This Article Abstract Full Text (PDF) Submit a related Letter to the Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Ciaraldi, T. P. Articles by Henry, R. R. Search for Related Content PubMed PubMed Citation Articles by Ciaraldi, T. P. Articles by Henry, R. R. Related Collections Diabetes and Insulin

Skeletal Muscle GLUT1 Transporter Protein Expression and Basal Leg Glucose Uptake Are Reduced in Type 2 Diabetes

Veterans Affairs San Diego HealthCare System, San Diego, California 92161; and Department of Medicine, University of California-San Diego, La Jolla, California 92093

Address all correspondence and requests for reprints to: Dr. Robert R. Henry, Veterans Affairs San Diego HealthCare System (111G), 3350 La Jolla Village Drive, San Diego, California 92161. E-mail: rrhenry{at}vapop.ucsd.edu.

	Abstract

Top Abstract Introduction Subjects and Methods Results Discussion References

 
To investigate the role of skeletal muscle tissue expression of the glucose transporter protein GLUT1 in mediating glucose disposal in the basal (fasting) state, skeletal muscle biopsies (vastus lateralis) were obtained from lean and obese nondiabetics and type 2 diabetic subjects. Basal and insulin-stimulated glucose uptakes were measured. Basal whole body glucose uptake was measured using isotope dilution, and arteriovenous catheterization limb balance was used to determine leg muscle glucose uptake. Basal (noninsulin-stimulated) whole body glucose uptake was higher in the type 2 group compared with the controls (2.26 ± 0.17 vs. 1.83 ± 0.15 mg/kg·min; P < 0.05). However, basal leg muscle glucose uptake was reduced in diabetic subjects (1.53 ± 0.56 vs. 3.89 ± 0.83 mg/100 ml·min; P < 0.025) despite basal hyperglycemia (230 ± 13 vs. 94 ± 2 mg/dl; P < 0.0005). Skeletal muscle GLUT1 protein expression was lower in the type 2 subjects (57 ± 12 vs. 91 ± 11 arbitrary units/10 µg protein; P < 0.05), although GLUT1 mRNA levels did not differ. In summary, 1) skeletal muscle tissue GLUT1 protein expression is reduced in type 2 diabetes and could contribute to impaired basal leg glucose uptake; and 2) elevated rates of basal whole body glucose uptake in type 2 diabetes are due to uptake in tissues other than skeletal muscle.

	Introduction

Top Abstract Introduction Subjects and Methods Results Discussion References

 
IT HAS LONG been known that insulin-mediated glucose uptake is impaired in type 2 diabetes (T2D) (1). However, less is known about glucose uptake in the basal, fasting state. Under basal conditions, approximately 75–85% of glucose is disposed of via noninsulin-mediated glucose uptake (2). Glucose uptake into tissues is mediated in large part by the members of a series of facilitated carrier proteins, designated GLUT1–12 (3). GLUT1 is nearly ubiquitous in its distribution and is thought to be primarily responsible for this basal, constitutive glucose transport (4). Although a major portion of noninsulin-mediated glucose uptake occurs in noninsulin-responsive tissues, such as brain and gut (5), the large proportion of total body mass represented by skeletal muscle suggests that uptake into this tissue is not an insignificant contributor to whole body basal glucose uptake. In previous studies using cultured skeletal muscle cells, we demonstrated that basal glucose uptake determined at normal ambient glucose levels is reduced in T2D (6). Thus, impaired basal glucose uptake into skeletal muscle could contribute to glucose intolerance and hyperglycemia independent of insulin resistance in T2D.

Type 2 diabetic subjects demonstrate increased whole body glucose uptake under basal hyperglycemic conditions compared with nondiabetic subjects (7). To date, it has not been fully elucidated which tissues are responsible for this increased disposal. Baron and colleagues (5) found that in somatostatin-infused (insulinopenic) normal control subjects, hyperglycemia is accompanied by a concomitant increase in skeletal muscle glucose uptake. Thus, one might predict that skeletal muscle accounts for some of the increased whole body glucose uptake in type 2 diabetic subjects at prevailing hyperglycemia. The mass action effect of the prevailing hyperglycemia could also mask or minimize an actual impairment of muscle glucose uptake. This postulate would be consistent with a decrease in GLUT1 expression in diabetic skeletal muscle. In the current study we investigated skeletal muscle GLUT1 expression and its possible relationship to skeletal muscle glucose uptake as well as the latter’s contribution to whole body glucose uptake in the basal state. In contrast to our expectation, basal leg muscle glucose uptake is reduced in type 2 diabetic patients despite hyperglycemia and increased rates of whole body glucose uptake. We also demonstrate that this impairment of muscle glucose uptake is associated with reductions in the expression of the constitutive basal transport protein GLUT1.

