On the origin of hyperglycaemia in the obese-hyperglycaemic mouse (obob): Effect of diet on blood glucose and serum insulin inobob and gold-thioglucose obese mice

Diabetologia, Oct 1971

Obese-hyperglycaemic mice (obob), goldthioglucose obese (GTG-obese) and normal control mice were fed a high-carbohydrate and a carbohydrate-free diet. Blood glucose and serum insulin levels were influenced by the diet in all animals studied, but more so in theobob and GTG-obese. Blood glucose levels were equally raised in both types of obesity. Serum insulin of GTG-obese showed a marked elevation above normal but failed to reach the serum insulin levels of theobob. The blood glucose ofobob and lean controls remained constant when fed the carbohydrate-free diet. Under these conditions, a trace amount of U-14C-glucose was injected intravenously. The rate of14C-glucose disappearance from the blood followed first-order kinetics and did not differ betweenobob and lean mice, suggesting a similar fractional rate of glucose uptake by tissues. The presence of a normal fractional rate of glucose extraction by the tissues ofobob, together with their hyperglycaemia and a normal volume of glucose distribution suggests an increased absolute rate of tissue glucose uptake. This increase is likely to be due to the expanded mass of adipose tissue in these animals.

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On the origin of hyperglycaemia in the obese-hyperglycaemic mouse (obob): Effect of diet on blood glucose and serum insulin inobob and gold-thioglucose obese mice

On the Origin of Hyperglycaemia in the 0bese-Hyperglycaemic Mouse (obob): Effect of Diet on Blood Glucose and Serum Insulin in obob and Gold-Thioglucose Obese Mice C. CHLOUVERAKIS 0 0 Department of Medicine, State University of New York at Buffalo and E . J . Meyer Memorial Hospital , Buffalo, New York , USA I/origine de l'hyperglyedmie ehez la souris ob~e-Iwperglyegmique (obob): Effet du rdgime sur le niveau de la glycdmie et de Z'insuline du sdrum ehez obob et chez la souris rendue obese par aurothioglucose R6sumd. U n r6gime fiche ou d @ o u r v u d'hydrates de carbone a 6td donn6 ~ u n t r o u p e de souris ob~se-hyperglyedmique (obob), ~ u n deuxiSme groupe de souris rendues ob~ses par aurothioglucose (GTG-ob@se) et ~ u n groupe de t6moins. La glycdmie et Finsulin6mie ont 6td modifi@es par le rdgime chez t o u s l e s a n i m a u x et particuli@rement chez les obob et GTG-ob~ses. Le niveau de la glyc6mie 6taft dlev6 de fa~on 6gale chez les deux groupes de souris ob@ses. L'insulin6mie des souris GTG-ob~ses draft consid6rablement augment6e mais n ' a t t c i g n a i t pus le niveau de l'insulindmie observ6e chez les obob. Le t a u x de glycdmie des obob et des tdmoins est demeur6 constant q u a n d les a n i m a u x recevaient le r6gime d6pourvu d'hydrates de carbone. A c e stade, nous avons inject6 par voie iutraveineuse une quantit6 traceuse de UA4C-glucose. La vitesse de disparition du traceur ob6issait ~ une cin6tique d'ordre u n et 6taft semblable chez les obob et les tgmoins, sugg6rant ainsi que l'utilisation fractionnelle du glucose 6taft semblable chez les deux groupes. L'existenec d'une utilisation fractionellc normale du glucose ehez les obob jointe ~ l'hypcrglycdmie ct ~ u n volume normal de distribution du glucose sugggre que l'utilisation absolue du glucose chcz ces a n i m a u x est augmentde. Cette augmentation est vraisemblablement due ~ l'expansion du tissu adipeux chcz les obob. - Key-words: obob mice, gold-thioglucose obese mice, dietary, carbohydrate, hyperglycaemia, serum insulin, insulin resistance, glucose disappearance. I t is still u n c l e a r to w h a t e x t e n t excessive food i n t a k e c o n t r i b u t e s to t h e h y p e r g l y c a e m i a of t h e obesehyperglycaemie mouse (obob). Thus, the blood glucose r e t u r n s to n o r m a l w h e n obob are fasted e v e n for a few hours [ 12 ]. F u r t h e r m o r e , a l t h o u g h obese-hyperglycaemic mice (obob) show a n o r m a l response to small a m o u n t s of oral glucose, t h e response is a b n o r m a l if t h e y are given a n a m o u n t of glucose p r o p o r t i o n a l to t h e i r b o d y weight [ 2 ]. E v i d e n c e a g a i n s t excessive food i n t a k e b e i n g solely responsible for the h y p e r g l y c a e m i a has b e e n p r o v i d e d m a i n l y b y