Although the primary defect responsible for the

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1 Rapid Publications Improved Glucose Tolerance Restores Insulin-Stimulated Akt Kiiiase Activity and Glucose Transport in Skeletal Muscle From Diabetic Goto-Kakizaki Rats Anna Krook, Yuichi Kawano, Xiao Mei Song, Suad Efendic, Richard A. Roth, Harriet Wallberg-Henriksson, and Juleen R. Zierath The serine/threonine kinase Akt (protein kinase B [PKB] or related to A and C protein kinase [RAC]) has recently been implicated to play a role in the signaling pathway to glucose transport. However, little is known concerning the regulation of Akt activity in insulinsensitive tissues such as skeletal muscle. To explore the role of hyperglycemia on Akt kinase activity in skeletal muscle, normal Wistar rats or Goto-Kakizaki (GK) diabetic rats were treated with phlorizin. Phlorizin treatment normalized fasting blood glucose and significantly improved glucose tolerance (P < 0.001) in GK rats, whereas in Wistar rats, the compound had no effect on glucose homeostasis. In soleus muscle from GK rats, maximal insulin-stimulated (120 nmol/1) Akt kinase activity was reduced by 68% (P < 0.01) and glucose transport was decreased by 39% (P < 0.05), compared with Wistar rats. Importantly, the defects at the level of Akt kinase and glucose transport were completely restored by phlorizin treatment. There was no significant difference in Akt kinase protein expression among the three groups. At a submaximal insulin concentration (2.4 nmol/1), activity of Akt kinase and glucose transport were unaltered. In conclusion, improved glucose tolerance in diabetic GK rats by phlorizin treatment fully restored insulin-stimulated activity of Akt kinase and glucose transport. Thus, hyperglycemia may directly contribute to the development of muscle insulin resistance through alterations in insulin action on Akt kinase and glucose transport. Diabetes 46: , 1997 From the Departments of Clinical Physiology (A.K., Y.K., X.M.S., H.W.-H., J.R.Z.) and Molecular Medicine (S.E.), Karolinska Hospital, Stockholm, Sweden; and the Department of Molecular Pharmacology, Stanford University School of Medicine (R.A.R.), Stanford, California. Address correspondence and reprint requests to Juleen R. Zierath, PhD, Department of Clinical Physiology, Karolinska Hospital, SE Stockholm, Sweden. jrz@klinfys.ks.se. Received for publication 31 July 1997 and accepted in revised form 18 September AUC, area under the curve; GS-K-3, glycogen synthase kinase 3; KHB, Krebs-Henseleit buffer; PDK1, phosphatidylinositol 3,4,5-trisphosphate-dependent protein kinase; PI, phosphatidylinositol; PKB, protein kinase B; RAC, related to A and C protein kinase Although the primary defect responsible for the development of NIDDM is unknown, a combination of genetic and environmental factors contribute to the manifestation of this progressive metabolic disorder (1,2). Early and intermediate steps in the insulin-signaling cascade including the insulin receptor (3), the insulin receptor substrate 1 (4-6), and phosphatidylinositol (PI)-3-kinase (5,6) are candidates for defects leading to insulin resistance in skeletal muscle. However, the extent to which these signaling defects are causative in the development of insulin resistance, or secondary to the altered metabolic state associated with NIDDM, is currently not known. The serine/threonine kinase Akt (also termed protein kinase B [PKB] or related to A and C protein kinase [RAC]) is stimulated by receptor tyrosine kinases and is a downstream target of PI-3-kinase (7-11), presumably through activation of the phosphatidylinositol 3,4,5-trisphosphate-dependent protein kinase (PDK-1) (12). Several lines of evidence show that PI-3-kinase is necessary and sufficient for growth factor-dependent activation of Akt (7-9). Furthermore, expression of a constitutively active Akt is sufficient to promote GLUT4 translocation