Fatty Acid Infusion Selectively Impairs Insulin Action on Akt1 and PKC / But Not on Glycogen Synthase Kinase-3

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1 JBC Papers in Press. Published on July 2, 2002 as Manuscript M Fatty Acid Infusion Selectively Impairs Insulin Action on Akt1 and PKC / But Not on Glycogen Synthase Kinase-3 Young-Bum Kim 1, Gerald I. Shulman 2 and Barbara B. Kahn 1 * 1Diabetes Unit, Division of Endocrinology and Metabolism, Department of Medicine, Beth Israel Deaconess Medical Center, and Harvard Medical School, Boston, MA and 2Howard Hughes Medical Institute and Department of Internal Medicine, Yale University School of Medicine, New Haven, CT Running title: Fatty acid infusion impairs Akt1and PKC / activation * Corresponding author: Barbara B. Kahn, M.D. Diabetes Unit, Beth Israel Deaconess Medical Center 99 Brookline Ave. Boston, MA Phone: (617) Fax: (617) bkahn@caregroup.harvard.edu 1 Copyright 2002 by The American Society for Biochemistry and Molecular Biology, Inc.

2 SUMMARY To determine the mechanism(s) for insulin resistance induced by fatty acids we measured the ability of insulin to activate PI3K and multiple distal pathways in rats. Following a 5 hr infusion of lipid or glycerol (control), rats underwent a euglycemic-hyperinsulinemic clamp. Insulin stimulated IRS-1-associated PI3K activity in muscle of glycerol-infused rats 2.4-fold but had no effect in lipid-infused rats. IRS-2- and phosphotyrosine-associated PI3K activity were increased 3.5- and 4.8-fold respectively by insulin in glycerol-infused rats but only 1.6- and 2.3-fold in lipid-infused rats. Insulin increased Akt1 activity 3.9-fold in glycerol-infused rats and this was impaired 41% in lipid-infused rats. Insulin action on Akt2 and p70s6k were not impaired whereas activation of PKC / activity was reduced 47%. Insulin inhibited GSK-3 activity by 30% and GSK-3 activity by ~65% and increased PP-1 activity by 40-47% in both glycerol- and lipid-infused rats. Insulin stimulated glycogen synthase activity 2.0-fold in glycerol-infused rats but only 1.4-fold in lipid-infused rats. Thus, 1) elevation of fatty acids differentially affects insulin action on pathways distal to PI3K, impairing activation of Akt1 and PKC /, 2) insulin action on glycogen synthase can be regulated independent of effects on GSK-3 and PP-1 activity in vivo. 2

3 INTRODUCTION A fundamental action of insulin is to control the plasma glucose concentration by stimulating glucose transport into muscle and adipose cells and inhibiting hepatic glucose output (1). Defects in glucose transport result in insulin resistance, which is a major factor in the development of type 2 diabetes (2,3). The molecular basis for this insulin resistance remains unknown. Insulin signaling defects have been identified in insulin-resistant states, but their full impact on glucose transport and metabolism in muscle is still unclear (4-6). Insulin signaling involves a cascade of events initiated by insulin binding to its cell surface receptor, followed by receptor autophosphorylation and activation of receptor tyrosine kinases which result in tyrosine phosphorylation of insulin receptor substrates (IRSs) (7,8). Binding of IRSs to the regulatory subunit of phosphoinositide 3-kinase (PI3K) results in activation of PI3K which is necessary for insulin action on glucose transport (9-13). The extent to which downstream effectors, Akt/protein kinase B (PKB) and PKC / mediate insulin action on glucose transport is still controversial (14-20). Three Akt isoforms have been cloned (21,22). Insulin has differential effects on Akt isoforms in a tissue-, isoform- and species-specific manner (23,24). In obese rats, insulinstimulated Akt1 activity was decreased in muscle and adipose tissue but increased in liver, while insulin-stimulated Akt2 activity was decreased in muscle and liver but increased in adipose tissue (23). In insulin resistant humans with obesity with or without type 2 diabetes, the effect of insulin on the activity of all Akt isoforms in muscle in vivo is normal (25). However, at very high insulin levels in vitro, Akt activity is diminished in muscle from lean type 2 diabetics (26). Furthermore, in adipocytes from obese type 2 diabetics, Akt2 phosphorylation is impaired (27). 3

