Glucose transport by osmotic shock and vanadate is impaired by glucosamine

In 3T3-L1 adipocytes, we previously reported that glucosamine impairs insulin stimulation of glucose transport, which is accompanied by impaired insulin stimulation of serine/threonine kinase Akt. To examine the role of Akt in glucosamine-induced insulin resistance, we investigated time course for insulin stimulation of Akt activity and glucose transport during recovery from glucosamine-induced insulin resistance. After induction of insulin resistance by glucosamine, we washed cells to remove glucosamine and incubated them for various times. After one hour, insulin stimulated-glucose transport was significantly increased and continued to increase up to 6-24 h. Insulin stimulation of Akt, however, did not increase after 1-3 h and began to slightly increase after 6 h. Next, we investigated effects of osmotic shock and vanadate on glucose transport in glucosamine-treated cells and found that glucosamine completely inhibited their actions in these cells. These data suggest that an Akt-independent mechanism is operative in glucosamine-induced insulin resistance and glucosamine impairs glucose transport stimulated by various stimuli involving and not involving Akt activation.     

Insulin sensitivity and responsiveness of epitrochlearis and soleus muscles from fed and starved rats. Recognition of differential changes in insulin sensitivities of protein synthesis and glucose incorporation into glycogen.

The insulin sensitivity of protein synthesis and glucose incorporation into glycogen by the soleus and epitrochlearis muscles from fed rats and 24 h-starved rats was determined in vitro during the first and second hours of incubation after isolation of the muscles. Rates of protein synthesis by both muscles from fed rats in the first hour of incubation were 2-fold higher than in the second hour and were not increased by insulin. Rates of protein synthesis during the first hour in the presence of 6000 microunits of insulin/ml were increased in soleus, but not in epitrochlearis, muscles from starved rats. Rates of protein synthesis in both muscles from fed and starved rats were increased significantly by insulin during the second hour. High concentrations of insulin caused a marked stimulation of the rates of glucose incorporation by both muscles from fed and starved rats in both the first and second hours of incubation. The insulin sensitivity of glucose incorporation during the second hour, defined as the concentration of insulin causing half-maximal stimulation, was increased 10-fold for both muscle types from starved rats (soleus, 65 microunits/ml; epitrochlearis, 45 microunits/ml) relative to muscles from fed rats (soleus, 600 microunits/ml; epitrochlearis, 500 microunits/m). The insulin sensitivity of protein synthesis in the second hour was greater for soleus muscles from starved rats (65 microunits/ml) than from fed rats (500 microunits/ml). In contrast, the insulin sensitivity of protein synthesis in epitrochlearis muscles from starved rats was significantly decreased (225 microunits/ml) compared with fed rats (25 microunits/ml Maximal rates achieved by high concentrations of insulin were not different from those in the same muscle from fed rats. It is suggested that protein synthesis, in distinction to glucose utilization, may be resistant to insulin stimulation during periods of acute starvation in muscles with fibre compositions similar to the epitrochlearis, but not in muscles with fibre compositions similar to the soleus. Partial reversal of the resistance observed in vitro for epitrochlearis muscles from starved rats may be due to the loss of factors which suppress the effect of insulin in vivo.     

Development of glucose intolerance and impaired plasma insulin response to glucose in obese hyperglycaemic (ob/ob) mice

The development of glucose intolerance in Aston ob/ob mice showed a gross exaggeration of the age-related changes of glucose tolerance in lean (+/+) mice. Intraperitoneal glucose tolerance in ob/ob mice was poor at 5 weeks, improved by 10 weeks, but markedly worsened by 20 weeks. A 24 hour fast further exaggerated the glucose intolerance of ob/ob mice. Unlike lean mice, tolerance improved in ob/ob mice at 40 weeks. Alterations of insulin sensitivity and the plasma insulin response to glucose accounted in part for these observations. Insulin sensitivity deteriorated until 20 weeks, but improved at 40 weeks in both fed and 24 hour fasted ob/ob mice. A positive plasma insulin response to glucose was lost after 5 weeks in fed ob/ob mice. The severity of this abnormality corresponded with the extent of the basal hyperinsulinaemia. A 24 hour fast reduced plasma insulin concentrations and restored a positive plasma insulin response to glucose in ob/ob mice. The results suggest that the plasma insulin response to glucose in ob/ob mice is related to the secretory activity of the B-cells prior to stimulation. Furthermore, it is evident that factors in addition to insulin insensitivity and the impaired plasma insulin response to glucose contribute to the development of glucose intolerance in these mice.     