and increase glucose transport in 3T3-L1 adipocytes (13,14). Whether defects in endogenous Akt activity contribute to reduced glucose transport in insulinresistant skeletal muscle remains to be elucidated. Substantial evidence has accumulated to suggest that chronic hyperglycemia can directly contribute to the development of peripheral insulin resistance (15-22). In partially pancreatectomized diabetic rats, correction of hyperglycemia by phlorizin treatment restored insulin-stimulated glucose transport in isolated adipocytes and improved wholebody insulin sensitivity (15,18,22). The extent to which restoration of glycemia can improve impaired insulin signaling in skeletal muscle from animals or humans, where NIDDM is known to be genetically inherited, is presently not known. The spontaneously diabetic Goto-Kakizaki (GK) rat is a nonobese model of NIDDM, developed by selective breeding of glucose-intolerant Wistar rats over several generations (23). Here, we investigate whether insulin-stimulated Akt kinase activity and glucose transport are altered in skeletal muscle from GK rats. To explore the possibility that potential defects at these sites may be caused by alterations DIABETES, VOL. 46, DECEMBER 1997

2 A. KROOK AND ASSOCIATES in the metabolic milieu, we treated diabetic GK rats with phlorizin to restore glycemia. RESEARCH DESIGN AND METHODS Animals. Male GK rats ( g) were obtained from our colony at the Karolinska Institute. Weight-matched male Wistar rats served as controls (B&K Universal, Sollentuna, Sweden). All rats were maintained under a 12-h light:dark cycle and had free access to water and standard rodent diet Four groups of animals were studied: vehicle-treated Wistar rats (n = 12), phlorizin-treated Wistar rats (n - 5), vehicle-treated GK rats (n = 12), and phlorizin-treated GK rats (n = 11). Phlorizin (0.8 g/kg body wt per day; as a 40% solution in propylene glycol) or vehicle (equal amounts of propylene glycol per kilogram) was administered as a subcutaneous injection in equal doses, at 12-h intervals for 4-5 weeks. Phlorizin was administered to the animals h before physiological testing. Glucose tolerance test. After a 15-h fast, glucose (2 g/kg body wt) was injected intraperitoneally in conscious rats. Blood was sampled from the tail before injection (time 0) and at 30,60, and 120 min after glucose injection. The glucose tolerance was performed on two occasions; before initiation of treatment (week 0) and after 3 weeks of phlorizin treatment. Muscle incubations. Fed rats were anesthetized with an intraperitoneal injection of pentobarbital sodium (5 mg/100 g body wt). Soleus muscles were removed, split into two equal portions, and incubated as described previously for the rat epitrochlearis muscle (24). All incubation media were prepared from a stock solution of pregassed (95% 0^5% CCy Krebs-Henseleit buffer (KHB) that contained 5 mmol/1 HEPES and 0.1% bovine serum albumin (BSA) (RIA Grade, Sigma, St. Louis, MO). The gas phase in the vials was maintained at 95% 0/5% CO 2 throughout all incubations. The muscles were incubated (30 C) for 15 min in KHB containing 2 mmol/1 pyruvate and 38 mmol/1 mannitol, and subsequently with or without insulin (2.4 or 120 nmol/1) for 30 min (glucose transport) or 6 min (Akt kinase). Glucose transport activity. The muscles were incubated at 30 C with or without insulin (2.4 or 120 nmol/1, respectively) for 10 min in KHB containing 8 mmol/1 [ 3 H]3-0-methylglucose (2.5 uci/mmol) and 32 mmol/1 [ 14 C]mannitol (26.3 uci/mmol). Glucose transport was assessed as described by Wallberg-Henriksson et al. (24). Glucose transport activity is expressed as micromoles of glucose analog accumulated per milliliter of intracellular water per hour. Akt kinase activity and protein expression. Soleus muscles were homogenized in an ice-cold buffer as described (25) and centrifuged at 150,000^ for 35 min (4 C). Protein was determined using