Ursodeoxycholic acid inhibits liver X receptor α-mediated hepatic lipogenesis via induction of the nuclear corepressor SMILE.

Small heterodimer partner interacting leucine zipper protein (SMILE) has been identified as a nuclear corepressor of the nuclear receptor (NRs) family. Here, we examined the role of SMILE in the regulation of nuclear receptor liver X receptor (LXR)-mediated sterol regulatory element binding protein-1c (SREBP-1c) gene expression. We found that SMILE inhibited T0901317 (T7)-induced transcriptional activity of LXR, which functions as a major regulator of lipid metabolism by inducing SREBP-1c, fatty acid synthase (FAS), and acetyl-CoA carboxylase (ACC) gene expression. Moreover, we demonstrated that SMILE physically interacts with LXR and represses T7-induced LXR transcriptional activity by competing with coactivator SRC-1. Adenoviral overexpression of SMILE (Ad-SMILE) attenuated fat accumulation and lipogenic gene induction in the liver of T7 administered or of high fat diet (HFD)-fed mice. Furthermore, we investigated the mechanism by which ursodeoxycholic acid (UDCA) inhibits LXR-induced lipogenic gene expression. Interestingly, UDCA treatment significantly increased SMILE promoter activity and gene expression in an adenosine monophosphate-activated kinase-dependent manner. Furthermore, UDCA treatment repressed T7-induced SREBP-1c, FAS, and ACC protein levels, whereas knockdown of endogenous SMILE gene expression by adenovirus SMILE shRNA (Ad-shSMILE) significantly reversed UDCA-mediated repression of SREBP-1c, FAS, and ACC protein levels. Collectively, these results demonstrate that UDCA activates SMILE gene expression through adenosine monophosphate-activated kinase phosphorylation, which leads to repression of LXR-mediated hepatic lipogenic enzyme gene expression.

The protective role of NAD(P)H:quinone oxidoreductase 1 on acetaminophen-induced liver injury is associated with prevention of adenosine triphosphate depletion and improvement of mitochondrial dysfunction.

An overdose of acetaminophen (APAP) causes hepatotoxicity due to its metabolite, N-acetyl-p-benzoquinone imine. NAD(P)H: quinone oxidoreductase 1 (NQO1) is an important enzyme for detoxification, because it catabolizes endogenous/exogenous quinone to hydroquinone. Although various studies have suggested the possible involvement of NQO1 in APAP-induced hepatotoxicity, its precise role in this remains unclear. We investigated the role of NQO1 against APAP-induced hepatotoxicity using a genetically modified rodent model. NQO1 wild-type (WT) and knockout (KO) mice were treated with different doses of APAP, and we evaluated the mortality and toxicity markers for cell death caused by APAP. NQO1 KO mice showed high sensitivity to APAP-mediated hepatotoxicity (as indicated by a large necrotic region) as well as increased levels of nitrotyrosine adducts and reactive oxygen species. APAP-induced cell death in the livers and primary hepatocytes of NQO1 KO mice, which was accompanied by an extensive reduction in adenosine triphosphate (ATP) levels. In accordance with this ATP depletion, cytosolic increases in mitochondrial proteins such as apoptosis-inducing factor, second mitochondria-derived activator of caspases/DIABLO, endonuclease G, and cytochrome c, which indicate severe mitochondrial dysfunction, were observed in NQO1 KO mice but not in WT mice after APAP exposure. Severe mitochondrial depolarization was also greater in hepatocytes isolated from NQO1 KO mice. Collectively, our data suggest that NQO1 plays a critical role in protection against energy depletion caused by APAP, and NQO1 may be useful in the development of therapeutic approaches to effectively diminish the hepatotoxicity caused by an APAP overdose.

Insulin directly regulates steroidogenesis via induction of the orphan nuclear receptor DAX-1 in testicular Leydig cells.

