Insulin Signaling and Glucose Transport in Skeletal Muscle From First-Degree Relatives of Type 2 Diabetic Patients
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糖尿病学杂志 2006年第5期
1 Department of Molecular Medicine and Surgery, Karolinska Hospital, Karolinska Institute, Stockholm, Sweden
2 Department of Physiology and Pharmacology, Karolinska Institute, Stockholm, Sweden
3 Division of Cardiology, Department of Medicine, Helsinki University Central Hospital and Biomedicum, Helsinki, Finland
IRS-1, insulin receptor substrate-1; KHB, Krebs-Henseleit buffer; NRF-1, nuclear respiratory factor-1; PGC, peroxisome proliferatoreCactivated receptor coactivator; PPAR, peroxisome proliferatoreCactivated receptor; UCP-3, uncoupling protein-3
ABSTRACT
Aberrant insulin signaling and glucose metabolism in skeletal muscle from type 2 diabetic patients may arise from genetic defects and an altered metabolic milieu. We determined insulin action on signal transduction and glucose transport in isolated vastus lateralis skeletal muscle from normal glucose-tolerant first-degree relatives of type 2 diabetic patients (n = 8, 41 ± 3 years, BMI 25.1 ± 0.8 kg/m2) and healthy control subjects (n = 9, 40 ± 2 years, BMI 23.4 ± 0.7 kg/m2) with no family history of diabetes. Basal and submaximal insulin-stimulated (0.6 and 1.2 nmol/l) glucose transport was comparable between groups, whereas the maximal response (120 nmol/l) was 38% lower (P < 0.05) in the relatives. Insulin increased phosphorylation of Akt and Akt substrate of 160 kDa (AS160) in a dose-dependent manner, with comparable responses between groups. AS160 phosphorylation and glucose transport were positively correlated in control subjects (R2 = 0.97, P = 0.01) but not relatives (R2 = 0.46, P = 0.32). mRNA of key transcriptional factors and coregulators of mitochondrial biogenesis were also determined. Skeletal muscle mRNA expression of peroxisome proliferatoreCactivated receptor (PPAR) coactivator (PGC)-1, PGC-1, PPAR, nuclear respiratory factor-1, and uncoupling protein-3 was comparable between first-degree relatives and control subjects. In conclusion, the uncoupling of insulin action on Akt/AS160 signaling and glucose transport implicates defective GLUT4 trafficking as an early event in the pathogenesis of type 2 diabetes.
Type 2 diabetes is a chronic metabolic disease and a major cause of morbidity and mortality, especially due to associated cardiovascular complications (1). Consequently, intense efforts are focused on defining the pathophysiological mechanisms for the conversion from normal glucose tolerance to type 2 diabetes. Impaired insulin-mediated whole-body glucose uptake is a characteristic feature of type 2 diabetes (2eC4), which arises from defects in glucose transport in skeletal muscle (2,5). In overt type 2 diabetic patients, the metabolic milieu is dramatically altered, and a constellation of features, including hyperglycemia, hyperlipidemia, hyperinsulinemia, and excessive cytokine production, develop. These secondary factors are known to influence insulin sensitivity and may obscure efforts to identify the primary cause of type 2 diabetes (6,7). Studies of insulin action in individuals at risk of developing type 2 diabetes, such as first-degree relatives of type 2 diabetic patients, are of particular relevance to dissect primary from secondary factors influencing glucose metabolism (8). First-degree relatives of type 2 diabetic patients with either normal or impaired glucose tolerance may already exhibit metabolic abnormalities characteristic of overt type 2 diabetes, such as insulin resistance and enhanced postprandial lipidmia without excessive hyperglycemia or hyperinsulipidemia (3,9eC12).
The molecular basis of peripheral insulin resistance in first-degree relatives to type 2 diabetic patients remains obscure, because few clinical studies have been performed in this population. Alterations in insulin signaling at the level of insulin receptor substrate-1 (IRS-1), phosphatidylinositol 3-kinase, and Akt have been observed in skeletal muscle from glucose-intolerant first-degree relatives of type 2 diabetic patients concomitant with impaired whole-body glucose uptake and metabolism (13). Because glucose-intolerant first-degree relatives to type 2 diabetic patients already manifest features of the type 2 diabetic phenotype (13), definite conclusions as to whether aberrant insulin signaling and glucose metabolism are intrinsic or secondary to the degree of metabolic dysregulation are difficult to obtain. Therefore, we determined whether insulin signaling and glucose transport are altered in skeletal muscle from normal glucose-tolerant first-degree relatives of type 2 diabetic patients.
RESEARCH DESIGN AND METHODS
Eight men with at least one first-degree relative with type 2 diabetes and nine men without a family history of diabetes (Table 1) were studied. One control subject had a grandfather with type 2 diabetes. Normal glucose tolerance was verified in all subjects with a 75-g oral glucose tolerance test. All subjects were instructed to avoid strenuous exercise for 72 h before the muscle biopsy. On the study days, the subjects reported to the laboratory after an overnight fast. The study protocol was approved by the ethical committee of the Karolinska Institutet, and informed consent was received from all subjects before participation.
Open muscle biopsy and in vitro incubations.
