Metabolic impact of overexpression of liver glycogen synthase with serine-to-alanine substitutions in rat primary hepatocytes

To investigate the effect of elevation of liver glycogen synthase (GYS2) activity on glucose and glycogen metabolism, we performed adenoviral overexpression of the mutant GYS2 with six serine-to-alanine substitutions in rat primary hepatocytes. Cell-free assays demonstrated that the serine-to-alanine substitutions caused constitutive activity and electrophoretic mobility shift. In rat primary hepatocytes, overexpression of the mutant GYS2 significantly reduced glucose production by 40% and dramatically induced glycogen synthesis via the indirect pathway rather than the direct pathway. Thus, we conclude that elevation of glycogen synthase activity has an inhibitory effect on glucose production in hepatocytes by shunting gluconeogenic precursors into glycogen. In addition, although intracellular compartmentation of glucose-6-phosphate (G6P) remains unclear in hepatocytes, our results imply that there are at least two G6P pools via gluconeogenesis and due to glucose phosphorylation, and that G6P via gluconeogenesis is preferentially used for glycogen synthesis in hepatocytes.     

Epidermal growth factor (urogastrone)-stimulated gluconeogenesis in isolated mouse hepatocytes

In freshly isolated mouse hepatocytes obtained from fasted animals, we have studied the receptors for epidermal growth factor urogastrone (EGF-URO) in terms of the electrophoretic profile, ligand affinity, and numbers of EGF-URO receptors present on the cells, and also in terms of the ability of EGF-URO to stimulate gluconeogenesis, as reflected by the increased incorporation of [3-14C]pyruvate into glucose. The effects of EGF-URO were compared with those of glucagon. Ligand-binding studies revealed that the mouse hepatocytes possess an unusually high number of EGF-URO receptors (about 3 X 10(6) binding sites/cell), with a ligand dissociation constant of 4.4 nM. The binding of EGF-URO by mouse hepatocytes was more than 10-fold higher than the previously measured binding of EGF-URO by rat hepatocytes. Crosslink-labeling studies, coupled with gel electrophoretic analysis, demonstrated the presence of intact EGF-URO receptors, although some receptor processing had occurred during the isolation procedure. EGF-URO was able to stimulate the incorporation of 3-14C-labeled pyruvate into glucose; glucagon was unable to do so. In contrast, in rat hepatocytes isolated and assayed under identical conditions, glucagon (10 nM) caused a marked (250%) stimulation of the incorporation of pyruvate into glucose. Maximally, EGF-URO caused a 34% increase in the incorporation of [3-14C]pyruvate into glucose; a half-maximal effect was observed at a concentration of 2.5 nM EGF-URO. The stimulatory effect of EGF-URO was not dependent on the concentration of pyruvate, lactate, glucose, or calcium in the incubation medium. Although raising the concentration of pyruvate in the incubation medium increased the incorporation of [3-14C]pyruvate into glycogen, EGF-URO did not cause any change in the incorporation of radioactivity into glycogen. Overall, our data point to marked differences between rat and mouse liver preparations, in terms of the hormonal regulation of glucose metabolism, and our work documents a potential role for the remarkably high number of mouse hepatocyte EGF-URO receptors in terms of the modulation of gluconeogenesis in the mouse.     

Antihyperglycemic effect of fraxetin on hepatic key enzymes of carbohydrate metabolism in streptozotocin-induced diabetic rats

