Antilipolytic actions of insulin on basal and hormone-induced lipolysis in rat adipocytes

The levels of insulin, free fatty acids (FFA), and triglycerides in rat sera increase with age. The increase in serum FFA levels accompanied the stimulation of basal lipolysis (i.e., lipolysis in the absence of lipolytic agents) in fat cells and enlargement of the diameter of the cells. An overnight fast resulted in a significant increase in basal lipolysis in fat cells from 6- and 8-week-old rats. Although insulin inhibited lipolysis induced by norepinephrine and ACTH at a concentration of 10-10 m, it failed to inhibit basal lipolysis even at a concentration of 10-6 m. Propranolol, another antilipolytic agent like insulin, also did not affect basal lipolysis. Insulin did not inhibit the accelerated basal lipolysis in enlarged fat cells, fasted fat cells, and sonicated cells. These results indicate that insulin inhibits only the lipolysis induced by lipolytic agents such as norepinephrine and ACTH but not the basal lipolysis found in the absence of lipolytic agents. The possibility that free fatty acids produced by enlarged fat cells initiate insulin resistance and diabetes mellitus, is discussed. —Morimoto, C., T. Tsujita, and H. Okuda. Antilipolytic actions of insulin on basal and hormone-induced lipolysis in rat adipocytes. J. Lipid Res. 1998. 38: 957–962. The levels of insulin, free fatty acids (FFA), and triglycerides in rat sera increase with age. The increase in serum FFA levels accompanied the stimulation of basal lipolysis (i.e., lipolysis in the absence of lipolytic agents) in fat cells and enlargement of the diameter of the cells. An overnight fast resulted in a significant increase in basal lipolysis in fat cells from 6- and 8-week-old rats. Although insulin inhibited lipolysis induced by norepinephrine and ACTH at a concentration of 10-10 m, it failed to inhibit basal lipolysis even at a concentration of 10-6 m. Propranolol, another antilipolytic agent like insulin, also did not affect basal lipolysis. Insulin did not inhibit the accelerated basal lipolysis in enlarged fat cells, fasted fat cells, and sonicated cells. These results indicate that insulin inhibits only the lipolysis induced by lipolytic agents such as norepinephrine and ACTH but not the basal lipolysis found in the absence of lipolytic agents. The possibility that free fatty acids produced by enlarged fat cells initiate insulin resistance and diabetes mellitus, is discussed. —Morimoto, C., T. Tsujita, and H. Okuda. Antilipolytic actions of insulin on basal and hormone-induced lipolysis in rat adipocytes. J. Lipid Res. 1998. 38: 957–962. Plasma FFA levels are affected by fat cell lipolysis which is regulated by hormone-dependent and hormone-independent lipolytic activities. The latter is termed basal lipolysis. Our previous study demonstrated that basal lipolysis is elevated in the enlarged fat cells of obese rats as a result of a reduction in the surface phosphatidylcholine concentration of endogenous lipid droplets (1Tsujita T. Morimoto C. Okuda H. Mechanism of increase in basal lipolysis of enlarged adipocytes in obese animals.Obesity Res. 1995; 3: 633s-636sGoogle Scholar, 2Okuda H. Morimoto C. Tsujita T. Role of endogenous lipid droplets in lipolysis in rat adipocytes.J. Lipid Res. 1994; 35: 36-44Google Scholar). An active hormone-sensitive lipase (HSL) is present in fat cells even in the absence of lipolytic hormones, and phosphatidylcholine on the surface of endogenous lipid droplets causes inhibition of the lipolytic action of HSL. The decrease in surface phosphatidylcholine concentrations in endogenous lipid droplets thus causes the increase