还原型谷胱甘肽对大鼠随意皮瓣成活影响的实验研究

目的通过经大鼠腹腔注射还原型谷胱甘肽(glutathione,GSH),观察其对大鼠随意皮瓣成活的影响,并初步探讨GSH影响皮瓣成活的作用机制。方法选择健康200~250gSD大鼠20只,于背部双侧肩胛下角连线设计制备面积为8cm×2cm随意皮瓣,原位回植。随机分成两组(n=10),实验组于皮瓣术后即刻及1、2d,腹腔注射GSH(250mg/kg),对照组腹腔注射等量生理盐水。术后7d测定皮瓣成活率,取大鼠皮瓣中轴距蒂部3cm处组织,检测超氧化物歧化酶(superoxide dismutase,SOD)和丙二醛(malonyldialdehyde,MDA)含量,对皮瓣远、中及近段进行组织学观察。结果术后7d,实验组皮瓣成活率为56.77%±10.67%,高于对照组的40.16%±7.12%,差异有统计学意义(P<0.05)。实验组SOD含量为306.06±84.87U/mgprot,高于对照组的224.79±27.12U/mgprot;而MDA含量为3.835±0.457nmol/mgprot,低于对照组的6.127±0.837nmol/mgprot,差异均有统计学意义(P<0.05)。组织学观察:实验组近、中及远段中性粒细胞浸润较对照组明显减轻,其血管、成纤维细胞、毛囊及附属腺体增生均明显优于对照组。结论腹腔注射GSH对大鼠随意皮瓣成活有明显促进作用,作用机制可能与清除自由基、降低脂质过氧化反应及减轻中性粒细胞浸润有关。     

内毒素/脂多糖对烫伤大鼠休克期肝脏脂肪代谢的影响

目的观察内毒素/脂多糖(LPS)对烫伤大鼠休克期肝脏脂肪代谢的影响。方法采用30%TBSAⅢ度烫伤大鼠模型,随机分为单纯烫伤组(烫伤组)和烫伤后LPS攻击组(攻击组),另设假烫对照组(对照组),每组20只。攻击组大鼠于烫伤后2h腹腔注射LPS,3.0mg/kg.伤后24、48h分别检测3组大鼠血浆LPS、肿瘤坏死因子(TNF)α、游离脂肪酸(FFA)含量和肝组织内腺苷三磷酸(ATP)、甘油三酯(TG)及丙二醛(MDA)含量,并进行肝组织形态学观察。结果对照组大鼠假烫后24、48h,血浆FFA水平分别为(0.4±0.3)、(0.5±0.3)mmol/L,烫伤组分别为(0.9±0.3)、(1.2±0.5)mmol/L,攻击组大鼠伤后增加至(2.3±0.3)、(2.5±0.4)mmol/L,后两组之间比较差异有统计学意义(P<0.01).伤后48h,烫伤组和攻击组的肝组织TG含量分别为(242±27)mmol/g和(530±30)mmol/g,ATP含量分别为(6.0±2.4)μmol/g和(1.7±0.5)μmol/g,组间比较差异有统计学意义(P<0.01).组织形态学检查见,烫伤组大鼠肝细胞脂肪变性和线粒体损伤程度明显轻于攻击组,对照组肝细胞形态正常。结论烧伤后早期LPS打击可引发脂源性能量利用障碍,并加剧肝脏脂肪变性。     

Intrahepatic mechanisms underlying the effect of metformin in decreasing basal glucose production in rats fed a high-fat diet.

The aim of this study was to understand by which intrahepatic mechanism metformin (Met) may inhibit basal hepatic glucose production (HGP) in type 2 diabetes. We studied rats that were fed for 6 weeks a high-fat (HF) diet, supplemented (HF-Met) or not (HF) with Met (50 mg x kg(-1) x day(-1)). Basal HGP, assessed by 3-[(3)H]glucose tracer dilution, was lower by 20% in HF-Met rats compared with HF-rats: 41.6 +/- 0.7 vs. 52 +/- 1.5 micromol x kg(-1) x min(-1) (means +/- SE, n = 5; P < 0.01). Glucose-6 phosphatase (Glc6Pase) activity, assayed in a liver lobe freeze-clamped in situ, was lower by 25% in HF-Met rats compared with HF-rats (7.9 +/- 0.4 vs. 10.3 +/- 0.9 micromol x min(-1) x g(-1) wet liver; P < 0.05). Glucose-6 phosphate and glycogen contents, e.g., 42 +/- 5 nmol/g and 3.9 +/- 2.4 mg/g, respectively, in HF-rats were dramatically increased by three to five times in HF-Met rats, e.g., 118 +/- 12 nmol/g and 19.6 +/- 4.6 mg/g (P < 0.05 and P < 0.01, respectively). Glucose-6 phosphate dehydrogenase activity was increased in HF-Met compared with HF rats (1.51 +/- 0.1 vs. 1.06 +/- 0.08 micromol x min(-1) x g(-1); P < 0.01). Intrahepatic lactate concentration tended to be lower in the Met-group (-30%; NS), whereas plasma lactate concentration was higher in HF-Met rats (1.59 +/- 0.15 mmol/l) than in HF rats (1.06 +/- 0.06 mmol/l; P < 0.05). We concluded that Met decreases HGP in insulin-resistant HF-fed rats mainly by an inhibition of hepatic Glc6Pase activity, promoting glycogen sparing. Additional mechanisms might involve the diversion of glucose-6 phosphate into the pentose phosphate pathway and an inhibition of hepatic lactate uptake.     

