by Springer-Verlag 1980

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1 Diabetologia 19, (198) by Springer-Verlag 198 Effect of a High Glucose Diet on nsulin Binding and nsulin Action in Rat Adipocytes A Longitudinal Study Y. ka, Y. Akanuma, M. Kasuga, and K. Kosaka Third Department of nternal Medicine, Faculty of Medicine, University of Tokyo, Hongo, Tokyo, Japan Summary. To elucidate the mechanisms whereby changes in dietary composition affect the action of insulin on glucose metabolism, insulin binding and glucose uptake and oxidation have been studied in epididymal fat pad adipocytes from rats fed high glucose diets for 5 and 1 days. After 5 days, insulin binding was increased, due mainly to an increased number of receptors ( vs sites per cell) in spite of increased plasma insulin levels ( vs ~tgl; p <.5). The maximal response of glucose oxidation to insulin was increased ( vs n moles2 15 cells2h; p <.1) and the dose-response curve of glucose uptake was shifted to the left. After 1 days, receptor number decreased to the control level and the effect of insulin on glucose uptake and oxidation (% basal) were similar to controls. Thus, in the early L stage of high glucose feeding, insulin receptor number, insulin sensitivity of glucose uptake, and insulin responsiveness of glucose oxidation were increased. Key words: High glucose diet, insulin receptor, 2-deoxyglucose uptake, glucose oxidation, insulin sensitivity, insulin responsiveness. A change in dietary composition has been shown to induce a major change in glucose metabolism and insulin action in man [1, 2, 3]. n rats high glucose diets lead to an increase in insulin effect on the glucose metabolism of adipocytes [4]. n spite of this increased insulin effect of high glucose diets, insulin binding has been reported to be decreased as compared with animals on standard rat chow after 1 days of feeding, and this decreased insulin binding was comparable to that of rats fed high fat diets for 1 days [4]. n the other hand, after 5 to 7 days of feeding with high fat diets there is a decrease in insulin binding as compared with high glucose diets [5]. However, the results in these studies were not compared with those from rats fed control diets. Thus, not only the dietary composition but also the duration of feeding may be important in respect of both insulin binding and the effect of insulin on glucose metabolism. The composition of the diets with which rats have been fed before the start of the experiments will also be important. n order to elucidate the mechanisms whereby changes in dietary composition affect the action of insulin upon glucose metabolism, we have used rats fed control diets after weaning and have studied insulin binding, glucose uptake, and glucose metabolism in isolated rat adipocytes from rats fed high glucose diets for 5 and 1 days and compared them with rats fed control diets. Methods Materials Porcine monocomponent insulin was a gift of Eli Lilly and o. (ndianapolis nd.) by courtesy of Dr. Horsley. 125f-labelled Na, [1-14]-D-glucose, 2-deoxy-[1-14]-D-glucose, and [U-14]-su - crose were purchased from New England Nuclear (Boston, Mass.), bovine serum albumin (BSA; fraction V) from Armour and o. (Phenix, Ariz.), collagenase (14Umg) from Worthington biochemicals (Freehold, N.J.), and lactoperoxidase from albiochem (San Diego, alif.). Animals and Diets Male Wistar rats fed a standard rat chow after weaning were used at a starting weight of g. They were fed high glucose diets or the standard rat chow ad libitum for five or ten days. The high glucose diets contained (by calories) 67% glucose and 33% casein, X819468$1.4

2 Y. ka et al.: High Glucose Diet and nsulin Action Table 1. haracteristics of experimental animals ontrol High glucose 5 days feeding Total body weight gain(g) 33.1, b Total food intake (al) _+ 16 Fat-pad weight(g).81_ b Fat-cell size(~tm) 51_ " Plasma glucose(mg1 ml) 154_+3 149,+3 Plasma insulin (gg1) 2.1, a 1 days feeding Total body weight gain(g) b Total food intake(al) _+ 17 Fat-pad weight(g) 1.17, Fat-cell size(~tm) Plasma glucose(mg1 ml) 153_+4 146_+3 Plasma insulin(pg1) 1.9_ _+.3 b Measurements were obtained when rats were sacrificed and are expressed as mean + SEM a p <.5 b p <.1 compared with controls and the standard rat chow contained 6% carbohydrate (derived as follows: 27% corn, 19% rice bran, 1% wheat flour, and 3% soyabean), 12% fat, and 28% protein. These diets were supplemented with vitamins and mineral mixtures. Preparation