Limitations to basal and insulin-stimulated skeletal muscle glucose uptake in the high-fat-fed rat

Amy E. Halseth, Deanna P. Bracy, and David H. Wasserman

Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Rats fed a high-fat diet display blunted insulin-stimulated skeletal muscle glucose uptake. It is not clear whether this is due solely to a defect in glucose transport, or if glucose delivery and phosphorylation are also impaired. To determine this, rats were fed standard chow (control rats) or a high-fat diet (HF rats) for 4 wk. Experiments were then performed on conscious rats under basal conditions or during hyperinsulinemic euglycemic clamps. Rats received primed constant infusions of 3-O-methyl-[3H]glucose (3-O-MG) and [1-14C]mannitol. Total muscle glucose concentration and the steady-state ratio of intracellular to extracellular 3-O-MG concentration [which distributes based on the transsarcolemmal glucose gradient (TSGG)] were used to calculate glucose concentrations at the inner and outer sarcolemmal surfaces ([G]im and [G]om, respectively) in soleus. Total muscle glucose was also measured in two fast-twitch muscles. Muscle glucose uptake was markedly decreased in HF rats. In control rats, hyperinsulinemia resulted in a decrease in soleus TSGG compared with basal, due to increased [G]im. In HF rats during hyperinsulinemia, [G]im also exceeded zero. Hyperinsulinemia also decreased muscle glucose in HF rats, implicating impaired glucose delivery. In conclusion, defects in extracellular and intracellular components of muscle glucose uptake are of major functional significance in this model of insulin resistance.

glucose delivery; glucose phosphorylation; 3-O-methylglucose; 2-deoxyglucose


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

IT IS WELL ESTABLISHED that dietary interventions in rodents are capable of significantly blunting insulin-stimulated glucose uptake by skeletal muscle (40). The question remains, however, as to the site or sites of functional limitations to muscle glucose uptake in vivo in this insulin-resistant state. A defect in glucose transport in dietary models of insulin resistance has been demonstrated numerous times (13, 27). There are reasons to believe that the other steps that comprise glucose uptake, delivery of glucose to the sarcolemma (15, 43), and glucose phosphorylation (38) may be impaired as well. One way to determine this is by assessing the magnitude of the transsarcolemmal glucose gradient (TSGG) in chow-fed and high-fat diet-fed (HF) rats under basal and insulin-stimulated conditions. Using a novel isotopic technique, we have previously demonstrated that physiological increments in insulin result in a narrowing of the TSGG in rat muscle, because the increase in sarcolemmal permeability is not accompanied by proportional increases in glucose delivery and/or phosphorylation (32). If the only defect in muscle glucose uptake in HF rats were at the glucose transport step, one would predict that the TSGG would exceed that seen in chow-fed rats during insulin stimulation. Therefore, we compared the maintenance of the TSGG under basal conditions and during a hyperinsulinemic euglycemic clamp in chow-fed (control) and HF rats by use of the glucose countertransport technique as has been described and applied previously (12, 32).


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animal maintenance and surgical procedures. Male Sprague-Dawley rats (Sasco, Omaha, NE) were individually housed at 23°C on a 0600-1800 light cycle and allowed free access to water and food. Rats were divided into two groups: control rats, which were fed Purina Rodent Chow (65% of calories from carbohydrate, 11% from fat, 24% from protein), and HF rats, which were fed a high-fat diet (37% of calories from carbohydrate, 41% from fat, 22% from protein) obtained from Dr. Catherine Field, University of Alberta and as described previously (27). The fat content of this diet, although comparable with that found in the typical human diet, is sufficient to induce a significant degree of insulin resistance in the rat. The rats were housed under these conditions for ~3 wk, by which time their weights had reached 250-300 g. During this period, the food conversion index (FCI; weight gained/food consumed) for each rat was calculated. Catheters were inserted under anesthesia in the left common carotid artery and the right jugular vein (12). After surgery, animal weights and food intake were monitored daily, and only animals in which presurgery weight and FCI were restored were used for experiments (>= 5 days). All procedures were preapproved by the Vanderbilt University Animal Care and Use Subcommittee and followed National Institutes of Health guidelines for the care and use of laboratory animals.

