A MDM2 inhibitor MX69 inhibits adipocytes adipogenesis and differentiation.

Adipose tissue, a key regulator of systemic energy homeostasis, can synthesize and store triglycerides to meet long-term energy demands. In response to nutrient overload, adipose tissue expands by hypertrophy or hyperplasia. As an oncogene, MDM2 has exerted diverse biological activities including human development, tissue regeneration, and inflammation, in addition to major oncogenic activities. Recently, some studies indicated that MDM2 plays an important role in adipose tissue function. However, the role of MX69, a MDM2 inhibitor, in adipose tissue function has not been fully elucidated. Here, we administered MX69 intraperitoneally to high-fat diet-induced obesity (DIO) wild type C57BL/6 mice and found that MX69 could promote the body weight and white adipose tissue weight of DIO mice. Moreover, MX69 had no effects on glucose tolerance and insulin sensitivity in DIO mice. And MX69 treatment decreased the size of adipocytes and fat deposition in adipose tissue and inhibited 3T3-L1 preadipocytes differentiation. Mechanistically, MX69 inhibited the protein levels of MDM2 and the mRNA levels of genes related to adipogenesis and differentiation. In summary, our results indicated that MDM2 has a crucial and complex role in regulating adipose tissue function.

Metformin impairs systemic bile acid homeostasis through regulating SIRT1 protein levels.

Metformin is widely used to treat hyperglycemia. However, metformin treatment may induce intrahepatic cholestasis and liver injury in a few patients with type II diabetes through an unknown mechanism. Here we show that metformin decreases SIRT1 protein levels in primary hepatocytes and liver. Both metformin-treated wild-type C57 mice and hepatic SIRT1-mutant mice had increased hepatic and serum bile acid levels. However, metformin failed to change systemic bile acid levels in hepatic SIRT1-mutant mice. Molecular mechanism study indicates that SIRT1 directly interacts with and deacetylates Foxa2 to inhibit its transcriptional activity on expression of genes involved in bile acids synthesis and transport. Hepatic SIRT1 mutation elevates Foxa2 acetylation levels, which promotes Foxa2 binding to and activating genes involved in bile acids metabolism, impairing hepatic and systemic bile acid homeostasis. Our data clearly suggest that hepatic SIRT1 mediates metformin effects on systemic bile acid metabolism and modulation of SIRT1 activity in liver may be an attractive approach for treatment of bile acid-related diseases such as cholestasis.

Spermidine ameliorates non-alcoholic fatty liver disease through regulating lipid metabolism via AMPK.

In this study, treatment of high-fat diet-induced obesity (DIO) C57BL/6J mice with spermidine decreased body weight and subcutaneous and visceral fat content, reversed the apparent hepatosteatosis, and reduced hepatic intracellular and serum triglyceride and total cholesterol concentrations. Moreover, spermidine treatment improved glucose tolerance and insulin sensitivity in DIO mice. The mechanism studies indicated that spermidine indeed increased the phosphorylation of hepatic AMP-activated protein kinase (AMPK), and inhibited the expression of lipogenic genes in vivo and in vitro. Moreover, these spermidine-mediated molecular effects were also abolished by compound C, an inhibitor of AMPK, in primary hepatocytes. In summary, spermidine protected against DIO-induced hepatosteatosis by decreasing lipogenic genes expression through an AMPK-mediated mechanism.

KLF10 transcription factor regulates hepatic glucose metabolism in mice.

