饥饿、糖尿病和肥胖状态对小鼠肝脏SOCS2基因表达的影响

目的确定小鼠肝脏SOCS2基因在饥饿、糖尿病和肥胖状态下的表达水平,并初步研究SOCS2对糖异生的影响。方法动物分3组:C57BL/6J小鼠、对照组(饱食)和实验组(饥饿24 h);糖尿病模型小鼠db/db及对照小鼠db/m饱食;肥胖模型小鼠ob/ob及其对照C57BL/6J小鼠(饱食)。处死小鼠后提取肝脏RNA做反转录PCR,荧光实时定量PCR检测小鼠肝脏SOCS2及糖异生相关基因在3组小鼠中的表达水平;使用腺病毒表达系统在C57BL/6J小鼠原代肝细胞中过表达SOCS2,Western blot检测SOCS2蛋白的表达,葡萄糖生成实验检测糖输出。结果饥饿使C57BL/6J小鼠肝脏中SOCS2 mRNA水平下调,db/db和ob/ob小鼠肝脏SOCS2基因表达比其对照小鼠均明显下降(P0.05),调节糖异生的关键基因PGC-1α、PEPCK和G6Pase的mRNA水平均上升。在C57BL/6J小鼠原代肝细胞中过表达SOCS2,得到大小为Mr 23 000的蛋白,糖输出受到明显抑制。结论初步认定SOCS2可抑制C57BL/6J小鼠原代肝细胞糖异生,可能是治疗糖尿病的一个新靶点。     

Glycogen: its mode of formation and contribution to hepatic glucose output in postabsorptive humans

Summary To assess the relative contributions of gluconeogenesis and glycogenolysis to overall hepatic glucose output in postabsorptive normal humans and those of the indirect and direct pathways for glycogen synthesis, we studied six normal volunteers, who had been fasted for 16 h to reduce their hepatic glycogen stores, and then ingested glucose (250 g over 10 h) enriched with [6-³H] glucose to replenish and label their hepatic glycogen. After a 10-h overnight fast, release of the [6-³H] glucose into the circulation was traced with [2-³H] glucose to estimate breakdown of glycogen that had been formed via the direct pathway while gluconeogenesis was simultaneously estimated by incorporation of infused [¹⁴C] lactate into plasma glucose. We found that release of [6-³H] glucose into plasma (6.79±0.69 mol · kg^–1 · min^–1) accounted for 46±5% of hepatic glucose output (15.0±0.7 mol · kg^–1· min^–1) while glucose formed from lactate (2.71±0.28 mol · kg^–1 · min^–1) accounted for 19±2% of hepatic glucose output. Since these determinations underestimate direct pathway glycogenolysis and overall gluconeogenesis, a maximal estimate for the contribution of indirect pathway glycogenolysis to hepatic glucose output is obtained by subtracting the sum of direct pathway glycogenolysis and lactate gluconeogenesis from hepatic glucose output. This amounted to a maximum of 36±5 % of hepatic glucose output and 44±6% of overall glycogenolysis. Assuming that the relative proportions of direct and indirect pathway glycogen breakdown would reflect the relative contributions of these pathways to glycogen formation, we conclude that under our experimental conditions the direct pathway is the predominant route for glycogen formation in man and that in overnight fasted humans, hepatic glucose output is mainly the result of glycogenolysis.     

Maternal di‐(2‐ethylhexyl) phthalate exposure alters hepatic insulin signal transduction and glucoregulatory events in rat F1 male offspring

Di‐(2‐ethylhexyl) phthalate (DEHP) is a commonly used plasticizer with endocrine disrupting properties. Its widespread use resulted in constant human exposure including fetal development and postnatal life. Epidemiological and experimental data have shown that DEHP has a negative influence on glucose homeostasis. However, the evidence regarding the effect of maternal DEHP exposure on hepatic glucose homeostasis is scarce. Hence, we investigated whether DEHP exposure during gestation and lactation disrupts glucose homeostasis in the rat F₁ male offspring at adulthood. Pregnant rats were divided into three groups and administered with DEHP (10 and 100 mg/kg/day) or olive oil from gestational day 9 to postnatal day 21 (lactation period) through oral gavage. DEHP‐exposed offspring exhibited hyperglycemia, impaired glucose and insulin tolerances along with hyperinsulinemia at postnatal day 80. DEHP exposure significantly reduced the levels of insulin signaling molecules such as insulin receptors, IRS1, Akt and its phosphorylated forms. GSK3β and FoxO1 proteins increased in DEHP‐exposed groups whereas its phosphorylated forms decreased. Treated groups showed decreased glycogen synthase activity and glycogen concentration. Glucose‐6‐phosphatase and phosphoenolpyruvate carboxykinase mRNA level and enzyme activity increased in DEHP‐treated groups. The interaction between FoxO1‐glucose‐6‐phosphatase and FoxO1‐phosphoenolpyruvate carboxykinase was also increased. This study suggests that DEHP exposure impairs insulin signal transduction and alters glucoregulatory events leading to the development of type 2 diabetes in F₁ male offspring.     