Testosterone level is low in insulin-resistant type 2 diabetes. Whether this is due to negative effects of high level of insulin on the testes caused by insulin resistance has not been studied in detail. In this study, we found that insulin directly binds to insulin receptors in Leydig cell membranes and activates phospho-insulin receptor-ß (phospho-IR-ß), phospho-IRS1, and phospho-AKT, leading to up-regulation of DAX-1 (dosage-sensitive sex reversal, adrenal hypoplasia critical region, on chromosome X, gene 1) gene expression in the MA-10 mouse Leydig cell line. Insulin also inhibits cAMP-induced and liver receptor homolog-1 (LRH-1)-induced steroidogenic enzyme gene expression and steroidogenesis. In contrast, knockdown of DAX-1 reversed insulin-mediated inhibition of steroidogenesis. Whether insulin directly represses steroidogenesis through regulation of steroidogenic enzyme gene expression was assessed in insulin-injected mouse models and high fat diet-induced obesity. In insulin-injected mouse models, insulin receptor signal pathway was activated and subsequently inhibited steroidogenesis via induction of DAX-1 without significant change of luteinizing hormone or FSH levels. Likewise, the levels of steroidogenic enzyme gene expression and steroidogenesis were low, but interestingly, the level of DAX-1 was high in the testes of high fat diet-fed mice. These results represent a novel regulatory mechanism of steroidogenesis in Leydig cells. Insulin-mediated induction of DAX-1 in Leydig cells of testis may be a key regulatory step of serum sex hormone level in insulin-resistant states.

In vivo imaging of islet transplantation using PLGA nanoparticles containing iron oxide and indocyanine green.

PURPOSE: We determined whether poly(lactic-co-glycolic acid) nanoparticles would be a useful reagent for the successful monitoring of isolated islets by magnetic resonance imaging and optical imaging systems, without clinically relevant toxicity in vitro or in vivo. METHODS: We used iron oxide for MR imaging and a cyanide dye approved by the Food and Drug Administration (indocyanine green) for optical imaging and estimated the in vivo detection of transplanted pancreatic islets. RESULTS: The poly(lactic-co-glycolic acid) nanoparticles were associated with the islets in vitro and were successfully detected by 4.7 T (MR) and optical imaging, without other toxic effects. When labeled islets were transplanted under the mouse kidney capsule, in vivo T2/ T2*-weighted scans with 4.7 T MR detected as few as 300 labeled islets by 4 weeks. Optical in vivo imaging revealed indocyanine green fluorescence by 2 and 4 days after transplantation of islets containing 250 and 500 µg/mL poly(lactic-co-glycolic acid) nanoparticles, respectively. These results were further supported by the immunohistochemical results for insulin and iron in the recipient mouse kidney and pancreas. CONCLUSIONS: Taken together, these data indicate that poly(lactic-co-glycolic acid) nanoparticles may be used to label transplanted islets and may be imaged with in vivo MR and optical imaging systems.

Phosphoenolpyruvate carboxykinase and glucose-6-phosphatase are required for steroidogenesis in testicular Leydig cells.

Cyclic AMP (cAMP) induces steroidogenic enzyme gene expression and stimulates testosterone production in Leydig cells. Phosphoenolpyruvate carboxykinase (PEPCK) is expressed in Leydig cells, but its role has not been defined. In this study, we found that PEPCK and glucose-6-phosphatase (Glc-6-Pase) are increased significantly following cAMP treatment of mouse Leydig cells. Moreover, cAMP treatment increased recruitment of the cAMP-response element-binding transcription factor and decreased recruitment of the corepressor DAX-1 on the pepck promoter. Furthermore, cAMP induced an increase in ATP that correlated with a decrease in phospho-AMP-activated protein kinase (AMPK). In contrast, knockdown or inhibition of PEPCK decreased ATP and increased phospho-AMPK. Treatment with an AMPK activator or overexpression of the constitutively active form of AMPK inhibited cAMP-induced steroidogenic enzyme promoter activities and gene expression. Liver receptor homolog-1 (LRH-1) was involved in cAMP-induced steroidogenic enzyme gene expression but was inhibited by AMPK activation in Leydig cells. Additionally, inhibition or knockdown of PEPCK and Glc-6-Pase decreased cAMP-mediated induction of steroidogenic enzyme gene expression and steroidogenesis. Finally, pubertal mouse (8-week-old) testes and human chorionic gonadotropin-induced prepubertal mouse testes showed increased PEPCK and Glc-6-Pase gene expression. Taken together, these results suggest that induction of PEPCK and Glc-6-Pase by cAMP plays an important role in Leydig cell steroidogenesis.