AIM/HYPOTHESIS: Abnormal activation of hepatic gluconeogenesis leads to hyperglycaemia. However, the molecular mechanisms underlying dysregulated hepatic gluconeogenesis remain to be fully defined. Here, we explored the physiological role of Krüppel-like factor 10 (KLF10) in regulating hepatic glucose metabolism in mice. METHODS: Hepatic KLF10 expression in wild-type C57BL/6J mice, the db/db mouse model of diabetes, the ob/ob mouse model of obesity and high-fat-diet-induced obese (DIO) mice was measured. Adenoviruses expressing Klf10 or Klf10-specific short-hairpin RNA were injected into wild-type C57BL/6J mice, db/db or DIO mice. Expression of gluconeogenic genes in the liver and blood glucose levels were measured. GTTs and pyruvate tolerance tests were performed. The molecular mechanism by which KLF10 regulates hepatic glucose metabolism was explored. RESULTS: Hepatic KLF10 expression was regulated by nutritional status in wild-type mice and upregulated in diabetic, obese and DIO mice. Overexpression of KLF10 in primary hepatocytes increased the expression of gluconeogenic genes and cellular glucose output. C57BL/6J mice with KLF10 overexpression in the liver displayed increased blood glucose levels and impaired glucose tolerance. Conversely, hepatic KLF10 knockdown in db/db and DIO mice decreased blood glucose levels and improved glucose tolerance. Furthermore, luciferase reporter gene assay and chromatin immunoprecipitation analysis indicated that KLF10 activates Pgc-1α (also known as Ppargc1a) gene transcription via directly binding to its promoter region. CONCLUSIONS/INTERPRETATION: KLF10 is an important regulator of hepatic glucose metabolism and modulation of KLF10 expression in the liver may be an attractive approach for the treatment of type 2 diabetes.

SIRT6 regulates metabolic homeostasis in skeletal muscle through activation of AMPK.

Because of the mass and functions in metabolism, skeletal muscle is one of the major organs regulating whole body metabolic homeostasis. SIRT6, a histone deacetylase, has been shown to regulate metabolism in liver and brain; however, its specific role in skeletal muscle is undetermined. In the present study we explored physiological function of SIRT6 in muscle. We generated a muscle-specific SIRT6 knockout mouse model. The mice with SIRT6 deficiency in muscle displayed impaired glucose homeostasis and insulin sensitivity, attenuated whole body energy expenditure, and weakened exercise performance. Mechanistically, deletion of SIRT6 in muscle decreased expression of genes involved in glucose and lipid uptake, fatty acid oxidation, and mitochondrial oxidative phosphorylation in muscle cells because of the reduced AMP-activated protein kinase (AMPK) activity. In contrast, overexpression of SIRT6 in C2C12 myotubes activates AMPK. Our results from both gain- and loss-of-function experiments identify SIRT6 as a physiological regulator of muscle mitochondrial function. These findings indicate that SIRT6 is a potential therapeutic target for treatment of type 2 diabetes mellitus.

The hepatic FOXQ1 transcription factor regulates glucose metabolism in mice.

AIM/HYPOTHESIS: Hepatic forkhead box q1 (FOXQ1) expression levels are regulated by nutritional and pathophysiological status. In this study we investigated the role of FOXQ1 in the regulation of hepatic gluconeogenesis. METHODS: We used multiple mouse and cell models to study the role of FOXQ1 in regulating expression of gluconeogenic genes, and cellular and hepatic glucose production. RESULTS: Expression of hepatic FOXQ1 was regulated by fasting in normal mice and was dysregulated in diabetic mice. Overexpression of FOXQ1 in primary hepatocytes inhibited expression of gluconeogenic genes and decreased cellular glucose output. Hepatic FOXQ1 rescue in db/db and high-fat diet-induced obese mice markedly decreased blood glucose level and improved glucose intolerance. In contrast, wild-type C57 mice with hepatic FOXQ1 deficiency displayed increased blood glucose levels and impaired glucose tolerance. Interestingly, studies into molecular mechanisms indicated that FOXQ1 interacts with FOXO1, thereby blocking FOXO1 activity on hepatic gluconeogenesis, preventing it from directly binding to insulin response elements mapped in the promoter region of gluconeogenic genes. CONCLUSIONS/INTERPRETATION: FOXQ1 is a novel factor involved in regulating hepatic gluconeogenesis, and the decreased FOXQ1 expression in liver may contribute to the development of type 2 diabetes.