Mayer, who reported t h a t mice r e n d e r e d C. Chlouverakis: On the Origin of Hyperglyeaemia in the Obese-Hyperglyeaemic Mouse obese b y injection of gold-thioglucose, except for v e r y old and obese animals, are normo-glycaemic despite an excessive food intake [ 13 ]. In addition, an increased rate of endogenous synthesis of glucose and activity of gluconeogenie enzymes has been reported in the obob [ 15, 16, 9 ]. I t is therefore conceivable t h a t the hyperglycaemia of the obob might result from an increased endogenous synthesis of glucose. These two factors, i.e. excessive dietary intake of carbohydrates and endogenous production of glucose (via gluconeogenesis), were evaluated in the present study. Materials and Methods Animals: The animals used in this s t u d y were purchased from the J a c k s o n Memorial L a b o r a t o r y (Bar Harbor, Maine) and consisted of male obese- hyperglycaemie mice (obob) and their lean littermates. All animals were brought into the laboratory at the age of 4 to 5 weeks and a we@ later a number of lean animals were injected intraperitoneally with a solution of gold-thioglucoseI in water (60 mg/ml, 0.6 mg goldthioglueose injected per g b o d y weight). The actual experimental s t u d y began when all animals were 4 months of age. F o u r t e e n obese-hyperglyeaemic mice 14 lean littermates and 14 gold-thioglueose obese mice (GTG-obese) were subjected to a dietary experiment which is described below. I. Dietary experiment. This experiment lasted 30 days and was divided into four periods, two control and two experimental, as follows. 1st control period (1st to 5 th day) : All animals were fed a pellet mouse/rat diet (Teklad Inc., Monmouth, Ill.). 1st experimental period (6th to 15th day) : Lean, obob and gold.thioglucose obese mice (GTGobese) were separated into two dietary groups. One group was fed a high-carbohydrate diet and the other a carbohydrate-flee diet (Nutritional Biochemicals Corp., Cleveland, Ohio). There were thus six groups of animals (each consisting of seven mice) three of which were fed the high-carbohydrate and the other three the carbohydrate-free diet, both in powder form. 2nd control period (16th to 20th day) : As in the first control period all animals were fed the pellet mouse/rat diet. 2nd experimental period (21st to 30th day) : During this period the groups of the first experimental period were reversed and animals t h a t had taken the highcarbohydrate diet were now fed the carbohydrate-free diet and vice-versa. Thus, each animal served as its own control for either of the two experimental diets. 1 Generously donated by Dr. Tabaehnick, Ph. D-, Schering Corp., Bloomfield, lq.g. The composition of the three diets is shown in Table 1. The carbohydrate content of the high-carbohydrate diet was less t h a n t h a t of the pellet mouse/rat diet (47.0 v. 55.7%). Food intake was measured daily and the average food intake was derived for individual mice during each control and experimental period. Animals were weighed on the first and last day of the four dietary periods and body weight changes during these periods were determined. Glucose and insulin were determined on whole blood and serum respectively. Blood was withdrawn from the ophthalmic venous plexus under light ether anaesthesia. Glucose was estimated on the last d a y of each control period and on the middle and last d a y of each experimental period on 20 ~/blood, using a commercially available method (Glucostat, Worthington, Biochem. Corp., Freehold, N.J.). The two blood glucose values of each experimental period were pooled for each animal. Serum immunoreaetive insulin (IRI) was determined on the last d a y of each period using a previously published adaptation [ 2 ] of the double-antibody immunoassay of Hales and Randle [ 7 ]. II. Rate of glucose disappearance from the circulation in obob and lean control mice fed a carbohydrate-free diet. To test the variability of blood glucose in obob and lean mice an experiment was performed in which 12 male obob, four months old, and an equal number of lean control mice were divided into three groups. One of these groups was fed ad libitum the pellet mouse/rat diet (vide supra) before and during the experiment. The second group was fed a similar diet b u t was fasted the night before and also during the experiment. The third group had free access to the carbohydrate-free diet for a period of four days before and during the experiment. 