of solated Adipocytes All the rats were killed by decapitation at 11 to 113 h without fasting. solated adipocytes were prepared from epididymal fat pads by shaking at 37 ~ for 6 rain in Krebs-Ringer-bicarbonate buffer (calcium 1.4 mmol1, ph 7.4) containing collagenase (2 mg ml) and bovine serum albumin (1mgml), according to the methods of Rodbell [6]. After osmium tetroxide fixation [7], adipocytes counts were performed with a oulter counter (model 2B) and adipocyte size was determined with a calibrated microscope. odination of nsulin Porcine insulin was iodinated by lactoperoxidase to a specific activity of 15-2,ai~tg, as described previously [8]. nsulin Binding Studies solated fat cells were suspended in Krebs-Ringer-bicarbonate buffer containing bovine serum albumin (1 mgml, ph 7.4) and incubated with 125-labelled insulin (.5 ngml) and unlabelled insulin in 3 ml plastic test tubes in a shaking water bath at 24 ~ A steady state for binding conditions was achieved after 45 min of incubation in both groups. Therefore, in all experiments, incubations were terminated after 45 min by removing 2 ~tl aliquots and by rapidly centrifuging the cells in plastic microcentrifuge tubes (capacity, 45 bl) containing 1 ~1 dinonyl phthalate [9]. After centrifugation, the fat cells were packed in a layer over the dinonyl phthalate oil, which had a specific gravity intermediate between those of the buffer and the fat cells. The tubes were cut through the oil layer, and the radioactivity present in the fat cells was determined. All the data were corrected for non-specific binding by subtracting the amount of radioactivity remaining bound in 469 the presence of 1 Bxgml porcine insulin from the amount of radioactivity in the cell layer at all other insulin concentrations. The non-specific binding in all experiments was 1-15% of the total amount bound. ell concentrations in experiments ranged from 4 to 7 15 cellsml. Since the data as a function of cell concentration was linear within the range of 4 to 7 15 cellsml, all data have been normalized to 5 15 cellsml for purposes of comparison. Glucose Uptake Studies Glucose uptake studies were performed using the same cell-centrifugation technique as described for the binding studies. solated adipocytes were preincubated with or without insulin for 45 min at 24~ and then were incubated with 2-deoxy-[1-14]-D-glucose (specific activity 1 imol) at a concentration of.2 mmol1 in Krebs-Ringer-bicarbonate buffer, ph 7.4, containing bovine serum albumin (1 mgml) at 24~ The assay was terminated at the end of a 3 min incubation by transferring 2 ~tl aliquots from the assay mixture into plastic microcentrifuge tubes containing 1 ~tl of dinonyl phthalate and rapidly centrifuging the cells as described by lefsky [1]. The incubation was considered terminated at the instant that centrifugation began. The amount of 2-deoxyglucose trapped in the extracellular water space of the cell layers was determined with [U-14]-sucrose [11]; all data for 2-deoxyglucose uptake were corrected for this factor. Studies of Glucose xidation and Lipogenesis The ability of adipocytes to oxidize glucose was measured essentially as described by Rodbell [6]. The adipocytes were incubated at 37~ with [1-14]glucose at a total glucose concentration of 3 mmol1 in Krebs-Ringer-bicarbonate buffer, ph 7.4, containing bovine serum albumin (1 mgml). After 2 h of incubation the 142 generated was absorbed onto filter paper steeped in Hyamin-1X and counted in a liquid scintillation counter. Glucose conversion to total lipids, glyceride-glycerol, and fatty acids was determined as described by Rodbell [6]. Glucose converted to glyceride-glycerol was calculated by subtracting the radioactivity in fatty acids from that in total lipids. Statistical Analysis The significance of the difference was examined by Student's t test for non-paired samples. P values of.5 or less were taken as significant, and the results are expressed as mean _+ SEM. Results Experimental Animals Some of the physiological characteristics of the experimental animals are given in Table 1. After five days of feeding, epididymal fat pad weight, fat-cell size, and plasma insulin levels were significantly increased in rats fed with high glucose diets but plasma glucose levels were similar. After ten days of feeding, no significant differences in fat-cell size and plasma glucose levels were found between the two groups, while plasma insulin levels were still elevated in the high glucose fed rats.

