Ghrelin在离体大鼠胰岛中抑制胰岛素分泌和上调Kir6．2表达

目的明确ghrelin在离体大鼠胰岛中对胰岛素分泌和内向整流钾通道（Kit6．2）表达的影响,并讨论两者的关系。方法离体大鼠胰岛以高浓度葡萄糖及不同浓度ghrelin和（或）其受体拮抗剂ED-lys^3]-GHRP-6孵育1h,采用放免法测定上清液的胰岛素,采用RT-PCR检测Kir6．2、磺酰脲受体1（SUR-1）、解偶联蛋白2（UCP-2）、葡萄糖转运子2（GluT-2）、胰十二指肠同源盒1（PDX-1）等基因的表达。结果10。～10“mol／Lghre｜in呈剂量依赖性抑制离体大鼠胰岛高浓度葡萄糖刺激的胰岛素释放,且剂量依赖性增加Kir6．2mRNA表达,但对SUR-1、UCP-2、GluT-2及PDX-1 mRNA表达则无显著影响。ED-lys^3]-GHRP-6可消除ghrelin对Kir6．2 mRNA表达的上调作用。结论在离体大鼠胰岛中,ghrelin通过作用于其受体,促进ATP敏感性钾通道组成成分Kir6．2的表达,改变钾通道功能状态。这可能是ghrelin抑制葡萄糖刺激的胰岛素分泌的机制之一。     

Glucolipotoxicity age-dependently impairs beta cell function in rats despite a marked increase in beta cell mass

Aims/hypothesis

Prolonged exposure of pancreatic beta cells to excessive levels of glucose and fatty acids, referred to as glucolipotoxicity, is postulated to contribute to impaired glucose homeostasis in patients with type 2 diabetes. However, the relative contribution of defective beta cell function vs diminished beta cell mass under glucolipotoxic conditions in vivo remains a subject of debate. We therefore sought to determine whether glucolipotoxicity in rats is due to impaired beta cell function and/or reduced beta cell mass, and whether older animals are more susceptible to glucolipotoxic condition.

Methods

Wistar rats (2 and 6 months old) received a 72 h infusion of glucose + intravenous fat emulsion or saline control. In vivo insulin secretion and sensitivity were assessed by hyperglycaemic clamps. Ex vivo insulin secretion, insulin biosynthesis and gene expression were measured in isolated islets. Beta cell mass and proliferation were examined by immunohistochemistry.

Results

A 72 h infusion of glucose + intravenous fat emulsion in 2-month-old Wistar rats did not affect insulin sensitivity, insulin secretion or beta cell mass. In 6-month-old rats by contrast it led to insulin resistance and reduced insulin secretion in vivo, despite an increase in beta cell mass and proliferation. This was associated with: (1) diminished glucose-stimulated second-phase insulin secretion and proinsulin biosynthesis; (2) lower insulin content; and (3) reduced expression of beta cell genes in isolated islets.

Conclusions/interpretation

In this in vivo model, glucolipotoxicity is characterised by an age-dependent impairment of glucose-regulated beta cell function despite a marked increase in beta cell mass.     

Immuno-localization of sulphonylurea receptor 1 in rat pancreas

Aims/hypothesis. A sulphonylurea receptor, SUR1, and an inward rectifier potassium channel, Kir6.2, reconstitute the ATP-sensitive K⁺ channel that mediates glucose-induced insulin secretion in pancreatic beta cells. We reported previously that Kir6.2 were localized at insulin-, glucagon-, and somatostatin-producing cells. In this new study we aimed to determine the distribution of SUR1 in rat pancreatic islets and to suggest the location of the ATP-sensitive K⁺ channels in the islet. Methods. Western blot analysis was carried out using two anti-SUR1 antibodies, which had been raised against different portions of rat SUR1. SUR1, Kir 6.2, and islet hormones were then localized by indirect immunofluorescence staining of the cryosections of rat pancreas. Results. In Western blot analysis, each of the anti-SUR1 antibodies detected a band at 140 kDa, which is close to the predicted molecular weight of SUR1, in the homogenate of isolated pancreatic islets. Double immunofluorescence staining of cryosections showed that SUR1 occurred all over the islets, and that SUR1 colocalized with insulin, glucagon, somatostatin, and pancreatic polypeptide. Kir6.2 was also shown to be present in pancreatic polypeptide cells. Conclusion/interpretation. Together with our previously reported data, the above findings indicate that K_ATP channels comprising SUR1 and Kir6.2 occur not only in beta cells but also in the alpha, delta, and pancreatic polypeptide cells of the pancreatic islets, suggesting that therapeutic sulphonylureas could act on these cells directly. [Diabetologia (1999) 42: 1204–1211] Received: 21 January 1999 and in final revised form: 21 May 1999     