	Subjects and Methods

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Subjects

The study group consisted of 14 T2D male and 16 age-matched nondiabetic (ND) male subjects. The clinical and metabolic profiles of the subjects are shown in Table 1. Both subject groups were equally distributed across a range of body mass indexes (23–37 in ND; 24–45 in T2D); the average body mass index did not differ between groups. The T2D subjects were hyperglycemic and hyperinsulinemic, although the differences in insulin values between groups did not attain statistical significance. All of the diabetic subjects were taking sulfonylureas before the study, and medications were discontinued at least 2 wk before studies were performed. The experimental protocol was approved by the committee on human investigation of University of California-San Diego. Informed written consent was obtained from all subjects after explanation of the protocol. All subjects were admitted to the Special Diagnostic and Treatment Unit at the Veterans Affairs Medical Center (San Diego, CA) and placed on a standardized weight maintenance diet (55% carbohydrate, 30% fat, and 15% protein) for at least 24 h before being studied. Studies were performed after a 12- to 14-h overnight fast.

BSA (Cohn fraction V) was supplied by Roche (Indianapolis, IN). [³H]3-Glucose was obtained from NEN Life Science Products (Boston, MA). Polyclonal antisera against GLUT1 (RaGLUTRANS) and GLUT4 (RaIRGT) were purchased from East Acres Biologicals (Cambridge, MA). An antirabbit immunoglobulin G conjugated with horseradish peroxidase and the enhanced chemiluminescence kit were obtained from Amersham Biosciences (Arlington Heights, IL). Electrophoresis reagents were obtained from Bio-Rad Laboratories (Richmond, CA). Pepstatin, leupeptin, phenylmethylsulfonylfluoride, and aprotinin were purchased from Sigma-Aldrich Corp. (St. Louis, MO).

Determination of whole body glucose uptake

Basal glucose uptake was determined at isoglycemia for each individual by infusion of [³H]3-glucose (0.6 µCi/min) in a continuous manner for 4–5 h. The glucose disposal rate was calculated during the final 30 min of each clamp study from the isotopically determined rate of glucose disappearance (Rd) corrected for changes in plasma glucose within its distribution space (8). To confirm whole body and leg muscle insulin resistance, glucose uptake was determined on a separate day using the hyperinsulinemic, euglycemic clamp technique as previously described (9). Insulin was infused in a primed continuous manner at 720 pmol/ml·min, and plasma glucose held constant at 90–99 mg/dl.

The Rd was calculated in the basal state for steady state conditions using Steele’s equation (10). Basal glucose uptake rates were determined from the Rd corrected for urinary glucose loss.

Leg glucose balance technique

Studies were performed in the postabsorptive state after a 12- to 14-h fast. At 0700 h on the morning of the study, catheters (Arrow, Reading, PA) were placed into a radial artery and a femoral vein for intermittent blood sampling, and simultaneous arterial and femoral venous blood was sampled at 10-min intervals for 30 min for plasma glucose measurements. After the last arteriovenous blood draw, leg blood flow was measured by venous occlusion plethysmography with a strain gauge apparatus (model 2560, UFI, Morro Bay, CA). Details of the study procedures have been described previously (11).

Determination of arteriovenous plasma glucose differences was made as described above, using catheters inserted in the radial artery and femoral vein for simultaneous sampling of arterial and femoral venous blood (8). Serum glucose values were converted to whole blood values by multiplying the plasma value by [1 – (0.30 x hematocrit)] (8). Blood flow to the leg was measured as described above. Two or three separate plethysmographic measurements were performed in each study to determine the mean blood flow.

Leg glucose uptake was calculated by the Fick principle (11) as the product of the arteriovenous difference for blood glucose and the leg blood flow and was expressed as milligrams per 100 ml leg tissue/min. Because precise quantitation of leg muscle mass is difficult, especially in obese subjects, the leg glucose uptake data are expressed as milligrams per 100 ml leg tissue/min. The leg volume was determined by water displacement.

Analytic methods

Blood drawn for glucose was immediately separated by an Eppendorf microcentrifuge (Brinkmann Instruments, Westbury, NY), and serum glucose was determined by the glucose oxidase method (model 23A, YSI, Inc., Yellow Springs, OH). Insulin was measured by a double antibody method (12).

Tissue biopsy

Immediately after completion of glucose turnover measurements in the basal state, percutaneous muscle biopsies were obtained from the lateral portion of the quadriceps femoris (vastus lateralis) muscle using a 5-mm side-cutting needle as previously described (13). Tissue was rinsed and rapidly frozen in liquid N₂. Total RNA was extracted from frozen tissue using the TRIzol reagent (Invitrogen Life Technologies, Grand Island, NY) according to the manufacturer’s protocol.

