Upsala J Med Sci 92: 47-58, 1987 Insulin Receptor Binding and Metabolic Effects of Insulin in Human Subcutaneous Adipose Tissue in Untreated Non-insulin Dependent Diabetes Mellitus Peter Arner, Peter Engfeldt, Einar Skarfors, Hans Lithell and Jan Bolinder The Department of Medicine, Huddinge Hospital, Karolinska Institute, Stockholm, Sweden, and the Department of Geriatrics, Kungsgardet’s Hospital, Uppsala University, Sweden A B S T R A C T Insulin a c t i o n a t the t a r g e t tissue l e v e l i n non-insulin dependent diabetes m e l l i t u s was investigated using human adipose tissue. Specific adipocyte r e c e p t o r binding o f insulin and t h e e f f e c t s o f the hormone on glucose o x i d a t i o n and lipolysis were d e t e r m i n e d in subcutaneous adipose tissue. The study included 25 p a t i e n t s w i t h u n t r e a t e d non- insulin dependent diabetes m e l l i t u s and 38 h e a l t h y c o n t r o l subjects m a t c h e d f o r age, sex and body weight. Insulin s t i m u l a t e d adipose tissue glucose o x i d a t i o n in a dose- dependent way in t h e c o n t r o l subjects. On the other hand. a m a r k e d i n h i b i t i o n o f t h i s insulin e f f e c t was observed in t h e diabetics. A weak s t i m u l a t i o n was observed only a t high unphysiological hormone concentrations [> 0.7 n m o l / l l and the m a x i m a l insulin response was 6 t i m e s l o w e r t h a n t h a t in t h e c o n t r o l subjects. However, n e i t h e r s p e c i f i c insulin r e c e p t o r b i n d i n g nor t h e a n t i l i p o l y t i c e f f e c t o f insulin were i n h i b i t e d in diabetes. S i m i l a r r e s u l t s w i t h insulin binding and t h e m e t a b o l i c e f f e c t s o f insulin were obtained in non-obese normoinsulinemic d i a b e t i c s as compared t o m o d e r a t e l y obese hyperin- sulinemic diabetics. It is concluded t h a t adipose tissue insulin resistance in non-insulin dependent diabetes m e l l i t u s only involves glucose m e t a b o l i s m and n o t antilipolysis. Furthermore. it m a y solely be due t o postreceptor d e f e c t s in insulin a c t i o n and seems n o t t o be influenced b y obesity o r oversecretion o f insulin. I N T R O D U C T I O N In p a t i e n t s w i t h non-insulin dependent diabetes m e l l i t u s t h e hypoglycemic response t o insulin i s o f t e n attenuated. Insulin resistance in t a r g e t tissues is an i m p o r t a n t cause o f h y p e r g l y c e m i a in t h i s f o r m o f diabetes. When t h e disease i s accompanied b y obesity the a c t i o n o f insulin m a y be f u r t h e r inhibited. Insulin resistance in non-insulin dependent diabetes m e l l i t u s is usually a t t r i b u t e d t o a c o m b i n a t i o n o f i m p a i r e d hormone b i n d i n g t o s p e c i f i c c e l l s u r f a c e r e c e p t o r s and i n h i b i t i o n o f t h e signals f r o m t h e r e c e p t o r t o i n t r a c e l l u l a r m e t a b o l i c processes [14,15.18.22.261. It is suggested t h a t a l t e r a t i o n o f the r e c e p t o r is an e a r l y e v e n t t h a t leads t o a m i l d f o r m o f insulin resistance. w h i c h is subsequently aggravated b y i m p a i r e d i n t r a c e l l u l a r hormone signal c20.221. The investi- g a t i o n o f t h e insulin a c t i o n in non-insulin dependent diabetes m e l l i t u s in m a n has been c e n t e r e d m a i n l y on hormone binding t o c i r c u l a t i n g blood cells, where insulin has l i t t l e . i f any, b i o l o g i c a l e f f e c t and on t h e over-all e f f e c t o f insulin on glucose m e t a b o l i s m 47 i n vivo. It is a t present unclear how insulin action i n this disease is altered i n the primary target tissues [fat, muscle and liver]. It is also not known i f insulin action i n target tissues d i f f e r s between obese and non-obese diabetics. The aim of the present study was accordingly t o establish whether insulin receptor binding and/or the metabolic e f f e c t s of the hormone were altered i n adipocytes i n non-insulin dependent diabetes mellitus. Subcutaneous adipose tissue was obtained f r o m 25 untreated patients w i t h this f o r m of diabetes: 12 of the patients were non-obese and the others were moderately obese. Comparison was made t o the finding i n 