3 47 Y. ka et al.: High Glucose Diet and nsulin Action nsufin Binding Studies Figure 1 shows the binding of insulin to adipocytes obtained from the two groups of rats after five days ~ 4o. of feeding. Adipocytes from the high glucose fed rats bound significantly more insulin at all insulin con- ~ 3oocentrations than those from the control rats. At the "-" "-(3 lowest insulin concentration investigated (.5 ngml), ~ 2- specific insulin binding was % on the con- n~ trol diet and % on the high glucose diet.c 1- (5 x 15cellsml, mean _ + SEM of ten separate -5 experiments, P <.5). Figure 2A shows the Scatch- _c ard plots for these insulin-binding data. The curve from the high glucose fed rats is placed above that of the control rats. Although the curve from the high glucose fed rats is somewhat less steep in the region of low insulin concentrations, the two curves were parallel at higher concentrations. These findings indicate that the increased insulin binding of adipocytes from the high glucose fed rats was due to an increase in the number of adipocyte insulin receptors with almost no change in receptor affinitiy for insulin. As calculated from the Scatchard plots, the mean number of receptor sites per cell is: control, [ ; and high glucose, Even when these data were corrected for cell surface area, the o. 4 number of insulin receptors was increased in the high R&'2~..._ glucose fed rats after 5 days of feeding (data not ~.3 shown). 12 After ten days of high glucose feeding, the \ " amount of insulin bound and the number of receptor ~ 2 sites per cell were decreased to the control levels (Fig. 2B). m 1 ""'""'"'" High Glucose ~ ontrol ~, i 2'5 nsulin oncentration (ngml) Fig. 1. Specific insulin binding to adipocytes from rats fed control (), or high glucose (9 diet for 5 days. eils ( cellsml) were incubated for 45 rain at 24~ with 125-labelled insulin (.5 ngml) plus unlabelled insulin to give the indicated total insulin concentration. Specific insulin binding was calculated as indicated in the text. Data are normalized to 5 15 cellsml and represent the mean _+ SEM of ten separate experiments. * P<.5 compared with controls.1 -~.. High Glucose Glucose Uptake Studies Glucose uptake was measured by using the glucose analogue 2-deoxyglucose which is transported by the same carrier as D-glucose and is phosphorylated but cannot be further metabolized. Time-course experiments have shown that uptake is linear with time for at least 7 min in both control and high glucose groups with 1 ngml of insulin or without insulin (data not shown). t has been reported that the phosphorylation step may not be rate-limiting when measuring 2-deoxyglucose uptake [12] and we have also ascertained this in adipocytes from both control and streptozitocin diabetic rats [13]. However, recently some questions have been raised about the relative rates of transport and phosphorylation [14]. We have therefore, designated this a measurement of glucose uptake rather than transport per se. Figure 3 A presents basal and insulin-stimulated 2-deoxyglucose uptake of cells obtained after 5 days of the diets. The absolute rates of basal and insulin- b_ \ " c" m nsulin Bound (pg5 X 15cells) Fig. 2. Scatchard analysis of the insulin binding data for rats fed control (), or high glucose (9 diet. A, Data represent the mean of ten separate experiments after 5 days of feeding. B, The mean of four separate experiments after 1 days

