/ . Biochem., 80, 929-934 (1976)
Further Studies on the Mechanism of Adrenaline-induced Lipolysis in Lipid Micelles Yasushi SAITO,*- l Nobuo MATSUOKA,*-» Hiromichi OKUDA,** and Setsuro FUJII* •Department of Enzyme Physiology, Institute for Enzyme Research, School of Medicine, Tokushima University, Tokushima, Tokushima 770, and **Department of Medical Biochemistry, School of Medicine, Ehime University, Matsuyama, Ehime 790 Received for publication, June 4, 1975
Lipase [EC 3.1.1.3] depleted lipid micelles, in which lipolysis was not elicited by adrenaline, were prepared from lipid micelles. When these lipase-depleted lipid micelles incubated with adipose tissue extract containing lipase activity, adrenalineinduced lipolysis was restored to almost the same level as that of native lipid micelles. Adrenaline-induced lipolysis was not restored when the lipase-depleted lipid micelles were homogenized or sonicated. Various tissue extracts from kidney, lung, liver, and pancreas, and post-heparin plasma, which contained lipase activity, restored adrenaline-induced lipolysis in lipase-depleted lipid micelles.
The mechanism of adrenaline-induced lipolysis has been explained in terms of the cyclic AMP theory, in which cyclic AMP plays a regulatory role {1—4). According to this mechanism, adrenaline stimulates adenylcyclase [EC 4.6.1.1] in the fat cell membrane and increases the cyclic AMP content of the cells. The increased level of cyclic AMP then stimulates protein kinase [EC 2.7.1.37] activity, which in turn activates the hormone-sensitive lipase [EC 3.1.1.3] (5, 6). The activated hormonesensitive lipase catalyzes the hydrolysis of triglyceride. However, recent experiments in our laboratory have shown that the presence of another mechanism is required to account
for adrenaline-induced lipolysis (7—9). In these experiments adrenaline-sensitive lipid micelles were prepared from isolated fat cells by disruption in a hypotonic medium. Incubation of these lipid micelles with adrenaline resulted in marked lipolysis, but no increase of protein kinase ( ° ) . These results indicate that adrenaline-induced lipolysis is not mediated by the action of protein kinase in these lipid micelles, but proceeds by some other mechanism. Experiments in our laboratory have indicated that adrenaline may elicit lipolysis by interaction with phospholipid which is located on the surface of the lipid micelles and by initiation of the reaction between lipase and triglyceride {8). In the present communication, further evidence for this hypothesis is presented.
1
Present address: The 2nd Department of Internal Medicine, School of Medicine, Chiba University, Chiba, Chiba 280. Vol. 80, No. 5, 1976
929
Y. SAITO, N. MATSUOKA, H. OKUDA, and S. FUJII
930
MATERIALS AND METHODS
Animals: Young, male albino rats of the Wistar-King strain were fed on standard laboratory diet and water, ad libitum. They were sacrificed by cervical' dislocation and the epididymal adipose tissue was quickly excised. Chemicals: Triton X-100 and pancreatic lipase were obtained from Wako Pure Chemical Co. Trypsin [EC 3. 4.21.4] and phospholipases A [EC 3.1.1.4], C [EC 3.1.4.3], and D [EC 3.1.4.4], which did not contain triglyceride - hydrolysing activity, were from Sigma. Triolein was purchased from Tokyo Kasei Co., and Ediol (50% coconut oil emulsion) from Calbiochem. Preparation of Lipid Micelles—Isolated fat cells were prepared by the method of Rodbell (10). Lipid micelles were prepared from fat cells as described previously (7). Preparation of Lipase-depleted Lipid Micells —Fat cells were suspended in 5 mM Tris-HCl buffer, pH 7.4, at a concentration equivalent to 400 mg of adipose tissue per ml. The suspension was mixed by slowly inverting the centrifuge tube several times and then centrifuged at 200 xg for 3 min at room temperature. The supernatant fraction was replaced by 5 mM Tris-HCl buffer, pH 7.4, containing 0.025% Triton X-100, the contents were mixed and centrifuged at 200xg for 3 min at room temperature, and then the latter procedures were repeated three times (i.e., the supernatant fraction was replaced by 5 mM Tris-HCl buffer, pH 7.4 (without Triton X-100), and the contents mixed and centrifuged). Finally, the fat layer was suspended in Krebs-Ringer phosphate buffer, pH 7.4, containing 5% bovine serum albumin at a concentration equivalent to 400 mg of adipose tissue per ml. This suspension is referred to as lipase-depleted lipid micelles, and contained about 0.6 mg of protein per ml. Estimation of Lipolysis in Lipase-depleted Lipid Micelles—The lipase-depleted lipid micelle suspension, 0.5 ml, was mixed with 0.5 ml of various tissue extracts. The mixture was incubated at 37° for 1 hr in the presence or absence of adrenaline (1 /*g/ml). After incubation, Dole's extraction mixture was added and