Epidemiological studies have demonstrated that the diabetes mellitus is a serious health burden for both governments and healthcare providers. The present study was hypothesized to evaluate the antihyperglycemic potential of fraxetin by determining the activities of key enzymes of carbohydrate metabolism in streptozotocin (STZ) – induced diabetic rats. Diabetes was induced in male albino Wistar rats by intraperitoneal administration of STZ (40 mg/kg b.w). Fraxetin was administered to diabetic rats intra gastrically at 20, 40, 80 mg/kg b.w for 30 days. The dose 80 mg/kg b.w, significantly reduced the levels of blood glucose and glycosylated hemoglobin (HbA1c) and increased plasma insulin level. The altered activities of the key enzymes of carbohydrate metabolism such as glucokinase, glucose-6-phosphate dehydrogenase, glucose-6-phosphatase, fructose-1,6-bisphosphatase and hepatic enzymes (aspartate transaminase (AST), alanine transaminase (ALT) and alkaline phosphatase (ALP)) in the liver tissues of diabetic rats were significantly reverted to near normal levels by the administration of fraxetin. Further, fraxetin administration to diabetic rats improved body weight and hepatic glycogen content demonstrated its antihyperglycemic potential. The present findings suggest that fraxetin may be useful in the treatment of diabetes even though clinical studies to evaluate this possibility may be warranted.     

骨钙素对糖代谢影响的研究进展

骨钙素,亦称γ-羧基骨蛋白、骨谷氨酸蛋白和骨依赖维生素K蛋白,是一种由非增殖期成骨细胞合成的分泌蛋白,经过翻译后加工生成羧化骨钙素而参与骨骼发育。既往研究认为它是骨形成和骨转化的标志。然而最新研究发现,未发生羧化修饰的骨钙素对糖代谢有一定的影响作用,可促进胰腺β细胞增殖和胰岛素分泌,减弱胰岛素抵抗。多项研究证实高血糖可影响骨钙素合成,骨钙素在糖尿病患者中降低,糖尿病患者因胰岛素分泌和作用缺陷对胰岛素受体作用减弱,影响成骨细胞摄取核酸、氨基酸、胶原纤维合成,使其合成分泌骨钙素减少。这些研究确立了骨钙素为一种可以影响糖代谢的重要激素,拓展了对骨骼功能影响的同时也为新型降血糖药物的开发提供了新靶点。本文针对骨钙素对糖代谢的影响做一综述。     

Dynamic changes in mouse lipoproteins induced by transiently expressed human phospholipid transfer protein (PLTP): importance of PLTP in preβ-HDL generation

The plasma phospholipid transfer protein (PLTP) plays an important role in the regulation of plasma high density lipoprotein (HDL) levels and governs the distribution of HDL sub-populations. In the present study, adenovirus mediated overexpression of human PLTP in mice was employed to investigate the distribution of PLTP in serum and its effect on plasma lipoproteins. Gel filtration experiments showed that the distributions of PLTP activity and mass in serum are different, suggesting that human PLTP circulated in mouse plasma as two distinct forms, one with high and the other with low specific activity. Our study further demonstrates that overexpression of PLTP leads to depletion of HDL and that, as PLTP activity declines, replenishment of the HDL fraction occurs. During this process, the lipoprotein profile displays transient particle populations, including apoA-IV and apoE-rich particles in the LDL size range and small particles containing apoA-II only. The possible role of these particles in HDL reassembly is discussed. The increased PLTP activity enhanced the ability of mouse sera to produce preβ-HDL. The present results provide novel evidence that PLTP is an important regulator of HDL metabolism and plays a central role in the reverse cholesterol transport (RCT) process.     

Paraoxonase 1 (PON1) attenuates diabetes development in mice through its antioxidative properties

Paraoxonase 1 (PON1) is a lipo-lactonase which is associated with HDL and possesses antioxidative properties. Diabetes is characterized by increased oxidative stress and by decreased PON1 activity. We aimed to analyze whether oxidative status and PON1 levels in mouse sera and macrophages could affect streptozotocin (STZ)-induced diabetes development. We have used two models of mice under low oxidative stress: STZ-injected apolipoprotein E-deficient mice supplemented with the antioxidant vitamin E, and P47(phox) knockout mice. In both mice models the decreased serum basal oxidative stress, was associated with a decreased rate of diabetes development, compared with control STZ-injected apolipoprotein E-deficient mice or with C57BL mice respectively. These data suggest that oxidative stress accelerates diabetes development. Next, we analyzed the effect of PON1 on macrophage oxidative stress and on diabetes development in STZ-injected C57BL mice, PON1 knockout mice, and PON1 transgenic mice. PON1 overexpression was associated with decreased diabetes-induced macrophage oxidative stress, decreased diabetes development, and decreased mortality, in comparison to C57BL mice, and even more so when compared to PON1KO mice. We thus concluded that on increasing PON1 expression in mice, diabetes development is attenuated, a phenomenon which could be attributed to the antioxidative properties of PON1, as decrement of oxidative stress significantly attenuated STZ-induced diabetes development.     