Membrane preparation

Frozen tissue (50 mg) was homogenized in a buffer containing 250 mmol/liter sucrose, 1 mmol/liter EDTA, 400 µmol/liter phenylmethylsulfonylfluoride, 10.5 µmol/liter leupeptin, 0.77 µmol/liter aprotinin, 1.46 µmol/liter pepstatin, and 30 mmol/liter HEPES, pH 7.4. All steps were performed at 4 C. The procedure was described by Hardin et al. (14). Briefly, initial homogenization was performed with four 5-sec pulses using a Polytron homogenizer (setting 5; Brinkmann Instruments). This was followed by 10 strokes in a Potter-Ejverham, Teflon-glass homogenizer. The homogenate was centrifuged for 15 min at 1000 x g, and the supernatant was centrifuged for 120 min at 338,000 x g. The final pellet, representing the postnuclear membrane fraction, was resuspended in homogenization buffer and stored at –70 C. Human erythrocyte membranes were prepared by the method of Steck and Kant (15).

Detection of glucose transporter proteins

Membrane preparations were diluted 1:1 in 2x Laemmli buffer without ß-mercaptoethanol (16) and heated for 5 min at 90 C. Proteins were separated on 10% SDS-PAGE gels, then transferred to nitrocellulose (17). GLUT1 was identified using a rabbit polyclonal antibody (RaGLUTRANS) that recognizes human GLUT1. A polyclonal antisera specific for GLUT4 (RaIRGT) was also employed. The second antibody was antirabbit immunoglobulin G conjugated with horseradish peroxidase. Immune complexes were detected using an enhanced chemiluminescence kit. Exposure was limited to the linear range of density, as determined by concentration curves established with human adipocyte low density microsome membranes (for GLUT4) or human erythrocyte membranes (for GLUT1), included as internal controls. These same standards were included in each analysis to permit normalization of the results. Quantitation was performed with a scanning laser densitometer (Stratoscan 7000, Stratagene, San Diego, CA).

Measurement of mRNA levels

A quantitative competitive PCR assay was developed for measurement of GLUT1 and GLUT4 mRNA levels.

cRNA construction. A synthetic gene containing GLUT1, GLUT4 MIMIC, and a polyadenylated tail was constructed by the RNA ligase-mediated MIMIC construction method (18). The synthetic gene was subcloned into a pGEM-T vector (Promega Corp., Madison, WI). This plasmid, termed pKSP-1, was used as a template for transcription by T7 polymerase according to the transcription protocol of Promega. The resulting KSP-1 cRNA product was purified with an Oligotex mRNA kit (Qiagen, Valencia, CA) and quantitated by absorbance at 260 nm.

PCR primers. Primers were synthesized on an automated solid phase DNA synthesizer, using standard phosphoramidite chemistry. Products were purified through Sep-Pak columns (Applied Biotechnology, Inc., Milford, MA). The following primers were employed: GLUT1 upstream, ATC ATC GGT GTG TAC TGC GG; GLUT1 downstream, GGT CAT GGG TCA CGT CAG CT; GLUT4 upstream, CAG AGC TAC AAT GAG ACG TGG; and GLUT4 downstream, CAT AGG AGG CAG CAG CGT TG.

RT-PCR. Total RNA (0.2–0.5 µg) was added to increasing concentrations of a cRNA construct that contained the MIMIC sequence. After the reverse transcriptase reaction, PCR was performed for GLUT4 (30 cycles at 55 C) or GLUT1 (35 cycles at 60 C). Products were separated on agarose gels and stained with ethidium bromide. Quantitation was performed on the photographed gels using NIH Image software. The ratio of each target product/cRNA standard was plotted against the number of copies of cRNA added, yielding the equivalence point between cRNA and target mRNA.

Statistical analysis

Data calculations and statistical analysis were performed using the StatView program (Abacus Concepts, Inc., Berkeley, CA). All data are expressed as the mean ± SE. Statistical significance was tested on normally distributed data with repeated measures ANOVA or two-tailed t test. A nonparametric equivalent of these tests was used when the data were not normally distributed.

	Results

Top Abstract Introduction Subjects and Methods Results Discussion References

 
In vivo glucose disposal

Whole body basal glucose uptake (Rd) was higher in T2D subjects (2.26 ± 0.17 mg/kg·min) than in the nondiabetic group (1.83 ± 0.15; P < 0.05; Fig. 1A). A portion of this difference could be due to the mass action effect of fasting hyperglycemia in diabetes, because the basal Rd was positively related to fasting glucose levels in both T2D (r = 0.673; P < 0.01) and nondiabetic groups (r = 0.562; P < 0.05). To evaluate the contribution of muscle glucose uptake to whole body glucose uptake, the leg balance technique was performed on a subset of the type 2 (n = 9) and nondiabetic (n = 9) subjects whose clinical characteristics were matched to those of the complete groups (Fig. 1B). Basal leg glucose uptake in diabetic subjects (1.53 ±. 0.56 mg/100 ml·min) was reduced to 40% of the value in the nondiabetic group (3.89 ± 0.83; P < 0.025). Maximally insulin-stimulated whole body glucose disposal, measured at euglycemia, was greatly reduced in the T2D subjects (Fig. 1A), confirming peripheral insulin resistance.

