DI-0253 Postreceptor Effects Skeletal Muscle Glyc of Sulfonylurea on ‘ogen Synthase Activity in Type II Diabetic Patients JENS FRIIS BAK, OLE SCHMITZ, NIELS SCHWARTZ SORENSEN, AND OLUF PEDERSEN To elucidate the subcallular mechanism of action of sulfonylurea on glucose utilization of skeletal muscle, ‘we studied nine newly diagnosed patients with type i {non-Inaulln-dependent) diabetes. Examinations were Performed betore and after 8 wk of gliciazide therapy. Glictazide trestment waa associated with Improved ‘glycemic control and enhanced pancreatic f-cell responses to meal stimulation. During euglycemic Insutin clamps, Insulin-inhibited endogenous glucose production was Improved after gilciazide therapy. Moreover, mean (=SE) glucose disposal rate Increased from 3.2 = 0.7 to 4.8 = 0.8 and from 7.9 = 0.9 to 10.4 = 0.9 mg - kg" - min" at In vivo pleama Insulin levels of ~75 and ~320 mU/L, In addition, synthase activity were analyzed in akeletal ‘muscle biopsies obtained in seven patients, The _ blopsies ware obtained during bass! Insulnemia and hyperinsulinemia (~320 mU/L) before and ater treatment, insulin receptors purified with wheat. ‘germ agglutinin showed unchanged ineulln-binding Properties and ur receptor kinase function ‘with reapect to basal and insulin-etimulated lation of exogenous peptige e Gliclazide treatment had no affect on the maximal” ‘ctivites of glycogen synthasa. Moreover, in blopsies oblained at basal inaulinemie, the halt ‘maximal activation constant for glucose &-phosphate (Aq) was Identical betore and afer (ost = In blopate obtained mt hypornauinemie, Aus in biopsies obtained at hyperinsulinemia, Aa, waa 0.30 = 0.05 va. 0.20 = 0.02 mM before and efter gilelazide therapy, P <.04. In conclusion, this study Indicates that gliciazide, in addition to hs splanchnle Gucose 1M = 18mg/a lee _1 pM = 0167 wi Flom ti Unieraiy Cine of Enaggwnoop ard inerai Vac ard Fe Deparment of Cncal hemi, Anue anyon: An. Geear ities coresgoncanes wore eureit oo Jag Bax Nec came teygeni. 8000 hn Denmark or puoscaton 2 Noverer 1008 ang accepted reed polyiGiurrtye). Fouseas eT? 06.02.1998 PU04311ENGO ouicuazise. IAAL fects, may enhance insulin-timutated peripheral ‘ghicose metabolism thraugh a potentiation of Insulin {ction on skeletal muncle glycogen aynthaes by ‘mechanism dlstal to the Inaulln-recaptoe kinase, Diabates 38:1343-50, 1909 (Ow sullonylureas mediate their glucose-iowering effects is stil debated. Although there appears 10 bbe no doubt about their acute stimulating effects Oninsuin secretion, numerous reports indicate that long-term effects of sulfonylurea eatment involve extrapan- reatic mechaniems. With eugiycemic insulin clamps in com ‘ination with. isotope-diltion techniques, the ‘extrapan- reatic effects seem lo be attributable to both a potentiation ==ot msulinnmediated: suppression of hepatic glucose output 412) and an enhancement of insuv-simulated peripheral ‘glucose disposal (1.3) Alter a carbohydrate load, most insulin regulated glucose Uptake takes place in skeletal muscle 4,5) In muscie biopsy specimens, measurements of glycogen synthase actives canbe used to assess insulin action. Insulin-mediated ac- tation of glycogen synthase i regulated covalent through _dephosphoryations by a phosphoprotein phosphatase, and therefore. glycogen synthase can remain in the activated slate after tissue extraction. Activated glycogen synthase is ‘Tore sensitve to-the-allosteic ‘activator glucose 6-phos- phate (GEP), and consequently. measurements of dose-re- ‘sponse characterisics ol GEP activation provide a senstive ‘estimate ofthe insuin effect on the muscle in vivo (6). Thus, ‘reduced activation of glycogen symthase in skeletal muscle has been found in insulinresistant patients such as nondi- abetic obese subjects (7.8) and type Il (non-insulr-cepen- ent) diabetic subjects (8). Moreover, insulin receptors can be purified trom skeletal muscle, and the insuinceceptor kinase-meciated phosphorylation of exogenous