Hindlimb ischemia-reperfusion increases complement deposition and glycolysis.

BACKGROUND: Hindlimb ischemia-reperfusion (HIR) impairs cellular energy metabolism and causes local muscle injury possibly through free radical or complement-mediated mechanisms. MATERIALS AND METHODS: To determine the relationship among myocellular energetics, histopathological injury, and mediator activity, male Wistar rats underwent 4 h of Sham (n = 8), Unilateral (n = 8), or Bilateral (n = 8) hindlimb ischemia followed by 4 h of reperfusion. All rats underwent 31P magnetic resonance spectroscopy of their right gastrocnemius muscle to determine various high-energy phosphate ratios including ATP to Pi (ATP/Pi, a measure of energy status) and phosphocreatine to Pi (PCr/Pi, a measure of thermodynamic capacity). Gastrocnemius muscles were then harvested to determine muscle damage and complement membrane attack complex (MAC) deposition by immunohistochemical staining [grade 0 (none) to 3 (very severe)] and to measure glutathione (GSH), DNA, and enzyme activities: beta-hydroxyacyl-CoA dehydrogenase, phosphofructokinase, and citrate synthetase. RESULTS: HIR was associated with significant declines in ATP/Pi and PCr/Pi (P < 0.001). Progressively more severe HIR (Sham, Unilateral, Bilateral) was associated with greater MAC deposition (0. 0 +/- 0.0, 1.0 +/- 0.3, 1.5 +/- 0.4, P = 0.06, mean +/- SEM) and histological damage (0.0 +/- 0.0, 0.9 +/- 0.3, 1.3 +/- 0.4, P < 0. 05). GSH levels, beta-hydroxyacyl-CoA dehydrogenase, and citrate synthetase activities were not affected by HIR, but phosphofructokinase activity increased (24.09 +/- 2.42, 35.16 +/- 5. 26, 59.29 +/- 9.82 mmol/mg of DNA/min, P < 0.05). Although GSH levels were not significantly altered, complement deposition was closely associated with skeletal muscle injury and compensatory changes in glycolysis. Alterations in myocellular bioenergetics after HIR closely paralleled complement deposition rather than GSH depletion. CONCLUSIONS: Therapeutic strategies aimed at controlling complement activity and assessment techniques based on bioenergetics may allow more precise determinations of the effects of HIR injury.     

Loss of the decrement in intraislet insulin plausibly explains loss of the glucagon response to hypoglycemia in insulin-deficient diabetes: documentation of the intraislet insulin hypothesis in humans

The intraislet insulin hypothesis for the signaling of the glucagon secretory response to hypoglycemia states that a decrease in arterial glucose --> a decrease in beta-cell insulin secretion --> a decrease in tonic alpha-cell inhibition by insulin --> an increase in alpha-cell glucagon secretion. To test this hypothesis in humans, a hyperinsulinemic- euglycemic ( approximately 5.0 mmol/l [90 mg/dl] x 2 h) and then a hypoglycemic ( approximately 3.0 mmol/l [55 mg/dl] x 2 h) clamp was performed in 14 healthy young adults on two occasions, once with oral administration of the ATP-sensitive potassium channel agonist diazoxide to selectively suppress baseline insulin secretion and once with the administration of a placebo. The decrement in plasma C-peptide during the induction of hypoglycemia was reduced by approximately 50% in the diazoxide clamps (from 0.3 +/- 0.0 to 0.1 +/- 0.0 nmol/l [0.8 +/- 0.1 to 0.4 +/- 0.1 ng/ml]) compared with the placebo clamps (from 0.4 +/- 0.0 to 0.1 +/- 0.0 nmol/l [1.2 +/- 0.1 to 0.4 +/- 0.1 ng/ml]) (P = 0.0015). This reduction of the decrement in intraislet insulin during induction of hypoglycemia caused an approximately 50% reduction (P = 0.0010) of the increase in plasma glucagon in the diazoxide clamps (from 29 +/- 3 to 35 +/- 2 pmol/l [102 +/- 9 to 123 +/- 8 pg/ml]) compared with the placebo clamps (from 28 +/- 2 to 43 +/- 5 pmol/l [98 +/- 7 to 151 +/- 16 pg/ml]). Baseline glucagon levels, the glucagon response to intravenous arginine, and the autonomic (adrenomedullary, sympathetic neural, and parasympathetic neural) responses to hypoglycemia were not altered by diazoxide. These data indicate that a decrease in intraislet insulin is a signal for the glucagon secretory response to hypoglycemia in healthy humans. The absence of that signal plausibly explains the loss of the glucagon response to falling plasma glucose concentrations, a key feature of the pathogenesis of iatrogenic hypoglycemia, in insulin-deficient (type 1 and advanced type 2) diabetes.     

