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Endocrinology Vol. 142, No. 4 1595-1605
Copyright © 2001 by The Endocrine Society

ARTICLES

Rosiglitazone, Insulin Treatment, and Fasting Correct Defective Activation of Protein Kinase C-{zeta}/{lambda} by Insulin in Vastus Lateralis Muscles and Adipocytes of Diabetic Rats1

Yoshinori Kanoh, Gautam Bandyopadhyay, Mini P. Sajan, Mary L. Standaert and Robert V. Farese

J. A. Haley Veterans Hospital Research Service and Department of Internal Medicine, University of South Florida College of Medicine Tampa, Florida 33612

Address all correspondence and requests for reprints to: Robert V. Farese, M.D., Research Service (VAR 151), J. A. Haley Veterans Hospital, 13000 Bruce B. Downs Boulevard, Tampa, Florida 33612. E-mail: rfarese{at}com1.med.usf.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Atypical protein kinases C (PKCs), {zeta} and {lambda}, and protein kinase B (PKB) are thought to function downstream of phosphatidylinositol 3-kinase (PI 3-kinase) and regulate glucose transport during insulin action in skeletal muscle and adipocytes. Insulin-stimulated glucose transport is defective in type II diabetes mellitus, and this defect is ameliorated by thiazolidinediones and lowering of blood glucose by chronic insulin therapy or short-term fasting. Presently, we evaluated the effects of these insulin-sensitizing modalities on the activation of insulin receptor substrate-1 (IRS-1)-dependent PI 3-kinase, PKC-{zeta}/{lambda}, and PKB in vastus lateralis skeletal muscles and adipocytes of nondiabetic and Goto-Kakizaki (GK) diabetic rats. Insulin provoked rapid increases in the activity of PI 3-kinase, PKC-{zeta}/{lambda}, and PKB in muscles and adipocytes of nondiabetic rats, but increases in IRS-1-dependent PI 3-kinase and PKC-{zeta}/{lambda}, but not PKB, activity were substantially diminished in GK muscles and adipocytes. Rosiglitazone treatment for 10–14 days, 10-day insulin treatment, and 60-h fasting reversed defects in PKC-{zeta}/{lambda} activation in GK muscles and adipocytes and increased glucose transport in GK adipocytes, without necessarily increasing IRS-1-dependent PI 3-kinase or PKB activation. Our findings suggest that insulin-sensitizing modalities, viz. thiazolidinediones, chronic insulin treatment, and short-term fasting, similarly improve defects in insulin-stimulated glucose transport at least partly by correcting defects in insulin-induced activation of PKC-{zeta}/{lambda}.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CLINICAL INSULIN resistance in type II diabetes mellitus is improved by a variety of treatment modalities, including thiazolidinediones (TZDs), which activate peroxisome proliferator-activated receptor-{gamma} (1), dietary restriction resulting in significant weight loss, and improvement in plasma glucose and/or fatty acid levels by intensive insulin treatment, prolonged dieting, and short-term fasting. Improvement in diabetes-related insulin resistance by these treatment modalities is at least partly due to enhanced effectiveness of insulin signaling to the glucose transport system in skeletal muscles, the major organ for insulin-stimulated glucose disposal, and, to a lesser extent, in adipocytes. Presently, there is only fragmentary understanding of the mechanisms that underlie both the defect in insulin signaling to the glucose transport system in diabetic states, and the subsequent improvement in such signaling after treatment with insulin-sensitizing modalities.

In contrast to skeletal muscle, adipocytes account for only a relatively small fraction of whole body glucose disposal in nonobese humans and animals. Nevertheless, adipocytes, like muscle, contain insulin-sensitive GLUT4 glucose transporters and, moreover, are useful for reflecting alterations in insulin signaling and insulin-stimulated glucose transport. For example, relative to the present studies, we recently reported that adipocytes of type II diabetic Goto-Kakizaki (GK) rats manifest defects in glucose transport and insulin signaling, and interestingly, the defects in glucose transport and signaling via protein kinases C-{zeta} and -{lambda} (PKC-{zeta}/{lambda}) are reversed by TZD treatment (2).

As alluded to above, there is considerable uncertainty about the mechanisms that cause defects in glucose transport in type II diabetes mellitus and reversal of such defects by insulin-sensitizing modalities. Concerning the latter, TZDs have been reported to 1) restore diminished GLUT4 glucose transporter levels in both muscles of obese Zucker rats in vivo (3) and tumor-necrosis factor-treated 3T3/L1 adipocytes in vitro (4), and 2) provoke increases in GLUT1 glucose transporter levels in 3T3/L1 adipocytes in vitro (5) and skeletal muscle preparations in vitro (6, 7, 8). 0n the other hand, TZDs can also enhance insulin-stimulated glucose transport in the absence of changes in levels of glucose transporters (2, 9), apparently by improving insulin signaling mechanisms. With respect to signaling factors used by insulin to stimulate GLUT4 glucose transporter translocation to the plasma membrane and subsequent glucose transport, phosphatidylinositol 3-kinase (PI 3-kinase) is generally recognized to be particularly important (10). Indeed, TZDs can increase the level or activity of PI3-kinase or reverse defects in insulin-induced activation of PI 3-kinase in certain situations (11, 12), but alterations in PI 3-kinase have not been observed (2) or reported in other studies. Compared with TZDs, there is even less available information on the effects of intensive insulin treatment, short-term fasting, and other insulin-sensitizing modalities on insulin signaling through PI 3-kinase to the glucose transport system.