Open biopsies were taken from vastus lateralis muscle under local anesthesia (5 mg/ml mepivakain chloride), as previously described (14,15). Briefly, a 4-cm incision was made 15 cm above the proximal border of patella, and the muscle fascia was exposed. A smaller muscle biopsy was also removed from the incision site using a Weil-Blakesley conchotome and immediately frozen and stored in liquid nitrogen for subsequent mRNA analysis. Thereafter four to five muscle fiber bundles were excised and placed in oxygenated Krebs-Henseleit buffer (KHB), which contained 5 mmol/l HEPES, 5 mmol/l glucose, 15 mmol/l mannitol, and 0.1% BSA (RIA Grade; Sigma, St. Louis, MO). Smaller skeletal muscle strips were dissected from the muscle biopsy specimen, mounted on Plexiglass clamps, and incubated in vitro in pregassed (95% O2 and 5% CO2) KHB in shaking water bath at 35°C. The gas phase in the vials was maintained during the incubation procedure. After a 30-min incubation in KHB, skeletal muscle strips were incubated for 30 min at 35°C in KHB without (basal) or with increasing concentrations of insulin (0.6, 1.2, and 120 nmol/l). The concentrations of insulin were maintained throughout the incubation procedures.
Glucose transport.
Skeletal muscle strips were transferred to fresh KHB containing 20 mmol/l mannitol and incubated at 35°C for 10 min. Thereafter, muscles were incubated 20 min in KHB containing 5 mmol/l 3-O-methyl [3H]glucose (800 e藽i/mmol) and 15 mmol/l [14C]mannitol (53 e藽i/mmol). Thus, muscle strips were exposed to insulin for a total of 60 min. After the incubation, the muscle strips were blotted of excess fluid, snap frozen in liquid nitrogen, and stored at eC80°C until analysis. Glucose transport was analyzed by the accumulation of intracellular 3-O-methyl [3H]glucose, as previously described (16).
Tissue processing.
Muscle strips (10eC20 mg) were freeze-dried overnight and subsequently dissected under a microscope to remove visible blood, fat, and connective tissue. Muscles were homogenized in ice-cold homogenization buffer (90 e蘬/e蘥 dry wt muscle) (20 mmol/l Tris [pH 7.8], 137 mmol/l NaCl, 2.7 mmol/l KCl, 1 mmol/l MgCl2, 1% Triton X-100, 10% [w/v] glycerol, 10 mmol/l NaF, 0.5 mmol/l Na3VO4, 1 e蘥/ml leupeptin, 0.2 mmol/l phenylmethyl sulfonyl fluoride, 1 e蘥/ml aprotinin, and 1 e蘭ol/l microcystin). Homogenates were rotated for 30 min at 4°C. Samples were subjected to centrifugation (12,000g for 15 min at 4°C), and protein concentration was determined in the supernatant using a BCA protein assay (Pierce, Rockford, IL). An aliquot of the homogenate was mixed with Laemmli buffer containing -mercaptoethanol and heated (60°C) for 30 min.
Western blot analysis.
Proteins were separated by SDS-PAGE, transferred to nitrocellulose membranes, and blocked with Tris-buffered saline with 0.02% Tween containing 5% milk for 2 h. Membranes were incubated overnight with antieCphospho-Akt (Ser473) (catalog no. 9271), antieCphospho-Akt (Thr308) (catalog no. 9275), antieCphospho-(Ser/Thr) Akt substrate (catalog no. 9611; Cell Signaling Technology, Beverly, MA), or antieCperoxisome proliferatoreCactivated receptor (PPAR) coactivator (PGC)-1 (catalog no. AB3242; Chemicon International) at 4°C or for 1.5 h with anti-GLUT4 (Geoffrey D Holman, University of Bath, Bath, U.K.) at room temperature. Membranes were washed in Tris-buffered saline with 0.02% Tween and incubated with appropriate secondary horseradish peroxidaseeCconjugated antibodies (Bio-Rad, Richmond, CA). Immunoreactive proteins were visualized by enhanced chemiluminescence (ECL plus; Amersham, Arlington Heights, IL) and quantified by densitometry using Molecular Analyst Software (Bio-Rad).
mRNA expression analysis.
A portion of the skeletal muscle biopsy (15eC20 mg) was homogenized using a polytron mixer. Total RNA was purified using TRIZOL reagent (Invitrogen, Carlsbad, CA), as specified by the manufacturer. Purified RNA was treated with DNase I using DNA-free kit (Ambion, Austin, TX), according to the manufacturer’s protocol. DNase-treated RNA served as a template for cDNA synthesis using oligo(dT) primers and SuperScript First Strand Synthesis System (Invitrogen). Real-time PCR (ABI-PRISM 7000 Sequence Detector; Perkin-Elmer Applied Biosystems, Foster City, CA) was used for quantification of specific mRNA. Data were collected and analyzed by ABI Prism 7000 SDS Software version 1.1. Oligonucleotide primers and TaqMan probes (FAM-MGB) for human PGC-1, PGC-1, PPAR, nuclear respiratory factor-1 (NRF-1), and uncoupling protein-3 (UCP-3) were purchased as Assays-on-Demand from Applied Biosystems (assay IDs: Hs00602622_m1, Hs00173304_m1, Hs00370186_m1, Hs00243297_m1, and Hs00602161_m1). mRNA expression of human -actin (part no. VIC-MGB 4326315E) was determined as an endogenous reference gene to correct for potential variation in cDNA loading and quantity.
Statistical analysis.
Data are presented as means ± SE. Differences within and between groups were determined by ANOVA. Fisher’s least significant difference post hoc analysis was used to identify significant differences. Pearson correlation analysis was applied to determine the existence of possible relationships between average glucose transport and average Akt substrate of 160 kDa (AS160) phosphorylation for each condition. Differences were considered significant at P < 0.05.