Oxidative stress during acute respiratory exacerbations in cystic fibrosis

BACKGROUND: Patients with cystic fibrosis experience chronic systemic oxidative stress. This is coupled with chronic inflammation of the lung involving bronchial polymorphonuclear neutrophil accumulation and activation. We hypothesised that, during periods of acute respiratory exacerbation, free radical activity and consequent damage would be most marked and that intensive treatment of the infection would result in improvement towards values found during stable periods. METHODS: Plasma and red blood cells were collected from 12 healthy normal volunteers and from 12 patients with cystic fibrosis with an acute respiratory exacerbation (increased respiratory symptoms, reduction in forced expiratory volume in one second (FEV1) of more than 10%, and a decision to treat with intravenous antibiotics). Further samples were collected from patients following two weeks of treatment. Samples were analysed for inflammatory markers, markers of free radical damage, and aqueous and lipid phase scavengers. RESULTS: During respiratory exacerbations FEV1 and forced vital capacity (FVC) were lower than in controls (mean differences -2.82 (95% CI -2.12 to -3.52) and -3. 79 (-3.03 to -4.55) l, respectively) but improved following treatment (mean change 0.29 (95% CI 0.18 to 0.40) and 0.33 (0.23 to 0.43) l, respectively). Inflammatory markers during exacerbations were significantly higher in patients than in controls with the following mean (95% CI) differences: C reactive protein (CRP), 46 (17 to 75) g/l; neutrophil elastase alpha1-antiprotease complexes (NEAPC), 4.4 (1.77 to 7.07) mg/l; white cell count (WCC), 5.3 (4.7 to 5.9) x 10(9)/l. These markers decreased significantly following treatment with the following mean (95% CI) changes: CRP -26 (-10 to -42) g/l; NEAPC -3.1 (-1.3 to -4.9) mg/l; WCC -1.5 (-1.3 to -1.7) x 10(9)/l. Malondialdehyde (MDA) as a marker of free radical activity was significantly higher in patients during exacerbations than in controls with a mean (95% CI) difference of 193 (107 to 279) which improved with treatment (mean change -56 (95% CI -28 to -84) nmol/mmol cholesterol). Red blood cell polyunsaturated fatty acids were significantly lower in patients than in controls with a mean difference of -4.4(95% CI -2.6 to -6.2) moles percent, but did not improve significantly after treatment. Protein carbonyls during exacerbations were not different from controls but did increase with treatment compared with levels during the exacerbation (mean change 0.39 (95% CI 0.11 to 0.67) micromol/g protein). Aqueous and lipid phase scavengers in patients during exacerbations were significantly lower than in controls with the following mean (95% CI) differences: ascorbate, -19.0 (-2.7 to -35.3) micromol/l; sulphydryls, -122 (-77 to -167) micromol/l; retinol, -237 (-47 to -427) nmol/mmol cholesterol; beta-carotene, -52.8 (-11.8 to -93.8) nmol/mmol cholesterol; luteine, -50.4 (-10.4 to -90.4) nmol/mmol cholesterol; lycopene, -90.1 (-30.1 to -150.1) nmol/mmol cholesterol. Treatment resulted in improvement with the following mean (95% CI) changes: sulphydryls, 50 (32 to 68) micromol/l; retinol, 152 (47 to 257) nmol/mmol cholesterol; alpha- and beta-carotene, 0.6 (0.0 to 1.2) and 7.6 (0.0 to 15.2) nmol/mmol cholesterol, respectively; alpha-tocopherol, 839 (283 to 1405) nmol/mmol cholesterol; and lycopene, 8.2 (0.0 to 16.2) nmol/mmol cholesterol. CONCLUSIONS: Abnormalities of markers of inflammation, free radical activity, and radical scavengers were significantly more extreme during acute respiratory exacerbations and showed improvement with treatment. The need to provide protection from inflammation and free radical damage should therefore be dynamic and related to the inflammatory and oxidative processes.     

Renal work, glutathione and susceptibility to free radical-mediated postischemic injury

Studies were performed to determine whether renal glutathione (GSH) is an important free-radical scavenger following ischemia and reperfusion, whether alterations in renal transport work affect renal GSH levels, and whether a decrease in renal work decreases susceptibility to postischemic renal injury via the first two effects. Following administration of either intravenous GSH to increase renal GSH or intraperitoneal diethylmaleate to decrease renal GSH, Sprague-Dawley rats underwent 60 minutes of renal ischemia. In animals with high renal GSH following GSH infusion, GFR 24 hours after ischemia was 0.43 +/- 0.08 ml/min compared to 0.15 +/- 0.02 ml/min in saline-infused control animals (P less than 0.01). When renal GSH was decreased by the administration of diethylmaleate postischemic renal dysfunction was accentuated. Twenty-four hours after ischemia GFR was 0.05 +/- 0.02 ml/min in diethylmaleate-treated animals and 0.28 +/- 0.06 ml/min in control animals (P less than 0.005). To test whether a decrease in renal transport work alters renal GSH the filtered load of sodium was reduced by producing unilateral renal artery stenosis. Alternatively, renal work was lessened when sodium reabsorption was interfered with by the infusion of a combination of natriuretic agents. Renal artery stenosis produced a 37% decrease in GFR. Renal GSH was 0.435 +/- 0.089 nmol/mg protein in intact kidneys and 0.804 +/- 0.239 nmol/mg protein in stenotic kidneys (P less than 0.05). The infusion of natriuretic agents produced no change in GFR or renal plasma flow but resulted in a striking elevation in renal GSH.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lipid peroxidation in vitamin E-deficient rats submitted to subtotal nephrectomy