View larger version (10K): [in this window] [in a new window]   FIG. 1. A, Rates of whole body glucose utilization in ND () and T2D () subjects. Rd values were determined from isoglycemic (basal) and hyperinsulinemic, euglycemic (insulin stimulated) clamps. B, Leg glucose uptake under basal conditions. Results are the average ± SEM. *, P < 0.025, T2D vs. ND.

Total muscle tissue membranes were prepared from biopsies of the vastus lateralis, and GLUT1 protein was identified by Western blotting. Figure 2A displays a representative chemiluminescence image of membranes from nondiabetic and T2D subjects; equal amounts of membrane protein were loaded for each subject. Quantitation of such blots revealed that total GLUT1 protein expression in muscle tissue from T2D subjects was only 60% of the level in nondiabetic muscle (Fig. 2B; P < 0.05). The extent of reduction in GLUT1 protein expression (40% decrease) was of the same general magnitude as the decrease in basal leg glucose uptake (60%). GLUT1 protein expression appeared to be independent of fasting glucose levels.

View larger version (17K): [in this window] [in a new window]   FIG. 2. GLUT1 protein expression in skeletal muscle total membranes from ND and T2D subjects. Western blotting procedures are detailed in Subjects and Methods. A, Representative Western blot. Equal amounts of membrane protein were loaded from ND (N) and T2D subjects. B, Quantitation of blots; the number of subjects is given in Table 1. Results are the average ± SEM. *, P < 0.05 vs. ND.

View larger version (10K): [in this window] [in a new window]   FIG. 3. Comparison of GLUT1 protein expression in skeletal muscle and erythrocyte membranes from ND () and T2D () subjects (n = 3). Results are the average ± SEM. *, P < 0.025, T2D vs. ND.

GLUT4 protein expression was measured in the same subjects. In agreement with results from other laboratories (19, 20, 21), total membrane GLUT4 protein expression was similar in skeletal muscle tissue from nondiabetic and T2D subjects (Fig. 4). GLUT4 mRNA levels were also comparable between the nondiabetic (25.4 ± 4.9 amol/µg) and T2D groups (20.5 ± 4.4).

View larger version (24K): [in this window] [in a new window]   FIG. 4. GLUT4 protein expression in skeletal muscle of ND and T2D subjects. A, Representative Western blot. Equal amounts of membrane protein were loaded from ND (N1–3) and T2D (D1–3) subjects. B, Quantitation of blots. Results are the average ± SEM; the number of subjects is given in Table 1.

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

Results from glucose clamp studies have confirmed that basal glucose uptake is elevated in T2D subjects (7). The strong correlation between this measurement and fasting glycemia indicates that these values are influenced by the mass-action effect of hyperglycemia, driving glucose into tissues. Modifications of the minimal model approach permit calculation of the glucose efficiency, a more direct measure of glucose uptake (23). Using this evaluation, several laboratories have reported that, when taking glucose levels into account, basal glucose uptake is indeed reduced in insulin-resistant subjects (24, 25), especially those with T2D (26). Our measurements of leg glucose uptake confirm that glucose uptake in skeletal muscle is impaired in diabetes. Furthermore, compared with that in nondiabetics, diabetic skeletal muscle studied under basal conditions continues to show decreased glucose uptake even in the presence of the prevailing hyperglycemia. This would also indicate that the mass action effect of high glucose to increase glucose uptake is impaired in diabetic muscle. Thus, the elevated basal rates of whole body glucose uptake in diabetes are not due to an increase in skeletal muscle glucose uptake. The current study indicates that type 2 diabetic skeletal muscle expresses at least two defects with regard to glucose uptake: insulin resistance and impaired basal uptake. Studies in cultured human skeletal muscle cells, where uptake can be measured under controlled glucose levels, have also found that basal glucose uptake is impaired in cells from type 2 diabetic subjects (6, 27).