Iron overload in Hepc1(-/-) mice is not impairing glucose homeostasis

Diabetes Mellitus is found with increasing frequency in iron overload patients with hemochromatosis. In these conditions, the pancreas shows predominant iron overload in acini but also islet beta-cells. We assess glucose homeostasis status in iron-overloaded hepcidin-deficient mice. These mice presented with heavy pancreatic iron deposits but only in the acini. The beta-cell function was found unaffected with a normal production and secretion of insulin. The mutant mice were not diabetic, responded as the control group to glucose and insulin challenges, with no alteration of insulin signalling in the muscle and the liver. These results indicate that, beta-cells iron deposits-induced decreased insulin secretory capacity might be of primary importance to trigger diabetes in hemochromatosic patients.     

重构肠道菌群Ⅱ型糖尿病大鼠造模血脂代谢变化及肝细胞线粒体超微结构观察

目的重构肠道菌群Ⅱ型糖尿病大鼠模型脂类代谢变化比较,探讨肠道菌群潜在的调脂作用。方法将64只SD雄性大鼠随机分为对照组、造模组、益生组和去污组。采用高糖高脂喂养,亚致病剂量（30mg／mL）链脲佐菌素腹腔一次性注射造模,分别于2周、4周后取腹腔主动脉血做血脂检测,同时处死大鼠取肝脏做电镜切片观察。结果注射4周后各组出现的胆固醇升高两两比较差异有统计学意义（P〈0．01）,LDL升高差异有统计学意义（P〈0．01）;益生组肝细胞线粒体受损明显;去污组血脂升高相对较轻,肝细胞内有大量脂肪堆积。结论益生菌对Ⅱ型糖尿病大鼠胆固醇升高有一定改善作用,肝线粒体损伤较明显;去污染组损伤相对较轻,而脂肪堆积明显;提示糖尿病脂类代谢与肠道菌群密切相关。     

Fluoxetine suppresses AMP-activated protein kinase signaling pathway to promote hepatic lipid accumulation in primary mouse hepatocytes

In the previous study, we demonstrated that fluoxetine (FLX) regulated lipogenic and lipolytic genes to promote hepatic lipid accumulation. On this basis, underlying mechanisms were investigated by focusing on the intracellular signaling transduction in the present study using primary mouse hepatocytes. The expression of lipogenesis- and lipolysis-related genes was evaluated with the application of specific activators and inhibitors. Activation status of respective signaling pathway and the lipid accumulation in hepatocytes were analyzed. We provided evidence that AMP-activated protein kinase (AMPK) activator AICAR (5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside) significantly suppressed the increased expression of representative lipogenesis-related genes, acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS) by FLX, while increased the repressed expression of lipolysis-related genes, carboxylesterases. In the meanwhile, FLX regulated the above genes in the same way as AMPK inhibitor Compound C did. Furthermore, AICAR inhibited the proteolytic activation of SREBP1c induced by FLX, resulting in the decreased level of nuclear SREBP1c. Further studies demonstrated that FLX significantly suppressed the phosphorylation of AMPK and subsequent phosphorylation of ACC, following the inhibited phosphorylation and nuclear export of liver kinase B1 (LKB1). As a functional analysis, FLX-induced lipid accumulation in hepatocytes was repeatedly abolished by AICAR. In conclusion, FLX-induced hepatic lipid accumulation is mediated by the suppression of AMPK signaling pathway. The findings not only provide new insight into the understanding of the mechanisms for selective serotonin reuptake inhibitors-mediated dyslipidemia effects, but also suggest a novel therapeutic target to interfere.     