Utilization of glucose during exercise in relation of meal-timing

The effect of glucose administration was studied on its utilization during exercise carried out in the hours 500-700, 1100-1300, 1700-1900, 2300-100. The control group comprised animals at rest which had one or two glucose loads. Circadian variability of blood glucose level was observed in response to glycaemic stimulation in control animals. In the animals during exercise the circadian changes of glucose level depended on the time after glucose administration and the duration of exercise. Glucose utilization during exercise was not identical at various times of the 24-hour period. The greatest fall of blood glucose was observed at 1800 after one as well as after two glucose loads. Glucose administration after one hour of exercise prevented hypoglycaemia development.     

Regulation of glycogenolysis in the liver of the newborn rat in vivo inhibitory effect of glucose

Newborn rats were injected immediately after delivery with glucose or glucose plus mannoheptulose, and the time-courses of liver glycogen, plasma glucose, insulin and glucagon concentration were studied. The administration of glucose prevented both liver glycogenolysis and the increase in plasma glucagon concentration which normally occurs immediately after delivery. In addition, the administration of glucose prevented the decrease of plasma glucose and insulin concentration which normally occurs during the first hour of extrauterine life. Supplementation of glucose with mannoheptulose prevented the increase of plasma insulin concentrations caused by the administration of glucose; liver glycogenolysis, however, was not stimulated in these circumstances. The increase in the rate of glycogenolysis caused by the administration of glucagon was prevented in newborn rats previously treated with glucose. These results suggest that glucose exerts an inhibitory effect on the stimulation of neonatal liver glycogenolysis by glucagon.     

Metabolism and distribution of cyclohexanecarboxylic Acid, a plant growth stimulant, in bush bean

A foliar spray of 10 mm cyclohexanecarboxylic acid (CHC), a component of the growth stimulant naphthenic acid, to primary leaves of 14-day-old plants of bush bean, Phaseolus vulgaris L., cv Top Crop, resulted in increased vegetative growth and pod production. One minute after the application of 0.5 mm CHC-7-(14)C (CHC(*)) to a primary leaf, CHC(*) was present within it. The chief conversion of the CHC(*) in the leaf during the interval 15 minutes to 4 hours after the acid had been applied appeared to be CHC(*) to its glucose conjugate (CHC(*)-G), and during 4 to 48 hours, CHC(*)-G to CHC(*)-aspartate and an unknown metabolite. Radioactivity was confined to the leaf for at least 1 hour, but by the 12th hour was detected throughout the plant. In the interval 1 to 4 weeks after CHC(*) application, the mean percentage distribution of radioactivity was: treated leaf, 65.3; roots, 18.8; stem, 7.7; trifoliate leaves, 5.9; flower buds-flowers-pods, 2.3. During this period CHC(*)-G was the most prominent metabolite in all organs; no free CHC(*) was detected. Light favored the movement of CHC(*) conjugates out of the leaf; glucose fed to dark-grown plants substituted for light to some extent but aspartate was relatively ineffective, suggesting the dependence of outward movement on ATP. The presence of the glucose and aspartate conjugates of the acid in all organs of CHC-treated plants and the absence of free CHC from them suggest that one or both conjugates, rather than the acid itself, are involved in growth stimulation.     