Long‐term enteral administration of melatonin reduces plasma insulin and increases expression of pineal insulin receptors in both Wistar and type 2‐diabetic Goto‐Kakizaki rats

Abstract: This paper represents an essential aspect of recent investigations into the functional and clinical implications of insulin–melatonin interrelationships. The aim of the study was to analyze whether melatonin reduces insulin secretion in an animal in a manner comparable to the pattern observed in previous in vitro experiments; to this end, we used two models: Wistar and type 2‐diabetic Goto‐Kakizaki (GK) rats. Thirty‐two Wistar and 32 GK rats were divided into two subgroups of 16 rats each; each subgroup was treated either with or without melatonin. The daily administration of melatonin, starting in 8‐ wk‐old rats, was adjusted to 2.5 mg/kg body weight. Melatonin was given daily during the dark period for 12 hr. After 9 wk of treatment, the rats were sacrificed in the middle of the dark period. Melatonin administration strongly enhanced the plasma melatonin level and diminished the expression of pancreatic melatonin receptor‐mRNA, whereas the expression of pineal AA‐NAT and HIOMT was unchanged. Furthermore, the experiments showed in agreement with recent in vitro results of pancreatic islets that plasma insulin levels were diminished after melatonin treatment. However, the pineal insulin receptor expression was increased after melatonin administration. The pancreatic expression of glucagon, GLUT2, and glucokinase was decreased in GK rats, whereas the glucose levels, as well as the parameters of glucose sensing, GLUT2‐mRNA, and glucokinase‐mRNA, were unchanged after melatonin administration in both Wistar and GK rats. In summary, the results show that melatonin administration decreases plasma insulin levels in vivo and, furthermore, that an insulin–melatonin antagonism exists.     

K(ATP) channel knockout mice crossbred with transgenic mice expressing a dominant-negative form of human insulin receptor have glucose intolerance but not diabetes

Impaired insulin secretion and insulin resistance are thought to be two major causes of type 2 diabetes mellitus. There are two kinds of diabetic model mice: one is a K(ATP) channel knockout (Kir6.2KO) mouse which is defective in glucose-induced insulin secretion, and the other is a transgenic mouse expressing the tyrosine kinase-deficient (dominant-negative form of) human insulin receptor (hIR(KM)TG), and which has insulin resistance in muscle and fat. However, all of these mice have no evidence of overt diabetes. To determine if the double mutant Kir6.2KO/hIR(KM)TG mice would have diabetes, we generated mutant mice by crossbreeding, which would show both impaired glucose-induced insulin secretion and insulin resistance in muscle and fat. We report here that: 1) blood glucose levels of randomly fed and 6 h fasted double mutant (Kir6.2KO/hIR(KM)TG) mice were comparable with those of wild type mice; 2) in intraperitoneal glucose tolerance test (ipGTT), Kir6.2KO/hIR(KM)TG mice had an impaired glucose tolerance; and 3) during ipGTT, insulin secretion was not induced in either Kir6.2KO/hIR(KM)TG or Kir6.2KO mice, while the hIR(KM)TG mice showed a more prolonged insulin secretion than did wild type mice; 4) hyperinsulinemic euglycemic clamp test revealed that Kir6.2KO, Kir6.2KO/hIR(KM)TG and hIR(KM)TG mice, showed decreased whole-body glucose disposal compared with wild type mice; 5) Kir6.2KO, but not Kir6.2KO/hIR(KM)TG mice had some obesity and hyperleptinemia compared with wild type mice. Thus, the defects in glucose-induced insulin secretion (Kir6.2KO) and an insulin resistance in muscle and fat (hIR(KM)TG) were not sufficient to lead to overt diabetes.     