Of the signaling factors that are known to operate downstream of PI 3-kinase, atypical PKC-{zeta}/{lambda} (13, 14, 15, 16, 17) and protein kinase B (PKB; or AKT) (18, 19, 20, 21) have been postulated to be important in mediating the effects of insulin on GLUT4 translocation and glucose transport. As alluded to, we recently reported that in vivo treatment over a 10- to 14-day period with the TZD, rosiglitazone, fully reverses defects in insulin-induced activation of PKC-{zeta}/{lambda} and insulin-stimulated glucose transport in isolated adipocytes of GK diabetic rats (2). In contrast, such rosiglitazone treatment did not alter GLUT4 or GLUT1 glucose transporter levels, insulin receptor substrate-1 (IRS-1)-dependent PI 3-kinase activation, which was markedly compromised, or PKB activation, which was only minimally, if at all, compromised in GK adipocytes (2). Thus, TZDs appeared to improve insulin-stimulated glucose transport in GK adipocytes largely via alterations in PKC-{zeta}/{lambda} signaling.

Presently, we examined the effects of three clinically effective modalities that have been used to treat diabetes-related insulin resistance, viz. 10- to 14-day TZD/rosiglitazone treatment, chronic 10-day insulin treatment, and short-term 60-h fasting, on insulin-induced activation of PI 3-kinase, PKC-{zeta}/{lambda}, and PKB in vastus lateralis muscles as well as adipocytes of GK rats. We found that there are 1) sizeable defects in insulin-induced activation of IRS-1-dependent PI 3-kinase and PKC-{zeta}/{lambda}, but not PKB, in vastus lateralis muscles as well as adipocytes of GK rats; and 2) each of the three insulin-sensitizing modalities reversed the defects in PKC- {zeta}/{lambda} activation in GK vastus lateralis muscles and adipocytes and concomitantly enhanced insulin-stimulated glucose transport in GK adipocytes despite having little or no apparent effect on either IRS-1-dependent PI 3-kinase or PKB activation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Experimental animals
Characteristics of nonobese, type II diabetic, insulin-resistant GK rats presently used have been described in previous reports (2, 22). As controls, we used nondiabetic Wistar rats (Harlan Sprague Dawley, Inc., Indianapolis, IN), which were fed the same diet and kept for at least 2 weeks before experimental use in our vivarium, which is temperature controlled and maintains daily 12-h light, 12-h dark cycles. GK and Wistar rats were used at 10–14 weeks of age (250–300 g BW). All experimental procedures were fully approved by the institutional animal care and use committee of the University of South Florida School of Medicine and the James A. Haley Veterans Administration Medical Center research and development committee.

In vivo treatments and muscle harvesting
Rosiglitazone (supplied by SmithKline Beecham, Philadelphia, PA) was given by oral gavage (4 mg/kg BW·day) for 10–14 days to GK diabetic and Wistar nondiabetic rats. This dose was selected because it is proportional to that found to be fully effective for restoring glucose transport responses over a comparable time course in a previous study of ob/ob mouse adipocytes (23) and is comparable to that dose found to rapidly induce peroxisome proliferator-activated receptor-{gamma}-dependent genes (24). As alternative in vivo treatments, GK rats were subjected to either a 60-h fast in which food, but not water, was withheld or were treated with 4–6 U intermediate-acting NPH insulin (dosage was adjusted in accordance with blood sugar determinations) given sc each day at approximately 1700 h over a 10-day period. As shown in Table 1Go, this relatively short 10- to 14-day rosiglitazone treatment schedule had little or no effect on serum glucose, insulin, or FFA levels in either Wistar nondiabetic or GK diabetic rats (group A); serum glucose levels 18–20 h after the last of 10 daily injections of NPH insulin were markedly improved in GK rats (group B), and GK rats fasted for 60 h (group C) had serum glucose levels comparable to those of fed Wistar nondiabetic rats (see group A) at the time of experimental use.


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  		Table 1. Serum glucose, insulin, and free fatty acid (FFA) levels

 
Between approximately 1000–1300 h on the day of the experiment, rats were injected im with saline or 0.25 U insulin in saline. This dose of insulin increased serum insulin levels to approximately 25–30 ng/ml 15 min after im injection. After 15 min, rats were killed, and vastus lateralis muscles were rapidly excised, chilled, and homogenized (Polytron, Brinkmann Instruments, Inc., Westbury, NY) in appropriate buffers. For studies of PKC-{zeta}/{lambda} activation, as previously described (15), buffer contained 250 mM sucrose, 20 mM Tris-HCl (pH 7.5), 1.2 mM EGTA, 20 mM ß-mercaptoethanol, 1 mM phenylmethylsulfonylfluoride (PMSF), 10 µg/ml aprotinin, 20 µg/ml leupeptin, 3 mM Na3VO4, 3 mM NaF, 3 mM Na4P2O7, and 1 µM LR-microcystin. For studies of PKB activation (see below), buffer contained 50 mM Tris-HCl (pH, 7.5), 1 mM EDTA, 1 mM EGTA, 1 mM NA3VO4, 0.1% ß-mercaptoethanol, 50 mM NaF, 5 mM NA4P207, 10 mM ß-glycerophosphate, 1 mM PMSF, 10 µg/ml aprotinin, 20 µg/ml leupeptin, and 1 µM LR-microcystin. For studies of PI 3-kinase activation, as previously described (15), buffer contained 255 mM sucrose, 20 mM Tris-HCl (pH, 7.4), 5 mM EDTA, 5 mM EGTA, 1 mM NA3V04, 1 mM NaF, 1 mM NA4P207, 1 mM PMSF, 10 µg/ml aprotinin, 20 µg/ml leupeptin, and 1 µM LR-microcystin. Homogenates were centrifuged for 10 min at 700 x g to remove nuclei, cellular debris, and floating fat. Supernatants were then supplemented with 1) 0.15 M NaCl, 1% Triton X-100, and 0.5% Nonidet and used for immunoprecipitation of PKC-{zeta}/{lambda} or IRS-1- or pY-dependent PI 3-kinase, or 2) with 1% Triton X-100 for immunoprecipitation of PKB as described below.

Note that 1) the same muscle and adipocyte homogenates were used for studies of immunoprecipitable IRS-1-dependent PI 3-kinase, PKC-{zeta}/{lambda}, and PKB; and 2) the same rats were used for in vivo muscle studies and in vitro adipocyte studies (however, see below regarding the complete loss of preexisting insulin effects on glucose transport in isolated adipocytes after collagenase digestion). Also note that adipocytes used in our previous rosiglitazone study (2) were partly derived from rats used in the present study of effects of rosiglitazone on skeletal muscle PKC-{zeta}/{lambda} activation.