衍生物Fla-CN的抗糖尿病作用及其作用机制研究

目的:体内外研究黄酮衍生物Fla-CN的抗糖尿病作用及其机制。方法:体外实验以人肝HepG2细胞为研究对象,不同浓度Fla-CN孵育后,利用荧光标记 2-脱氧葡萄糖（2-NBDG）,通过检测HepG2 细胞内2-NBDG的荧光强度,观察Fla-CN对肝细胞葡萄糖摄取的影响;采用硫酸-蒽酮比色法检测HepG2细胞内糖原含量;通过检测一定时间内葡萄糖生成量,观察Fla-CN对肝细胞糖异生过程的影响。体内实验采用2型糖尿病模型db/db小鼠为研究对象,给予Fla-CN和二甲双胍干预4 周后,测定db/db小鼠空腹血糖、糖耐量以及胰岛素耐量,计算糖耐量、胰岛素耐量实验血糖-曲线下面积（AUC）。通过Western印迹法检测化合物干预后小鼠肝组织中磷酸化AMP活化蛋白激酶 (AMPK)、糖异生关键酶磷酸烯醇式丙酮酸羧激酶（PEPCK）、葡萄糖-6-磷酸酶（G6Pase）及转录调控因子过氧化物酶体增殖物激活受体γ协同刺激因子1α（PGC-1α）的表达。进一步通过Western 印迹检测AMPK抑制剂Compound C或AMPK激动剂AICAR 与Fla-CN联用后,HepG2细胞内PEPCK、G6Pase、PGC-1α的蛋白表达。结果:Fla-CN能够促进HepG2细胞对葡萄糖的摄取（F=33.50,P<0.01）和糖原合成（F=93.63,P<0.01）,抑制糖异生（F=44.19,P<0.01）。同时,Fla-CN显著降低2型糖尿病db/db小鼠空腹血糖以及时间-血糖曲线下面积（F=98.40,P<0.05）。Western印迹显示Fla-CN能够激活db/db小鼠肝组织中 AMPK,抑制PEPCK、G6Pase 以及PGC-1α 的表达,并呈现一定剂量依赖性。Compound C能在一定程度上阻断Fla-CN对AMPK的激活和对PEPCK、G6Pase 以及PGC-1α的抑制作用。结论:黄酮衍生物Fla-CN通过激活AMPK,有效调节肝 HepG2 细胞糖代谢,抑制肝糖异生,降低2型糖尿病db/db小鼠空腹血糖,改善其受损的糖耐量,提高胰岛素敏感性。     

Failure of nocturnal changes in growth hormone to alter carbohydrate tolerance the following morning

Summary To determine whether the increases in growth hormone that occur during sleep alter carbohydrate tolerance the following morning, two groups of volunteers were studied on two occasions. In one group saline alone was injected and infused (i. e. no octreotide) on one occasion and on the other octreotide was injected at 23.00 hours to inhibit endogenous growth hormone secretion followed by saline infusion to create a state of relative nocturnal growth hormone deficiency. In the other group the octreotide injection was followed on one occasion by a constant growth hormone infusion designed to maintain growth hormone concentrations at “basal” levels throughout the night whereas on the other it was followed by a constant infusion plus two supplemental growth hormone infusions given at midnight and 02.30 hours to mimic the normal nocturnal rise in growth hormone. The next morning, subjects were fed a radiolabelled mixed meal. The differences in the nocturnal growth hormone concentrations had no effect on the glucose, insulin, C-peptide and glucagon concentrations following breakfast ingestion nor did they alter postprandial rates of glucose production, disappearance or substrate oxidation. Thus, the normal nocturnal rise in growth hormone does not appear to be an important regulator of carbohydrate tolerance the following morning. [Diabetologia (1998) 41: 1064–1072] Received: 15 January 1998 and in revised form: 27 April 1998     

褪黑素通过Akt抑制胰岛素抵抗HepG2细胞糖内生

目的探讨褪黑素(melatonin,MLT)对胰岛素抵抗(insulin resistance,IR)肝HepG2细胞葡萄糖内生的影响及其机制。方法HepG2 IR细胞模型采用高糖(25 mmol·L-1)联合高胰岛素(1μmol·L-1)培养诱导建立。MLT(10 nmol·L-1)处理模型细胞6 h后检测糖消耗及糖原含量,GSK-3β、Akt和FoxO1蛋白磷酸化水平检测采用Western blot,免疫荧光法检测FoxO1蛋白核外排情况。结果IR HepG2细胞经MLT处理后,葡萄糖的摄取和糖原合成增加,p-GSK-3β和p-Akt蛋白水平分别增高约66%和48%,FoxO1磷酸化水平明显提高且细胞质含量增加。结论MLT可能通过Akt/GSK-3β及Akt/FoxO1信号通路促进胰岛素抵抗HepG2细胞的糖原合成和抑制糖异生,从而改善糖代谢。     