20 ~/blood was withdrawn from animals for blood glucose estimation five times in the course of 6 ~ h, the first blood sample having been obtained at 9:00 a.m. No glycosuria was present in any of the animals fasted overnight or fed the carbohydrate-free diet. I n this experiment it was found (see Results) t h a t while on a carbohydrate-free diet blood glucose showed little variation and, therefore, in the absence of exogenous carbohydrates, the rate of glucose disappearance from the circulation was equal to that Vol. 7, No. 5, 1971 of endogenously produced glucose entering the blood stream. A tracer a m o u n t of radioactive glucose was injected intravenously into obese-hyperglyeaemic and lean control mice under these conditions of constancy of blood glucose, and the rate of disappearance of 14Cglucose was measured. The experiment involving determination of the rate of U-14C-glucose from the circulation of obesehyperglycaemic mice (obob) and their lean littermates was performed as follows: Results The order in which the two experimental diets were fed had no effect on the variables measured, i.e. mean food intake, b o d y weight, blood glucose and serum insulin levels. Similarly, these variables did not differ between the two control periods. Accordingly, the data were pooled for animals in each of the three groups (i. e. lean, obob and gold-thioglucose obese mice) on the same diet. Eight male obese-hyperglycaemic mice (obob) four months of age and an equM number of lean control mice, were fed the carbohydrate-free diet for a period of four days, at the end of which a fixed volume (0.2 ml) of a trace a m o u n t of U-14C-glucose in saline (5 ~a/ml, spec. activity 0.3 mci/mM, the RadiochemicM Centre, Amersham, England) was injected rapidly into the tail vein. Thus, each mouse received approximately 1 9a of radioactivity and 60 ~g glucose. At 5, 10, 15, 20 and 30 rain following the injection, 20 9/blood was t a k e n from each animal for determination of blood glucose radioactivity. 14C-glucose in the blood was determined b y an adaptation of the method of Delisle and Fritz [ 6 ] as follows: the blood was deproteinized with 3 ml of ZnSO4/Ba(OH)2 and an aliquot (1.5 ml) of the filtrate was mixed with 0.5 g of a mixed bed resin (AG 501--X8, 20--50 Mesh, Bio R a d Laboratories, Richmond, Cal.), pre-equilibrated with 0.5 ml of a glucose solution, 250 mg/ml. The mixture was incubated for 90 rain at 37~C in a shaking metabolic incubator. After centrifugation, 1 ml of the clear supernatant was pipctted into 10 ml of Bray's scintillator [1] and counted in a Nuclear-Chicago Liquid scintillation spectrometer using channel's ratio to correct for quenching. The data of dpm versus time were subjected to covariante analysis after logarithmic transformation of dpm, and the best fitting straight regression line was calculated separately for obob and lean mice [ 17 ]. A summary of these pooled data is shown in Table 2. While on the high-carbohydrate diet the daily food consumption was approximately the same for the three groups of animals. Lean mice, however, consumed significantly less quantities of the carbohydrate-free diet t h a n the two obese groups (p < 0.001). Obese-hyperglycaemic and gold-thioglucose obese mice consumed similar quantities of either experimental diet. The lower caloric intake of lean animals on the carbohydrate-free diet was accompanied b y a small b u t significant decrease in body weight compared with their weight on the high-carbohydrate diet (p < 0.01). There was no other, statistically significant, diet-effect on the animal's b o d y weight. This relative lack of effect of carbohydrate cont e n t of the diet on the animal's b o d y weight was in contrast to its marked effect on the blood glucose and serum insulin levels. Thus, the carbohydrate-free diet caused a significant decrease of the blood glucose and serum insulin in all three groups of animals. The blood glucose and serum insulin levels of the lean animals were significantly lower t h a n those of either groups of obese mice. I n addition, blood glucose level was almost identical in obob and gold-thioglucose obese mice fed the same diet, b u t serum insulin was significantly higher in the former (Table 2). The variation of blood glucose with time in the three