4 Y. ka et al.: High Glucose Diet and nsulin Action 471 6' High Glucose High Glucose, {3. X 9 ~ 4. o~-~ _~~ co o.2 c~l c-, ontrol r _ D u~ x a ', X v 4.,i:l ontrol 8. '~176 1, ,,,1 2 ' nsulin oncentration (ngml) Fig. 3. Effect of insulin on 2-deoxyglucose uptake by adipocytes after 5 days of feeding. A, Adipocytes from rats fed control (), or high glucose (9 diet were preincubated for 45 rain, then uptake of 2-deoxyglucose was measured following a 3-rain incubation as indicated in the text. Data represent the mean _+ SEM of six separate experiments. B, Data in figure 3A were plotted as % of maximal insulin effect. This is calculated by dividing the increment in the 2-deoxyglucose uptake (absolute value - basal) at the indicated insulin concentration by the maximum increment in the uptake (at 25 ngml). *P <.5, **P<.1 compared with controls.e_ ~ioo X..d c" ~s ~5.~ c" c" ) n B S i 2'.5 lb,, 25 nsulin oncentration (ngml) Fig. 4. Effect of insulin on 2-deoxyglucose uptake by adipocytes after 1 days of feeding. A, The uptake by adipocytes from rats fed control (), or high glucose ((3) diet was measured as indicated in the text. Data represent mean _+ SEM of four separate experiments. B, Data in Figure 4 A were plotted as % of maximal insulin effect. This was calculated as explained in the legend to Figure 3 B. *P<.5 compared with controls stimulated 2-deoxyglucose uptake were significantly increased in adipocytes from the high glucose fed rats. To examine sensitivity to insulin, the data in Figure 3 A was calculated as % maximal insulin effect (Fig. 3 B). The dose-response curve was shifted to the left. Figure 4 A represents the 2-deoxyglucose uptake after 1 days of feeding. When analyzed as % maximal insulin effect (Fig. 4B), no significant differences were found between the two groups. After 1 days of feeding both basal and insulinstimulated glucose oxidation were increased (Fig. 5 B). However, when the data were analyzed as percent of basal, there was no significant difference between the two groups. Table 2 shows the incorporation of [1J4]glucose into total lipids, glyceride-glycerol, and fatty acids after five days of the diets. nsulin-stimulated fattyacid synthesis was markedly increased on the high glucose diet, even when analyzed as percent of basal. Studies of Glucose xidation and Lipogenesis Figure 5A shows basal and insulin-stimulated glucose oxidation in adipocytes from the two groups after 5 days of feeding. There was no significant difference in basal glucose oxidation, but the absolute rates of insulin-stimulated glucose oxidation were markedly increased on the high-glucose diet at insulin concentrations of 1. ngml or more. Discussion When compared to rats on a control diet, animals after 5 days of feeding with a high glucose diet showed significant elevations of both plasma insulin levels and insulin binding to adipocytes. The latter is probably due solely to an increased number of insulin

5 472 d'2 Pc,, l 2~ ox _ze4 1. 5" High Glucose ~** i ontrol Y. ka et al.: High Glucose Diet and nsulin Action Table 2. ncorporation of [1-14]glucose into lipids by rat adipocytes after 5 days of feeding with control or high glucose diets Total lipids Glyceride- Fatty acids glycerol ontrol Basal 51_ _+6 nsulin (25 ngml) 356_ _+58 High glucose Basal nsulin (25ngml) 89_ _+13 a 47_ a 42_ " Values represent mean + SEM of three experiments and are expressed as nmoles of glucose incorporated into total lipids, glyceride-glycerol, and fatty acids per 2 x 15 cells during a 2 h incubation with 3 mmol1 glucose a p <.5 compared with controls - i E ~1-.9 o 5" B High Glucose ~,._-m... ~** i,, ontrol 125 o'.5 1;o 2'5 nsulin oncentration (ngml) Fig. 5. Effect of insulin on [1-14]glucose oxidation by adipocytes. A, Adipocytes from rats fed control (), or high glucos (9 diet for 5 days were incubated for 2 h at 37~ with a final glucose concentration of 3 mmoll with or without insulin and generated 142 was measured as indicated in the text. Data represent mean + SEM of six separate experiments. B, Generated ~4 2 by adipocytes from rats fed control (), or high glucose (9 diet for 1 days. Data represent mean _+ SEM of four separate experiments. 