a commercial kit (Bio-Rad, Richmond, CA). The Akt-a antibody was raised in rabbit using a fusion protein consisting of the PH domain of human Akt-a and glutathione S-transferase (GST). Aliquots of the supernatant (600 ug) were immunoprecipitated with anti-akt-a antibody, and Akt kinase activity was measured against a peptide substrate (GRPRTSSFAEG), based on a motif from glycogen synthase kinase-3 (GSK-3) (26). Briefly, Akt immunoprecipitates were collected on protein-a Sepharose beads and washed four times in buffer A (25 mmol/1 HEPES, 10% glycerol, 1% Triton X100,1 mol/1 NaCl, 1 mmol/1 DTT, 0.1% BSA), twice in kinase buffer (50 mmol/1 Tris-HCl ph 7.5, 10 mmol/1 MgCl 2,1 mmol/1 DTT), and resuspended in 30 ul of kinase buffer supplemented with 100 umol/1 ATP, 100 umol/1 GSK-3 peptide, and 2 uci [^-^PJATP. The reaction was terminated after a 30-min incubation at 30 C, and pp] incorporation into the peptide substrate was determined by resolving the reaction products on a 40% acrylamide gel. The gel was visualized on a Phospholmager (Bio-Rad), and the band corresponding to the peptide substrate was quantitated. Aliquots (30 ug) of the supernatant were solubilized in Laemmli sample buffer, separated by SDS-polyacrylamide gel electrophoresis (10% resolving gel), and transferred to nitrocellulose membranes. Immunodetection of Akt protein was performed using the polyclonal Akt-a antibody described above. The nitrocellulose sheets were washed and incubated with appropriate secondary antibodies. Akt kinase protein was visualized by enhanced chemiluminescence (Amersham, Arlington Heights, IL) and quantified by densitometry. Analytical determinations. Blood glucose levels were measured with a Medisense Sensors Electrodes Plus glucose meter (Birmingham, U.K.). Plasma insulin concentrations were determined by radioimmunoassay (Linco Research, St. Louis, MO), using rat insulin standards. Statistics analysis. Data are presented as means ± SE. Statistical differences were determined by a two-way analysis of variance (ANOVA) for glucose tolerance, and by a one-way ANOVA for glucose transport and Akt kinase activity. When the ANOVA resulted in a significant F-ratio (P < 0.05), the location of the significance was determined with the Fisher-LSD test. RESULTS Animal characteristics. Body weight of GK rats was not significantly different from Wistar rats (Table 1), consistent with previously published work presenting the GK rat as a nonobese model of NTDDM (23). The phlorizin-treated GK rats weighed 10% less than Wistar rats (P < 0.01). Blood glucose levels were elevated in the untreated GK rats compared with Wistar rats (Table 1). Phlorizin treatment decreased the fasting blood glucose concentration in GK rats to a level that was not different from Wistar rats; however, this improvement was not noted in the fed state. Glucose tolerance. Glucose tolerance was markedly impaired in GK rats compared with Wistar rats (P < 0.001; Fig. 1.). Phlorizin treatment significantly improved glucose tolerance in GK rats (area under the curve [AUC], 410 ± 66 vs. 1,212 ± 67 mmol/1 min" 1 above Wistar levels for treated vs. untreated rats, respectively, P < 0.001). Glucose tolerance was not fully restored in phlorizin-treated GK rats (P < vs. Wistar); however, blood glucose level 2 h post-glucose administration was fully normalized. Phlorizin had no effect on glucose tolerance in the Wistar rats. Glucose transport. No significant difference in basal glucose transport was observed between the groups (Fig. 2). In GK rats, maximal (120 nmol/1) insulin-stimulated 3-Omethylglucose transport activity (increase over basal glucose transport) was reduced by 39% (P < 0.05), whereas submaximal (2.4 nmol/1) insulin-stimulated glucose transport was similar to Wistar rats (Fig. 2). Phlorizin treatment completely restored maximal insulin-stimulated glucose transport (4.49 ± 0.32 vs ± 0.44 umol ml" 1 h" 1, for Wistar