the free fatty acid released was estimated by the method of Dole (11). Preparation of Various Tissue Extracts— Extracts containing lipase activity were prepared from' various tissues by homogenizing slices of the tissue with Krebs-Ringer phosphate buffer (KRP) (pH 7.4) using ten strokes of a Teflon homogenizer to give a 20% homogenate (w/v). The homogenates were centrifuged at 4°, 8,000xg for 30 min, and the resulting supernatants were used after adjusting the lipase activity (Ediol-hydrolyzing activity) to 3.5 ^Eq/ml/hr. Plasma was collected from a rat 5 min after injecting 600 /Jg of heparin and used as post-heparin plasma after adjusting the lipase activity to 3.5 /iEq/ml/hr. Concentrations of protein of adipose tissue, lung, liver, and kidney extracts and post-heparin plasma were 6.9, 17.6, 10.4, 8.0, and 6 mg per ml, respectively. Purified pancreatic lipase was obtained from Wako Pure Chemicals and dissolved in KRP at a concentration of 200 /*g/ ml, adjusting the Ediol-hydrolyzing activity to 3.5 /iEq/ml/hr. Estimation of Lipase Activity—Lipase activity in tissue extracts was assayed with Ediol as a substrate, as described previously (7). RESULTS AND DISCUSSION In our previous experiments, lipid micelles were prepared from fat cells by hypotonic shock with as little damage to the intracellular structure as possible (12). On treatment of the lipid micelles with various concentrations of Triton X-100, the induction of lipolysis by adrenaline was found to be reduced to almost nil at a concentration of 0.025% Triton X-100 (Fig. 1). Treatment of the lipid micelles with Triton X-100 accelerated the removal of lipase from them, 46% being removed with 0.025% Triton X-100 and 70% with 0.1% Triton X-100 (Fig. 2). When these lipase-depleted lipid .micelles were incubated with adipose tissue extract containing lipase activity (Ediol-hydrolyzing activity, 3.5 /*Eq/ml/hr), adrenaline-induced lipolysis was restored to almost the same level as that of untreated lipid micelles, as shown in Table I. / . Biochem.
MECHANISM OF ADRENALINE-INDUCED LIPOLYSIS IN LIPID MICELLES
8Hj 00
6-
i —
42-
n
None 0.005 0.01 0.025 0.05 0.1 TRITON X-100 (%) Fig. 1. Effect of Triton X-100 on adrenaline-induced lipolysis. Lipase-depleted lipid micelles were prepared using various concentrations of Triton X-100. Lipolysis was estimated as described in " MATERIALS AND METHODS."
931
When these lipase-depleted lipid micelles were first homogenized or sonicated, lipolysis was remarkably stimulated by the addition of adipose tissue extract even in the absence of adrenaline, and further increase of lipolysis was not observed on the addition of this hormone. The lipase-depleted lipid micelles might have some inhibitory action interfering with the association between. lipase and the interface of the micelles. Destruction of the lipasedepleted lipid micelles or addition of adrenaline to the micelles might remove this inhibitory effect and accelerate the association between lipase and the interface of the micelles, resulting in acceleration of the lipolytic reaction. In the following section, this hypothesis will be further discussed.
TABLE I. Effect of the substrate state on adrenaline-induced lipolysis. Lipase-depleted lipid micelles were homogenized by ten strokes of a Teflon homogenizer or sonicated at 20 kc three times for 3 min each. The concentrations of Ediol and triolein were 5% and 25%, respectively. Triolein was sonicated at 20 kc for 3 min. Adipose tissue extract was prepared by homogenization of adipose tissue in a Teflon homogenizer to give a 20% homogenate (w/v). The homogenate was centrifuged at 8,000 xg for 30 min, and the resulting supernatant was used as adipose tissue extract, adjusted to 3.5 //Eq/ml/hr of Ediol-hydrolyzing activity (6.9 mg of protein per ml). A volume of 0.5 ml of fat cells, lipid micelles or lipase-depleted lipid micelles, equivalent to 200 mg of adipose tissue (the protein concentrations were about 1.4, 0.6, and 0.3 mg per 0.5 ml, respectively) and 0.5 ml of Ediol or triolein were used as a substrate. These substrates were mixed with 0.5 ml of Krebs-Ringer phosphate buffer (pH 7.4) or 0.5 ml of adipose tissue extract. The mixtures were incubated at 37° for 60 min in the presence or absence of adrenaline (1 /*g/ml). Estimation of lipolysis is described in "MATERIALS AND METHODS." Adrenaline (1 /Jg/ml) Substrate
Addition -
+
A
(A
Further Studies on the Mechanism of Adrenaline-induced Lipolysis in Lipid Micelles Yasushi SAITO,*- l Nobuo MATSUOKA,*-» Hiromichi OKUDA,** and Setsuro FUJII* •Department of Enzyme Physiology, Institute for Enzyme Research, School of Medicine, Tokushima University, Tokushima, Tokushima 770, and **Department of Medical Biochemistry, School of Medicine, Ehime University, Matsuyama, Ehime 790 Received for publication, June 4, 1975
Lipase [EC 3.1.1.3] depleted lipid micelles, in which lipolysis was not elicited by adrenaline, were prepared from lipid micelles. When these lipase-depleted lipid micelles incubated with adipose tissue extract containing lipase activity, adrenalineinduced lipolysis was restored to almost the same level as that of native lipid micelles. Adrenaline-induced lipolysis was not restored when the lipase-depleted lipid micelles were homogenized or sonicated. Various tissue extracts from kidney, lung, liver, and pancreas, and post-heparin plasma, which contained lipase activity, restored adrenaline-induced lipolysis in lipase-depleted lipid micelles.