Liposome encapsulated tetracaine lowers blood glucose

Tetracaine, a local anesthetic, was previously shown to block hormonal stimulation of gluconeogenesis and glycogenolysis ( Friedmann , N. and Rasmussen, H. (1970) Biochim. Biophys. Acta 222, 41-52). In the present studies tetracaine incorporated into liposomes (phospholipid vesicles) was injected into intact rats and epinephrine was administered an hour later. Liposomal tetracaine blocked 50% of the hyperglycemic response. When tetracaine, incorporated into liposomes, was injected into diabetic rats it reduced transiently, but significantly, blood glucose levels. Equivalent doses of free tetracaine were toxic. These studies indicate that liposomal drug administration might be developed into a tool to influence hepatic metabolism and, consequently, blood glucose levels.     

Pertussis toxin reversal of the antilipolytic action of insulin in rat adipocytes in the presence of forskolin does not involve cyclic AMP

Insulin inhibition of lipolysis in the presence of forskolin was reversed by a four hour exposure of adipocytes to pertussis toxin. In contrast, the antilipolytic action of insulin against lipolysis due to theophylline was unaffected by pertussis toxin as was the ability of insulin to lower cyclic AMP in the presence of either forskolin or theophylline. The stimulation of adenylate cyclase by norepinephrine in crude plasma membranes obtained from rat adipocytes was inhibited by N6-(Phenylisopropyl)adenosine (PIA) and abolished by pretreating rat adipocytes with pertussis toxin. The stimulation of glucose metabolism by insulin was not altered by pertussis toxin pretreatment of rat adipocytes. These findings suggest that pertussis toxin selectively abolishes the antilipolytic effect of insulin in the presence of forskolin through a cyclic AMP independent mechanism.     

Activation of glucose transport in muscle by prolonged exposure to insulin. Effects of glucose and insulin concentrations

Glucose transport activity was found to increase over 5 h in rat epitrochlearis muscle in response to a moderate concentration (50-100 microunits/ml) of insulin. This process was examined using 3-methylglucose. The increase in permeability to 3-methylglucose was 2- to 4-fold greater after 5 h than after 1 h in muscles incubated with 50 microunits/ml of insulin and 1 or 8 mM glucose. The increase in permeability to 3-methylglucose during the period between 1 and 5 h of exposure to 50 microunits/ml of insulin and 1 mM glucose was due to an increase in the apparent Vmax of sugar transport. There were two components to this activation of glucose transport. One, which was not influenced by inhibition of protein synthesis, resulted in activation of sugar transport to the same extent by 50 microunits/ml as by 20,000 microunits/ml of insulin; however, this activation took approximately 20 times longer with 50 microunits/ml insulin. The other, which was blocked by cycloheximide, resulted in a further activation of sugar transport to a level higher than that attained in response to 20,000 microunits/ml of insulin. Glucose had no effect on activation of sugar transport during the first hour, but a high concentration (20-36 mM) of glucose prevented the further activation of glucose transport during prolonged treatment with 50 microunits/ml of insulin. It appears from these results that prolonged exposure to a moderate concentration of insulin has previously unrecognized effects that include: a progressive activation of glucose transport over a long time that eventually results in as great a response as a "supramaximal" insulin concentration, and in the presence of low glucose concentration, further activation of glucose transport by an additional, protein synthesis-dependent mechanism. The results also show that a high concentration of glucose can, under some conditions, inhibit stimulation of its own transport.     

High Variability in Oral Glucose Tolerance among 1,128 Patients with Cystic Fibrosis: A Multicenter Screening Study

Background

In cystic fibrosis, highly variable glucose tolerance is suspected. However, no study provided within-patient coefficients of variation. The main objective of this short report was to evaluate within-patient variability of oral glucose tolerance.

Methods

In total, 4,643 standardized oral glucose tolerance tests of 1,128 cystic fibrosis patients (median age at first test: 15.5 [11.5; 21.5] years, 48.8% females) were studied. Patients included were clinically stable, non-pregnant, and had at least two oral glucose tolerance tests, with no prior lung transplantation or systemic steroid therapy. Transition frequency from any one test to the subsequent test was analyzed and within-patient coefficients of variation were calculated for fasting and two hour blood glucose values. All statistical analysis was implemented with SAS 9.4.