RESULTS
Clinical characteristics.
First-degree relatives and control subjects were matched with respect to age, BMI, and adiposity (Table 1). VO2max tended to be lower in the first-degree relatives; however, this difference was not significant (P = 0.21). Fasting plasma glucose and insulin concentration and HbA1c were comparable between first-degree relatives and control subjects (Table 1). Serum total and HDL cholesterol and triglyceride concentration were also comparable between first-degree relatives and control subjects. Plasma glucose was measured at 30-min intervals during a 120-min oral glucose tolerance test. All subjects had normal glucose tolerance. Total glucose concentration expressed as area under the curve (770 ± 28 vs. 769 ± 36 mmol · leC1 · mineC1) or as an incremental area under the curve (114 ± 30 vs. 111 ± 33 mmol · leC1 · mineC1) was comparable between first-degree relatives and control subjects, respectively (Fig. 1). However, the 2-h plasma insulin concentration was 50% higher in the first-degree relatives (128 ± 19 vs. 86 ± 17 pmol/l, for relatives vs. control subjects, P = 0.12), providing evidence for mild insulin resistance compared with control subjects. The 2-h plasma glucose concentration was comparable between the subjects (5.3 ± 0.3 vs. 5.5 ± 0.3 mmol/l for relatives and control subjects, respectively, NS). Systolic blood pressure was increased in the first-degree relatives (P < 0.05).
Glucose transport and GLUT4 expression.
Basal glucose transport in isolated skeletal muscle strips from first-degree relatives and control subjects was identical (Fig. 2). Insulin-stimulated glucose transport in isolated muscle strips was similar between first-degree relatives and control subjects at physiological (0.6 nmol/l) and low pharmacological (1.2 nmol/l) insulin concentrations. Insulin-stimulated (120 nmol/l) glucose transport was reduced 38% (P < 0.05) in skeletal muscle from first-degree relatives, compared with the control subjects. Skeletal muscle GLUT4 protein expression was similar between control subjects and first-degree relatives (1.06 ± 0.06 vs. 1.02 ± 0.09 arbitrary units, respectively).
Insulin signaling: Akt phosphorylation at Ser473 (Fig. 3A) and Thr308 (Fig. 3B).
Basal phosphorylation of Akt on Ser473 and Thr308 was similar between first-degree relatives and control subjects. In vitro exposure of isolated skeletal muscle to insulin was associated with a concentration-dependent increase in Akt phosphorylation at Ser473 and Thr308, with similar responses noted between first-degree relatives and control subjects.
The AS160 is the insulin signaling component most proximal to glucose transporter translocation that has been identified to date (17,18). Basal phosphorylation of AS160 was comparable between first-degree relatives and control subjects (Fig. 4). Insulin increased AS160 phosphorylation in a concentration-dependent manner, with similar responses observed between first-degree relatives and control subjects. AS160 phosphorylation was positively correlated with glucose transport in control subjects (R2 = 0.97, P = 0.01; Fig. 5A), whereas this association was not observed in the first-degree relatives (R2 = 0.46, P = 0.32; Fig. 5B).
mRNA expression analysis.
mRNA of key transcriptional factors and coregulators of mitochondrial biogenesis were determined in skeletal muscle biopsies from first-degree relatives and control subjects (Table 2). Skeletal muscle mRNA expression of PGC-1, PGC-1, PPAR, NRF-1, and UCP-3 was comparable between first-degree relatives and control subjects. Protein expression of PGC-1 was similar between first-degree relatives and control subjects (1.95 ± 0.01 vs. 1.88 ± 0.01, means ± SE arbitrary units, respectively). Similar results between the genotypes were obtained when data were normalized against the expression of glyceraldehyde-3-phosphate dehydrogenase.
DISCUSSION
The resolution of the molecular mechanisms accounting for skeletal muscle insulin resistance and the conversion from normal glucose tolerance to type 2 diabetes can offer insight into disease pathogenesis. In vivo studies provide evidence that normal glucose-tolerant first-degree relatives of type 2 diabetic patients are insulin resistant at the level of skeletal muscle (3,9eC12), but the molecular mechanisms are undefined. Because reduced insulin-stimulated glucose transport in skeletal muscle has been proposed as one of the earliest metabolic abnormalities observed in the natural course of type 2 diabetes (19,20), we hypothesized that people at risk for the development of type 2 diabetes, namely first-degree relatives of type 2 diabetic patients, would present impairments in insulin signaling and glucose transport in skeletal muscle. Here, we provide evidence that insulin responsiveness for glucose transport is impaired in first-degree relatives of type 2 diabetic patients, despite intact insulin signaling, implicating GLUT4 trafficking defects in contributing to the insulin-resistant phenotype.
One advantage of using isolated muscle strips obtained either from elective surgery (21) or from a surgical open muscle biopsy (14) over in vivo studies is that insulin action can be studied under strictly controlled conditions and skeletal muscle from one individual can be subjected to multiple perturbations. Studies using this approach have revealed glucose transport defects under insulin-stimulated conditions in skeletal muscle from type 2 diabetic patients (21,22,23) and women with gestational diabetes (24). Women with a history of gestational diabetes and first-degree relatives of type 2 diabetic patients have an increased risk of developing diabetes later in life (8,25). Although we did not directly measure insulin sensitivity by means of the euglycemic-hyperinsulinemic clamp technique, the first-degree relatives in this study had mild insulin resistance, as evidenced by a trend for increased insulin concentration at 2 h during the glucose tolerance test. Nevertheless, the 2-h plasma glucose concentration, fasting insulin, triglyceride and cholesterol (total, LDL, and HDL) concentration, and glucose tolerance were normal, indicating that any metabolic dysregulation was mild in this cohort.