Gastric inhibitory polypeptide is the major insulinotropic factor in K(ATP) null mice

OBJECTIVE: ATP-sensitive K(+) (K(ATP)) channels in pancreatic beta-cells are crucial in the regulation of glucose-induced insulin secretion. Recently, K(ATP) channel-deficient mice were generated by genetic disruption of Kir6.2, the pore-forming component of K(ATP) channels, but the mice still showed a significant insulin response after oral glucose loading in vivo. Gastric inhibitory polypeptide (GIP) is a physiological incretin that stimulates insulin release upon ingestion of nutrients. To determine if GIP is the insulinotropic factor in insulin secretion in K(ATP) channel-deficient mice, we generated double-knockout Kir6.2 and GIP receptor null mice and compared them with Kir6.2 knockout mice. METHODS: Double-knockout mice were generated by intercrossing Kir6.2-knockout mice with GIP receptor-knockout mice. An oral glucose tolerance test, insulin tolerance test and batch incubation study of pancreatic islets were performed on double-knockout mice and Kir6.2-knockout mice. RESULTS: Fasting glucose and insulin levels were similar in both groups. After oral glucose loading, blood glucose levels of double-knockout mice became elevated compared with Kir6.2-knockout mice, especially at 15 min (345+/-10 mg/dl vs 294+/-20 mg/dl, P<0.05) and 30 min (453+/-20 mg/dl vs 381+/-26 mg/dl, P<0.05). The insulin response was almost completely lost in double-knockout mice, although insulin secretion from isolated islets was stimulated by another incretin, glucagon-like peptide-1 in the double-knockout mice. Double-knockout mice and Kir6.2-knockout mice were similarly insulin sensitive as assessed by the insulin tolerance test. CONCLUSION: GIP is the major insulinotropic factor in the secretion of insulin in response to glucose load in K(ATP) channel-deficient mice.     

Defective insulin secretion and enhanced insulin action in KATP channel-deficient mice

ATP-sensitive K⁺ (K_ATP) channels regulate many cellular functions by linking cell metabolism to membrane potential. We have generated K_ATP channel-deficient mice by genetic disruption of Kir6.2, which forms the K⁺ ion-selective pore of the channel. The homozygous mice (Kir6.2^−/−) lack K_ATP channel activity. Although the resting membrane potential and basal intracellular calcium concentrations ([Ca²⁺]_i) of pancreatic beta cells in Kir6.2^−/− are significantly higher than those in control mice (Kir6.2^+/+), neither glucose at high concentrations nor the sulfonylurea tolbutamide elicits a rise in [Ca²⁺]_i, and no significant insulin secretion in response to either glucose or tolbutamide is found in Kir6.2^−/−, as assessed by perifusion and batch incubation of pancreatic islets. Despite the defect in glucose-induced insulin secretion, Kir6.2^−/− show only mild impairment in glucose tolerance. The glucose-lowering effect of insulin, as assessed by an insulin tolerance test, is increased significantly in Kir6.2^−/−, which could protect Kir6.2^−/− from developing hyperglycemia. Our data indicate that the K_ATP channel in pancreatic beta cells is a key regulator of both glucose- and sulfonylurea-induced insulin secretion and suggest also that the K_ATP channel in skeletal muscle might be involved in insulin action.     

Ghrelin inhibits insulin release by regulating the expression of inwardly rectifying potassium channel 6.2 in islets

IntroductionThe objective is to investigate the influence of ghrelin administration on both the insulin secretion and the expression of ATP-sensitive K⁺ channels in islet.MethodsGhrelin and [D-Lys³] growth hormone releasing peptide-6 were administered via intraperitoneal injection in Wistar rats at the doses 10 and 10 μmol/kg/d for 2 weeks, respectively. Then glucose tolerance tests were performed and plasma insulin concentrations were measured. Islets were isolated for insulin release experiments. Single β cells were isolated for electrophysiological experiments. Determination of the Kir6.2 and SUR1 mRNA and protein expression levels in islets was performed by polymerase chain reaction and western blotting.ResultsIntraperitoneal administration of exogenous ghrelin significantly (P < 0.05) increased blood glucose concentrations, attenuated insulin responses during glucose tolerance tests, reduced insulin release from the isolated islets induced by 11.1 and 16.7 mmol/L glucose, hyperpolarized the resting membrane potential and increased the Kir6.2 mRNA and protein expression levels. In contrast, counteraction of ghrelin by intraperitoneal injection of [D-Lys³] growth hormone releasing peptide-6 significantly (P < 0.05) attenuated the aforementioned changes. SUR1 expression levels were not altered in this study.ConclusionsGhrelin via pancreatic growth hormone secretagogue receptor up-regulates the Kir6.2 expression in islet by hyperpolarizing the resting membrane potential which results in the inhibition of insulin release.     