9 P <.5, **P <.1 compared with controls receptors. This finding is in contrast to the generally recognized concept that the number of insulin receptors is reciprocally regulated by insulin in vivo [13, 15-17] and in vitro [18]. Recently, some exceptions have been reported [19-21]. We have found increased insulin receptors per cell in spite of increased insulin levels in ventromedial hypothalamus (VMH) lesioned rats which are markedly hyperinsulinaemic (unpublished observations). ncreased insulin receptors per cell were also found in adipocytes from Zucker obese rats which are markedly hyperinsulinaemic at 6 weeks [22] and 1 1 weeks [23] of age. Although insulin receptor density per cell surface area and sensitivity of glucose transport to insulin has been reported to be decreased in 6 and 1 week old Zucker obese rats, we have found increased receptor density per cell surface area and increased sensitivity of glucose uptake to insulin after 5 days of feeding with a high glucose diet. This appa r- ent relationship between sensitivity of glucose transport or uptake to insulin and insulin receptor density per cell surface area seems reasonable, since glucose transport is thought to be a cell membrane function. After 5 days of feeding with a high glucose diet, insulin-stimulated glucose oxidation was significantly increased without any change in the basal rate. These findings represent an increased responsiveness of glucose oxidation to insulin according to Kahn [24]. After 1 days of feeding with a high glucose diet, the effect of insulin on glucose oxidation, when analyzed as percent of basal because of different basal rates, was similar to that seen on the control diet. As shown in our study, during the early phase of high glucose feeding (in this study, 5 days), plasma insulin levels, insulin-receptor number, and the absolute rates of insulin-stimulated glucose uptake and glucose oxidation are all increased. We have found the same trends in adipocytes from rats developing obesity seven days after receiving ventromedial hypothalamic lesions (unpublished observations). These results suggest that adipocytes exposed to hyperinsulinaemia for a certain period of time, whether caused by diet or by VMH lesions, show increased response to insulin in respect of prereceptor, receptor and post-receptor in the early stage of developing adipocity. n fact, after 5 days of feeding with the high glucose diet, insulin stimulated incorporation of glucose into cell lipids was significantly increased and fat-pad weight and fat cell size were greater than with the control diet.

6 Y. ka et al.: High Glucose Diet and nsulin Action Recently, the effects of short term (5 days) and long term (2 weeks) high carbohydrate diets on insulin binding by human adipocytes have been reported [25]. nsulin binding was decreased due to a decrease in affinity after 5 days of feeding. ur results are not consistent with this report. t may be due to the differences of species, age (we used relatively young rats) and dietary composition (we used high glucose, no fat diets). Since adipocytes from rats fed high glucose no fat diets do not have a dietary source of fatty acids, increased fatty acids synthesis in the high-glucose groups might be due to an adaptative mechanism. t has been reported that fatty acid synthesis plays a primary role in insulin sensitivity of rat adipocytes [26]. Therefore, it may be speculated that zero fat content in the diet may have relevance to our findings through such a mechanism. However, more studies will be needed to clarify this point. ur results after 1 days of feeding with the high glucose diet are in almost complete agreement with those of the previous report [4] except for the insulin binding data. n that report, the number of receptors on adipocytes after a high glucose diet for 1 days was less than after a control diet. This difference in results might be due to the different dietary compositions of the control diets. The present study demonstrated that the insulin-receptor number was decreased after 1 days of the diet when compared to after 5 days of the diet, a change which may be due to "down regulation" by