in basal lipolysis. One of the most important metabolic actions of insulin is the inhibition of lipolytic activity in fat cells (3Rahn T. Ridderstråle M. Tornqvist H. Manganiello V. Fredrikson G. Belfrage P. Degerman E. Essential role of phosphatidylinositol 3-kinase in insulin-induced activation and phosphorylation of the cGMP-inhibited cAMP phosphodiesterase in rat adipocytes.FEBS Lett. 1994; 350: 314-318Google Scholar). Activation of the adipocyte cGMP-inhibited cAMP phosphodiesterase by insulin is believed to be the major mechanism whereby insulin reduces cellular cAMP which then leads to inactivation of the cAMP-dependent protein kinase, net dephosphorylation of HSL and antilipolysis (3Rahn T. Ridderstråle M. Tornqvist H. Manganiello V. Fredrikson G. Belfrage P. Degerman E. Essential role of phosphatidylinositol 3-kinase in insulin-induced activation and phosphorylation of the cGMP-inhibited cAMP phosphodiesterase in rat adipocytes.FEBS Lett. 1994; 350: 314-318Google Scholar, 4Manganiello V. Vaughan M. An effect of insulin on cyclic adenosine 3′:5′-monophosphate phosphodiesterase activity in fat cells.J. Biol. Chem. 1973; 248: 7164-7170Google Scholar, 5Londos C. Honnor R.C. Dhillon G.S. cAMP-dependent protein kinase and lipolysis in rat adipocytes.J. Biol. Chem. 1985; 260: 15139-15145Google Scholar, 6Strålfors P. Honnor R.C. Insulin-induced dephosphorylation of hormone-sensitive lipase-correlation with lipolysis and cAMP-dependent protein kinase activity.Eur. J. Biochem. 1989; 182: 379-385Google Scholar). This mechanism of insulin-mediated antilipolysis presupposes that HSL activity is reduced. However, there is no direct evidence as to whether insulin-induced antilipolysis accompanies reduction in HSL activity. Further experiments are therefore needed to clarify the precise mechanism of the antilipolytic action of insulin. Although insulin is known to inhibit hormone-induced lipolytic activity in fat cells, there have been no studies that have examined the effect of insulin on hormone-independent or basal lipolysis. The present investigation was designed to clarify whether insulin inhibits basal lipolysis in rat fat cells. Young male Wistar-King rats, 6, 8 and 10 weeks of age, were given a standard laboratory diet (Oriental Yeast Co. Ltd) and water ad libitum. They were anesthetized by intraperitoneal injection of pentobarbital (50 mg/kg) and venous blood samples were obtained at 10:00 am; then their epididymal adipose tissues were quickly removed. Collagenase (type IV) was purchased from Worthington Biochemical Corp. (Freehold, NJ). Norepinephrine was obtained from Sankyo Co. Ltd. ACTH was from Daiichi Seiyaku Co. Ltd. (Tokyo, Japan). Bovine serum albumin, TES, Glucose B-test wako, NEFA C-test wako, triglyceride E-test wako, cholesterol E-test wako and GLAZYME Insulin-EIA TEST were purchased from Wako Pure Chemical Industries (Osaka, Japan). Bovine serum albumin was extracted by the method of Chen (7Chen R.F. Removal of fatty acids from serum albumin by charcoal treatment.J. Biol. Chem. 1967; 242: 173-181Google Scholar) to remove free fatty acids. The levels of serum insulin, glucose, FFA, triglyceride and cholesterol in rats were estimated using commercial kits from Wako Pure Chemical Industries. Isolated fat cells were obtained from rat epididymal adipose tissue by the method of Rodbell (8Rodbell M. Metabolism of isolated fat cells.J. Biol. Chem. 1964; 239: 375-380Google Scholar). The fat cells (50 μl packed volume) were incubated for 1 h at 37°C in 250 μl of buffer A (25 mm TES, pH 7.4, containing 135 mm NaCl, 5 mM KCl and 1 mM MgCl2) supplemented with 2.5% bovine serum albumin and the test samples. Then 3 ml of chloroform–heptane 1:1 (v/v) containing 2% (v/v) methanol was added and any FFA released were measured with copper reagent and bathocuproine as described by Okuda, Tsujita, and Kinutani (9Okuda H. Tsujita T. Kinutani M. Studies on a protein kinase inhibitor-insensitive, phospholipase C-sensitive pathway of lipolysis in rat adipocytes.Pharmacol. Res. Commun. 