Forkhead Box P1 (FOXP1) Transcription Factor Regulates Hepatic Glucose Homeostasis.

Dysregulation of hepatic gluconeogenesis contributes to the pathogenesis of diabetes, yet the detailed molecular mechanisms remain to be fully elucidated. Here we show that FOXP1, a transcriptional repressor, plays a key role in the regulation of systemic glucose homeostasis. Hepatic expression levels of FOXP1 are decreased in diabetic mice. Modest hepatic overexpression of FOXP1 in mice inhibited the expression of gluconeogenic genes, such as peroxisome proliferators-activated receptor γ coactivator-1α (PGC-1α), phosphoenolpyruvate carboxykinase (PEPCK), and glucose-6-phosphatase (G6PC), leading to a decrease in hepatic glucose production and fasting blood glucose levels in normal mice and different mouse models of diabetes, including db/db diabetic and high-fat diet-induced obese mice. FOXP1 physically interacted with FOXO1 in vivo and competed with FOXO1 for binding to the insulin response element in the promoter region of gluconeogenic genes, thereby interfering expression of these genes. These results identify a previously unrecognized role for FOXP1 in the transcriptional control of hepatic glucose homeostasis.

Mst1 regulates hepatic lipid metabolism by inhibiting Sirt1 ubiquitination in mice.

Previous study showed mammalian Ste20-like kinase (Mst1) may serve as target for the development of new therapies for diabetes. However, the function of Mst1 involved in liver lipid metabolism has remained elusive. In this study, we report that the liver of Mst1 knockout (Mst1(-/-)) mice showed more severe liver metabolic damage under fasting and high-fat diet than that of control mice. And fasting induced hepatic Mst1 expression. Mst1 overexpression inhibited Srebp-1c expression and increased the expression of antioxidant genes in primary hepatocytes. We also found that fasting-induced expression of hepatic Sirt1 was attenuated in Mst1(-/-) mice. Mst1 overexpression promoted Sirt1 expression, probably due to inhibiting Sirt1 ubiquitination. In summary, our study suggests that Mst1 regulates hepatic lipid metabolism by inhibiting Sirt1 ubiquitination in mice.

Hepatic differentiated embryo-chondrocyte-expressed gene 1 (Dec1) inhibits sterol regulatory element-binding protein-1c (Srebp-1c) expression and alleviates fatty liver phenotype.

Hepatic steatosis, characterized by ectopic hepatic triglyceride accumulation, is considered as the early manifestation of non-alcoholic fatty liver diseases (NAFLD). Increased SREBP-1c level and activity contribute to excessive hepatic triglyceride accumulation in NAFLD patients; however, negative regulators of Srebp-1c are not well defined. In this study, we show that Dec1, a critical regulator of circadian rhythm, negatively regulates hepatic Srebp-1c expression. Hepatic Dec1 expression levels are markedly decreased in NAFLD mouse models. Restored Dec1 gene expression levels in NAFLD mouse livers decreased the expression of Srebp-1c and lipogenic genes, subsequently ameliorating the fatty liver phenotype. Conversely, knockdown of Dec1 expression by an adenovirus expressing Dec1-specific shRNA led to an increase in hepatic TG content in normal mouse livers. Correspondingly, expression levels of lipogenic genes, including Srebp-1c, Fas, and Acc, were increased in livers of mice with Dec1 knockdown. Moreover, a functional lipogenesis assay suggested that Dec1 overexpression repressed lipid synthesis in primary hepatocytes. Finally, a luciferase reporter gene assay indicates that DEC1 inhibits Srebp-1c gene transcription via the E-box mapped to the promoter region. Chromatin immunoprecipitation confirmed that DEC1 proteins bound to the identified E-box element. Our studies indicate that DEC1 is an important regulator of Srebp-1c expression and links circadian rhythm to hepatic lipogenesis. Activation of Dec1 can alleviate the nonalcoholic fatty liver phenotype.

