Effect of an α-glycosidase inhibitor on experimentally-induced obesity in mice

Diabetologia, Jan 1990

The effect of prolonged treatment with acarbose, an inhibitor of α-glycosidase, has been studied in mice made obese and hyperinsulinaemic by goldthioglucose. After the onset of obesity, one month after goldthioglucose administration, mice were then treated, with or without a 10% sucrose supplement, for four months with acarbose, added to the diet at 50 mg/100 g food. When mice received a standard diet, acarbose had no effect on body weight, blood glucose or insulin levels. In contrast, in the control obese mice receiving a 10% sucrose-enriched diet, it decreased the body weight gain, and prevented the rise in glycaemia and insulinaemia. Basal (non insulin-stimulated) glucose uptake, which is decreased in isolated soleus muscle from untreated obese mice, returned to normal values under acarbose treatment. However, muscle insulin resistance was not improved in acarbose-treated obese mice at maximal and submaximal effective concentrations, despite a higher insulin binding in muscles of acarbose-treated obese than in control obese animals. Furthermore, insulin receptor autophosphorylation and tyrosine kinase activity were altered similarly in treated and untreated obese mice compared to lean mice.

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Effect of an α-glycosidase inhibitor on experimentally-induced obesity in mice

Diabetologia 0 INSERM U145, Facult6 de Mddecine , Nice , France Summary. The effect of prolonged treatment with acarbose, an inhibitor of c~-glycosidase, has been studied in mice made obese and hyperinsulinaemic by goldthioglucose. After the onset of obesity, one month after goldthioglucose administration, mice were then treated, with or without a 10% sucrose supplement, for four months with acarbose, added to the diet at 50 mg/100 g food. When mice received a standard diet, acarbose had no effect on body weight, blood glucose or insulin levels. In contrast, in the control obese mice receiving a 10% sucrose-enriched diet, it decreased the body weight gain, and prevented the rise in glycaemia and insulinaemia. Basal (non insulin-stimulated) glucose uptake, which is decreased in isolated soleus muscle from untreated obese mice, Insulin receptor; tyrosine kinase; goldthioglueose obese mice; insulin resistance; muscle; hyperinsulinaemia; acarbose 9 Springer-Verlag1990 Effect o f an a - g l y c o s i d a s e inhibitor o n e x p e r i m e n t a l l y - i n d u c e d obesity in m i c e Y. L e M a r c h a n d - B r u s t e l , N. R o c h e t , T. G r 6 m e a u x , I. M a r o t a n d E. V a n O b b e r g h e n H u m a n and animal obesities are usually associated with insulin resistance, which in vivo is characterized by hyperinsulinaemia and normo- or hyperglycaemia [ 1, 2 ]. Insulin resistance is also present in vitro, in insulin target tissues such as skeletal muscle [ 3-6 ] or adipocytes [ 7 ]. In the muscle of obese animals and humans, this resistance results f r o m different abnormalities b o t h at the level of insulin receptor, i.e. decreased r e c e p t o r n u m b e r [ 3, 8 ] and altered r e c e p t o r tyrosine kinase activity [ 8, 9 ], and at steps i n d e p e n d e n t from the receptor, such as glucose and amino acid uptake or glycogen synthase activation [ 3, 4, 6, 10 ]. Acarbose is a competitive inhibitor of intestinal c~-glycosidase and thus inhibits starch and sucrose digestion at the level of the small intestine [ 11, 12 ]. T h e unabsorbed carbohydrates are subsequently degraded by microorganisms in the large bowel. This leads to a decrease in the postprandial rise of b l o o d glucose and, consequently, to a decreased insulin secretion. In genetically obese animals (fa/fa rats or db/db mice), t r e a t m e n t with acarbose was able to p r e v e n t hyperglycaemia and to decrease insulin levels. It also diminished the prevalence of diabetic nephropathy in acarbose-treated db/db mice [ 12-15 ]. Hyperinsulinaemia plays a k e y role not only in the d e v e l o p m e n t of the obese syndrome, since it p r o m o t e s adipose and liver lipogenesis, but also in the establishment and maintenance