1986; 18: 877-893Google Scholar). The isolated fat cells were mixed with buffer A containing 2% osmium chloride. The mixture stood for 3 h at 4°C and was then subjected to scanning electron micrography (Hitachi H-500). The diameters of the fat cells were estimated from the electron micrographs with an image analyzer. Statistical analysis was performed by one-way ANOVA and multiple comparisons were tested with Scheffe's method. The levels of insulin, FFA, and triglyceride in the non-fasted rat sera increased with age as shown in Table 1. It is likely that the increase in FFA caused a rise in triglyceride levels through an elevation in VLDL formation in the liver. The increase in serum FFA levels accompanied stimulation of basal lipolysis (lipolysis in the absence of lipolytic agents) in fat cells and enlargement of the diameter of the cells (Fig. 1A). The levels of serum FFA increased and the levels of serum glucose and insulin decreased with overnight fasting as compared with non-fasted rats (Table 2). The increase in serum FFA levels accompanied stimulation of basal lipolysis in fat cells by overnight fasting; for rats 6 weeks of age, fasting resulted in a marked rise of serum FFA levels (4.6-fold) and basal lipolysis (7.8-fold) (Fig. 1B). Taken together, it seems likely that the rise in serum FFA levels is due to stimulation of basal lipolysis in fat cells.TABLE 1.Characteristics of non-fasted ratsAgeBody weightInsulinGlucoseFree fatty acidsTriglycerideCholesterolweeksgmU/lmg/lμEq/lmg/lmg/l6178 ± 131.0 ± 3.12110 ± 63131 ± 16581 ± 39712 ± 158289 ± 5aaP < 0.0162.4 ± 6.5aaP < 0.011980 ± 16209 ± 14bbP < 0.05 vs. value in rats at 6 weeks.1250 ± 85aaP < 0.01728 ± 1610360 ± 13aaP < 0.0149.1 ± 2.1bbP < 0.05 vs. value in rats at 6 weeks.2370 ± 40aaP < 0.01324 ± 17aaP < 0.011290 ± 130aaP < 0.01697 ± 50Values are means ± SE of four separate experiments.a aP < 0.01b bP < 0.05 vs. value in rats at 6 weeks. Open table in a new tab TABLE 2.Characteristics of overnight fasted ratsAgeBody weightInsulinGlucoseFree Fatty AcidsTriglycerideCholesterolweeksgmU/lmg/lμEq/lmg/lmg/l6173 ± 29.7 ± 1.1742 ± 79606 ± 78764 ± 136746 ± 358273 ± 6aaP < 0.01 vs. value in rats at 6 weeks.17.0 ± 3.9773 ± 68775 ± 4594 ± 60668 ± 4510349 ± 3aaP < 0.01 vs. value in rats at 6 weeks.17.1 ± 3.1941 ± 16758 ± 20654 ± 45680 ± 33Values are means ± SE of four separate experiments.a aP < 0.01 vs. value in rats at 6 weeks. Open table in a new tab Values are means ± SE of four separate experiments. Values are means ± SE of four separate experiments. Figure 2 shows antilipolytic actions of insulin on lipolysis in fat cells from overnight fasted rats. Although insulin inhibited lipolysis elicited by norepinephrine and ACTH at a concentration of 10-10 m, it failed to inhibit basal lipolysis in fat cells isolated from rats who were 6 weeks old even at a concentration of 10-6 m. Similar results were observed in fat cells isolated from 6-week-old non-fasted rats (data not shown). Figure 3 shows the effect of time on basal lipolysis in fat cells from overnight fasted rats. The accumulation of released FFA increased time-dependently for at least 1 h. Insulin did not inhibit the accelerated basal lipolysis in enlarged fat cells isolated from non-fasted rats who were 8 and 10 weeks of age (Fig. 4A). Insulin