vs. phlorizin-treated GK rats; ND). Phlorizin treatment had no effect on 3-O-methylglucose transport in soleus muscle in Wistar rats (data not shown). Akt kinase activity and protein expression. We next assessed whether improvement of glycemia would affect Akt kinase activity in soleus muscle (Fig. 3). Insulin (120 nmol/1) induced a 5.7-fold increase in Akt kinase activity in soleus muscle from Wistar rats (P < 0.001). Maximal insulin-stimulated Akt activity was 68% lower (P < 0.01) in GK versus Wistar rats. In phlorizin-treated GK rats, improvement of gly- TABLE1 Animal characteristics Treatment Weight (g) Fasted glucose (mmol/1) Fed glucose (mmol/1) Fed insulin (pmol/1) Wistar Wistar-phlorizin GK GK-phlorizin 281 ±3 278 ±7 274 ±6 252 ± ± ± ± 0.2t 3.9 ± ± ± ± 0.6* 7.3 ± 0.5* 424 ± ± ± 17* 248 ± 34* Data are means ± SE for vehicle-treated Wistar rats (n = 12), phlorizin-treated Wistar rats (n = 5), vehicle-treated GK rats (n = 12), and phlorizin-treated GK rats (n = 11). *P < 0.05, tp < 0.01, P < 0.001, significantly different from Wistar rats. DIABETES, VOL. 46, DECEMBER

3 REGULATION OF Akt KINASE AND GLUCOSE TRANSPORT Time (min) FIG. 1. Glucose tolerance test in Wistar or GK rats. Wistar (n = 12), Phlorizin (Phl)-treated Wistar (n = 5), GK (w = 12), or phlorizintreated (n = 11) GK rats were fasted for 15-h, and D-glucose (2 g/kg body wt) in saline was injected intraperitoneally in conscious rats. Glucose levels were determined in blood samples collected from the tail vein. Data are presented as means ± SE. The glucose excursion was significantly greater in treated and untreated GK rats vs. Wistar rats {P < 0.001), and in untreated vs. treated GK rats (P < 0.001). Blood glucose level between 0 and 120 min was significantly greater in untreated GK rats (P < 0.001), whereas no difference was observed for all other groups. cemia resulted in a full normalization of insulin-stimulated (120 nmol/l) Akt kinase activity. Akt kinase activity was not significantly different between Wistar and GK rats in response to 2.4 nmol/l insulin. Protein content of Akt kinase was assessed in soleus muscle lysate to determine whether changes in insulin-stimulated Akt kinase activity observed in GK rats was due to changes in Akt kinase protein expression. In contrast to the results for Akt kinase activity, Akt-a protein expression was not significantly different between control and diabetic rats (Fig. 4). DISCUSSION Here, we show for thefirsttime impaired insulin-signaling at the level of Akt kinase in skeletal muscle from diabetic ani- Akt i Wistar GK GK-Phl FIG. 3. Insulin-stimulated Akt kinase activity in isolated soleus muscle from Wistar, GK, or phlorizin-treated GK rats. Muscles were incubated at 30 C for 6 min in the absence (D) or presence of 2.4 nmol/l ( ) or 120 nmol/l ( ) insulin. Akt kinase activity in muscle lysates was measured against a peptide substrate based on a motif from GSK3 as described in METHODS. Results are expressed as means ± SE for four muscles per group. Values are reported as arbitrary Phospholmager units. *P < vs. Wistar rats. Wistar GK GK-Phl FIG O-methylglucose transport in isolated soleus muscle from Wistar, GK, or phlorizin-treated GK rats. Muscles were incubated at 30 c C for 30 min in the absence ( ) or presence of 2.4 nmol/l ( ) or 120 nmol/l ( ) insulin. 3-O-methylglucose transport was assessed as described in METHODS. Results are expressed as means ± SE for 5-10 muscles per group. *P < 0.05 vs. Wistar rats; fp < 0.01 vs. phlorizintreated GK rats. mals. Interestingly, the defect in insulin action on Akt kinase activity in GK rats was not related to reduced protein expression of Akt kinase. Moreover, we show that restoration of glycemia is sufficient to restore the impaired insulin stimulation of glucose transport and Akt kinase activity in skeletal muscle of the lean diabetic GK rat. Whether restoration of Akt kinase activity in soleus muscle from phlorizintreated