Sirt1 mediates the effect of the heme oxygenase inducer, cobalt protoporphyrin, on ameliorating liver metabolic damage caused by a high-fat diet.

BACKGROUND & AIMS: Heme oxygenase 1 (HO-1)-mediated increases in adiponectin, ameliorate the deleterious effects of obesity and metabolic syndrome; however, the effect of HO-1 on hepatic lipid metabolism remains elusive. The aim of this study is to evaluate the role of HO-1 in hepatic lipid metabolism. METHODS: Functional studies were performed using C57BL/6J (WT) mice and Sirt1 liver specific mutant (Sirt1-deficient) mice. The molecular mechanism was explored in primary hepatocytes and mouse liver. RESULTS: Chronic exposure to high-fat diet (HFD) induced hepatic steatosis in WT mice. Treatment of WT mice on HFD with cobalt protoporphyrin (CoPP), an inducer of HO-1 activity, decreased body weight and visceral fat content, reduced intracellular hepatic triglyceride and serum total cholesterol concentrations, and decreased liver lipid droplet formation. Compared with WT mice, the administration of CoPP to Sirt1-deficient mice on HFD increased visceral fat content, and slightly promoted liver lipid droplet formation. CoPP improved glucose tolerance and insulin sensitivity in WT mice on HFD, but compromised insulin sensitivity in Sirt1-deficient mice on HFD. Furthermore, CoPP-induced Sirt1 expression and decreased sterol regulatory element binding protein 1c (SREBP-1c) expression in WT mice on HFD. However, CoPP promoted SREBP-1c expression in Sirt1-deficient hepatocytes, which was reversed by a protein tyrosine phosphatase 1b inhibitor. Additionally, while the administration of CoPP to WT mice on HFD improved antioxidant and anti-inflammatory states, these CoPP-mediated effects were abolished in Sirt1-deficient mice. CONCLUSIONS: Sirt1 mediates the effect of CoPP on ameliorating liver metabolic damage caused by HFD.

MicroRNA-29a-c decrease fasting blood glucose levels by negatively regulating hepatic gluconeogenesis.

BACKGROUND & AIMS: The expression levels of microRNA-29 (miR-29) family members (miR-29a, miR-29b, miR-29c, here denoted collectively as miR-29a-c) are increased in livers of Goto-Kakizaki diabetic rats and db/db diabetic mice. However, the functional consequences of miR-29a-c upregulation in diabetic livers are not explored. The objective of this study was to evaluate the roles of miR-29a-c in the regulation of hepatic glucose production and blood glucose levels using different mouse models. METHODS: db/m, db/db diabetic and diet-induced obese (DIO) mice were injected with adenovirus expressing miR-29a-c through the tail vein. Blood glucose levels were measured and glucose-tolerance tests and pyruvate-tolerance tests were performed. To explore the molecular mechanism by which miR-29a-c regulate hepatic glucose metabolism, gain or loss of miR-29a-c function studies were performed in primary mouse hepatocytes and the direct effectors of miR-29-mediated effects on glucose metabolism were identified. RESULTS: Adenovirus-mediated overexpression of miR-29a-c in the livers of db/m, db/db, and DIO mice decreased fasting blood glucose levels and improved glucose tolerance. Overexpression of miR-29a-c in primary hepatocytes and mouse livers decreased the protein levels of PGC-1α and G6Pase, the direct targets of miR-29a-c, thereby reducing cellular, and hepatic glucose production. In contrast, loss of miR-29a-c function in primary hepatocytes increased the protein levels of PGC-1α and G6Pase and increased cellular glucose production. Finally, enforced expression of PGC-1α increased miR-29a-c expression levels in primary hepatocytes, thus forming a negative feedback regulation loop. CONCLUSIONS: miR-29a-c can regulate hepatic glucose production and glucose tolerance in mice.

Mouse KLF11 regulates hepatic lipid metabolism.

BACKGROUND & AIMS: Missense mutations in human Krüppel-like factor 11 (KLF11) lead to the development of diabetes, as a result of impaired insulin synthesis in the pancreas. However, the role of KLF11 in peripheral tissues is largely unknown. The aim of this study is to evaluate the role of KLF11 in the regulation of hepatic lipid homeostasis using different mouse models. METHODS: Adenoviruses expressing KLF11 (Ad-KLF11) or KLF11-specific shRNA (Ad-shKLF11) were injected into db/db diabetic, high-fat diet-induced obese (DIO), or normal C57BL/6J mice. Histological analysis of the fatty liver phenotype and biochemical analysis of hepatic and serum TG levels in these mice were performed. The molecular mechanism by which KLF11 regulates lipid metabolism in primary hepatocytes and mouse livers was explored. RESULTS: The expression of the transcription factor KLF11 gene is dysregulated in the livers of db/db and DIO mice. Adenovirus-mediated overexpression of KLF11 in the livers of db/db and DIO mice activates the PPARα signaling pathway, subsequently markedly improving the fatty liver phenotype. Conversely, knockdown of KLF11, by adenovirus (Ad-shKLF11) in livers of wild type C57BL/6J and db/m mice, increases hepatic triglyceride (TG) levels, owing to decreased fatty acid oxidation. Finally, the treatment of diabetic mice with Ad-shPPARα abolishes KLF11 stimulatory effects on the expression of genes involved in fatty acid oxidation and inhibitory effects on hepatic TG content. In contrast, PPARα rescue restores the increased hepatic TG levels in Ad-shKLF11-infected db/m mice to normal levels. CONCLUSIONS: KLF11 is an important regulator of hepatic lipid metabolism.

Mouse patatin-like phospholipase domain-containing 3 influences systemic lipid and glucose homeostasis.

UNLABELLED: Human patatin-like phospholipase domain-containing 3 (PNPLA3) is associated with increased liver fat content and liver injury. Here, we show that nutritional status regulates PNPLA3 gene expression in the mouse liver. Sterol response element binding protein-1 (SREBP-1) activated PNPLA3 gene transcription via sterol regulatory elements (SREs) mapped to the promoter region. Chromatin immunoprecipitation and electrophoretic mobility shift assays confirmed that SREBP-1 proteins bound to the identified SREs. Furthermore, SREBP-1c mediated the insulin and liver X receptor agonist TO901317-dependent induction of PNPLA3 gene expression in hepatocytes. Adenovirus-mediated overexpression of mouse PNPLA3 increased intracellular triglyceride content in primary hepatocytes, and knockdown of PNPLA3 suppressed the ability of SREBP-1c to stimulate lipid accumulation in hepatocytes. Finally, the overexpression of PNPLA3 in mouse liver increased the serum triglyceride level and impaired glucose tolerance; in contrast, the knockdown of PNPLA3 in db/db mouse liver improved glucose tolerance. CONCLUSION: Our data suggest that mouse PNPLA3, which is a lipogenic gene directly targeted by SREBP-1, promotes lipogenesis in primary hepatocytes and influences systemic lipid and glucose metabolism.

Decreased miR-29 suppresses myogenesis in CKD.

The mechanisms underlying the muscle wasting that accompanies CKD are not well understood. Animal models suggest that impaired differentiation of muscle progenitor cells may contribute. Expression of the myogenesis-suppressing transcription factor Ying Yang-1 increases in muscle of animals with CKD, but the mechanism underlying this increased expression is unknown. Here, we examined a profile of microRNAs in muscles from mice with CKD and observed downregulation of both microRNA-29a (miR-29a) and miR-29b. Because miR-29 has a complementary sequence to the 3'-untranslated region of Ying Yang-1 mRNA, a decrease in miR-29 could increase Ying Yang-1. We used adenovirus-mediated gene transfer to express miR-29 in C2C12 myoblasts and measured its effect on both Ying Yang-1 and myoblast differentiation. An increase in miR-29 decreased the abundance of Ying Yang-1 and improved the differentiation of myoblasts into myotubes. Similarly, using myoblasts isolated from muscles of mice with CKD, an increase in miR-29 improved differentiation of muscle progenitor cells into myotubes. In conclusion, CKD suppresses miR-29 in muscle, which leads to higher expression of the transcription factor Ying Yang-1, thereby suppressing myogenesis. These data suggest a potential mechanism for the impaired muscle cell differentiation associated with CKD.

Protocol to knock in or out target genes in mouse epidydimal white adipose tissue by spot injecting adeno-associated viruses.

Visceral fat obesity is more strongly associated with ectopic fat deposition, lipotoxicity, and metabolic disease compared to generalized obesity. To study the function of visceral fat tissue, we describe steps to knock in or out target genes by spot injecting adeno-associated viruses (AAV) in visceral fat tissue. We provide details on anesthesia, incision, and spot injection into the epidydimal white adipose tissue (eWAT) of live anesthetized mice. Furthermore, we detail an efficient technique for expressing exogenous protein in mouse eWAT. For complete details on the use and execution of this protocol, please refer to Zhao et al. (2022).

Murine double minute 2 aggravates adipose tissue dysfunction through ubiquitin-mediated six-transmembrane epithelial antigen of prostate 4 degradation.

Healthy adipose tissue is crucial to maintain normal energy homeostasis. Little is known about the role of murine double minute 2 (MDM2), an E3 ubiquitin ligase and has been highlighted in oncopathology, in adipose tissue. Our results indicated that MDM2 expression was associated with nutritional status. Mdm2 adipocyte-specific knock-in (Mdm2-AKI) mice exhibited exacerbated weight gain, insulin resistance, and decreased energy expenditure. Meanwhile, chronic high-fat diet (HFD) exposure caused obvious epididymal white adipose tissue (eWAT) dysfunction, such as senescence, apoptosis, and chronic inflammation, thereby leading to hepatic steatosis in Mdm2-AKI mice. Mechanically, MDM2 could interact with six-transmembrane epithelial antigen of prostate 4 (STEAP4) and inhibit STEAP4 expression through ubiquitin-mediated STEAP4 degradation. Thereinto, the K18 and K161 sites of STEAP4 were ubiquitin-modificated by MDM2. Finally, STEAP4 restoration in eWAT of Mdm2-AKI mice on a HFD rescued MDM2-induced adipose dysfunction, insulin resistance, and hepatic steatosis. Summary, the MDM2-STEAP4 axis in eWAT plays an important role in maintaining healthy adipose tissue function and improving hepatic steatosis.

Sterol-regulatory-element-binding protein 1c mediates the effect of insulin on the expression of Cidea in mouse hepatocytes.

Members of the Cide [cell death-inducing DFFA (DNA fragmentation factor-alpha)-like effector] gene family have been reported to be associated with lipid metabolism. In the present study, we show that Cidea mRNA levels are markedly reduced by fasting and are restored upon refeeding in mouse livers. To elucidate the molecular mechanism, the promoter region of the mouse Cidea gene was analysed and a putative SRE (sterol-regulatory element) was identified. Studies using luciferase reporter constructs together with electrophoretic mobility-shift assays and chromatin immunoprecipitation confirmed the binding of SREBP-1c (SRE-binding protein 1c) to the putative SRE. Furthermore, adenovirus-mediated overexpression of SREBP-1c led to a dramatic increase in Cidea mRNA. In contrast with the induction of Cidea expression by insulin and TO901317 in wild-type mouse hepatocytes, the stimulatory effects were lost in hepatocytes prepared from SREBP-1c-null mice. Adenovirus-mediated overexpression of Cidea in hepatocytes promoted lipid accumulation and triacylglycerol (triglyceride) storage; however, knockdown of Cidea compromised the ability of SREBP-1c to stimulate lipid accumulation. Taken together, these results suggest that SREBP-1c directly mediates the effect of insulin on Cidea in hepatocytes and that Cidea, at least in part, mediates SREBP-1c-dependent lipid accumulation.

PPARγ co-activator-1α co-activates steroidogenic factor 1 to stimulate the synthesis of luteinizing hormone and aldosterone.

The orphan nuclear receptor SF-1 (steroidogenic factor 1) is highly expressed in the pituitary, gonad and adrenal glands and plays key roles at all levels of the hypothalamic-pituitary-steroidogenic tissue axis. In the present study, we show that PGC-1α [PPARγ (peroxisome-proliferator-activated receptor γ) co-activator 1α] interacts with and co-activates SF-1 to induce LHß (luteinizing hormone ß) and αGSU (α-glycoprotein subunit) gene expression, subsequently leading to the increased secretion of LH in pituitary gonadotrope-derived αT3-1 cells. PGC-1α co-activation of LHß expression requires an SF-1-binding element [GSE (gonadotrope-specific element)] mapped to the promoter region of LHß. Mammalian two-hybrid and co-immunoprecipitation assays, as well as GST (glutathione transferase) pull-down experiments demonstrated that PGC-1α interacts with SF-1 in vivo and in vitro. Additionally, PGC-1α stimulates the expression of Cyp11b2 (aldosterone synthase gene), Cyp11b1 (steroid 11ß-hydroxylase gene) and P450scc (cholesterol side-chain cleavage enzyme), and the synthesis of aldosterone in adrenal-cortex-derived Y-1 cells. Chromatin immunoprecipitation assays confirmed that endogenous PGC-1α co-localizes with SF-1 in the LHß and Cyp11b2 promoter region. Knockdown of endogenous SF-1 by siRNA (small interfering RNA) abolished the PGC-1α induction of LHß and Cyp11b2 gene expression in αT3-1 and Y-1 cells respectively. Finally, we demonstrated that PGC-1α induces SF-1 gene expression in both αT3-1 and Y-1 cells. Taken together, our findings reveal the potential role of PGC-1α and suggest that it may play important roles in steroidogenesis, gonad development and sex differentiation through SF-1.

[Peroxisome proliferator activated receptor gamma Pro12Ala: old topic of conversation and new question].

Comments concerning Meta analysis for relationship between peroxisome proliferator activated receptor gamma Pro12Ala polymorphism and type 2 diabetes susceptibility in different cohorts in this mini review were given. The comments pointed out existent problems and presented suggestions for genetic analysis of diseases in Chinese populations.

[Adenovirus construction of expression and its function of connective tissue growth factor].

OBJECTIVE: To construct and identify a adenovirus vector of the expression of connective tissue growth factor (CTGF) and to explore the role of CTGF in the metabolism of glucose and lipid. METHODS: The over-expressed plasmid of CTGF was cloned, and then the CTGF sequences were cloned into pAdTrack-CMW vector. The reformed E. coli BJ5183-sensitive bacteria that contain pAdEasy-1 were transformed with lined vector cut by Pme I enzyme. The recombinant adenovirus vector was cut with Pac I enzyme and obtained, then transfected 293A cells to produce virus. Through three times of amplification, the adenovirus infected the primary hepatocytes to determine the infection efficiency and CTGF expression. The mice were starved for several time periods, and then the liver RNA was extracted for real-time PCR to detect the expressions of CTGF under different nutritional conditions. RESULTS: The adenovirus of CTGF was successfully produced with an infection efficiency of 90%. The expressions of the CTGF were different under different nutritional conditions and showed a coincidence with the expression of peroxisome proliferators-activated receptor gamma coactivator 1 alpha. After the mice were starved for 24h, the expression of CTGF increased by (2.38 +/- 0.51) folds; after the mice were starved for 48 h, the expression of CTGF increased by (2.95 +/- 0.57) folds (P < 0.05). CONCLUSION: CTGF is speculated to be involved in the metabolism of glucose and lipids.