also did not inhibit the accelerated basal lipolysis in fat cells isolated from overnight fasted rats (Fig. 4B).Fig. 3.Effect of time on basal lipolysis in fat cells isolated from the overnight fasted rats. Basal lipolysis (lipolysis in the absence of lipolytic agents) was examined using fat cells isolated from rats of 6 (□), 8 (▵) and 10 weeks (○) of age. Each point represents the means ± SE of four separate assays.View Large Image Figure ViewerDownload (PPT)Fig. 4.Effect of insulin on basal lipolysis in fat cells isolated from the non-fasted (A) and the overnight fasted (B) rats. Basal lipolysis (lipolysis in the absence of lipolytic agents) was examined using fat cells isolated from rats of 6 (□), 8 (▵), and 10 weeks (○) of age, respectively, in the presence and absence of insulin. Each point represents the means ± SE of four separate assays.View Large Image Figure ViewerDownload (PPT) In 1994, we first reported that sonication of fat cells elicited marked lipolysis in the absence of epinephrine (2Okuda H. Morimoto C. Tsujita T. Role of endogenous lipid droplets in lipolysis in rat adipocytes.J. Lipid Res. 1994; 35: 36-44Google Scholar). In other words, the sonication of fat cells caused a marked increase in basal lipolysis. As shown in Fig. 5, insulin also failed to inhibit the accelerated basal lipolysis in sonicated cells. These results clearly indicate that insulin only inhibited the lipolysis induced by lipolytic agents such as norepinephrine and ACTH but did not affect the basal lipolysis found in the absence of lipolytic agents. It is well known that the β-blocker, propranolol, inhibits lipolysis induced by norepinephrine, whereas the α-blocker phenoxybenzamine does not inhibit lipolysis. Figure 6 shows the effect of β-blocker on lipolysis in fat cells isolated from overnight fasted rats. Propranolol completely inhibited the lipolysis elicited by norepinephrine at a concentration of 10-5 m whereas it failed to inhibit basal lipolysis at a concentration of 10-4 m. Similar results were observed in fat cells isolated from 6-week-old non-fasted rats (data not shown). Propranolol also failed to inhibit the basal lipolysis accelerated by sonication of the cells (data not shown). Although insulin inhibited lipolysis induced by norepinephrine or ACTH in fat cells, it did not affect basal lipolysis in these cells (Figs. Fig. 2., Fig. 4.). Propranolol, another antilipolytic agent, also failed to inhibit basal lipolysis (Fig. 6). It has been reported that an enlargement in fat cell size causes an increase in basal lipolysis (1Tsujita T. Morimoto C. Okuda H. Mechanism of increase in basal lipolysis of enlarged adipocytes in obese animals.Obesity Res. 1995; 3: 633s-636sGoogle Scholar). Aging caused an enlargement in fat cell size and an increase in basal lipolysis, which possibly induced elevation in serum FFA levels (Table 1 and Fig. 1). It has been suggested that elevation in basal lipolysis in enlarged fat cells is induced by the reduction in the surface phosphatidylcholine concentration of the enlarged lipid droplets in these cells (1Tsujita T. Morimoto C. Okuda H. Mechanism of increase in basal lipolysis of enlarged adipocytes in obese animals.Obesity Res. 1995; 3: 633s-636sGoogle Scholar). Surface phosphatidylcholine is known to inhibit the lipolytic reaction between lipid droplets and HSL (1Tsujita T. Morimoto C. Okuda H. Mechanism of increase in basal lipolysis of enlarged adipocytes in obese animals.Obesity Res. 1995; 3: 633s-636sGoogle Scholar, 2Okuda H. Morimoto C. Tsujita T. Role of endogenous lipid droplets in lipolysis in rat adipocytes.J. Lipid Res. 1994; 35: 36-44Google Scholar). It is well known