP-1)-based diabetes drugs. Semaglutide possesses a longer half-life. Utilizing relatively lower doses, we compared the beneficial metabolic effects of these 2 drugs in mice fed a high-fat diet (HFD), aiming to deepen our mechanistic understanding on their energy homeostatic functions. METHODS: Male C57BL/6J mice were fed an HFD for 10 weeks, followed by daily phosphate-buffered saline (PBS, as control); liraglutide (150 µg/kg body weight); or semaglutide (12 µg/kg body weight, low dose [LD]; or 60 µg/kg body weight, high dose [HD]) injection for 4 weeks. Metabolic tolerance and other tests were conducted within the 4-week period. Expression of metabolism-related genes, including Fgf21 in the liver and adipose tissues, was assessed after mice were euthanized. RESULTS: HFD-induced body weight gain, increasing inguinal fat tissue mass, glucose defects and insulin intolerance were effectively and comparably attenuated in the 3 experimental groups. HD semaglutide showed an even better effect on attenuating hyperleptinemia. Liraglutide but not semaglutide treatment enhanced hepatic fibroblast growth factor 21 (FGF21) protein level. All 3 experimental groups showed elevated expression of genes that encode pyruvate dehydrogenase kinase 4 and enoyl-CoA hydratase and 3-hydroxyacyl-coenzyme A dehydrogenase, associated with reduced plasma triglyceride levels. Finally, the plasma "GLP-1" level in HD semaglutide-treated mice was 14-fold higher than in HFD-fed control mice. CONCLUSIONS: Liraglutide, but not semaglutide, increased hepatic FGF21 protein level, whereas semaglutide had a greater effect on attenuating hyperleptinemia. Thus, these 2 GLP-1-based diabetes drugs may target metabolic organs, including liver and adipose tissue, with differing levels of efficacy.

Liraglutide stimulates the ß-catenin signaling cascade in mouse epididymal fat tissue.

Although canonical Wnt signaling pathway activation was shown to negatively regulate adipogenesis, recent investigations suggest that Wnt pathway effectors TCF7L2 and ß-catenin (ß-cat) in adipose tissues are also involved in energy homeostasis during adulthood. In assessing the metabolic beneficial effect of GLP-1-based diabetes drugs in high-fat diet (HFD)-challenged mice, we observed that liraglutide treatment affected the expression of a battery of adipose tissue-specific genes, including those that encode adiponectin and leptin, mainly in epididymal white adipose tissue (eWAT). Fourteen-week HFD challenge repressed TCF7L2 and ß-cat S675 phosphorylation in eWAT, while such repression was reversed by liraglutide treatment (150 µg/kg body weight daily) during weeks 10-14. In Glp1r-/-mice, liraglutide failed in stimulating TCF7L2 or ß-cat in eWAT. We detected Glp1r expression in mouse eWAT and its level is enriched in its stromal vascular fraction (SVF). Mouse eWAT-SVF showed reduced expression of Tcf7l2 and its Tcf7l2 level could not be stimulated by liraglutide treatment; while following adipogenic differentiation, rat eWAT-SVF showed elevated Tcf7l2 expression. Direct in vitro liraglutide treatment in eWAT-SVF stimulated CREB S133, ß-cat S675 phosphorylation, and cellular cAMP level. Thus, cAMP/ß-cat signaling cascade can be stimulated by liraglutide in eWAT via GLP-1R expressed in eWAT-SVF.

GABA requires GLP-1R to exert its pancreatic function during STZ challenge.