Adipocyte studies
As described previously (2), adipocytes were prepared by collagenase digestion of epididymal fat pads, incubated in glucose-free Krebs-Ringer phosphate medium containing 1% BSA with or without 10 nM insulin for 10 min in studies of enzyme activation or with the indicated concentrations of insulin for 30 min in studies of [3H]2-deoxyglucose uptake. After incubation, cells were 1) homogenized by sonication (see no. 2 below) in appropriate buffers (see above) for subsequent measurement of immunoprecipitable enzyme activity (see below), or 2) subjected to measurement of [3H]2-deoxyglucose (0.1 µCi; medium concentration, 50 µM) uptake over a 1-min period as previously described (2). As indicated above, the adipocytes used presently and previously (2) were prepared by collagenase digestion of epididymal fat pads taken, in most cases, from the same rats that were used for in vivo studies of the vastus lateralis muscle. However, as alluded to above, prior insulin effects on glucose transport were completely lost during collagenase treatment of adipocytes in vitro. Also note that neither 10-day insulin treatment nor 60-h fasting had a significant effect on the number of adipocytes recovered in a unit volume of packed cells, and each incubation tube used in studies of glucose transport contained 300 µl of a 7% cell suspension, or approximately 150,000–180,000 cells.

PKC-{zeta}/{lambda} activation. As described previously (15, 25), cell lysates (250 µg adipocyte protein and 500 µg muscle protein) were immunoprecipitated overnight at 0-4 C with a polyclonal antiserum (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) that recognizes the C-termini of both PKC-{zeta} and PKC-{lambda} (compared with mouse tissues that are rich in PKC-{lambda}, rat tissues are rich in PKC-{zeta}, and, as shown below, contain only small amounts of PKC-{lambda}). Precipitates were collected on protein AG-Sepharose beads, washed, and incubated for 8 min at 30 C in 100 µl buffer containing 50 mM Tris-HCl (pH 7.5), 5 mM MgCl2, 1 mM EGTA, 100 µM Na3VO4, 100 µM Na4P2O7, 1 mM NaF, 100 µM PMSF, 50 µM ATP, 3–5 µCi [{gamma}-32P]ATP (NEN Life Science Products), 4 µg phosphatidylserine, and 40 µM serine-159 analog of the PKC-{epsilon} pseudosubstrate (amino acids 153–164; Biosource Technologies, Inc.), as described previously (15, 25). After incubation, 32P-labeled peptide was trapped on p81 filter paper and counted in a liquid scintillation counter.

PKB activation
As described previously (25), cell lysates (500 µg protein) were immunoprecipitated and assayed for PKB enzyme activity using reagents and directions supplied in a kit obtained from Upstate Biotechnology, Inc. (Lake Placid, NY). PKB activation was also assessed by immunoblotting for phosphorylation of serine 473 (25).

PI 3-kinase activation. Cell lysates (500 µg protein) were immunoprecipitated overnight at 0-4 C with anti-IRS-1 antibodies (provided by Dr. Alan Saltiel) and assayed for PI 3-kinase enzyme activity as previously described (2, 15). 32P radioactivity in TLC-purified PI-3-PO4 was quantified with a phosphorimager (Bio-Rad Laboratories, Inc., Hercules, CA).

Western analyses
As described previously (15, 16, 25), cell lysates and immunoprecipitates were boiled and stored in Laemmli buffer, subjected to SDS-PAGE, transferred to nitrocellulose membranes, and blotted with the following: 1) rabbit polyclonal anti-PKC-{zeta}/{lambda} antiserum that recognizes the C-termini of both PKC-{zeta} and PKC-{lambda} (Santa Cruz Biotechnology, Inc.); 2) sheep polyclonal anti-PKB antiserum (Upstate Biotechnology, Inc.); 3) rabbit polyclonal anti-p85{alpha}/PI 3-kinase subunit antiserum (Upstate Biotechnology, Inc.); 4) sheep polyclonal anti-3-phosphoinositide-dependent kinase-1 (anti-PDK-1) antiserum (Upstate Biotechnology, Inc.); 5) rabbit polyclonal anti-phosphoserine 473-PKB antiserum (New England Biolabs, Inc., Beverly, MA); 6) rabbit polyclonal anti-GLUT1 antiserum (provided by Dr. Ian Simpson); 7) rabbit polyclonal anti-GLUT4 antiserum (Biogenesis, Cambridge, MA); 8) goat polyclonal anti-PKC-{zeta} antiserum that recognizes the specific N-terminus of PKC-{zeta}; 9) mouse monoclonal anti-PKC-{lambda} antibodies that recognize a specific internal sequence in PKC-{lambda}; and 10) rabbit polyclonal antisera that recognize IRS-1 or IRS-2 (supplied by Dr. Morris White). Blots were quantitated by measurement of extended chemiluminescence in a Molecular Analyst Chemiluminescence/Phosphor-Imaging System (Bio-Rad Laboratories, Inc.).

Other analyses
Serum insulin levels were measured by RIA using a kit obtained from Linco Research, Inc. (St. Charles, MO). Serum FFA were measured with a kit obtained from Roche (Indianapolis, IN).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effects of rosiglitazone treatment on insulin signaling in muscles of Wistar nondiabetic and GK diabetic rats
Insulin treatment over a 15-min period provoked 2- to 3-fold increases in immunoprecipitable PKC-{zeta}/{lambda} activity that were readily apparent in the vastus lateralis muscles of both fed and fasting (over 24 h) Wistar nondiabetic rats (Fig. 1AGo). In all in vivo muscle studies, the vastus lateralis muscle was used, because it was large and could be rapidly sampled, and because in vivo effects of insulin on PKC-{zeta} appeared to be greater in this muscle compared with those in soleus and gastrocnemius muscles (data not shown). Also, as our primary goal was to compare insulin signaling mechanisms in muscles of untreated maximally hyperglycemic diabetic GK rats to those of either normoglycemic Wistar nondiabetic rats or to treated GK rats, all subsequent studies were conducted in fed rats, except in the case of GK diabetic rats that were subjected to the 60-h fast, which was required to bring their serum glucose levels reasonably close to those found in nondiabetic fed rats, viz. approximately 140–160 mg/dl (see below).



