Interactions of conjugated linoleic acid and lipoic acid on insulin action in the obese Zucker rat

The fatty acid conjugated linoleic acid (CLA) and the antioxidant R-(+)-alpha-lipoic acid (R-ALA) individually enhance glucose tolerance and insulin action on skeletal muscle glucose transport in the insulin-resistant obese Zucker rat. To date, no study has assessed the potential interactions between these 2 interventions in treating insulin resistance. The present study was designed to determine whether chronic treatment with CLA and R-ALA in combination would enhance skeletal muscle glucose transport to a greater extent than either intervention individually. CLA, R-ALA, or a combination treatment of R-ALA and CLA were administered to female obese Zucker rats for 20 days at low or high doses. Whereas low-dose R-ALA (10 mg/kg body weight) alone did not alter muscle glucose transport, low-dose CLA (0.3 g/kg) induced a significant increase (38%, P <.05) in insulin-mediated glucose transport in epitrochlearis, but not in soleus. Low-dose combination therapy brought about the greatest enhancement of insulin-mediated glucose transport in epitrochlearis (77%) and soleus (54%), with the latter effect being associated with a 50% reduction in protein carbonyls (an index of tissue oxidative stress) and a 33% diminution in muscle triglycerides. High-dose treatments with CLA (1.5 g/kg), R-ALA (50 mg/kg), and the combination of CLA and R-ALA elicited increases in insulin-mediated glucose transport in epitrochlearis (57%, 58%, and 77%) and soleus (32%, 35%, and 54%). However, whereas the individual high-dose treatments with CLA and R-ALA reduced protein carbonyls (63% and 49%) and triglycerides (29% and 28%) in soleus, no further reductions were observed with the high-dose combination treatment groups. These findings support a significant interaction between low doses of CLA and R-ALA for enhancement of insulin action on skeletal muscle glucose transport, possibly via reductions in muscle oxidative stress and in lipid storage.     

α-linolenic acid supplementation prevents exercise-induced improvements in white adipose tissue mitochondrial bioenergetics and whole-body glucose homeostasis in obese Zucker rats

Aims/hypothesis

While the underlying mechanisms in the development of insulin resistance remain inconclusive, metabolic dysfunction in both white adipose tissue (WAT) and skeletal muscle have been implicated in the process. Therefore, we investigated the independent and combined effects of α-linolenic acid (ALA) supplementation and exercise training on whole-body glucose homeostasis and mitochondrial bioenergetics within the WAT and skeletal muscle of obese Zucker rats.

Methods

We randomly assigned obese Zucker rats to receive a control diet alone or supplemented with ALA and to remain sedentary or undergo exercise training for 4 weeks (CON-Sed, ALA-Sed, CON-Ex and ALA-Ex groups). Whole-body glucose tolerance was determined in response to a glucose load. Mitochondrial content and bioenergetics were examined in skeletal muscle and epididymal WAT (eWAT). Insulin sensitivity and cellular stress were assessed by western blot.

Results

Exercise training independently improved whole-body glucose tolerance as well as insulin-induced signalling in muscle and WAT. However, the consumption of ALA during exercise training prevented exercise-mediated improvements in whole-body glucose tolerance. ALA consumption did not influence exercise-induced adaptations within skeletal muscle, insulin sensitivity and mitochondrial bioenergetics. In contrast, within eWAT, ALA supplementation attenuated insulin signalling, decreased mitochondrial respiration and increased the fraction of electron leak to reactive oxygen species (ROS).

Conclusions/interpretation

These findings indicate that, in an obese rodent model, consumption of ALA attenuates the favourable adaptive changes of exercise training within eWAT, which consequently impacts whole-body glucose homeostasis. The direct translation to humans, however, remains to be determined.