4 Whether these impairments in Akt1 or Akt2 activity are sufficient to play a role in insulin resistant states is unclear. Akt1 knockout mice do not have insulin resistance, although their growth retardation or developmental effects could mask this (28,29). Akt2 knockout mice have impaired insulin action on liver and modest effects in muscle and adipocytes which could possibly be secondary to the liver defect (29). Thus, the potential impact of altered Akt activity in muscle and fat is not certain. Studies suggest that activation of the atypical PKC isoforms and is required for insulin stimulation of glucose uptake and Glut4 translocation (18-20). Overexpression of a dominant negative mutant of PKC or PKC abrogated insulin-stimulated glucose transport and Glut4 translocation in adipose (18,30) and muscle cells (19,31). Overexpression of constitutively activated PKC in adipocytes (18) or wild type PKC in muscle in vivo (32) enhanced both basal and insulin-stimulated glucose transport. In addition, insulin-stimulated PKC / activity was impaired in skeletal muscle and adipose tissue of diabetic rats (33,34), in muscle of obese type 2 diabetic humans (35) and in myotubes of obese humans (36). It is therefore possible that PKC / could play an important role in insulin resistance in skeletal muscle and adipose tissue. Glucose transport is the rate-controlling step for insulin-stimulated muscle glycogen synthesis under normal conditions and in type 2 diabetes (37,38). Insulin stimulates glycogen synthase by both activating protein phosphatase-1 (PP-1) and inhibiting glycogen synthase kinase 3 (GSK-3) (39). Two isoforms of GSK-3 has been identified: GSK-3 and GSK-3 (40). GSK- 3 is a serine/threonine kinase that inhibits glycogen synthase activity by phosphorylating glycogen synthase (39). Insulin inactivates GSK-3 in skeletal muscle of rats and humans (41-4

5 43), which leads to activation of glycogen synthase and therefore increased glycogen synthesis (39). The upstream signaling pathways necessary for insulin activation of glycogen synthase include PI3K (44,45). Activation of PI3K by insulin is impaired in muscle of humans with type 2 diabetes (25), and both basal and insulin-stimulated glycogen synthase activity are reduced (25), but the important intermediary pathways are still unknown. Plasma fatty acids are often elevated in obesity and type 2 diabetes (3,46) and most likely contribute to the insulin resistance in these states. We previously demonstrated that fatty acid infusion decreases whole body glucose uptake and glycogen synthesis and that this is associated with a defect in insulin-stimulated PI3K activity associated with IRS-1 in muscle (47,48). In this study we sought to determine the mechanism(s) for the fatty acid-induced impairment in insulin action by measuring key steps downstream of PI3K leading to GSK-3 inhibition in skeletal muscle. We found differential regulation of Akt isoforms and discordance between insulin action on GSK-3, PP-1 and glycogen synthase. In addition, PKC / activation is impaired in this insulin-resistant state. 5

6 EXPERIMENTAL PROCEDURES Animals. All animal studies were conducted in accordance with the principles and procedures outlined in the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Male Sprague-Dawley rats (Charles River, Wilmington, MA) weighing between 250 and 300 g were housed in environmentally controlled conditions with 12 hr light-dark cycle and fed standard rat food. Rats were catheterized in the right jugular vein and carotid artery, and the catheters were externalized through an incision in the skin flap behind the head. After surgery, rats recuperated until they reached preoperative weight (~5-7 days). All rats were fasted hr before each infusion study. Protocols. Three groups of rats were studied using a 5 hr preinfusion protocol of saline, lipid [Liposyn II (Abbot laboratories, North Chicago, IL)] combined with heparin (continuous infusion IU/min) or glycerol (1:3 vol/vol) followed by a 30 min hyperinsulinemic-euglycemic clamp. Heparin activates lipoprotein lipase that catalyzes the breakdown of lipoproteins to fatty acids. Lipid/heparin or glycerol infusion was continued during the 30 min insulin clamp. Hyperinsulinemic-euglycemic clamps (10 mu/kg/min) were performed maintaining glucose concentration at 5.5 mm using a variable 20% glucose infusion (47). Humulin regular insulin (Eli Lilly, Indianapolis, IN) was infused during the clamps. Control rats were infused only with saline for 5.5 hours. After clamps, gastrocnemius muscle was rapidly removed, frozen in liquid nitrogen, and stored at -80 C until analysis. All studies were approved by the Yale Animal Use and Care Committee. 6

7 Preparation of muscle lysates. 50 mg of muscle was homogenized using a polytron at half maximum speed for 1 min on ice in 500 l buffer A (20 mm Tris ph 7.5, 5 mm EDTA, 10 mm Na 4 P 2 O 7, 100 mm NaF, 2 mm Na 3 VO 4 ) containing 1% NP-40, 1mM PMSF, 10 g/ml aprotinin and 10 g/ml leupeptin. For GSK-3 activity, we changed 100 mm NaF to 1 mm NaF in buffer A. Tissue lysates were solubilized by continuous stirring for 1 hr at 4 C, and centrifuged for 10 min at 14,000 g. The supernatants were stored at -80 C until analysis. Determination of PI3K activity. Muscle lysates (500 g protein) were subjected to immunoprecipitation with 5 l of a polyclonal IRS-1 antibody or IRS-2 antibody (1:100 dilution; gift from Dr. Morris White, Joslin Diabetes Center) or a monoclonal antiphosphotyrosine (1:50 dilution, Santa Cruz) coupled to protein A- sepharose (Sigma, St. Louis, MO) or protein G-Sepharose beads. The immune complex was washed and PI3K activity was determined as described (23). Determination of Akt activity. Muscle lysates (500 g protein) were subjected to immunoprecipitation for 4 hr at 4 C with 5 l of a polyclonal Akt1 (1:100 dilution, gift from T. Franke, Columbia University) (23), Akt2 (1:100 dilution, gift from T. Franke, Columbia University) (23) or Akt3 specific antibodies (1:100 dilution, Upstate Biotechnology, Lake Placid, NY), coupled to protein A or protein G- Sepharose beads (Amersham Pharmacia Biotechnology, Piscataway, NJ). Immune pellets were washed and Akt activity was determined as previously described (23). 7