Clinical and experimental evidence has indicated that chronic renal failure (CRF) is related to increased free radical production. CRF patients show increased lipid peroxidation after a progressive reduction in vitamin E, one of the most important antioxidants. In the present study the role of vitamin E deficiency in lipid peroxidation was investigated in rats submitted to subtotal nephrectomy. Male Wistar rats were divided into groups receiving different diets for a period of 45 days: SND - sham rats submitted to a regular diet containing vitamin E; ERD nephrectomized rats submitted to a regular diet containing vitamin E; SDD - sham rats submitted to a vitamin E-deficient diet; EDD nephrectomized rats submitted to a vitamin E-deficient diet. After 30 days the Experimental animals were submitted to 5/6 nephrectomy and the Controls were submitted to sham operation. The vitamin E levels of the SDD and EDD groups were significantly reduced (p < 0.05) in plasma (4.92 +/- 1.22 and 8.37 +/- 2.09 mmol/L, respectively), liver (7.57 +/- 2.72 and 9.44 +/- 2.55mg/g tissue, respectively) and kidney (8.17 +/- 2.38 and 9.40 +/- 3.10 mg/g tissue, respectively) when compared to the SRD and ERD groups. In contrast, in the EDD group the levels of thiobarbituric acid-reactive substances, expressed as nmol/mg protein, were significantly increased (p < 0.05) in the liver (1.41 +/- 0.27) and kidney (1.67 +/- 0.47), and superoxide dismutase activity was significantly increased in the erythrocytes (4455.80 +/- 1322.63 Ug/Hb) compared to all other groups. The vitamin E-deficient diet associated with subtotal nephrectomy determined an increase in lipid peroxidation, suggesting an important role of free radicals in the development of chronic renal failure.     

Growth hormone secretion from pituitary cells in chronic renal insufficiency.

To examine whether growth hormone (GH) secretion is adversely affected by chronic renal insufficiency (CRI), the GH secretory response of dispersed anterior pituitary cells perifused with GH-releasing hormone (GHRH) was investigated in 5/6 nephrectomized (CRI, N = 18) and sham-operated (N = 18) rats. Two weeks after nephrectomy, during a period of stable uremia, CRI rats had significantly higher serum concentrations (mean +/- SEM) of urea nitrogen and creatinine than sham rats, 16.8 +/- 1.4 mmol/liter (47 +/- 4 mg/dl) and 79.6 +/- 0.0 mumol/liter (0.9 +/- 0.0 mg/dl) versus 6.1 +/- 0.4 mmol/liter (17 +/- 1 mg/dl) and 35.4 +/- 0.0 mumol/liter (0.4 +/- 0.0 mg/dl), respectively (P less than 0.0001). Incremental gains in body weight and nose to tail-tip length of CRI rats over two weeks were also significantly depressed, 53.3 +/- 5.38 g (CRI) versus 87.0 +/- 3.78 g (sham; P less than 0.0001) and 3.2 +/- 0.2 cm (CRI) versus 3.6 +/- 0.1 cm (sham; P less than 0.05). The cumulative food intake as well as food efficiency (g food consumed/g weight gain) were also adversely influenced by the uremic state: food intake 304 +/- 1 g (CRI) versus 397 +/- 6 g (sham; P less than 0.0001) and food efficiency 0.173 +/- 0.013 g/g of weight gain (CRI) versus 0.219 +/- 0.008 g/g of weight gain (sham). No significant difference in GH secretory rate (ng/min/10(7) cells) was found between the uremic and sham animals under basal conditions, 65.2 +/- 2.1 (CRI) and 67.9 +/- 2.2 (sham) or in response to GH-releasing hormone, 282.8 +/- 42.4 (CRI) versus 306.2 +/- 42.6 (sham).(ABSTRACT TRUNCATED AT 250 WORDS)     

Oral aluminum administration to uremic, hyperparathyroid, or vitamin D-supplemented rats

In the present study, the role of factors was investigated that could possibly lead to changes in plasma and tissue aluminum (A1) concentrations following oral A1 exposure. In chronically uremic rats that received an oral A1 supplementation of 150 mumol/g diet during 4 weeks, a significant increase in mean (+/- SEM) liver A1 content was observed when compared to sham-operated, pair-fed control rats (9.9 +/- 2.0 versus 4.8 +/- 0.65 nmol/g wet weight, p less than 0.02). No such difference was found in non-A1-supplemented rats. Plasma A1 and the A1 content of other organs studied except muscle were not increased in uremic as compared to control animals. In rats with hyperparathyroidism secondary to a calcium-poor diet, mean liver and bone A1 content was significantly decreased when not A1-exposed (2.5 +/- 0.13 and 62 +/- 5.5 nmol/g, respectively) and normal when A1-supplemented (4.7 +/- 0.59 and 120 +/- 23 nmol/g, respectively) as compared to normal control rats without A1 supplementation (5.1 +/- 1.5 and 170 +/- 17 nmol/g, respectively). However, in the hyperparathyroid rats, mean plasma A1 concentration was higher than in control, euparathyroid rats. In rats with exogenous hyperparathyroidism (parathyroid extract) a significant increase in liver A1 content was observed when compared to control rats (8.1 +/- 0.95 versus 5.3 +/- 0.53 nmol/g, p less than 0.05). In A1-supplemented normal rats treated with 1,25(OH)2 vitamin D3 during 4 weeks, liver A1 content was significantly lower than in control rats receiving vehicle solution only (2.9 +/- 0.76 versus 5.7 +/- 0.59 nmol/g, p less than 0.02).(ABSTRACT TRUNCATED AT 250 WORDS)     

The influence of cryogenic brain injury on the pharmacodynamics of pentobarbital. Evidence for a serotonergic mechanism.