that fasting results in a decrease in serum concentration of insulin and glucose, and a marked rise in FFA. In our experiment, the serum glucose and insulin levels decreased and the serum FFA levels increased with overnight fasting (Tables TABLE 1., TABLE 2.). The increased FFA levels were accompanied by the accelerated basal lipolysis (Fig. 1). However, in rats at 10 weeks of age, the levels of basal lipolysis did not increase with overnight fasting (Fig. 1). Insulin may play a pivotal role in the hormonal control of metabolic adaptation of fasting. The known ability of insulin to inhibit the lipolysis suggests that it should have an important role in controlling the serum FFA levels: fasting causes a decrease in serum insulin levels, which induces lipolysis, and results in an increase in serum FFA levels. However, the accelerated basal lipolysis induced by fasting was not affected by insulin (Fig. 4). Further experiments are needed to clarify the mechanism of acceleration of basal lipolysis by fasting. Sonication of fat cells is known to increase basal lipolysis (1Tsujita T. Morimoto C. Okuda H. Mechanism of increase in basal lipolysis of enlarged adipocytes in obese animals.Obesity Res. 1995; 3: 633s-636sGoogle Scholar, 2Okuda H. Morimoto C. Tsujita T. Role of endogenous lipid droplets in lipolysis in rat adipocytes.J. Lipid Res. 1994; 35: 36-44Google Scholar). Sonication of fat cells decreased the average diameter (56.6 ± 0.7 μm) of the lipid droplets to 4.6 ± 0.2 μm, with an increase in the droplet number. The increase in the surface area of the droplets was directly proportional to the ratio of the average diameter of the intact to the sonicated lipid droplets. The average surface area of the sonicated lipid droplets was calculated to be about 12 times that of intact lipid droplets. Thus, the concentration of phosphatidylcholine on the surface of the lipid droplets was reduced by about 12-fold by sonication. The reduction in phosphatidylcholine concentration accelerated the association between the sonicated lipid droplets and HSL, resulting in elevated basal lipolysis (2Okuda H. Morimoto C. Tsujita T. Role of endogenous lipid droplets in lipolysis in rat adipocytes.J. Lipid Res. 1994; 35: 36-44Google Scholar). The present study clearly shows that insulin fails to inhibit the elevated basal lipolysis found in sonicated fat cells (Fig. 5). Taken together, the results show that insulin inhibits lipolysis in the presence of lipolytic agents such as norepinephrine and ACTH, but does not affect lipolysis in the absence of these agents (basal lipolysis). Norepinephrine- and ACTH-induced lipolysis was not completely inhibited by insulin, whereas it was completely inhibited by β-blocker (Figs. Fig. 2., Fig. 6.). Some proportion of hormone-stimulated lipolysis was “insulin-resistant.” Further experiments are needed to clarify this lipolysis. In general, obesity is often associated with insulin resistance; indeed, circulating FFA levels are chronically elevated roughly in proportion to the degree of obesity (10DeFronzo R.A. Insulin secretion, insulin resistance, and obesity.Int. J. Obes. 1982; 6: 73-79Google Scholar, 11Olefsky J.M. Kolterman O.G. Scarlett J.A. Insulin action and resistance in obesity and noninsulin-dependent type II diabetes mellitus.Am. J. Physiol. 1982; 243: E15-E30Google Scholar). Randle et al. (12Randle P.J. Garland P.B. Hales C.N. Newsholme E.A. The glucose fatty acid cycle: its role in insulin sensitivity and the metabolic disturbances of diabetes mellitus.Lancet. 