Helen Chang

Additive Hypoglycemic Effects of Drugs That Modify Free-Fatty Acid Metabolism by Different Mechanisms in Rats With Streptozocin-Induced Diabetes

In this study the effect of two drugs [etomoxir and nicotinic acid (NA)] on plasma glucose, free–fatty acid (FFA), and triglyceride (TG) concentrations was determined in rats with streptozocin (STZ)-induced diabetes. The two compounds modify FFA metabolism by different mechanisms, etomoxir (ethyl-2-[6-(4-cholorophenoxyl)-hexyl]oxirane-2-carboxylate) by inhibiting hepatic fatty acid oxidation, and NA by inhibiting lipolysis in adipose tissue. Diabetes was induced in male Sprague-Dawley rats, weighing ∼ 400 g, by STZ injection (30 mg/kg i.v.), and the metabolic effects of the two drugs were studied 7–10 days later. The acute administration of either etomoxir or NA lowered plasma glucose concentrations in diabetic rats by ∼150 mg/dl (P < .001) in 4 h. However, the two drugs differed dramatically in their effects on plasma FFA and TG concentrations. Specifically, etomoxir produced striking increases in plasma FFA and TG concentrations, whereas NA administration caused a marked decrease. However, when NA was given in conjunction with etomoxir, NA prevented the increase in plasma FFA and TG concentration seen with etomoxir; the combination of NA and etomoxir approximately doubled the decrease in plasma glucose concentration produced by NA or etomoxir when given alone. Because plasma insulin concentrations did not change in response to either drug, whether administered singly or in combination, these metabolic effects do not result from a change in insulin secretion. These results suggest that modulation of FFA metabolism at the level of the adipocyte or the liver can have dramatic effects on carbohydrate and lipid metabolism. Also, NA prevented the potentially adverse effects that etomoxir had on FFA and TG. The observation that the two drugs given in combination were capable of lowering plasma glucose from <400 mg/dl to essentially normal levels within 4 h raises the possibility that this pharmacologic approach may have relevance to the treatment of diabetes.

Resistance to insulin-stimulated glucose uptake in adipocytes isolated from spontaneously hypertensive rats.

The ability of insulin to stimulate glucose uptake and inhibit catecholamine-induced lipolysis was measured in adipocytes of similar size isolated from SHR and WKY rats. The results indicate that glucose transport was decreased in adipocytes from SHR rats; both basal (19 ± 2 vs. 32 ± 2 fmol · cell−1 · s−1 P < .001) and maximal (207 ± 30 vs. 373 ± 20 fmol · cell−1 · s−1 P < .01) insulin-stimulated glucose transport were lower in SHR than in WKY rats. In addition, the EC50 of insulin-stimulated glucose uptake was higher (921 ± 82 vs. 557 ± 69 pM insulin, P < .05) in adipocytes from SHR rats than from WKY rats. The ability of phenylisopropyladenosine (PIA) to modulate basal and maximal insulin-stimulated glucose uptake was compared in adipocytes from SHR and WKY rats. These results also demonstrated that glucose uptake was decreased in adipocytes from SHR rats and that PIA similarly enhanced both basal and maximal insulin-stimulated glucose uptake in adipocytes from both groups. Although maximal isoproterenol-stimulated lipolysis was decreased in adipocytes from SHR rats, the ability of insulin to inhibit catecholamine-stimulated lipolysis was at least as great in adipocytes from SHR as from WKY rats. Despite the decrease in insulin-stimulated glucose transport in isolated adipocytes from SHR rats, total number of insulin receptors, their affinity for insulin, and the ability of insulin to stimulate receptor-associated tyrosine kinase activity were similar in adipocytes from SHR and WKY rats. These results demonstrate a defect in both basal and insulin-stimulated glucose uptake and a loss of sensitivity of the adipocyte glucose-transport system to insulin in adipocytes from SHR rats and suggest that the site of the abnormality is distal to the insulin receptor.

Abnormalities of carbohydrate and lipid metabolism in Dahl rats.

Plasma glucose, insulin, and triglyceride concentration, blood pressure, and insulin action on isolated adipocytes were determined in weight-matched Sprague-Dawley, Dahl salt-resistant, and Dahl salt-sensitive rats. Blood pressure and plasma glucose concentrations were not significantly different in the three groups. However, Dahl salt-sensitive rats had significantly higher plasma insulin (39±2 microunits/ml) and triglyceride (213±11 mg/dl) concentrations than did Sprague-Dawley rats (27±2 microunits/ml and 101±6 mg/dl, respectively). Values for insulin (34±4 microunits/ml) and triglyceride (159 ±11 mg/dl) were intermediate in Dahl salt-resistant rats. In contrast, maximal insulin-stimulated glucose transport was significantly lower in adipocytes isolated from Dahl salt-sensitive as compared with Sprague-Dawley rats (400 ±16 versus 523 ±14 ft/cell/sec), with Dahl salt-resistant rats again having intermediate values. However, the ability of insulin to maximally inhibit catecholamine-stimulated lipolysis was similar in all three groups, averaging –20% of the activity present in the absence of insulin. All of these differences were seen when the rats were eating conventional chow and did not change in Dahl rats after 2 weeks of an 8% NaCl diet On the other hand, the predicted rise in blood pressure took place in Dahl salt-sensitive rats, increasing from 147±4 to 181 ±6 mm Hg. These data indicate that Dahl rats have higher values for plasma triglyceride and insulin concentration than control Sprague-Dawley rats, associated with a defect in insulin-stimulated glucose uptake by isolated adipocytes. These metabolic changes are not dependent on Dahl rats eating a high salt diet and do not vary as a function of salt intake.

