0O13-7227/78/1036-2252$O2.0O/0 Endocrinology Copyright © 1978 by The Endocrine Society

Vol. 103, No. 6

Printed in U.S.A.

The Effects of Dietary Carbohydrate Content on Insulin Binding and Glucose Metabolism by Isolated Rat Adipocytes* JERROLD M. OLEFSKYf AND MARK SAEKOW Department of Medicine, University of Colorado School of Medicine, University of Colorado, Denver, Colorado 80262 ABSTRACT. The effects of changes in the amount of dietary carbohydrate (CHO) on cellular insulin and glucose metabolism have been assessed in rat adipocytes. Feeding animals a 67% CHO (fat-free) diet resulted in decreased insulin binding but enhanced activity of both the glucose transport system and intracellular

aspects of insulin's cellular action were depressed. On the other hand, at first approximation, the increased in vivo insulin response caused by a high CHO diet appears contradictory to the observed decrease in insulin binding. However, a probable explanation for this apparent paradox is provided by the enhanced activity of the

pathways of glucose metabolism. Feeding rats a 67% fat

cellular insulin effector systems distal to the insulin

(CHO-free) diet resulted in decreased insulin receptors as well as decreased activity of the glucose transport system and intracellular glucose metabolism. Therefore, the in vivo insulin resistance caused by a high fat, low CHO diet seems to be adequately explained, since all

receptor. Therefore, the increased in vivo insulin responsiveness after high CHO feedings is most likely due to post receptor increases in various aspects of glucose metabolism. (Endocrinology 103: 2252, 1978)

T

HE CARBOHYDRATE (CHO) composition of the diet has a major effect on overall in vivo insulin action (1,2). It has been found that high CHO diets lead to enhanced insulin effects (3, 4), while low CHO diets lead to insulin resistance (5-7). Although these diet-induced changes in insulin response are well described, the mechanisms underlying these effects remain unclear. Therefore, in an attempt to elucidate mechanisms for the effects of dietary CHO content on the cellular response to insulin, we have assessed insulin receptors, glucose transport, and glucose metabolism in isolated adipocytes from rats fed either high CHO (fat-free) or high fat (CHOfree) diets.

Materials and Methods Materials Porcine monocomponent insulin was generously supplied by Dr. Ronald Chance of the Eli Lilly and Co. (Indianapolis, IN). [125I]NaI was purchased from New England Nuclear (Boston, MA); bovine serum albumin (BSA; fraction V) was obtained from Armour Pharmaceutical Co. (Phoenix, AZ); collagenase was obtained from Worthington Biochemical Corp. (Freehold, NJ); 2-deoxy[l-14C]glucose, [14Clglucose, [14C]sucrose, and [l-14C]inulin were purchased from New England Nuclear Co.; and Dowex (1-X8) was obtained from Dow Chemical Co. All experimental diets were prepared by the Nutritional Biochemicals Co. (Cleveland, OH). Experimental animals

Male Sprague-Dawley rats, whose initial weights were 115-135 g, were fed high CHO or high fat diets Received March 31,1978. Address all correspondence and requests for reprints ad libitum for 10 days. The high CHO diet conto: Jerrold M. Olefsky, M.D., University of Colorado tained (by calories) 67% glucose and 33% protein, Medical Center, Department of Medicine, Room 4629, and the high fat diet contained 67% lard and 33% 4200 East Ninth Avenue, Denver, Colorado 80262. protein. The protein was derived from a casein * This work was supported by funds from the Medical hydrolysate and each diet was supplemented with Research Service of the Veterans Administration and by a vitamin and mineral mixture. Control animals NIH-NIAMDD Grants 19905 and 20993. f Clinical Investigator with the Veterans Administra- were fed a standard rat chow diet containing 60% CHO, 22% fat, and 18% protein. The CHO consisted tion. 2252

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DIETARY CARBOHYDRATE AND GLUCOSE METABOLISM of starch; protein was derived from a casein hydrolysate; and fat consisted of a mixture of animal and vegetable oils having a polyunsaturated to saturated ratio of —1.0. Preparation of isolated adipocytes All studies were begun at 0900 h. Animals were stunned by a blow to the head and decapitated, and epididymal fat pads were removed. Isolated fat cells were prepared by shaking at 37 C for 60 min in Krebs-Ringer bicarbonate buffer containing collagenase (3 mg/ml) and BSA (40 mg/ml) according to the method of Rodbell (8). Cells were filtered through 250-ju.m nylon mesh, centrifuged at 400 rpm for 4 min, and washed three times in buffer (9). Adipocyte counts were performed according to a modification of method III of Hirsch and Gallian (10) in which the cells were fixed in 2% osmium tetroxide in 0.05 M coUidine buffer (made isotonic with saline) for 24 h at 37 C and then taken up in a known volume of 0.154 M NaCl for counting with a Coulter counter (model ZB). Adipocyte size was determined with a calibrated microscope by the method of DiGirolamo (11). Iodination of insulin [125I]Insulin was prepared at a specific activity of 100-150 juCi/jug, according to Freychet et al.'s modification (12) of the method of Hunter and Greenwood (13), as previously described (14). Binding studies