Antiatherogenic effect of antioxidant polyphenols from Phellinus baumii in apolipoprotein E-deficient mice.

AIMS: The present study was carried out to investigate the antiatherosclerotic effect of antioxidant polyphenols from Phellinus baumii (PBE) in apolipoprotein E-deficient (apoE-/-) mice. METHODS AND RESULTS: apoE-/- mice were randomly divided into three groups: mice on a normal chow diet comprised the normal group, mice on an atherogenic diet plus vehicle were the control group, and mice on an atherogenic diet plus PBE (500 mg/kg) comprised the PB500 group. After 8 weeks of treatment, the plasma lipids and cytokine levels were measured. Although no significant differences were found in cholesterol levels among groups, the triglyceride level was significantly decreased in the PBE-treated group compared with the control group. Plasma tumor necrosis factor (TNF)-α and interleukin (IL)-6 levels were reduced by PBE treatment. Real-time PCR analysis of the aorta showed that PBE significantly prevented the upregulation of the vascular cell adhesion molecule (VCAM)-1, intercellular adhesion molecule (ICAM)-1, TNF-α, IL-6, and IL-1ß expression. Furthermore, reduced macrophage infiltration, lipid accumulation and atherosclerotic lesions were observed in the aortic sinus and en face of the whole aorta in PBE-fed apoE-/- mice compared with atherogenic diet-fed control mice. CONCLUSIONS: Collectively, the findings of the present study suggest that the antiatherosclerotic effect of PBE is probably related to the inhibition of adhesion molecule and cytokine expression resulting in amelioration of lesion development.

Hepatoprotective effect of Platycodon grandiflorum against chronic ethanol-induced oxidative stress in C57BL/6 mice.

AIMS: This study was carried out to evaluate the hepatoprotective effect of Platycodon grandiflorum (PG) in ethanol (EtOH)-induced liver damage. METHODS AND RESULTS: PG treatment (both the total extract and saponin fraction) significantly blocked EtOH-induced oxidative stress through the preservation of activities of antioxidant enzymes in HepG2 cells. Furthermore, while the administration of EtOH to C57BL/6 mice for 6 weeks induced liver damage, along with a significant increase in plasma glutamic oxalacetic transaminase, glutamic pyruvic transaminase, hepatic triglyceride and thiobarbituric acid reactive substance levels, PG treatment significantly decreased glutamic oxalacetic transaminase, glutamic pyruvic transaminase, hepatic triglyceride and thiobarbituric acid reactive substance levels compared with the EtOH-treated control group (p < 0.05). Histological observation by hematoxylin-eosin and oil red O staining in the liver showed more effective inhibition of lipid accumulation in PG-treated groups, as compared to the EtOH-treated control group. Additionally, PG treatments appeared to enhance the activities of superoxide dismutase and catalase in the liver (p < 0.05). CONCLUSION: These results suggest that PG has a protective effect against EtOH-induced oxidative damage, possibly by inhibition of lipid accumulation and peroxidation through the enhancement of the antioxidant defense system. PG might be useful as a therapeutically potent natural ingredient for the prevention of chronic EtOH-induced oxidative stress and liver damage.

Preventative effects of Platycodon grandiflorum treatment on hepatic steatosis in high fat diet-fed C57BL/6 mice.