侧脑室注射共轭亚油酸(CLA)对大鼠糖脂代谢的影响

观察侧脑室注射共轭亚油酸(Conjugated linoleic acid,CLA)对SD大鼠糖脂代谢的影响及其可能的机制。方法:向正常SD大鼠侧脑室内注射共轭亚油酸(CLA),分别于注射后2、4、8、12、24小时采血,试剂盒法测血糖、胰岛素、瘦素、血甘油三脂、血胆固醇、血高密度脂蛋白。48小时后处死,分离动物的脂肪(皮下、内脏、肾周、睾周)进行称重,计算体脂比。结果:与对照组相比,侧脑室注射CLA48小时后,大鼠脂体比、皮下脂肪、睾周脂肪均下降。术后2-12h血糖降低,血清胰岛素浓度也降低,而且持续的时间较长(48h)。侧脑室注射CLA对脂代谢有影响,2h时血清甘油三酯升高、胆固醇降低、4h时高密度脂蛋白升高。结论:共轭亚油酸能够通过中枢神经系统调节外周糖脂代谢,这可能与其能减轻体重的机制有关。     

Adipocyte enhancer-binding protein 1 (AEBP1) (a novel macrophage proinflammatory mediator) overexpression promotes and ablation attenuates atherosclerosis in ApoE (-/-) and LDLR (-/-) mice

Atherogenesis is a long-term process that involves inflammatory response coupled with metabolic dysfunction. Foam cell formation and macrophage inflammatory response are two key events in atherogenesis. Adipocyte enhancer-binding protein 1 (AEBP1) has been shown to impede macrophage cholesterol efflux, promoting foam cell formation, via peroxisome proliferator-activated receptor (PPAR)-γ1 and liver X receptor α (LXRα) downregulation. Moreover, AEBP1 has been shown to promote macrophage inflammatory responsiveness by inducing nuclear factor (NF)-κB activity via IκBα downregulation. Lipopolysaccharide (LPS)-induced suppression of pivotal macrophage cholesterol efflux mediators, leading to foam cell formation, has been shown to be mediated by AEBP1. Herein, we showed that AEBP1-transgenic mice (AEBP1(TG)) with macrophage-specific AEBP1 overexpression exhibit hyperlipidemia and develop atherosclerotic lesions in their proximal aortas. Consistently, ablation of AEBP1 results in significant attenuation of atherosclerosis (males: 3.2-fold, P = 0.001 [en face]), 2.7-fold, P = 0.0004 [aortic roots]; females: 2.1-fold, P = 0.0026 [en face], 1.7-fold, P = 0.0126 [aortic roots]) in the AEBP1(-/-)/low-density lipoprotein receptor (LDLR )(-/-) double-knockout (KO) mice. Bone marrow (BM) transplantation experiments further revealed that LDLR (-/-) mice reconstituted with AEBP1(-/-)/LDLR (-/-) BM cells (LDLR (-/-)/KO-BM chimera) display significant reduction of atherosclerosis lesions (en face: 2.0-fold, P = 0.0268; aortic roots: 1.7-fold, P = 0.05) compared with control mice reconstituted with AEBP1(+/+)/LDLR (-/-) BM cells (LDLR (-/-)/WT-BM chimera). Furthermore, transplantation of AEBP1(TG) BM cells with the normal apolipoprotein E (ApoE) gene into ApoE (-/-) mice (ApoE (-/-)/TG-BM chimera) leads to significant development of atherosclerosis (males: 2.5-fold, P = 0.0001 [en face], 4.7-fold, P = 0.0001 [aortic roots]; females: 1.8-fold, P = 0.0001 [en face], 3.0-fold, P = 0.0001 [aortic roots]) despite the restoration of ApoE expression. Macrophages from ApoE (-/-)/TG-BM chimeric mice express reduced levels of PPARγ1, LXRα, ATP-binding cassette A1 (ABCA1) and ATP-binding cassette G1 (ABCG1) and increased levels of the inflammatory mediators interleukin (IL)-6 and tumor necrosis factor (TNF)-α compared with macrophages of control chimeric mice (ApoE (-/-)/NT-BM ) that received AEBP1 nontransgenic (AEBP1(NT) ) BM cells. Our in vivo experimental data strongly suggest that macrophage AEBP1 plays critical regulatory roles in atherogenesis, and it may serve as a potential therapeutic target for the prevention or treatment of atherosclerosis.     