Results

A diabetic glucose tolerance was confirmed in 41.2% by the subsequent test. A regression to normal glucose tolerance at the subsequent test was observed in 21.7% and to impaired fasting glucose, impaired glucose tolerance or both in 15.2%, 12.0% or 9.9%. The average within-patient coefficient of variation for fasting blood glucose was 11.1% and for two hour blood glucose 25.3%.

Conclusion

In the cystic fibrosis patients studied, a highly variable glucose tolerance was observed. Compared to the general population, variability of two hour blood glucose was 1.5 to 1.8-fold higher.     

Enhancement of whole body glucose uptake during and after human skeletal muscle low-frequency electrical stimulation.

There is considerable evidence to suggest that electrical stimulation (ES) activates glucose uptake in rodent skeletal muscle. It is, however, unknown whether ES can lead to similar metabolic enhancement in humans. We employed low-frequency ES through surface electrodes placed over motor points of quadriceps femoris muscles. In male subjects lying in the supine position, the highest oxygen uptake was obtained by a stimulation pattern with 0.2-ms biphasic square pulses at 20 Hz and a 1-s on-off duty cycle. Oxygen uptake was increased by approximately twofold throughout the 20-min stimulation period and returned to baseline immediately after stimulation. Concurrent elevation of the respiratory exchange ratio and blood lactate concentration indicated anaerobic glycogen breakdown and utilization during ES. Whole body glucose uptake determined by the glucose disposal rate during euglycemic clamp was acutely increased by 2.5 mg. kg(-1). min(-1) in response to ES and, moreover, remained elevated by 3-4 mg. kg(-1). min(-1) for at least 90 min after cessation of stimulation. Thus the stimulatory effect of ES on whole body glucose uptake persisted not only during, but also after, stimulation. Low-frequency ES may become a useful therapeutic approach to activate energy and glucose metabolism in humans.     

Stimulation by glucose of gluconeogenesis in hepatocytes isolated from starved rats.

Control properties of the gluconeogenic pathway in hepatocytes isolated from starved rats were studied in the presence of glucose. The following observations were made. (1) Glucose stimulated the rate of glucose production from 20 mM-glycerol, from a mixture of 20 mM-lactate and 2 mM-pyruvate, or from pyruvate alone; no stimulation was observed with 20 mM-alanine or 20 mM-dihydroxyacetone. Maximal stimulation was obtained between 2 and 5 mM-glucose, depending on the conditions. At concentrations above 6 mM, gluconeogenesis declined again, so that at 10 mM-glucose the glucose production rate became equal to that in its absence. (2) With glycerol, stimulation of gluconeogenesis by glucose was accompanied by oxidation of cytosolic NADH and reduction of mitochondrial NAD+ and was insensitive to the transaminase inhibitor amino-oxyacetate; this indicated that glucose accelerated the rate of transport of cytosolic reducing equivalents to the mitochondria via the glycerol 1-phosphate shuttle. (3) With lactate plus pyruvate (10:1) as substrates, stimulation of gluconeogenesis by glucose was almost additive to that obtained with glucagon. From an analysis of the effect of glucose on the curves relating gluconeogenic flux and the steady-state intracellular concentrations of gluconeogenic intermediates under various conditions, in the absence and presence of glucagon, it was concluded that addition of glucose stimulated both phosphoenolpyruvate carboxykinase and pyruvate carboxylase activity.     

Serum fructosamine concentrations in patients with type II (non-insulin-dependent) diabetes mellitus during changes in management.

The serum fructosamine concentration was examined as a new means to monitor metabolic control in non-insulin-dependent diabetes during changes in management. Weekly fructosamine estimations were compared with glycosylated haemoglobin (HbA1c), 24 hour urinary glucose, and fasting plasma glucose concentrations in a 17 week study entailing withdrawal and reinstitution of oral treatment. The serum fructosamine concentration was more sensitive than the other measurements in detecting a deterioration in diabetic control after stopping oral hypoglycaemic drugs. The response to reinstitution of treatment was not significant in the first three weeks (p = 0.266), despite a highly significant reduction in fasting plasma glucose (p = 0.001) and 24 hour urinary glucose concentrations (p = 0.012). Compared with HbA1c, concentrations of fructosamine appeared more useful in monitoring short term (three to six weeks) changes after alterations in management of diabetes. Additional advantages were lower cost and technical simplicity of measurement.     