The conjecture that GLUT1 is involved in basal transport is supported by studies in transgenic mice, where GLUT1 is overexpressed in skeletal muscle (28). Basal glucose uptake in isolated muscles of these transgenic mice was increased 3- to 4-fold over that in nontransgenic littermates. The increase in muscle uptake was reflected at the whole body level by reductions in fasting glycemia and improvements in disposal of an oral glucose load (28). Overexpression of GLUT1 in L6 myoblasts also resulted in augmented basal rates of glucose uptake (29). Thus, basal glucose uptake into skeletal muscle may be regulated at least in part by the level of GLUT1 expression in that tissue. Conflicting evidence about the relative roles of GLUT1 and GLUT4 in basal skeletal muscle glucose uptake comes from studies in transgenic mice with a muscle-specific knockout of GLUT4 (30, 31). Both groups found GLUT1 expression to be unaltered. Basal glucose uptake into isolated soleus muscle was normal in one report (30), but reduced by 50% in the other (31). Reasons for this discrepancy could involve the strains of mice involved or the approaches employed to manipulate GLUT4 expression. Both studies suggest that GLUT1 plays some role in basal glucose uptake into skeletal muscle tissue. It should be noted that the relevance of conclusions drawn from muscle-specific GLUT4 knockout mice to T2D in humans may be limited, because the first is a condition with normal GLUT1 expression and a near total lack of GLUT4, whereas in humans, muscle GLUT4 expression is normal (19, 20, 21), and GLUT1 is, in the current report, reduced.

Given the results of this study and the potential role of GLUT1 as an important transporter under basal conditions, one could postulate depressed GLUT1 expression in diabetic muscle. Our study indicates that diabetic skeletal muscle tissue does indeed express lower levels of GLUT1 protein; furthermore, the magnitude of this decrease is of the same general magnitude as the decrease in glucose disposal by leg muscle under basal conditions. A previous study addressed the expression of GLUT1 in skeletal muscle and did not find a difference between diabetic and matched nondiabetic controls (20). One possible reason for the discrepancy with the current findings could be that in the earlier study a number of the diabetic subjects (seven of 19) were newly diagnosed and untreated (20).

It should be noted that the specific cellular expression of GLUT1 in skeletal muscle tissue has been a topic of some debate (32). Although a number of investigators have observed that GLUT1 is expressed in muscle cells (33, 34, 35, 36), a muscle tissue biopsy contains a mixture of cell types, and others have demonstrated the primary localization of GLUT1 to nonmuscle cell types (36, 37). Besides myotubes, major components of a muscle biopsy include nerves and erythrocytes, both of which contain high levels of GLUT1 (32), any of which could be responsible for the observed differences between nondiabetic and diabetic groups. We tested for one aspect of this possibility and found no effect of diabetes on GLUT1 expression in erythrocytes. Handberg et al. (33) reported that GLUT1 expression in perineural sheaths was not influenced, at least in rodents, by different states of glucose intolerance. Taken together, these results suggest that the contributions of nerves and erythrocytes to GLUT1 levels in a muscle biopsy could represent a constant component between groups. We reported previously (6) that total membrane GLUT1 protein levels in cultured human skeletal muscle cells were also lower in cells from T2D subjects. Because these cultures contain a single cell type, there is no contribution from other cell types to the measured GLUT1 levels. The results in cultured cells and biopsies suggest that the defect in GLUT1 protein expression in T2D is an intrinsic property of diabetic muscle that persists in culture. Regardless of the exact cellular distribution of GLUT1 protein in skeletal muscle tissue, the biopsy samples analyzed for GLUT1 expression are reflective of the tissue bed, where leg glucose uptake was measured.

Several laboratories have reported on GLUT1 mRNA in skeletal muscle from T2D subjects and found it to be normal (20, 38), a result confirmed in our subjects. Decreases in GLUT1 protein expression in the face of normal mRNA levels suggests that the problem in T2D resides at a posttranscriptional level. Indeed, there are multiple examples of insulin-resistant conditions where rates of translation and protein synthesis are impaired, especially in response to insulin (reviewed in Ref. 39), suggestive of insulin resistance for control of translation as well as regulation of glucose metabolism.

Although defects in skeletal muscle GLUT1 protein expression in T2D could occur together with impaired glucose uptake under basal conditions, these findings do not indicate which event is the cause and which is the consequence. The most obvious candidates are the metabolic abnormalities (hyperinsulinemia, hyperglycemia, elevated free fatty acids, etc.) characteristic of this condition. In biopsies from nondiabetic subjects, the correlation between fasting insulin and GLUT1 protein levels approached significance (r = 0.525; P = 0.054), suggesting that some relationship might exist in this population. There is no such relationship in diabetic subjects (r = 0.054; P = 0.72). In studies of cultured muscle cells, insulin treatment increased GLUT1 expression (40), rather than lowered it. We obtained similar results in cultured human skeletal muscle cells, where insulin treatment up-regulated GLUT1 protein (6) and was equally effective in cells from nondiabetic and diabetic subjects. This suggests that 1) the hyperinsulinemia usually associated with T2D may actually be masking a greater GLUT1 deficit; and/or 2) the in vivo GLUT1 response to insulin is blunted in diabetes.