Inhibition of lipolysis by adenosine is potentiated with age.

The ability of a variety of hormones to activate cells declines with age. We have investigated the mechanism for the reduced ability of beta adrenergic stimulation to activate lipolysis in fat cells from older rats. Previously, we have found that these cells have an intact lipolytic response to a cAMP analogue but diminished cAMP accumulation after isoproterenol stimulation, suggesting that the blunted cAMP response is rate limiting. In the present study we have tested the hypothesis that enhanced inhibition of lipolysis by endogenously released adenosine accounts for the diminished lipolysis. Adenosine deaminase was added to media containing the adipocytes from older rats to remove endogenous adenosine. Under these conditions beta adrenergic stimulation of lipolysis is intact in fat cells from older rats. The adenosine analogue N6-phenylisopropyladenosine more effectively inhibited lipolysis in the older group (77 +/- 6%) than in the younger group (46 +/- 5%), suggesting that enhanced efficacy of endogenous adenosine may account for the reduced lipolytic response to catecholamines. When pertussis vaccine was used to functionally inactivate adenosine receptors in adipocytes from the younger and older rats, the ability of isoproterenol to activate lipolysis was restored in the older group. All the data are consistent with the hypothesis that enhanced inhibitory effects of adenosine explain the diminished ability of beta adrenergic agonists to activate lipolysis. It is possible that enhanced inhibitory pathways may be involved in blunting responses to stimulatory hormones in other tissues from older animals.

Desensitization of adenosine receptor-mediated inhibition of lipolysis. The mechanism involves the development of enhanced cyclic adenosine monophosphate accumulation in tolerant adipocytes.

Adipocytes contain adenosine receptors, termed A1 receptors, which inhibit lipolysis by decreasing adenylate cyclase activity. The inhibition of lipolysis by adenosine agonists in vivo acutely suppresses the plasma concentrations of free fatty acids (FFA) and triglycerides. We have found that infusions of the adenosine receptor agonist phenylisopropyladenosine (PIA) initially decreases plasma FFA concentrations; however, with prolonged exposure (6 d), rats become very tolerant to the effects of the drug. Adipocytes isolated from epididymal fat pads from PIA-infused rats have altered lipolytic responses. When lipolysis is stimulated with a relatively high concentration of isoproterenol (10(-7) M), PIA does not inhibit lipolysis in adipocytes from the infused animals. However, PIA inhibits isoproterenol-stimulated cyclic AMP (cAMP) accumulation in adipocytes from the infused rats although with decreased sensitivity compared with controls. The explanation for the impaired antilipolytic effect appears to be due to the fact that isoproterenol-stimulated cAMP accumulation is markedly increased in cells from infused rats. Indeed, basal lipolysis and lipolysis stimulated with lower concentrations of isoproterenol (10(-9), 10(-8) M) are effectively inhibited by PIA. cAMP accumulation is greatly increased in adipocytes from infused rats when stimulated by isoproterenol, ACTH, and forskolin. The results have some striking analogies to changes induced in nerve cells by prolonged exposure to narcotics. These data suggest that tolerance to PIA develops in adipocytes as a consequence of enhanced cAMP accumulation.

Disparate effects of prazosin and propranolol on lipid metabolism in a rat model

The effects of 14 days of daily intraperitoneal injections of prazosin hydrochloride (3.0 or 0.3 mg/kg) or propranolol (5 mg/kg) on various aspects of the lipid metabolism of normal rats was studied. The results indicate that plasma triglyceride and cholesterol concentrations were lower and the ratio of plasma HDL-cholesterol to total cholesterol concentration higher in prazosin-treated rats compared with propranolol-treated rats. The decline in plasma triglyceride levels in prazosin-treated rats was associated with a commensurate reduction in triglyceride secretion rates. Plasma free fatty acid levels were also lower in prazosin-treated rats, and this change may have contributed to the fall in triglyceride secretion rates. These data demonstrate that the disparate effects of alpha and beta receptor antagonists, previously shown to occur in hypertensive humans, can be duplicated in normal rats.

