Interaction between insulin and thyroid hormones on the control of carbohydrate and lipid metabolism in rat adipose tissue

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Interaction between insulin and thyroid hormones on the control of carbohydrate and lipid metabolism in rat adipose tissue
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  BIOCHEMICAL MEDICINE AND METABOLIC BIOLOGY 38, 81-87 (1987) Interaction between Insulin and Thyroid Hormones on the Control of Carbohydrate and Lipid Metabolism in Rat Adipose Tissue’ MARGARET L. MCKENZIE, K. G. THOMASKUTTY, MARY L. SWIFT, AND RICHARD H. POINTER’ Department of Biochemistry, Howard University College o Medicine, Washington, DC 2OtM Received August 25, 1986, and in revised form December 3, 1986 Evidence of the coexistence and interaction of diabetes mellitus with thyroid disease has been accumulating for more than 40 years (reviewed in Ref. (1)). The pioneering experiments of Houssay (2) demonstrated that hyperglycemia could be induced in partially pancreatectomized dogs by the administration of thyroid extracts. Clinically, it has been demonstrated that hyperthyroidism in diabetic patients often results in both increased hyperglycemia and insulin demand while hypothyroidism in diabetic patients can lead to a decrease both in the hyperglycemic response and in the need for insulin (1). In addition, the admin- istration of thyroid hormones to patients suffering from hypothyroidism and diabetes mellitus can result in the aggravation of the glycemic response (1). Despite these observations, the exact mechanism of thyroid hormone action on glucose homeostasis is yet to be elucidated. The multiple effects of thyroid hormones in the intact animal (3) make it difficult to draw conclusions about possible interactions of thyroid hormones and insulin at target cells. As it has become evident that insulin and thyroid hormones have as one common denominator the regulation of carbohydrate and lipid metabolism (l), the effects of thyroid hormones on basal and insulin-stimulated glucose metabolism in vitro are controversial. The arguments result from the interpretation of data obtained from the very few existing studies of glucose and lipid metabolism in isolated fat pads or cells in response to thyroid status and the action of insulin. Attempts to produce a unifying concept to define the cellular target which is responsive to both thyroid hormones and insulin have led to two possibilities. One is the membrane-associated insulin effector system and the other is the insulin-responsive intracellular metabolic step(s). The present study was designed therefore to correlate the effects of thyroid hormones and insulin on glucose oxidation and lipogenesis with the action of these hormones on the catalytic ’ Supported by NSF Grant PRM 8110709 and DRR/NiH/ MBRS 506 RR 08016 Division of Research Resources, NIH. ’ To whom correspondence should be addressed. 81 0885-4505187 3.00 Copyright 0 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.  82 MC KENZIEETAL. activities of the pyruvate dehydrogenase complex and the fatty acid synthetase system. MATERIALS AND METHODS Thyroid Status Male rats Sprague-Dawley C-D strain were obtained from Charles River Breeding Laboratories (Wilmington, MA) and randomly divided into three groups. On Day 1, all rats weighed 100-120 g. One group was rendered hypothyroid by maintenance on an iodine-deficient diet (U.S. Biochemical Corp., diet No. 17700) and tap water containing 0.00625% 6-N-propyl-2-thiouracil (Sigma Chemical Co.) for 21 days. This protocol has been demonstrated to lead to a reproducible hypothyroid state as determined by circulating thyroid hormone levels (45) and confirmed by our own measurements (Immunochem. Covalent CoA+ Radioimmunoassay for T,; Immunochem. Corp., Carson, CO). In the second group, the euthyroid rats (controls) were littermates and of the same weight range as the experimental rats and were maintained on the tap water and the same test diet, to which normal iodine had been readded by the commercial supplier. The third group of rats from the same group of littermates was maintained on the same regimen as the control rats for 21 days and then given daily injections of (-)3,3’,5’-triio- dothyronine (T3) at 30 pg/lOO g body wt subcutaneously for 5 days prior to their sacrifice. T3 (Sigma) was dissolved in 0.01 N NaOH. Control rats received injections of 0.01 N NaOH in 0.1~ml doses. On the day of sacrifice, animals were anesthetized with Nembutal sodium (Abbott Laboratories) and