Insulin stimulates glucose uptake in skeletal muscle by inducing translocation of GLUT4, the predominant glucose transporter isoform expressed in skeletal muscle, from an intracellular pool to the plasma membrane (26). Recent evidence suggests that the PKB/Akt substrate AS160 is the insulin signaling step most proximal to GLUT4 trafficking events (17,18,27). AS160 is phosphorylated by PKB/Akt in response to insulin (18). Studies in cultured adipocytes provide evidence that insulin stimulation of GLUT4 exocytosis, at a step before fusion with the plasma membrane, is dependent on AS160 phosphorylation (18). We have recently reported that insulin-stimulated phosphorylation of AS160 is impaired in skeletal muscle from type 2 diabetic patients (28) and from healthy subjects in response to tumor necrosis factor- infusion (29). Impaired AS160 phosphorylation is associated with reduced skeletal muscle glucose uptake (28,29). Here, we reveal insulin increases the phosphorylation of Akt at Ser473 and Thr308 and AS160 in a concentration-dependent manner, with a similar response observed between first-degree relatives of type 2 diabetic patients and control subjects. Moreover, AS160 phosphorylation was positively related with insulin-stimulated glucose transport in healthy control subjects, providing correlative evidence that these two events are linked. In contrast, maximal insulin-mediated glucose transport was impaired in skeletal muscle from first-degree relatives, and the relationship with AS160 phosphorylation was disassociated, providing evidence that at higher insulin concentrations, GLUT4 traffic and possibly fusion of GLUT4 vesicles with the plasma membrane may be impaired. This is consistent with previous evidence whereby defects in the trafficking and translocation of GLUT4 glucose transporters in skeletal muscle have been linked to insulin resistance in human skeletal muscle (15,19,30). This issue will be challenging to directly resolve in human skeletal muscle. However, based on the evidence that insulin-independent glucose transport elicited in response to either hypoxia (30) or the AMP-activated protein kinase activator 5-amino-imidazole carboxamide riboside (15) is also impaired in skeletal muscle from type 2 diabetic subjects, defects in insulin signaling are unlikely to fully account for the impaired glucose transport in type 2 diabetes.
A recent study of first-degree relatives of type 2 diabetic patients provides evidence that insulin action on Ser473 phosphorylation and peripheral glucose uptake is impaired, concomitant with increased intramyocellular lipid content, elevated IRS-1 serine phosphorylation, and decreased mitochondrial content, independent of changes in expression of genes important for mitochondrial biogenesis (31). Although these subjects had normal glucose tolerance, the mean plasma concentration of glucose and insulin during the oral glucose tolerance test was significantly elevated in the first-degree relatives (31). In contrast, the glucose tolerance curves from the control and first-degree relatives in the present study were superimposable (Fig. 1), thus the insulin resistance phenotype in our cohort is milder. Moreover, insulin signaling at the level of Akt and AS160 in our cohort was unaltered. Thus, defects in glucose transport appear to precede impairments in insulin signaling at the level of Akt and AS160.
Key transcriptional factors and coregulators such as PGC-1, PGC-1, PPAR, and NRF-1 are known to regulate mitochondrial biogenesis (32). Several studies have provided evidence for a coordinated reduction of genes important for oxidative metabolism in humans with insulin resistance and type 2 diabetes and have implicated a role for PGC-1 (33,34). PGC-1 and -1 are coactivators involved in the regulation of mitochondrial metabolism and the maintenance of glucose and lipid metabolism and energy homeostasis (32). Microarray studies performed in skeletal muscle from type 2 diabetic subjects (33,34) and first-degree relatives (33) show PGC-1, as well as the genes they encode, are decreased compared with insulin-sensitive control subjects. These studies provide evidence to suggest that a primary defect in genes encoding proteins important for oxidative phosphorylation contributes to the development of insulin resistance and type 2 diabetes. However, in our study of glucose-tolerant first-degree relatives, expression of PGC-1, PGC-1, PPAR, NRF-1, and UCP-3 was unaltered. This is consistent with observations in young, lean, insulin-resistant glucose-tolerant first-degree relatives of type 2 diabetic patients, whereby skeletal muscle expression level of PGC-1, PGC-1, NRF-1, NRF-2, and mitochondrial transcription factor A is unaltered, despite reduced mitochondrial density (31).
In conclusion, insulin-mediated glucose uptake in skeletal muscle from glucose-tolerant first-degree relatives of type 2 diabetic patients is impaired, despite unaltered phosphorylation of Akt and AS160. The uncoupling of insulin action on AS160 and glucose transport may constitute an early defect in the pathogenesis of type 2 diabetes. Future longitudinal studies may be of value to clarify the extent of the disease progression in this cohort of glucose-tolerant first-degree relatives to type 2 diabetic patients.
ACKNOWLEDGMENTS
H.A.K. has received support from the Finnish Cultural Foundation, the Finnish Diabetes Research Foundation, the Finnish Foundation for Advancement of Laboratory Science, the Finnish Foundation for Cardiovascular Research, the Sigrid Juselius Foundation, the Paulo Foundation, and the Novo Nordisk Foundation. This work was supported by grants from the Swedish Research Council, the Swedish Diabetes Association, the Foundation for Scientific Studies of Diabetology, the Strategic Research Foundation, the Swedish Centre for Sports Research, and the Commission of the European Communities (contract nos. LSHM-CT-2004-005272 EXGENESIS and LSHM-CT-2004-512013).