Platycodon grandiflorum (PG) (Korean name, Doraji; Chinese name, Jiegeng; and Japanese name, Kikyo) is a perennial plant in the Campanulaceae family that contains triterpenoid saponins, carbohydrates, and fibers. This study was carried out to investigate effects of root of PG on fatty liver inhibition in high fat diet (HFD)-fed C57BL/6 mice. C57BL/6 mice were divided into control, total extract of PG (T-PG, 500 mg/kg) and saponin fraction (S-PG, 50 mg/kg)-treated groups. Significant decreases in body weight, associated with fat mass reduction, were observed in PG-treated groups (p<0.05). Hepatic lipid content and score index calculated from morphometric observations on fatty liver were significantly decreased in the PG-treated groups (p<0.05). Moreover, activities of fatty acid synthase (FAS) and carnitine palmitoyl-transferase (CPT) were significantly suppressed and increased as compared with the control group, respectively (p<0.05). mRNA expressions of the sterol regulatory element binding protein (SREBP1c) and stearoyl-CoA desaturase (SCD1) gene were suppressed in the T-PG and S-PG groups (p<0.05). From these findings, we speculate that fatty liver inhibition effects of PG extract and its saponins appear to be conferred by hepatic lipogenesis and acceleration of energy expenditure, along with modulation of liver FAS and CPT activities in HFD-fed C57BL/6 mice.

High-performance dendritic contrast agents for X-ray computed tomography imaging using potent tetraiodobenzene derivatives.

The use of computed tomography (CT) for vascular imaging is critical in medical emergencies requiring urgent diagnostic decisions, such as cerebral ischemia and many cardiovascular diseases. Small-molecule iodinated contrast media are often injected intravenously as radiopaque agents during CT imaging to achieve high contrast enhancement of vascular systems. The rapid excretion rate of these agents is overcome by injecting a significantly high dose of iodine, which can have serious side effects. Here we report a simple method to prepare blood-pool contrast agents for CT based on dendrimers for the first time using tetraiodobenzene derivatives as potent radiopaque moieties. Excellent in vivo safety has been demonstrated for these small (13-22nm) unimolecular water-soluble dendritic contrast agents, which exhibit high contrast enhancement in the blood-pool and effectively extend their blood half-lives. Our method is applicable to virtually any scaffold with suitable surface groups and may fulfill the current need for safer, next-generation iodinated CT contrast agents.

NAD(P)H:quinone oxidoreductase 1 activation reduces blood pressure through regulation of endothelial nitric oxide synthase acetylation in spontaneously hypertensive rats.

BACKGROUND: Endothelial nitric oxide synthase (eNOS) is involved in blood pressure (BP) regulation through the production of nitric oxide. Sirtuin I (SIRT1), an NAD-dependent protein deacetylase, promotes vascular relaxation through deacetylation and activation of eNOS. ß-Lapachone (ßL) increases the cellular NAD(+)/NADH ratio by activating NAD(P)H: quinone oxidoreductase 1 (NQO1). In this study, we verified whether activation of NQO1 by ßL modulates BP through regulation of eNOS acetylation in a hypertensive animal model. METHODS: Spontaneously hypertensive rats (SHRs) and an endothelial cell line (bEnd.3 cells) were used to investigate the hypotensive effect of ßL and its mechanism of action. RESULTS: ßL treatment significantly lowered the BP in SHRs, but this hypotensive effect was completely blocked by eNOS inhibition with ω-nitro-l-arginine methyl ester. In vitro studies revealed that ßL activated eNOS, which was accompanied by an increased NAD(+)/NADH ratio. Moreover, ßL significantly decreased acetylation of eNOS; however, this reduced eNOS acetylation was completely precluded by inhibition of SIRT1 in the bEnd.3 cells and in the aorta of the SHRs. Consistent with these effects, ßL-induced reduction in BP was also abolished by SIRT1 inhibition in the SHRs. CONCLUSIONS: To the best of our knowledge, this is the first study to demonstrate that eNOS acetylation can be regulated by NQO1 activation in an SIRT1-dependent manner, which is correlated with the relief of hypertension. These findings provide strong evidence that NQO1 might be a new therapeutic target for hypertension.

Protection of NAD(P)H:quinone oxidoreductase 1 against renal ischemia/reperfusion injury in mice.