insulin. A minimal duration of hyperinsulinaemia may be necessary for "down regulation" of the number of insulin receptors to occur [27]. The duration of 1 days of feeding in our study may not have been long enough to decrease the insulin-receptor number to below control levels. Many of the previous studies upon the effects of dietary manipulation upon insulin binding and action were cross-sectional. However, it has been shown that time is an important factor in the establishment of insulin resistance in hypothalamic obese mice [28]. ur results show that the duration of feeding is also important in studying the effect of dietary composition upon insulin binding and action. The present study has not elucidated the mechanisms of changes in the number of adipocyte insulin receptors caused by high-glucose feeding and whether changes in the effect of insulin are also found in other metabolic pathways affected by insulin, such as protein and nucleic acid metabolism; these remain to be determined in the future. Acknowledgement. The authors wish to thank Dr. Wilfred Y. Fujimoto for his critical reading of this manuscript and also thank Miss K. Kimura for excellent technical assistance. References Beck-Nielsen H, Pedersen, S6rensen NS, (1978) Effect of diet on the cellular insulin binding and the insulin sensitivity in young healthy subjects. Diabetologia 15: Brunzell JD, Lerner RL, Hazzard WR, Porte D Jr, Bierman EL (1971) mproved glucose tolerance with high carbohydrate feeding in mild diabetes. N Engl J Med 284: Salans LB, Brays GA, ushman SW, Danforth E Jr, Glennon JA, Horton ES, Sims EAH (1974) Glucose metabolism and the response to insulin by human adipose tissue in spontaneous and experimental obesity. Effect of dietary composition and adipose cell size. J lin nvest 53: lefsky JM, Saekow M (1978) The effect of dietary carbohydrate content on insulin binding and glucose metabolism by isolated rat adipocytes. Endocrinology 13: p, Tepperman HM, Holohan P, Tepperman J (1976) nsulin binding and insulin response of adipocytes from rats adapted to fat feeding. J Lipid Res 17: Rodbell M (1964) Metabolism of isolated fat cells. 1. 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7 474 monocyte receptor binding of [125]insulin in infants of gestational diabetic mothers. J lin Endocrinol Metab 47: Schoenle E, Zapf J, Froesch ER (1979) Receptor binding and effects of insulin and NSLA-S on glucose transport and metabolism in adipocytes from hypophysectomized rats. Diabetologia 16: ushman SW, Zarnowski MJ, Franzusoff A J, Salans LB (1978) Alterations in glucose metabolism and its stimulation by insulin in isolated adipose cells during the development of genetic obesity in the Zucker fatty rat. Metabolism 27 (Suppl 2): zech MP, Richardson DK, Becker SG, Walters G, Gitomer W, Heinrich J (1978) nsulin response in skeletal muscle and fat cells of the genetically obese Zucker rat. Metabolism 27 (Suppl 2): Kahn R (1978) nsulin resistance, insulin insensitivity and insulin unresponsiveness: A necessary distinction. Metabolism 27 (Suppl 2): Kolterman G, Greenfield M, Reaven GM, lefsky JM (1979) Effect of a high carbohydrate diet on insulin binding to adipocytes and on insulin action in vivo in man. Diabetes 28: Y. ka et al.: High Glucose Diet and nsulin Action 26. Richardson DK, zech MP (1978) Primary role of decreased fatty-acid synthesis in insulin resistance of large rat adipocytes. Am J Physiol 234:E Kobayashi M, lefsky JM (1978) Effect of experimental hyperinsulinemia on insulin binding and glucose transport in isolated rat adipocytes, Am J Physiol 235:E Le Marchand Y, Freychet P, Jeanrenaud B (1978) Longitudinal study on the establishment of insulin resistance in hypothalamic obese mice. Endocrinology 12:47-85 Received: February 25, 198 and in revised form: June 26, 198 Dr. Yoshitomo ka Third Department of nternal Medicine Faculty of Medicine University of Tokyo Hongo Tokyo 113 Japan