Normal insulin receptor tyrosine kinase activity and glucose transporter (GLUT 4) levels in the skeletal muscle of hyperinsulinaemic hypertensive rats

Summary The spontaneous hypertensive rat is an animal model characterized by a syndrome of hypertension, insulin resistance and hyperinsulinaemia. To elucidate whether in analogy to other insulin resistant animal models an inactivity of the insulin receptor kinase or an alteration of the glucose transporter (GLUT 4) level in the skeletal muscle might contribute to the pathogenesis of insulin resistance we determined insulin receptor kinase activity and GLUT 4 level in the hindlimbs of spontaneous hypertensive rats and normotensive control rats. Normotensive normoinsulinaemic Lewis and Wistar rats were used as insulin sensitive controls, obese Zucker rats were used as an insulin resistant control with known reduced skeletal muscle insulin receptor kinase activity. Binding of ¹²⁵I-insulin, crosslinking of ¹²⁵I-B²⁶-insulin, autophosphorylation in vitro with ³²P-ATP and phosphorylation of the synthetic substrate Poly (Glu 4: Tyr 1) were performed after partial purification of solubilized receptors on wheat germ agglutinin columns. GLUT 4 levels were determined by Western blotting of subcellular muscle membranes. Insulin receptors from spontaneous hypertensive rats compared to those from Lewis and Wistar rats showed no difference of the binding characteristics or the in vitro auto- and substrate phosphorylation activity of the receptor, while in the Zucker rats the earlier described insulin receptor kinase defect was clearly evident. Western blots of subcellular muscle membrane fractions with antibodies against GLUT 4 revealed no difference in transporter levels. These data suggest that insulin resistance in spontaneous hypertensive rats is caused neither by an insulin receptor inactivity nor by a decreased number of glucose transporters in the skeletal muscle.     

Blockade of tumor necrosis factor (TNF) receptor type 1-mediated TNF-alpha signaling protected Wistar rats from diet-induced obesity and insulin resistance

TNF-alpha plays an important role in the pathogenesis of obesity and insulin resistance in which the effect of TNF-alpha signaling via TNF receptor type 1 (TNFR1) largely remains controversial. To delineate the role of TNFR1-mediated TNF-alpha signaling in the pathogenesis of this disorder, a TNFR1 blocking peptide-Fc fusion protein (TNFR1BP-Fc) was used for the present study. Wistar rats were fed a high-fat/high-sucrose (HFS) diet for 16 wk until obesity and insulin resistance developed. In comparison with increased body weight and fat weight, enlarged adipocytes, and hypertriglyceridemia in the obese state, the subsequent 4-wk treatment with TNFR1BP-Fc resulted in significant weight loss characterized by decreased fat pad weight and adipocyte size and reduced plasma triglycerides. Furthermore, obesity-induced insulin resistance, including hyperinsulinemia, elevated C-peptide, higher degree of hyperglycemia after glucose challenge, and less hypoglycemic response to insulin, was markedly improved, and the compensatory hyperplasia and hypertrophy of pancreatic islets were reduced. Interestingly, treatment with TNFR1BP-Fc markedly suppressed systemic TNF-alpha release and its local expression in pancreatic islets and muscle and adipose tissues. In addition, blockage of TNFR1-mediated TNF-alpha signaling in obese rats significantly enhanced tyrosine phosphorylation of insulin receptor substrate 1 (IRS-1) in the muscle and fat tissues. Our results strongly suggest a pivotal role for TNFR1-mediated TNF-alpha signaling in the pathogenesis of obesity and insulin resistance. Thus, TNFR1BP-Fc may be a good candidate for the treatment of this disease.     