Ischemia/reperfusion (I/R) is the most common cause of acute renal injury. I/R-induced reactive oxygen species (ROS) are thought to be a major factor in the development of acute renal injury by promoting the initial tubular damage. NAD(P)H: quinone oxidoreductase 1 (NQO1) is a well-known antioxidant protein that regulates ROS generation. The purpose of this study was to investigate whether NQO1 modulates the renal I/R injury (IRI) associated with NADPH oxidase (NOX)-derived ROS production in an animal model. We analyzed renal function, oxidative stress, and tubular apoptosis after IRI. NQO1(-/-) mice showed increased blood urea nitrogen and creatinine levels, tubular damage, oxidative stress, and apoptosis. In the kidneys of NQO1(-/-) mice, the cellular NADPH/NADP(+) ratio was significantly higher and NOX activity was markedly higher than in those of NQO1(+/+) mice. The activation of NQO1 by ß-lapachone (ßL) significantly improved renal dysfunction and reduced tubular cell damage, oxidative stress, and apoptosis by renal I/R. Moreover, the ßL treatment significantly lowered the cellular NADPH/NADP(+) ratio and dramatically reduced NOX activity in the kidneys after IRI. From these results, it was concluded that NQO1 has a protective role against renal injury induced by I/R and that this effect appears to be mediated by decreased NOX activity via cellular NADPH/NADP(+) modulation. These results provide convincing evidence that NQO1 activation might be beneficial for ameliorating renal injury induced by I/R.

Enhanced activation of NAD(P)H: quinone oxidoreductase 1 attenuates spontaneous hypertension by improvement of endothelial nitric oxide synthase coupling via tumor suppressor kinase liver kinase B1/adenosine 5'-monophosphate-activated protein kinase-mediated guanosine 5'-triphosphate cyclohydrolase 1 preservation.

AIMS: Guanosine 5'-triphosphate cyclohydrolase-1 (GTPCH-1) is a rate-limiting enzyme in de-novo synthesis of tetrahydrobiopterin (BH4), an essential cofactor for endothelial nitric oxide synthase (eNOS) coupling. Adenosine 5'-monophosphate-activated protein kinase (AMPK) is crucial for GTPCH-1 preservation, and tumor suppressor kinase liver kinase B1 (LKB1), an upstream kinase of AMPK, is activated by NAD-dependent class III histone deacetylase sirtuin 1 (SIRT1)-mediated deacetylation. ß-Lapachone has been shown to increase cellular NAD/NADH ratio via NAD(P)H: quinone oxidoreductase 1 (NQO1) activation. In this study, we have evaluated whether ß-lapachone-induced NQO1 activation modulates blood pressure (BP) through preservation of GTPCH-1 in a hypertensive animal model. METHODS AND RESULTS: Spontaneously hypertensive rats (SHRs), primary aortic endothelial cells, and endothelial cell line were used to investigate the hypotensive effect of ß-lapachone and its action mechanism. ß-Lapachone treatment dramatically lowered BP and vascular tension in SHRs and induced eNOS activation in endothelial cells. Consistent with these effects, ß-lapachone treatment also elevated levels of both aortic cGMP and plasma nitric oxide in SHRs. Meanwhile, ß-lapachone-treated SHRs showed significantly increased levels of aortic NAD, LKB1 deacetylation, and AMPK Thr phosphorylation followed by increased GTPCH-1 and tetrahydrobiopterin/dihydrobiopterin ratio. In-vitro study revealed that AMPK inhibition by overexpression of dominant-negative AMPK nearly abolished GTPCH-1 protein conservation. Enhanced LKB1 deacetylation and AMPK activation were also elicited by ß-lapachone in endothelial cells. However, inhibition of LKB1 deacetylation by blocking of NQO1 or SIRT1 blunted AMPK activation by ß-lapachone. CONCLUSION: This is the first study demonstrating that eNOS coupling can be regulated by NQO1 activation via LKB1/AMPK/GTPCH-1 modulation, which is possibly correlated with relieving hypertension. These findings provide strong evidence to suggest that NQO1 might be a new therapeutic target for hypertension.