GK rats directly improves insulin-stimulated glucose transport remains to be elucidated. Inhibitors of PI-3-kinase block insulin-stimulated Akt kinase activity (7-9). Furthermore, dominant-negative mutants of the p85 regulatory subunit of PI-3-kinase prevent stimulation of Akt kinase activity by platelet-derived growth factor (8), whereas overexpression of a constitutively active PI-3-kinase activates Akt kinase (10,11). The latter studies provide evidence that Akt kinase is a major target of PI-3-kinase-generated signals. In skeletal muscle, insulin increases Akt kinase activity in a time course that parallels the activation of PI-3-kinase (27). However, activation of Akt kinase is also linked to a PI-3-kinase independent pathway known to regulate glucose transport. For example, Akt kinase can be stimulated by okadaic acid, a substance known to mediate GLUT4 translocation by a PI-3- kinase-independent pathway (28). More importantly, overexpression of Akt in 3T3-L1 adipocytes directly promotes glucose transport and translocation of GLUT1 and GLUT4 to the plasma membrane (13,14). Although these studies indicate that Akt is sufficient to promote glucose transport, further studies are required whereby a pharmacological inhibitor of Akt or a dominant negative construct of Akt is used to ascertain whether activation of glucose transport requires activation of Akt kinase. Hyperglycemia may directly contribute to the development of insulin resistance in NIDDM patients through alterations in insulin signaling in peripheral tissues (20,21,29). We have previously shown that restoration of glycemia normalizes the reduced capacity for insulin-stimulated glucose transport in skeletal muscle from NIDDM patients (20). Phlorizin inhibits the renal reabsorption of glucose and restores normoglycemia (15,22). Thus the phlorizin-treated GK rat provides a good model in which to study the effect of long DIABETES, VOL. 46, DECEMBER 1997

4 A. KROOK AND ASSOCIATES kda GK GK P W GK GK P Akt FIG. 4. Akt-a expression in soleus muscle from Wistar, GK, and phlorizin-treated GK rats. Muscle samples were prepared as described in the METHODS. An aliquot of the lysate (30 ug) was subjected to SDS-PAGE and immunoblotted with anti-akt-a antibody. A representative autoradiograph is presented for Wistar (W; n = 6), GK (n = 6), and GK P (phlorizin-treated GK; n = 5). Relative molecular weights are indicated on the left. term changes in the glycemic milieu on insulin action in skeletal muscle. Our results show that in the lean GK diabetic rat, long-term improvement of glycemia has profound effects on glucose tolerance, and significantly improves insulin action at the level of Akt kinase and glucose transport in skeletal muscle. Interestingly, decreased insulin-stimulated activity of Akt kinase and glucose transport in GK rats was only observed with maximal insulin stimulation. Nevertheless, the GK diabetic rat presents marked glucose intolerance. Thus, impaired insulin secretion (30,31) and insulin resistance in the liver (31) may also contribute to the pathogenesis of diabetes in the GK rat. In conclusion, our results suggest that hyperglycemia may have deleterious effects on intermediate and final components of the insulin signaling pathway in skeletal muscle. Whether the decrease in insulin-stimulated glucose transport in skeletal muscle from NIDDM patients is coupled to a defect in insulin action on Akt kinase remains to be investigated. ACKNOWLEDGMENTS This study was supported by grants from the Swedish Medical Research Council (34, 9517, 11823, 12211), the Novo- Nordisk Foundation, and the Swedish Diabetes Association to S.E., H.W.-H., and J.R.Z., a Junior Individual Grant from the Foundation for Strategic Research to J.R.Z., and National Institutes of Health Grant DK to R.A.R. We thank Dr. Akhtar Khan for valuable comments throughout the study. 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