Stimulation of AMP-activated protein kinase (AMPK) is associated with enhancement of Glut1-mediated glucose transport

In cells expressing only the Glut1 isoform of glucose transporters, we have shown that glucose transport is markedly stimulated in response to hypoxia or inhibition of oxidative phosphorylation, conditions that would be expected to cause a stimulation of AMP-activated protein kinase (AMPK) activity. In the present study we tested the hypothesis that the stimulation of AMPK activity might be accompanied by an enhancement of Glut1-mediated glucose transport. Exposure of Clone 9 cells, 3T3-L1 preadipocytes, and C(2)C(12) myoblasts (cells that express only the Glut1 isoform) to 5-aminoimidazole-4-carboxamideribonucleoside (AICAR), an adenosine analog that stimulates AMPK activity, resulted in a marked increase in the rate of glucose transport (ranging from four- to sixfold) that was accompanied by activation of AMPK. This stimulation of AMPK activity was associated with an increase in the phosphorylation of threonine 172 on the activation loop of its alpha subunit, with the predominant change being in the alpha-2 isoform. Exposure of Clone 9 cells to 5-iodotubercidin, an inhibitor of adenosine kinase, abolished the accumulation of AICAR-5'-monophosphate (ZMP), stimulation of AMPK, and the enhancement of glucose transport in response to AICAR. There was no significant increase in the content of Glut1 in plasma membranes of Clone 9 cells exposed to AICAR. We conclude that stimulation of AMPK activity is associated with enhancement of Glut1-mediated glucose transport, and that the glucose transport response is mediated by activation of Glut1 transporters preexisting in the plasma membrane.     

Effect of tetracaine and lidocaine on insulin release in isolated mouse pancreatic islets

The effect of tetracaine and lidocaine on insulin secretion and glucose oxidation by islets of ob/ob-mice was measured. Tetracaine, at a concentration of 1 microM to 0.1 mM, did not markedly influence the basal (3 mM glucose) insulin secretion, whereas 0.5-3.5 mM induced a marked increase. At 7 mM glucose, there was a dose-dependent increase with 0.1-2.5 mM tetracaine. Insulin release induced by 20 mM glucose was potentiated by 0.1 mM and 0.5 mM tetracaine, but this effect disappeared at 1 mM tetracaine. The stimulatory effect of 0.5-1 mM tetracaine on basal insulin release was blocked by the secretory inhibitors, adrenaline (1 microM), clonidine (1 microM) and by Ca2+-deficiency, but the stimulation by 3.5 mM tetracaine was not reduced by 1 microM clonidine or Ca2+ deficiency. Atropine (10 microM) did not affect the stimulation by 0.5 mM tetracaine at 3 mM glucose or by 0.25 mM tetracaine at 20 mM glucose. Tetracaine, at 0.1 mM, potentiated the secretory stimulation of 20 mM L-leucine, 20 mM D-mannose, or 1 microM glibenclamide. Mannoheptulose, 10 mM, abolished the combined effects of 0.1 mM tetracaine and 10 mM glucose. Lidocaine, 1-5 mM, stimulated basal insulin release, but 1 microM-1 mM of the drug did not affect glucose-induced (20 mM glucose) insulin release and 5 mM lidocaine inhibited glucose stimulation. The oxidation of 10 mM D-[U-14C]glucose was slightly enhanced by 0.1 and 1 mM tetracaine. The results indicate that tetracaine and lidocaine, at certain concentrations, can induce insulin release and that tetracaine potentiates secretion induced by other secretagogues. It is concluded that these effects may be associated with beta-cell functions related to the adrenergic receptors but probably not to cholinergic receptors.     

Blood glucose and serum insulin levels in lean and genetically obese mice.