There were no significant correlations between fasting glucose and GLUT1 protein in either nondiabetic (r = 0.169; P = 0.56) or diabetic (r = 0.327; P = 0.33) subjects. The effects of in vivo hyperglycemia on muscle GLUT1 protein are variable, but in vitro, in multiple cell types, there is a consistent reduction in expression with exposure to high glucose levels (38, 41). Hyperglycemia can also down-regulate glucose transport activity in skeletal muscle cells of diabetic subjects (6); transporter levels were not measured under those conditions. Thus, there are data to support a possible regulatory role for glucose on GLUT1 activity or expression in muscle tissue.

Impaired basal transport of glucose may play a role in metabolic disorders of insulin resistance. It is known that in the basal state, the majority of glucose disposal is into the central nervous system, with a small percentage accounted for by skeletal muscle. Glucose that is not taken up into muscle or not disposed of into the urine must be taken up into other tissues. The exact tissue sites of this increased glucose uptake remains to be elucidated, although prime candidate tissues include adipose and liver. An increased uptake into intraabdominal adipose tissue could contribute to insulin resistance in other tissues by increasing lipogenesis and fatty acid stores (42). Elevations in hepatic glucose uptake could have the potential to disturb lipid homeostasis. Given the characterization of the metabolic syndrome and its accompanying insulin resistance and dyslipidemia, basal glucose metabolism is an area that deserves additional investigation. The current studies did not investigate the influence of circulating triglyceride and free fatty acid levels on basal glucose uptake or GLUT1 expression. However, the role of increased skeletal muscle fatty acid uptake or utilization, driven by the hyperlipidemia also present in T2D, to modulate basal glucose uptake is an area that merits additional study.

In summary, our study has shown that 1) skeletal muscle tissue GLUT1 protein expression is reduced in T2D in the face of unaltered levels of mRNA; and 2) the elevated basal glucose disposal displayed in diabetes is not into leg muscle tissue. Defects in GLUT1 protein expression occur in association with lower rates of glucose transport activity. This defect is an intrinsic property of T2D skeletal muscle and is not secondary to the metabolic environment.

	Footnotes

 
This work was supported by grants from the Medical Research Service, Department of Veterans Affairs and Veterans Affairs San Diego Healthcare System, American Diabetes Association (to T.P.C.), an American Diabetes Association Mentor-Based Fellowship (to R.R.H.), NIH Grant RO1-DK-58291 (to R.R.H.), and Grant MO1-RR-00827 from the General Clinical Research Branch, Division of Research Resources, NIH.

First Published Online October 13, 2004

Abbreviations: ND, Nondiabetic; Rd, rate of glucose disappearance; T2D, type 2 diabetes.

Received March 17, 2004.

Accepted September 27, 2004.

	References

Top Abstract Introduction Subjects and Methods Results Discussion References

 