Low levels of glucose transporters and K+ATP channels in human pancreatic beta cells early in development

AIMS/HYPOTHESIS: Although cells expressing insulin are detected early in human fetal development, islets isolated from fetal pancreases show poor insulin secretory responses to glucose, which may be the result of deficient glucose sensing. We have used dual and triple immunolabelling of human fetal and adult pancreas sections to investigate the presence of proteins that participate in glucose sensing in the pancreatic beta cell, namely glucose transporter 1 (GLUT 1, also known as SLC2A1), glucose transporter 2 (GLUT2, also known as SLC2A2), glucokinase (GCK) and inwardly rectifying K+ channel (KIR6.2, also known as KCNJ11) and sulphonylurea receptor 1 (SUR1, also known as ABCC8) subunits of ATP-sensitive K+ channels (K+(ATP) channels). MATERIALS AND METHODS: Pancreases obtained with ethical approval from human fetuses from 11 to 36 weeks of gestation, from infants and from adults were formalin-fixed and embedded in paraffin. Sections were labelled with antibodies to proteins of interest. Co-production of antigens was examined by dual and triple immunolabelling. RESULTS: GLUT2 and K+(ATP) channel labelling was detected in the 11-week pancreas, but largely within the pancreatic epithelium, whereas no labelling for GLUT1 was observed. From 15 weeks, GLUT1, GCK and K+(ATP) channel labelling was detected in an increasing proportion of insulin-positive cells and epithelial labelling with K+(ATP) channel antibodies diminished. GLUT2 was seen in the majority of beta cells only after 7 months of age. CONCLUSIONS/INTERPRETATION: The results demonstrate that only a subpopulation of beta cells in the human fetal pancreas produce all key elements of the glucose-sensing apparatus, which may contribute to poor secretory responses in early life.     

The contribution of skeletal muscle tumor necrosis factor-alpha to insulin resistance and hypertension in fructose-fed rats

OBJECTIVE: The aim of this study was to determine the role of tumor necrosis factor-alpha (TNF-alpha) in skeletal muscle tissue in insulin resistance and hypertension and the effect of anti-hypertensive medicine on skeletal muscle TNF-alpha in fructose-induced insulin-resistant and hypertensive rats (fructose-fed rats: FFR). DESIGN AND METHODS: Six-week-old male Sprague-Dawley rats were fed either normal rat chow or fructose-rich chow. For the last 2 weeks of a 6-week period of either diet, the rats were treated with a vehicle (control or FFR); temocapril, an angiotensin converting enzyme inhibitor (ACEI); or CS-866, an angiotensin II type 1 receptor blocker (ARB). The euglycemic hyperinsulinemic glucose clamp technique was performed to evaluate insulin sensitivity (M value). TNF-alpha levels in soleus and extensor digitorum longus (EDL) muscles and epididymal fat pads were measured. We also measured the TNF-alpha concentration in an incubated medium secreted from soleus muscle strips with or without angiotensin II. RESULTS: TNF-alpha levels were significantly higher in the soleus and EDL muscles, but not in the epididymal fat, in the FFRs compared with the control rats. Temocapril and CS-866 lowered systolic blood pressure, improved insulin resistance, and reduced TNF-alpha in both skeletal muscles. There were significant negative correlations between M values and TNF-alpha levels in both soleus and EDL muscles. Also, the soleus muscle strip incubation with 10(-7) mol/l angiotensin II increased TNF-alpha secreted into the incubation medium compared to the incubation without angiotensin II. These results suggest that skeletal muscle TNF-alpha is linked to insulin resistance and hypertension and that angiotensin II may be one of the factors that regulate skeletal muscle TNF-alpha.     