Quercitrin,a bioflavonoid improves glucose homeostasis in streptozotocin‐induced diabetic tissues by altering glycolytic and gluconeogenic enzymes

The present study is an investigation into the role of quercitrin on carbohydrate metabolism in normal and streptozotocin (STZ)‐induced diabetic rats. Administration of STZ leads to a significant increase (P < 0.05) in fasting plasma glucose and a decrease in insulin levels. The content of glycogen is significantly decreased (P < 0.05) in liver and muscle, but increased in the kidney. The activity of hexokinase decreased whereas the activities of glucose 6‐phosphatase and fructose 1,6‐bisphosphatase significantly increased (P < 0.05) in the tissues. Oral administration of quercitrin (30 mg/kg) to diabetic rats for a period of 30 days resulted in significant (P < 0.05) alterations in the parameters studied but not in normal rats. A decrease of plasma glucose and increase in insulin levels were observed along with the restoration of glycogen content and the activities of carbohydrate metabolic enzymes in quercitrin‐treated diabetic rats. The histopathological study of the pancreas revealed the protective role of quercitrin. There was an expansion of the islets and decreased fatty infiltrate of the islets in quercitrin treated diabetic rats. In normal rats treated with quercitrin, we could not observe any significant change in all the parameters studied. Combined, these results show that quercitrin plays a positive role in carbohydrate metabolism and antioxidant status in diabetic rats.     

Glucose production pathways by 2H and 13C NMR in patients with HIV-associated lipoatrophy.

Patients with HIV taking protease inhibitors were selected for the presence (five subjects) or absence (five subjects) of lipoatrophy. Following an overnight fast, subjects were given oral (2)H(2)O in divided doses (5 mL/kg body water), [U-(13)C(3)] propionate (10 mg/kg), and acetaminophen (1000 mg). Glucose (from plasma) or acetaminophen glucuronide (from urine) were converted to monoacetone glucose for (2)H NMR and (13)C NMR analysis. The fraction of plasma glucose derived from gluconeogenesis was not significantly different between groups. However, flux from glycerol into gluconeogenesis relative to glucose production was increased from 0.20 +/- 0.13 among subjects without lipoatrophy to 0.42 +/- 0.12 (P < 0.05) among subjects with lipoatrophy, and the TCA cycle contribution was reduced. Lipoatrophy was associated with an abnormal profile of glucose production as assessed by (13)C and (2)H NMR of plasma and urine.     

Tumor necrosis factor alpha stimulates gluconeogenesis from alanine in vivo

An increase in gluconeogenesis contributes to the cachexia seen in severe injury, sepsis, and malignancy by converting amino acids from skeletal muscle to glucose. Since tumor necrosis factor α (TNFα) may mediate this cachexia, we examined the effect of this cytokine on gluconeogenesis. Twenty-eight male Fischer rats were injected intraperitoneally with TNFα (250 μg/kg) or saline, and after 4 hours, isolated hepatocytes were obtained by in situ collagenase liver perfusion. Hepatocytes were incubated with alanine (10 mM), and rates of gluconeogenesis were determined. Plasma lactate, glucose, insulin, glucagon, cortisol, and amino acids were measured. TNFα administration resulted in a 50% increase in gluconeogenesis from alanine (P <0.05) and a three-fold increase in plasma glucagon (P = 0.01). Total and glucogenic plasma amino acids decreased with TNFα injection (P < 0.05). In vivo TNFα causes an increase in hepatic gluconeogenesis associated with increased plasma glucagon.     

Contribution to the Pathogenesis of Tumour Hypoglycaemia

Abstract In a patient with severe hypoglycaemia associated with an intrahepatic fibrosarcoma, hyperinsulinism could be excluded, since normal serum insulin and insulinlike activity (ILA) levels were measured in the fasting state and during glucose, glucagon, and tolbutamide tolerance tests. Treatment with methylprednisolone led to an improvement of the clinical condition of the patient. Diazoxide treatment was ineffective. Blood glucose levels were higher during corticoid therapy, but total glucose assimilation remained unchanged. Studies of the metabolism of [1-¹⁴C] glucose before and during treatment with prednisolone showed that an inhibition of hepatic glycogenolysis was not significant for the pathogenesis of the hypoglycaemia. The differences of glucose and blood lactate after glucose i. v. before and during prednisolone suggested, however, that gluconeogenesis was increased as a result of corticoid therapy. Measurements of hepatic enzyme activities also favoured this hypothesis: activities of FDP'ase and G-6-P'ase were low before treatment and increased by a factor of 1.5 and 5 respectively during treatment, indicating a stimulation of the gluconeogenetic processes in the liver. It is concluded that the combination of tumoral glucose overutilization with hepatic glucose underproduction due to impaired gluconeogenesis was the pathogenetic mechanism responsible for the hypoglycaemia of this patient. Electron microscopic studies of the tumour cells revealed abundant mitochondria and a highly active ergastoplasm. In addition, secretory granules were found in the cytoplasm and some even in the mitochondria.