Protective role of NAD(P)H:quinone oxidoreductase 1 (NQO1) in cisplatin-induced nephrotoxicity.

Although cisplatin is widely used as an anti-cancer agent, its use is significantly limited because of its tendency to induce nephrotoxicity through poorly understood mechanisms. NAD(P)H:quinone oxidoreductase 1 (NQO1) is well known to regulate ROS generation. The purpose of this study was to investigate whether NQO1 modulates cisplatin-induced renal failure associated with NADPH oxidase (NOX)-derived ROS production in an animal model. NQO1-/- mice were treated with cisplatin (18 mg/kg) and renal function, oxidative stress, and tubular apoptosis were assessed. NQO1-/- mice showed increased blood urea nitrogen and creatinine levels, tubular damage, oxidative stress, and apoptosis. In accordance with these results, the cellular NADPH/NADP ratio and NOX activity were markedly increased in the kidneys of NQO1-/- mice compared to NQO1+/+ mice. In addition, activation of NQO1 by ßL treatment significantly improved renal dysfunction and reduced tubular cell damage, oxidative stress, and apoptosis. This study demonstrates that NQO1 protects cells against renal failure induced by cisplatin, and that this effect is mediated by decreased NOX activity via cellular NADPH/NADP modulation. These results provide convincing evidence that NQO1 might be beneficial for ameliorating renal failure induced by cisplatin.

Increased vulnerability to ß-cell destruction and diabetes in mice lacking NAD(P)H:quinone oxidoreductase 1.

NAD(P)H:quinone oxidoreductase 1 (NQO1) has been known to protect cells against stressors, including the diabetogenic reagent streptozotocin (STZ). The present study demonstrated that NQO1 deficiency resulted in increased pancreatic ß-cell death induced by multiple low dose of STZ (MLDS) injections. NQO1 knockout (KO) mice showed hyperglycemia, body weight loss, impaired glucose clearance rate and a lower plasma insulin level after MLDS treatment. Moreover, ß-cell mass and pancreatic insulin content were significantly lower in KO mice than in wild-type (WT) mice after MLDS treatment. Five days after the first STZ treatment, the islets of KO mice had substantially more TUNEL-positive ß-cells than those of WT mice, but there was no difference in the regeneration of ß-cells between KO mice and WT mice. At the same time, MLDS-treated KO mice showed significantly increased apoptotic markers in ß-cells, including cleaved caspase 3, Smac/DIABLO and AIF (apoptosis inducing factor) in the cytoplasm. These results suggest that mice deficient in NQO1 are vulnerable to MLDS-induced ß-cell destruction and diabetes, caused by increase of ß-cell apoptosis in pancreas.

Scoparone inhibits adipocyte differentiation through down-regulation of peroxisome proliferators-activated receptor γ in 3T3-L1 preadipocytes.

This study was performed to investigate the effect of scoparone on the differentiation of 3T3-L1 preadipocytes. Scoparone inhibited triglyceride (TG) accumulation in the mature adipocytes, evidenced by Oil-red O staining and intracellular quantification. Real time-PCR analysis showed that scoparone significantly down-regulated the mRNA expression of key adipogenic transcription factors, PPARγ, C/EBPα, compared with mature adipocytes. Scoparone appeared to reduce mRNA expression of SREBP1c and FAS being related to the late stage of adipogenesis. Furthermore, aP2 and CD36/FAT, as adipocyte-specific genes, were decreased in mature adipocytes by scoparone treatment. Moreover, scoparone inhibited the up-regulated expression of PPARγ target genes by rosiglitazone to near that observed in cells treated with GW9662. The luciferase assay revealed that scoparone negatively regulates the transcriptional activity of PPARγ. Chromatin immunoprecipitation assay also showed that participation of scoparone in the regulation of PPARγ. Collectively, scoparone has a PPARγ antagonic effect and suppresses differentiation through down-regulation of adipogenic genes by PPARγ inhibition in 3T3-L1 preadipocytes.