     

Effects of stevioside on glucose transport activity in insulin-sensitive and insulin-resistant rat skeletal muscle

Stevioside (SVS), a natural sweetener extracted from Stevia rebaudiana, has been used as an antihyperglycemic agent. However, little is known regarding its potential action on skeletal muscle, the major site of glucose disposal. Therefore, the purpose of the present study was to determine the effect of SVS treatment on skeletal muscle glucose transport activity in both insulin-sensitive lean (Fa/-) and insulin-resistant obese (fa/fa) Zucker rats. SVS was administered (500 mg/kg body weight by gavage) 2 hours before an oral glucose tolerance test (OGTT). Whereas the glucose incremental area under the curve (IAUC(glucose)) was not affected by SVS in lean Zucker rats, the insulin incremental area under the curve (IAUC(insulin)) and the glucose-insulin index (product of glucose and insulin IAUCs and inversely related to whole-body insulin sensitivity) were decreased (P<.05) by 42% and 45%, respectively. Interestingly, in the obese Zucker rat, SVS also reduced the IAUC(insulin) by 44%, and significantly decreased the IAUC(glucose) (30%) and the glucose-insulin index (57%). Muscle glucose transport was assessed following in vitro SVS treatment. In lean Zucker rats, basal glucose transport in type I soleus and type IIb epitrochlearis muscles was not altered by 0.01 to 0.1 mmol/L SVS. In contrast, 0.1 mmol/L SVS enhanced insulin-stimulated (2 mU/mL) glucose transport in both epitrochlearis (15%) and soleus (48%). At 0.5 mmol/L or higher, the SVS effect was reversed. Similarly, basal glucose transport in soleus and epitrochlearis muscles in obese Zucker rats was not changed by lower doses of SVS (0.01 to 0.1 mmol/L). However, these lower doses of SVS significantly increased insulin-stimulated glucose transport in both obese epitrochlearis and soleus (15% to 20%). In conclusion, acute oral SVS increased whole-body insulin sensitivity, and low concentrations of SVS (0.01 to 0.1 mmol/L) modestly improved in vitro insulin action on skeletal muscle glucose transport in both lean and obese Zucker rats. These results indicate that one potential site of action of SVS is the skeletal muscle glucose transport system.     

Nebivolol improves insulin sensitivity in the TGR(Ren2)27 rat

Hypertension is often associated with increased oxidative stress and systemic insulin resistance. Use of β-adrenergic receptor blockers in hypertension is limited because of potential negative influence on insulin sensitivity and glucose homeostasis. We sought to determine the impact of nebivolol, a selective vasodilatory β₁-adrenergic blocker, on whole-body insulin sensitivity, skeletal muscle oxidative stress, insulin signaling, and glucose transport in the transgenic TG(mRen2)27 rat (Ren2). This rodent model manifests increased tissue renin angiotensin expression, excess oxidative stress, and whole-body insulin resistance. Young (age, 6-9 weeks) Ren2 and age-matched Sprague-Dawley control rats were treated with nebivolol 10 mg/(kg d) or placebo for 21 days. Basal measurements were obtained for glucose and insulin to calculate the homeostasis model assessment. In addition, insulin metabolic signaling, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity, reactive oxygen species, and ultrastructural changes as evaluated by transmission electron microscopy were examined ex vivo in skeletal muscle tissue. The Ren2 rat demonstrated systemic insulin resistance as examined by the homeostasis model assessment, along with impaired insulin metabolic signaling in skeletal muscle. This was associated with increased oxidative stress and mitochondrial remodeling. Treatment with nebivolol was associated with improvement in insulin resistance and decreased NADPH oxidase activity/levels of reactive oxygen species in skeletal muscle tissue. Nebivolol treatment for 3 weeks reduces NADPH oxidase activity and improves systemic insulin resistance in concert with reduced oxidative stress in skeletal muscle in a young rodent model of hypertension, insulin resistance, and enhanced tissue RAS expression.     

Exercise training increases components of the c-Cbl-associated protein/c-Cbl signaling cascade in muscle of obese Zucker rats

The purpose of this investigation was to determine whether alterations in the c-Cbl-associated protein/c-Cbl pathway and/or p38-mitogen-activated protein kinase (p38 MAP kinase) were associated with improved skeletal muscle insulin responsiveness in exercise-trained obese Zucker rats. Obese Zucker rats ran 5 d/wk on a motorized treadmill for 90 minutes over a 7-week period. Age-matched obese Zucker rats (OB-SED) and their lean littermates (LN-SED) were obtained to serve as nontrained controls. Twenty-four (OB-EX-24 h) or 48 hours (OB-EX-48 h) after the last exercise bout, the trained rats were studied via the hind limb perfusion technique in the presence of insulin. Insulin-stimulated glucose uptake was significantly decreased across the skeletal muscle of OB-SED rats compared with LN-SED, but was normalized in the obese rats by 7 weeks of training. The insulin-stimulated plasma membrane protein concentrations of TC10 and glucose transporter 4 were reduced in the sedentary Zuckers, but both proteins were increased by the training protocol. Training did not increase insulin-stimulated p38 MAP kinase protein concentration, nor did it have an effect on insulin-stimulated p38 MAP kinase phosphorylation at the plasma membrane. These results suggest that skeletal muscle insulin resistance is associated with reduced expression of TC10 and that this deficiency can be corrected with exercise training.     