of insulin resistance [ 4 ]. T h e use of a model of experimentally-induced obesity makes it possible to p e r f o r m longireturned to normal values under acarbose treatment. However, muscle insulin resistance was not improved in acarbosetreated obese mice at maximal and submaximal effective concentrations, despite a higher insulin binding in muscles of acarbose-treated obese than in control obese animals. Furthermore, insulin receptor autophosphorylation and tyrosine kinase activity were altered similarly in treated and untreated obese mice compared to lean mice. tudinal studies on the d e v e l o p m e n t of obesity and on the effect of treatment before obesity has plateaued. Taking advantage of the potential lowering effect of acarbose on insulinaemia, the present work was u n d e r t a k e n to study the effects of prolonged o~-glycosidase inhibition on the d e v e l o p m e n t of the obese syndrome, and on the state of insulin resistance in skeletal muscle [ 4 ]. Materials and methods Materials Acarbose was a gift of Bayer laboratories (Wttppertal, FRG). Insulin (monocomponent) was purchased from Novo (Copenhagen, Denmark); Triton X-100, N-acetylglucosamine, goldthioglucose, and defatted bovine serum albumin were from Sigma Chemicals (St.Louis, Mo, USA). Wheat germ-agglutinin-agarose was from Miles Scientific. [7-32p]ATP(triethylammonium salt; aqueous solution, 3,000Ci/mmol) was from the Radiochemical Centre (Amersham, Bucks, UK). 1-[~4C]-deoxyglucosewas from New England Nuclear (Dreich, FRG) and ~z~Iwas from Commissariat ~tl'6nergie atomique (Paris, France). All reagents for sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) were from BioRad Laboratories (Richmond, Calif,USA). Serum from patients B5 or B7 with autoantibodies to insulin receptors was a gift from Dr R Gorden (Bethesda, Md, USA) and photoreactive insulin (Bz-[2nitro, 4azidophenylacetyl] desPhe Blinsulin) was from Dr D. Brandenburg (Aachen, FRG). .o ( > 0.3 g/day). They were then divided in a control group, which received chow without drug (which will be referred to as "control obese") and a treated group receiving chow containing acarbose ("acarbose-treated obese") for a period of four months. Animals were weighed weekly. In some experiments, age-matched lean mice, which received the sucrose supplemented diet, were also used. At least 30 mice were included in each experimental group and all the experiments were repeated with two-four different groups. Blood determinations Monthly blood samplings (100 p.1) were performed between 10.00 and 12.00 hours from the retroorbital sinus under diethylether anaesthesia, and plasma stored at - 20~ C. Plasma glucose and immunoreactive insulinwere then determined as previously described [ 3,16 ]. Deoxyglucose uptake and insulin binding to isolated soleus muscles Soleus muscles were isolated and incubated as described previously [ 3, 5 ]. One soleus muscle was preincubated for 60 min at 37~ C in i ml Krebs-Ringer bicarbonate buffer (pH 7.35), containing 10 mg defatted bovine serum albumin, 2 mmol/1 pyruvate, in the absence or presence of insulin at the concentration indicated in the figures. Deoxyglucose uptake was then measured after a 15 min incubationwith 2-deoxy-D-[1-14C]-glucose(0.5 mmol/1, 0.1 btCi/ml). At the end of incubation, muscles were washed, dissolved in 0.3 ml of i mol/1NaOH, and an aliquot sample used for determination of 14Ccontent [ 3 ]. Insulin binding was measured after a 4 h incubation at 20~ C as previously described [ 3 ]. Preparation o f partially purified insulin receptors and measurement o f insulin binding Muscles (5 g) from hindlegs were dissected and used for preparation of partially purified insulin receptors [ 9, 17, 18 ]. Aliquot samples (10g l) of receptor preparations were incubated in Hepes (50 mmol/1), NaC1 (150 mmol/1) buffer pH 7.6 containing lZSf-labelled insulin (10-11 tool/l, 250 mCi/mg) in the absence or presence Mice injected with goldthioglucose were treated without or with acarbose for the number of days indicated. The changes in body weight from the initialweight at the beginning of treatment (overall) and per month are indicated. Values are mean _+SEM of 30-35 mice