垂体功能减退症患者和低促性腺激素性性腺功能减退症患者糖脂代谢的研究

目的:比较垂体功能减退症患者(Hypopituitarism,Hypo-Pit)与低促性腺激素性性腺功能减退症(Hypogonadotropic hypogonadism,HH)的糖脂代谢状况。方法:收集从2013年6月至2016年9月在西京医院内分泌代谢科确诊的Hypo-Pit及HH男性患者的临床资料,分别有32例Hypo-Pit患者和43例HH患者纳入本研究,对照组为22例年龄、体质指数(BMI)与研究组相匹配,且无糖尿病等慢性疾病家族史的男性。采集空腹静脉血后测定血脂水平,行3h口服葡萄糖耐量试验(OGTT),测定血糖及胰岛素水平,计算葡萄糖、胰岛素曲线下面积、胰岛素抵抗指数等。3组间计量资料组间的比较应用LSD方差分析检验,非正态分布的数据经自然对数转换(ln)后进行分析。结果:各组间年龄、BMI均无统计学差异(P0.05);与正常对照组相比,Hypo-Pit患者及HH患者的高密度脂蛋白水平下降(均P0.05),甘油三酯水平升高(P0.05);与HH患者相比,Hypo-Pit患者的胆固醇、甘油三酯、低密度脂蛋白升高(P0.05);与正常对照组相比,HH患者的稳态模型胰岛素抵抗指数及胰岛素敏感指数升高(P0.05);与对照组比较,Hypo-Pit患者及HH患者OGTT 3h血糖水平升高,差异有统计学意义(均P0.05);HH患者空腹、2h、3h血清胰岛素水平均明显升高,差异有统计学意义(均P0.05);与HH组相比,Hypo-pit患者的HOMA-IR水平降低(P0.05)。结论:Hypo-Pit患者存在糖脂代谢紊乱,且脂代谢紊乱较HH患者更差,而HH患者胰岛素抵抗水平高于Hypo-Pit患者。     

Possible mode of action for insulinomimetic activity of vanadyl(IV) compounds in adipocytes

Vanadyl(IV) ions (+4 oxidation state of vanadium) and their complexes have been shown to have in vitro insulinomimetic activity and to be effective in treating animals with diabetes mellitus. Although, researchers have proposed many vanadyl compounds for the treatment of diabetes patients, the mode of action of vanadyl compounds remains controversial. In order to evaluate the mode of action of these compounds, we examined the insulinomimetic activity of VOSO4, bis(picolinato)oxovanadyl(IV), and bis(maltolato)oxovanadyl(IV) in the presence of several inhibitors relevant to the glucose metabolism. After confirming that these vanadyl compounds were incorporated in the adipocytes as estimated by ESR method, we evaluated the mode of action by examining free fatty acids (FFA) release in the adipocytes. Inhibition of FFA release by these vanadyl compounds was found to be reversed by the addition of inhibitors, typically by cytochalasin B (glucose transporter 4 (GLUT4) inhibitor), cilostamide (phosphodiesterase inhibitor), HNMPA-(AM)3 (tyrosine kinase inhibitor), and wortmannin (PI3-k inhibitor), indicating that these compounds affect primarily GLUT4 and phosphodiesterase, as named "ensemble mechanism". Based on these results, we suggest that vanadyl compounds act on at least four sites relevant to the glucose metabolism, and on GLUT4 and phosphodiesterase in particular in rat adipocytes, which in turn normalizes the blood glucose levels of diabetic animals. The obtained results provide evidence for the role of vanadyl ion and its complexes in stimulation of the uptake and degeneration of glucose.     