dietary groups of lean mice was small, Similarly, blood glncose changed little in obob fed the carbohydrate-free diet or fasted overnight, b u t showed marked variation in obob fed the standard pellet diet (Fig. 1). The mean standard deviation derived from the standard deviations of o Fasting - Pellel diet 9 CliO free die1 T / 9 ~ T "~ "]" L~3~':: ""9.,.".................. o~...... lean..... 1 ~1 4 Time (hours) Fig. 1. Variability of blood glucose concentration in obob and lean control mice, fed a standard pellet diet, a carbohydrate-free diet, or following an overnigh~ fas$. Each point is the mean of four animals. Vertieat lines indicate standard error of mean. . . . . . . . . I I.. ! 15 2o 3o Time (minutes) Fig. 2. Disappearaaace rate of a trace amount of U-~Cglucose injected intravenously into obob and lean control mice. Each point is the mean of eight animals. Vertical lines indicate standard error of mean. the mean blood glucose of each mouse was greatest in mice fed the pellet diet, b u t did not differ significantly between mice fed the carbohydrate-free diet or fasted overnight (1o> 0.05). This was true for both obob and lean mice. While on a carbohydrate-free diet the dis_ T "r" I 6 9 9 leon o obob appearance of a trace amount of 14C-glucose from the circulation of b o t h obese and lean mice approached first-order kinetics, and the F-ratio for non-paralMism of the two regression lines [ 17 ] failed to achieve statistical significance (p = 0.2). These data suggest t h a t the apparent rate of glucose disappearance from the circulation is proportional to its concentration, and t h a t the proportionality constant is the same in lean and obese-hyperglycaemic mice (Fig. 2). Discussior~ The present data fail to support the contention made b y Mayer t h a t hy~erglycaemia characterizes mice with metabolic obesity and is not encountered in animals with regulatory obesity [ 13 ]. I n the present. experiments, obese.hyperglyeaemic and gold-thioglucase obese mice had similar blood glucose levels, regardless of nutritional status. Serum insulin was raised in b o t h obob and GTG-obese mice and was markedly decreased on feeding the carbohydrate-free diet. The raised serum insulin levels of the GTG-obese mice in association with their hyperglycaemia implies the presence of insulin resistance. These data, then, are in basic agreement with earlier reports indicating increased levels of both glucose and insulin in mice wif~ gold-thioglueose-induced obesi~r [t0, 5], and also with reports suggesting the presence of insulin resistance in adult rats with electrolytic lesions in the ventremedial nucleus of the hypothalamus [ 11, 8 ]. The blood glucose levels were similar in obob and GTGobese mice despite much higher insulin levels in the former suggesting a greater degree of insulin resistance in the obob compared with GTG-obese mice. This, however, should be expected if, as suggested b y previous studies, insulin resistance is secondary to obesity [3]. The obese-hyInarglyeaemie mice used in the present studies were considerably heavier t h a n the GTG-obese mice and the average value of their carcass fat was 39.5 g compared with 25.3 in the gold-thioglucose obese mice [4]. The present d a t a also indicate t h a t the nutritional status influences blood glucose more in obese-hyperglyeaemie t h a n in lean mice. Thus, obob respond to an overnight fast and to a carhohydrate-free diet b y a marked decrease of blood glucose. Despite this decrease, however, the blood glucose level of the obob is still bJgher t h a n t h a t of the lean libtermates. These findings are in basic agreement with previous reports showing an increased sensitivity of the blood glucose of the obob to fasting [ 12 ]. I n this context, an overnight fast and feeding a carbohydrate-free diet are basicMly similar in their effects on b o d y glucose homeostasis, in t h a t blood glucose is derived from endogenous synthesis in both. I t is of interest, therefore, t h a t there was no signfficant difference in the blood glucose of the obob mice fasted overnight or those fed a carbohydrate-free d i e t . Vol. 7, .No. 5, 1971 Two m a i n findings do not support the hypothesis t h a t increased caloric intake is solely responsible for the hyperglycaemia of obob and GTG-obese mice. First, while on the high-carbohydrate diet, food intake was comparable in lean and obese mice and yet, blood gincose levels were significantly higher in the obese animal. Second, following overnight fast and when fed a carbohydrate-free diet, obob mice still displayed a mild b u t significant hyperglycaemia. I t can, thus, be argued t h a t the allegedly increased gluconeogenie capacity of the obob mice [ 15, 16, 9 ] m i g h t be responsible for the hyperglycaemia of these animals when fasted overnight or fed a carbohydrate-free diet. However, under these two conditions the blood glucose was shown to be constant, implying t h a t the rate of glucose e n t r y into the blood stream was equal to t h a t of glucose removal b y the tissues. Thus, an increased rate of glucose e n t r y into the blood stream (resulting from increased gluconeogenesis) will lead to normoglyeaemia if accompanied b y a comparable rise in the rate of glucose disappearance from the blood. If tissues, however, fail to increase the u p t a k e of glucose, the blood glucose will rise until a new steady-state is achieved. Elucidation of the mechanism involved in the production of hyperglycaemia of the obese-hyperglycaemie mice is provided b y the present experiment involving the administration of a trace a m o u n t of 14C-glucose to mice fed a carbohydrate-free diet. The fractional rate of glucose-loss to the tissues was similar in obob mice and lean controls. This, in face of a normal volume of glucose distribution [ 2 ] and of a raised blood glucose level in the obob mice, suggests a greater absolute rate of glucose u p t a k e b y the tissues. Such an increased rate should be expected in these animals due to their expanded b o d y mass. The tissues of the obob mice, however, are capable of maintaining a normal fractional rate of glucose extraction at the expense of a m a r k e d increase in serum insulin since, even on a carbohydrate.free diet, there is a 20-fold increase of circulating immunoreact i r e insulin concentration s. Thus, on a carbohydrate-free diet (and possibly after an overnight fast) blood glucose and serum insulin levels in the obob mice stabilize at levels allowing an optimal rate of glucose extraction b y their expanded adipose tissue. The contrast between glucose-insulin homeostasis in h u m a n diabetics and obese.hyperglycaemic mice is obvious, since in the former the absolute glucose u p t a k e b y the tissues is normal and the fractional is low [ 18, 14 ], whereas in the obob mice the fractional glucose u p t a k e is maintained at a normallevel. Thus, fasting hyperglycaemia in h u m a n diabetes is caused b y a failure of tissues to m a i n t a i n a normal fractional rate of glucose uptake, whereas the hyperglycaemia of obesehyperglycaemic mice, subjected to overnight fast or to a carbohydrate-free diet, appears to be a consequence of their expanded adipose tissue, which is also respon2 I n the present discussion the assumption is made that the insulin of the obob mice is of normal biological activity [19]. Dtabetologia,Vol. 7 sible for their insulin resistance [ 3 ]. I n b o t h cases the hyperglycaemia represents a compensatory physiological response. Obese-hyperglycaemic mice could become euglycaemic only b y increasing the fractional rate of glucose extraction above normal. Such an increase, however, would require a further rise in the already elevated serum insulin levels, and might have the undesirable effect of suppressing the production of endogenous glucose [ 20 ]. These data, thus, are consistent with previous observations t h a t hyperglycaemia, hyperinsulinaemia, and insulin resistance in the obesehyperglycaemie mice are secondary to obesity [ 2, 3 ]. Aclcnowledgements. This work was supported in part by a Buswell Fellowship from the State University of New York at Buffalo and a Diabetes Training Program NIAMD AM-05507-04. The author is indebted to Mr. David Kolody for his excellent technical assistance. _~e_,fe.?'enc68 I ,, l,,, I.... I 2 I. Bray , G.A. : A simple efficient liquid scintillator for counting aqueous solutions in a liquid scintillation counter . Analyt. Biochem. l, 279 -- 285 ( 1960 ). 2. 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C. Chlouverakis. On the origin of hyperglycaemia in the obese-hyperglycaemic mouse (obob): Effect of diet on blood glucose and serum insulin inobob and gold-thioglucose obese mice, Diabetologia, 1971, 373-378, DOI: 10.1007/BF01219473