1. Hunter SJ, Garvey WT 1998 Insulin action and insulin resistance: diseases involving defects in insulin receptors, signal transduction and the glucose transport effector system. Am J Med 105:331–345[CrossRef][Medline]
2. Gottesman I, Mandarino LJ, Gerich J 1983 Estimation and kinetic analysis of insulin dependent glucose uptake in human subjects. Am J Physiol 244:E6322–E6325
3. Joost HG, Thorens B 2001 The extended GLUT-family of sugar/polyol transport facilitators: nomenclature, sequence characteristics, and potential function of its novel members. Mol Membr Biol 18:247–256[CrossRef][Medline]
4. Olson AL, Pessin JE 1996 Structure, function and regulation of the mammalian facilitative glucose transporter gene family. Annu Rev Nutrition 16:235–256[CrossRef][Medline]
5. Baron AD, Brechtel G, Wallace P, Edelman SV 1988 Rates and tissue sites of non-insulin- and insulin-mediated glucose uptake in humans. Am J Physiol 255:E769–E774
6. Ciaraldi TP, Abrams L, Nikoulina S, Mudaliar S, Henry RR 1995 Glucose transport in cultured human skeletal muscle cells. Regulation by insulin and glucose in nondiabetic and non-insulin-dependent diabetes mellitus subjects. J Clin Invest 96:2820–2827
7. Baron AD, Kolterman OG, Bell J, Mandarino LJ, Olefsky JM 1985 Rates of noninsulin-mediated glucose uptake are elevated in type II diabetic subjects. J Clin Invest 76:1782–1788
8. DeFronzo RA, Tobin JD, Andres R 1979 Glucose clamp technique: a method for quantifying insulin secretion and resistance. Am J Physiol 237:E214–E223
9. Thornburn AW, Gumbiner B, Bulacan F, Wallace P, Henry RR 1990 Intracellular glucose oxidation and glycogen synthase activity are reduced in non-insulin dependent (type II) diabetes independent of impaired glucose uptake. J Clin Invest 85:522–529
10. Steele R 1959 Influence of glucose loading and injected insulin on hepatic glucose output. Ann NY Acad Sci 82:522–529
11. Zierler KL 1961 Theory of the use of arteriovenous concentration differences for measuring metabolism in steady and non-steady states. J Clin Invest 40:2111–2125
12. Desbuquois B, Aurbach GD 1971 Use of polyethylene glycol to separate free and antibody-bound peptide hormones in radio-immunoassays. J Clin Endocrinol Metab 33:732–738[Abstract/Free Full Text]
13. Henry RR, Abrams L, Nikoulina S, Ciaraldi TP 1995 Insulin action and glucose metabolism in nondiabetic control and NIDDM subjects. Comparison using human skeletal muscle cell cultures. Diabetes 44:936–946[Abstract]
14. Hardin DS, Domingeuz JH, Garvey WT 1993 Muscle-group specific regulation of GLUT4 glucose transporters in control, diabetic and insulin-treated diabetic rats. Metabolism 42:1310–1315[CrossRef][Medline]
15. Steck TL, Kant JA 1974 Preparation of impermeable ghosts and inside out vesicles from human erythrocyte membranes. Methods Enzymol 31:172–180[CrossRef][Medline]
16. Laemmli UK 1970 Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 27:680–685
17. Towbin H, Staehelin T, Gordon J 1979 Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76:4350–4354[Abstract/Free Full Text]
18. Borriello F, Lederer J 1998 Construction of quantitative RT-PCR MIMICs. Biotechniques 19:580–584
19. Garvey WT, Maianu L, Hancock JA, Golichowski AM, Baron A 1992 Gene expression of GLUT4 in skeletal muscle from insulin-resistant patients with obesity, IGT, GDM, and NIDDM. Diabetes 41:465–475[Abstract]
20. Pedersen O, Bak JF, Andersen PH, Lund S, Moeller DE, Flier JS, Kahn BB 1990 Evidence against altered expression of GLUT1 or GLUT4 in skeletal muscle of patients with obesity or NIDDM. Diabetes 39:865–870[Abstract]
21. Lund S, Vestergaard H, Andersen PH, Schmitz O, Gotzsche LBH, Pedersen O 1993 GLUT-4 content in plasma membrane of muscle from patients with non-insulin-dependent diabetes mellitus. Am J Physiol 265:E889–E897
22. DeFronzo RA 1988 Lilly lecture. The triumvirate: ß-cell, muscle, liver. A collusion responsible for NIDDM. Diabetes 37:667–687[Medline]
23. Saad MF, Anderson RL, Laws A, Watanabe RM, Kodes WW, Chen Y-DI, Sands RE, Pei D, Savage PJ, Bergman RN 1994 A comparison between the minimal model and the glucose clamp in the assessment of insulin sensitivity across the spectrum of glucose tolerance. Diabetes 43:1114–1121[Abstract]
24. Taniguchi A, Nakai Y, Fukushima M, Imura H, Kawamura H, Nagata I, Florant GL, Tokuyama K 1994 Insulin sensitivity, insulin secretion, and glucose effectiveness in subjects with impaired glucose tolerance: a minimal model analysis. Metabolism 43:714–718[CrossRef][Medline]
25. Martin BC, Warram JH, Krolewski AS, Bergman RN, Soeldner JS, Kahn CR 1992 Role of glucose and insulin resistance in development of type 2 diabetes mellitus: results of a 25-year follow-up study. Lancet 340:925–929[CrossRef][Medline]
26. Taniguchi A, Nakai Y, Fukushima M, Kawamura H, Imura H, Nagata I, Tokuyama K 1992 Pathogenic factors responsible for glucose intolerance in patients with NIDDM. Diabetes 41:1540–1546[Abstract]
27. Gaster M, Petersen I, Hojlund K, Poulsen P, Beck-Nielsen H 2002 The diabetic phenotype is conserved in myotubes established from diabetic subjects: evidence for primary defects in glucose transport and glycogen synthase activity. Diabetes 51:921–927[Abstract/Free Full Text]
28. Marshall BA, Ren J-M, Johnson DW, Gibbs EM, Lillquist JS, Soeller WC, Holloszy JO, Mueckler M 1993 Germline manipulation of glucose homeostasis via alteration of glucose transporter levels in skeletal muscle. J Biol Chem 268:18442–18445[Abstract/Free Full Text]
29. Robinson R, Robinson LJ, James DE, Lawrence JC 1993 Glucose transport in L6 myoblasts overexpressing GLUT1 and GLUT4. J Biol Chem 268:22119–22126[Abstract/Free Full Text]
30. Ryder JW, Kawano Y, Chibalin AV, Rincon J, Tsao T-S, Stenbit AE, Combatsiaris T, Yang J, Holman GD, Charron MJ, Zierath JR 1999 In vitro analysis of the glucose-transport system in GLUT4-null skeletal muscle. Biochem J 342:321–328
31. Zisman A, Peroni OD, Abel ED, Michael MD, Mauvais-Jarvis F, Lowell BB, Wojtaszewski JFP, Hirshman MF, Viramaki A, Goodyear LJ, Kahn CR, Kahn BB 2000 Targeted disruption of the glucose transporter 4 selectively in muscle causes insulin resistance and glucose intolerance. Nat Med 6:924–928[CrossRef][Medline]
32. Gaster M, Handberg A, Beck-Nielsen H, Schroder HD 2000 Glucose transporter expression in human skeletal muscle fibers. Am J Physiol 279:E529–E538
33. Handberg A, Kayser L, Hoyer PE, Micheelsen J, Vinten J 1994 Elevated GLUT1 level in crude muscle membranes from diabetic Zucker rats despite a normal GLUT1 level in perineurial sheaths. Diabetologia 37:443–448[CrossRef][Medline]
34. Marette A, Richardson JM, Ramlal T, Balon TW, Vranic M, Pessin JE, Klip A 1992 Abundance, localization, and insulin-induced translocation of glucose transporters in red and white muscle. Am J Physiol 263:C443–C452
35. Kainulainen H, Breiner M, Schurmann A, Marttinen A, Virjo A, Joost HG 1994 In vivo glucose uptake and glucose transporter proteins GLUT1 and GLUT4 in heart and various types of skeletal muscle from streptozotocin-diabetic rats. Biochim Biophys Acta 1225:275–282[Medline]
36. Handberg A, Kayser L, Hoyer PE, Vinten J 1992 A substantial part of GLUT-1 in crude membranes from muscle originates from perineurial sheaths. Am J Physiol 262:E721–E727
37. Kahn BB, Rossettti L, Lodish HF, Charron MF 1991 Decreased in vivo glucose uptake but normal expression of GLUT1 and GLUT4 in skeletal muscle of diabetic rats. J Clin Invest 87:2197–2206
38. McGowan KM, Long SD, Pekala PH 1995 Glucose transporter gene expression: regulation of transcription and mRNA stability. Pharmacol Ther 66:465–505[CrossRef][Medline]
39. Shi Y, Taylor SI, Tan S-L, Sonenberg N 2003 When translation meets metabolism: multiple links to diabetes. Endocr Rev 24:91–101[Abstract/Free Full Text]
40. Sargent R, Mitsumoto Y, Sarabia V, Shillabeer G, Klip A 1993 Hormonal regulation of glucose transporters in muscle cells in culture. J Endocrinol Invest 16:147–162[Medline]
41. Klip A, Tsakiridis T, Marette A, Ortiz PA 1994 Regulation of expression of glucose transporters by glucose: a review of studies in vivo and in cell cultures. FASEB J 8:43–53[Abstract]
42. Abel ED, Peronl O, Kim JK, Kim Y-B, Boss O, Hadro E, Minnemann T, Shulman GI, Kahn BB 2001 Adipose-selective targeting of the GLUT4 gene impairs insulin action in muscle and liver. Nature 409:729–733[CrossRef][Medline]