Effects of in vitro antagonism of endocannabinoid‐1 receptors on the glucose transport system in normal and insulin‐resistant rat skeletal muscle

Objective: We determined the direct effects of modulating the endocannabinoid‐1 (CB1) receptor on the glucose transport system in isolated skeletal muscle from insulin‐sensitive lean Zucker and insulin‐resistant obese Zucker rats. Methods: Soleus strips were incubated in the absence or presence of insulin, without or with various concentrations of the CB1 receptor antagonist SR141716 or with the CB1 receptor agonist arachidonyl‐2‐chloroethylamide (ACEA). Results: CB1 receptor protein expression in visceral adipose (57%), soleus (40%) and myocardial (36%) tissue was significantly (p < 0.05) decreased in obese compared to lean animals, with a trend for a reduction (17%, p = 0.079) in the liver. In isolated soleus muscle from both lean and obese Zucker rats, CB1 receptor antagonism directly improved glucose transport activity in a dose‐dependent manner. Basal glucose transport activity was maximally enhanced between 100 and 200 nM SR141716 in lean (26–28%) and obese (22–31%) soleus. The maximal increase in insulin‐stimulated glucose transport for lean muscle (～30%) was achieved at 50 nM SR141716 and for obese muscle (～30%) at 100 nM SR141716. In contrast, CB1 receptor antagonism did not alter hypoxia‐stimulated glucose transport activity. CB1 receptor agonism (1 mM ACEA) significantly decreased both basal (15%) and insulin‐stimulated (22%) glucose transport activity in isolated lean soleus. This effect was reversed by 200 nM SR141716. In both lean and obese muscle, the functionality of key signalling proteins (insulin receptor β‐subunit, Akt, glycogen synthase kinase‐3β (GSK‐3β), AMP‐dependent protein kinase (AMPK), p38 mitogen‐activated protein kinase (p38 MAPK)) was not altered by either CB1 receptor agonism or antagonism. Conclusion: These results indicate that the engagement of CB1 receptor can negatively modulate both basal and insulin‐dependent glucose transport activity in lean and obese skeletal muscles, and that these effects are not mediated by the engagement of elements of the canonical pathways regulating this process in mammalian skeletal muscle.     

过氧钒烟酸络合物（POR）逆转Zucker肥胖大鼠胰岛素抵抗的作用

目的：探讨过氧钒烟酸络合物（POR）对胰岛素抵抗的逆转作用,POR促进葡萄糖摄取与胰岛素促进作用在机制上的异同,方法方法：以皮下渗透式输注POR,观察POR对Zucker大鼠胰岛素抵抗的缓解作用,检测POR对具有胰岛素严重抵抗的Zucker肥胖大鼠睾丸脂肪垫脂肪细胞对葡萄糖提取的促进作用,以及对PL－3K P110亚单位酷氨酸磷酸化的影响,比较POR和胰岛素促进GLUT4的转位对PI－3K的依赖性,结果：POR可以使肥胖伴胰岛素抵抗大鼠的空腹血糖明显降低,对糖负荷的处理能力明显提高,GTT曲线下胰岛素的水平明显降低,提示对胰岛素的的敏感性提高,葡萄糖载体的转位水平与PI－3K活性并不平行,胰岛素促进的葡萄糖摄取作用对PI－3K有依赖性,过氧钒络合物（POR）对葡萄糖载体转位的促进作用不依赖于PI－3K,表现为POR促进的葡萄糖摄取下被PI－3K抑制剂（Wortmannin)抑制,结论：POR在1／10钒酸盐剂量下能逆转Zucker大鼠的胰岛素抵抗,胰岛素对葡萄糖载体的激活作用是PI－3K依赖性的,而过氧钒（POR）则可通过其他途径激活葡萄糖载体,具体机制可能与载体的直接激活有关。     

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.     