38 healthy control subjects w i t h normal glucose tolerance, who were matched w i t h the diabetics f o r age, sex and body weight. M A T E R I A L A N D METHODS Patients: The series f o r the study consisted of 25 patients w i t h recently diagnosed and untreated non-insulin dependent diabetes mellitus. Twelve were non-obese and 13 were moderately obese. Moderate obesity was defined as a r e l a t i v e body weight of 115-150% of the average body weight. which was obtained f r o m tables i n Documenta Geigy [131. Glucosuria was present i n a l l diabetics but ketoacidosis i n none. They were otherwise healthy and there were no signs o f physical i n a c t i v i t y secondary t o the illness. A f t e r the investigation the diabetics received diet therapy, either alone or i n combina- t i o n w i t h sulphonylurea. Thirty-eight healthy volunteers w i t h normal values f o r either intravenous glucose tolerance [25 g glucosel or oral glucose tolerance [ l o 0 g glucosel served as control subjects: 18 were classed as moderately obese and 20 as non-obese. Some o f these subjects have been included i n previous reports as control subjects. The obese subjects had not followed any f o r m of slimming program during the six months preceeding the study. Clinical data o f diabetic patients and control subjects are present in Table 1 . They were matched w i t h respect t o age. sex and body-weight. The patients and control subjects consumed a diet consisting o f 40% carbohydrate, 40% fat, 20% protein and 7-8.5 MJ, according a 24-h recall. A l l subjects and patients were informed i n detail about the study and their consent was obtained. The study was approved by the Hospital's Ethical Committee. The investigation was performed a t 8 a.m. a f t e r an overnight fast a t the Out-Patient Department. Venous blood samples were taken f o r the determination of glucose [ 1 1 1 and serum immunoreactive insulin [301. Gluteal specimens o f subcutaneous adipose tissue were obtained surgically. Local anesthesia was induced w i t h prilocaine chloride. which was given i n such a way that it did not a f f e c t the metabolism o f adipose tissue [ll. Adipocyte insulin receptor binding: Isolated f a t cells were prepared by Rodbell's method [291. They were incubated i n 0.5 m l o f Krebs-Henseleit bicarbonate b u f f e r [ p H 7.41 containing dialyzed bovine serum albumin [40 g/ll, glucose [5 m m o l / l l mono 1251-[Tyr A ]-insulin L0.05 nmol/ll, and unlabelled insulin [O-50 nmol/ll. The f i n a l cell concentration was 8 % [vol/voll. A f t e r incubation f o r 60 m i n a t 24OC i n triplicate, 10 ml of ice-cold saline was added and the c e l l s were centrifuged through 1.2 ml of 14 48 a, 5 t .- a, 5 e 0 h m + I (D h 0, N + I m c : 2 + I c J + I m c - N + I N m c c \ J c m N rn U .- a, n 0 .- 1 d h J + I I0 0 c c c + I 0 c c d + I [9 J m + I - c c N + I m J m - \ 0 N m m rn - E Y c 0 N - h J + I + I m m a - J h ( D m + I m h. + I h. 0 c - N m + I 0 h 0 c c m + I + I - N h m N + I +I m c n h h N m +I + I o m m a - N O - N m + I m N - m + I m m 1D \ h m - w Lo w 8 I\ (D + I - m - - N + I c N c d + I m. a- N + I I\ N c rn + I 0 m m \ 0 - m c rn 0 c - Y 6 N 0 V 9 7 0 0 V 9 R 4-878571 49 silicone oil: they were then removed f r o m the o i l surface for r a d i o a c t i v i t y determination. Non-specific binding was measured as the amount o f 1251-insulin remaining in the cell layer in the presence o f unlabelled insulin [20 umol/ll. A l l the values presented were corrected for non-specific binding. which amounted t o 2 4 % . The method has been described in detail C5.71. It was previously shown t h a t [a1 insulin binding reaches a maximum a f t e r 30 m i n and remains constant for a t least 90 min, [ b l insulin degradation i n the incubation medium is insignificant and [ c l the coefficient o f variation w i t h i n one subject for insulin binding is 8 % C5.71. / Adipose tissue metabolism. Explants o f adipose tissue [about 100 m g l were preincubat- ed f o r 30 m i n and then incubated i n t r i p l i c a t e f o r 2 h a t 37OC in 1 m l o f Krebs-Henseleit bicarbonate buffer CpH 7.41 t o which dialyzed bovine serum albumin [40 g/ll. glucose 1 4 9 [5 mmol/ll. [U Cl-glucose [2 x 10 cpm/ll and insulin [O-7 nmol/ll were added. A mixture o f [CO :O 1 [5:951 was used as gas phase. A f t e r incubation glycerol release and production were determined as described i n detail previously L5.71. These measures were used as indices o f lipolysis and glucose oxidation, respectively. The c o e f f i c i e n t o f variance within one subject f o r the metabolic studies was 8%. Insulin degradation in the incubation medium. as determined by the tricholoroacetic acid precipitation method [51. was 5%. We have discussed i n detail previously why the metabolic e f f e c t s o f insulin are preferably detemined on segments o f f a t rather than on isolated f a t cells [71. 