The mechanism of adrenaline-induced lipolysis has been explained in terms of the cyclic AMP theory, in which cyclic AMP plays a regulatory role {1—4). According to this mechanism, adrenaline stimulates adenylcyclase [EC 4.6.1.1] in the fat cell membrane and increases the cyclic AMP content of the cells. The increased level of cyclic AMP then stimulates protein kinase [EC 2.7.1.37] activity, which in turn activates the hormone-sensitive lipase [EC 3.1.1.3] (5, 6). The activated hormonesensitive lipase catalyzes the hydrolysis of triglyceride. However, recent experiments in our laboratory have shown that the presence of another mechanism is required to account
for adrenaline-induced lipolysis (7—9). In these experiments adrenaline-sensitive lipid micelles were prepared from isolated fat cells by disruption in a hypotonic medium. Incubation of these lipid micelles with adrenaline resulted in marked lipolysis, but no increase of protein kinase ( ° ) . These results indicate that adrenaline-induced lipolysis is not mediated by the action of protein kinase in these lipid micelles, but proceeds by some other mechanism. Experiments in our laboratory have indicated that adrenaline may elicit lipolysis by interaction with phospholipid which is located on the surface of the lipid micelles and by initiation of the reaction between lipase and triglyceride {8). In the present communication, further evidence for this hypothesis is presented.
1
Present address: The 2nd Department of Internal Medicine, School of Medicine, Chiba University, Chiba, Chiba 280. Vol. 80, No. 5, 1976
929
Y. SAITO, N. MATSUOKA, H. OKUDA, and S. FUJII
930
MATERIALS AND METHODS
Animals: Young, male albino rats of the Wistar-King strain were fed on standard laboratory diet and water, ad libitum. They were sacrificed by cervical' dislocation and the epididymal adipose tissue was quickly excised. Chemicals: Triton X-100 and pancreatic lipase were obtained from Wako Pure Chemical Co. Trypsin [EC 3. 4.21.4] and phospholipases A [EC 3.1.1.4], C [EC 3.1.4.3], and D [EC 3.1.4.4], which did not contain triglyceride - hydrolysing activity, were from Sigma. Triolein was purchased from Tokyo Kasei Co., and Ediol (50% coconut oil emulsion) from Calbiochem. Preparation of Lipid Micelles—Isolated fat cells were prepared by the method of Rodbell (10). Lipid micelles were prepared from fat cells as described previously (7). Preparation of Lipase-depleted Lipid Micells —Fat cells were suspended in 5 mM Tris-HCl buffer, pH 7.4, at a concentration equivalent to 400 mg of adipose tissue per ml. The suspension was mixed by slowly inverting the centrifuge tube several times and then centrifuged at 200 xg for 3 min at room temperature. The supernatant fraction was replaced by 5 mM Tris-HCl buffer, pH 7.4, containing 0.025% Triton X-100, the contents were mixed and centrifuged at 200xg for 3 min at room temperature, and then the latter procedures were repeated three times (i.e., the supernatant fraction was replaced by 5 mM Tris-HCl buffer, pH 7.4 (without Triton X-100), and the contents mixed and centrifuged). Finally, the fat layer was suspended in Krebs-Ringer phosphate buffer, pH 7.4, containing 5% bovine serum albumin at a concentration equivalent to 400 mg of adipose tissue per ml. This suspension is referred to as lipase-depleted lipid micelles, and contained about 0.6 mg of protein per ml. Estimation of Lipolysis in Lipase-depleted Lipid Micelles—The lipase-depleted lipid micelle suspension, 0.5 ml, was mixed with 0.5 ml of various tissue extracts. The mixture was incubated at 37° for 1 hr in the presence or absence of adrenaline (1 /*g/ml). After incubation, Dole's extraction mixture was added and