健脾理气化痰法针刺对高脂饲养胰岛素抵抗大鼠骨骼肌葡萄糖转运体4蛋白表达的影响

[目的]探讨健脾理气化痰法针刺对高脂饲养胰岛素抵抗大鼠骨骼肌葡萄糖转运体4（GLuT4）蛋白表达及胰岛素敏感性的影响。[方法]40只Wistar大鼠分别用高脂饲料、普通饲料喂养。经高胰岛素-正常葡萄糖钳夹术确定高脂饲养制备IR模型成功后,将模型大鼠随机分为模型组和电针组（取足三里、中脘、丰隆、关元穴）,普通饲料喂养组为正常组。治疗6周后,行腹腔糖耐量、腹腔胰岛素耐量实验测量各组大鼠血糖水平;治疗8周后,采用免疫组化法检测各组大鼠股四头肌GLUT4蛋白的表达。[结果]与模型组比较,电针组胰岛素降低血糖的效果显著增强（P〈o．01）;与模型组比较,电针组股四头肌GLUT4蛋白的表达水平增加（P〈o．05）。[结论]健脾理气化痰法针刺具有提高胰岛素敏感性的作用,并能显著提高股四头肌GLUT4蛋白的表达水平。     

胰岛素抵抗大鼠视黄醇结合蛋白4骨骼肌磷脂酰肌醇3激酶的表达及吡格列酮的干预作用

目的 观察胰岛素抵抗(IR)大鼠体内视黄醇结合蛋白4(RBP4)、骨骼肌磷脂酰肌醇3激酶( P13K)和晚期氧化蛋白产物(AOPP)的水平,以及给予吡格列酮干预后其活性的变化,探讨RBP4与IR的关系及其可能的机制。 方法 将SPF级雄性Wistar大鼠35只随机分为2组,正常对照组（对照组）11只,饲以普通饲料;模型组24只,饲以高糖高脂饲料。模型组造模成功后再随机分为2个亚组,IR组和IR+吡格列酮干预组（干预组）,每组12只;IR组和干预组继续饲以高糖高脂饲料,干预组大鼠同时给予吡格列酮20mg·kg 1·d-1灌胃,持续8周。第16周末处死大鼠取血检测三酰甘油(TG)、高密度脂蛋白(HDL-C)、低密度脂蛋白(LDL-C)及空腹血糖(FBG)、空腹胰岛素(FINS),计算胰岛素抵抗指数(HOME-IR);酶联免疫吸附法(ELISA)检测血清RBP4水平,RT-PCR测定附睾脂肪组织RBP4表达;免疫组织化学染色检测骨骼肌PI3K水平;紫外分光光度计法测定AOPP水平;并取大鼠腹腔内肠系膜、附睾、腹膜反折处的脂肪组织称质量,计算腹部脂肪含量与体质量的比值。 结果 (1)IR组大鼠体质量16周后明显增加,TG、LDL-C、FINS以及脂体比较对照组均明显升高,而HDL-C则明显降低;吡格列酮干预后,干预组体质量、TG、LDL-C、FINS以及脂体比较IR组有明显下降,HDL-C明显升高;(2)IR组大鼠血清及附睾脂肪组织RBP4水平和血清AOPP水平明显高于对照组,干预组水平明显降低;(3)IR组大鼠骨骼肌组织PI3K表达水平明显低于对照组,吡格列酮干预其表达水平升高;(4)相关分析表明大鼠血清RBP4与FINS、脂体比、LDL-C呈正相关;与HDL-C、骨骼肌组织PI3K水平呈负相关。 结论 (1)IR大鼠RBP4、AOPP水平升高,RBP4是致IR的脂肪细胞因子,并可引起机体脂代谢紊乱及氧化应激增强;(2)RBP4降低大鼠胰岛素敏感性可能与其削弱胰岛素信号转导作用有关;(3)吡格列酮可降低IR大鼠RBP4及血清AOPP水平,增加骨骼肌PI3K表达,从而提高机体对胰岛素的敏感性。     

A low protein diet alters the balance of islet cell replication and apoptosis in the fetal and neonatal rat and is associated with a reduced pancreatic expression of insulin-like growth factor-II.

A programmed turnover of pancreatic beta cells occurs in the neonatal rat involving a loss of beta cells by apoptosis, and their replacement by islet cell replication and neogenesis. The timing of apoptosis is associated with a loss of expression of a survival factor, insulin-like growth factor-II (IGF-II), in the pancreatic islets. Offspring from rats chronically fed a low protein isocalorific diet (LP) exhibit a reduced pancreatic beta cell mass at birth and a reduced insulin secretion in later life. This study therefore investigated the impact of LP on islet cell ontogeny in the late fetal and neonatal rat, and any associated changes in the presence of IGFs and their binding proteins (IGFBPs). Pregnant Wistar rats were fed either LP (8% protein) or normal (C) (20% protein) chow from shortly after conception until the offspring were 21 days postnatal (PN). Bromo-deoxyuridine (BrdU) was administered 1 h before rats were killed and pancreata removed from animals between 19.5 days fetal life and postnatal day 21. Offspring of rats given LP diet had reduced birthweight, pancreatic beta cell mass, and pancreas insulin content, with smaller islets compared with control fed animals, which persisted to weaning. Histological analysis showed that islets from pups given LP diet had a lower nuclear labeling index with BrdU in the beta cells, although, paradoxically, more beta cells showed immunoreactivity for proliferating cell nuclear antigen (PCNA). Because PCNA is present in G1 as well as S phase of the cell cycle, we quantified the number of beta cells immunopositive for cyclin D1, a marker of G1, and NEK2, an indicator of cells in G2 and mitosis. More beta cells in islets from LP-fed animals contained cyclin D1, but less contained NEK2 than did those in controls. This suggests that the beta cell cycle may have a prolonged G1 phase in LP-fed animals in vivo. Offspring of rats given C diet had a low rate of islet cell apoptosis detected by the TUNEL method in fetal and neonatal life (1-2%), with a transient increase to 8% at PN day 14. Offspring of rats receiving LP diet demonstrated a significantly greater level of islet cell apoptosis at every age, rising to 15% at PN 14. IGF-II mRNA was quantified in whole pancreas and was significantly reduced in LP-fed animals at ages up to PN day 10. IGF-II immunoreactivity within the islets of LP-fed rats was also less apparent, but no changes were seen in immunoreactive IGF-I or IGFBPs-2 to -5. These findings show that LP diet changes the balance of beta cell replication and apoptosis in fetal and neonatal neonatal life, which may involve an altered length of beta cell cycle, and contribute to the smaller islet size and impaired insulin release seen in later life. A reduced pancreatic expression of IGF-II may contribute to the lower beta cell proliferation rate and increased apoptosis seen in the fetus and neonate after feeding LP diet.     

增龄对大鼠骨骼肌细胞葡萄糖转运蛋白的影响

目的　探讨增龄对大鼠骨骼肌细胞葡萄糖转运蛋白4(glucose transporter 4,GLUT4)的影响。　方法　SD实验大鼠分为2组青年组（3月龄）和老年组（24月龄）各式各样0只。制务GLUT4羧基端正2肽的多克隆抗体,利用Western印迹法检测2组大鼠骨骼肌细胞GLUT4蛋白含量,并检测大鼠尾静脉血糖。　结果　老年组大鼠的血糖（5.6±0.5mmol/L)略高于青年组大鼠（4.5±0.5mmol/L),但差异无显著性;青年组大鼠骨骼肌细胞内GLUT4相对含量为109.62±12.25,而老年组为86.46±8.25,差异有显著性（P<0.05)。　结论　增龄可引起大鼠骨骼肌细胞GLUT4蛋白含量明显减少,使骨骼肌细胞对葡萄糖的转运发生障碍,这可能是老年人易产生胰岛素抵抗的机制之一。     

The effects of high-fat diet on exercise-induced changes in metabolic parameters in Zucker fa/fa rats

The objectives of this study were to document the effects of moderate aerobic exercise on insulin secretion and other metabolic indices in fa/fa rats and to determine if a high-fat (HF) diet altered these effects. Six-week-old fa/fa and lean Zucker rats were either sedentary or exercised by daily swimming for 4 weeks. Half of the exercised and sedentary rats were fed a diet with 16% fat and 44% carbohydrate, while the control groups were fed a diet with 4.5% fat and 49% carbohydrate. At the end of 4 weeks, caloric intake, weight gain, plasma hormone and nutrient levels, and oral glucose tolerance were measured. The pancreatic islet beta-cell function was assessed by measuring glucose-stimulated insulin secretion, glucose phosphorylating activity, and free fatty acid (FFA) oxidation in cultured islets. In fa/fa rats fed the control diet, exercise reduced weight gain, caloric intake, and fasting plasma triglyceride (TG) concentrations without affecting fasting glucose and insulin concentrations. HF diet blocked the effects of exercise on weight gain and food intake and worsened insulin resistance of fa/fa rats. In vitro, neither exercise nor HF diet alone affected islet beta-cell function. However, in combination, exercise and high dietary fat reduced glucokinase sensitivity to glucose and increased islet cell response to mannoheptulose inhibitory actions. We conclude that beneficial effects of moderate exercise on metabolism are not mediated by effects on pancreatic beta cells. Diets elevated in fat decrease the beneficial effects of exercise on metabolic indices in vivo.     