Expression of the adiponectin receptors AdipoR1 and AdipoR2 in lean rats and in obese Zucker rats

The adiponectin receptors, AdipoR1 and AdipoR2, are thought to transmit the insulin-sensitizing effects of adiponectin, an adipokine secreted by adipocytes. Modifications of their expression in insulin-sensitive tissues (skeletal muscle, liver, and adipose tissue) could therefore play a role in the control of insulin sensitivity and the development of insulin resistance. Recent data in mice supported this possibility. We examined whether the expression of adiponectin receptors (messenger RNA [mRNA] concentrations) is controlled in vivo in rats (Wistar) by nutritional factors (high-fat [HF] vs high-carbohydrate diet, fasting vs fed state) and whether this expression is decreased in an experimental model of insulin resistance, the obese Zucker rat. In Wistar rats, neither an HF diet nor fasting modified the mRNA concentrations of AdipoR1 in muscle, liver, or adipose tissue; the only modification observed was a decrease (P < .05) in AdipoR2 mRNA level in the liver of rats fed with an HF diet. In obese Zucker rats compared with their lean controls, neither AdipoR1 nor AdipoR2 expression was modified in muscle. AdipoR2 expression was slightly decreased in adipose tissue, whereas the expression of both AdipoR1 and AdipoR2 was increased (P < .05) in the liver of obese Zucker rats. In conclusion, contrary to what was reported in mice, the expression of adiponectin receptors in rats is poorly responsive to changes in nutritional conditions and is not decreased in a model of insulin resistance. These results do not support an important role for the expression of AdipoR1 and AdipoR2 in the modulation of sensitivity to insulin.     

Insulin Resistance Impairs Endothelial Function but not Adrenergic Reactivity or Vascular Structure in Fructose‐fed Rats

Obesity and diabetes are major risk factors for the development of vascular disease in the lower limbs. Previous studies have demonstrated reduced nitric oxide (NO)‐mediated vasodilation, increased adrenergic constriction, and inward, atrophic remodeling in the limb circulation of obese Zucker rats, but the component of the “metabolic syndrome” driving these changes is unclear. Because insulin resistance precedes the state of frank diabetes, the current study hypothesized that insulin resistance independent of obesity induced by fructose feeding would impair microvascular function in the skeletal muscle circulation in lean Zucker rats (LZR). A 66% fructose diet impaired glucose tolerance and induced moderate insulin resistance with no changes in whole‐body hemodynamics of anesthetized rats (FF‐LZR), compared to control LZR. NO‐mediated vasodilation of isolated gracilis arteries, assessed in vitro with acetylcholine and sodium nitroprusside, was reduced ～20% in FF‐LZR vs. LZR. NO‐independent cGMP‐mediated vasodilation was unimpaired. Pretreatment of isolated vessels with the superoxide scavenger, tempol, improved responses to both vasodilators. Reactivity to adrenergic stimulation was unaltered in FF‐LZR vs. LZR, although constriction to endothelin was increased. Structural and passive mechanical characteristics of isolated gracilis arteries were similar in both LZR and FF‐LZR. Taken together, these findings indicate that moderate insulin resistance is sufficient to impair endothelial function in an oxidant‐dependent manner in the rat hindlimb circulation. Other aspects of skeletal muscle vascular function documented in obese models, specifically adrenergic tone and inward remodeling, must reflect either severe insulin resistance or other aspects of obesity. The factors accounting for nonendothelial vasculopathies remain unknown.     

Intracellular fatty acid metabolism in skeletal muscle and insulin resistance

Triglyceride accumulation in skeletal muscle is increased in subjects with insulin resistance. Increased intracellular lipolysis from stored triglyceride may induce insulin resistance in skeletal muscle by activating the glucose-fatty acid cycle. However, inconsistent with this hypothesis, intracellular lipolysis from skeletal muscle is decreased in high fat-fed, insulin resistant rats. Therefore, it is suggested that an increase in triglyceride accumulation is the result of decreased mitochondrial fatty acid oxidation in the cells. As evidence, fenofibrate (a PPARalpha activator), rosiglitazone (a PPARgamma activator) and alpha-lipoic acid completely prevented the development of diabetes in obese diabetes-prone rats. All three drugs increased fatty acid oxidation and decreased triglyceride accumulation in skeletal muscle. Administration of ALA activated AMPK and increased fatty acid oxidation. It is suggested that decreased fatty acid oxidation in skeletal muscle is one of the major factors leading to an accumulation of lipid metabolites and insulin resistance.     