the free fatty acid released was estimated by the method of Dole (11). Preparation of Various Tissue Extracts— Extracts containing lipase activity were prepared from' various tissues by homogenizing slices of the tissue with Krebs-Ringer phosphate buffer (KRP) (pH 7.4) using ten strokes of a Teflon homogenizer to give a 20% homogenate (w/v). The homogenates were centrifuged at 4°, 8,000xg for 30 min, and the resulting supernatants were used after adjusting the lipase activity (Ediol-hydrolyzing activity) to 3.5 ^Eq/ml/hr. Plasma was collected from a rat 5 min after injecting 600 /Jg of heparin and used as post-heparin plasma after adjusting the lipase activity to 3.5 /iEq/ml/hr. Concentrations of protein of adipose tissue, lung, liver, and kidney extracts and post-heparin plasma were 6.9, 17.6, 10.4, 8.0, and 6 mg per ml, respectively. Purified pancreatic lipase was obtained from Wako Pure Chemicals and dissolved in KRP at a concentration of 200 /*g/ ml, adjusting the Ediol-hydrolyzing activity to 3.5 /iEq/ml/hr. Estimation of Lipase Activity—Lipase activity in tissue extracts was assayed with Ediol as a substrate, as described previously (7). RESULTS AND DISCUSSION In our previous experiments, lipid micelles were prepared from fat cells by hypotonic shock with as little damage to the intracellular structure as possible (12). On treatment of the lipid micelles with various concentrations of Triton X-100, the induction of lipolysis by adrenaline was found to be reduced to almost nil at a concentration of 0.025% Triton X-100 (Fig. 1). Treatment of the lipid micelles with Triton X-100 accelerated the removal of lipase from them, 46% being removed with 0.025% Triton X-100 and 70% with 0.1% Triton X-100 (Fig. 2). When these lipase-depleted lipid .micelles were incubated with adipose tissue extract containing lipase activity (Ediol-hydrolyzing activity, 3.5 /*Eq/ml/hr), adrenaline-induced lipolysis was restored to almost the same level as that of untreated lipid micelles, as shown in Table I. / . Biochem.
MECHANISM OF ADRENALINE-INDUCED LIPOLYSIS IN LIPID MICELLES
8Hj 00
6-
i —
42-
n
None 0.005 0.01 0.025 0.05 0.1 TRITON X-100 (%) Fig. 1. Effect of Triton X-100 on adrenaline-induced lipolysis. Lipase-depleted lipid micelles were prepared using various concentrations of Triton X-100. Lipolysis was estimated as described in " MATERIALS AND METHODS."
931
When these lipase-depleted lipid micelles were first homogenized or sonicated, lipolysis was remarkably stimulated by the addition of adipose tissue extract even in the absence of adrenaline, and further increase of lipolysis was not observed on the addition of this hormone. The lipase-depleted lipid micelles might have some inhibitory action interfering with the association between. lipase and the interface of the micelles. Destruction of the lipasedepleted lipid micelles or addition of adrenaline to the micelles might remove this inhibitory effect and accelerate the association between lipase and the interface of the micelles, resulting in acceleration of the lipolytic reaction. In the following section, this hypothesis will be further discussed.
TABLE I. Effect of the substrate state on adrenaline-induced lipolysis. Lipase-depleted lipid micelles were homogenized by ten strokes of a Teflon homogenizer or sonicated at 20 kc three times for 3 min each. The concentrations of Ediol and triolein were 5% and 25%, respectively. Triolein was sonicated at 20 kc for 3 min. Adipose tissue extract was prepared by homogenization of adipose tissue in a Teflon homogenizer to give a 20% homogenate (w/v). The homogenate was centrifuged at 8,000 xg for 30 min, and the resulting supernatant was used as adipose tissue extract, adjusted to 3.5 //Eq/ml/hr of Ediol-hydrolyzing activity (6.9 mg of protein per ml). A volume of 0.5 ml of fat cells, lipid micelles or lipase-depleted lipid micelles, equivalent to 200 mg of adipose tissue (the protein concentrations were about 1.4, 0.6, and 0.3 mg per 0.5 ml, respectively) and 0.5 ml of Ediol or triolein were used as a substrate. These substrates were mixed with 0.5 ml of Krebs-Ringer phosphate buffer (pH 7.4) or 0.5 ml of adipose tissue extract. The mixtures were incubated at 37° for 60 min in the presence or absence of adrenaline (1 /*g/ml). Estimation of lipolysis is described in "MATERIALS AND METHODS." Adrenaline (1 /Jg/ml) Substrate
Addition -
+
A
(A