1963; 1: 785-789Google Scholar) initially reported that FFA inhibit glucose utilization in cardiac and diaphragmatic muscles and Garland, Newsholme, and Randle (13Garland P.B. Newsholme E.A. Randle P.J. Regulation of glucose uptake by muscle. Effects of fatty acids and ketone bodies, and of alloxan-diabetes and starvation, on pyruvate metabolism and on lactate/pyruvate and l-glycerol-3-phosphate/dihydroxyacetone phosphate concentration ratios in rat heart and rat diaphragm muscles.Biochem. J. 1964; 93: 665-678Google Scholar) showed that FFA inhibit muscle glucose utilization by decreasing glucose transport and inhibiting glycolysis and pyruvate oxidation. FFA also stimulate gluconeogenesis from lactate, alanine and pyrurate (14Friedman B. Goodman Jr., S. Weinhouse E.H. Effects of insulin and fatty acids on gluconeogenesis in the rat.J. Biol. Chem. 1967; 242: 3620-3627Google Scholar). Lee et al. (15Lee K.U. Lee H.K. Koh C.S. Min H.K. Artificial induction of intravascular lipolysis by lipid-heparin infusion leads to insulin resistance in man.Diabetologia. 1988; 31: 285-290Google Scholar) examined the effect of a lipid–heparin infusion on glucose metabolism in ten normal subjects by the euglycemic glucose clamp technique and isotopic determination of glucose turnover to test the hypothesis that an increase in lipolysis is related to insulin resistance. Their conclusion was that artificial induction of intravascular lipolysis by a lipid–heparin infusion led to a state of insulin resistance in humans (15Lee K.U. Lee H.K. Koh C.S. Min H.K. Artificial induction of intravascular lipolysis by lipid-heparin infusion leads to insulin resistance in man.Diabetologia. 1988; 31: 285-290Google Scholar). Based on these results, it seems likely that insulin resistance associated with obesity may be caused by an increase in basal lipolysis. Although the elevation in basal lipolysis induces hyperinsulinemia in humans and rodents (16Yalow R.S. Berson S.A. Plasma insulin concentrations in nondiabetic and early diabetic subjects. Determinations by a new sensitive immunoassay technique.Diabetes. 1960; 9: 254-260Google Scholar, 17Karam J.H. Grodsky G.M. Forsham P.H. Excessive insulin response to glucose in obese subjects as measured by immunochemical assay.Diabetes. 1963; 12: 197-204Google Scholar, 18Bray G.A. York D.A. Hypothalamic and genetic obesity in experimental animals: an autonomic and endocrine hypothesis.Physiol. Rev. 1979; 59: 719-809Google Scholar), insulin does not inhibit the lipolysis (Figs. Fig. 2., Fig. 4.). Milburm et al. (19Milburn Jr., J.L. Hirose H. Lee Y.H. Nagasawa Y. Ogawa A. Ohneda M. BeltrandelRio H. Newgard C.B. Johnson J.H. Unger R.H. Pancreatic β-cells in obesity.J. Biol. Chem. 1995; 270: 1295-1299Google Scholar) indicated that FFA produced by the increase in basal lipolysis induced β-cell hyperplasia and enhanced low Km glucose metabolism in the islet cells. Thus, FFA levels elevated by the increase in basal lipolysis cause insulin resistance resulting in compensatory hyperinsulinemia. However, the hyperinsulinemia fails to reduce FFA levels produced by the increase in basal lipolysis. In summary, therefore, it seems probable that the increase in basal lipolysis induced by the enlargement of lipid droplets in fat cells may initiate insulin resistance and result in diabetes mellitus. Further experiments are needed to prove this hypothesis. The work was supported by Research Grants from Fuji-Bio Co. Ltd. (Shizuoka, Japan). The authors thank Mr. Manabu Miyoshi (the Laboratory Animal Center) and Mr. Masachika Shudo (Central Research Laboratory) at Ehime University, School of Medicine, for their technical assistance. free fatty acids hormone-sensitive lipase adrenocorticotropic hormone N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid.