Mutations of key hydrophobic surface residues of 11β‐hydroxysteroid dehydrogenase type 1 increase solubility and monodispersity in a bacterial expression system

11β‐Hydroxysteroid dehydrogenase type 1 (11β‐HSD1) is a key enzyme in the conversion of cortisone to the functional glucocorticoid hormone cortisol. This activation has been implicated in several human disorders, notably the metabolic syndrome where 11β‐HSD1 has been identified as a novel target for potential therapeutic drugs. Recent crystal structures have revealed the presence of a pronounced hydrophobic surface patch lying on two helices at the C‐terminus. The physiological significance of this region has been attributed to facilitating substrate access by allowing interactions with the endoplasmic reticulum membrane. Here, we report that single mutations that alter the hydrophobicity of this patch (I275E, L266E, F278E, and L279E in the human enzyme and I275E, Y266E, F278E, and L279E in the guinea pig enzyme) result in greatly increased yields of soluble protein on expression in E. coli. Kinetic analyses of both reductase and dehydrogenase reactions indicate that the F278E mutant has unaltered K_m values for steroids and an unaltered or increased k_cat. Analytical ultracentrifugation shows that this mutation also decreases aggregation of both the human and guinea pig enzymes, resulting in greater monodispersity. One of the mutants (guinea pig F278E) has proven easy to crystallize and has been shown to have a virtually identical structure to that previously reported for the wild‐type enzyme. The human F278E enzyme is shown to be a suitable background for analyzing the effects of naturally occurring mutations (R137C, K187N) on enzyme activity and stability. Hence, the F278E mutants should be useful for many future biochemical and biophysical studies of the enzyme.     

Sex‐Dependent Dietary Obesity,Induction of UCPs,and Leptin Expression in Rat Adipose Tissues

Objective: The aim of this study was to determine the sex‐dependent differences in the response of key parameters involved in thermogenesis and control of body weight in brown adipose tissue (BAT) and white adipose tissue (WAT) in postcafeteria‐fed rats, a model of dietary obesity. Research Methods and Procedures: BAT and WAT were obtained from male and female control and postcafeteria‐fed Wistar rats. Postcafeteria‐fed rats were initially fed with cafeteria diet from day 10 of life until day 110 (cafeteria period) and with standard chow diet from then until day 180 of life (postcafeteria period). Body mass and energy intake were evaluated. Biometric parameters were analyzed in interscapular BAT (IBAT). Levels of uncoupling protein 1 (UCP1), α₂‐adrenergic receptor (AR), and β₃‐AR proteins and UCP1, UCP2, UCP3, β₃‐AR, and leptin mRNAs, in IBAT or WAT, were studied by Western blot and Northern blot analyses, respectively. Results: Rats attained 59% (females) and 39% (males) increase in body weight at the end of the cafeteria period. During the postcafeteria period, the rats showed a loss of body weight, which was higher in females. Postcafeteria‐fed female rats also presented higher activation of thermogenic parameters in IBAT, including UCP1, UCP2, and UCP3 mRNAs. Female control rats showed lower levels of both α2 and β₃‐ARs in BAT compared with male rats, but these levels in postcafeteria‐fed female and male rats were the same, because males tended to down‐regulate them. Levels of leptin mRNA in response to the postcafeteria state depended on gender and the specific WAT depot studied. Discussion: It is suggested that in postcafeteria‐fed female rats, BAT thermogenic capacity becomes more efficiently activated than in males. Female rats also showed a bigger weight loss. The parallel regulation of the levels of UCP2 and UCP3 mRNAs, with respect to UCP1 mRNA, with higher activation in female postcafeteria‐fed rats, suggests a possible role of both UCP2 and UCP3 in the regulation of energy expenditure and in the control of body weight. The distinct responses to overweight of α2 and β₃‐ARs—which were sex dependent—and leptin mRNA—which depended on both sex and WAT depot—also support the different response of thermogenesis‐related parameters between overweight males and females.     

Effects of Dietary Korean Proso-Millet Protein on Plasma Adiponectin,HDL Cholesterol,Insulin Levels,and Gene Expression in Obese Type 2 Diabetic Mice

We investigated the effect of dietary Korean proso-millet protein concentrate (PMP) on glycemic responses, plasma lipid levels, and the plasma level and gene expression of adiponectin in obese type 2 diabetic mice under normal and high-fat feeding conditions. The findings were that the feeding of PMP clearly elevated plasma high-density lipoprotein cholesterol (HDL cholesterol) and adiponectin levels and brought about effective reduction in the levels of glucose and insulin in mice under high-fat diet conditions as compared with a control diet. Gene expression study revealed that the diet up-regulated expression of adiponectin and down-regulated tumor necrosis factor-α (TNF-α). Considering the central role of adiponectin and HDL cholesterol in improving and ameliorating type 2 diabetes, obesity, and cardiovascular disease, our findings imply that PMP may have potential for therapeutic intervention in type 2 diabetes.     

Cortisone induces insulin resistance in C2C12 myotubes through activation of 11beta‐hydroxysteroid dehydrogenase 1 and autocrinal regulation

The enzyme 11β‐hydroxysteroid dehydrogenase 1 (11β‐HSD1) is known to catalyse inactive glucocorticoids into active forms, and its dysregulation in adipose and muscle tissues has been implicated in the development of metabolic syndrome. To delineate the molecular mechanism by which active cortisol has an antagonizing effect against insulin, we optimized the metabolic production of cortisol and its biological functions in myotubes (C2C12). Myotubes supplemented with cortisone actively catalysed its conversion into cortisol, which in turn abolished phosphorylation of Akt in response to insulin treatment. This led to diminished uptake of insulin‐induced glucose. This was corroborated by the application of 11β‐HSD1 inhibitor glycyrrhetinic acid and a glucocorticoid receptor antagonist RU‐486, which reversed completely the antagonizing effects of cortisol on insulin action. Therefore, development of specific inhibitors targeting 11β‐HSD1 might be a promising way to improve impaired insulin‐stimulated glucose uptake. Copyright © 2013 John Wiley & Sons, Ltd.     

Gender Effects on Adrenergic Receptor Expression and Lipolysis in White Adipose Tissue of Rats

Objective: To investigate the effects of short‐term (15 days) cafeteria‐diet feeding on the expression of α‐ and β‐adrenergic receptors (AR) and its association with lipolytic stimulation in isolated retroperitoneal white adipocytes. Research Methods and Procedures: Six female and 6 male Wistar rats (4 weeks old) were fed a cafeteria diet plus standard diet for 15 days. The remaining 12 age‐ and sex‐matched rats received a standard diet only. White retroperitoneal adipose tissue was isolated and used for the determination of both α₂ and β‐AR expression and for in vitro studies of lipolytic activity. Results: In female control rats, we found higher lipolytic capacities located at the postreceptor level and a lower α₂/β₃‐AR ratio than male rats. Cafeteria‐diet feeding for 15 days decreased lipolytic activity in both male and female rats and altered the α_2A‐ and β₃‐AR protein levels with an increase of α_2A‐AR in males and a β₃‐AR decrease in females. Discussion: Our results indicate that a 15‐day cafeteria‐diet feeding induced an increase in the α₂/β₃‐AR balance and impaired adipose tissue lipolytic activity, which was higher in males and may contribute to the development of increased fat mass. The higher functionality of α₂‐AR, together with the minor role developed by β₃‐AR and lower lipolytic capacities located at the postreceptor level in cafeteria‐diet‐fed male rats compared with female rats, may be responsible for the gender‐dependent differences observed in this study.     