8 Determination of PKC / activity. Muscle lysates (500 g protein) were subjected to immunoprecipitation for 4 hr at 4 C with 2 ug of a polyclonal PKC antibody (Santa Cruz Biotechnology, Santa Cruz, LA) that recognizes both PKC and PKC, coupled to protein A-Sepharose beads (Amersham Pharmacia Biotechnology, Piscataway, NJ). Immune pellets were washed and PKC / activity was determined as previously described (33). Determination of GSK-3 activity. Muscle lysates (500 g protein) were subjected to immunoprecipitation for 4 hr at 4 C with either GSK-3 (1:100 dilution: Santa Cruz) or GSK-3 -specific antibody (1:100 dilution: gift from Dr. Hagit Eldar-Finkelman, Tel-Aviv University, Israel), coupled to protein A-Sepharose or protein G-Sepharose beads. The immune complexes were washed twice with 50 mm Tris, ph 7.4, 500 mm LiCl and 1 mm DTT and twice with 50 mm Tris, ph 7.4. The kinase assay was performed on the immunoprecipitates in reaction mixtures including 50 mm Tris, 10 mm MgCl2, 0.5 mg/ml BSA, 100 M ATP (0.25 mci/ml), and 100 M PGS-1 peptide substrate and incubation was carried out for 20 min at 30C. The reaction mixtures were spotted on p81 phosphocellulose paper and washed with 75mM Phosphoric acid and counted for radioactivity (49). Determination of Glycogen synthase activity. 20 mg of muscle were homogenized using a polytron at half maximum speed for 1 min on ice in 0.5 ml extraction buffer (50 mm Hepes, 10 mm EDTA, 100 mm NaF, 5 mm DTT, 1 µm leupeptin, 1 µm pepstatin, and 200 µm PMSF, ph 7.5). Homogenates were used to measure 8

9 glycogen synthase (GS) activity as described previously (50). GS activity was determined at a physiologic concentration of substrate (0.3 mm UDP-glucose), calculated as nanomoles of UDPglucose incorporated into glycogen per minute per milligram of total protein and expressed as the ratio of activity assayed at 0 mm glucose-6-phosphate divided by the activity at 7.2 mm glucose- 6-phosphate. This is an indicator of the change in phosphorylation state of glycogen synthase in response to insulin (51). Determination of PP-1 activity. 20 mg of muscle were homogenized using a polytron at half maximum speed for 1 min on ice in 0.5 ml PP-1 homogenization buffer (50 mm Tris, 2 mm EDTA, 0.2% -mecaptoethanol, 2mg/ml glycogen, ph 7.4) containing 0.1 mm PMSF, 1 mm benzamidine and 10 mg/ml aprotinin. Muscle Homogenates (10 g) were preincubated with PP-1 homogenization buffer containing 4.5 nm okadaic acid for 2 min at 37 C. The reaction was initiated by adding 15 g of 32 P-labeled phosphorylase a in the presence of 3 nm okadaic acid and 5 mm caffeine. Phosphate release was determined as described previously (52). Determination of MAPK activity. Muscle lysates (500 g protein) were subjected to immunoprecipitation for 4 hr at 4 C with 1 l of a MAPK (Erk1 and Erk2) antibody (1:500 dilution: gift from Dr. John Blenis, Harvard Medical School), coupled to protein A-Sepharose beads. The immune complexes were washed three times with Buffer A and twice with 20 mm Tris, ph 7.4, 20 mm MgCl 2, 1 mm DTT and 1 mm EGTA. The kinase assay was performed on the immunoprecipitates in reaction mixtures including 20 mm Tris, 10 mm MgCl 2, 1 mm DTT, 1 mm EGTA, 10 M protein kinase 9

10 inhibitor, 10 mm ATP (0.25 mci/ml), and 10 mg/ml myelin basic protein substrate and incubation was carried out for 20 min at 30 C. The reaction mixtures were spotted, washed and counted as described for GSK-3 activity. Determination of p70s6 kinase mobility shift and amounts of signaling proteins g of tissue lysates protein per lane were resolved by SDS-PAGE (8% and 10% gel) and transferred to nitrocellulose membranes (Schleicher & Schuell). The membranes were blocked with 5% nonfat dry milk for 1 hr at room temperature and incubated with the following antibodies: Akt1 or Akt2 specific polyclonal antibody (gift from T. Franke, Columbia University or Akt1 antibody from Santa Cruz and Akt2 antibody from Upstate Biotechnology which gave similar results to our antibodies), a polyclonal antibody of PKC / (Santa Cruz Biotechnology), a monoclonal antibody for GSK-3 (Upstate Biotechnology) which recognizes both GSK-3 and GSK-3, a polyclonal antibody against the p85 subunit (Upstate Biotechnology) which recognizes all isoforms of the regulatory subunit of PI3K or p110 subunit of PI3K (Santa Cruz Biotechnology), a polyclonal anti-glut4 (gift from H. Haspel, Henry Ford Hospital, Detroit, MI) or a polyclonal PP-1 antibody (Upstate Biotechnology) or (a polyclonal glycogen synthase antibody (gift from J. Lawerence, University of Virginia), or a polyclonal IRS-1 or IRS-2 antibody (gift from M. White, Joslin Diabetes Center) in 1% nonfat dry milk overnight at 4 C. The membranes were washed and bands were visualized as described previously (23). Statistical analysis. 10