The Effects of stingless bee (Tetragonula biroi) honey on streptozotocin-induced diabetes mellitus in rats

Diabetes mellitus (DM) is a metabolic disease characterised by chronic hyperglycaemia with impaired carbohydrate, fat and protein metabolism caused by defects in insulin secretion or action. Based on our previous research, stingless bee honey (SLBH) from Tetragonula biroi and T. laeviceps can inhibit alpha-glucosidase activities. Therefore, the aim of the present study was to determine the effects of daily oral administration of SLBH on body weight (BW) and fasting blood glucose (FBG) levels of male rats with streptozotocin (STZ)-induced DM. Thirty-six male Sprague Dawley rats were divided into six groups of six rats each. One group of normal non-diabetic rats served as a positive control. The diabetic groups were intraperitoneally (i.p.) injected with STZ (50 mg/kg BW) for induction of DM and divided into five equal subgroups of six animals each: an untreated group as a negative control; a group treated with 0.6 mg/kg BW of glibenclamide as a positive control and three SLBN treatment groups that had daily oral administration of 0.5, 1.0 or 2.0 g/kg BW, respectively, for 35 days. The results showed that SLBH significantly reduced loss of BW in diabetic rats. FBG levels in diabetic rat blood, collected from the tail, were measured using Accu-Chek test strips. The FBG levels in diabetic rats that have oral administered intake with glibenclamide and SLBH were stable. There were no changes in serum FBG levels in SLBH-treated diabetic rats for 35 days. Pancreatic histopathology results from all groups showed no abnormalities or tissue damage in either diabetic or non-diabetic rats. The results of this study show that administration of SLBH reduced BW loss or improved BW of rats with STZ-induced DM. Meanwhile, the reduction in loss of BW that occurred in diabetic rats after 35 days of SLBH administration was the result of reduced formation of fats and proteins, which are broken down into energy. Further research is needed to determine the antidiabetic effects of honey from other stingless honeybee species.     

Interrelationship between oxidative stress,DNA damage and cancer risk in diabetes (Type 2) in Riyadh,KSA

Type 2 Diabetes Mellitus (T2DM) is the most widely known type of disorder of the endocrine system marked by hyperglycemia resulting either due to deficiency of insulin and or resistance. Persistent hyperglycemia induces oxidative stress and is suggested to play a prominent role in the pathophysiology underlying T2DM. Besides, oxidative stress can result in DNA damage leading to high cancer risk. Current study aimed to evaluate status of oxidative damage, damage to DNA and cancer biomarkers in regard to increased glucose in T2DM patients and to correlate the glycemic state with cancer. A total of 150 subjects consisting of control (50) and T2DM patients (1 0 0) were enrolled. Additionally, three tertiles were created among the two groups based on levels of HbA1c (Tertile I = 5.37 ± 0.34, n = 50; Tertile II = 6.74 ± 0.20, n = 50; Tertile III = 9.21 ± 1.47, n = 50). Oxidative stress parameters including malondialedehyde (MDA) and antioxidant enzymes were measured. Damage to DNA was analyzed by measuring the levels of DNA damage adduct-8 hydroxy deoxy Guanosine (8-OHdG). To detect cancer resulting from oxidative stress, cancer biomarkers CEA, AFP, CA125, CA-15, CA19-9, prolactin were measured in these subjects. All measurements were analysed by SPSS software. Levels of MDA and antioxidant enzymes altered significantly in T2DM group at p < 0.001 and p < 0.05 level of significance. Significant DNA damage accompanied with elevated levels of CEA, CA19-9 and decreased CA125, AFP and prolactin were noted in T2DM group. CA 19-9 and CEA levels increased at p < 0.05, whereas levels of prolactin decreased significantly (p < 0.001) in T2DM group compared to control. Additionally the mean values of DNA damage adduct 8-OHdG differ significantly at P < 0.01 between the two groups. However, no significant correlation in oxidative stress parameter, antioxidant enzymes, DNA damage and neither with the highest tertile of HbA1c (>7.5%) was noted. Based on the results obtained in the present study, we conclude that there is considerable change in oxidative stress and DNA damage in T2DM patients. Hence, assumption that the oxidative stress could cause cancer in T2DM as a result of hyperglycemic state was not speculated in this study.