Selection and optimization of MCF‐7 cell line for screening selective inhibitors of 11β‐hydroxysteroid dehydrogenase 2

An 11β‐hydroxysteroid dehydrogenase type 1 (11β‐HSD1) produces glucocorticoid (GC) from 11‐keto metabolite, and its modulation has been suggested as a novel approach to treat metabolic diseases. In contrast, type 2 isozyme 11β‐HSD2 is involved in the inactivation of glucocorticoids (GCs), protecting the non‐selective mineralocorticoid receptor (MR) from GCs in kidney. Therefore, when 11β‐HSD1 inhibitors are pursued to treat the metabolic syndrome, preferential selectivity of inhibitors for type 1 over type 2 isozyme is rather important than inhibitory potency. Primarily, to search for cell lines with 11β‐HSD2 activity, we investigated the expression profiles of enzymes or receptors relevant to GC metabolism in breast, colon, and bone‐derived cell lines. We demonstrated that MCF‐7 cells had high expression for 11β‐HSD2, but not for 11β‐HSD1 with its cognate receptor. Next, for the determination of enzyme activity indirectly, we adopted homogeneous time resolved fluorescence (HTRF) cortisol assay. Obviously, the feasibility of HTRF to cellular 11β‐HSD2 was corroborated by constructing inhibitory response to an 11b‐HSD2 inhibitor glycyrrhetinic acid (GA). Taken together, MCF‐7 that overexpresses type 2 but not type 1 enzyme is chosen for cellular 11β‐HSD2 assay, and our results show that a nonradioactive HTRF assay is applicable for type 2 as well as type 1 isozyme. Copyright © 2010 John Wiley & Sons, Ltd.     

Gender-specific links between hepatic 11beta reduction of cortisone and adipokines

Objective: Reduction of cortisone to cortisol is mediated by 11β‐hydroxysteroid dehydrogenase type 1 (11βHSD1), a putative key enzyme in obesity‐related complications. Experimental studies suggest that adipokines, notably leptin and tumor necrosis factor‐α (TNF‐α), are of importance for 11βHSD1 activity. We hypothesized that the regulation of hepatic preceptor glucocorticoid metabolism is gender‐specific and associated with circulating levels of leptin and TNF‐α receptors and/or sex hormones. Research Methods and Procedures: A total of 34 males and 38 women (14 premenopausal and 22 postmenopausal) underwent physical examination and fasting blood sampling. Insulin sensitivity was tested by euglycemic hyperinsulinemic clamps, and hepatic 11βHSD1 enzyme activity was estimated by the conversion of orally‐ingested cortisone to cortisol. Results: Hepatic 11βHSD1 activity was negatively associated with leptin and soluble TNF (sTNF) r1 and sTNFr2 in males. These correlations remained significant after adjustment for age and insulin sensitivity, and for sTNF‐α receptors also after adjustment of BMI and waist circumference. In contrast, 11β reduction of cortisone was positively associated to leptin in females after adjustment for BMI and waist circumference. Discussion: Hepatic 11β reduction shows different links to circulating adipocyte‐derived hormones in males and females. This emphasizes the need for further studies on tissue‐specific regulation of 11βHSD1 in both genders.     

Effects of hydroxy‐delta‐5‐steroid dehydrogenase, 3 beta‐ and steroid delta‐isomerase 1 polymorphisms on fat androstenone level and gene expression in Duroc pigs

A high level of androstenone in porcine adipose tissue is a major factor contributing to boar taint. Porcine hydroxy‐delta‐5‐steroid dehydrogenase, 3 beta‐ and steroid delta‐isomerase 1 (3β‐HSD, also known as HSD3B1) plays a key role in the hepatic metabolism that catalyzes androstenone to β‐androstenol. Therefore, 3β‐HSD is a candidate gene for boar taint. This study aimed to investigate functional 3β‐HSD polymorphisms in Duroc pigs. We found eight single nucleotide polymorphisms (SNPs) in the full‐length porcine 3β‐HSD. Four of the SNPs had restriction enzyme sites, and we genotyped them in 147 uncastrated male Duroc pigs using a polymerase chain reaction–restriction fragment length polymorphism method. Pigs with the GG genotype at the g.165262G>A locus (SNP5) had significantly lower androstenone levels than did those with other genotypes (P = 0.030). SNP5 also was associated with differences in 3β‐HSD mRNA levels: pigs with the GG genotype had higher levels than those with other genotypes (P = 0.019). The SNP5 polymorphism could affect the hepatic catabolism of androstenone and consequently impact androstenone accumulation in the adipose tissue. Therefore, SNP5 in the 3β‐HSD of Duroc pigs could be a useful selective marker for decreasing boar taint.     