11 Data are presented as mean ± SEM. Statistical analyses were performed using the Stat View program (Abacus Concepts, Inc., Berkeley, CA). Statistical significance was tested with the unpaired Student's t-test. 11

12 RESULTS PI3K activity. After 30 min of insulin stimulation, IRS-1-associated PI3K activity in skeletal muscle of glycerol-infused rats was stimulated 2.4 fold above basal levels in saline-infused rats (Fig. 1A). In contrast, in lipid-infused rats, IRS-1-associated PI3K activity did not respond to insulin and was similar to that in saline control rats. Insulin also stimulated IRS-2-associated PI3K activity 3.5 fold in muscle of glycerol-infused rats but only 1.6 fold in lipid-infused rats (Fig. 1B). Because of the possibility that PI3K activity associated with other phosphoproteins could contribute to the effect of insulin on glucose uptake, we also measured antiphosphotyrosineassociated PI3K (Fig. 1C). Insulin increased antiphosphotyrosine-associated PI3K activity 4.8- fold in glycerol-infused rats but only 2.3 fold in lipid-infused rats. Akt isoforms activity and protein level. Insulin resistant states are associated with differential effects on Akt1, Akt2 and Akt3 isoforms. (23). Fig 2 shows that insulin stimulated Akt1 activity in skeletal muscle 3.9-fold in glycerolinfused rats and 2.3-fold in lipid-infused rats compared with saline-infused control rats. The insulin-stimulated increment in Akt1 activity above the saline value is reduced 55% in lipidinfused rats compared to glycerol-infused rats (p<0.05) (Fig. 2A). Insulin also increased Akt2 activity 1.8-fold in glycerol-infused rat and 2.3-fold in lipid-infused rats compared with control rats (Fig. 2B). However, Akt3 activity did not respond to insulin in either group (Fig. 2C). These results suggest that fatty acid infusion selectively impaired Akt1 isoform activity in skeletal muscle. Fig. 2D shows that the protein levels of Akt1 and Akt2 in skeletal muscle were not different among saline-infused, glycerol-infused and lipid-infused rats. 12

13 PKC / activity and protein level. To determine whether impaired activation of atypical PKC plays a role in FA-induced insulin resistance in rats, we measured the activity and protein levels of PKC / in muscle. Fig 3 shows that insulin stimulated PKC / activity in skeletal muscle 2.0-fold in glycerol-infused rats and 1.35-fold in lipid-infused rats compared with saline-infused rats (Fig. 3A). The increment in insulin-stimulated PKC / activity over saline was reduced 65% in lipid-infused rats compared with glycerol-infused rats (p<0.05). Fig. 3B shows that the protein levels of PKC / in skeletal muscle were not different among saline-infused, glycerol-infused or lipid-infused rats. GSK-3 isoform activity and protein levels. To determine whether the insulin resistance induced by lipid infusion involves an impairment in insulin's effect to inhibit GSK-3 activity, we measured the activity of the two known GSK-3 isoforms, and GSK-3 activity in skeletal muscle was reduced 30% in response to insulin in glycerol- and lipid-infused rats (p<0.05) compared with saline-infused rats (Fig. 4A). In contrast, insulin inhibited GSK-3 activity 63% in muscle of glycerol-infused rats (p<0.01 vs saline) and 68% in lipid-infused rats (p<0.001) compared with non-insulin infused control rats (Fig. 4B). The magnitude of decrease in GSK-3 activity was greater than the decrease in GSK- 3 activity, suggesting greater susceptibility of GSK-3 to insulin regulation in rat skeletal muscle. Fig. 4C shows the protein levels of GSK-3 and GSK-3 in skeletal muscle from the 3 study groups. We detected a 46 kda band for GSK-3 and a 51 kda band for GSK-3 using an 13