Exercise training improves insulin sensitivity in the obese Zucker rat

The effect of exercise on in vivo insulin sensitivity was examined in lean and obese Zucker rats. Rats (6 to 7 weeks of age) were swum two hours per day or kept sedentary for 8 weeks. Exercise decreased body weight gain as well as percent of fat in both genotypes. Sedentary obese rats had 62% higher gastrocnemius citrate synthase activity per gram of muscle than did lean rats. Exercise increased activity of this oxidative enzyme similarly in both genotypes. Compared to lean rats, obese rats had higher plasma-insulin levels and were less sensitive to insulin during an insulin tolerance test. Although training had no effect on plasma-insulin levels, exercise trained obese rats showed a greater drop in plasma glucose relative to sedentary controls following intravenous injection of three concentrations of insulin. It was concluded that moderate exercise training improved the insulin sensitivity of the obese Zucker rat.     

α-硫辛酸减轻糖尿病大鼠肾脏损伤的机制

研究α-硫辛酸对链脲佐菌素致糖尿病大鼠肾脏损伤的作用和机制.α-硫辛酸减轻糖尿病大鼠肾皮质氧化应激水平,降低糖基化终末产物[(0.087±0.003对0.103±0.014)pg/mg蛋白,P<0.05]及其受体蛋白(1.81±0.04对2.67±0.01,P<0.01)和转化生长因子β mRNA(1.51±0.20对2.04+0.08,P<0.05)表达,降低肾小球系膜区面积和Ⅳ型胶原水平,改善肾功能.     

Selective angiotensin II receptor antagonism reduces insulin resistance in obese Zucker rats

Effects of oral administration of the angiotensin II receptor antagonist (selective AT(1)-subtype) irbesartan on glucose tolerance and insulin action on skeletal-muscle glucose transport were assessed in the insulin-resistant obese Zucker rat. In the acute study, obese rats received either vehicle (water) or irbesartan 1 hour before the experiment. Although irbesartan had no effect on glucose transport (2-deoxyglucose uptake) in the epitrochlearis muscle, which consists mainly of type IIb fibers, acute angiotensin II receptor antagonism led to a dose-dependent increase in insulin action in the predominantly type I soleus muscle. Irbesartan at 25 and 50 mg/kg induced significant increases (41% and 50%, respectively; P<0.05) in insulin-mediated glucose transport. Moreover, these acute irbesartan-induced improvements in soleus-muscle glucose transport were associated with enhancements in whole-body insulin sensitivity (r=-0.732; P<0.05), as assessed during an oral glucose tolerance test. After chronic administration of irbesartan (21 days at 50 mg. kg(-1). d(-1)), glucose tolerance was enhanced further, and insulin-mediated glucose transport was significantly elevated in both epitrochlearis (32%) and soleus (73%) muscle. Chronic angiotensin II receptor antagonism was associated with significant increases in glucose transporter-4 (GLUT-4) protein expression in soleus (22%) and plantaris (20%) muscle and myocardium (15%). Chronic irbesartan-induced increases in whole-body insulin sensitivity were associated with increased insulin-mediated glucose transport in both epitrochlearis (r=-0.677; P<0.05) and soleus (r=-0.892; P<0.05) muscle. In summary, angiotensin II receptor (AT(1)-subtype) antagonism, either acutely or chronically, improves glucose tolerance, at least in part because of an enhancement in skeletal-muscle glucose transport, and the effect of chronic angiotensin II receptor antagonism on type I skeletal-muscle glucose uptake is associated with an increase in GLUT-4 protein expression.     