Mechanistic insights into the effects of SREBP1c on hepatic stellate cell and liver fibrosis

Sterol regulatory element‐binding protein 1c (SREBP1c) plays key roles in maintenance of hepatic stellate cell (HSC) quiescence. The present researches investigated the mechanisms underlying the effects of SREBP1c on HSCs and liver fibrogenesis by HSC‐targeted overexpression of the active SREBP1c using adenovirus in vitro and in vivo. Results demonstrated that SREBP1c exerted inhibitory effects on TAA‐induced liver fibrosis. SREBP1c down‐regulated TGFβ1 level in liver, reduced the receptors for TGFβ1 and PDGFβ, and interrupted the signalling pathways of Smad3 and Akt1/2/3 but not ERK1/2 in HSCs. SREBP1c also led to the decreases in the protein levels of the bromodomain‐containing chromatin‐modifying factor bromodomain protein 4, methionine adenosyltransferase 2B (MAT2B) and TIMP1 in HSCs. In vivo activated HSCs did not express cyclin D1 and cyclin E1 but SREBP1c down‐regulated both cyclins in vitro. SREBP1c elevated PPARγ and MMP1 protein levels in the model of liver fibrosis. The effect of SREBP1c on MAT2B expression was associated with its binding to MAT2B1 promoter. Taken together, the mechanisms underlying the effects of SREBP1c on HSC activation and liver fibrosis were involved in its influences on TGFβ1 level, the receptors for TGFβ1 and PDGFβ and their downstream signalling, and the molecules for epigenetic regulation of genes.     

Synthesis of β‐Ketoamide Curcumin Analogs for Anti‐Diabetic and AGEs Inhibitory Activities

Two different series of novel β‐ketoamide curcumin analogs enriched in biological activities have been synthesized. The synthesized compounds were screened for their in vitro anti‐diabetic and AGEs inhibitory activities and exhibited potent to good anti‐diabetic and AGEs inhibitory activities. The molecular docking study was also performed with the α‐amylase enzyme.     

脂联素基因单核苷酸多态性与Ⅱ型糖尿病合并肥胖及胰岛素抵抗相关联

脂联素是近年新发现的脂肪组织特异性的细胞因子,其mRNA是脂肪组织中含量最丰富的基因转录产物,该因子可通过多种途径影响个体对胰岛素的敏感性。脂联素基因多态性与肥胖、胰岛素抵抗和2型糖尿病密切相关,而与冠心病相关性研究的报道较少。本研究以中国汉族人群1,098例为对象,其中304例冠心病(CHD)患者,389例糖尿病患者(T2DM),及405例性别年龄相匹配的正常对照,采用PCR-RFLP技术对脂联素基因-4522C/T进行基因分型,并分别对血脂水平、胰岛素抵抗、体重指数等临床数据进行分析比较。研究结果显示,脂联素基因-4522C/T各基因型及等位基因在CHD组与对照组、T2DM组与对照组中的分布差异无显著性;经分组分析发现,T2DM合并肥胖患者BMI≥25kg/m2TT基因型及T等位基因明显多于对照组,差异有显著性,P=0.014和P=0.034;TT基因型T2DM患者胰岛素抵抗指数(HOMA-IR)显著高于携带有C等位基因的T2DM患者,P=0.0069。本研究提示脂联素基因-4522C/T与中国汉族人群T2DM合并肥胖的发生及T2DM患者胰岛素抵抗相关,是引发糖尿病患者肥胖和胰岛素抵抗的重要候选基因,而与冠心病的发生无关联。