2253

Glucose transport studies Transport studies were performed using the same cell centrifugation technique as described for the binding studies, and the details of this method have been previously reported (19, 20). Unless otherwise stated, isolated adipocytes were incubated with 2-deoxy-D-[l-14C]glucose (SA, 2 mCi/mM) at a concentration of 0.1 mM in Krebs-Ringer bicarbonate, pH 7.4, containing BSA (10 mg/ml) at 24 C. This assay measures the total uptake of the radioactive 2-deoxy-glucose and is based on the principle that while 2-deoxy-glucose is transported and phosphorylated by the same processes as D-glucose, it cannot be further metabolized (21). The assay was terminated at the end of 3 min by transferring 200fi\ aliquots from the assay mixture to plastic microtubes containing 100 /xl silicone oil. The tubes were centrifuged for 30 sec in a Beckman microfuge, and the assay was considered to be terminated when the centrifugation began. In experiments in which the stimulatory effect of insulin on uptake was measured, the cells were preincubated with insulin for 60 min at 24 C. The amount of sugar trapped in the extracellular water space of the cell layers was determined using [14C]inulin, according to the method of Gliemann (22). Extracellular water space was measured in each experiment, and all data on sugar uptake were corrected for this factor. The percentage of the total amount of sugar available which was trapped in the extracellular water space averaged 0.033 ± 0.001 at a concentration of 2 X 105 cells/ml. The amount of trapped sugar ranged from 2-10% of the total sugar uptake depending on the conditions of incubation.

Isolated fat cells were suspended in a buffer containing 35 mM Tris, 120 mM NaCl, 1.2 mM Determination of intracellular 2-deoxy-glucose MgSO4, 2.5 mM KC1, 10 mM glucose, 1 mM EDTA, and 2-deoxy-glucose-6-phosphate 1% BSA (15), pH 7.6, and incubated with [125I]Intracellular 2-deoxy-glucose and 2-deoxy-gluinsulin and unlabeled insulin in plastic vials in a cose-6-phosphate were measured by a modification 24 C shaking water bath, as previously described of the method of Tsuboi et al. (23). The details of (9, 16, 17). Optimal steady state binding conditions this method are reported elsewhere (24). Cells were are achieved at 24 C after 90 min of incubation. incubated with 2-deoxy[l-14C]glucose, and uptake The incubations were terminated, as described by was stopped by addition of iced Krebs-Ringer Gammeltoft and Gliemann (18), by removing 200- buffer containing 1 mM phloretin. Cells were isojul aliquots from the ceU suspension and rapidly lated and disrupted in boiling water, and intracelcentrifuging the cells in plastic microtubes to which lular hexose was determined by anion exchange 100 /ill silicone oil had been added. Silicone oil has chromatography using a Dowex 1-X8 column. a. specific gravity intermediate between buffer and [14C]Sucrose was used to correct for extracellular cells and therefore, after centrifugation, three layers water trapped in the cell layer in these experiments. result: cells on top, oil in the middle, and buffer on the bottom. The cells were then removed, and the Analytical methods radioactivity was determined. All studies were done Plasma glucose was determined by the glucose in triplicate.

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Endo • 1978 Vol 103 • No 6

OLEFSKY AND SAEKOW

2254

oxidase method using a Beckman glucose analyzer, and plasma insulin was determined by the technique of Desbuquois and Aurbach (25).

centrations the decrease is more marked for rats fed the high fat diet. Figure IB presents the Scatchard plots of these insulin-binding

Statistical analysis All P values were obtained by use of a nonpaired t test.

Results Experimental groups Table 1 summarizes some of the physiological characteristics of the experimental animals. As can be seen, the initial and final weights were quite comparable for each group. Daily food intake was comparable for the control and high CHO-fed rats, and was somewhat less for the high fat-fed animals. However, the fat diet was calorically more dense than the other diets and therefore, all groups had the same intake on a caloric basis. Fat cell size was the same for animals on the control (89 ± 2.0) and high CHO diet (90 ± 1.6), but was significantly greater for animals fed the high fat diet (124 ± 7.4). Intracellular water space (picoliter per cell) was increased on the high CHO and fat diets. Plasma insulin levels were comparable for controls and rats on the high fat diet and were strikingly elevated for animals fed the high CHO diet. Plasma glucose levels were the same for the three diet groups. Insulin binding studies The ability of adipocytes from all three study groups to bind insulin is seen in Fig. 1A. The amount of insulin bound is less for cells from experimental animals at each insulin concentration, but at the higher insulin con-