substrates ‘appears to be equally reduced in obese subjects and type 'ciabetic patients (10,11), whereas receptor autophospho- ‘Mlation of the B-subunit is normal (11) In contrast, in adie 130.ocyte-derived insulin recepiors. autophosphoryltion and exogenous substrate phospharylaion are reduced in type It Giabetic patients but normal in obese subjects without cia betes (12). Tis siUay was designed to examine ne impact ot the sulfonylurea giclazide on insulin action in skeletal muscle of type II diabetic patents. The protocol included measur ‘ments of in vivo glucose metabolism, insulin-receptor func- tion, and glycogen synthase actly in needle biopsies of ‘skeletal muscle obtained at fasting insulinemia and hyper- insulinemia betore and atter acive drug therapy. RESEARCH DESIGN AND METHODS Patients were included in the study according tothe folowing criteria. newly diagnosed diabetes melitus; aye 30-70 yr, ‘acceptable C-peptide response to an intravenous f-mg glu- ‘cagon load (i... >0.50 nM atter 6 min) no signs of hepatic, renal, cardiac. or metabolic disease: and absence of late iabetic complications. Al patients had been observed dur- ing treatment with an individualized fow-lat high-starch diet for >1 mo. Those who obtained acceptable glycemic control ‘during this Giet were excluded. Thus, the nine patients who were includes stil hac tasting plasma glucose >7 mM and/ oF presence of glucosuria >7 mmol/26h after diet treatment. ‘According to the eocond Declaration of Helsinki, verbal in- formed consent was obtained tom all the patients before the investigation. The patients are characterized in Table 1. Because glucose-owering treatment may have tong- standing effects on diabetic control even after cessation of therapy. we desisted from performing a double-blinded ‘cross-over examination of gicazice therapy. Patients were ‘examined forthe frst time after 3 wk of placebo treatment (1 tablet twice daily) and then again after 8 wk of glclazide {weatment (Diamicron, kindly provided by Servier, Paris; 80 mq twice daly) The participants were informed about the pplacebo-contralied design of he study but not about which of the treatment periods consisted ol active drug therapy. Alter the fist 3 wk of active drug treatment, giclazide dose was fixed at 80 mg 2-4 times/ .1), However, insulin-stimulated glucose dsposal was significantly potentiated by giclazide treatment (3.2 + 0.7 ys, 48 = 08 mg» kg"! min, P< 05, at ~75 mU/L Plasma insulin and 7.9 + 09 vs. 10.4 = 9 mg kg 16 Endogenous glucose production (mg/kg/d mmin-* at ~320 mU/L plasma insulin, P < .06: Fig. 2). In addition, suifonyivrea potentiated the insuin-mediated ‘suppression of the estimated hepaic glucose production rates at ~75 mU/L hyperinsulinemia (P <.08: Fig. 2). Plasma ftee-taty acid levels inthe basal period ware timiae belore ‘and after giclazide therapy, and they were suppressed equally during insuin infusions (data not shown). ‘Skeletal muscie biopsies. n seven ofthe patients, sutficient amounts of skeletal muscle tissue (>200 mg) were obtained in all four biopsies to analyze insulin-receptor function ang glycogen synthase activity according to the protocol, One Of the patients retused to have the fourth muscle biopsy taken, and in another subject. data were excluded due to technical diticuities during the preparation. Insulin binding, Scatchard analysis cl bincing data showed ‘ronlinear plots, The estimated binding constants of high- affinity binding sites were similar belore and ater treat ‘ment in the muscle biopsies obtained at basal insulinemia (335 = 47 vs. 375 + 67 pM insulin) and in the biopsies ‘obtained at hyperinsulinemia (35.9 = 5.7 vs. 96.9 = 6.7 pM 12, 10: Gluce: (eng /kg/mmin) 2 o 160 ‘plasms insulin (mU/i) 200 300-400 i. 2. Rate of geese caposa! and endogenous glucose production sion vam dem tore () ar ai (@) # mo tase I f OBETES. VOL. 38, NOVEMBER 1969,{R. 3. Sestohard plots for aula bing 1. 