Gamma-aminobutyric acid (GABA) administration attenuates streptozotocin (STZ)-induced diabetes in rodent models with unclear underlying mechanisms. We found that GABA and Sitagliptin possess additive effect on pancreatic ß-cells, which prompted us to ask the existence of common or unique targets of GLP-1 and GABA in pancreatic ß-cells. Effect of GABA on expression of thioredoxin-interacting protein (TxNIP) was assessed in the INS-1 832/13 (INS-1) cell line, WT and GLP-1R-/- mouse islets. GABA was also orally administrated in STZ-challenged WT or GLP-1R-/- mice, followed by immunohistochemistry assessment of pancreatic islets. Effect of GABA on Wnt pathway effector ß-catenin (ß-cat) was examined in INS-1 cells, WT and GLP-1R-/- islets. We found that GABA shares a common feature with GLP-1 on inhibiting TxNIP, while this function was attenuated in GLP-1R-/- islets. In WT mice with STZ challenge, GABA alleviated several 'diabetic syndromes', associated with increased ß-cell mass. These features were virtually absent in GLP-1R-/- mice. Knockdown TxNIP in INS-1 cells increased GLP-1R, Pdx1, Nkx6.1 and Mafa levels, associated with increased responses to GABA or GLP-1 on stimulating insulin secretion. Cleaved caspase-3 level can be induced by high-glucose, dexamethasone, or STZ in INS-1 cell, while GABA treatment blocked the induction. Finally, GABA treatment increased cellular cAMP level and ß-cat S675 phosphorylation in WT but not GLP-1R-/- islets. We, hence, identified TxNIP as a common target of GABA and GLP-1 and suggest that, upon STZ or other stress challenge, the GLP-1R-cAMP-ß-cat signaling cascade also mediates beneficial effects of GABA in pancreatic ß-cell, involving TxNIP reduction.

Alterations in methylation and expression levels of imprinted genes H19 and Igf2 in the fetuses of diabetic mice.

The study aimed to reveal alterations in expression and methylation levels of the growth-related imprinted genes H19 and Igf2 in fetuses of diabetic mice. Diabetes was induced in female mice by intraperitoneal injection of streptozotocin. DNA and total RNA were extracted from fetuses obtained from diabetic and control dams on embryonic day (E) 14. Real-time RT-PCR analysis revealed that the mRNA expression of Igf2 in fetuses from diabetic mice was 0.65-fold of the control counterparts. Bisulfite genomic sequencing demonstrated that the methylation level of the H19-Igf2 imprint control region was 19.1% higher in diabetic fetuses than in those of control dams. In addition, the body weight of pups born to diabetic dams was 26.5% lower than that of the control group. The results indicate that maternal diabetes can affect fetal development by means of altered expression of imprinted genes. The modified genomic DNA methylation status of imprinting genes may account for the change in gene expression.

The LIM homeodomain protein ISL1 mediates the function of TCF7L2 in pancreatic beta cells.

Pancreatic ß-cell Tcf7l2 deletion or its functional knockdown suggested the essential role of this Wnt pathway effector in controlling insulin secretion, glucose homeostasis and ß-cell gene expression. As the LIM homeodomain protein ISL1 is a suggested Wnt pathway downstream target, we hypothesize that it mediates metabolic functions of TCF7L2. We aimed to determine the role of ISL1 in mediating the function of TCF7L2 and the incretin hormone GLP-1 in pancreatic ß-cells. The effect of dominant negative TCF7L2 (TCF7L2DN) mediated Wnt pathway functional knockdown on Isl1 expression was determined in ßTCFDN mouse islets and in the rat insulinoma cell line INS-1 832/13. Luciferase reporter assay and chromatin immunoprecipitation were utilized to determine whether Isl1 is a direct downstream target of Tcf7l2 TCF7L2DN adenovirus infection and siRNA-mediated Isl1 knockdown on ß-cell gene expression were compared. Furthermore, Isl1 knockdown on GLP-1 stimulated ß-catenin S675 phosphorylation and insulin secretion was determined. We found that TCF7L2DN repressed ISL1 levels in ßTCFDN islets and the INS-1 832/13 cell line. Wnt stimulators enhanced Isl1 promoter activity and binding of TCF7L2 on Isl1 promoter. TCF7L2DN adenovirus infection and Isl1 knockdown generated similar repression on expression of ß-cell genes, including the ones that encode GLUT2 and GLP-1 receptor. Either TCF7L2DN adenovirus infection or Isl1 knockdown attenuated GLP-1-stimulated ß-catenin S675 phosphorylation in INS-1 832/13 cells or mouse islets and GLP-1 stimulated insulin secretion in INS-1 832/13 or MIN6 cells. Our observations support the existence of TCF7L2-ISL1 transcriptional network, and we suggest that this network also mediates ß-cell function of GLP-1.

Dietary Curcumin Intervention Targets Mouse White Adipose Tissue Inflammation and Brown Adipose Tissue UCP1 Expression.