111 co2 2 2 Adipocyte cellularity: The f a t cell volume and surface area. and the number o f f a t cells incubated were determined as described i n detail previously r5.71. Chemicals: Crystalline. glucagon-free porcine insulin was a generous g i f t f r o m V i t r u m AB [Sweden]. Mono 1251-[Tyr A I-insulin [specific a c t i v i t y about 200 Ci/gl. was f r o m Novo [Denmarkl. U C-glucose [specific a c t i v i t y 270 m C i / m m o l l was f r o m the Radio- chemical Centre [England]. Bovine serum albumin [ f r a c t i o n V1 was f r o m Armour Pharma- ceutical Company [England]. 14 1 4 Statistical methods: The reported values are the mean SE. Student's unpaired t- test was used f o r statistical comparison. RESULTS F a t cell size was enhanced i n the obese diabetic and obese non-diabetic groups: the values for diabetics and control subjects were however, similar [Table 11. The fasting serum insulin level was t w i c e as high [p<0.021 for the obese diabetics than for the obese control subjects and 5 0 % higher [p 150%1 P i m a Indians 1191 and in a small group o f p a t i e n t s w i t h n o r m a l f a s t i n g insulin levels (71. However. the influence o f obesity and f a s t i n g hyperinsulinemia on insulin a c t i o n was n o t considered in these studies. Non-insulin dependent diabetics are o f t e n obese and hyperinsulinemic. B o t h these f a c t o r s m a y induce insulin resistance independently o f diabetes 1223. In t h i s study a s i m i l a r degree o f adipose tissue insulin resistance was observed i n non-obese diabetics w i t h n o r m a l f a s t i n g insulin levels as in obese diabetics w i t h f a s t i n g hyperinsulinemia. These r e s u l t s i n d i c a t e t h a t n e i t h e r obesity nor enhanced insulin s e c r e t i o n are o f i m p o r t a n c e f o r adipose tissue insulin resistance in non-insulin dependent diabetes mellitus. The present results w i t h insulin a c t i o n on lipolysis in diabetics d i f f e r e d m a r k e d l y f r o m those w i t h insulin-induced glucose oxidation. The a n t i l i p o l y t i c e f f e c t o f insulin was n o t i n h i b i t e d i n adipose tissue o f u n t r e a t e d non-insulin dependent diabetics. In t h e whole m a t e r i a l insulin a c t i o n on lipolysis was almost i d e n t i c a l in diabetes and t h e c o n t r o l s t a t e b o t h regarding insulin s e n s i t i v i t y and insulin responsiveness [ m a x i m u m hormone e f f e c t ] . Furthermore. n e i t h e r non-obese nor obese diabetics were r e s i s t a n t t o t h e a n t i l i p o l y t i c e f f e c t o f insulin. These d a t a s t r o n g l y support our o r i g i n a l hypothesis 12.71 t h a t a n t i l i p o l y s i s is n o t involved i n diabetes associated insulin resistance. F u r t h e r support f o r t h i s t h e o r y is t h e r e c e n t f i n d i n g o f n o r m a l a n t i l i p o l y t i c e f f e c t o f insulin in adipose tissue o f non-insulin dependent diabetics on long t e r m t r e a t m e n t w i t h d i e t plus sulphonylurea 1211. The f i r s t step i n the a c t i o n o f insulin is i t s binding t o s p e c i f i c c e l l s u r f a c e receptors. F r o m r e c e n t r e v i e w a r t i c l e s it would appear t o be a c o m m o n b e l i e f t h a t reduced r e c e p t o r number is an i m p o r t a n t f a c t o r underlying insulin resistance in insulin sensitive tissues in non-insulin-dependent diabetes m e l l i t u s [14.15.18.22,261. However. in t h e present study adipocyte insulin r e c e p t o r binding in a large group o f non-obese and obese p a t i e n t s w i t h u n t r e a t e d non-insulin dependent diabetes m e l l i t u s was n o t l o w e r t h a