1. The Hill coeticients were ‘comparable, and they were equally reduced during insulin infusion belore and ater treatment (1.92 = 0.06 vs. 1.20. Insulin (aM) seerBASAL INSULINEMIA HYPERINSULINEMIA 100: (0.02 betore treatment, P < 05, and 1.90 + 0.04 vs, 1.18 = 005 attor ueatmant, P < 06), DISCUSSION ~ ‘The patients in his study were all newly diagnosed. ie. they were examined within the tst yr after diagnosis and alter a let treatment thal had been unsuccessful. In accorcance with previous studies, we found thatthe improved glycemic 1 contol obtained during 2 mo of sufonyturea treatment is followed by unchanged fasting plasma levels cf insulin and C-peptide (1) but elevated postprandial levels (1.3). How ‘ever, his improved meal-simulated insulin secretory pattern might a leat in pan be secondary tote eiminned "gi ose toxcaton” of ihe pancreate B-cels (22.23). ‘The notion that sulonyurea has exrapancrealic effects in addition tots pancreatic efects was iially based on stud- ies of long-term administration of these drugs showing In- tensifed efects of insuin (24) However. vivo insulin en sity canbe estimated more precisely wit the euglycemic insun clamp in combination wih an iotope-dition tech. nique to quantity total glucose tumover. Thus, our findings that suforyivrea enhanced insul-stulated but not basal in vwo Qlucase dispasal conf stber airing (325 26) However, due tothe compex and integrated nature of giv ose and insutin metabolism in vivo, the exact mechanism ‘of the hypogycemic acto of sulfonylurea treatment i si a matter of debale. For instance, the enhances insuln-me- diated peripheral glucose uptake ater sulfonylurea therapy might be secondary to the improved diumal glucose levels ei ‘We examined peripheral effects of sulonyiurea by ana- Iyzing insulivreceptoe function and glycogen synthase ac twvkyin biopsies of skeletal muscle obtained before and ater @ hyperinsulinemic-eugiycemic clamp. Giciazice therapy dic not change the yield of skeletal muscle-derived WGA- puried insuin receptors significant, in accordance with stucies.ofasuln nding to human at eels (1). in contrast, inguin Dincing to monccytes has been shown Yo be in’ ‘teased afte sulonyutea therapy in some stucies (28.28) Dut notin al (25.28). However, slucies on blood cels may be oflimited importance forthe interpretation of inulin action in peripheral tissues. Our insuln-binding data trom sohb ‘ized insulin receptors mht not be comparable with those of studies tat used intact cals and should probabiy be ‘rterproted wih some caution. In addon, in his study, we measured the insuli-receptor kinase-meciated phosphoryation of a synthetic substrate Similar kinase actwvites wer found betore and ater gicla- ide treatment, indicating thatthe potentiating effects of sullonyiutea treaiment on insulin acon at penpheral issues |S not meciated ttrough enhanced responsiveness of the insulineceptor kinase to nsuin stimulation. A simar con- clusion was drawn by Jacobs and associates (90-32). who tncubated isoated rat auipocyles fr 20 nin tne absence oF presence ot suonyurea. Thus, sullonyreahas been shown to enhance the-suln-stimulaied glucase uptake in acipo™ _eftes (30), de to & potemiaing ettect onthe insulins “Gla reeittmint of glucosétrarsperters trom an itracel: lular pool to the piasma membrane (31). Ths efect of sulfonyturea did not appear to be secondary to an increased: actviy ofthe insulin-receptor kinase (32). The importance of muscle glycagen synthase for nsulin- stimulated glucose disposal is sll iscussed. A correlation Between in vive glucose cisposel rote and glycogen ayn ‘nase activity in skeletal musce biopsies nas been found in numerous studies (33-36). Glycogen synthase regulates ‘lycogen depostion in muscle cals, and it is activated in an allosteric manner by GEP When musci cols are expoced to insulin. ghycogen synthase is dephosphorylated by a jnespnoproten phosphalase. Te less phesphoryiales frm OIABETES, VOX. 98, NOVEWEER 1989by increased rates of giycogen eynthesis al subsal- rating GEP