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  		Figure 1. Effects of insulin on PKC-{zeta}/{lambda} activity in vastus lateralis muscles of fed and fasted Wistar nondiabetic rats (A) and effects of insulin and rosiglitazone (RSGZ) treatment on immunoprecipitable PKC-{zeta}/{lambda} enzyme activity in vastus lateralis muscles of Wistar-nondiabetic and GK diabetic rats. A, Rats were fed standard rat chow ad libitum or were deprived of food for 24 h, then were treated for 15 min with 0.25 U insulin given im. After death, vastus lateralis muscles were rapidly removed and assayed for immunoprecipitable PKC-{zeta}/{lambda} enzyme activity. Values are the mean ± SE of the number of determinations shown in parentheses. B, Rats were treated first with rosiglitazone for 10–14 days and then with 0.25 U insulin for 15 min as described in Materials and Methods. Vastus lateralis muscles were then removed and analyzed for immunoprecipitable PKC-{zeta}/{lambda} enzyme activity. Values are expressed relative to the mean Wistar nondiabetic control value, which was set at unity. Bar graphs and brackets depict the mean ± SE of the number of determinations shown in parentheses in five separate experiments. P was determined by 2 x 2 x 2 factorial ANOVA. See A for representative data from a typical experiment.

 
As shown in Fig. 1BGo, insulin treatment in vivo provoked, on the average, 2.5-fold increases in immunoprecipitable PKC-{zeta}/{lambda} activity in the vastus lateralis muscles of fed Wistar nondiabetic rats. Compared with Wistar nondiabetic controls, insulin-induced activation of PKC-{zeta}/{lambda} in vastus lateralis muscles of fed GK diabetic rats was reduced by approximately 65%, on the average. Of particular interest, rosiglitazone treatment for 10–14 days completely reversed this defect (i.e. relative to nondiabetic Wistar rats) in insulin-induced activation of PKC-{zeta}/{lambda} in vastus lateralis muscles of GK diabetic rats, but had little or no significant effect on PKC-{zeta}/{lambda} activation in vastus lateralis muscles of Wistar nondiabetic rats (Fig. 1Go). Note that basal PKC{zeta}/{lambda} activity was not altered substantially by either the GK diabetic state or rosiglitazone treatment. Treatment with rosiglitazone for a shorter time period (6 days) was partially effective in reversing the defect in PKC-{zeta}/{lambda} activation by insulin in GK vastus lateralis muscles (data not shown).

As shown in Fig. 2Go, the recovery of combined PKC-{zeta} and PKC-{lambda} in immunoprecipitates prepared with the anti-C-terminal antiserum was not altered as a consequence of the GK diabetic state, insulin treatment, or rosiglitazone treatment. Similarly, the GK diabetic state, acute insulin treatment, and rosiglitazone treatment did not have significant effects on the level of PKC-{lambda}, as assessed in immunoprecipitates with a specific mouse monoclonal antibody (Fig. 2Go). It was necessary to use relatively large amounts of anti-C-terminal immunoprecipitates and relatively long periods of chemiluminescence to detect significant amounts of immunoreactive PKC-{lambda} in rat muscles (and rat adipocytes) (2), as the levels of immunoreactive PKC-{lambda} recovered in whole lysates of rat vastus lateralis muscles were very low compared with the level of PKC-{lambda} found in mouse vastus lateralis muscles, which contained virtually the same amount of total atypical PKC (as per anti-C-terminal antibodies) as the rat vastus lateralis muscle (Fig. 2Go).



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  		Figure 2. Levels of total combined PKC-{zeta} plus PKC-{lambda} and PKC-{lambda} in vastus lateralis muscles of Wistar nondiabetic and GK diabetic rats. A, Comparison of levels of immunoreactive combined PKC-{zeta}/{lambda} (as per rabbit anti-C-terminal antiserum that recognizes both PKC-{zeta} and PKC-{lambda}), PKC-{zeta} (as per goat anti-N-terminal antiserum that specifically recognizes PKC-{zeta}), and PKC-{lambda} (as per mouse monoclonal antibody that specifically recognizes an internal sequence in PKC-{lambda}) in 75 µg protein obtained from whole cell lysates of rat and mouse vastus lateralis muscles. B, Wistar nondiabetic and GK diabetic rats were treated as described in Fig. 1BGo, with or without RSGZ and/or insulin, and 1 mg cell lysate protein was immunoprecipitated with anti-C-terminal antiserum and blotted with the indicated antibodies ({alpha}).

 
In contrast to PKC-{zeta}/{lambda} activity, insulin-induced increases in PKB activity appeared to be diminished only slightly (~15%), but not significantly, in vastus lateralis muscles of GK diabetic rats (Fig. 3AGo). Moreover, PKB activity in the vastus lateralis muscle was not altered significantly by rosiglitazone treatment in either Wistar nondiabetic or GK diabetic rats (Fig. 3AGo). Insulin-induced increases in phosphorylation of serine 473 in PKB were, if anything, slightly higher in GK vastus lateralis muscles and, as with PKB enzyme activity, were not significantly changed by rosiglitazone treatment in vastus lateralis muscles of either Wistar nondiabetic or GK diabetic rats (Fig. 3BGo).



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  		Figure 3. Effects of insulin and rosiglitazone treatment on immunoprecipitable PKB enzyme activity (A) and phosphorylation of serine 473 in PKB (B) in vastus lateralis muscles of Wistar nondiabetic and GK diabetic rats. Experiments were conducted as described in Fig. 1BGo, except that immunoprecipitable PKB enzyme activity and immunoreactive phosphoserine 473-PKB were assayed as described in Materials and Methods. Values are the mean ± SE of the number of determinations shown in parentheses. Also see Fig. 5Go for representative blots of PKB and phosphoserine 473-PKB.

 
In association with decreases in PKC-{zeta}/{lambda} activation, insulin-induced increases in IRS-1-dependent PI 3-kinase activity were diminished in vastus lateralis muscles of GK diabetic rats (Fig. 4Go). On the other hand, rosiglitazone treatment had no significant effect on IRS-1-dependent PI 3-kinase activation in either Wistar nondiabetic or GK diabetic rats (Figs. 4Go and 5Go).



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  		Figure 4. Effects of insulin on IRS-1-dependent PI 3-kinase in vastus lateralis muscles of Wistar nondiabetic and GK diabetic rats treated or untreated with rosiglitazone. Rats were treated as described in Fig. 1BGo, after which cellular lysates (0.5–1 mg protein) were subjected to immunoprecipitation with anti-IRS-1 antibodies, and precipitates were assayed for PI 3-kinase activity. 32P-Labeled PI-3-PO4 was separated by TLC and quantitated in a phosphorimager (Bio-Rad Laboratories, Inc.). A, Mean ± SE values of the indicated treatment values compared with the mean Wistar-nondiabetic control value for each TLC plate; the number of determinations is shown in parentheses. B, Representative autoradiogram. P was determined by t test comparison of insulin-stimulated PI 3-kinase activity in muscles of Wistar nondiabetic and GK diabetic rats.

 


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  		Figure 5. Effects of insulin and rosiglitazone (RSGZ) treatment on levels of immunoreactive p85{alpha}/PI 3-kinase subunit, PDK-1, PKB, phosphoserine 473- PKB, PKC-{zeta}/{lambda}, GLUT1, and GLUT4 in vastus lateralis muscles of Wistar nondiabetic and GK diabetic rats. Tissues from Figs. 1BGo, 3Go, and 4Go were analyzed as described in Materials and Methods. Shown here are blots that are representative of at least four determinations. See Table 2Go for results of multiple comparisons. Aside from shifts in PKB and increases in phosphoserine 473-PKB after insulin treatment, there were no significant alterations in proteins that could be attributed either to the GK diabetic state or to acute insulin or 10- to 14-day rosiglitazone treatment.

 
Levels of IRS-1, the p85{alpha}/PI 3-kinase subunit, PDK-1, PKB, phosphoserine 473-PKB, PKC-{zeta}/{lambda}, GLUT1, and GLUT4 in GK vastus lateralis muscles, both basally and following 15-min insulin treatment, were not altered significantly by either GK diabetes or rosiglitazone treatment (Figs. 4Go and 5Go and Table 2Go).


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  		Table 2. Levels of immunoreactive substances in vastus lateralis of muscles of Wistar nondiabetic and GK-diabetic rats treated with or without rosiglitazone (RSGZ) and/or insulin (INS)

 
Effects of chronic (10-day) insulin treatment on insulin signaling in muscles and adipocytes of GK diabetic rats
After treatment of GK rats with daily injections of 4–6 U intermediate-acting NPH insulin at 1700 h over a 10-day period, serum glucose was diminished to essentially normal levels (see Table 1Go) at the time of the experiment, i.e. approximately 18–20 h after the last injection of NPH insulin. Subsequent acute treatment with 0.25 U rapid-acting regular insulin for 15 min caused serum glucose to decrease from 113 to approximately 60 mg/dl, and this was attended by substantial enhancement of acute insulin effects on PKC-{zeta}/{lambda} activity in the vastus lateralis muscle compared with acute insulin effects in muscles of GK rats not pretreated with NPH insulin (Fig. 6BGo). In contrast, acute effects of insulin on IRS-1-dependent PI 3-kinase activity in the vastus lateralis muscle were not increased after 10-day NPH insulin treatment (Fig. 6AGo), nor were effects on PKB activation (Fig. 6CGo) or PKB phosphorylation (Fig. 8Go). Baseline levels of IRS-1-dependent PI 3-kinase and PKC-{zeta}/{lambda} activity were mildly increased in muscles of GK rats treated for 10 days with NPH insulin, and this probably reflects residual increases in serum insulin levels after injection of NPH insulin on the preceding day (see Table 1Go). Despite these baseline alterations, it seems clear that subsequent increases in PKC-{zeta}/{lambda} activity after acute administration of 0.25 U regular insulin cannot be ascribed to alterations in IRS-1-dependent PI 3-kinase activation.



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  		Figure 6. Effects of acute insulin treatment on activation of IRS-1-dependent PI 3-kinase (A), PKC-{zeta}/{lambda} (B), and PKB (C) in vastus lateralis muscles of GK diabetic rats treated or untreated chronically with 4–6 U NPH insulin given sc daily at 1700 h for 10 days. Acute insulin treatment (0.25 U regular insulin, im, 15 min before death) was given 18–20 h after the last injection of NPH insulin. The inset in A shows a representative autoradiogram for PI-3-PO4 after separation by TLC. Values are the mean ± SE of of the number of determinations shown in parentheses. P was determined by t test comparison of insulin-stimulated PKC-{zeta}/{lambda} activity in muscles of chronically untreated and chronically insulin-treated GK diabetic rats.

 


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  		Figure 8. Levels of immunoreactive p85{alpha}/PI 3-kinase subunit, PKB, phosphoserine 473-PKB, PKC-{zeta}/{lambda}, and GLUT4 glucose transporters in vastus lateralis muscles and adipocytes of GK rats treated or not treated chronically with 4–6 U NPH daily for 10 days and subsequently treated or not treated acutely with 0.25 U regular insulin, as indicated. Treatments are described in Figs. 6Go and 7Go. Shown here are representative immunoblots. Similar results were obtained at least four times.

 
As described above, we have reported that isolated adipocytes of GK rats have defects in insulin-stimulated IRS-1-dependent PI 3-kinase activation, PKC-{zeta}/{lambda} activation, and glucose transport in vitro, and moreover, these defects in PKC-{zeta}/{lambda} and glucose transport, but not PI 3-kinase, are fully reversed by 10- to 14-day rosiglitazone treatment in vivo (2). [Defective glucose transport in GK adipocytes have also been reported by Begum et al. (26).] It was therefore of interest to find that chronic 10-day NPH insulin treatment in vivo, like 10- to 14-day rosiglitazone treatment, increased in vitro effects of acute insulin treatment on PKC-{zeta}/{lambda} activation (Fig. 7BGo) and glucose transport (Fig. 7AGo), without increasing IRS-1-dependent PI 3-kinase activation (Fig. 7CGo) or PKB phosphorylation (Fig. 8Go) in isolated GK adipocytes. Basal PKC-{zeta}/{lambda} activity, but not basal glucose transport or basal IRS-1-dependent PI 3-kinase activity, was increased in adipocytes isolated from rats treated with NPH insulin for 10 days (Fig. 7Go, A–C). In view of the fact that preexisting insulin effects on glucose transport are completely lost during collagenase digestion (i.e. during adipocyte preparation), the failure of such basal increases in PKC-{zeta}/{lambda} activity to increase glucose transport in isolated adipocytes probably reflects the fact that the activation of glucose transport most likely requires PKC-{zeta}/{lambda} to be activated at specific cellular sites, e.g. as defined by activation of the insulin receptor and certain intermediate downstream signaling factors, or requires another factor.



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  		Figure 7. Effects of acute insulin treatment on [3H]2-deoxyglucose uptake (A), PKC-{zeta}/{lambda} activation (B), and IRS-1-dependent PI 3-kinase activation (C) in adipocytes of GK rats treated or not treated chronically with 4–6 U NPH insulin given sc daily at 1700 h for 10 days. A, Adipocytes were incubated for 30 min with the indicated concentrations of insulin before measurement of [3H]2-deoxyglucose uptake over 1 min. B and C, Adipocytes were incubated for 10 min with or without 10 nM insulin before measurement of immunoprecipitable PKC-{zeta}/{lambda} and IRS-1-dependent PI 3-kinase activity. Values are the mean ± SE of the number of determinations shown in parentheses. The inset in C shows a representative autoradiogram for PI-3-PO4 after separation by TLC. P was determined by t test comparison of acute insulin-stimulated PKC-{zeta}/{lambda} activity in adipocytes of chronically untreated and chronically insulin-treated GK rats.

 
Acute increases in insulin-induced activation of PKC-{zeta}/{lambda} after chronic 10-day insulin treatment could not be explained by changes in the levels of PKC-{zeta}/{lambda} in either vastus lateralis muscles or adipocytes of GK rats (Fig. 8Go). Similarly, there were no significant changes in levels of IRS-1, p85{alpha}/PI3K subunit, or GLUT4 in vastus lateralis muscles and adipocytes after chronic 10-day insulin treatment (Fig. 8Go). PKB levels appeared to be increased in adipocytes and perhaps to a lesser extent in vastus lateralis muscles after chronic insulin treatment (Fig. 8Go), but this did not increase PKB activation (Fig. 6Go) or phosphorylation (Fig. 8Go; perhaps not surprising, as PKB activation and phosphorylation were not significantly compromised to begin with in these GK adipocytes and vastus lateralis muscles).

Effects of fasting on insulin signaling in muscles of GK rats
Restriction of food intake for 60 h caused serum glucose levels to decrease substantially in GK rats (Table 1Go). This fasting was attended by a significant increase in insulin-stimulated, but not basal, PKC-{zeta}/{lambda} activity in the vastus lateralis muscle of fasted GK rats (Fig. 9BGo). Insulin-stimulated PKB activity (Fig. 9CGo), but not PKB phosphorylation (Fig. 10Go), in the GK vastus lateralis muscle was also increased modestly, but not significantly, after short-term fasting. Absolute levels of insulin-stimulated IRS-1-dependent PI 3-kinase activity in GK vastus lateralis muscles were not altered by short-term fasting, but basal PI 3-kinase activity was decreased, and relative effects of insulin on PI 3-kinase activity were accordingly increased (Fig. 9AGo). In this regard, it may be noted that the lower basal PI 3-kinase activity in vastus lateralis muscles of fasted GK rats may have been due to fasting-induced decreases in circulating levels of glucose and insulin (see serum levels in Table 1Go). In any event, it is possible, but nevertheless uncertain, that increases in insulin-induced increases in PKC-{zeta}/{lambda} and possibly PKB activity were caused by increases in relative increases in IRS-1-dependent PI 3-kinase activation.



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  		Figure 9. Effects of insulin (0.25 U regular insulin, im, 15 min before death) on activation of IRS-1-dependent PI 3-kinase (A), PKC-{zeta}/{lambda} (B), PKB (C), and phosphoserine 473-PKB (C) in vastus lateralis muscles of GK rats fed ad libitum or fasted for 60 h. Values are the mean ± SE of the number of determinations shown in parentheses. The inset in A shows a representative autoradiogram for PI-3-PO4 after separation by TLC. P was determined by t test comparison of insulin-stimulated PKC-{zeta}/{lambda} activity in muscles of fed and fasted GK diabetic rats. NS, P > 0.05.

 


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  		Figure 10. Levels of immunoreactive p85{alpha}/PI 3-kinase subunit, PDK-1, PKB, phosphoserine 473-PKB, PKC-{zeta}/{lambda}, and GLUT4 glucose transporters in vastus lateralis muscles of GK rats fed ad libitum or fasted for 60 h and subsequently treated or not treated with 0.25 U regular insulin 15 min before death, as indicated. Treatments are described in Fig. 9Go. Shown here are representative immunoblots. Similar results were obtained at least four times.

 
Fasting-induced alterations in insulin- and IRS-1-dependent PI 3-kinase, PKC-{zeta}/{lambda}, and PKB activation could not be explained by changes in the levels of IRS-1, PKC-{zeta}/{lambda}, p85{alpha}/PI 3-kinase subunit, PDK-1, or PKB in GK vastus lateralis muscles (Fig. 10Go). Similarly, GLUT4 glucose transporter levels in GK vastus lateralis muscles were not altered by fasting (Fig. 10Go).

Short-term fasting also increased insulin-induced PKC-{zeta}/{lambda} activation in adipocytes of GK rats (Fig. 11BGo), and this was attended by increases in insulin-stimulated glucose transport (Fig. 11AGo). We did not examine changes in PI 3-kinase or PKB activity in adipocytes of fasted GK rats.



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  		Figure 11. Effects of insulin on [3H]2-deoxyglucose uptake (A) and PKC-{zeta}/{lambda} activation (B) in adipocytes of GK rats fed ad libitum or fasted for 60 h. A, Adipocytes were incubated for 30 min with the indicated concentrations of insulin before measurement of [3H]2-deoxyglucose uptake over 1 min. B, Adipocytes were incubated for 10 min with or without 10 nM insulin before measurement of immunoprecipitable PKC-{zeta}/{lambda} activity. Values are the mean ± SE of the number of determinations shown in parentheses.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Presently, we found that insulin treatment in vivo provoked increases in the activity of PKC-{zeta}/{lambda} in rat vastus lateralis skeletal muscles that were comparable in magnitude and rapidity to those observed in isolated rat adipocytes in vitro (15, 16). Although it was not possible to examine directly in the present in vivo studies, we suspect that insulin effects on PKC-{zeta}/{lambda} in vastus lateralis muscles are dependent upon activation of PI 3-kinase and PDK-1, as has been observed in isolated rat adipocytes (15, 25, 27), L6 myotubes (14), and 3T3/L1 adipocytes (17).

It was particularly interesting to find that the activation of PKC-{zeta}/{lambda} by insulin was impaired in the vastus lateralis muscles of GK diabetic rats. This defect in PKC-{zeta}/{lambda} activation could not be explained by altered levels of PKC-{zeta}, PKC-{lambda}, PDK-1, or the p85{alpha} subunit of PI 3-kinase, but, on the other hand, may have been caused by a defect in insulin-induced activation of IRS-1-dependent PI 3-kinase, as observed both presently and previously in GK diabetic skeletal muscles (28, 29) and as observed previously in GK adipocytes (2). In this scenario, our finding of only slight, if any, diminution in PKB activation in GK muscles and adipocytes suggested that, compared with PKC-{zeta}/{lambda}, PKB may be activated to a greater extent by factors other than IRS-1, or PKB may be more completely activated at lower levels of IRS-1-dependent PI 3-kinase activation.

Of further interest, as in GK adipocytes (2), rosiglitazone corrected the defect in insulin-stimulated PKC-{zeta}/{lambda} activation in GK vastus lateralis muscles despite 1) failing to improve insulin effects on IRS-1-dependent PI 3-kinase activation, 2) having little or no effect on PI 3-kinase/PDK-1-dependent PKB activation, and 3) having little or no effect on serum glucose, FFA, or insulin levels. Clearly, the effects of rosiglitazone on insulin-induced PKC-{zeta}/{lambda} activation in GK muscles and adipocytes cannot be explained by improvement of glucotoxicity or by alterations in IRS-1-dependent PI 3-kinase. Further, although we did not specifically measure IRS-2-dependent PI 3-kinase activation, IRS-2 levels, like IRS-1 levels, and total pY-dependent PI 3-kinase activation were not altered by rosiglitazone treatment (data not shown). It therefore seems clear that rosiglitazone did not provoke a generalized increase in PI 3-kinase and/or PDK-1 activity, but it is possible that rosiglitazone may have acted on a pool of PI 3-kinase/PDK-1 that selectively regulates PKC-{zeta}/{lambda}, rather than PKB. Alternatively, rosiglitazone may have down-regulated a phosphatase that selectively dephosphorylates and inactivates PKC-{zeta}/{lambda}. Further studies are needed to evaluate these possibilities.

Two treatments that effectively decrease blood glucose levels and improve clinical insulin resistance in human diabetics, viz. short-term fasting and chronic insulin treatment, like rosiglitazone, enhanced insulin-induced activation of PKC-{zeta}/{lambda} in both vastus lateralis muscles and adipocytes of GK diabetic rats. Whereas the effects of rosiglitazone on PKC-{zeta}/{lambda} activation could not be explained by improvement in blood glucose levels, the effects of 10-day insulin treatment and 60-h fasting on PKC-{zeta}/{lambda} activation may have been caused at least in part by a reversal of hyperglycemia-dependent alterations in insulin signaling. In this regard, note that serum FFA levels, despite being elevated in GK rats, were not altered significantly by rosiglitazone or chronic insulin treatment and were, in fact, increased in fasted GK rats. Accordingly, alterations in serum FFA levels could not account for the observed increases in insulin-stimulated PKC-{zeta}/{lambda} activation after 10- to 14-day rosiglitazone treatment, 10-day insulin treatment, or 60-h short-term fasting.

As with rosiglitazone, the mechanisms underlying the observed increases in insulin-induced activation of PKC-{zeta}/{lambda} in GK vastus lateralis muscles and adipocytes after chronic 10-day insulin treatment and short-term 60-h fasting remain uncertain. Nevertheless, regardless of the underlying mechanism, it is of considerable interest that chronic insulin treatment and short-term fasting were capable of inducing changes in PKC-{zeta}/{lambda} activation that were for the most part similar to those of rosiglitazone treatment.

As discussed, GK adipocytes have defects in insulin-stimulated IRS-1-dependent PI 3-kinase activation, PKC-{zeta}/{lambda} activation, and glucose transport, and the defects in PKC-{zeta}/{lambda} activation and glucose transport are reversed by 10- to 14-day rosiglitazone treatment, with little or no change in IRS-1-dependent PI 3-kinase or PKB activation (2). Similarly, we observed increases in insulin-stimulated PKC-{zeta}/{lambda} activation and glucose transport in GK adipocytes after 10-day insulin treatment and 60-h fasting, and at least in the case of 10-day insulin treatment, there were no significant increases in insulin-stimulated IRS-1-dependent PI 3-kinase activation or PKB phosphorylation/activation. It is therefore reasonable to suggest that observed increases in insulin-stimulated glucose transport in isolated GK adipocytes after 10-day insulin treatment and short-term fasting, like those after rosiglitazone treatment, were at least partly due to increases in PKC-{zeta}/{lambda} activation and moreover, at least in the case of 10-day insulin treatment, appeared to be independent of alterations in IRS-1-dependent PI 3-kinase and PKB.

Although it was presently not possible to examine insulin effects on glucose transport in vastus lateralis muscles, the defects in PKC-{zeta}/{lambda} activation and patterns of reversal of defects by each of the three treatments appeared to be similar in both adipocytes and vastus lateralis muscles. It is therefore reasonable to suggest that insulin-stimulated glucose transport in GK vastus lateralis muscles, like glucose transport in GK adipocytes, was improved by each of the three treatments. Nevertheless, more definitive studies are needed to test this possibility, e.g. euglycemic-hyperinsulinemic clamp studies in vivo or glucose transport studies in vitro, to determine whether these treatments repair the known defects in glucose disposal (22, 30) and glucose transport (28, 29) in GK muscle.

Ten- to 14-day rosiglitazone treatment had little or no effect on serum glucose levels in fed GK rats despite improving PKC-{zeta}/{lambda} activation and glucose transport in adipocytes and, presumably, in skeletal muscle. This contrasts with modest, but significant, decreases in fasting blood glucose levels in GK rats treated with troglitazone for longer periods of time (31). The latter apparent improvement in glycemia may reflect differences in fasting vs. fed states, effects of different thiazolidinediones, more chronic alterations in levels or activity of glucose transporters with longer thiazolidinedione treatment, and/or improvement in insulin secretion and/or hepatic glucose output with more prolonged thiazolidinedione treatment. Along the latter lines, it is possible that defects in glucose transport may not be as important as defects in insulin secretion and hepatic glucose output in determining blood glucose levels in type II diabetic GK rats, wherein the primary defect appears to reside in insulin secretion (32). In keeping with this possibility is the observation that muscle-specific knockout of the insulin receptor has little or no effect on plasma glucose levels and glucose tolerance (33), suggesting that muscle and, accordingly, insulin receptor-dependent glucose transport into muscle may not be as important as other tissues and other cellular processes in determining plasma glucose levels. On the other hand, muscle-specific knockout of the GLUT4 glucose transporter leads to severe insulin resistance and glucose intolerance (34), suggesting that defective glucose transport in muscle is required for normal glucose homeostasis. Another factor to consider in evaluating the failure of plasma glucose levels to diminish significantly despite improvement in insulin-stimulated glucose transport in the present studies is the fact that basal total body glucose utilization is, in fact, paradoxically increased in hyperglycemic/mildly hyperinsulinemic GK diabetic rats (30), most likely as a result of hyperglycemia, which can alone increase glucose uptake by "mass action" and perhaps other mechanisms in a variety of tissues. Despite such increases in glucose utilization at high glucose levels in GK rats (30), it is important to note that insulin-stimulated glucose disposal, uptake, and storage in muscle have also been shown to be diminished during euglycemic/hyperinsulinemic clamp studies of GK rats (22, 30).

Assuming that PKC-{zeta}/{lambda} activation is important for insulin-stimulated glucose transport, our findings suggest that each of the presently used insulin-sensitizing modalities can effectively repair the observed defects in insulin signaling to PKC-{zeta}/{lambda} and the glucose transport system in GK diabetic rats. Although it is not entirely certain, it seems likely that a sizeable portion of the insulin resistance observed in GK rats is acquired or secondary in nature, i.e. caused at least in part by hyperglycemia, fatty acidemia, sustained mild hyperinsulinemia, and/or increased tissue levels of diacylglycerol and subsequent activation of diacylglycerol-sensitive PKCs (35). Presumably, these known down-regulating factors led to the observed defects in insulin-induced activation of IRS-1, PI 3-kinase, and PKC-{zeta}/{lambda} as well as other signaling substances in the present studies of GK rats. Accordingly, insulin-stimulated PKC-{zeta}/{lambda} activation and glucose transport can be improved by these insulin-sensitizing regimens, even in the absence of substantial increases in IRS-1-dependent PI 3-kinase activation or PKB activation and, in the case of rosiglitazone, even in the absence of improvement in serum levels of glucose and FFA levels. Further studies are needed to characterize the mechanisms for enhancing the activation of PKC-{zeta}/{lambda}.

In summary, insulin-induced activation of IRS-1-dependent PI 3-kinase and PKC-{zeta}/{lambda} were found to be defective in vastus lateralis muscles as well as adipocytes of type II diabetic GK rats. Moreover, the defect in PKC-{zeta}/{lambda} activation was fully reversed or ameliorated by 10- to 14-day rosiglitazone treatment, chronic 10-day insulin treatment, and 60-hshort-term fasting even in the absence of changes in IRS-1-dependent PI 3-kinase activation. Although further studies are needed to identify the factors responsible for stimulatory effects of these insulin-sensitizing treatment modalities on PKC-{zeta}/{lambda} activation, the defect in PKC-{zeta}/{lambda} activation seems likely to at least partly explain the observed defects in insulin-stimulated glucose transport in tissues of GK diabetic rats.


   Acknowledgments
 
We thank Sara M. Busquets for her invaluable secretarial assistance.


   Footnotes
 
1 This work was supported by funds from the Department of Veterans Affairs Merit Review Program and NIH Research Grant 2R01-DK-38079–09A1. Back

Received October 5, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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