To study of the influence of brain injury on the pharmacodynamics of pentobarbital, the authors examined the effect of a focal cortical freezing lesion in rats on the brain concentration of pentobarbital associated with lack of response to tail clamp. The freezing lesion was made with a probe (-50 degrees C) applied through a craniotomy to the intact dura over the left parietal cortex. Three days after injury the rats were anesthetized with a continuous intravenous infusion of pentobarbital until they first did not respond to tail clamp stimulation. The brains were then removed for determination of pentobarbital by high-performance liquid chromatography. The brain pentobarbital concentration required to prevent response to tail clamp (EC50) was reduced from 209 +/- 39 nmol/g (mean +/- standard deviation) in rats without brain injury to 149 +/- 28 nmol/g in the injured animals (P = 0.005). The cortical serotonin (5-HT) concentration was increased from 1904 +/- 358 pmol/g in uninjured rats to 2513 +/- 598 pmol/g (P less than 0.01) in injured animals ipsilateral to the lesion. Pretreatment of the rats with p-chlorophenylalanine (PCPA, 200 mg/kg by intraperitoneal injection) to inhibit 5-HT synthesis abolished both the increase in 5-HT concentration associated with the injury (left cortex, 708 +/- 389 pmol/g; right cortex, 911 +/- 979 pmol/g) and the effect of the lesion on EC50 (uninjured, EC50 = 186 +/- 24 nmol/g; injured, EC50 = 179 +/- 47 nmol/g). Prevention of the decrease in EC50 by inhibition of 5-HT synthesis provides support for a functional role for 5-HT in the influence of cold injury on the pharmacodynamics of pentobarbital.     

Effect of a sustained reduction in plasma free fatty acid concentration on intramuscular long-chain fatty Acyl-CoAs and insulin action in type 2 diabetic patients

To investigate the effect of a sustained (7-day) decrease in plasma free fatty acid (FFA) concentrations on insulin action and intramyocellular long-chain fatty acyl-CoAs (LCFA-CoAs), we studied the effect of acipimox, a potent inhibitor of lipolysis, in seven type 2 diabetic patients (age 53 +/- 3 years, BMI 30.2 +/- 2.0 kg/m2, fasting plasma glucose 8.5 +/- 0.8 mmol/l, HbA 1c 7.5 +/- 0.4%). Subjects received an oral glucose tolerance test (OGTT) and 120-min euglycemic insulin (80 mU/m2 per min) clamp with 3-[3H]glucose/vastus lateralis muscle biopsies to quantitate rates of insulin-mediated whole-body glucose disposal (Rd) and intramyocellular LCFA-CoAs before and after acipimox (250 mg every 6 h for 7 days). Acipimox significantly reduced fasting plasma FFAs (from 563 +/- 74 to 230 +/- 33 micromol/l; P < 0.01) and mean plasma FFAs during the OGTT (from 409 +/- 44 to 184 +/- 22 micromol/l; P < 0.01). After acipimox, decreases were seen in fasting plasma insulin (from 78 +/- 18 to 42 +/- 6 pmol/l; P < 0.05), fasting plasma glucose (from 8.5 +/- 0.8 to 7.0 +/- 0.5 mmol/l; P < 0.02), and mean plasma glucose during the OGTT (from 14.5 +/- 0.8 to 13.0 +/- 0.8 mmol/l; P < 0.05). After acipimox, insulin-stimulated Rd increased from 3.3 +/- 0.4 to 4.4 +/- 0.4 mg x kg(-1) x min(-1) (P < 0.03), whereas suppression of endogenous glucose production (EGP) was similar and virtually complete during both insulin clamp studies (0.16 +/- 0.10 vs. 0.14 +/- 0.10 mg x kg(-1) x min(-1); P > 0.05). Basal EGP did not change after acipimox (1.9 +/- 0.2 vs. 1.9 +/- 0.2 mg x kg(-1) x min(-1)). Total muscle LCFA-CoA content decreased after acipimox treatment (from 7.26 +/- 0.58 to 5.64 +/- 0.79 nmol/g; P < 0.05). Decreases were also seen in muscle palmityl CoA (16:0; from 1.06 +/- 0.10 to 0.75 +/- 0.11 nmol/g; P < 0.05), palmitoleate CoA (16:1; from 0.48 +/- 0.05 to 0.33 +/- 0.05 nmol/g; P = 0.07), oleate CoA (18:1; from 2.60 +/- 0.11 to 1.95 +/- 0.31 nmol/g; P < 0.05), linoleate CoA (18:2; from 1.81 +/- 0.26 to 1.38 +/- 0.18 nmol/g; P = 0.13), and linolenate CoA (18:3; from 0.27 +/- 0.03 to 0.19 +/- 0.02 nmol/g; P < 0.03) levels after acipimox treatment. Muscle stearate CoA (18:0) did not decrease after acipimox treatment. The increase in R(d) correlated strongly with the decrease in muscle palmityl CoA (r = 0.75, P < 0.05), oleate CoA (r = 0.76, P < 0.05), and total muscle LCFA-CoA (r = 0.74, P < 0.05) levels. Plasma adiponectin did not change significantly after acipimox treatment (7.9 +/- 1.8 vs. 7.5 +/- 1.5 microg/ml). These data demonstrate that the reduction in intramuscular LCFA-CoA content is closely associated with enhanced insulin sensitivity in muscle after a chronic reduction in plasma FFA concentrations in type 2 diabetic patients despite the lack of an effect on plasma adiponectin concentration.     