n in h e a l t h y subjects w i t h n o r m a l glucose tolerance, who were m a t c h e d f o r age. sex and body weight. These results are a t variance w i t h numerous studies w h i c h have demonstrated a de- 54 creased number o f insulin receptors on circulating lymphocytes. monocytes and erythro- cytes i n non-insulin dependent diabetes mellitus 13.12.16.23.24.27.281. However. evidence of decreased insulin binding t o circulating blood cells i s probably irrelevant since these cells are not natural target cells for insulin action. The results d i f f e r also f r o m those published by Kolterman and co-workers who observed decreased adipocyte insulin binding i n untreated non-insulin dependent diabetics [ZOI. In that investigation. however. the diabetic patients were significantly older than the control subjects. Age matching o f the groups is probably important in any comparison i n respect o f adipocyte insulin binding. since several reports have demonstrated that insulin binding t o human f a t cells decrease w i t h age [8.21.251. It is also possible that regional variations in adipocyte insulin binding play a role. We investigated gluteal f a t cells and Kolterman and co- workers investigated abdominal f a t cells. We have recently observed that insulin binding varies between d i f f e r e n t adipose sites in man 14.51. O n the other hand, normal insulin binding t o adipocytes has recently been observed i n some rare groups of untreated non-insulin dependent diabetics. namely ones w i t h normal insulin secretion [71 and young hyper-obese Pima Indians [191. Furthermore, normal insulin binding t o f a t cells was also observed i n non-insulin dependent diabetics receiving long-term diet and sulpho- nylurea therapy [9.211. Thus. when previous and present findings are considered together the adipocyte insulin receptor appears not t o be impaired i n any f o r m o f non-insulin dependent diabetes mellitus. This would seem t o call into question the role o f the insulin receptor i n the development o f insulin resistance i n this disease. It should be noted that i n the present binding experiments f a t cells were incubated a t 24OC. A t this temperature some of the insulin bound is internalized. In theory the l a t t e r f r a c t i o n may d i f f e r between diabetics and non-diabetics. However. i n human f a t cells we have recently observed [5.61 an excellent correlation [r>0.91 between insulin binding a t 24OC and 37OC [where a large portion of insulin is internalized] and between insulin binding a t 24OC and 16OC [where no insulin is internalized]. This indicates that differences in insulin internalization between diabetics and non-diabetics have l i t t l e bearing upon the present results w i t h insulin receptor binding. Furthermore, the observa- t i o n o f normal sensitivity o f the a n t i l i p o l y t i c e f f e c t of insulin argues further f o r t h a t the insulin receptor was normal i n our diabetics. since insulin sensitivity is thought t o r e f l e c t insulin action a t the receptor level [171. Apparently, the presently observed inhibition of insulin-induced glucose metabolism is solely due t o a post-binding alteration o f insulin action. Furthermore. this resistance t o insulin appears not t o involve the a n t i l i p o l y t i c e f f e c t of the hormone. The molecular mechanisms behind defects in insulin action on glucose metabolism a t the postreceptor level i n f a t cells of non-insulin diabetics are unclear. However, decreased a b i l i t y o f insulin t o Stimulate adipocyte glucose transport has recently been observed i n this f o r m o f diabetes [10.191. Such a defect may in p a r t explain the inhibition o f insulin-induced adipose tissue glucose metabolism found in this study. However, additional defects i n the intracellular action o f insulin may also cause the presently observed insulin resistance. particularly in the non-obese subjects where we also found a marked reduction o f basal glucose oxidation. In the l a t t e r respect it is o f interest t o compare adipose tissue metabolism i n our two diabetes groups. 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Address f o r reprints: Jan Bolinder D e p a r t m e n t o f Medicine Huddinge H o s p i t a l S-1 4 1 86 Huddinge SWEDEN 58