Davallialactone protects against acetaminophen overdose-induced liver injuries in mice.

Oxidative stress is closely associated with acetaminophen (APAP)-induced toxicity. Davallialactone (DAVA), a hispidin analog derived from the mushroom Inonotus xeranticus, has antioxidant properties. This study evaluated whether DAVA plays protective roles against APAP hepatotoxicity in mice. Pretreatments with DAVA (10 mg/kg) prior to exposures of mice to a hepatotoxic dose of 600 mg/kg APAP significantly increased survival rate compared to APAP alone. To verify this effect, mice were treated with 400 mg/kg APAP 30 min after DAVA administration and were then sacrificed after 0.5, 1, 3, and 6 h. APAP alone caused severe liver injuries as characterized by increased plasma GOT and GPT levels, ATP and GSH depletion, and peroxynitrite and 4-HNE formations. These liver damages induced by APAP were significantly attenuated by DAVA pretreatments. The GSH/GSSG ratio nearly recovered to the levels observed in non-APAP-treated mice at 6h after APAP treatment in DAVA-pretreated mice. Furthermore, while hepatic ROS levels were increased by APAP exposures, pretreatments with DAVA completely blocked ROS formation. In addition, APAP-induced sustained activations of JNK and ERK were remarkably reduced by DAVA pretreatment. In conclusion, these results suggest that DAVA plays protective roles against APAP-mediated hepatotoxicity through function as ROS scavenger.

Inverse agonist of nuclear receptor ERRγ mediates antidiabetic effect through inhibition of hepatic gluconeogenesis.

Type 2 diabetes mellitus (T2DM) is a progressive metabolic disorder with diverse pathological manifestations and is often associated with abnormal regulation of hepatic glucose production. Many nuclear receptors known to control the hepatic gluconeogenic program are potential targets for the treatment of T2DM and its complications. Nevertheless, the therapeutic potential of the estrogen-related receptor γ (ERRγ) in T2DM remains unknown. In this study, we show that the nuclear receptor ERRγ is a major contributor to hyperglycemia under diabetic conditions by controlling hepatic glucose production. Hepatic ERRγ expression induced by fasting and diabetic conditions resulted in elevated levels of gluconeogenic gene expression and blood glucose in wild-type mice. Conversely, ablation of hepatic ERRγ gene expression reduced the expression of gluconeogenic genes and normalized blood glucose levels in mouse models of T2DM: db/db and diet-induced obesity (DIO) mice. In addition, a hyperinsulinemic-euglycemic clamp study and long-term studies of the antidiabetic effects of GSK5182, the ERRγ-specific inverse agonist, in db/db and DIO mice demonstrated that GSK5182 normalizes hyperglycemia mainly through inhibition of hepatic glucose production. Our findings suggest that the ability of GSK5182 to control hepatic glucose production can be used as a novel therapeutic approach for the treatment of T2DM.

The orphan nuclear receptor small heterodimer partner negatively regulates pancreatic beta cell survival and hyperglycemia in multiple low-dose streptozotocin-induced type 1 diabetic mice.

The small heterodimer partner (SHP; NR0B2) regulates the transcription of a variety of target genes and controls a variety of physiological functions in various tissues. However, the role of SHP in beta cell has not been fully determined yet. We used SHP knockout (SHP KO) mice to investigate the role of SHP in multiple low-dose streptozotocin (MLDS)-induced diabetes. Blood glucose and insulin levels were measured until 20 days, and intraperitoneal glucose tolerance and glucose-stimulated insulin secretion tests were performed. The expression of apoptotic genes and beta cell markers were detected by quantitative realtime-polymerase chain reaction, immunostaining and western blot analysis. SHP KO mice showed significantly lower blood glucose, higher insulin levels, and enhanced glucose tolerance compared with wild type (WT) mice after MLDS treatment. Moreover, beta cell mass and pancreatic insulin content were remarkably increased in SHP KO mice. In the response to glucose stimulation, islets of SHP KO showed increased insulin secretion via up-regulation of beta cell enriched transcription factors compared to WT mice after streptozotocin (STZ) treatment. In quantification for beta cell apoptosis at day 1 post STZ treatment, the SHP KO mice showed significantly increased anti-apoptotic gene expression and decreased release of apoptotic markers cytochrome c, smac/diablo, and only a few apoptotic beta cells were found in SHP KO pancreas through inactivation of caspase-3, compared to those of WT. These data demonstrate that SHP deficiency ameliorates hyperglycemia and preserves islet function by inhibiting apoptosis of pancreatic beta cells and up-regulating of their enriched transcriptional factors.