Portal vein afferents are critical for the sympathoadrenal response to hypoglycemia

We sought to elucidate the role of the portal vein afferents in the sympathetic response to hypoglycemia. Laparotomy was performed on 27 male Wistar rats. Portal veins were painted with either 90% phenol (denervation group [PDN]) or 0.9% saline solution (sham-operated group [SHAM]). Rats were chronically cannulated in the carotid artery (sampling), jugular vein (infusion), and portal vein (infusion). After a recovery period of 5 days, animals were exposed to a hyperinsulinemic-hypoglycemic clamp, with glucose infused either portally (POR) or peripherally (PER). In all animals, systemic hypoglycemia (2.48+/-0.09 mmol/l) was induced via jugular vein insulin infusion (50 mU x kg(-1) x min(-1)). Arterial plasma catecholamines were assessed at basal (-30 and 0 min) and during sustained hypoglycemia (60, 75, 90, and 105 min). By design, portal vein glucose concentrations were significantly elevated during POR versus PER (4.4+/-0.14 vs. 2.5+/-0.07 mmol/l; P<0.01, respectively) for both PDN and SHAM. There were no significant differences in arterial glucose or insulin concentration between the four experimental conditions at any point in time. When portal glycemia and systemic glycemia fell concomitantly (SHAM-PER), epinephrine increased 12-fold above basal (3.75+/-0.34 and 44.56+/-6.1 nmol/l; P<0.001). However, maintenance of portal normoglycemia (SHAM-POR) caused a 50% suppression of the epinephrine response, despite cerebral hypoglycemia (22.2+/-3.1 nmol/l, P<0.001). Portal denervation resulted in a significant blunting of the sympathoadrenal response to whole-body hypoglycemia (PDN-PER 27.6+/-3.8 nmol/l vs. SHAM-PER; P<0.002). In contrast to the sham experiments, there was no further suppression in arterial epinephrine concentrations observed during PDN-POR versus PDN-PER (P = 0.8). These findings indicate that portal vein afferent innervation is critical for hypoglycemic detection and normal sympathoadrenal counterregulation.     

Impaired response of the denervated kidney to endothelin receptor blockade in normotensive and spontaneously hypertensive rats

BACKGROUND: As yet, there are only limited data available on the exact role of endothelin (ET) acting through endothelin-A (ETA) receptors in renal sodium and water regulation and the potential functional implications of an interaction of the renal ET system with renal nerves in normotensive and spontaneously hypertensive rats. METHODS: Experiments were carried out in 64 male conscious spontaneously hypertensive rats and in 56 normotensive Wistar-Kyoto (WKY) rats. Bilateral renal denervation (BRD) was performed in 32 spontaneously hypertensive rats and 28 WKY rats 7 days before the experiments. The ETA receptor antagonist, BQ-123 (16.4 nmol/kg x min intravenously) or the endothelin-B (ETB) receptor antagonist, BQ-788 (25 nmol/kg x min intravenously) were infused at a rate of 25 microL/min for 50 minutes. RESULTS: Renal papillary ET-1 concentration in intact spontaneously hypertensive rats was 67.8% lower than in intact WKY rats (154 +/- 40 fmol/mg protein vs. 478 +/- 62 fmol/mg protein, P < 0.01). BRD decreased papillary ET-1 by 73.5% in WKY rats to 127 +/- 19 fmol/mg protein (P < 0.001), but had no effect in spontaneously hypertensive rats (122 +/- 37 fmol/mg protein). BRD, BQ-123, or BQ-788 did not affect glomerular filtration rate (GFR) or renal blood flow (RBF) in any of the groups. In intact WKY, BQ-123 decreased urine flow rate (V) from 4.65 +/- 0.44 microL/min.100 g body weight to 2.44 +/- 0.35 microL/min.100 g body weight (P < 0.01), urinary excretion of sodium (UNaV) from 238.2 +/- 27.4 to 100.2 +/- 17.0 (P < 0.01) and potassium (UKV) from 532.1 +/- 62.6 nmol/min.100 g body weight to 243.0 +/- 34.2 nmol/min.100 g body weight (P < 0.001), whereas BQ-788 decreased only V and UNaV. In renal denervated WKY, BQ-123 or BQ-788 did not alter V, UNaV, or UKV. In intact spontaneously hypertensive rats BQ-123 but not BQ-788 decreased V from 3.94 +/- 0.48 microL/min.100 g body weight to 2.55 +/- 0.44 microL/min.100 g body weight (P < 0.05). In renal denervated spontaneously hypertensive rats neither BQ-123 nor BQ-788 affected V, UNaV, or UKV. CONCLUSION: An interaction between ET and renal nerves is involved in the control of renal function. Moreover, renal nerves participate in the regulation of ET-1 production within the kidney. Finally, decreased synthesis of ET-1 in the renal papilla of spontaneously hypertensive rats may contribute to development and/or maintenance of hypertension due to modulation of renal excretory function.     