14 antibody which recognizes both isoforms. The total amounts of GSK-3 and GSK-3 protein were unchanged by lipid infusion (Fig. 4C). Glycogen synthase activity and protein level. To determine the effects of lipid infusion on glycogen synthase activity in rats, we measured glycogen synthase activity in skeletal muscle after 30 min hyperinsulinemic euglycemic clamp following preinfusion with glycerol or lipid for 5 hr. The ratio of glycogen synthase I-form (without glucose-6-phosphate) over total activity (with glucose-6-phosphate) increased 2.0-fold in glycerol-infused rats but only 1.4-fold in lipid-infused rats compared with saline-infused rats (Fig. 5A). There is no significant difference in total glycogen synthase activity (with glucose-6- phosphate) in either group (data not shown). Fig. 5B shows the protein levels of glycogen synthase in skeletal muscle of salineinfused, glycerol-infused or lipid-infused rats. There were no significant differences in the amount of glycogen synthase protein in muscle among the 3 groups (Fig. 5B). PP-1 activity and protein level. To determine whether PP-1 activity is impaired in skeletal muscle of lipid-induced insulin resistance in rats, we measured the activity and protein levels of PP-1 in muscle. Fig 6A shows that insulin stimulated PP-1 activity in skeletal muscle 40% in glycerol-infused rats and 47% in lipid-infused rats compared with saline-infused rats (p<0.05). Fig. 6B shows a representative blot indicating that the amount of PP-1 protein was unaltered in muscle of saline-infused, glycerol-infused or lipid-infused rats. MAPK activity and p70s6 kinase gel mobility shift. 14

15 To determine whether other downstream signaling steps are impaired by lipid infusion, we measured MAPK activity and p70s6 kinase phosphorylation in skeletal muscle using the same protocol of 30 min insulin clamp following preinfusion with glycerol or lipid for 5 hr. With this protocol, MAPK activity was not activated by insulin in skeletal muscle of either glycerolinfused or lipid-infused rats (Fig. 7A). We saw a similar result in the phosphorylation of MAP kinase using a phospho-specific antibody (not shown). We routinely find that more extended periods of fasting are necessary to reduce basal MAPK activity in skeletal muscle of salineinfused rats enough to detect an insulin effect. Panel 7B shows the levels and insulin-stimulated hyperphosphorylation of p70s6 kinase in skeletal muscle of glycerol-infused and lipid-infused rats. In the basal state, we detect a single ~66 kda band for p70s6 kinase in muscle. After insulin stimulation of rats for 30 min, p70s6 kinase shifts to a hyperphosphorylated state in both glycerol-infused and lipid-infused rats. There is no significant change in the phosphorylation of p70s6 kinase in muscle of lipid-infused rats compared with glycerol-infused rats (Fig. 7B). Signaling protein levels. The amount of the p85, p55 and p50 regulatory subunit and p110 catalytic subunit of PI3K and of IRS-1 and IRS-2 was unaltered in muscle of lipid-infused rats compared with glycerolinfused rats (Fig. 8). Also, the total amount of Glut4 protein was not significantly altered in skeletal muscle of lipid-infused rats compared with glycerol-infused rats (Fig. 8). 15

16 DISCUSSION In insulin resistant states such as obesity and type 2 diabetes, plasma fatty acids are elevated (3,46). Lipid infusion in rats and humans results in reduced insulin-stimulated rates of muscle glycogen synthesis, whole body glucose oxidation and glucose uptake (53-56). These changes are accompanied by a defect in insulin-stimulated PI3K activity associated with IRS-1 in skeletal muscle (47,48). In the present study, we investigated the effects of lipid infusion on the downstream mediators of PI3K, with particular focus on whether impaired insulin action on Akt, PKC /, GSK-3 isoforms or PP-1 contributes to this insulin-resistant state. Here we show that 5 hrs of fatty acid infusion specifically impairs insulin-stimulated Akt1 activity in skeletal muscle but insulin action on Akt2, GSK-3 and PP-1 is unaltered. Akt1 in rat skeletal muscle is highly responsive to insulin and the time course of its stimulation parallels that of inhibition of GSK-3 activity (24). Combined with in vitro data showing phosphorylation of GSK-3 by Akt1 (57), this makes it possible that Akt1 could be important for insulin action on glycogen synthesis. However, our study shows that Akt1 does not appear to be the major upstream regulator of GSK- 3 activity in the insulin-resistant state induced by lipid infusion or that other Akt isoforms can substitute when Akt1 activation is defective. Furthermore, lipid infusion leads to impaired insulin-induced glycogen synthase activity but no defect in insulin action on GSK-3 or PP-1, indicating there is dominant regulation of glycogen synthase activity by a non-gsk-3 and non- PP-1 mediated pathway(s) (58,59). Finally, this insulin-resistant state also involves impaired insulin activation of PKC / activity in skeletal muscle which could contribute to the metabolic defects. There is increasing evidence that key steps in the insulin signaling pathway, including the insulin receptor, IRSs, and PI3K, are candidates for defects leading to insulin resistance in 16

17 skeletal muscle of rodents and humans (23,25,60-62). Activation of PI3K is necessary for insulin-induced glucose transport and Glut4 translocation (9-13). In addition to the defect in insulin-stimulated IRS-1-associated PI3K activity in skeletal muscle of lipid-infused rats (47), we found that insulin-stimulated PI3K activity associated with IRS-2 or phosphotyrosine is also decreased by ~50%. Similar results have been demonstrated in type 2 diabetic subjects as well as in obese nondiabetic subjects (25,63) and a number of genetic models of diabetes and insulin resistance (23,60,61). In view of the evidence that lipid-induced insulin resistance involves a defect of glucose transport in skeletal muscle (37,53), it is likely that the reduction of insulinstimulated PI3K activity leads to an impairment of Glut4 translocation which plays a major role in the decreased glucose disposal. The inhibition of Akt1 activation with lipid infusion contrasts with observations in insulin-resistant muscle from obese humans with type 2 diabetes (25) and glucosamine-infused rats, in which Akt activation was normal (64). However, insulin-stimulated Akt activity is diminished in some insulin-resistant states such as in nonobese type 2 diabetic humans (26) and GK rats (65). The difference may be due to differences in the metabolic disturbances, the degree of insulin resistance or the dose, duration or route of insulin treatment in these studies. It is not clear whether a defect in Akt1 activation in vivo could contribute to the development of FAinduced insulin resistance. Mice with knockout of Akt1 are not insulin resistant (28,29). However, these mice are growth retarded and developmental abnormalities or reduced fat mass could mask insulin resistance in muscle. Akt is required for insulin s effect to inhibit to GSK-3 in cell culture (57). However, we find that insulin-induced inactivation of GSK-3 isoforms is normal despite decreased 17

18 activation of Akt1 in skeletal muscle of lipid-infused rats. We see a similar discrepancy in skeletal muscle of lactate-infused rats (66) and in Zucker fa/fa obese rats (YBK, BBK unpublished data). Taken together, these data suggest that either maximal Akt1 activation is not required for maximal inhibition of GSK-3 or that Akt1 is not a major upstream regulator of GSK-3 in this insulin-resistant state in vivo. This raises the possibility of other Akt isoforms or other pathway downstream of PI3K playing a role in insulin action on glycogen synthesis. The atypical PKC isoforms, PKC and, are also downstream targets of PI3K (see Introduction) (18-20). Here we show that insulin-stimulated PKC / activation is impaired in skeletal muscle of lipid-infused rats compared with glycerol-infused rats. Consistent with our current results are data from several insulin-resistant models including GK diabetic rats (33), high fat fed rats (67) and obese Zucker rats (YBK, BBK unpublished data) as well as obese nondiabetic and obese diabetic humans (35), all of which have decreased activation of PI3K and PKC /. This raises the possibility that defective activation of atypical PKCs in muscle could contribute to the decreased glucose uptake and metabolism in those states. A recent study demonstrates that PKC enhances Akt-mediated GSK-3 phosphorylation in skeletal muscle cells in vitro (68), indicating that both PKC and Akt are required for the potent inhibition of GSK-3 activation. In our study, lipid infusion compromises insulinstimulated PKC / activity, but GSK-3 inhibition is normal. Since the impairment of insulinstimulated PKC / activity is partial, the remaining PKC / activity may be sufficient to enable GSK-3 phosphorylation in vivo. Since insulin action on Akt1, but not Akt2 or Akt3, is 18

19 selectively impaired in skeletal muscle of lipid-infused rats, other Akt isoforms may also contribute to GSK-3 phosphorylation and inhibition in this model. Another discrepancy revealed by the lipid-infused model is insulin s effects on GSK-3 and glycogen synthase activity. GSK-3 is thought to be a major regulator of glycogen synthase activity. Inhibition of GSK-3 blocks the ability of insulin to inactivate GSK-3 and increases glycogen synthase activity in cultured muscle cells (69,70) and adipocytes (71). Additionally, overexpression of GSK-3 in 293 cells causes a reduction in basal glycogen synthase activity (72). However, lipid infusion results in a reduction of glycogen synthase activity in skeletal muscle despite the fact that insulin-stimulated GSK-3 activation is normal, suggesting that glycogen synthase activity can be regulated independent of GSK-3 in vivo. Support for this comes from studies showing that GSK-3 is not essential for glycogen synthase activation by insulin in adipocytes (58), rat-1 fibroblasts (58) and hepatocytes (59). Here we demonstrate that two other possible pathways, MAPK and p70s6 kinase, are not involved in the modulation of glycogen synthase in this insulin-resistant state. Glycogen synthase has nine phosphorylation sites, which are located at both NH2- and COOH-termini (73). GSK-3 phosphorylates sites 4, 3c, 3b and 3a (39), but sites 3a and 3b can also be phosphorylated by currently unidentified protein kinases or unknown factors (73,74). These two sites are the most important for regulation of glycogen synthase (73). It is therefore conceivable that lipid-infusion increases phosphorylation of sites 3a and 3b directly or through unidentified protein kinases, leading to a decrease in glycogen synthase activity. Alternatively, altered PP-1 activity could be involved. However, our results demonstrate that PP-1 activation and expression is normal in the lipid-induced insulin resistant state. 19

20 In conclusion, the impaired insulin-stimulated glucose disposal in skeletal muscle induced by lipid infusion is associated with a decrease in insulin-stimulated PI3K activation. Several observations challenge the recent linear concept of the insulin signaling cascade regulating glycogen synthesis. For example, lipid infusion impairs insulin action on Akt1 in skeletal muscle but action on GSK-3 is preserved. This may be due to the fact that other Akt isoforms can regulate GSK-3 activity or that only submaximal activation of Akt1 is required to fully inhibit GSK-3. Interestingly, Akt isoforms are differentially affected in this insulinresistant state. In spite of a marked impairment in insulin action on PI3K, Akt2 activation is normal, suggesting possible alternative upstream pathways for Akt2 activation. Furthermore, insulin action on glycogen synthase is impaired with no defect in action on GSK-3 inhibition or PP-1 activation, also suggesting alternative pathways. Lipid infusion is associated with an impairment of PKC / activation by insulin in skeletal muscle which could contribute to the development of insulin resistance. Thus, lipid infusion induces insulin resistance by impairing insulin signaling in a manner that selectively affects specific downstream mediators of the PI3K pathway. Understanding the molecular mechanisms for this selectivity and the potential alternative pathways for regulating glucose metabolism could lead to new therapeutic targets for obesity and type 2 diabetes. 20

21 Acknowledgments This work was supported by the National Institute of Diabetes and Digestive and Kidney Disease Grant NIH DK43051 and a research grant from the American Diabetes Association (B.B. Kahn) and RO1 DK40936 and DERC P30 DK45735 (G.I. Shulman). We thank M.E. Griffin and N. Barucci for critical experimental contributions and Drs. M.J. Brady, K. Ueki and M. Noguchi for technical advice and helpful discussions and Drs. M.F. White, T. Franke, J. Blenis and H. Eldar- Finkelman for antibodies. 21

22 FIGURE LEGENDS Fig. 1. IRS-1-associated (A), IRS-2-associated (B) and phosphotyrosine-associated (C) PI3K activities in muscle of rats after hyperinsulinemic-euglycemic clamp following preinfusion with either glycerol or intralipid for 5 hr. Control rats were infused only with saline for 5.5 hours. PI3K activities were measured in IRS-1, IRS-2 or phosphotyrosine immunoprecipitates and were quantitated using a PhosphorImager. Results are means±sem for 3 rats for saline and for 7-8 rats for glycerol or lipid infusion. *p<0.01 vs. glycerol. Fig. 2. Akt1 (A), Akt2 (B) and Akt3 (C) activities and protein levels (D) in muscle of rats after hyperinsulinemic-euglycemic clamp following preinfusion with either glycerol or intralipid for 5 hr. Control rats were infused only with saline for 5.5 hours. Muscle lysates (500 g) were subjected to immunoprecipitation with antibodies specific for Akt1, Akt2 or Akt3. The immune pellets were assayed for kinase activity using Crosstide as substrate. Panel D: Proteins in muscle lysates (100 g) were separated by SDS/PAGE on 8% gels and transferred to nitrocellulose membranes. Akt1 and Akt2 were visualized by immunoblotting with specific Akt1 and Akt2 antibodies. Results are means±sem for 3 rats for saline and for 7-8 rats for glycerol or lipid infusion. *p<0.01 vs. glycerol. Fig. 3. PKC / activities (A) and protein levels (B) in muscle of rats after hyperinsulinemiceuglycemic clamp following preinfusion with either glycerol or intralipid for 5 hr. Control rats were infused only with saline for 5.5 hours. Muscle lysates (500 g) were subjected to immunoprecipitation with an antibody that recognizes PKC or PKC. The immune pellets were assayed for kinase activity using a PKC- pseudosubstrate as substrate. Panel B: Proteins in 22

23 muscle lysates (50 g) were separated by SDS/PAGE on 8% gels and transferred to nitrocellulose membrane. PKC / was visualized by immunoblotting with a PKC / antibody. Results are means±sem for 3 rats for saline and for 6-7 rats for glycerol or lipid infusion. *p<0.05 vs. glycerol. Fig. 4. GSK-3 and GSK-3 activities (B) and protein levels (C) in muscle of rats after hyperinsulinemic-euglycemic clamp following preinfusion with either glycerol or intralipid for 5 hr. Control rats were infused only with saline for 5.5 hours. Muscle lysates (500 g) were subjected to immunoprecipitation with antibodies specific for GSK-3 or GSK-3. The immune pellets were assayed for kinase activity using PGS-1 as substrate. Panel C: Proteins in muscle lysates (50 g) were separated by SDS/PAGE on 8% gels and transferred to nitrocellulose membrane. GSK-3 isoforms were visualized by immunoblotting with a GSK-3 antibody that recognizes GSK-3 or GSK-3. Results are means±sem for 3 rats for saline and for 7-8 rats for glycerol or lipid infusion. *p<0.05 vs. saline. **p<0.01 vs. saline. Fig. 5. Glycogen synthase activity (A) and protein level (B) in muscle of rats after hyperinsulinemic-euglycemic clamp following preinfusion with either glycerol or intralipid for 5 hr. Control rats were infused only with saline for 5.5 hours. Glycogen synthase activity was measured as described in Methods. Panel A: The ratio of glycogen synthase activity represents the activity measured in the absence divided by that in the presence of glucose-6-phosphate. Panel B: Proteins in muscle lysates (5 g) were separated by SDS/PAGE on 8% gels and transferred to nitrocellulose membranes. Glycogen synthase was visualized by immunoblotting 23

24 with a glycogen synthase antibody. Results are means±sem for 3 rats for saline and for 7-8 rats for glycerol or lipid infusion. *p<0.05 vs. glycerol. Fig. 6. PP-1 activity (A) and protein level (B) in muscle of rats after hyperinsulinemiceuglycemic clamp following preinfusion with either glycerol or intralipid for 5 hr. Control rats were infused only with saline for 5.5 hours. PP-1 activity in muscle homogenates (10 g) was measured using 32 P-labeled phosphorylase a as substrate. Panel B: Proteins in muscle lysates (50 g) were separated by SDS/PAGE on 8% gels and transferred to nitrocellulose membrane. PP-1 was visualized by immunoblotting with a PP-1 antibody. Results are means±sem for 3 rats for saline and for 6-7 rats for glycerol or lipid infusion. *p<0.05 vs. saline. Fig. 7. MAPK activity (A) and p70s6 kinase mobility shift (B) in muscle of rats after hyperinsulinemic-euglycemic clamp following preinfusion with either glycerol or intralipid for 5 hr. Control rats were infused only with saline for 5.5 hours. Panel A: Muscle lysates (500 g) were subjected to immunoprecipitation with MAPK antibody. The immune pellets were assayed for kinase activity using MBP as substrate. Results are means±sem for 3 rats for saline and for 7-8 rats for glycerol or lipid. Panel B: Proteins in muscle lysates (50 g) were separated by SDS/PAGE on 8% gels and transferred to nitrocellulose membranes. p70s6 kinase was visualized by immunoblotting with a p70s6 kinase antibody. This blot is representative of 3 blots on 3-8 rats per group. Fig. 8. Signaling protein levels in muscle of rats after hyperinsulinemic-euglycemic clamp following preinfusion with either glycerol or intralipid. Control rats were infused only with 24

25 saline for 5.5 hours. Proteins in muscle lysates ( g) were separated by SDS/PAGE on 8 or 10% gels and transferred to nitrocellulose membranes. p85, p55, p50, p110, IRS-1, IRS-2 and Glut4 were visualized by immunoblotting with specific antibodies. This blot is representative of 3 blots on 3-10 rats per group. 25

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31 (A) IRS1 (B) IRS2 (C) Phosphotyrosine % of saline Insulin: * 200 * Insulin: Insulin: Saline Glycerol Lipid Saline Glycerol Lipid Saline Glycerol * Lipid Fig 1. A,B,C Downloaded from by guest on May 11, 2018

32 (A) Akt1 (B) Akt2 (C) Akt % of saline Insulin: 0 0 Insulin: Insulin: Saline Glycerol Lipid Saline Glycerol Lipid Saline Glycerol Lipid Fig. 2 A,B,C Downloaded from by guest on May 11, 2018

33 (D) Saline Glycerol Lipid Insulin: Akt1 Akt2 Fig. 2 D

34 250 (A) * 0 Saline Glycerol Lipid (B) Saline Glycerol Lipid Insulin: PKC λ/ζ Fig. 3 A,B PCKλ/ζ activity (% of saline) Insulin:

35 (A) GSK3 α (B) GSK3 β * * ** 25 % of saline 0 Insulin: Insulin: Saline Glycerol Lipid 0 Saline Glycerol Lipid Fig. 4 A,B

36 (C) Saline Glycerol Lipid Insulin: GSK3α GSK3β Fig. 4 C

37 (A) (B) Saline Glycerol Lipid Insulin: * Glycogen synthase activity ratio (-/+ G-6-P) Saline Glycerol Lipid Saline Glycerol Lipid GS Fig. 5 A,B % of saline Insulin: Insulin:

38 (A) PP-1 Activity 150 * * Insulin: Saline Glycerol Lipid (B) Saline Glycerol Lipid Insulin: PP-1 Fig. 6 A,B % of saline

39 (A) 150 MAPK Activity % of saline 0 Insulin: Saline Glycerol Lipid (B) Saline Glycerol Lipid Insulin: p70s6k Fig. 7 A,B

40 Saline Glycerol Lipid Insulin: p85α p55α p50α p110α IRS1 IRS2 Glut4 Fig. 8

41 Fatty acid infusion selectively impairs insulin action on Akt1 and PKC<IMG SRC="/math/lambda.gif" ALIGN="BASELINE" ALT="lambda ">/<IMG SRC="/math/zeta.gif" ALIGN="BASELINE" ALT="zeta "> but not on glycogen synthase kinase-3 Young-Bum Kim, Gerald I. Shulman and Barbara B. Kahn J. Biol. Chem. published online July 2, 2002 Access the most updated version of this article at doi: /jbc.M Alerts: When this article is cited When a correction for this article is posted Click here to choose from all of JBC's alerts