Classical PKC is not associated with defective insulin signaling in patients with impaired glucose tolerance

diabetes research and clinical practice 83 (2009) 334–340

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Diabetes Research and Clinical Practice journal homepage: www.elsevier.com/locate/diabres

Classical PKC is not associated with defective insulin signaling in patients with impaired glucose tolerance Do Min Kim b, Hyun Ju Jang a, Seung Jin Han a, Eun Suk Ha a, Yun Kyung Kim a, Jee Won Park a, Kyoung Eun Song a, Sun Hye Jung a, Sang Mi Ahn a, Sung E. Choi a, Hae Jin Kim a, Dae Jung Kim a, Hyun Chul Lee c, Kwan Woo Lee a,* a

Department of Endocrinology and Metabolism, Ajou University, School of Medicine, Suwon, South Korea Department of Internal Medicine, Hanil General Hospital, Seoul, South Korea c Department of Internal Medicine, Yonsei University, School of Medicine, Seoul, South Korea b

article info

abstract

Article history:

Background and aim: To investigate the role of insulin signaling defects in impaired glucose

Received 22 May 2008

tolerance (IGT), we assessed the functionality of the insulin signaling cascade before and

Received in revised form

after insulin stimulation in both IGT group and control group.

20 November 2008

Methods: Ten IGT subjects and 15 control subjects were recruited for this study. Whole-body

Accepted 25 November 2008

insulin-mediated glucose uptake was determined using a euglycemic hyperinsulinemic

Published on line 4 January 2009

clamp test. Muscle biopsies were obtained from the vastus lateralis muscle before and after insulin stimulation, to assess the insulin signaling cascade.

Keywords:

Results: The insulin-stimulated incremental changes in phosphorylated IR-beta, IRS, Akt,

Insulin resistance

and GSK-3 beta and in the membrane-associated PKC-zeta protein level were reduced in the

Impaired glucose tolerance (IGT)

IGT group compared with those in the control group ( p < 0.05). The membrane-associated

Insulin signaling

PKC-lambda protein level was also reduced in the IGT group, but not significantly so ( p = 0.08). The incremental changes in the protein levels of PKC-alpha, -beta, and -theta were not significantly different between the two groups. Conclusion: The subjects with IGT showed decreased membrane-associated PKC-zeta/ lambda activity in response to insulin stimulation, as well as defects in early insulin signaling. Our results suggest that membrane-associated PKC-alpha and -beta may not be associated with insulin resistance in IGT. Crown Copyright # 2008 Published by Elsevier Ireland Ltd. All rights reserved.

1.

Introduction

Insulin resistance, i.e., a loss in insulin sensitivity, is the major characteristic of impaired glucose tolerance (IGT) and type 2 diabetes mellitus (T2DM). Target tissues such as skeletal muscle account for the majority of insulin-stimulated glucose uptake and are therefore the major sites of insulin resistance [1]. Impairment of glucose uptake and metabolism has been

shown to play a key role in the pathogenesis of insulin resistance [2–4]. The binding of insulin to the extracellular alpha-subunit of its receptor results in the autophosphorylation of tyrosine residues in the receptor beta-subunit. The activation of the beta-subunit then leads to insulin receptor substrate (IRS) 1-4 phosphorylation and IRS-1-associated phosphatidylinositol-3 phosphate kinase (PI3 kinase) activation [5]. PI3 kinase activation is required for stimulation of the

* Corresponding author at: Department of Endocrinology and Metabolism, Ajou University School of Medicine, San-5 Wonchon Dong, Yongtong Gu, Suwon 443-721, South Korea. Tel.: +82 31 219 4526; fax: +82 31 219 4497. E-mail address: [email protected] (K.W. Lee). 0168-8227/$ – see front matter . Crown Copyright # 2008 Published by Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.diabres.2008.11.035

diabetes research and clinical practice 83 (2009) 334–340

glucose transporter GLUT4, which is then translocated to the plasma membrane where it imports glucose into the cell. Recent reports indicate that PI3 kinase downstream effectors, e.g., serine/threonine kinase AKT (PKB), play an important role in this process by linking GLUT4 [6,7]. Additionally, some PKC isoforms (PKC-lambda/zeta) may be involved in insulinstimulated glucose transport, as recent data indicate that PKC activity is decreased in insulin resistance and T2DM [8]. Glycogen synthase kinase-3 (GSK-3) is a serine/threonine kinase with multiple functions, including the regulation of glycogen synthase. GSK-3 remains active at resting state and is inactivated by protein kinases such as AKT [9]. Insulininduced glycogen synthase is inactivated by GSK-3. In addition, GSK-3-dependent phosphorylation of IRS-1 serine residues decreases insulin action via the PI3 kinase pathway in obese diabetic subjects [10]. PKC activity was recently shown to play a role in insulin resistance in cultured cells [11–13]. Kim et al. [14] demonstrated that membrane-bound PKC-lambda/zeta activation and decreased PI3 kinase activity are both involved in insulinmediated glucose disposal in an insulin-resistant rat model. In addition, insulin-stimulated PKC-alpha, -beta, and -theta translocate from the cytosol to the plasma membrane [15]. These data suggest that defects in the insulin signaling pathway leads to insulin resistance. Our previous reports demonstrated that IRS, AKT, and GSK-3 phosphorylation were lower in type 2 diabetic patients than in controls [16]. However, the underlying mechanisms mediating this effect are currently unknown. In the present study, we characterized insulin-mediated glucose uptake signaling cascades in Korean IGT patients, demonstrating that PKC-alpha, -beta, and -theta are not involved in insulin signaling in IGT patients.

2.

Subjects and methods

2.1.

Subjects

The IGT group consisted of 10 patients, and the control group consisted of 15 patients (without distinction of age, sex, or body mass index). All participants gave written informed consent for participation, and the institutional medical ethics committee approved all methods used in this study.

2.2.

Oral glucose tolerance test (OGTT)

Before the OGTT, all subjects fasted for 12 h, and blood glucose was measured after fasting. A beverage containing 75 g of glucose was consumed by each subject, and blood glucose was measured every half-hour afterward, for a total of 2 h. The subjects were classified into control groups and IGT groups based on the degree of glucose tolerance measured by the OGTT, in accordance with the National Diabetes Data Group.

2.3.

Measurements and biochemical markers

All participants received a complete physical examination that included measurements of height, weight, total body fat (using dual-energy X-ray absorptiometry), and waist and hip circumferences (waist-to-hip ratio; WHR). Fasting blood

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samples were drawn for measuring the concentrations of total cholesterol, triglycerides, high-density lipoproteins, HbA1c, insulin, and C-peptide.

2.4. Euglycemic hyperinsulinemic clamp test and muscle biopsy Whole-body insulin-mediated glucose uptake was determined using a euglycemic hyperinsulinemic clamp test [17]. The plasma insulin concentration is acutely raised and maintained at approximately 100 mU/ml by a prime-continuous infusion of insulin. The plasma glucose concentration is held constant at basal levels by a variable glucose infusion using the negative feedback principle. Briefly, a percutaneous biopsy sample of the vastus lateralis muscle was obtained from 15 to 20 cm above the knee, using a Bergstrom needle. Biopsy samples were immediately blotted free of blood and were frozen and stored in liquid nitrogen for later assay. A second muscle biopsy was performed at a site 4 cm from the first site at 120 min after the start of the insulin infusion. Glucose disposal rates were calculated using steadystate equations. To assess pancreatic beta-cell function, homeostasis model assessment (HOMA) of beta-cell function was calculated as follows: HOMA beta-cell function = 20  fasting insulin (mU/ml)/[fasting glucose (mmol/l)  3.5].

2.5. Analytical methods for measuring the insulin signal pathway (Western blot) In this study, we determined the levels of phosphorylated IRbeta, IRS, Akt, and GSK-3beta. A 50-mg sample of muscle tissue was homogenized at 4 8C in 500 ml of lysis buffer (20 mM Tris, pH 8.0, 137 mM NaCl, 1 mM MgCl2, 2 mM CaCl2, 1% NP-40, 2 mM vanadate, 1 mM DTT, and 2.5 mM phenylmethylsulfonylfluoride), and the lysate was centrifuged at 12,000 rpm for 20 min at 4 8C. Aliquots of the supernatant were removed for protein analysis by the Bradford method. The supernatant was heated at 95 8C for 5 min in SDS sample buffer containing 100 mM DTT, 100 mg of the sample was resolved by SDS-PAGE (10% resolving gel), and the separated proteins were transferred to a polyvinylidene fluoride membrane. To detect phosphorylated IR-beta, IRS, Akt, and GSK-3beta, the membranes were incubated with the following antibodies, respectively: anti-phospho-IR beta (Tyr1162/1163) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-phospho-IRS1(Tyr612) (Upstate Biotechnology, Lake Placid, NY, USA), polyclonal anti-phospho-Akt(Ser473), polyclonal anti-phospho-GSK-3 beta (Cell Signaling Technology, Minneapolis, MN, USA). Immunoreactive IR-beta, IRS were detected using HRPconjugated anti-rabbit IgG as the secondary antibody; for Akt and GSK-3beta, HRP-conjugated anti-rabbit IgG was used as the secondary antibody. The immunoreactive proteins were visualized and quantified with a chemiluminescence system.

2.6. Determination of membrane-associated PKC-alpha, -beta, -theta, -epsilon, -zeta, -lambda, and phospho-PKC-zeta/lambda protein levels Muscle tissue was lysated with buffer A [20 mm Tris–HCl, pH 7.5, containing 250 mM sucrose, 0.5 mM EDTA, 2 mM EGTA,

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diabetes research and clinical practice 83 (2009) 334–340

protease inhibitor cocktail (Roche Applied Science, Mannheim, Germany)], then incubated at 4 8C for 30 min, and followed by centrifugation at 100,000  g for 1 h. The supernatant was designated as the cytosolic fraction. The pellet was extracted with buffer B [20 mm Tris–HCl, pH 7.5, containing 1% SDS, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, protease inhibitor cocktail (Roche Applied Science, Mannheim, Germany)], and centrifuged at 100,000  g for 15 min. We determined that this supernatant was a membrane fraction by Western blot analysis using an anti-voltagedependent anion channel antibody (Calbiochem, La Jolla, CA, USA). We quantified the protein levels of PKC-alpha, beta, -theta, -epsilon, -zeta, -lambda, and phospho-PKCzeta/lambda on Western blots as described above, with the exception that the primary antibodies were anti-PKC-theta, -alpha, -beta, -lambda (BD Transduction, San Diego, CA, USA), anti-PKC-epsilon, -zeta (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and anti-phospho-PKC-zeta/lambda(Thr410/403) (Cell Signaling Technology, Minneapolis, MN, USA).

2.7.

Statistical analyses

All data are presented as the means  standard error. Statistics were performed using SPSS Version 13.0 (SPSS, Chicago, IL, USA). Groups were compared using the Mann– Whitney U-test. A value of p < 0.05 was considered to be statistically significant.

3.

Results

3.1.

Clinical and biochemical characteristics

Table 1 summarizes the characteristics of the study subjects. The subjects in the two groups were matched for age, BMI, and WHR. Patients with IGT had significantly higher fasting blood glucose and HbA1c levels in comparison with control subjects. Total-cholesterol, triglyceride, and high-density lipoprotein levels were not significantly different between the groups.

3.2.

Insulin-stimulated glucose infusion rates

The beta-cell function did not differ between the two groups; however, glucose utilization rates were approximately 39% lower in the IGT group than in the control group. These results suggest that the IGT group exhibited insulin resistance (control: 7.33  0.5 vs. IGT: 4.54  0.6, p < 0.01) (Table 2).

3.3.

Insulin signaling cascade

3.3.1.

IR-beta phosphorylation

The binding of insulin to the extracellular alpha-subunit of its receptor results in the autophosphorylation of tyrosine residues in the receptor beta-subunit. Baseline IR-beta phosphorylation did not differ between the groups. However, following insulin stimulation, the incremental percentage of IR-beta phosphorylation was lower in the IGT group (16.3%) than in the control group (43.6%; Fig. 1A; Control: before

Table 1 – Clinical characteristics and biochemical data of the subjects (means W S.E.). Characteristics Age (year) Sex (M/F) BMI (kg/m2) WHR HbA1c (%) Cholesterol (mmol/l) TG (mmol/l) HDL-C (mmol/l) LDL-C (mmol/l) FBG (mmol/l) Fasting insulin (mU/ml)

Control (n = 15) 38.9  1.9 15/5 23.6  0.6 0.85  0.01 5.2  0.1 4.65  0.16 1.11  0.11 1.44  0.09 2.76  0.11 5.42  0.09 5.63  0.42

IGT (n = 10) 42.9  2.0 0/10 24.4  1.0 0.88  0.01 5.7  0.1* 4.54  0.20 1.31  0.12 1.39  0.66 2.54  0.18 6.26  0.15* 6.11  0.79

BMI: body mass index; WHR: waist-to-hip ratio; TG: triglyceride; HDL-C: high-density lipoprotein cholesterol; LDL-C: low-density lipoprotein cholesterol; FBG: fasting blood glucose. * p < 0.01 vs. Control.

Table 2 – Beta-cell function and insulin resistance of the subjects (means W S.E.). Control (n = 15) Beta-cell function (%) M: glucose utilization rate (mg/kg min) *

66.95  5.9 7.33  0.5

IGT (n = 10) 54.4  6.1 4.54  0.6*

p < 0.01 vs. Control.

insulin stimulation 95.3  13.8, after insulin stimulation 136.3  28.3; IGT: before insulin stimulation 106.0  16.4, after insulin stimulation 121.8  24.5, p < 0.01).

3.3.2.

IRS phosphorylation

The activation of the IR-beta then leads to insulin receptor substrate (IRS) phosphorylation. Baseline IRS phosphorylation did not differ between the groups. The incremental percentage of IRS phosphorylation after insulin stimulation was lower in the IGT group than in the control group (Fig. 1B; Control: before insulin stimulation 91.7  7.9, after insulin stimulation 129.4  16.3; IGT: before insulin stimulation 103.3  15.6, after insulin stimulation 123.3  14.9, p < 0.01).

3.3.3.

Akt phosphorylation

The activation of IRS-1 then leads to PI3 kinase activation. PI3 kinase downstream effectors, e.g., serine/threonine kinase AKT (PKB), play an important role in this process by linking GLUT4. The incremental percentage of insulin-stimulated Akt phosphorylation was significantly decreased in the IGT group (33%) compared with that in the control group (50%; Fig. 1C; Control: before insulin stimulation 114.9  17.4, after insulin stimulation 146.0  34.9; IGT: before insulin stimulation 99.4  32.1, after insulin stimulation 133.9  18.5, p < 0.05).

3.3.4.

GSK-3 beta phosphorylation

Increased GSK-3 beta activity may be linked to pathology in diseases such as diabetes mellitus. GSK-3 is inactivated by protein kinases such as AKT. We observed elevated baseline

diabetes research and clinical practice 83 (2009) 334–340

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Fig. 1 – IR-beta, IRS-tyrosine, Akt, and GSK-3beta phosphorylation in control and IGT subjects before and after insulin stimulation. Representative immunoblots under basal conditions (S) and after insulin stimulation (+) are shown for one subject from each group. The bar graphs present the summary data (means W S.E.) for each group. (A) and (B) The incremental percentages of IR-beta and IRS phosphorylation in response to insulin in the IGT group were significantly lower than the control value (*p < 0.01 vs. control). (C) The incremental percentage of Akt phosphorylation in response to insulin in the IGT group was significantly lower than the control value (yp < 0.05 vs. control). (D) The incremental percentage of GSK-3beta phosphorylation in response to insulin in the IGT group was significantly lower than the control value (**p < 0.001 vs. control).

GSK-3 beta phosphorylation levels in the IGT group. Insulinstimulated GSK-3 beta phosphorylation increased 4.3% in the IGT group as compared with 25.7% in the control group.

3.3.5. Membrane-associated PKC-alpha, -beta, -theta, -epsilon, -zeta, -lambda, and phospho-PKC-zeta/lambda protein levels The basal membrane-associated levels of PKC-alpha, -beta, and -epsilon protein were similar in the IGT and control group, while the basal membrane-associated levels of PKC-theta, zeta, and -lambda, and phospho-PKC-zeta/lambda protein were significantly greater in the IGT group ( p < 0.05). Insulin-stimulated membrane-associated PKC-alpha, beta, and -theta activation did not differ between the groups (Fig. 2A–C). The IGT group showed higher incremental percentages of membrane-associated PKC-epsilon activation in response to insulin than the control group (38.8% vs. 3.5%, p < 0.05; Fig. 2D). The incremental percentages of membrane-associated PKC-zeta proteins and phospho-PKCzeta/lambda proteins in response to insulin were significantly lower in the IGT group than in the control group (12.7% vs. 32.9%, p < 0.05; 6.5% vs. 36.9%, p < 0.05; Fig. 2E and G). The IGT group tended to show lower incremental percentages of membrane-associated PKC-lambda activation in response to insulin than the control group (12.4% vs. 27.3%, p = 0.08; Fig. 2F).

4.

Discussion

The insulin signaling cascade leads to GLUT4 translocation and glucose uptake, and defects in this cascade play an important role in the pathogenesis of insulin resistance and impaired glucose tolerance in skeletal muscle, which is a major site of insulin resistance [8]. Recent reports indicate that the GLUT4 expression level does not differ between diabetic and non-diabetic patients, yet normal translocation of GLUT4 in the muscle of T2DM patients does not occur with insulin [18–20]. Therefore, we hypothesized that defects in the early steps of the insulin signaling cascade lead to the impaired glucose transport and may play a key role in the pathogenesis of insulin resistance. Most studies addressing these questions have been performed in patients with type 2 diabetes, and little is known regarding signaling defects in pre-diabetic stages such as IGT. In this study, we compared insulin signaling between control and IGT groups by determining the phosphorylation or expression of molecules involved in the insulin signaling pathway. Consistent with our hypothesis, we found that PKC isoforms are involved in the manifestation of IGT. Compared with the control subjects, the patients with IGT had significantly higher fasting blood glucose levels and had

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Fig. 2 – Membrane-associated protein levels of PKC-alpha, -beta, -theta, -epsilon, -zeta, -lambda, and phospho PKC-zeta/ lambda in control and IGT subjects before and after insulin stimulation. Representative immunoblots under basal conditions (S) and after insulin stimulation (+) are shown for one subject from each group. The bar graphs present the summary data (means W S.E.) for each group. (A) The incremental percentage of PKC-alpha activation in response to insulin did not differ between the groups. (B) The incremental percentage of PKC-beta activation in response to insulin did not differ between the groups. (C) The incremental percentage of PKC-theta activation in response to insulin did not differ between the groups. (D) The incremental percentage of PKC-epsilon activation in response to insulin in the IGT group was significantly higher than the control value (yp < 0.05 vs. control). (E) The incremental percentage of PKC-zeta activation in response to insulin in the IGT group was significantly lower than the control value (yp < 0.05 vs. control). (F) The incremental percentage of PKC-lambda activation in response to insulin in the IGT group was lower than the control value ( p = 0.08 vs. control). (G) The incremental percentage of phospho-PKC-zeta/lambda in response to insulin in the IGT group was significantly lower than the control value (yp < 0.05 vs. control).

glucose utilization rates that were about 39% lower, demonstrating the presence of insulin resistance in the IGT group. We next analyzed the phosphorylation or expression levels of proteins in the insulin signaling pathway. Baseline IR-beta and IRS phosphorylation did not differ between the groups; however, the incremental percentages of phosphorylated IR-beta and IRS after glucose stimulation were lower in the IGT group than in the control group. These data suggest that decreased activation of the insulin signaling cascade at the level of the insulin receptor may decrease PI3 kinase activation and thereby impair GLUT4 translocation.