Experimental procedures. Food was removed from control and HF rats ~5 h before the beginning of a study. Rats were studied either under basal conditions or during a hyperinsulinemic euglycemic clamp. At t = -100 min, an infusion of saline or insulin (1.0 mU · kg-1 · min-1) was begun. Also at t = -100 min, primed infusions of [1-14C]mannitol ([14C]MN; 3.5 µCi primer and 60 nCi/min infusion) and 3-O-methyl-[3H]glucose (3-O-MG; 25 µCi primer and 150 nCi/min infusion) were initiated. 3-O-MG is a substrate for glucose transport but is not further metabolized in skeletal muscle, whereas [14C]MN is restricted to the extracellular space and thus can be used to measure this space. From t = 0 to 40 min, a constant-rate infusion of 2-[1,2-3H]deoxyglucose (2-DG; 900 nCi/min) was given. 2-DG is transported into the cell and phosphorylated, yielding 2-DG-6-phosphate (2-DG-6-P). This 2-DG-6-P can undergo further metabolism to yield small amounts of other radiolabeled phosphorylated metabolites and glycogen. All radioisotopes were obtained from New England Nuclear (Boston, MA). Arterial blood samples ranging from 150 to 500 µl for tracer and/or insulin analyses were taken at -100, -70, -40, -10, 1, 2.5, 5, 7.5, 10, 15, 20, 25, 30, and 40 min. Washed erythrocytes from the experimental animal and whole blood from a donor rat were used to maintain hematocrit, but neither was infused after -10 min. On average, hematocrit fell from 42 to 37 by the end of the experiments, with no significant differences between groups. At 40 min, rats were anesthetized with intravenous pentobarbital sodium, and the soleus (comprised of slow-twitch oxidative fibers), gastrocnemius (gastroc; comprised of fast-twitch oxidative and fast-twitch glycolytic fibers), and superficial vastus lateralis (vastus; comprised of fast-twitch glycolytic fibers) muscles were rapidly excised and frozen in liquid N2 within 10 s of removal from the animal. In separate basal experiments in which the equilibration period was extended to 300 min, soleus muscle [14C]MN and 3-O-MG contents did not differ from those in the 140-min basal experiments, indicating that steady-state conditions were attained (data not shown). The 100-min isotope equilibration period used in these experiments was not long enough to reach steady-state conditions in the gastroc and vastus muscles, because the 3-O-MG content in muscle was higher when the equilibration period was extended to 300 min (12). Therefore, only data not dependent on 3-O-MG equilibration are presented for the gastroc and vastus muscles.

Processing of blood and muscle samples. Plasma glucose concentrations were measured by the glucose oxidase method with the use of an automated glucose analyzer (Beckman Instruments, Fullerton, CA), and immunoreactive insulin was measured using a double antibody method (29). Methods used to distinguish radioactivity from [14C]MN, 3-O-MG, and 2-DG in muscle and plasma have been described previously (12). Briefly, the glucose analogs were distinguished on the basis of on whether or not they were precipitated with Ba(OH)2 and ZnSO4 and phosphorylated by yeast hexokinase (~10 units/sample). Tests in our laboratory revealed that treatment of samples with 10 units of hexokinase resulted in phosphorylation of ~25% of the 3-O-MG. Because both muscle and plasma samples were similarly affected, this did not change the calculated value of the steady-state ratio of 3- O-MG concentration in intracellular to extracellular water. One advantage of using Ba(OH)2 and ZnSO4 precipitation over the anion exchange method of separation (22) is that any 2-DG-6-phosphate (2-DG-6-P) incorporated into muscle glycogen (5, 42) will be counted in the same fraction as free 2-DG-6-P (12). This is due to the fact that all phosphorylated products of 2-DG as well as glycogen are recovered in the Ba(OH)2 and ZnSO4 precipitate (12). Muscle was deproteinized by homogenization in ice-cold 0.5% perchloric acid (PCA) and centrifuged at 4°C to remove insoluble proteins, and supernatants were kept on ice until neutralization with 5 M KOH. We have demonstrated in tests in our laboratory that, under these conditions, 2-DG-1-phosphate is preserved in its phosphorylated form instead of being degraded to 2-DG, as occurs in more concentrated PCA (7). The use of 0.5% PCA, therefore, prevents an overestimate of free 2-DG. Tests in our laboratory also showed that this concentration of PCA is also sufficient to prevent metabolism of glucose and 2-DG/2-DG-6-P by the muscle homogenate. Tissue glucose was measured in neutralized PCA extracts by an enzymatic method (28) and is expressed as millimoles per liter of muscle water, with a muscle water content of 0.75 ml/g used for all calculations. Nonesterified fatty acid (NEFA) concentration in plasma was determined spectrophotometrically using an automated analyzer (Monarch 2000 Centrifugal Analyzer, Instrumentation Laboratory, Lexington, MA) and a kit obtained from Wako Chemicals (NEFA-C, Richmond, VA).