OBJECTIVE: This study aimed to determine whether dietary curcumin intervention targets both white adipose tissue (WAT) inflammation and brown adipose tissue (BAT)-mediated energy expenditure. METHODS: C57BL/6J mice were fed with a low-fat diet, high-fat diet (HFD), or HFD plus curcumin. In addition to assessing the effect of curcumin intervention on metabolic profiles, this study assessed WAT macrophage infiltration and composition and inflammatory cytokine production. Metabolic cages were applied for determining energy expenditure. Raw264.7 (ATCC, Manassas, Virginia) and other cell models were utilized to test the in vitro effect of curcumin treatment. RESULTS: Curcumin intervention reduced WAT macrophage infiltration and altered macrophage functional polarity, as the ratio of M2-like versus M1-like macrophages increased after curcumin intervention. Curcumin treatment reduced M1-like macrophage markers or proinflammation cytokine expression in both macrophages and adipocytes. Curcumin intervention also increased energy expenditure and body temperature in response to a cold challenge. Finally, the in vivo and in vitro investigations suggested that curcumin increased expression of uncoupling protein 1 (UCP1), possibly involving PPAR-dependent and -independent mechanisms. CONCLUSIONS: Curcumin intervention targets both WAT inflammation and BAT UCP1 expression. These observations advanced our knowledge on the metabolic beneficial effects of the curry compound curcumin, bringing us a novel perspective on dietary polyphenol research.

Tissue-specific effect of dietary cysteamine on expression of adiponectin receptors in rats.

Adiponectin is synthesized by adipocytes and affects glucose and lipid metabolism by binding to its receptors, AdipoR1 and AdipoR2. Cysteamine, a naturally existing intermediate metabolite of sulfur amino acid, has been reported to modulate metabolism and growth in various species of animals; however, whether the action of cysteamine involves adiponectin and its receptors is unknown. The objective of the present study was therefore to investigate the effect of dietary cysteamine on the expression of AdipoR1/R2 in different tissues, in association with the alterations in endocrine and metabolic status. Rats were fed either of the diets supplemented with 0 or 700 mg/kg cysteamine feed additive (containing 30% of cysteamine hydrochloride) for 4 weeks, and the expression of adiponectin and its receptors in adipose tissue, AdipoR1 and AdipoR2 in liver, gastrocnemius, and soleus muscle was determined, in association with the growth performance and serum concentrations of hormones and metabolites. A temporal trend of increase in growth rate and the ratio of feed consumption relative to body weight gain was observed in the second week of cysteamine supplementation. Serum concentrations of insulin and TNF-alpha increased, while serum levels of triglycerides, FFA, and total cholesterol decreased significantly 4 weeks after cysteamine treatment. Leptin and GH remained unaffected. Cysteamine supplementation increased mRNA expression of AdipoR1 in adipose tissue, gastrocnemius, and soleus muscle as well as that of AdipoR2 in soleus muscle and adipose tissue. Nevertheless, hepatic expression of AdipoR1 and AdipoR2 was not influenced. Despite a numeric increase, no significant alteration in adiponectin mRNA expression in adipose tissue was observed. In conclusion, dietary supplementation of cysteamine modulates the endocrine and metabolic status of rats, which may involve the tissue-specific responses of adiponectin receptors at the level of mRNA transcription.

Exposure of preimplantation embryos to insulin alters expression of imprinted genes.

Insulin promotes early embryonic development, but whether this action affects postimplantation fetal development and alters the expression of imprinted genes remain to be determined. This study analyzed the expression and methylation levels of the growth-related imprinted genes H19 and insulin-like growth factor 2 (Igf2) in fetuses exposed to insulin before implantation. We cultured 2-cell embryos in either 0 or 0.25 microg/ml insulin until the blastocyst stage and then transferred them into pseudopregnant recipient mice. The number of embryos developing to blastocysts after insulin exposure was 16.4% higher than that of the control, and the birth body weight of the insulin-exposed group was 17.8% higher than that of the control group. Real-time reverse transcription-polymerase chain reaction analysis revealed that exposure of preimplantation embryos to insulin increased the mRNA expression of both Igf2 and H19 in embryonic day (E) 14 fetuses. Bisulfite genomic sequencing demonstrated that the methylation level of the H19-Igf2 imprint control region was 19.3% lower in insulin-exposed E14 fetuses than in controls. The present study indicates that insulin exposure during the preimplantation stage alters the expression of imprinted genes and affects fetal development.