Effect of Exercise Training and Sucrose Feeding on Insulin-stimulated Glucose Uptake in Rats with Streptozotocin-induced Insulin-deficient Diabetes

The effect of exercise training and a sucrose-rich diet on insulin-stimulated glucose disposal was studied in rats with streptozotocin-induced insulin deficiency. Rats were injected with streptozotocin (40 mg/kg), and 3 days later divided into three groups with equal degrees of hyperglycemia. One group of rats was allowed to run spontaneously on exercise wheels, another group remained sedentary but ate a sucrose-rich diet (66% sucrose), and the third also remained sedentary but consumed conventional rat chow. Three weeks later, we determined the effect of these various programs on postabsorptive plasma glucose and insulin levels, as well as on the ability of exogenous insulin to stimulate disposal of a glucose load during a period in which endogenous insulin was suppressed by epinephrine and propanolol. Basal plasma insulin levels were the same in all three groups, but plasma glucose levels were significantly lower (P < 0.001) in exercise-trained rats, and significantly higher (P < 0.05) in sucrose-fed rats, than in chow-fed diabetic rats. The inference that exercise training markedly enhanced insulin action in rats with insulin deficiency was borne out by direct estimation of insulin-stimulated glucose disposal. In contrast, sucrose-fed diabetic rats seemed to be more insulin-resistant than chow-fed diabetic rats. These results provide direct evidence that spontaneous exercise can dramatically attenuate the severity of diabetes in insulin-deficient rats by enhancing insulin action.

Age-related Changes in Rat Muscle Glycogen Synthase Activity

Abstract This study was designed to evaluate effects of aging on glycogen synthase activity in rat skeletal muscle. Total enzyme activity was shown to be significantly, (p < .001) lower in tensor fascia latae, biceps femoris, and soleus muscle obtained from 24-month-old compared with 2-month-old rats. Similarly, values for the active form of enzyme were significantly lower, (p < .001) in all three muscle types of 24-month-old compared with 2-month-old rats. This age-related decline in glycogen synthase activity was not due to a reduction in the affinity of the enzyme for its activator (glucose-6-phosphate) and was independent of the concentration of substrate (UDP-glucose) in the assay system. Because similar age-related changes were seen when enzyme activity was expressed per milligram of muscle protein or per gram of muscle tissue, the fall in enzyme activity was not a simple function of an age-related decline in muscle mass. Glycogen levels also were reduced significantly in tensor fascia latae, biceps femoris, and soleus of 24-monthold rats compared with 2-month-old rats, p < .001. These results document an age-related change in a key enzyme regulating glycogen metabolism in muscle.

Tissue-dependent activation of protein kinase C in fructose-induced insulin resistance

Rats fed a fructose-enriched diet develop increases in blood pressure and resistance to insulin-mediated glucose disposal, but the underlying biochemical alterations have not been clearly defined. Since protein kinase C (PKC) has been implicated in the pathogenesis of insulin resistance, as well as blood pressure (BP) regulation, the present study was initiated to see whether changes in PKC signaling are present in rats with fructose-induced insulin resistance and hypertension. Consequently, liver, muscle, and adipose tissues were collected from fructose (n = 13) and chow (n = 12) fed Sprague-Dawley rats. PKC enzyme activity, and expression of classical PKC isozymes, were measured in cytosol and membrane fractions, and 1, 2-diacylglycerol (DAG), an endogenous stimulator of PKC, was measured by radio-enzymatic assay. Fructose feeding was associated with significant increases in fasting plasma insulin (140%) and triglyceride (400%) levels, and increased BP (20 mmHg). PKC activity was increased in the membrane fraction of adipose tissue (234 ± 38 (SE)vs 85 ± 30 pmol/min/mg protein,P< 0.007), without evidence of increased translocation or activation by DAG. Thus, fructose-induced insulin resistance has no effect on conventional PKC activity and subcellular distribution in liver and muscle, but the 3-fold increase in membraneassociated kinase activity in fat may be relevant to the mechanism of hypertriglyceridemia associated with fructose feeding.

Effect of Exercise and Diet on Triglyceride Metabolism in Rats with Moderate Insulin Deficiency

The ability of exercise and diet to modify the effects of moderate streptozotocin-induced insulin deficiency on triglyceride metabolism has been studied in the rat. Insulin-deficient rats allowed to run spontaneously in exercise wheel cages had significantly lower (P < 0.001) plasma glucose levels (187 ± 19 mg/dl) than either sedentary (374 ± 24 mg/dl) or sucrose-fed (450 ± 13 mg/dl) diabetic rats, despite the fact that plasma insulin levels were comparable in all these groups. Plasma triglyceride (TG) levels in exercise-trained rats with diabetes (51 ± 5 mg/dl) were actually lower than in control rats with normal glucose tolerance (90 ± 14 mg/dl). In contrast, plasma TG levels were higher than control levels in diabetic sedentary rats (128 ± 11 mg/dl), and severe hypertriglyceridemia developed in sucrose-fed diabetic rats (369 ± 35 mg/dl). The ability of exercise training to attenuate diabetic hypertriglyceridemia, which was observed in both chow-fed and sucrose-fed rats, was secondary to a decrease in TG secretion, and appeared to be related to lower plasma FFA concentrations. In contrast, the accentuation of diabetic hypertriglyceridemia seen in sucrose-fed rats was related to a defect in TG catabolism. Adipose tissue lipoprotein lipase (LPL) activities were essentially identical in all diabetic rats, suggesting that the observed difference in TG kinetics could not be attributed to concomitant increases or decreases in adipose tissue LPL activity. These results emphasize the powerful impact of exercise and diet on TG metabolism in rats with moderate degrees of insulin deficiency.