In humans with obesity or type 2 diabetes, insulin target tissues are resistant to many actions of insulin. The atypical protein kinase C (PKC) isoforms λ and ζ are downstream of phosphatidylinositol-3 kinase (PI3K) and are required for maximal insulin stimulation of glucose uptake. Phosphoinositide-dependent protein kinase-1 (PDK-1), also downstream of PI3K, mediates activation of atypical PKC isoforms and Akt. To determine whether impaired PKCλ/ζ or PDK-1 activation plays a role in the pathogenesis of insulin resistance, we measured the activities of PKCλ/ζ and PDK-1 in vastus lateralis muscle of lean, obese, and obese/type 2 diabetic humans. Biopsies were taken after an overnight fast and after a 3-h hyperinsulinemic-euglycemic clamp. Obese subjects were also studied after weight loss on a very-low-calorie diet. Insulin-stimulated glucose disposal rate is reduced 26% in obese subjects and 62% in diabetic subjects (both comparisons P < 0.001). Insulin-stimulated insulin receptor substrate (IRS)-1 tyrosine phosphorylation and PI3K activity are impaired 40–50% in diabetic subjects compared with lean or obese subjects. Insulin stimulates PKCλ/ζ activity ∼2.3-fold in lean subjects; the increment above basal is reduced 57% in obese and 65% in diabetic subjects. PKCλ/ζ protein amount is decreased 46% in diabetic subjects but is normal in obese nondiabetic subjects, indicating impaired insulin action on PKCλ/ζ. Importantly, weight loss in obese subjects normalizes PKCλ/ζ activation and increases IRS-1 phosphorylation and PI3K activity. Insulin also stimulates PDK-1 activity approximately twofold with no impairment in obese or diabetic subjects. In contrast to our previous data on Akt, reduced insulin-stimulated PKCλ/ζ activity could play a role in the pathogenesis of insulin resistance in muscle of obese and type 2 diabetic subjects.
Impaired insulin-stimulated glucose transport in muscle and adipose tissue is a major contributor to the pathogenesis of insulin-resistant states such as obesity and type 2 diabetes (1). Insulin stimulates glucose transport by activating a cascade of tyrosyl phosphorylation events, initiated by binding of insulin to its receptor (2,3). The phosphorylated insulin receptor binds to and activates insulin receptor substrates (IRSs), resulting in stimulation of critical downstream pathways such as phosphatidylinositol 3-kinase (PI3K). PI3K is necessary, albeit not sufficient, for insulin stimulation of glucose uptake (4–7). Insulin-stimulated PI3K activity is impaired in skeletal muscle of obese type 2 diabetic subjects (8,9) and nonobese type 2 diabetic subjects (10,11). However, activation of Akt, a serine/threonine kinase downstream of PI3K involved in some of the metabolic actions of insulin (12,13), is normal in these same obese type 2 diabetic subjects (8), although not when muscle from lean type 2 diabetic subjects is incubated with a very high insulin concentration (14). Hence, the pathways downstream of PI3K that are responsible for the impairment in insulin-stimulated glucose metabolism are unclear.
Studies suggest that the atypical protein kinase C (PKC) isoforms λ and ζ are downstream mediators of PI3K and that their activation is required for insulin stimulation of glucose uptake, which involves translocation of the major insulin-responsive glucose transporter GLUT4 from intracellular sites to the cell membrane (15–17). Overexpression of a dominant-negative mutant of PKCλ or PKCζ abrogates insulin-stimulated glucose transport and GLUT4 translocation in adipose (15,18) and muscle cells (16,19). Overexpression of constitutively active PKCλ in adipocytes (15) or wild-type PKCζ in muscle in vivo (20) enhances both basal and insulin-stimulated glucose transport. Furthermore, PKCλ and -ζ appear to function interchangeably, since overexpression of wild-type PKCλ restores the inhibitory effects of a dominant-negative mutant of PKCζ on insulin-stimulated GLUT4 translocation and vice versa (21). The possibility that PKCλ/ζ could play an important role in insulin resistance in vivo is supported by studies showing impaired insulin-stimulated PKCλ/ζ activity in skeletal muscle and adipose tissue of nonobese type 2 diabetic Goto-Kakizaki (GK) rats (22,23) and normalization of PKCλ/ζ activity by the insulin-sensitizing drug rosiglitazone (22,23). Insulin-stimulated PKCλ/ζ activity is also decreased in cultured myotubes of obese humans with impaired glucose tolerance (24) and in muscle of obese diabetic monkeys (25). However, there are no data regarding the activation of PKCλ/ζ in vivo in humans.
Phosphoinositide-dependent protein kinase-1 (PDK-1) is a serine/threonine kinase, activated by a lipid product of PI3K phosphatidylinositol 3,4,5-triphosphate [PI(3,4,5)P3] (26–29). PDK-1 directly phosphorylates and activates downstream effectors of PI3K, such as Akt and atypical PKCs, that mediate physiological actions of insulin (27,29–31). PDK-1 appears to be a rate-limiting step since overexpression of wild-type PDK-1 in adipocytes is sufficient to stimulate GLUT4 translocation to the plasma membrane in the absence of insulin (32–34). Overexpression of kinase-inactive PDK-1 inhibits insulin-stimulated GLUT4 translocation (34). These data suggest that PDK-1 plays an important role in the regulation of glucose transport. It is unknown whether insulin activates PDK-1 in skeletal muscle in humans or whether activation of PDK-1 is impaired in insulin-resistant states. Therefore, to determine the mechanism(s) for insulin resistance in obese nondiabetic and type 2 diabetic subjects, we investigated the ability of insulin to stimulate PKCλ/ζ and PDK-1 activity in skeletal muscle. Furthermore, we determined whether defects in PKCλ/ζ activation were reversed with weight loss therapy in obese humans.
RESEARCH DESIGN AND METHODS
For the initial study, 15 lean and 16 obese nondiabetic subjects and 21 obese subjects with type 2 diabetes participated. Nearly all subjects were male (Table 1) except for the three women in the diabetic group. There was no difference in the insulin action parameters when the three women were excluded from the analyses, so we included them. The experimental protocol was approved by the Committee on Human Investigation of the University of California, San Diego. Informed written consent was obtained after explanation of the protocol. All nondiabetic subjects had normal glucose tolerance (75-g oral glucose load) as defined by fasting glucose <7.0 mmol/l and 2-h glucose <7.8 mmol/l (35). Except for diabetes, the subjects were healthy and on no medications known to influence glucose metabolism. Up until several weeks before the study, the treatment of diabetic subjects was as follows: metformin for two subjects, metformin plus insulin for three, metformin plus sulfonylurea for five, insulin for one, insulin plus sulfonylurea and metformin for one, sulfonylurea for three, exercise and diet for three, and no therapy for three subjects. Hypoglycemic agents were withdrawn at least 2 weeks before studies were performed.
Weight loss protocol.
Six obese subjects (BMI 30–45 kg/m2) (calculated as weight in kilograms divided by the height in meters squared) with normal glucose tolerance defined by oral glucose tolerance test (OGTT) criteria, who were weight-stable for at least 3 months before the study, entered the Special Diagnostics and Treatment Unit for baseline characterization: OGTT, hyperinsulinemic-euglycemic clamp, and muscle biopsy. Subjects were then placed on a very-low-calorie diet (VLCD) of 600–800 calories/day for up to 24 weeks or until ∼10–15% of initial body weight was lost. Subjects were inpatients during the first 4 weeks of diet therapy and were then monitored at weekly visits. Once the weight loss goal was met, subjects were introduced to a weight-maintenance diet. Metabolic characterization studies were repeated after subjects were weight-stable at their new level for at least 2–3 weeks.
Subjects were admitted to the Special Diagnostic and Treatment Unit. After an overnight fast, they underwent a 3-h hyperinsulinemic (300 mU · m−2 · min−1)-euglycemic (5.0–5.5 mmol/l) clamp (8). The glucose disposal rate (GDR) was determined during the last 30 min of the clamp. Percutaneous needle biopsies of vastus lateralis muscle were performed before insulin infusion and at the end of the clamp (8), and muscle tissue was immediately frozen in liquid nitrogen. Plasma glucose and insulin levels were determined before each biopsy.
Tyrosine phosphorylation of the insulin receptor and IRS-1.
Muscle lysates were prepared (8), and insulin receptor and IRS-1 phosphorylation were determined as described (36). IRS-1 phosphorylation in obese subjects before and after weight loss was determined using a polyclonal IRS-1 [pY 612] phosphospecific antibody (Biosource International, Camarillo, CA).
Determination of PI3K activity.
Muscle lysates (500 μg protein) were subjected to immunoprecipitation with a polyclonal IRS-1 antibody, and PI3K activity was determined as described (8).
Determination of PKCλ/ζ and PDK-1 activity.
Muscle lysates (200 μg protein) were subjected to immunoprecipitation with a polyclonal PKCζ antibody (Santa Cruz Biotechnology, Santa Cruz, CA) that recognizes both PKCλ and -ζ or a polyclonal PDK-1 antibody (Upstate Biotechnology, Lake Placid, NY) coupled to protein A-Sepharose beads (Sigma) or G-Sepharose beads (Amersham Pharmacia Biotechnology, Piscataway, NJ). Immune pellets were washed, and PKCλ/ζ activity was determined (22). PDK-1 activity was determined according to the manufacturer’s instructions (Upstate Biotechnology). With this protocol the efficiency of immunoprecipitation of PKCλ/ζ and PDK-1 is ∼90% (not shown).
PKCλ/ζ, PKCα, PKCι, PKCε, PKCθ, and PDK-1 protein levels.
Tissue lysate protein (50 μg per lane) was resolved by SDS-PAGE and transferred to nitrocellulose membranes. The nitrocellulose membranes were blocked with 5% nonfat dry milk and incubated with antibodies against PKCλ/ζ (recognizes both PKCλ and PKCζ; Santa Cruz Biotechnology), PDK-1 (Upstate Biotechnology), PKCα (Santa Cruz Biotechnology), PKCι (Transduction laboratories, San Jose, CA; recognizes human PKCι and mouse PKCλ, which are 97% homologous), PKCε (Santa Cruz Biotechnology), or PKCθ (Transduction Laboratories). The membranes were washed and bands visualized with enhanced chemiluminescence (Amersham) and quantified by densitometry (Molecular Dynamics) (37).
Statistical analyses including correlation coefficients were performed using the Stat View program (Abacus Concepts, Berkeley, CA). Statistical significance was tested with ANOVA and paired Student’s t test when appropriate.
The mean age was similar in all groups (Table 1). BMI was increased in obese nondiabetic and obese type 2 diabetic subjects compared with lean subjects. BMI was not different between obese nondiabetic and diabetic subjects. HbA1c and fasting serum glucose were normal in obese nondiabetic subjects but elevated in type 2 diabetic subjects. Fasting plasma insulin levels tended to be increased in obese nondiabetic subjects and were 2.9-fold elevated in diabetic subjects compared with lean subjects (Table 1). Clamp insulin levels were the same in lean and obese nondiabetic subjects. Although they were reduced in diabetic subjects, the concentration was still in the maximally stimulating range. Fasting plasma FFA levels were normal in obese nondiabetic subjects but were elevated in diabetic subjects. GDR was reduced 26% in obese nondiabetic subjects compared with lean subjects, 62% in obese diabetic subjects compared with lean subjects, and 48% in obese diabetic compared with obese nondiabetic subjects (Table 1). Thus, obese nondiabetic subjects were mildly insulin resistant, whereas diabetic subjects were more severely insulin resistant.
Insulin receptor and IRS-1 tyrosine phosphorylation.
Insulin stimulated insulin receptor tyrosine phosphorylation 2.8- to 3.7-fold in muscle of all groups, with no impairment in obese nondiabetic or diabetic subjects (Fig. 1A). Insulin also stimulated IRS-1 tyrosine phosphorylation ∼2.5-fold in lean and obese nondiabetic subjects. IRS-1 phosphorylation was decreased 41–48% in diabetic subjects compared with lean or obese nondiabetic subjects. The total amounts of insulin receptor and IRS-1 proteins were unaltered among the groups (Fig. 1A). These data confirm other previous findings (9–11).
Insulin increased IRS-1-associated PI3K activity 5.7-fold in lean, 3.5-fold in obese nondiabetic, and only 2.1-fold in obese diabetic subjects (Fig. 1B). Insulin-stimulated IRS-1-associated PI3K activity tended to be lower in muscle from obese nondiabetic subjects, but this did not reach statistical significance. PI3K was reduced 51% in obese diabetic subjects compared with lean subjects and 37% compared with obese nondiabetic subjects. The insulin-stimulated increment in PI3K activity above basal was decreased 70% in diabetic compared with lean subjects and 60% in diabetic compared with obese nondiabetic subjects. This is most likely due to decreased IRS-1 tyrosine phosphorylation (Fig. 1A). These data are also consistent with the previous findings (9–11). However, we felt it was important to quantitate the degree of impairment in these proximal signaling steps in the same samples in which we measured PKCλ/ζ activity and PDK-1 activity.
PKCλ/ζ activity and protein.
Basal activity of PKCλ/ζ was not different among the three groups (Fig. 2A). Insulin stimulated PKCλ/ζ activity 2.3-fold in muscle of lean subjects but only 1.6-fold in obese and 1.5-fold in diabetic subjects. The insulin-stimulated increment in PKCλ/ζ activity above basal was decreased 57% in obese and 65% in diabetic subjects compared with lean subjects (Fig. 2A). This suggests that reduced PKCλ/ζ activity may contribute to insulin resistance in muscle in humans. PKCλ/ζ protein amount was normal in obese subjects but was reduced 46% in diabetic subjects compared with lean subjects (Fig. 2B). When PKCλ/ζ activity was normalized for the protein amount, the increment of insulin-stimulated PKCλ/ζ activity over basal was still reduced 33% in diabetic subjects [lean 144 ± 28 and diabetic 96 ± 17 increase over basal (arbitrary units), P < 0.05].
A positive relationship was seen between insulin-stimulated PKCλ/ζ activity and insulin-stimulated GDR (r = 0.70, P < 0.08) in lean subjects. This correlation was not present in obese nondiabetic or obese diabetic subjects (data not shown). We found a similar correlation between the amounts of PKCλ/ζ protein and insulin-stimulated GDR in lean subjects (r = 0.53, P < 0.002).
PDK-1 activity and protein.
Insulin increased PDK-1 activity in muscle of nearly every subject. Neither basal nor insulin-stimulated PDK-1 activity was different among the three groups (Fig. 3A). In addition, the insulin-stimulated increment above basal was unchanged with obesity or diabetes. There was also no difference in the amount of PDK-1 protein in muscle among the groups (Fig. 3B).
PKCα, PKCι, PKCε, and PKCθ protein levels.
To determine the specificity of the effect to decrease PKCλ/ζ in muscle of obese diabetic subjects, we measured protein levels for specific PKC isoforms in the same samples. PKCα, PKCι/λ, PKCε, and PKCθ protein amounts were not altered by obesity or diabetes (Fig. 4). PKCι is the human homologue of the mouse PKCλ; these proteins are 97% homologous (38,39). The total amount of PKCι/λ was not decreased in diabetic subjects (Fig. 4), whereas immunoreactivity with the antibody that recognizes PKCλ and -ζ showed a 43% decrease (Fig. 2B). Transfection studies in COS-7 cells showed equal affinity of this latter antibody for PCKλ and PKCζ (not shown). Therefore, these data imply that PKCζ is specifically reduced in muscle of diabetic subjects.
Only obese nondiabetic subjects were treated with VLCD. Subjects lost 4.3–14.4 kg. After 24 weeks of VLCD treatment, body weight, BMI, and skinfold thickness were decreased (P < 0.01), but HbA1c, glucose, insulin, clamp insulin, and free fatty acid concentrations were unaltered (Table 2). Triglyceride concentrations tended to be reduced and GDR increased by 24% after VLCD treatment (P < 0.05) (Table 2).
VLCD treatment significantly increased IRS-1 tyrosine phosphorylation compared with pretreatment levels (Fig. 5A). In parallel, insulin-stimulated IRS-1-associated PI3K activity was increased ∼2.4-fold (P < 0.05) posttreatment with VLCD compared with pretreatment (Fig. 5B). The total amount of IRS-1 protein was unaltered by the diet treatment (Fig. 5A).
Basal activity of PKCλ/ζ was not altered by VLCD treatment, but insulin-stimulated activity was increased nearly twofold over pretreatment levels (P < 0.01) and was similar to the effect in lean control subjects (Figs. 2A and 6A). The amount of PKCλ/ζ protein in muscle was unchanged between both groups (Fig. 6B), indicating that the ability of insulin to activate PKCλ/ζ was improved by VLCD. This could be due to increased signaling upstream of PKCλ/ζ (Fig. 5B).
Because PI3K is important for insulin action to maintain glucose homeostasis (2,3) and because activation of PI3K is defective in obese (8,9) and nonobese type 2 diabetic humans (10,11), elucidation of the potential downstream mediators of PI3K that contribute to the insulin resistance of obesity and type 2 diabetes is of great interest. While recent studies in knockout mice indicate that Akt2 is important for the metabolic effects of insulin at least in the liver, it is not clear whether the mild defect in skeletal muscle is directly due to the absence of Akt2 in muscle or is secondary to the insulin-resistant state (13). Furthermore, we found no impairment in insulin-stimulated Akt1, Akt2, or Akt3 activation in skeletal muscle of obese nondiabetic or obese diabetic humans despite decreased PI3K activation in diabetic humans (8). However, a study in muscle of lean type 2 diabetic subjects incubated with very high insulin concentrations showed decreased Akt activation (14). In the current study we investigated the potential role of the atypical PKC isoforms, PKCλ/ζ, and of PDK-1, which are also downstream targets of PI3K.
Our data are the first to demonstrate that in vivo insulin administration in humans activates PKCλ/ζ and PDK-1 in skeletal muscle. Importantly, insulin-stimulated PKCλ/ζ activity is reduced in muscle of obese nondiabetic and obese diabetic subjects. This contrasts with our previous findings that Akt activation is normal in obese and diabetic subjects with similar metabolic characteristics (8). Thus, although IRS-1-associated PI3K activity is reduced in diabetic subjects, not all downstream pathways are similarly affected. Consistent with our current results in humans, several studies in insulin-resistant rodent models including GK diabetic rats (22), high-fat-fed rats (40), free fatty acid-infused rats (41), and Zucker obese rats (Y.-B.K., B.B.K., unpublished data) and in obese diabetic monkeys (25) also show decreased insulin-stimulated PKCλ/ζ activity in skeletal muscle. Taken together, these data suggest that reduced insulin-stimulated atypical PKC activity may play an important role in insulin resistance in vivo. Importantly, the impaired PKCλ/ζ activation in obese humans is reversed with weight reduction. This, combined with the fact that rosiglitazone treatment of diabetic subjects reverses the defect in PKCλ/ζ activity (42), suggests that reversal of the PKCλ/ζ defect might enhance insulin sensitivity in insulin-resistant states.
In obese subjects, the amount of PKCλ/ζ protein is normal, indicating that the entire defect in PKCλ/ζ activation is due to impaired insulin action on the enzyme. In our diabetic subjects, PKCλ/ζ protein in muscle is reduced. This may explain part of the defect in PKCλ/ζ activation, but the reduction in the insulin-stimulated increment in PKCλ/ζ activation above basal levels is greater than the reduction in PKCλ/ζ protein. Therefore, there appears to also be a defect in insulin action on PKCλ/ζ activity. The fact that basal PKCλ/ζ activity is not reduced in diabetic subjects in spite of lower PKCλ/ζ protein levels could be due to the elevated fasting insulin levels (Table 1). We see a similar reduction in PKCλ/ζ protein in muscle of obese Zucker rats with severe insulin resistance (Y.-B.K., B.B.K., unpublished data).
The reduction in PKCλ/ζ protein in diabetic humans appears to be relatively specific because protein levels of PKCα, PKCι, PKCε, and PKCθ, as well as Akt isoforms (8), are not affected. We did not measure the activity of these other PKC isoforms because the aim of our study was to identify pathways downstream of PI3K that have been implicated in mediating insulin-stimulated glucose transport. Our data do not exclude the possibility that the activity or membrane localization of other PKC isoforms could also be altered and could possibly impair insulin action at other steps in the pathway.
The impairment in PKCλ/ζ activation in diabetic subjects appears to be due to, at least in part, decreased upstream signaling including PI3K. Interestingly, PKCλ/ζ activity is impaired in obese subjects who have no defect in PI3K activity. This discrepancy may reflect the fact that a PI3K-independent pathway can be involved in PKCλ/ζ stimulation by insulin. The potential for metabolic regulation of PKCλ/ζ activation by alternate pathways is demonstrated by the fact that glucose at high concentration rapidly activates PKCλ/ζ in vitro in skeletal muscles through proline-rich tyrosine kinase-2, extracellular signal-related kinase-1/2 and phospholipase D pathways that are distinct from the PI3K pathway (43).
Because PI3K activates PKCλ/ζ by producing PI(3,4,5)P3, which binds directly to and activates PDK-1 (26–29), another possible mechanism for discordance between normal activation of PI3K and impaired PKCλ/ζ activity could be impaired PDK-1 activation. In the present study, we show that insulin stimulates PDK-1 activity in human skeletal muscle in vivo. Support for an insulin effect comes from in vitro studies showing that insulin increases phosphorylation and catalytic activity of PDK-1 in isolated rat adipocytes (32) and skeletal muscle cells (44). However, some studies indicated that PDK-1 is a constitutively active enzyme whose catalytic activity is not stimulated by growth factors such as insulin or IGF-1 in 293 cells or CHO cells or 3T3-L1 adipocytes (45–49). The difference may be due to differences in the type of cells or assay methodology in these studies. In addition, more recent studies in HeLa cells and 293 cells show insulin-stimulated translocation of PDK-1 to the plasma membrane, where it binds lipid products of PI3K and activates downstream signaling (50,51). In our study, insulin-dependent activation of PDK-1 is normal in muscle of obese nondiabetic and obese diabetic individuals. This combined with the impaired PKCλ/ζ activation in obese and diabetic subjects suggests that PKCλ/ζ activity can be regulated independent of PDK-1. Possibly, a defect in activation of another PDK isoform might explain the decrease in PKCλ/ζ activation or altered subcellular localization of PDK-1 could modify PKCλ/ζ activation. Although recent studies suggest that PDK-1 activity may be important for insulin stimulation of glucose transport and GLUT4 translocation in adipocytes and muscle cells (32–34), our data suggest that PDK-1 activation is not involved in the pathogenesis of insulin resistance in obesity and type 2 diabetes in humans.
In summary, in vivo administration of insulin in humans stimulates PKCλ/ζ and PDK-1 activity. In insulin-resistant obese subjects with or without type 2 diabetes and with the characteristic impairments in glucose disposal in skeletal muscle, insulin-stimulated PKCλ/ζ activity is reduced. Whereas PKCλ/ζ protein content is normal in obese nondiabetic subjects, it is reduced in diabetic subjects. However, this cannot fully account for the defect in PKCλ/ζ activation in diabetic subjects, and an impairment in insulin action on PKCλ/ζ appears to also be present. This impairment can be reversed in obese subjects treated with a VLCD. Thus, reduced PKCλ/ζ activity due to impaired activation and/or decreased expression may be an important factor in insulin resistance in muscle of obese nondiabetic and obese type 2 diabetic humans. Targeting PKCλ/ζ itself could potentially have side effects, since atypical PKCs mediate other biologic processes such as cell growth and proliferation (52–54). However, the emergence of PKCλ/ζ as a potentially important step in the pathogenesis of insulin resistance could lead to new treatment approaches for obesity and type 2 diabetes.
This work was supported by grants from the National Institute of Diabetes and Digestive and Kidney Diseases (NIH DK-43051), the American Diabetes Association, the Medical Research Service, the Department of Veterans Affairs and VA San Diego Healthcare System, and the General Clinical Research Branch, Division of Research Resources (NIH MO1 RR-00827).