FOOTNOTES
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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2 Department of Physiology and Pharmacology, Karolinska Institute, Stockholm, Sweden
3 Division of Cardiology, Department of Medicine, Helsinki University Central Hospital and Biomedicum, Helsinki, Finland
IRS-1, insulin receptor substrate-1; KHB, Krebs-Henseleit buffer; NRF-1, nuclear respiratory factor-1; PGC, peroxisome proliferatoreCactivated receptor coactivator; PPAR, peroxisome proliferatoreCactivated receptor; UCP-3, uncoupling protein-3
ABSTRACT
Aberrant insulin signaling and glucose metabolism in skeletal muscle from type 2 diabetic patients may arise from genetic defects and an altered metabolic milieu. We determined insulin action on signal transduction and glucose transport in isolated vastus lateralis skeletal muscle from normal glucose-tolerant first-degree relatives of type 2 diabetic patients (n = 8, 41 ± 3 years, BMI 25.1 ± 0.8 kg/m2) and healthy control subjects (n = 9, 40 ± 2 years, BMI 23.4 ± 0.7 kg/m2) with no family history of diabetes. Basal and submaximal insulin-stimulated (0.6 and 1.2 nmol/l) glucose transport was comparable between groups, whereas the maximal response (120 nmol/l) was 38% lower (P < 0.05) in the relatives. Insulin increased phosphorylation of Akt and Akt substrate of 160 kDa (AS160) in a dose-dependent manner, with comparable responses between groups. AS160 phosphorylation and glucose transport were positively correlated in control subjects (R2 = 0.97, P = 0.01) but not relatives (R2 = 0.46, P = 0.32). mRNA of key transcriptional factors and coregulators of mitochondrial biogenesis were also determined. Skeletal muscle mRNA expression of peroxisome proliferatoreCactivated receptor (PPAR) coactivator (PGC)-1, PGC-1, PPAR, nuclear respiratory factor-1, and uncoupling protein-3 was comparable between first-degree relatives and control subjects. In conclusion, the uncoupling of insulin action on Akt/AS160 signaling and glucose transport implicates defective GLUT4 trafficking as an early event in the pathogenesis of type 2 diabetes.
Type 2 diabetes is a chronic metabolic disease and a major cause of morbidity and mortality, especially due to associated cardiovascular complications (1). Consequently, intense efforts are focused on defining the pathophysiological mechanisms for the conversion from normal glucose tolerance to type 2 diabetes. Impaired insulin-mediated whole-body glucose uptake is a characteristic feature of type 2 diabetes (2eC4), which arises from defects in glucose transport in skeletal muscle (2,5). In overt type 2 diabetic patients, the metabolic milieu is dramatically altered, and a constellation of features, including hyperglycemia, hyperlipidemia, hyperinsulinemia, and excessive cytokine production, develop. These secondary factors are known to influence insulin sensitivity and may obscure efforts to identify the primary cause of type 2 diabetes (6,7). Studies of insulin action in individuals at risk of developing type 2 diabetes, such as first-degree relatives of type 2 diabetic patients, are of particular relevance to dissect primary from secondary factors influencing glucose metabolism (8). First-degree relatives of type 2 diabetic patients with either normal or impaired glucose tolerance may already exhibit metabolic abnormalities characteristic of overt type 2 diabetes, such as insulin resistance and enhanced postprandial lipidmia without excessive hyperglycemia or hyperinsulipidemia (3,9eC12).
The molecular basis of peripheral insulin resistance in first-degree relatives to type 2 diabetic patients remains obscure, because few clinical studies have been performed in this population. Alterations in insulin signaling at the level of insulin receptor substrate-1 (IRS-1), phosphatidylinositol 3-kinase, and Akt have been observed in skeletal muscle from glucose-intolerant first-degree relatives of type 2 diabetic patients concomitant with impaired whole-body glucose uptake and metabolism (13). Because glucose-intolerant first-degree relatives to type 2 diabetic patients already manifest features of the type 2 diabetic phenotype (13), definite conclusions as to whether aberrant insulin signaling and glucose metabolism are intrinsic or secondary to the degree of metabolic dysregulation are difficult to obtain. Therefore, we determined whether insulin signaling and glucose transport are altered in skeletal muscle from normal glucose-tolerant first-degree relatives of type 2 diabetic patients.
RESEARCH DESIGN AND METHODS
Eight men with at least one first-degree relative with type 2 diabetes and nine men without a family history of diabetes (Table 1) were studied. One control subject had a grandfather with type 2 diabetes. Normal glucose tolerance was verified in all subjects with a 75-g oral glucose tolerance test. All subjects were instructed to avoid strenuous exercise for 72 h before the muscle biopsy. On the study days, the subjects reported to the laboratory after an overnight fast. The study protocol was approved by the ethical committee of the Karolinska Institutet, and informed consent was received from all subjects before participation.
Open muscle biopsy and in vitro incubations.
Open biopsies were taken from vastus lateralis muscle under local anesthesia (5 mg/ml mepivakain chloride), as previously described (14,15). Briefly, a 4-cm incision was made 15 cm above the proximal border of patella, and the muscle fascia was exposed. A smaller muscle biopsy was also removed from the incision site using a Weil-Blakesley conchotome and immediately frozen and stored in liquid nitrogen for subsequent mRNA analysis. Thereafter four to five muscle fiber bundles were excised and placed in oxygenated Krebs-Henseleit buffer (KHB), which contained 5 mmol/l HEPES, 5 mmol/l glucose, 15 mmol/l mannitol, and 0.1% BSA (RIA Grade; Sigma, St. Louis, MO). Smaller skeletal muscle strips were dissected from the muscle biopsy specimen, mounted on Plexiglass clamps, and incubated in vitro in pregassed (95% O2 and 5% CO2) KHB in shaking water bath at 35°C. The gas phase in the vials was maintained during the incubation procedure. After a 30-min incubation in KHB, skeletal muscle strips were incubated for 30 min at 35°C in KHB without (basal) or with increasing concentrations of insulin (0.6, 1.2, and 120 nmol/l). The concentrations of insulin were maintained throughout the incubation procedures.
Glucose transport.
Skeletal muscle strips were transferred to fresh KHB containing 20 mmol/l mannitol and incubated at 35°C for 10 min. Thereafter, muscles were incubated 20 min in KHB containing 5 mmol/l 3-O-methyl [3H]glucose (800 e藽i/mmol) and 15 mmol/l [14C]mannitol (53 e藽i/mmol). Thus, muscle strips were exposed to insulin for a total of 60 min. After the incubation, the muscle strips were blotted of excess fluid, snap frozen in liquid nitrogen, and stored at eC80°C until analysis. Glucose transport was analyzed by the accumulation of intracellular 3-O-methyl [3H]glucose, as previously described (16).
Tissue processing.
Muscle strips (10eC20 mg) were freeze-dried overnight and subsequently dissected under a microscope to remove visible blood, fat, and connective tissue. Muscles were homogenized in ice-cold homogenization buffer (90 e蘬/e蘥 dry wt muscle) (20 mmol/l Tris [pH 7.8], 137 mmol/l NaCl, 2.7 mmol/l KCl, 1 mmol/l MgCl2, 1% Triton X-100, 10% [w/v] glycerol, 10 mmol/l NaF, 0.5 mmol/l Na3VO4, 1 e蘥/ml leupeptin, 0.2 mmol/l phenylmethyl sulfonyl fluoride, 1 e蘥/ml aprotinin, and 1 e蘭ol/l microcystin). Homogenates were rotated for 30 min at 4°C. Samples were subjected to centrifugation (12,000g for 15 min at 4°C), and protein concentration was determined in the supernatant using a BCA protein assay (Pierce, Rockford, IL). An aliquot of the homogenate was mixed with Laemmli buffer containing -mercaptoethanol and heated (60°C) for 30 min.
Western blot analysis.
Proteins were separated by SDS-PAGE, transferred to nitrocellulose membranes, and blocked with Tris-buffered saline with 0.02% Tween containing 5% milk for 2 h. Membranes were incubated overnight with antieCphospho-Akt (Ser473) (catalog no. 9271), antieCphospho-Akt (Thr308) (catalog no. 9275), antieCphospho-(Ser/Thr) Akt substrate (catalog no. 9611; Cell Signaling Technology, Beverly, MA), or antieCperoxisome proliferatoreCactivated receptor (PPAR) coactivator (PGC)-1 (catalog no. AB3242; Chemicon International) at 4°C or for 1.5 h with anti-GLUT4 (Geoffrey D Holman, University of Bath, Bath, U.K.) at room temperature. Membranes were washed in Tris-buffered saline with 0.02% Tween and incubated with appropriate secondary horseradish peroxidaseeCconjugated antibodies (Bio-Rad, Richmond, CA). Immunoreactive proteins were visualized by enhanced chemiluminescence (ECL plus; Amersham, Arlington Heights, IL) and quantified by densitometry using Molecular Analyst Software (Bio-Rad).
mRNA expression analysis.
A portion of the skeletal muscle biopsy (15eC20 mg) was homogenized using a polytron mixer. Total RNA was purified using TRIZOL reagent (Invitrogen, Carlsbad, CA), as specified by the manufacturer. Purified RNA was treated with DNase I using DNA-free kit (Ambion, Austin, TX), according to the manufacturer’s protocol. DNase-treated RNA served as a template for cDNA synthesis using oligo(dT) primers and SuperScript First Strand Synthesis System (Invitrogen). Real-time PCR (ABI-PRISM 7000 Sequence Detector; Perkin-Elmer Applied Biosystems, Foster City, CA) was used for quantification of specific mRNA. Data were collected and analyzed by ABI Prism 7000 SDS Software version 1.1. Oligonucleotide primers and TaqMan probes (FAM-MGB) for human PGC-1, PGC-1, PPAR, nuclear respiratory factor-1 (NRF-1), and uncoupling protein-3 (UCP-3) were purchased as Assays-on-Demand from Applied Biosystems (assay IDs: Hs00602622_m1, Hs00173304_m1, Hs00370186_m1, Hs00243297_m1, and Hs00602161_m1). mRNA expression of human -actin (part no. VIC-MGB 4326315E) was determined as an endogenous reference gene to correct for potential variation in cDNA loading and quantity.
Statistical analysis.
Data are presented as means ± SE. Differences within and between groups were determined by ANOVA. Fisher’s least significant difference post hoc analysis was used to identify significant differences. Pearson correlation analysis was applied to determine the existence of possible relationships between average glucose transport and average Akt substrate of 160 kDa (AS160) phosphorylation for each condition. Differences were considered significant at P < 0.05.
RESULTS
Clinical characteristics.
First-degree relatives and control subjects were matched with respect to age, BMI, and adiposity (Table 1). VO2max tended to be lower in the first-degree relatives; however, this difference was not significant (P = 0.21). Fasting plasma glucose and insulin concentration and HbA1c were comparable between first-degree relatives and control subjects (Table 1). Serum total and HDL cholesterol and triglyceride concentration were also comparable between first-degree relatives and control subjects. Plasma glucose was measured at 30-min intervals during a 120-min oral glucose tolerance test. All subjects had normal glucose tolerance. Total glucose concentration expressed as area under the curve (770 ± 28 vs. 769 ± 36 mmol · leC1 · mineC1) or as an incremental area under the curve (114 ± 30 vs. 111 ± 33 mmol · leC1 · mineC1) was comparable between first-degree relatives and control subjects, respectively (Fig. 1). However, the 2-h plasma insulin concentration was 50% higher in the first-degree relatives (128 ± 19 vs. 86 ± 17 pmol/l, for relatives vs. control subjects, P = 0.12), providing evidence for mild insulin resistance compared with control subjects. The 2-h plasma glucose concentration was comparable between the subjects (5.3 ± 0.3 vs. 5.5 ± 0.3 mmol/l for relatives and control subjects, respectively, NS). Systolic blood pressure was increased in the first-degree relatives (P < 0.05).
Glucose transport and GLUT4 expression.
Basal glucose transport in isolated skeletal muscle strips from first-degree relatives and control subjects was identical (Fig. 2). Insulin-stimulated glucose transport in isolated muscle strips was similar between first-degree relatives and control subjects at physiological (0.6 nmol/l) and low pharmacological (1.2 nmol/l) insulin concentrations. Insulin-stimulated (120 nmol/l) glucose transport was reduced 38% (P < 0.05) in skeletal muscle from first-degree relatives, compared with the control subjects. Skeletal muscle GLUT4 protein expression was similar between control subjects and first-degree relatives (1.06 ± 0.06 vs. 1.02 ± 0.09 arbitrary units, respectively).
Insulin signaling: Akt phosphorylation at Ser473 (Fig. 3A) and Thr308 (Fig. 3B).
Basal phosphorylation of Akt on Ser473 and Thr308 was similar between first-degree relatives and control subjects. In vitro exposure of isolated skeletal muscle to insulin was associated with a concentration-dependent increase in Akt phosphorylation at Ser473 and Thr308, with similar responses noted between first-degree relatives and control subjects.
The AS160 is the insulin signaling component most proximal to glucose transporter translocation that has been identified to date (17,18). Basal phosphorylation of AS160 was comparable between first-degree relatives and control subjects (Fig. 4). Insulin increased AS160 phosphorylation in a concentration-dependent manner, with similar responses observed between first-degree relatives and control subjects. AS160 phosphorylation was positively correlated with glucose transport in control subjects (R2 = 0.97, P = 0.01; Fig. 5A), whereas this association was not observed in the first-degree relatives (R2 = 0.46, P = 0.32; Fig. 5B).
mRNA expression analysis.
mRNA of key transcriptional factors and coregulators of mitochondrial biogenesis were determined in skeletal muscle biopsies from first-degree relatives and control subjects (Table 2). Skeletal muscle mRNA expression of PGC-1, PGC-1, PPAR, NRF-1, and UCP-3 was comparable between first-degree relatives and control subjects. Protein expression of PGC-1 was similar between first-degree relatives and control subjects (1.95 ± 0.01 vs. 1.88 ± 0.01, means ± SE arbitrary units, respectively). Similar results between the genotypes were obtained when data were normalized against the expression of glyceraldehyde-3-phosphate dehydrogenase.
DISCUSSION
The resolution of the molecular mechanisms accounting for skeletal muscle insulin resistance and the conversion from normal glucose tolerance to type 2 diabetes can offer insight into disease pathogenesis. In vivo studies provide evidence that normal glucose-tolerant first-degree relatives of type 2 diabetic patients are insulin resistant at the level of skeletal muscle (3,9eC12), but the molecular mechanisms are undefined. Because reduced insulin-stimulated glucose transport in skeletal muscle has been proposed as one of the earliest metabolic abnormalities observed in the natural course of type 2 diabetes (19,20), we hypothesized that people at risk for the development of type 2 diabetes, namely first-degree relatives of type 2 diabetic patients, would present impairments in insulin signaling and glucose transport in skeletal muscle. Here, we provide evidence that insulin responsiveness for glucose transport is impaired in first-degree relatives of type 2 diabetic patients, despite intact insulin signaling, implicating GLUT4 trafficking defects in contributing to the insulin-resistant phenotype.
One advantage of using isolated muscle strips obtained either from elective surgery (21) or from a surgical open muscle biopsy (14) over in vivo studies is that insulin action can be studied under strictly controlled conditions and skeletal muscle from one individual can be subjected to multiple perturbations. Studies using this approach have revealed glucose transport defects under insulin-stimulated conditions in skeletal muscle from type 2 diabetic patients (21,22,23) and women with gestational diabetes (24). Women with a history of gestational diabetes and first-degree relatives of type 2 diabetic patients have an increased risk of developing diabetes later in life (8,25). Although we did not directly measure insulin sensitivity by means of the euglycemic-hyperinsulinemic clamp technique, the first-degree relatives in this study had mild insulin resistance, as evidenced by a trend for increased insulin concentration at 2 h during the glucose tolerance test. Nevertheless, the 2-h plasma glucose concentration, fasting insulin, triglyceride and cholesterol (total, LDL, and HDL) concentration, and glucose tolerance were normal, indicating that any metabolic dysregulation was mild in this cohort.
Insulin stimulates glucose uptake in skeletal muscle by inducing translocation of GLUT4, the predominant glucose transporter isoform expressed in skeletal muscle, from an intracellular pool to the plasma membrane (26). Recent evidence suggests that the PKB/Akt substrate AS160 is the insulin signaling step most proximal to GLUT4 trafficking events (17,18,27). AS160 is phosphorylated by PKB/Akt in response to insulin (18). Studies in cultured adipocytes provide evidence that insulin stimulation of GLUT4 exocytosis, at a step before fusion with the plasma membrane, is dependent on AS160 phosphorylation (18). We have recently reported that insulin-stimulated phosphorylation of AS160 is impaired in skeletal muscle from type 2 diabetic patients (28) and from healthy subjects in response to tumor necrosis factor- infusion (29). Impaired AS160 phosphorylation is associated with reduced skeletal muscle glucose uptake (28,29). Here, we reveal insulin increases the phosphorylation of Akt at Ser473 and Thr308 and AS160 in a concentration-dependent manner, with a similar response observed between first-degree relatives of type 2 diabetic patients and control subjects. Moreover, AS160 phosphorylation was positively related with insulin-stimulated glucose transport in healthy control subjects, providing correlative evidence that these two events are linked. In contrast, maximal insulin-mediated glucose transport was impaired in skeletal muscle from first-degree relatives, and the relationship with AS160 phosphorylation was disassociated, providing evidence that at higher insulin concentrations, GLUT4 traffic and possibly fusion of GLUT4 vesicles with the plasma membrane may be impaired. This is consistent with previous evidence whereby defects in the trafficking and translocation of GLUT4 glucose transporters in skeletal muscle have been linked to insulin resistance in human skeletal muscle (15,19,30). This issue will be challenging to directly resolve in human skeletal muscle. However, based on the evidence that insulin-independent glucose transport elicited in response to either hypoxia (30) or the AMP-activated protein kinase activator 5-amino-imidazole carboxamide riboside (15) is also impaired in skeletal muscle from type 2 diabetic subjects, defects in insulin signaling are unlikely to fully account for the impaired glucose transport in type 2 diabetes.
A recent study of first-degree relatives of type 2 diabetic patients provides evidence that insulin action on Ser473 phosphorylation and peripheral glucose uptake is impaired, concomitant with increased intramyocellular lipid content, elevated IRS-1 serine phosphorylation, and decreased mitochondrial content, independent of changes in expression of genes important for mitochondrial biogenesis (31). Although these subjects had normal glucose tolerance, the mean plasma concentration of glucose and insulin during the oral glucose tolerance test was significantly elevated in the first-degree relatives (31). In contrast, the glucose tolerance curves from the control and first-degree relatives in the present study were superimposable (Fig. 1), thus the insulin resistance phenotype in our cohort is milder. Moreover, insulin signaling at the level of Akt and AS160 in our cohort was unaltered. Thus, defects in glucose transport appear to precede impairments in insulin signaling at the level of Akt and AS160.
Key transcriptional factors and coregulators such as PGC-1, PGC-1, PPAR, and NRF-1 are known to regulate mitochondrial biogenesis (32). Several studies have provided evidence for a coordinated reduction of genes important for oxidative metabolism in humans with insulin resistance and type 2 diabetes and have implicated a role for PGC-1 (33,34). PGC-1 and -1 are coactivators involved in the regulation of mitochondrial metabolism and the maintenance of glucose and lipid metabolism and energy homeostasis (32). Microarray studies performed in skeletal muscle from type 2 diabetic subjects (33,34) and first-degree relatives (33) show PGC-1, as well as the genes they encode, are decreased compared with insulin-sensitive control subjects. These studies provide evidence to suggest that a primary defect in genes encoding proteins important for oxidative phosphorylation contributes to the development of insulin resistance and type 2 diabetes. However, in our study of glucose-tolerant first-degree relatives, expression of PGC-1, PGC-1, PPAR, NRF-1, and UCP-3 was unaltered. This is consistent with observations in young, lean, insulin-resistant glucose-tolerant first-degree relatives of type 2 diabetic patients, whereby skeletal muscle expression level of PGC-1, PGC-1, NRF-1, NRF-2, and mitochondrial transcription factor A is unaltered, despite reduced mitochondrial density (31).
In conclusion, insulin-mediated glucose uptake in skeletal muscle from glucose-tolerant first-degree relatives of type 2 diabetic patients is impaired, despite unaltered phosphorylation of Akt and AS160. The uncoupling of insulin action on AS160 and glucose transport may constitute an early defect in the pathogenesis of type 2 diabetes. Future longitudinal studies may be of value to clarify the extent of the disease progression in this cohort of glucose-tolerant first-degree relatives to type 2 diabetic patients.
ACKNOWLEDGMENTS
H.A.K. has received support from the Finnish Cultural Foundation, the Finnish Diabetes Research Foundation, the Finnish Foundation for Advancement of Laboratory Science, the Finnish Foundation for Cardiovascular Research, the Sigrid Juselius Foundation, the Paulo Foundation, and the Novo Nordisk Foundation. This work was supported by grants from the Swedish Research Council, the Swedish Diabetes Association, the Foundation for Scientific Studies of Diabetology, the Strategic Research Foundation, the Swedish Centre for Sports Research, and the Commission of the European Communities (contract nos. LSHM-CT-2004-005272 EXGENESIS and LSHM-CT-2004-512013).
FOOTNOTES
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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