Prolonged elevation of plasma free fatty acids desensitizes the insulin secretory response to glucose in vivo in rats

Prolonged exposure of pancreatic islets to free fatty acids (FFAs) inhibits glucose-stimulated insulin secretion (GSIS) in vitro. However, FFA inhibition of GSIS has not been clearly demonstrated in vivo. We examined the in vivo effect of prolonged elevation of plasma FFAs on GSIS using a two-step hyperglycemic clamp in rats treated with a 48-h intravenous infusion of either 20% Intralipid plus heparin (INT) (5 microl/min plus heparin, 0.1 U/min; n = 8), oleate (OLE) (1.3 microEq/min; n = 6), saline (SAL) (n = 6), or bovine serum albumin (BSA) (vehicle for OLE; n = 5). Because there was no difference in any of the parameters between BSA and SAL rats, these groups were combined as control rats (CONT) (n = 11). At the end of the 48-h OLE/INT/CONT infusions, after an overnight fast, plasma glucose was clamped for 2 h at 13 mmol/l and for another 2 h at 22 mmol/l. Preclamp plasma FFAs were elevated twofold (P < 0.01) versus CONT with both INT and OLE (NS, INT vs. OLE). Preclamp glucose, insulin, and C-peptide levels were higher in INT than in CONT rats (P < 0.05), suggesting insulin resistance, but they were not different in OLE and CONT rats. The insulin and C-peptide responses to the rise in plasma glucose from basal to 13 mmol/l were lower in OLE (336 +/- 72 pmol/l and 1.2 +/- 0.1 nmol/l, P < 0.01 and P < 0.05, respectively) than in CONT (552 +/- 54 and 1.9 +/- 0.1) rats, but they were not different between CONT and INT rats (648 +/- 150 and 2.0 +/- 0.4). The insulin and C-peptide responses to the rise in plasma glucose from 13 to 22 mmol/l were lower in both INT (1,188 +/- 204 pmol/l and 3.0 +/- 0.3 nmol/l, P < 0.01 and P < 0.001) and OLE (432 +/- 60 and 1.7 +/- 0.2, P < 0.001 vs. CONT or INT) rats than in CONT rats (1,662 +/- 174 and 5.0 +/- 0.6). In summary, 1) both INT and OLE decreased GSIS in vivo in rats, and 2) the impairing effect of INT on GSIS was less than that of OLE, which might be due to the different type of fatty acid (mostly polyunsaturated in INT versus monounsaturated as OLE) and/or to differential effects of INT and OLE on insulin sensitivity. In conclusion, prolonged elevation of plasma FFAs can desensitize the insulin secretory response to glucose in vivo, thus inducing a beta-cell defect that is similar to that found in type 2 diabetes.     

Testicular oxidative stress. Effects of experimental varicocele in adolescent rats

OBJECTIVE: This present study was undertaken to determine the levels of malondialdehyde (MDA), nitric oxide (NO) and total antioxidant status (TAS) in testes of adolescent rats with experimental bilateral varicocele and to determine the effects of oxidative stress on testis produced by varicocele. METHODS: 6-week-old, male Wistar rats, weighing 146-334 g (228.37 +/- 41.34 g), were randomly allocated into two groups. The first group underwent selective and bilateral partial ligation of the spermatic vein (n = 28), and the second group underwent a sham operation and served as the controls (n = 15). Animals were sacrificed 12 weeks after surgery and dilatation of the spermatic veins was observed in the first study group. Bilateral orchiectomy was performed in all rats, and MDA, NO and TAS levels were measured. RESULTS: In the study group, the mean MDA (SEM) level was 15.58 +/- 6.07 micromol/g protein, and in the control group, it was 11.59 +/- 3.86 micromol/g protein, respectively; this difference was statistically significant (p < 0.05). The mean NO level was 82.73 +/- 77.84 nmol/g protein in the study group, whereas 28.65 +/- 20.18 nmol/g protein in the control group, this difference was also statistically significant (p < 0.005). The mean TAS levels of the study and control groups were 0.91 +/- 0.32 and 1.78 +/- 0.46 nmol/g tissue, respectively, and this difference was also statistically significant (p < 0.001). But there was no correlation between these three parameters (MDA<-->TAS: r = -0.103, p > 0.05; MDA<-->NO: r = -0.104, p > 0.05; NO<-->TAS: r = -0.123, p > 0.05). CONCLUSION: These findings suggest that varicocele may change the testicular oxidative status and may play a role in testicular dysfunction that causes infertility.     

Effect of supplemental dietary glutamine on methotrexate concentrations in tumors.

This study evaluated the effects of supplemental dietary glutamine (GLN) on methotrexate sodium concentrations in tumors and serum of sarcoma-bearing rats following the initiation of methotrexate. After randomization to a GLN diet (+GLN) or GLN-free diet (-GLN), tumor-bearing rats received 20 mg/kg of methotrexate sodium by intraperitoneal injection. The provision of supplemental GLN in the diet increased methotrexate concentrations in tumor tissues at 24 and 48 hours (38.0 +/- 0.20 nmol/g for the +GLN group vs 28.8 +/- 0.10 nmol/g for the -GLN group and 35.6 +/- 0.18 nmol/g for the +GLN group vs 32.5 +/- 0.16 nmol/g for the -GLN group, respectively). Arterial methotrexate levels were elevated only at 48 hours (0.147 +/- 0.007 microns/L for the +GLN group vs 0.120 +/- 0.006 microns/L for the -GLN group). Tumor morphometrics were not different between the groups but significantly greater tumor volume loss was seen even at 24 hours (-2.41 +/- 1.3 cm3 for the +GLN group vs -0.016 +/- 0.9 cm3 for the -GLN group). Tumor glutaminase activity was suppressed in both groups at 48 hours, but more so in the +GLN group (0.94 +/- 0.13 mumol/g per hour for the +GLN group vs 1.47 +/- 0.22 mumol/g per hour for the -GLN group). This study suggests that GLN may have therapeutic as well as nutritional benefit in oncology patients.     