This article has been cited by other articles:

				R. Jani, M. Molina, M. Matsuda, B. Balas, A. Chavez, R. A. DeFronzo, and M. Abdul-Ghani Decreased Non-Insulin-Dependent Glucose Clearance Contributes to the Rise in Fasting Plasma Glucose in the Nondiabetic Range Diabetes Care, February 1, 2008; 31(2): 311 - 315. [Abstract] [Full Text] [PDF]

				H. E. Silber, P. M. Jauslin, N. Frey, R. Gieschke, U. S. H. Simonsson, and M. O. Karlsson An Integrated Model for Glucose and Insulin Regulation in Healthy Volunteers and Type 2 Diabetic Patients Following Intravenous Glucose Provocations J. Clin. Pharmacol., September 1, 2007; 47(9): 1159 - 1171. [Abstract] [Full Text] [PDF]

				L. Gnudi, S. M. Thomas, and G. Viberti Mechanical Forces in Diabetic Kidney Disease: A Trigger for Impaired Glucose Metabolism J. Am. Soc. Nephrol., August 1, 2007; 18(8): 2226 - 2232. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Submit a related Letter to the Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Ciaraldi, T. P. Articles by Henry, R. R. Search for Related Content PubMed PubMed Citation Articles by Ciaraldi, T. P. Articles by Henry, R. R. Related Collections Diabetes and Insulin