Effect of a high fat diet on rat adipocyte lipolysis: responses to epinephrine, forskolin, methylisobutylxanthine, dibutyryl cyclic AMP, insulin and nicotinic acid.

An earlier report from this laboratory showed that feeding rats a high fat diet decreased epinephrine-stimulated lipolysis in their adipose tissue. Experiments were designed to explore further the effects of such diets on adipocyte response to epinephrine and to several other lipolytic and antilipolytic agents. Rats were fed diets with 67% of energy consisting of glucose or lard for 5 to 7 d. Adipocytes were prepared from epididymal fat pads and lipolysis measured by the release of glycerol into the medium during 1-h incubations. The cells from the rats fed the high fat diet showed lower lipolytic responses to stimulation by epinephrine, forskolin and dibutyryl cyclic AMP than those from rats fed the high glucose diet. The lard diet effect on the lipolytic response to isobutylmethylxanthine varied among experiments, but it also decreased it in some of them. However, the high fat diet did not induce decreased sensitivity or responsiveness to the antilipolytic effect of insulin, although previous reports have demonstrated resistance to other actions of insulin in rats fed a high fat diet. The antilipolytic effect of nicotinic acid was also similar in cells from rats fed a high fat diet to that found for cells from rats fed the high glucose diet.

Effect of acute exercise on insulin generation of pyruvate dehydrogenase activator by rat liver and adipocyte plasma membranes.

Groups of young adult rats with body weights of 125-135 g (group A) or 300-400 g (group B) were subjected to one bout of prolonged exercise to exhaustion on a treadmill and were studied 2 h postexercise. Liver glycogen levels were markedly depleted in the exercised rats. Adipocytes from group A exercised rats showed a significantly greater increase in pyruvate dehydrogenase (PDH) activity in response to insulin than those from sedentary controls. Incubation with insulin of liver particulate fractions from exercised group A rats resulted in an increased production of a mitochondrial PDH activator compared with preparations from sedentary controls. The tissues of both exercised and sedentary group B rats were less responsive to insulin than those of the smaller rats. A significant effect of exercise on increased production of a PDH activator in response to insulin was found only in experiments in which adipocyte plasma membranes were coincubated with mitochondria and insulin. For group B rats exercise provided no significant enhancement of insulin activation of intact adipocyte PDH or stimulation of the production of a PDH activator by liver particulate preparations. Insulin binding to fat cells was not affected by exercise. Group A rats made insulin resistant by a high-fat diet did not respond to exercise by significantly increasing the insulin stimulation of PDH activator by liver membranes. The enhancing effect of a single bout of exercise on insulin response was not readily demonstrable in rats resistant to insulin either in association with age and weight or with a high-fat diet.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of trifluoperazine on the action of insulin in rat adipocytes.

Trifluoperazine (TFP), a potent inhibitor of calmodulin action, at a concentration of 12 microM decreased the stimulating effects of insulin on 1) fat cell pyruvate dehydrogenase (PDH) activation, 2) generation/action of PDH activator by adipocyte plasma membranes, and 3) insulin-induced loss of insulin receptors, without altering spermine-induced activation of fat cell PDH or preventing insulin stimulation of glucose oxidation. In addition to these effects on insulin action, TFP abolished several biological actions of the insulin-generated PDH stimulator from liver particulate fractions. These actions include fat cell PDH activation and decrease in receptors. These data indicate that TFP inhibits both membrane-associated and intracellular components of insulin action. The results suggest involvement of calcium-binding protein (calmodulin) and/or phospholipid dependent-calcium activated protein kinase C in some of the actions of insulin in fat cells. The insulin effect on glucose oxidation appears to be less dependent on these mediators.

Insulin-induced internalization and replacement of insulin receptors in adipocytes of rats adapted to fat feeding.