Administration of ghrelin to young uraemic rats increases food intake transiently, stimulates growth hormone secretion and does not improve longitudinal growth.

BACKGROUND: Ghrelin administration stimulates appetite and growth hormone (GH) secretion. Whether these effects are preserved in young individuals with chronic renal failure (CRF) and their potential benefit on growth is questioned. METHODS: Three experiments were performed in subtotally nephrectomized young rats (Nx). (i) Food intake was monitored in CRF rats receiving saline intraperitoneally or a ghrelin dose (30 nmol) shown to increase food intake over 2 and 24 h in rats with normal renal function. (ii) Plasma levels of GH were measured after a dose of intravenous ghrelin (3 nmol) was given to three groups of five rats each: Nx, sham-operated fed ad libitum (SAL) and sham-operated pair-fed with Nx (SPF). (iii) Growth of Nx rats treated with intraperitoneal ghrelin (3 nmol) for 7 days (Nx-Ghr) was compared with that of SAL and Nx groups receiving saline (n=8-10 per group). RESULTS: In CRF rats, the dose of 30 nmol of ghrelin increased food consumption for 2 h (1.3+/-0.2 g vs 0.5+/-0.2 g, P<0.05) but not 24-h food intake (12.5+/-0.6 g vs 12.2+/-0.5 g). Ghrelin (3 nmol) increased plasma levels of GH, which were maximal 10 min after injection, no differences being observed among groups (SAL: 666.2+/-104.6 ng/ml; Nx: 691.6+/-90.7 ng/ml; SPF: 577.8+/-125.4 ng/ml). Return to basal GH levels was delayed in Nx. Ghrelin did not improve body length and weight gains, longitudinal bone growth rate or food intake in the Nx-Ghr group. CONCLUSIONS: In young uraemic rats, ghrelin increases appetite but not 24-h food intake, stimulates GH secretion and does not improve growth.     

Endotoxin and renal glutamine metabolism.

The effect of endotoxin on renal glutamine metabolism and ammoniagenesis was investigated in vivo in the rat to gain further insight into the altered glutamine flow that characterizes critical illness. Studies were done 15 hours following a single dose of Escherichia coli lipopolysaccharide (10 mg/kg). Renal blood flow and arterial glutamine concentration were similar in control and study rats, but the kidney switched from an organ of slight glutamine uptake in controls (129 +/- 52 nmol/100 g of body weight per minute) to net release in the endotoxin-treated animals (-273 +/- 170 nmol/100 g of body weight per minute). Simultaneously, the specific activity of renal glutamine synthetase increased by almost 50% (374 +/- 40 nmol/mg of protein per hour in rats given endotoxin vs 253 +/- 12 nmol/mg of protein per hour in controls), while glutaminase was unchanged. Urinary ammonia excretion was reduced by 35% in the endotoxin-treated animals (47 +/- 6 mumol/12 h in endotoxin-treated animals vs 70 +/- 8 mumol/12 h in controls) despite a 10% fall in the arterial bicarbonate value. Endotoxin alters the net flux of glutamine across the kidney which appears to be partially regulated enzymatically. This may impair the kidneys' ability to maintain acid/base homeostasis.     

Protective action of glycine in cisplatin nephrotoxicity

Because glycine is cytoprotective for kidney cells in vitro, we investigated its possible action in vivo to protect rats against cisplatin nephrotoxicity, a well-established experimental model of renal tubular injury. Glycine was infused at a dose of 1 mmol per 100 g body weight per hour for 75 minutes, starting 15 minutes before cisplatin, 5 mg per kg, was injected intravenously. Plasma concentration of glycine rose to 3.5 mmol per liter at the time cisplatin was injected. These rats were compared with cisplatin-treated animals treated with L-alanine or with isotonic saline. After five days plasma creatinine of saline-treated rats given cisplatin had risen threefold to 2.6 +/- 1.5 mg per 100 ml (mean +/- SD), as creatinine clearance fell to 25% of baseline (0.14 +/- 0.05 ml/min/100 g). Morphological evaluation disclosed extensive damage involving all S3 segments in the outer medulla as well as the medullary rays of the cortex. In contrast, in rats treated with glycine, plasma creatinine rose only to 1.2 +/- 0.2 mg/100 ml and creatinine clearance was maintained at 75% of baseline (0.35 +/- 0.05 ml/min/100 g). Glycine also attenuated the weight loss, polyuria, increased fractional excretion of sodium and potassium, decreased urinary osmolality, and renal glycosuria observed in control, saline-treated rats after cisplatin, while substantially decreasing the percentage of S3 tubules with evident morphological injury. Renal platinum content was unaffected by glycine. The administration of L-alanine or the delayed infusion of glycine, starting one hour after cisplatin was given, did not prevent cisplatin toxicity.(ABSTRACT TRUNCATED AT 250 WORDS)