revels (Hractional velocities) and a decrease in the concentration of GEP required to hat maximaty activate the enzyme (44, for GAP: 19) Alineugh giclazide Rael na etfect on the insui-receptor function. potentiated insulin effects on the glycogen synthase. Thus. in the muscle bi0p sigs obtained during insulin infusion, hall: maximal activation ‘of glycogen synthase was achieved at 0:30 = 0.05 mM GSP before treatment compared to 0.20 = 0.02 mM GEP after gliclazice treatment. This corresponds to recent findings by ‘Johnson et al. (37). The kinetics of skeletal muscle glycogen synthase activation by GP exhibit positive cooperativity as demonstrated by a Hil coefficient >1. This cooperativity tends to diminish after physical exertion (38) and meal sim- Ulation (10). Gliclazide did not infuence the basal positive cooperativity or the insulin effect on this From this data, we cannot exclude the possibilty that the peripheral effects of giclazide are secondary fo the long- term improvements in metabolic control. However, severa feports suggest that the insuin-meciated activation (de- Phosphorylation) of glycogen synthase is independent of ‘glucose flux into muscle cells (34.39.40). Moreover, it has Deen reported that improved glycemic control during long- term inguin treatment of type ll diabetic patents does not affect insulin activation of glycogen synthase (41). The pa- tients in out study had significantly higher diurnal insulin levels after sufonyistea therapy. However, chronic overn- ‘sulinization does not appesr to increase the sensitivity of glycogen synthase to acute insulin stimulation (42). indeed, fasting plasma insulin levels in our patients were not signi- ‘cantly diferent betore and after sulfonylurea treatment. ‘According to these considerations, the potentiated insulin ‘tfect on glycogen synthase is not ikely to be an indirect ctfect of improved glycemic control or higher diumal plasma Insulin levels ater Qiciazice treatment. Therefore. a primary ostreceptor kinase effect of giclazide on the muscie cell: ‘might be suggested. Such an effect would be expected 19 involve the phosphoprotein phosphatase or one of the kin= ‘ases that regulate phosphorylation of glycogen synthase. To ‘speculate on how sulfonylureas might accomplish their cel> lular etfects, the mechanism of action in cultured insulin= [producing cell ines should be considered. Sulfonyiureas bind to high-atfinty receptors, blocking an ATP-sensitive ‘channel (43). Thus, K* efflux's reduced. The resulting de- Polarization allows entty of Ca, which Wiggers insulin release from B-cells (4448). Because the ATP. dependent K* channels nave also"been demonstrated in skeletal muscle (46). any direct peripheral action of suifo- ‘ylurea might be hypothesized to involve enhanced cellular ‘depolarization, intiated by binding of sulfonylurea to a re- Cceporike structure in the plasma membrane of the muscle. Obviously, to settle these questions, further research inthis field is needed, - Whether the regulation of hepatic glycogen synthase is atfected by ghilazide treatment ic not known. The regulation (of hepatic glycogen synthase by glucose and insulin is cit- ferent from the regulation of skeletal muscle glycogen syn- ‘hase (29). Consequently, itis not possibie trom our data to deduce an effect of giciazide on glycogen synthase activity In hepatic tissue. In conclusion, these data indicate thal giclazide, in ad- OWBETES, VOL 38, NOVEMBER 1989 ition to its pancreatic elects, improves insulin elfecis on the iver and peripheral tissues in type Il ciabetic patients, In skeletal muscle, giclazide appears to potentiate nsukn- ‘mediated activation o! glycogen synthase—directly oF ind- tmetiy— by a mechanism ciatata the insulin receptor kina ‘ACKNOWLEDGMENTS, We thank Anette Mengel, Lisbet ak, Tove Skrumsager, and Pemilie Sonne for excelent technical assistance. This study was supported by the Institut De Recherches. 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