Fed and 24 hour fasted lean and genetically obese mice (ob/ob) were given a fixed glucose load per gm body weight by intraperitoneal and intragastric administration. Intraperitoneal glucose injection into the obese mice produced a prolonged elevated blood glucose level with a concomitant significant decrease of circulating insulin. Possible interpretations of this observation are discussed. In those obese animals in which glucose was administered intragastrically the fed obese mice had a blood glucose concentration of 450-500 mg% for a period of one hour but there was no increase in circulating insulin, however, in the fasted obese mice in which the glucose concentration was about 350 mg% for one hour, there was a significant increase in the circulating insulin levels. The fed and fasted lean mice showed normal glucose tolerance curves and the expected increase in circulating insulin following either intraperitoneal orintragastric glucose loads. It is concluded that hyperglycaemia in the ob/ob mice is unlikely to be the principal cause of hyperinsulinaemia.     

Comparison of intraduodenal and intravenous glucose metabolism under clamp conditions in humans

To determine whether the uptake and metabolic partition of glucose are influenced by its delivery route, 12 normal volunteers underwent two 3-h euglycemic (approximately 93 mg/dl) hyperinsulinemic (approximately 43 mU/l) clamps at a 3- to 5-wk interval, one with intravenous (i.v.) and the other with intraduodenal (i.d.) glucose labeled with [3-3H]- and [U-14C]glucose. Systemic glucose was traced with [6,6-2H2]glucose in eight subjects. During the last hour of the clamps, the average glucose infusion rate (5.85 +/- 0.37 vs. 5.43 +/- 0.43 mg.kg(-1).min(-1); P = 0.02) and exogenous glucose uptake (5.66 +/- 0.37 vs. 5.26 +/- 0.41 mg.kg(-1).min(-1); P = 0.04) were borderline higher in the i.d. than in the i.v. studies. The increased uptake was entirely accounted for by increased glycolysis (3H2O production), which was attributed to the stimulation of gut metabolism by the absorptive process. No difference was observed in glucose storage whether it was calculated as glucose uptake minus glycolysis (i.d. vs. i.v.: 2.44 +/- 0.28 vs. 2.40 +/- 0.31 mg.kg(-1).min(-1)) or as glucose uptake minus net glucose oxidation (2.86 +/- 0.33 vs. 2.81 +/- 0.35 mg.kg(-1).min(-1)). Because peripheral tissues were exposed to identical glucose, insulin, and free fatty acid levels under the two experimental conditions, we assumed that their glucose uptake and storage were similar during the two tests. We therefore suggest that hepatic glycogen storage (estimated as whole body minus peripheral storage) was also unaffected by the route of glucose delivery. On the other hand, in the i.d. tests, the glucose splanchnic extraction ratio calculated by the dual-isotope technique averaged 4.9 +/- 2.3%, which is close to the figures published for i.v. glucose. Despite the limitations related to whole body measurements, these two sets of data do not support the idea that enteral glucose stimulates hepatic uptake more efficiently than i.v. glucose.     

Transient extreme insulin resistance in shock during diabetic ketoacidosis.

Transient extreme insulin resistance was encountered during an episode of diabetic ketoacidosis (DKA) in an insulin-treated diabetic patient. On admission, the plasma glucose level was 1241 mg dl-1 and arterial blood pH 6.895 with HCO3- 4.7 mEql-1. An intravenous bolus injection of 20 units, followed by continuous infusion of 20 units h-1 of short-acting regular human insulin, was instituted. Ischemic myocardial changes were noted on the initial electrocardiogram, therefore fluid replacement was limited to 1,000 ml of 0.9% saline solution in the first hour. As the plasma glucose level declined by only 203 mg dl-1 (41 mg dl-1 h-1) in the first 5 h, the insulin dose was doubled every 2 h. At hour 4, the patient developed circulatory shock which required vasopressor support and respiratory assistance. A plasma glucose level of 300 mg dl-1 was not achieved until the total dosage of insulin amounted to 91,580 units at hour 25. Insulin resistance was not observed from that point on. The patient had neither insulin antibodies nor anti-insulin receptor antibodies in serologic testing. The insulin binding characteristics of the patient's erythrocytes were similar to those from healthy controls both with and without experimental acidosis and with a high level of beta-hydroxybutyrate. Among multiple potential factors, the severe shock associated with DKA has been considered as a primary cause of the transient severe insulin resistance in this case.     