Calculations. The fraction of extracellular to total water space in biopsies (Fe) was calculated with [14C]MN as described previously (12). An index of muscle glucose uptake (Rg) (22) was calculated from muscle accumulation of 2-DG-6-P and other phosphorylated 2-DG metabolites and glycogen, the integrated plasma 2-DG concentration over the infusion period, and the plasma glucose concentration.

In these experiments, the steady state ratio of 3-O-MG concentration in intracellular to extracellular water (Si/So), was determined to calculate the glucose concentration at the outer face of the sarcolemma ([G]om), the glucose concentration at the inner face of the sarcolemma ([G]im), and the TSGG, as has been described previously (12). Si/So is calculated by the equation
S<SUB>i</SUB><IT>/</IT>S<SUB>o</SUB><IT>=</IT>([<IT>3-O-</IT>MG]<SUB>m</SUB><IT>−</IT>[<IT>3-O-</IT>MG]<SUB>p</SUB><IT>·</IT>F<SUB>e</SUB>)<IT>/</IT> (1)

((<IT>1−</IT>F<SUB>e</SUB>)<IT>·</IT>[<IT>3-O-</IT>MG]<SUB>p</SUB>)
where the subscripts m and p refer to muscle and arterial plasma. Because 3-O-MG is not metabolized by muscle, extracellular gradients and intracellular gradients of this analog do not exist at steady state, and [3-O-MG]p equals the interstitial 3-O-MG concentration. Si/So can be used to calculate the TSGG, because the steady-state distribution of 3-O-MG across the sarcolemma is determined by competition between glucose and 3-O-MG for binding sites on the transporters they share. The relationship between Si/So and the TSGG is defined by the equation
S<SUB>i</SUB><IT>/</IT>S<SUB>o</SUB><IT>=</IT>(<IT>K</IT><SUB>m</SUB><IT>+</IT>[G]<SUB>im</SUB>)<IT>/</IT>(<IT>K</IT><SUB>m</SUB><IT>+</IT>[G]<SUB>om</SUB>) (2)
where Km is the Michaelis-Menten constant for glucose transport. This equation has been described and applied previously (6, 10, 12, 30, 32). Assumptions of the method have been discussed in detail (10, 32).

Knowledge of Si/So tells us the relationship between [G]om and [G]im, but not the actual values for each of these concentrations. Because of the presence of glucose gradients, it is impossible to directly measure the true glucose concentration at the sarcolemma or anywhere in the interstitial or intracellular space. Therefore, we have developed a novel approach for calculating limits for the average [G]om, based on two theoretical glucose distributions. In the first calculation of [G]om, [G]im is assumed to be localized to such a small volume of the intracellular water that it contributes only negligibly to the total muscle glucose mass (denoted with superscript alpha ). The second approach for the calculation of the mean [G]om assumes that [G]im is distributed evenly throughout the intracellular water (denoted with superscript beta ). Neither of these calculated values, or any other single value, will be identical to the actual [G]om at all places along the sarcolemma; however, the current approach allows us to express the mean of these values within a known range. [G]omalpha was calculated as
[G]<SUP><IT>&agr;</IT></SUP><SUB>om</SUB><IT>=</IT>[G]<SUB>m</SUB><IT>/</IT>F<SUB>e</SUB> (3)
where [G]m is the muscle glucose concentration, and Fe is the fraction of muscle water that is extracellular. [G]imalpha was then calculated from Eq. 2. [G]ombeta was calculated as
[G]<SUP><IT>&bgr;</IT></SUP><SUB>om</SUB><IT>=</IT>[[G]<SUB>m</SUB><IT>−</IT>[G]<SUP><IT>&bgr;</IT></SUP><SUB>im</SUB><IT>·</IT>(<IT>1−</IT>F<SUB>e</SUB>)]<IT>/</IT>F<SUB>e</SUB> (4)
The solution to Eq. 4 can be substituted into Eq. 2 and then solved for [G]imbeta . TSGGalpha and TSGGbeta are calculated with the values of [G]imalpha and [G]omalpha , and [G]imbeta and [G]ombeta , respectively
TSGG<IT>=</IT>[G]<SUB>om</SUB><IT>−</IT>[G]<SUB>im</SUB> (5)
A range of 2-5 mM has been reported for the Km of GLUT-4 in vitro (33). A value of 3 mM was used in these calculations because it yielded a value for [G]im of ~0 under basal conditions, which is generally assumed to exist (e.g., Refs. 4, 9, 16), and because it approximates estimates obtained from muscle venous drainage in vivo (8, 45). Although the use of different values for Km in calculations has a quantitative effect on the calculated data, no qualitative effects exist, and the magnitude of the quantitative differences decreases as Si/So increases, which has been described in detail previously (32). It is notable that the Km for muscle glucose transport is unchanged by insulin stimulation (11, 31, 35).