NQO1 activation regulates angiotensin-converting enzyme shedding in spontaneously hypertensive rats.

AIMS: Angiotensin-converting enzyme (ACE) plays a key role in blood pressure (BP) homeostasis via regulation of angiotensin II. Active ACE ectodomain is enzymatically cleaved and released into body fluids, including plasma, and elevated plasma ACE levels are associated with increased BP. ß-lapachone (ßL) has been shown to increase cellular NAD(+)/NADH ratio via activation of NAD(P)H:quinone oxidoreductase 1 (NQO1). In this study, we evaluated whether NQO1 activation by ßL modulates BP through regulation of ACE shedding in an animal model of hypertension. METHODS AND RESULTS: Spontaneously hypertensive rats (SHR) and a human ACE-overexpressing rat lung microvascular endothelial cell line (RLMVEC-hACE) were used to investigate the mechanism by which ßL exerts a hypotensive effect. In vitro studies revealed that ßL significantly increased intracellular Ca(2+) ([Ca(2+)]i) levels and CaMKII Thr(286) phosphorylation, followed by diminished ACE cleavage secretion into culture media. Inhibition of ßL-induced [Ca(2+)]i level changes through intracellular Ca(2+) chelation, Nqo1-specific siRNA or ryanodine receptor blockade abolished not only ßL-induced increase in [Ca(2+)]i levels and CaMKII phosphorylation, but also ßL-mediated decrease in ACE shedding. The effect of ßL on ACE shedding was also blocked by inhibition of CaMKII. In SHR, ßL reduced BP following increase of CaMKII Thr(286) phosphorylation in the lung and decrease of ACE activity and angiotensin II levels in plasma. CONCLUSION: This is the first study demonstrating that ACE shedding is regulated by NQO1 activation, which is possibly correlated with relieving hypertension in SHR. These findings provide strong evidence suggesting that NQO1 might be a new target for ACE modulation and BP control.

Susceptibility to gold nanoparticle-induced hepatotoxicity is enhanced in a mouse model of nonalcoholic steatohepatitis.

Although the safety of gold nanoparticle (AuNP) use is of growing concern, most toxicity studies of AuNPs had focused on their chemical characteristics, including their physical dimensions, surface chemistry, and shape. The present study examined the susceptibility of rodents with healthy or damaged livers to AuNP-induced hepatotoxicity. To induce a model of liver injury, mice were fed a methionine- and choline-deficient (MCD) diet for 4 weeks. Sizes and biodistribution of 15-nm PEGylated AuNPs were analyzed by transmission electron microscopy. Levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were estimated with an automatic chemical analyzer, and liver sections were subjected to pathological examination. Activities of antioxidant enzymes were determined by biochemical assay. Lateral tail vein injection of MCD diet-fed mice with 5 mg kg(-1) AuNPs significantly elevated the serum ALT and AST levels compared to MCD diet-fed mice injected with mPEG (methylpolyethylene glycol). Similarly, severe hepatic cell damage, acute inflammation, and increased apoptosis and reactive oxygen species (ROS) production were observed in the livers of AuNP-injected mice on the MCD diet; these liver injuries were attenuated in mice fed a normal chow diet. The results suggest that AuNPs display toxicity in a stressed liver environment by stimulating the inflammatory response and accelerating stress-induced apoptosis. These conclusions may point to the importance of considering health conditions, including liver damage, in medical applications of AuNPs.