blood samples were taken from the left ventricle of the heart, to assay T3 levels. All animals were housed in individual cages in air-conditioned rooms with 12-hr day and night light controls. At the time of sacrifice, the euthyroid rats weighed 341 + 50.4 g. The hyperthyroid and hypothyroid rats weighed 236.9 + 16.9 g and 323.9 2 63.9 g, respectively. Fat Pads Upon sacrifice, the epididymal fat pads were removed and floated in 0.15 M NaCl and the blood vessels and epididymis were dissected out and discarded. The remaining segments were washed twice by flotation on fresh 0.15 M NaCl, blotted, divided, weighed, and distributed among the incubation tubes. Pyruvate Dehydrogenase For studies in which the activity of pyruvate dehydrogenase (PDH) was assayed, fat pads (150-200 mg) were incubated in Krebs-Henseleit buffer as described by Taylor et al. (6) in the absence and presence of insulin, 1.0 mu/ml (Novo Laboratories), for 30 min in an atmosphere of 95% 02/5% CO2 at 37°C in a metabolic shaker bath. At the end of this time period, the tissue was homogenized in an Ultra-Turrax homogenizer (Tekmar Co.) for 1.0 min (20~set intervals at full setting). The homogenizing buffer and subsequent assay of the catalytic activity of the pyruvate dehydrogenase complex were as described by Taylor et al. (6).  THYROID HORMONE-INSULIN INTERACTION IN ADIPOSE TISSUE 83 Glucose Oxidation Glucose oxidation was quantified by measuring the 14C02 released from fat pads incubated with n[U-r4C]glucose, 1 O @/pmole (7). Approximately 150 mg of tissue was placed in plastic tubes containing “Pardee bicarbonate buffer” (8). The tubes were equipped with rubber stoppers containing plastic center wells (Kontes Co.) with 10 x 2.5cm filter papers (Whatman No. 1) for the entrapment of CO;?. The total incubation volume was 2.0 ml and each tube was allowed to incubate for 2 hr in a metabolic shaker in the presence and absence of insulin in an atmosphere of 95% 02/5% CO* at 37°C. The reaction was terminated by the addition of 0.25 ml of 1 O N H,SO,. Aliquots of 0.20 ml of phenylmethylamine were added to each filter paper by injection through the center wells of the rubber stoppers. Following incubation, the filter papers were removed and the 14C0, content was determined in a liquid scintillation counter. Total Lipid Analysis For total lipid analysis, the adipose tissue fragments were removed from the tubes after 14C0, analysis, rinsed three times in 120-ml portions of ice-cold 0.15 M NaCl, and homogenized using the Ultra-Turrax in 20 vol of chloroform methanol, 2: 1 (v: v) (9). The resulting homogenate was centrifuged at 10,OOOg for 10 min, and the infranatant was washed with 0.2 times its volume using 0.15 M NaCl and 1 O ml of this washed chloroform-methanol extract was assayed for radioactive glucose incorporated into total lipid. Fatty Acid and Glycerol-Glyceride Analysis For fatty acid analysis, the remaining chloroform-methanol extract was evap- orated and the residue was saponified in 0.946 ml of 1.1 N NaOH in 89% ethanol for 15 min at 50°C. This digest was then acidified with H2S04 to pH 3 and extracted three times with diethyl ether. The combined ether extracts were analyzed for the presence of 14C-labeled fatty acids. The remaining aqueous layer, after ether extraction, was neutralized with 1.0 N NaOH. One millilter of this layer was used for the determination of glyceride- glycerol radioactivity. Fatty Acid Synthetase Approximately 150 mg of tissue was incubated in either the presence or absence of insulin (1 mu/ml) in 2 ml of Pardee buffer for 30 min. Following incubation, the tissue was homogenized in fresh buffer (2 ml) and centrifuged for 10 min at 10,OOOg. Aliquots (0.36 ml) of the supernatant were analyzed for fatty acid synthetase activity by measuring the linear rate of decrease in the extinction of NADPH at 340 nm at 28°C (10). Materials n-[U-‘4C]Glucose and [l-‘4C]Pyruvate were obtained from ICN and New England Nuclear Corp., respectively. Phenylmethlamine, acetyl CoA, malonyl CoA, and NADPH were obtained from Sigma. All other chemicals including solvents were of reagent grade.  