Effect of the parenteral administration of energy substrates in different postoperation phases on the initiation and development of liver regeneration in rats subjected to partial hepatectomy

DNA specific activity in the liver, the total DNA content of the liver and the mitotic index of the hepatocytes were studied after the infusion of glucose or lipid emulsions in female laboratory rats with a mean pre-operation weight of 250 +/- 30 g after partial (65-70%) hepatectomy (PH). The infusions were administered in the early prereplication phase (the 1st to 6th hour after the operation), in the late prereplication phase (the 7th to 12th hour after the operation), or continuously from the 1st to the 12th, or the 1st to the 24th, hour after partial hepatectomy. The effect of these parenterally administered energy substrates on the initiation of liver regeneration was evaluated 18 and 24 hours after partial hepatectomy. The results indicate that the infusion of glucose, in any interval after the operation, inhibited the initial phases of liver DNA synthesis (18 h after PH), but not its further development (24 h after PH). Neither the mitotic index of the hepatocytes, nor the total DNA content of the liver differed from the control groups in the case of rats given a glucose infusion. In the experimental groups given lipid emulsions, inhibition of liver DNA synthesis was recorded 18 h after PH only when the infusions were given from the 1st to the 12th or the 1st to the 18th hour after PH. The total DNA content of the liver 18 h after PH was raised in all the experimental groups.(ABSTRACT TRUNCATED AT 250 WORDS)     

热量限制对SH-SY5Y细胞氧化损伤的影响

目的观察热量限制培养条件下,SH-SY5Y细胞抗氧化应激损伤的能力。方法建立过氧化氢诱导的SH-SY5Y细胞损伤模型。体外培养SH-SY5Y细胞,分为对照组、损伤组（50、100、250、500、1 000μmol/L H2O2）、低糖组（2 g/L）、低糖＋损伤组,进行细胞形态观察、测定各组细胞的噻唑蓝（MTT）代谢率、乳酸脱氢酶（LDH）漏出率。结果与对照组比较,（50、100、250、500、1 000）μmol/L H2O2损伤1 h后MTT代谢率测定细胞活力,50μmol/L组与对照组比较差异无统计学意义（P〉0.05）;其他组与对照组比较,随着H2O2浓度的增加,细胞活力呈递减趋势,差异具有显著性（P〈0.01）;选定250μmol/L H2O2组为损伤应激源。用低糖预处理细胞24 h,给与250μmol/L H2O2损伤1 h后测定MTT代谢率显示,与对照组比较,损伤组活力明显下降,低糖组活力上升（P〈0.01）;与损伤组比较,低糖＋损伤组活力明显上升（p〈0.01）;继续培养至7 h发现,与对照组比较,低糖组活力上升（P〈0.01）;与损伤组比较,低糖＋损伤组活力明显上升（P〈0.01）。进一步检测LDH漏出率显示,损伤1 h后结果显示,与对照组比较,损伤组漏出率明显增加（P〈0.05）,低糖组漏出率稍有减少（P〉0.05）;与损伤组比较,低糖＋损伤组漏出率明显减少（P〈0.01）;继续培养7h显示,低糖7h组与低糖1 h组比较,漏出稍有增多（P〉0.05）,低糖＋损伤组7 h组与低糖＋损伤组1 h比较漏出率稍有增加（P〈0.05）;细胞形态学观察显示,未加损伤之前,低糖组的细胞形态,与对照组比较无明显改变。加入损伤药物1h后的细胞形态与对照组比较无明显改变。加入损伤药物7 h后的细胞形态,低糖组和对照组细胞突起伸展良好细长,损伤组可见细胞数目明显减少,死细胞多,突起回缩,细胞明显变圆,贴壁性不好,透光性差。结论热量限制能提高神经细胞的抗氧化应激能力,增加细胞生存率,降低死亡率。