Glucose uptake and perfusion in subcutaneous and visceral adipose tissue during insulin stimulation in nonobese and obese humans

To elucidate the role of adipose tissue glucose uptake in whole-body metabolism, sc and visceral adipose tissue glucose uptake and perfusion were measured in 10 nonobese and 10 age-matched obese men with positron emission tomography using [(18)F]-2-fluoro-2-deoxy-D-glucose, and [(15)O]-labeled water during normoglycemic hyperinsulinemia. Whole-body and skeletal muscle glucose uptake rates per kilogram were lower in obese than in nonobese subjects (P < 0.01). Compared with nonobese, the obese subjects had 67% lower abdominal sc and 58% lower visceral adipose tissue glucose uptake per kilogram of fat. In both groups, insulin stimulated glucose uptake per kilogram fat was significantly higher in visceral fat depots than in sc regions (P < 0.01). Both sc and visceral adipose tissue blood flow expressed per kilogram and minute was impaired in the obese subjects, compared with the nonobese (P < 0.05). Fat masses measured with magnetic resonance images were higher in obese than in nonobese individuals. If regional glucose uptake rates were expressed as per total fat mass, total glucose uptake rates per depot were similar in obese and nonobese subjects and represented 4.1% of whole-body glucose uptake in obese and 2.6% in nonobese subjects (P < 0.02 between the groups). In conclusion, insulin-stimulated glucose uptake per kilogram fat is higher in visceral than in sc adipose tissue. Glucose uptake and blood flow in adipose tissue exhibit insulin resistance in obesity, but because of the larger fat mass, adipose tissue does not seem to contribute substantially to the reduced insulin stimulated whole-body glucose uptake in obesity.     

Glycation and insulin resistance: novel mechanisms and unique targets?

Multiple biochemical, metabolic, and signal transduction pathways contribute to insulin resistance. In this review, we present evidence that the posttranslational process of protein glycation may play a role in insulin resistance. The posttranslational modifications, the advanced glycation end products (AGEs), are formed and accumulated by endogenous and exogenous mechanisms. AGEs may contribute to insulin resistance by a variety of mechanisms, including generation of tumor necrosis factor-α direct modification of the insulin molecule, thereby leading to its impaired action, generation of oxidative stress, and impairment of mitochondrial function, as examples. AGEs may stimulate signal transduction via engagement of cellular receptors, such as receptor for AGEs. AGE-receptor for AGE interaction perpetuates AGE formation and cellular stress via induction of inflammation, oxidative stress, and reduction in the expression and activity of the enzyme glyoxalase I that detoxifies the AGE precursor, methylglyoxal. Once set in motion, glycation-promoting mechanisms may stimulate ongoing AGE production and target tissue stresses that reduce insulin responsiveness. Strategies to limit AGE accumulation and action may contribute to the prevention of insulin resistance and its consequences.     

Abnormal insulin and β-adrenergic modulation of lipoprotein lipase during refeeding after prolonged fasting in the Zucker rat

Aims/hypothesis. To characterise the response of tissue lipoprotein lipase to refeeding after prolonged (24 h) fasting in lean and obese Zucker rats, and to verify whether lipoprotein lipase in obese rats is resistant to the short-term action of insulin and escapes modulation by the β-adrenergic pathway.¶Methods. Lean Fa/? and obese fa/fa male Zucker rats fasted for 24 h and refed at will. Lipoprotein lipase activity in adipose and muscle tissues was assessed in the freely fed and fasted states and at various times during refeeding, with or without β-adrenergic blockade (propranolol).¶Results. The 24-h fast erased the phenotype-related differences in insulinaemia and adipose lipoprotein lipase activity present in freely fed rats. Adipose lipoprotein lipase increased twofold in obese rats 1 h after refeeding, whereas no change occurred at that time in lean rats. Activity remained at that level for at least 6 h after refeeding in obese rats, whereas in lean animals it was increased fivefold after 6 h of refeeding. In muscle of obese rats, lipoprotein lipase decreased in response to refeeding, but paradoxically increased twofold in lean animals. Giving propranolol to lean rats before refeeding abolished the atypical response of muscle lipoprotein lipase to food intake and restored the early (1 h after refeeding) increase in adipose lipoprotein lipase but had no effect in obese rats.¶Conclusion/interpretation. Refeeding after prolonged fasting activates the β-adrenergic pathway in lean rats, which transiently counteracts insulin-mediated modulation of lipoprotein lipase. The β-adrenergic pathway is not activated by refeeding in adipose tissue and muscle of the obese Zucker rat. In the obese Zucker rat, the early modulation of adipose lipoprotein lipase activity is abnormal upon refeeding after prolonged fasting, suggesting short-term resistance to the action of insulin. [Diabetologia (2000) 43: 866–874]     