84 MC KENZIE ET AL. TABLE 1 Effects of Insulin on n-[U-‘4C]Glucose Oxidation and Pyruvate Dehydrogenase Activity during Altered States Condition o-[U-‘4C]Cluose Pyruvate oxidation to CO2 dehydrogenase activity Euthyroid Control Insulin (1 mu/ml) Hypothyroid Control Insulin (1 mu/ml) Hyperthyroid Control Insulin (1 mu/ml) 27.8 k 3.9 9.8 k 1.6 62.3 f 11.9 20.7 f 2.3 12.4 2 2.1 4.10 f 1.5 27.7 f 4.9 7.3 f 0.4 77.9 f 14.1 16.8 f 3.1 200.6 r 47.1 36.4 f 5.2 Note. Isolated fat pads were incubated and assayed as described in the text. The values for glucose oxidation represent the mean +- SEM of five experiments performed in duplicate on separate days and expressed as nmoles of glucose oxidized/hr/g wet weight. The values for pyruvate dehydrogenase activity represent the k SEM for three experiments done on different days in duplicate and are expressed as nmoles of pyruvate oxidized/min/g wet weight. RESULTS To confirm that the protocol for altering thyroid status was effective, serum T3 levels were measured in all animals at sacrifice. The values observed in nanograms per decaliter of rat serum were 141.5 + 22.3 for euthyroid rats, 80.5 + 25.4 for hypothyroid rats, and 536.3 + 153.0 for hyperthyroid rats. Thus it was presumed that adipose tissue in the intact animal would be exposed to the stated circulating levels of Tj. Shown in Table 1 are the results of experiments in which the effect of insulin on glucose oxidation and pyruvate dehydrogenase (PDH) complex activities in adipose tissue fragments excised from eu-, hypo-, and hyperthyroid rats were measured. In the absence of insulin, glucose oxidation to CO2 and the catalytic activity of the PDH complex decreased by 50% when serum T3 levels were low and increased by more than twofold when serum T3 levels were high (Table 1). The rate of glucose oxidation to CO2 and PDH activity more than doubled in the presence of insulin regardless of thyroid status. These results suggested that thyroid status may not alter the response of adipose tissue to insulin but instead to regulate basal activities. To further pursue this concept, it was of interest to see what effect T3 levels in the intact animal have on other insulin-responsive parameters studied in vitro. Table 2 shows that insulin increases the rate of glucose incorporation into total lipid as well as increases the activity of the fatty acid synthetase system irrespective of thyroid status. However, when serum T3 levels were low, fatty acid synthetase activity markedly decreased and increased by more than twofold when serum T3 levels were high (Table 2). With respect to glucose incorporation into total lipid, high serum T3 levels resulted in more than twofold increases in basal activity, while at low serum T3, basal activity remained unchanged (Table 2).  THYROID HORMONE-INSULIN INTERACTION IN ADIPOSE TISSUE 85 TABLE 2 Effects of Insulin on o-[U-‘4C]Glucose Conversion to Total Lipids and on the Activity of Fatty Acid Synthetase Condition D-[U-‘~C]Glucose conversion to total lipid Fatty acid synthetase activity Euthyroid Control 10.9 + 1.6 14.95 z I 10.25 Insulin (1 mu/ml) 26.3 k 2.7 151.40 f 5.45 Hypothyroid Control 7.6 f 1.8 13.16 f 0.59 Insulin (1 mu/ml) 19.7 ? 1.8 41.52 f. 2.75 Hyperhtyroid Control 34.7 f 9.6 166.50 + 17.42 Insulin (1 mu/ml) 82.8 2 23.4 256.40 2 16.77 Note. All incubations and assays were as described in the text. All values represent the mean * SEM for five experiments done in duplicate on different days. The values for glucose conversion to total lipid are expressed as nmoles of glucose incorporated into lipid/hr/g wet weight. The values for fatty acid synthetase activity are expressed as mU/g wet weight. The lack of an effect of low serum T3 on basal glucose incorporation into total lipid prompted us to examine its effect on glucose incorporation into glycerol- glyceride and fatty acids. Those results (Table 3) show that basal glucose in- corporation into glycerol-glyceride and fatty acids was not affected by low serum T3 levels, while high serum T3 levels resulted in marked increases in these parameters. The response to insulin in all thyroid states remained at least twofold (Table 3). TABLE 3 Effects of Insulin on D-[U-“C] Glucose Incorporation into Glyceride-Glycerol and Fatty Acids during Altered Thyroid States Condition D-[U-‘4C]GhJCOSe o-[U-‘4C]Glucose conversion to glycerol-glyceride converion to fatty acids Euthyroid Control Insulin (1 mu/ml) Hypothyroid Control Insulin (1 mu/ml) Hyperthyroid Control Insulin (1 mu/ml) 0.57 -c 0.14 1.1 2 0.2 1.14 2 0.30 2.5 f 0.5 0.48 * 0.17 1.3 2 0.4 1.02 2 0.23 3.7 t 1.3 3.0 t 0.62 4.1 f 0.5 6.4 k 1.8 14.1 + 3.0 Note. All incubations and assays were as described in the text. The values seen represent the mean ? SEM for five experiments performed in duplicate on separate days and are expressed as nmoles of glucose converted to either glyceride-glycerol or fatty acids hr/g wet weight.
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