胰岛α细胞胰岛素抵抗与炎症通路激活的关系及机制

目的探讨高脂饲养大鼠胰岛α细胞炎症通路分子基因的表达变化及吡格列酮干预的影响。方法8周龄雄性SD大鼠随机分为3组（每组15只）：正常饲养组（NC）、高脂饲养组（HF）、高脂＋吡格列酮组（HP）。喂养20周后检测空腹血胰岛素（Fins）、胰高血糖素（Glc）、游离脂肪酸（FFA）、高敏C反应蛋白（hsCRP）水平;正常血糖高胰岛素钳夹试验评价外周胰岛素抵抗程度;离体胰岛细胞表面灌注检测高糖状态Glc分泌的动态变化,同时3组大鼠各随机人组8只给予大剂量链脲菌素去β细胞处理,分为正常去β细胞组（NC-B）,高脂去β细胞组（HF-B）,高脂＋吡格列酮去母细胞组（HP—B）,采用定量PCR方法比较3组去母细胞大鼠α细胞NF-κB、NF-κB抑制蛋白α（IκBα）mRNA表达的情况。结果（1）HF组葡萄糖输注率（GIR）明显低于NC组,血Fins、Glc、FFA及hsCRP水平均显著高于NC组;而HP组以上各项指标较HF组均明显改善。（2）胰岛细胞表面灌注,HF组基础Glc的分泌高于NC组（P〈0．01）,16．7mmol／L葡萄糖灌注后HF组胰岛的Glc分泌未受抑制,HP组与NC组比较差异无统计学意义。（3）与NC-B组相比,HF-B组α细胞NF-κB mRNA的表达增高20．5％,IκBα mRNA表达降低24．3％（P值均〈0．01）。HP-B组较HF．B组NF-κB、IκBα mRNA分别改善78．3％、58．8％。（4）HF组血FFA水平与GIR呈负相关（r=-0．675,P〈0．01）;与NF-κB mRNA表达呈正相关（r=0．775,P〈0．05）。结论高脂饲养导致胰岛α细胞胰岛素抵抗,同时激活了α细胞炎症通路基因的表达且与FFA升高有关。吡格列酮干预能改善上述变化。     

[Val12] HRAS downregulates GLUT2 in beta cells of transgenic mice without affecting glucose homeostasis.

Glucose-induced insulin release from pancreatic beta cells depends on the beta-cell metabolism of glucose, which generates intracellular signals for secretion. The beta-cell glucose transporter isotype GLUT2 and the glucose phosphorylating enzyme glucokinase have both been implicated in coupling insulin secretion to extracellular glucose levels. Here we present evidence that a pronounced decrease in beta-cell GLUT2 has no immediate effect on glucose homeostasis. Analysis of transgenic mice overexpressing human [Val12]HRAS oncoprotein under control of the insulin promoter reveals a great reduction in plasma-membrane GLUT2 levels. These mice are nonetheless able to maintain normal fed and fasting plasma glucose and insulin levels for a period of several months. Insulin secretion studied in isolated islets and the perfused pancreas is characterized by a normal incremental response to increasing glucose concentrations. Glucose metabolism, as measured by glucose phosphorylation and oxidation in isolated islets, shows a normal dose dependence on extracellular glucose concentrations. These findings suggest that normal GLUT2 expression in beta cells is not essential for glucose sensing. The transgenic mice provide an experimental system for studying the role of glucose phosphorylation in regulation of insulin release in the absence of GLUT2.