in each group ] 90 I 120 Day of treatment Male Swiss albino mice were fed ad libitum and maintained at 22~ C on a 12 h light cycle until the time of death. They were rendered obese by goldthioglucose (GTG) injection at 3 weeks of age [ 16 ]. The composition of the laboratory chow (Us d'Alimentation Rationelle, Villemoisson, Epinay/Orge, France) was as follows (% weight): protein 22%, lipids 5%, starch 30%, hem cellulose and other carbohydrates 25%, salts 6%, water 12% (3000 cal/kg food). Where indicated, food was supplemented with 10% sucrose. Acarbose (50 mg/100 g food) was added and thoroughly mixed with the powdered mouse chow before it was reconstituted into pellets. Experimental protocol Mice received goldthioglucose at 3 weeks of age [ 3, 16 ] and were then weighed twice weekly. Since goldthioglucose treatment does not produce obesity in all injected mice, "preobese" mice were selected at 8 weeks of age on the basis of their body weight gain 30 60 90 120 30 60 90 120 6.3 10.0 12.7 14.4 11.4 18.1 ~ . 7  25.7 3.7 2.7 1.7 6.7 4.6 3.0 E ._= b 12.5 10 7.5 50 25 0 { I I I C ( I I I I ~...~r...~ I I I 90 I 120 d I 20 I 50 I 80 of unlabelled insulin (10-6 mol/1) in a final volume of 200 gl. After 2 h at 20~ C, bound insulin was separated from u n b o u n d hormone using polyethylene glycol [ 19 ] and the radioactivity of the pellet measured in a gamma spectrometer. To minimize interexperimental variations, the receptor preparations to be compared were always assayed in parallel, wheat germ agglutinin (WGA) -columns were identical, insulin binding was measured in the same assay and phosphorylation was carried out on the same day [ 20 ]. Cell-free phosphorylation and immunoprecipitation o f partially purified insulin receptors To evaluate, on the same gel, both the 13-subunit receptor phosphorylation and insulin binding to the a-subunit, the receptor subunits were labelled according to their functions [ 9, 20 ]. Receptors were first labelled on the c~-subunit using photoreactive insulin. After incubation in the dark for 3 h at 15~ C with 5 nmol/1 12SI-labelled B2Napa-insulin [21], samples were irradiated for 5 min at 4 ~ C using a mercury lamp. The receptors were then used in a cell-free phosphorylation system [ 9, 20 ] in order to label the ~-subunit. Finally, the receptors were immunoprecipitated with antibodies against the insulin receptor and analysed by one dimensional SDS/7.5% P A G E according to Laemmli [22]. The gels were stained, dried, and autoradiographed. The labelled bands were cut and their radioactivity determined for lasI (c~-subunit) and 32p radioactivity (13-subunit). Determination o f insulin receptor kinase activity using a synthetic substrate Partially purified insulinreceptor preparations (30 gl) were preincubated for 60 min at 20~ C in 140 ~tlHepes buffer (50 mmol/1, pH 7.6), containing NaC1 (150 mmol/1), bovine serum albumin (0.2 mg/ml) and insulin (10 -11 tool/1 to 10 7mol/1). The substrate [poly(glutamate-tyrosine, 4-1), final concentration 0.25 mg/ml] was then added for an additional 30 rain. Phosphorylation was initiated by adding 20 gl of a solution containing [7-32p]ATP (20 ~tmol/1, 3,000 cpm/ pmol), MnC12 (4 mmol/1) and MgC12 (8 mmol/1), final concentrations. After 30 min the reaction was stopped by applying 75 gl samples to filter papers (Whatman E T 31) and soaking the papers in a solution of 10% trichloracetic acid, and 10 mmol/1 sodium pyrophosphate. After extensive washing, radioactivity of the trichloracetic precipitable material adsorbed on papers was measured using Cerenkov radiation [ 9, 23 ]. Statistical analysis Statistical significance was assessed by Student's t-test for unpaired comparisons [ 24 ]. R e s u l t s Effect o f acarbose treatment on body weight, glycaemia and insulinaemia o f obese mice A s s h o w n i n F i g u r e 1, u p p e r p a n e l , a c a r b o s e t r e a t m e n t h a d n o e f f e c t o n b o d y w e i g h t o f g o l d t h i o g l u c o s e o b e s e m i c e w h e n n o r m a l f o o d w a s g i v e n t o t h e a n i m a l s . B y c o n Mice injected with goldthioglucosc were treated without or with acarbose, food being supplemented with 10% sucrose. Food intake was measured daily during the first, fifth and ninth weeks of treatment. Values are mean  SEM of 15-18 mice Day 80 80 Fig.3. Effect of acarbose treatment on individual insulin levels of obese mice. Obese mice receiving diet enriched with 10% sucrose were sampled after 80 days of treatment without (control) or with acarbose (50 rag/100g food). Each circle represents an individual insulin value. The bar represents the median value in each group. The hatched area corresponds to the range of insulin levels in normal lean mice ) " . J " .---U. . . . . . . . . L_//.J 0 0.5 I 1.5 i 5 E E trast, when f o o d was supplemented with 10% sucrose, acarbose p r e v e n t e d the additional body weight gain due to sucrose (Figure 1, lower panel, Table 1). Acarbose was affecting b o d y weight change mainly during the first month, the body weight gain being similar in control and treated groups during the second, third and fourth months of treatment (Table 1). This reduction in b o d y weight gain was observed without any change in f o o d intake (Table 2). Blood glucose and insulin determinations were perforlned monthly in the same groups of mice. W h e n mice received the normal diet, acarbo~e did not modify glucose levels (Fig. 2 a) but delayed sligfftly the increase in insulin levels which occurred in control obese mice (Fig. 2 b). However, it was no longer effective after three months of treatment. W h e n f o o d was supplemented with 10% sucrose, acarbose p r e v e n t e d the rise in blood glucose (Fig. 2 c) and the huge increase in insulin levels (Fig. 2 d) which were observed in control obese mice. Insulin levels were thus similar, at approximately 15 ng/ml, in acarbosetreated obese mice, whether they received the normal food or the diet enriched in sucrose. The insulin lowering effect of acarbose is even m o r e evident when individual insulin levels are presented (Fig.3). In the control obese group, all animals had insulin levels higher than the normal range (which is indicated by the shaded area), half of the animals having insulin levels higher than 30 ng/ml. By contrast, when animals received acarbose, one third of the animals had insulin levels in the normal range, and 90% of the animals presented levels lower than 20 ng/ml. It should be n o t e d that a small percentage of obese mice seemed totally resistant to the treatment, insulin levels remaining higher than 25 ng/ml. Effect o f acarbose treatment on glucose uptake and insulin binding in isolated soleus muscle Since acarbose was effective only when the food was supplemented with 10% sucrose, the following series of experiments were p e r f o r m e d in mice receiving this diet. An additional group of age-matched lean mice receiving the same diet was studied in parallel. Deoxyglucose uptake was measured as a function of insulin concentration in soleus muscles from mice which had b e e n treated for four months (Fig.4). In muscles f r o m acarbose-treated obese animals, basal glucose uptake, which was significantly (p <0.05) decreased in untreated obese mice, returned to normal. T h e insulin effect was similarly altered in u n t r e a t e d and treated obese mice both at submaximal and maximal insulin concentrations. Insulin binding to soleus muscles has b e e n determined with 125I-insulin in the absence (total binding) and in the presence (non-specific-binding) of unlabelled insulin (1 gmol/1) (Table 3, upper panel). Specific insulin binding was decreased by 35% and 22% in muscles of untreated and acarbose-treated obese mice, respectively, c o m p a r e d to lean animals. Similar results were obtained in partially purified insulin r e c e p t o r preparations (Table 3, lower panel). Effect o f acarbose treatment on insulin receptor autophosphorylation and kinase activity The first step in insulin action following h o r m o n e binding to the r e c e p t o r ~-subunit is the activation of the ~3-subunit tyrosine kinase [ 25-27 ]. This enzymatic p r o p e r t y was then studied in partially purified p r e p a r a t i o n s of receptors obtained f r o m lean, u n t r e a t e d and a c a r b o s e - t r e a t e d obese mice. Insulin receptors were incubated with 125I-photoreactive insulin and subsequently submitted to p h o s p h o r y lation, b e f o r e i m m u n o p r e c i p i t a t i o n with n o r m a l s e r u m (Fig. 5, lanes A, C, E) or antireceptor antibodies (Fig. 5, lanes B, D, F). The a u t o r a d i o g r a m s clearly show that less p h o s p h o r y l a t i o n of the insulin r e c e p t o r 13-subunit occurred b o t h in control obese and a c a r b o s e - t r e a t e d obese mice c o m p a r e d with lean mice. This observation was confirmed by measuring the radioactivity present in the two bands: for an equal a m o u n t of insulin binding, 22% less radioactivity was found in the ~-subunit of control obese or a c a r b o s e - t r e a t e d obese c o m p a r e d to lean animals. T h e ability of insulin receptors to catalyse the p h o s p h o r y l a t i o n of an exogenous substrate, a c o p o l y m e r of glutamate and tyrosine residues in a 4:1 ratio, was then investigated (Fig. 6). In four different insulin r e c e p t o r preparations, c o m p a r e d to lean mice p r e p a r a t i o n s where the insulin effect on kinase activity was expressed as 100%, stimulation was found to be 47.2 _+5.7% and 47.7 + 2.5%, while insulin binding was 70.7 + 4.3% and 85.3 + 3.2% in control and a c a r b o s e - t r e a t e d obese, respectively ( m e a n + SEM). Discussion The results p r e s e n t e d in this study show that acarbose t r e a t m e n t partially p r e v e n t e d the d e v e l o p m e n t of obesity, h y p e r g l y c a e m i a and hyperinsulinaemia in goldthioglucose obese mice which received a diet enriched with 10% Acarbose-treated obese 6.6 + 0.6 Insulin binding was measured in soleus muscles in the presence of 125I-insulinin the absence (total) or in the presence (non-spedfic) of insulin (10-6 mol/1). Insulin binding to solubilized receptors was measured as described in Materials and methods. Values are the mean _+SEM of six (soleus muscles) or five (partially purified receptor preparations) Fig.S. Effect of acarbose treatment on insulin receptor autophosphorylation of obese mice. Muscle insulin receptors were partially purified from lean (lanes A and B), control obese (lanes C and D), and acarbose-treated obese mice (lanes E and F) at the end of four months of treatment, on a 10% sucrose-enriched diet. Insulin receptors were incubated in the dark for 3 h at 15~ C with 125I-labelled photoreactive insulin (10 nmol/1).After ultraviolet irradiation, samples were phosphorylated with 7-[32p-ATP](15 gmol/1)for 15 min at 20~C. Samples were immunoprecipitated with normal serum (lanes A, C, E) or serum containing antibodies to the insulin receptor (lanes B, D, F), solubilized and analysed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis as described in Materials and methods. Each band was cut out, radioactivity measured, and the following results were obtained: c~-subunit, lane B, 11,710, lane D, 10,046, lane F 8949 cpm; ~-subunit, lane B, 1899, lane D, 1281, lane F, 1051 cpm sucrose. Similar to the observations m a d e in db/db mice [ 14 ], food intake was not modified by acarbose. The significantly reduced b o d y weight of the t r e a t e d - o b e s e animals can t h e r e f o r e be attributed only to the m e t a b o l i c effects of the drug. Following oc-glycosidase inhibition, the rate of intestinal c a r b o h y d r a t e absorption is reduced [ 11, 13 ] and consequently, b l o o d insulin levels are significantly lowered resulting in decreased rates of liver and adipocyte lipogenesis c o m p a r e d to control obese mice. T h e effect of acarbose in preventing the b o d y weight changes was noticeable only during the first m o n t h of treatment. It is possible that the efficacy of the drug was less i m p o r t a n t after some weeks of t r e a t m e n t since p r o l o n g e d a-glycosidase inhibition induces significant changes in small intestine disaccharidase activity in treated animals [28]. H o w ever, this adaptation is not likely to be very large since lowering effects of acarbose t r e a t m e n t on insulin levels persisted even after three or four months. It should be noted that a small p r o p o r t i o n of the mice did not display any beneficial effect of acarbose therapy, since they p r e s e n t e d v e r y high insulin levels during the whole study, .>m .~ "7 s e~ E g -5 E 0 0 o./'X:" A//~///. / a finding which could be explained by elevated contents of disaccharidases in their intestinal mucosa. T h e lowering effect of acarbose on insulin levels was clearly not sufficient to p r e v e n t all the alterations in muscle glucose metabolism. Indeed, when glucose u p t a k e was studied in muscles of mice which had b e e n treated for four months, only basal u p t a k e returned to n o r m a l values. D e c r e a s e d basal (non-insulin-stimulated) glucose u p t a k e has b e e n substantiated in different tissues such as skeletal muscle [ 3, 5, 6, 18 ] or adipocytes [ 7, 29 ] of obese animals and humans. This alteration is thought to play a m a j o r role in the d e v e l o p m e n t of hyperglycaemia and insulin resistance. This diminution results, at least in part, f r o m the reduction in the n u m b e r of glucose transporters [ 28, 30 ]. In adipocytes f r o m fasted and refed rats, the n u m b e r of glucose transporters strongly correlates with the specific m R N A a b u n d a n c e [31]. It is tempting to hypothesize that insulin resistance is also associated with changes in the level of the glucose transporter m R N A and that acarbose is able to prevent both the decrease in transporter m R N A and in glucose transport activity which occurs in obese animals. Clearly, m o r e studies are n e e d e d to clarify the mechanism of its action, but this effect of acarbose therapy p r o b a b l y participates in the prevention of hyperglycaemia in obese mice. A c a r b o s e t r e a t m e n t also partially p r e v e n t e d the decrease in insulin binding, as m e a s u r e d b o t h in intact soleus muscles or in partially purified insulin r e c e p t o r p r e p a r a tions, a result which was anticipated f r o m the modifications in insulin levels. Scatchard analysis of the data shows that this reflects changes in insulin r e c e p t o r n u m b e r (data sufficient to p r e v e n t insulin resistance in muscle tissue. Indeed, muscle f r o m acarbose-treated mice was still markedly resistant to insulin b o t h at s u b m a x i m a l and maximal effective concentrations. F u r t h e r m o r e , when autophosphorylation of insulin receptors and their tyrosine kinase activity were studied, the alterations were similar in preparations of control obese and acarbose-treated obese mice. Such a defect in the insulin r e c e p t o r tyrosine kinase activity has also b e e n r e p o r t e d in skeletal muscles and adipocytes of obese Type 2 (non-insulin-dependent) diabetic patients [ 8, 9, 32, 33 ] and p r o b a b l y plays a key role in the insulin resistant state. Although the exact m e c h a n i s m of the alteration of the kinase activity has not yet b e e n determined [17], it is clear f r o m this study that it does not directly relate to the level of insulin. In conclusion, this study suggests that acarbose m a y be of use in reducing the d e v e l o p m e n t of obesity and hyperinsulinaemia, in conjunction with high intake of carbohydrates. However, the drug is insufficient to reverse or prevent muscle insulin resistance. Acknowledgements. We thank Dr. Bischoff, Bayer AG (FRG) for supplying acarbose and for his scientific comments throughout this study, and Bayer Pharma, France, for financial support. We thank Dr. G. W.G. Sharp for his critical reading of the manuscript. We are greatly indebted to Ms. N. Gr6meaux for technical assistance, and to Mr. C. Minghelli and Ms. A. Grima for illustrations. The generous gifts of antireceptor antibodies by Dr. R Gorden (Bethesda, Md, USA) and of photoreactive insulin by Dr. D. Brandenburg (Aachen, FRG) are gratefully acknowledged. R e f e r e n c e s Dr. Y. Le Marchand-Brustel INSERM U145 Facult6 de Mddecine Avenue de Valombrose F-06034 Nice Cddex France 1. 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Y. Le Marchand-Brustel, N. Rochet, T. Grémeaux, I. Marot, E. Van Obberghen. Effect of an α-glycosidase inhibitor on experimentally-induced obesity in mice, Diabetologia, 1990, 24-30, DOI: 10.1007/BF00586457