Statistical analyses. Statistical significance for variables with only one value was determined with one-factor ANOVA, using Fisher's PLSD as a post hoc test. For comparison of ranges defined by alpha - and beta -boundaries ([G]im, [G]om, and TSGG), one-factor ANOVA was used to compare data sets consisting of both alpha - and beta -values for each group. Also presented in the text in specific instances are P values for between-group comparisons (one-factor ANOVA) made between alpha - or beta -values instead of entire ranges. We took this statistical approach so as not to exclude the possibility that either the alpha - or beta -calculation is more representative of the actual distribution of glucose. Differences were considered statistically significant at P < 0.05. Data are expressed as means ± SE.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animal characteristics. There were no differences between control and HF rats in body weight, 5-h-fasted arterial plasma glucose, or insulin concentrations (Table 1). Insulin concentrations were similarly elevated in control and HF rats by insulin infusion. The arterial plasma glucose concentration was maintained between 7.3 and 8.0 mM in all groups over the last 40 min of experiments. The glucose infusion rate necessary to maintain euglycemia during hyperinsulinemia was decreased ~70% in HF rats compared with control rats.

                              
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Table 1.   Arterial plasma glucose concentration, insulin concentration, and glucose infusion rate

Arterial plasma nonesterified fatty acid concentration. At -100 min, plasma nonesterified fatty acid (NEFA) concentrations were not different between control (1.0 ± 0.1 mM) and HF (1.1 ± 0.1 mM) rats. NEFA concentrations were not altered by 140 min of saline infusion in control or HF rats. The response of plasma NEFAs to hyperinsulinemia differed between control and HF rats. In control rats, the plasma NEFA concentration decreased significantly to 0.6 ± 0.1 mM at 40 min, whereas the NEFA concentration was maintained in HF rats (1.4 ± 0.1 mM at 40 min; Fig. 1). This suggests that diet-induced insulin resistance is not limited to muscle glucose uptake but is also manifested by an inability of insulin to inhibit lipolysis at the adipocyte.


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Fig. 1.   Arterial plasma nonesterified fatty acid (NEFA) concentration (in mM) at the end of experiments in control and high-fat diet-fed (HF) rats under basal and hyperinsulinemic conditions. Data represent means ± SE; n = 7, 8, 6, and 8 for control basal, control insulin, HF basal and HF insulin, respectively. * Significant differences from basal within a dietary treatment; dagger  significant differences from control within an experimental treatment.

Muscle glucose concentration and Rg. Glucose concentrations in soleus and vastus were similar in control rats under basal and hyperinsulinemic conditions, but glucose concentration in gastroc fell in response to hyperinsulinemia (P < 0.05). Muscle glucose concentrations were not significantly different between HF and control rats in the basal state; however, during hyperinsulinemia, there was a significant decrease in glucose concentration in soleus and vastus in HF rats. Rg tended to be lower in HF rats under basal conditions, but this did not reach statistical significance. Under hyperinsulinemic conditions, Rg was significantly blunted in HF rats compared with control rats in all three muscles studied, indicating insulin resistance (Table 2). It is interesting to note that the falls in muscle glucose concentration in HF rats during hyperinsulinemia in soleus and vastus occurred despite the fact that the Rg values for these muscles were less than one-half of that measured in control rats; therefore, the fall is not due to greater glucose utilization.

                              
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Table 2.   Muscle glucose concentration and muscle Rg in soleus, gastroc and vastus

Soleus Si/So, [G]om, [G]im, and the TSGG. Soleus Si/So, the ratio of 3-O-MG concentration in intracellular to extracellular water, was 0.55 ± 0.13 in control rats in the basal state. This value was significantly (P < 0.05) increased to 0.79 ± 0.07 by hyperinsulinemia in control rats. In HF rats, soleus Si/So in the basal state (0.82 ± 0.06) was significantly (P < 0.05) increased compared with that in control rats, indicating a narrower basal TSGG. In HF rats during hyperinsulinemia, Si/So was 0.84 ± 0.09, which was not different from the Si/So obtained in HF rats in the basal state or control rats during hyperinsulinemia.