The role of PI3 kinase in the impairment of GLUT4 translocation remains unclear; however, Akt and the atypical PKC-zeta and -lambda isoforms may be the downstream effectors of PI3 kinase [21–23]. AKT is a serine/ threonine kinase that is activated via a tyrosine kinase receptor [24–26]. Recent reports have demonstrated decreased AKT activity and IRS protein/PI3 kinase interactions in non-insulin dependent diabetic subjects [27]. Other studies have reported no difference in Akt activation between obese diabetic patients and normal subjects, despite the presence of decreased glucose transport and glycogen synthase activity [7]. Our findings showed a

diabetes research and clinical practice 83 (2009) 334–340

significantly decreased incremental change in insulinstimulated Akt phosphorylation in the IGT group compared with the change in the control group. The role of Akt in insulin resistance remains controversial, and further largescale studies are needed. Nikoulina et al. [28] found that both insulin-stimulated and unstimulated GSK-3 protein levels were significantly increased in obese diabetic subjects, but the specific activity (activity per molecule of enzyme) did not differ from control. Thus, although the specific activity of GSK-3 was not greater in the diabetic subjects, the total activity was greater owing to a higher level of GSK-3 in the muscle tissue of diabetic subjects as compared with controls. We observed elevated baseline GSK-3beta phosphorylation levels and reduced insulin-stimulated GSK-3beta phosphorylation in the IGT group. Defects in glucose transport and IRS-2-dependent PI-3 kinase and PKC-lambda/zeta activation were observed in cultured myotubes from obese subjects with IGT [8]. In addition, the insulin-stimulated PKC-lambda/zeta protein level was significantly lower in obese diabetic subjects [29]. Beeson et al. showed that PKC-theta/lambda activation after insulin infusion diminished in IGT and type 2 diabetic subjects [30]. In our study, the baseline level of membraneassociated PKC-zeta, -lambda, and phospho-PKC-zeta/ lambda protein was higher in the IGT group than in the control group. However, the incremental percentage of insulin-stimulated membrane-associated PKC-zeta protein and phospho-PKC-zeta/lambda protein decreased significantly in the IGT group; these findings are consistent with studies on IGT subjects. Previous studies have shown that insulin increases membrane-associated PKC-theta, as well as PKC-alpha and beta in rat skeletal muscle [31,32]. Griffin et al. [33] found decreased IRS-1-associated PI3 kinase activity and significantly increased membrane-bound PKC-theta in free fatty acid-induced insulin resistance in a rat model. The insulinstimulated membrane-associated PKC-alpha, -beta, and theta protein levels did not differ between the groups in our study. This is most likely explained by the normal range of blood lipid concentrations in our subjects, given that lipids activate PKC. Basal membrane-associated protein levels of PKC-theta, which is thought to contribute to insulin resistance [34], were significantly higher in the IGT group. Our study showed that insulin increased membrane-associated PKCepsilon in the IGT group. Activation of muscle PKC-epsilon has been associated with dietary-induced models of insulin resistance in normal rats [35]. PKC-epsilon exerts negative regulatory effects on insulin action, especially in skeletal muscle. In conclusion, patients with IGT demonstrated insulin resistance resulting from decreased insulin-stimulated glucose utilization. Furthermore, the insulin resistance was associated with impairments in IRS-1 activity and the insulin signaling cascade. In addition, our data suggest that decreased AKT, GSK-3 beta, and membraneassociated PKC-lambda/zeta activities are involved in insulin signaling, while membrane-associated PKC-alpha, -beta, and -theta are not likely involved in insulin resistance in IGT.

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Acknowledgements This work was supported by 2003 grant from ‘‘Department of Medical Sciences, The Graduate School, Ajou University’’ and a grant of the Korean Health 21 R&D Project, Ministry of Healthe and Welfare, Republic of Korea (A050463).

Conflict of interest There are no conflicts of interest.

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