To elucidate the mechanisms of the previously observed decrease in adipocyte surface insulin binding in fat diet-induced insulin resistance (Ip et al., J. Lipid Res. 1976; 17:588-99), we performed studies on the distribution of insulin receptors between the cell surface and cell interior, and the extent of internalization and recycling of insulin receptors in adipocytes obtained from fat-fed and glucose-fed rats. Intact cell binding and binding to solubilized cells (total) was decreased by 50% in rats fed fat for 7 days when compared with glucose-fed rats. Incubation of adipocytes with insulin in Tris buffer (100 ng/ml) resulted in a 40-60% decrease in cell surface insulin binding capacity. In two separate experiments, it was found that this insulin-induced receptor loss (%) in fat-fed rat adipocyte preparations was either comparable to that of glucose-fed rats or somewhat decreased. The degradation of the receptors was not affected, as seen by the lack of difference in the chloroquine effect between the two groups. Incubation of fat cells with insulin in tissue culture medium promoted complete reinsertion of receptors into the cell membrane in glucose-fed rat adipocytes, while fat-fed rat preparations demonstrated a significant decrease (37%) in the extent of reinsertion of insulin receptors. Thus, the decrease in cell surface insulin binding and receptor number in fat-fed rat adipocytes is related to an adaptive decrease in the total receptor content coupled with an impairment in the ability to reinsert insulin receptors from the cell interior after insulin-induced internalization.

Studies on the effects of protease substrate analogues on some of the actions of insulin.

Added TAME (N alpha-p-tosyl-1-anginine methyl ester) or BAME (benzoyl-anginine methyl ester) inhibited insulin induced activation of glucose oxidation and fat cell PDH activation without affecting spermine action on PDH activation and glucose oxidation in fat cells. BAME inhibited insulin-induced generation of both PDH stimulator and PDH inhibitor from liver particulate fraction. In contrast, insulin-induced internalization of insulin receptors and negative cooperativity of insulin receptors were not affected by protease substrate inhibitors. These results suggest that certain actions of insulin (glucose oxidation, generation of PDH regulators) are mediated by proteolytic events, while insulin-induced down regulation and negative cooperativity of insulin receptors are not mediated by activation of endogenous proteases.

Studies on the possible involvement of prostaglandins in insulin generation of pyruvate dehydrogenase activator.

Insulin-exposed liver particulate fraction supernatants from control rats stimulated mitochondrial pyruvate dehydrogenase (PDH) activity by 26%, while the stimulation by similar preparations from indomethacin-injected rats (5 mg/kg twice daily, i.p., for 2 days) was 4%. In vitro addition of indomethacin to the particulate fraction during insulin exposure also inhibited stimulation of PDH by insulin. This inhibitory effect of indomethacin was completely overcome by the in vitro addition of prostaglandin E2 (PGE2) to the liver particulate incubation mixture. Intact adipocytes showed a similar (62%) decrease in insulin activation of PDH in the presence of indomethacin. In a cell-free adipocyte system (co-incubation of mitochondria and plasma membrane), indomethacin addition resulted in 90% decrease in insulin-stimulated PDH response. PGE2 addition completely reversed this inhibition. In contrast to its effects on PDH activation, indomethacin had no effect on insulin-stimulated glucose oxidation. In vitro incubation of fat cells with dexamethasone (1 microM) also resulted in decreased insulin activation of PDH. Inclusion of arachidonic acid during dexamethasone exposure of fat cells resulted in partial restoration of the insulin effect on PDH in fat cells and in cell-free preparations. However, addition of PGE2 during insulin exposure of plasma membranes from dexamethasone-treated preparations showed no significant restoration of the insulin effect on PDH. These studies suggest that: (1) PG metabolism is involved in insulin's generation of the second messenger, and (2) the mechanism of dexamethasone-induced inhibition of insulin effect on PDH is a complex phenomenon involving the synthesis and action of eicosanoids.

Effect of dexamethasone on adipose tissue and liver pyruvate dehydrogenase and its stimulation by insulin-generated chemical mediator.

Rats were treated with dexamethasone (50 micrograms/day, sc) for 4 days. Total pyruvate dehydrogenase (PDH) and insulin-stimulated PDHa activities were decreased in fat pads from dexamethasone-treated rats compared to control values. Coincubation experiments with adipocyte mitochondria, plasma membrane, and insulin demonstrated decreased stimulation of PDH in preparations from dexamethasone-treated rats. The responsiveness of the mitochondrial PDH system to insulin and control rat plasma membranes was not different in glucocorticoid-treated adipocyte preparations compared to controls. Liver mitochondria from dexamethasone-treated rats demonstrated decreased basal enzyme activity and a decreased percentage of stimulation of PDH when supernatants from insulin-exposed liver particulate fractions were tested. These experiments suggest that insulin resistance produced by glucocorticoid treatment, like that resulting from fat feeding, is accompanied by a decrease in the capacity of adipocyte and liver plasma membranes to generate PDH activator in response to insulin.

Decreased insulin-generation of pyruvate dehydrogenase inhibitor in insulin resistant states.

Insulin resistance produced in rats by feeding a high fat diet or by dexamethasone administration (50 micrograms/day, sc for 4 days) resulted in 50-70% decrease in the generation of pyruvate dehydrogenase inhibitor by insulin exposed liver particulate fractions. The inhibition was dose dependent. Treatment of insulin mediator preparations with neuraminidase and B-D-galactosidase resulted in inactivation of the pyruvate dehydrogenase inhibitor. Presence of exogenous enzyme substrates during enzyme digestion partially protected the inhibitor from inactivation. Protease treatment did not affect the inhibitor while the stimulatory activity of the insulin mediator was abolished by trypsin treatment. These results together with the previous report suggest that insulin resistance results in a decrease in the generation of both of the mediators of insulin action. This may result from a decrease in insulin binding, shown earlier, or from a decrease in precursor availability.

The effects of streptozotocin diabetes on the activities of rat liver glycosyltransferases.

The effects of streptozotocin diabetes on the activities of rat liver glycosyltransferase enzymes have been investigated. Liver microsomal fractions were prepared from rats that had been injected with streptozotocin (65 mg/kg, i.v.) 3 wk to 2 mo earlier. Preparations from diabetic rats had decreased activities of N-acetylglucosaminyl transferase compared with those of age-matched controls (0.98 +/- 0.11 nmol transferred per mg protein in 30 min versus 3.19 +/- 0.34 for controls, P less than 0.001). Galactosyltransferase activity was also lower in diabetic rat livers (1.48 +/- 0.26 nmol transferred per mg protein in 30 min versus 3.32 +/- 0.56 for controls, P less than 0.025). Sialytransferase activities were not significantly different between diabetic and control rat livers. There were no significant differences between the diabetic and control rat liver microsomes in the activities of UDP N-acetylglucosamine pyrophosphatase, UDP galactose pyrophosphatase, or CMP sialic acid phosphatase. The glycosidases, N-acetylglucosaminidase and galactosidase, had similar activities in the livers of both groups of rats. Sialidase activity could not be detected in microsomal preparations from either diabetic or control rat livers. These results are discussed in relation to our previously reported alterations in glycosyltransferase activities, and plasma membrane glycoprotein composition in the livers of rats made insulin-resistant by a carbohydrate-free, high-fat diet and to the observation of Carter and his colleagues (FEBS Lett. 1979; 104:389-92.) that streptozotocin diabetes alters the glycoprotein composition of rat liver plasma membranes.

Effects of high fat and high carbohydrate diets on liver pyruvate dehydrogenase and its activation by a chemical mediator released from insulin-treated liver particulate fraction: effect of neuraminidase treatment on the chemical mediator activity.

Rats were fed a high fat diet or a high glucose diet for 5-7 days. Basal pyruvate dehydrogenase activity (both the active form and the total enzyme activity) was decreased in liver homogenates from fat diet-adapted rats as compared to those fed the glucose diet. Supernatants from insulin-exposed liver particulate fractions from fat-fed rats showed decreased stimulation of pyruvate dehydrogenase activity as compared to those from glucose-fed rats. There was no difference in the response of the mitochondria from the two groups when they were stimulated by supernatants from insulin-treated liver particulate fractions from stock diet-fed rats. Liver particulate fractions from fat-fed rats showed decreased generation of the chemical activator in response to Concanavalin A and trypsin stimulation. This suggests that fat feeding results in a decrease in membrane protease substrate availability. Treatment of the insulin mediator with neuraminidase and beta-D-galactosidase resulted in inactivation of the mediator. Presence of exogenous enzyme substrates during enzyme digestion protected the mediator from inactivation, suggesting that carbohydrate residues are important in the action of the insulin mediator. This fat diet-induced decrease in the generation of a chemical mediator of insulin action may result from 1) a decrease in insulin binding, shown earlier; 2) a decrease in the amount of protease substrate; and 3) an alteration in its carbohydrate composition, which is important in its ability to activate pyruvate dehydrogenase.

Effects of high carbohydrate and high fat diets on rat adipose tissue pyruvate dehydrogenase responses to concanavalin A and spermine.

Rats were fed a high lard diet or a high glucose diet for 5-7 days. Basal and Concanavalin A (Con A)-stimulated epididymal fat pad pyruvate dehydrogenase (PDH) activities were decreased in fat diet-adapted rats compared to those fed the glucose diet. When adipocyte plasma membranes and mitochondria were coincubated with and without Con A, it was found that the lectin stimulation of PDH activity was lower in preparations from fat-fed rats. These results are comparable to our earlier observations with insulin on adipose tissue PDH. Spermine also stimulated PDH in whole adipose tissue pieces in both the absence and presence (0.5 mM) of medium glucose. The spermine stimulation of PDH in adipose tissue was decreased in fat-fed rats. In contrast to Con A, spermine failed to stimulate PDH in a cell-free system. This suggests that spermine activation of PDH in adipose tissue does not involve the generation of the second messenger responsible for the effects of insulin and Con A. The hypothesis was further substantiated by the findings that (1) the insulin and spermine effects were additive in whole adipose tissue and also in adipocytes, and (2) the spermine effect on fat cells was not significantly inhibited by protease inhibitors, which abolish the effects of insulin on fat cell PDH. The fat-induced decreases in response to Con A and spermine involve not only an adaptive change in the ability of the plasma membrane to generate the chemical modulator of PDH but are also related to postreceptor events.

Effect of high fat and high carbohydrate diets on adipose tissue pyruvate dehydrogenase and its activation by a plasma membrane-enriched fraction and insulin.

Rats were fed a high lard diet or a high glucose diet for 5--7 days. Basal and insulin-stimulated epididymal fat pad pyruvate dehydrogenase (PDH) activities were decreased in fat diet-adapted rats compared to those fed the glucose diet. When adipocyte plasma membranes and mitochondria were incubated together with and without insulin, it was found that the insulin stimulation of PDH activity was lower in preparations from fat-fed rats on both an absolute and percentage basis. Supernatant fractions from insulin-stimulated glucose-fed rat plasma membranes activated mitochondrial PDH to a greater extent than those from lard-fed rat preparations. There was no difference in the response of mitochondria from the two groups when they were stimulated by insulin-treated plasma membranes from stock diet-fed rat adipose tissue. These experiments suggest that fat feeding results in adaptive changes in adipocyte plasma membranes which are involved in the generation of the insulin-stimulated chemical activator of PDH. This adaptive change is in addition to those described earlier.

Effects of high glucose and high lard diets on the activities of rat liver glycosyltransferases.

The activities of several enzymes involved in glycoprotein synthesis were measured in the livers of rats (L) fed diets with 67% of calories as lard and compared with those of rats (G) fed 67% glucose diets for 5-9 days. Glucosamine synthetase activity was not influenced by diet, but the activities of UDP-N-acetylglucosaminyl, galactosyl and sialyltransferases were significantly greater in the livers of the rats fed the glucose diet than in L rat livers. The content of UDP-N-acetylglucosamine was also higher in G livers than in the L group. Activities of glycosidases and of sugar nucleotide pyrophosphatases and phosphatases were the same on both diets. Serum total hexosamine was higher in L than in G rats. These findings are discussed in relation to earlier reports that liver plasma membranes from G rats contained more glycoprotein carbohydrate than L membranes.

Control of endogenous triglyceride breakdown in the mouse diaphragm.

The control of endogenous triglyceride breakdown was studied in vitro, in the incubated intact mouse diaphragm. Isoproterenol (2 microgram/ml) produced parallel increases in glycerol and free fatty acid release, and in tissue cyclic AMP levels, suggesting that cyclic AMP mediates the action of the catecholamine on triglyceride mobilization. In addition to cyclic AMP, calcium seems to be involved in the action of isoproterenol because preincubation of hemidiaphragms in the presence of the calcium ionophore A23187 decreased the lipolytic effect of the drug. Insulin (12.5 mU/ml) antagonized the action of isoproterenol on triglyceride breakdown (it decreased glycerol and free fatty acid release) without altering its stimulatory effect on cyclic AMP levels. On the other hand, no detectable effect on lipolysis was observed with carbachol in control and denervated hemidiaphragms, although the latter possess acetylcholine receptors over the entire surface area of the muscle. It was concluded that catecholamines control triglyceride breakdown in muscle while the cholinergic system does not seem to be involved. Cyclic AMP, calcium, and insulin all affect lipolysis in muscle and the interrelationships remain to be elucidated.

Lipolysis and cyclic AMP response to isoproterenol in diaphragms from control and dystrophic mice.

A comparison was made of the sensitivity of lipolysis (glycerol and free fatty acid release) and of cyclic AMP production to the action of isoproterenol in diaphragms from control and dystrophic Bar Harbor mice at 7 weeks of age. An increased lipolytic response was observed in diaphragms from dystrophic mice that was more apparent in the males, and was demonstrable when cyclic AMP was used instead of isoproterenol. The increased glycerol and free fatty acid release in response to isoproterenol and cyclic AMP cannot be explained by a higher triglyceride content of diaphragms from dystrophic mice, because it was found to be similar to that of controls when it was estimated by biochemical and light microscopic techniques. The increased lipolytic response was not paralleled by changes in cyclic AMP levels, which were found to be similar in diaphragms from control and dystrophic mice, whether in the basal or the stimulated state. It was concluded that the lipolytic apparatus in muscles from dystrophic mice shows an increased sensitivity to isoproterenol that seems to be related to events more intracellular than the cAMP production step.

Effects of high-glucose and high-fat diets on concanavalin A binding to rat liver plasma membranes and on the amount and pattern of their glycoprotein carbohydrates.

Purified liver plasma membranes were prepared from rats fed a high-fat, carbohydrate-free diet or a high-glucose, fat-free diet. Membranes from rats fed the high-fat diet bound significantly less 125I-labeled concanavalin A (Con A) than did those from rats fed the fat-free diets. The magnitude of the binding difference increased with increasing concentrations of Con A. Neither association nor dissociation rates of the lectin-receptor complex was affected by diet. The extent of degradation of Con A by liver plasma membrane preparations from rats fed either diet was the same. Chemical analysis of delipidated liver plasma membrane showed that membranes prepared from high-fat diet-adapted rats had significantly lower values for all carbohydrate components measured with the exception of galactose. The results indicate that, in liver cells, a change in plasma membrane glycoproteins is part of the complex adaptation to altered diet composition.

Studies on the utilization and mobilization of lipid in skeletal muscles from streptozotocin-diabetic and control rats.

Several aspects of lipid metabolism in the soleus and diaphragm muscles of streptozotocin-diabetic and control rats were investigated. The triglyceride content of both muscles was elevated in the diabetic state and the presence of increased intracellular lipid was confirmed by electron microscopy. In vitro glucose and palmitate oxidation studies showed that both types of muscle from the diabetic animals metabolized more fat than did the soleus and diaphragm from control rats. While isoproterenol alone produced a significant lipolytic response in both the soleus and diaphragm from control and diabetic animals, there was no difference in the percent increase in fatty acids released from muscles of diabetic rats compared to controls. However, the absolute difference was greater when the diaphragms were compared. Muscles from experimental and control animals showed a marked reduction in the amount of free fatty acids released in response to insulin. In addition, in the presence of the hormone, both the absolute and percent isoproterenol-stimulated increases in fatty acids were significantly greater for both diaphragm and soleus muscles from diabetic rats. The effects of insulin, isoproterenol, and the combination of these two hormones on the amount of glycerol released into the incubation medium were similar to those found on free fatty acid release. The results of these experiments show that there is an apparent increase in fat utilization in skeletal muscle of diabetic rats. Furthermore, measurements of triglyceride concentration and the enhanced response to isoproterenol stimulation in the muscles from these animals suggests that they may have an increased capacity for mobilization of intracellular lipids. Finally, in the diabetic state, both the soleus and diaphragm appear to demonstrate an increased response to the antilipolytic effect of insulin as measured by the decreased amount of fatty acid released into the incubation medium, the percent change also being significant for the soleus muscle.-Stearns, S. B., H. M. Tepperman, and J. Tepperman. Studies on the utilization and mobilization of lipid in skeletal muscles from streptozotocin-diabetic and control rats.

Lipid composition of liver plasma membranes isolated from rats fed a high glucose or a high fat diet.

Several plasma membrane associated functions have been shown to differ in rats fed a high lard, carbohydrate free diet (L rats) from those observed in preparations from rats fed a high glucose, fat free diet (G rats). To explore the possibility that differences in the lipid components of the plasma membranes might contribute to these functional changes, groups of rats were fed each diet for 5 days and the lipids of their plasma membranes were separated and analyzed. The major differences found were a greater cholesterol content in the plasma membranes from L rats compared to those of G rats (12.2 +/- 2.20 versus 7.8 +/- 1.40 mg/100 mg membrane protein) and more than twice as much phosphatidyl serine and phosphadidyl inositol in the plasma membranes of the former group. Of several variations in fatty acid distribution found in the phospholipid and neutral lipid fractions, the most notable quantitatively were an increase in phospholipid oleic acid (18:1) and a decrease in phospholipid stearic acid (18:0) in L rat plasma membranes. Whether these changes are related to altered membrane function is not known.