In control rats studied in the basal state, the calculated range for soleus [G]om was between 3.6 ± 0.5 mM ([G]omalpha ) and 3.7 ± 0.8 mM ([G]ombeta ), as shown in Fig. 1. Glucose concentration at the inner face of the membrane did not differ significantly from zero in this group, resulting in TSGG values that ranged between 3.1 ± 0.9 mM (TSGGalpha ) and 3.6 ± 1.1 mM (TSGGbeta ) in control rats.

In control rats during hyperinsulinemia, the calculated range for soleus [G]om was not significantly different from that under basal conditions (Fig. 1). Soleus [G]im was significantly increased by hyperinsulinemia in these rats, with a calculated range between 2.3 ± 0.5 mM ([G]imalpha ) and 1.0 ± 0.2 mM ([G]imbeta ). The increase in [G]im resulted in a significant decrease in the TSGG during hyperinsulinemia in control rats to values ranging between 1.9 ± 0.5 mM (TSGGalpha ) and 1.2 ± 0.5 mM (TSGGbeta ; P < 0.05 compared with control rats under basal conditions).

When HF rats were studied under basal conditions, interpretation of results for [G]om, [G]im and TSGG was dependent on whether the alpha - or beta -condition was assumed to exist, because the range for these values was quite wide (Fig. 1). The entire range for soleus [G]om in HF rats in the basal state was not significantly different from the range in control rats under basal conditions. However, [G]ombeta was significantly lower in HF than in control rats in the basal state (P < 0.05); so if the beta -assumption more accurately represents the glucose distribution, glucose at the outer face of the sarcolemma may in fact be lower in HF than in control rats under these conditions. Regardless of assumptions regarding the distribution volume of glucose, [G]im in HF rats in the basal state was significantly increased compared with that in control rats. Consequently, the TSGG in HF rats under basal conditions was significantly decreased compared with that in control rats.

During hyperinsulinemia in HF rats, the entire range for soleus [G]om was unchanged compared with that for HF rats in the basal state (Fig. 2). In this case, however, [G]omalpha was significantly decreased by hyperinsulinemia in HF rats (P < 0.005). The range for [G]im was significantly decreased compared with the range for [G]im in HF rats under basal conditions and was similar to that calculated in control rats during hyperinsulinemia. The calculated range for TSGG in HF rats during hyperinsulinemia ranged between 1.8 ± 0.3 mM (TSGGalpha ) to 1.0 ± 0.5 mM (TSGGbeta ), which was similar to the range of TSGG in HF rats in the basal state and in control rats during hyperinsulinemia.


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Fig. 2.   Calculated glucose concentrations (in mM) at the outer and inner surfaces of the sarcolemma ([G]om and [G]im, respectively) and the transsarcolemmal glucose gradient (TSGG) in soleus muscle from control and HF rats under basal and hyperinsulinemic conditions. Data represent means ± SE for the ranges bounded by alpha - and beta -values; n = 7, 8, 6, and 8 for control basal, control insulin, HF basal, and HF insulin, respectively. The alpha - and beta -values represent the top and bottom of each of the ranges, respectively, except for [G]om and TSGG in the control basal group, where the beta -values are the upper limit. * Significant differences from basal within a dietary treatment; dagger  significant differences from control within an experimental treatment.

Muscle water and muscle glucose to plasma glucose ratio. Fe, the fraction of muscle water that is extracellular, was significantly increased by hyperinsulinemia in control rats in soleus, but this was not observed in gastroc or vastus. Fe was not altered by hyperinsulinemia in HF rats in any muscle studied, nor was Fe different between control and HF rats in the basal state in any muscle studied (Table 3).

                              
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Table 3.   Fe and muscle glucose to plasma glucose ratio

Also shown in Table 3 is muscle glucose normalized to the prevailing arterial plasma glucose concentration. By comparing Fe to the muscle glucose-to-plasma ratio, one can assess the extraction of glucose by the muscle, with a larger difference in these two percentages reflecting a lower average interstitial glucose concentration. The fact that Fe and the muscle-to-plasma glucose ratio are similar under basal conditions in gastroc and vastus indicates that no large gradient between the arterial plasma glucose concentration and the interstitial glucose concentration exists in these two fast-twitch muscles, in contrast to the situation in the soleus.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The consumption of a high-fat diet for ~4 wk resulted in a dramatic decrease in the ability of skeletal muscle to dispose of glucose, as has been demonstrated a number of times previously (23, 27). In this series of experiments, insulin-stimulated whole body glucose disposal was decreased by ~70% in HF rats, whereas muscle Rg was decreased by 50-70%. It has been demonstrated repeatedly that a defect in glucose transport exists in this model of insulin resistance (27, 37). Presumably, this occurs because of an impairment in insulin signaling to the GLUT-4-containing vesicles (44), because high-fat diet consumption has been demonstrated to impair both expression (20) and activation (44) of a number of insulin-signaling pathway constituents. The data presented in this work provide evidence that a defect at the level of glucose transport is not the sole cause of this diet-induced insulin resistance but that pre- and posttransport steps also make significant functional contributions to this impairment.