10 100 Insulin concentration (ng/rnl) 200-

Insulin bound (ng)

FIG. 1. A, Ability of adipocytes from control (•), high CHO (O), and high fat (A) diet rats to specifically bind insulin. Cells were incubated for 90 min at 24 C with 0.2 ng/ml [125I]insulin plus unlabeled insulin to give the indicated total insulin concentrations. All data are corrected for nonspecific binding by subtracting the amount of radioactivity remaining bound at an insulin concentration of 200 fig/ml from the amount of radioactivity in the cell pellet at all other insulin concentrations. Nonspecific binding averaged 6 ± 1%, 4 ± 1%, and 7 ± 2% of the total amount bound at 0.2 ng/ml [125I]insulin for the control, high CHO, and high fat studies, respectively. Results represent the mean (±SE) of 12 separate experiments for each group and are normalized to 2 x 105 cells. B, Scatchard plots of the insulin binding data for control (•), high CHO (O), and high fat (A) diet rats. The ratio of bound to free insulin (B/F) is plotted on the ordinate and bound insulin is on the abscissa.

FABLE 1. Characteristics of experimental animals

Initial BW (g) Final BW (g) Average food intake in last 4 days Wt of epididymal fat pads (g) Adipocyte volume (pi) Intracellular H2O space (pl/cell) Plasma insulin (fdJ/ml)" Plasma glucose (mg/100 ml)°

Normal

High CHO

High fat

122 ± 0.9 198 ± 2.7 20.1 ± 0.4 1.23 ± 0.1 89 ± 1.9 1.6 ± 0.2 38 ± 5.3 132 ± 2.8

128 ± 2.4 196 ± 2.6 19.9 ± 0.3 1.54 ± 0.1 90 ± 1.6 4.2 ± 0.2 82 ± 6.1 152 ± 4.1

130 ± 3.9 200 ± 3.5 13.5 ± 0.6 1.79 ± 0.1 124 ± 7.4 2.8 ± 0.4 39 ± 5.6 146 ± 4.8

Values represent data obtained at 0900 h.

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DIETARY CARBOHYDRATE AND GLUCOSE METABOLISM

data. The curves from the normal and high fat-fed rats are generally parallel, while the xintercept is lower for the group on the high fat diet. This indicates that the decreased insulin binding caused by high fat feeding is due to a decrease in the number of adipocyte insulin receptors with no change in the affinity of the receptor for insulin. When the curves from the control and high CHO groups are compared, the differences are more complicated. The curve from the high CHO group is less steep than control, and the x-intercept is lower. This indicates a decrease in both receptor affinity and number. These findings are further illustrated in Fig. 2 in which the data are presented on an average affinity plot, as described by De Meyts and Roth (26). As can be seen, there is no difference in the average affinity profiles of the control and high fat groups, while the average affinity is appreciably lower for the high CHO group. Additionally, as calculated from the Scatchard plots (Fig. IB), the mean number of receptor sites per cell is: control, 2.6 X 105; high CHO, 2.1 X 105; and high fat, 1.4 X 105 sites/cell. Thus, the insulin-binding data show that high fat feedings lead to decreased insulin receptor number, and high CHO feedings lead to both a decrease in receptor number and a decrease in the affinity of the receptor for insulin.

Insulin degradation Not only do adipocytes bind insulin, but they also degrade the hormone. Using solubility in trichloroacetic acid (TCA) as a criterion

7 (B/Ro)

FIG. 2. Average affinity profile (control, • ; high CHO, O; high fat, • ) of the three diet groups. K, Average affinity (calculated as (B/F)/(Ro - B), is plotted as a function of the log of the fractional occupany, y [log (B/Ro)].

2255

FIG. 3. Time course of insulin degradation. Degradation of [l25I]insulin by fat cells was measured by TCA precipitation of the radioactivity remaining in the infranatant fraction of the binding incubation mixture (15, 17, 18).

for insulin degradation (15,17,18), this process was determined for cells from each study group. The time course of insulin degradation is presented in Fig. 3. Degradation is linear over time for each group, but the rate of degradation is greater on the high CHO and high fat diets. This enhanced ability to degrade insulin is most striking for the high CHO-fed rats. At a medium insulin concentration of 0.2 ng/ml, the mean (±SE) percentage of the [125I]insulin which is degraded at 90 min of incubation was: control (n = 6), 7.5 ± 0.3; high fat (n = 12), 13.6 ± 1.1; and high CHO (n = 11), 25 ± 3%/2 x 105 cells. To be certain that this degradative process represented a cellular function and was not simply due to proteolytic activity in the buffer, cell-free infranates were obtained after 90 min of incubation with adipocytes and incubated for an additional 90 min with [125I]insulin (0.2 ng/ml). The percentage of degradation was always