Insulin in Combination with Vanadate Stimulates Glucose Transport in Isolated Cardiomyocytes from Obese Zucker Rats

Insulin stimulates glucose uptake in muscle cells via activation of protein kinase B (PKB). The protein tyrosine phosphatase (PTP) inhibitor vanadate, is a known insulin mimetic agent but the mechanism whereby vanadate exerts its effect is not clearly understood. Vanadate also has beneficial effects in the diabetic myocardium. The aim of this study was to correlate insulin stimulation of glucose uptake and PKB activation with that induced by vanadate in adult ventricular myocytes from lean and obese Zucker fa/fa rats. In lean Zucker rats, 100 nM insulin and 5 mM vanadate stimulated myocardial 2-deoxy-D-[³H]glucose (2-DG) uptake from 27.17 ± 1.72 to 96.52 ± 10.87 and 43.86 ± 4.02 pmole/mg protein p/30 min respectively while a combination of insulin and vanadate could not improve the maximal response of insulin. In obese Zucker hearts, basal as well as insulin and vanadate stimulated glucose uptake were severely impaired (15.49 ± 1.44 vs 25.51 ± 3.11 and 20.11 ± 1.68 pmole/mg protein/30 min respectively). A combination of insulin and vanadate, resulted in a response significantly improved from the maximal response of insulin. This stimulation of 2-DG uptake was, in all instances, blocked by the PI 3-kinase inhibitors wortmannin and LY 294002.Insulin could not activate PKB, as measured by the Ser⁴⁷³ phosphorylated form of the enzyme, in the obese Zucker rats to the same extent as in lean controls. Similar to glucose uptake, activation of PKB by vanadate plus insulin was significantly more than that accomplished by insulin alone in obese rats. Both insulin and vanadate activation of PKB was prevented by wortmannin and LY 294002. Thus, the present study demonstrates that: (i) in cardiomyocytes from lean and obese Zucker rats, both insulin and vanadate stimulate glucose uptake and PKB activation through a PI-3-kinase sensitive pathway. (ii) In obese Zucker rats, neither insulin nor vanadate could induce glucose uptake or activation of PKB to the same extent as in lean controls. (iii) A combination of insulin with vanadate may be beneficial to increase glucose uptake in diabetic hearts, as this gives a better response than insulin alone.     

The effect of insulin and exercise on c-Cbl protein abundance and phosphorylation in insulin-resistant skeletal muscle in lean and obese Zucker rats

Aims/hypothesis Recruitment of the protein c-Cbl to the insulin receptor (IR) and its tyrosine phosphorylation via a pathway that is independent from phosphatidylinositol 3-kinase is necessary for insulin-stimulated GLUT4 translocation in 3T3-L1 adipocytes. The activation of this pathway by insulin or exercise has yet to be reported in skeletal muscle.Methods Lean and obese Zucker rats were randomly assigned to one of three treatment groups: (i) control, (ii) insulin-stimulated or (iii) acute, exhaustive exercise. Hind limb skeletal muscle was removed and the phosphorylation state of IR, Akt and c-Cbl measured.Results Insulin receptor phosphorylation was increased 12-fold after insulin stimulation (p<0.0001) in lean rats and threefold in obese rats. Acute exercise had no effect on IR tyrosine phosphorylation. Similar results were found for serine phosphorylation of Akt. Exercise did not alter c-Cbl tyrosine phosphorylation in skeletal muscle of lean or obese rats. However, in contrast to previous studies in adipocytes, c-Cbl tyrosine phosphorylation was reduced after insulin treatment (p<0.001).Conclusions/interpretation We also found that c-Cbl associating protein expression is relatively low in skeletal muscle of Zucker rats compared to 3T3-L1 adipocytes and this could account for the reduced c-Cbl tyrosine phosphorylation after insulin treatment. Interestingly, basal levels of c-Cbl tyrosine phosphorylation were higher in skeletal muscle from insulin-resistant Zucker rats (p<0.05), but the physiological relevance is not clear. We conclude that the regulation of c-Cbl phosphorylation in skeletal muscle differs from that previously reported in adipocytes.Abbreviations IR insulin receptor - PI 3-kinase phosphatidylinositol 3-kinase - CHO-IR Chinese hamster ovary cells expressing 3×10⁶ human insulin receptors per cell - CAP c-Cbl associating protein - IRS insulin receptor substrate