Three muscles were studied in these experiments, one comprised almost solely of slow-twitch oxidative fibers (soleus), from which most of the results were obtained, and two muscles comprised primarily of fast-twitch fibers, the gastroc and vastus. This allowed for a first assessment of how diet-induced insulin resistance may affect specific types of muscle fibers. It is difficult to measure fiber-specific effects in humans, because most human muscles are comprised of a mixture of fiber types. One advantage of the rat model is that the soleus and superficial vastus lateralis are relatively homogeneous for fiber type; thus glucose gradients between arterial plasma and the sarcolemma should display less regional heterogeneity. A technical disadvantage to muscles comprised of fast-twitch fibers is that, presumably because of their decreased capillary density and perfusion, the isotope equilibration period used in these experiments was not of sufficient duration to reach steady-state conditions (see MATERIALS AND METHODS). Therefore, only analyses not dependent on 3-O-MG accumulation were performed on these muscles. Conclusions regarding the intracellular contribution to impaired muscle glucose uptake must be limited to the soleus. It should be noted that, because of its role as a postural muscle and its associated slow-twitch fiber type composition, the soleus is not necessarily representative of human muscle overall.

Data from all three muscles support an impairment in glucose delivery in HF rats. Despite the much lower rate of glucose flux in muscle from HF rats, all three muscles were characterized by a fall in the muscle glucose-to-plasma glucose ratio in response to hyperinsulinemia, which was apparent only in gastroc in control rats. It is possible that a structural or functional defect may exist within the microcirculation of HF rats, resulting in decreased muscle capillary perfusion. For example, a decrease in capillary density has been reported in insulin-resistant human (24, 26) and rat muscle after 6-8 wk of poorly controlled insulin-deficient diabetes (21). Another possibility is that there is a decrease in insulin-stimulated muscle blood flow in HF rats. Although the ability of insulin at physiological concentrations to stimulate muscle blood flow is controversial (1, 41), a significant amount of data demonstrates that insulin does have effects on arteriole resistance (15, 43). The fall in muscle glucose concentration with hyperinsulinemia in HF rats is consistent with the demonstration that insulin-induced arteriole vasodilation is impaired in two rat models of insulin resistance, the fructose-fed rat (15) and the genetically obese Zucker rat (43). In addition, in the spontaneously hypertensive rat, the ability of insulin to increase Doppler-measured hindlimb blood flow is blunted (34). It is therefore possible that insulin resistance of the vasculature, in which the insulin-stimulated increase in glucose delivery is blunted or prevented, is a contributor to the impairment in muscle glucose uptake.

Under basal conditions, the TSGG in the soleus was reduced in HF compared with control rats. The magnitude of the TSGG is directly related to the degree to which glucose transport limits glucose uptake. The fact that the TSGG was small in the soleus of HF rats under basal conditions means that the sarcolemma functions as less of a relative barrier to glucose uptake than it does in control rats. This does not necessarily mean that the sarcolemmal permeability is greater in HF than in control rats under these conditions; instead, it is more likely that the processes that maintain the TSGG (glucose delivery and/or glucose phosphorylation) are insufficient to maintain the larger TSGG observed in chow-fed rats. Because the distribution volume of glucose in intracellular water is unknown, [G]om, [G]im, and the TSGG were calculated on the basis of two theoretical distributions (referred to as alpha  and beta ), as described in MATERIALS AND METHODS. Under conditions where both the Si/So and the muscle glucose concentration are high, as was the case for the soleus in HF rats under basal conditions, the ranges spanned by the alpha - and beta -values for [G]om and [G]im are wide. Thus precise conclusions regarding the roles of glucose delivery and glucose phosphorylation in limiting muscle glucose uptake cannot be determined. Regardless of these assumptions, however, [G]im was elevated in soleus in HF rats in the basal state, indicating a limitation at glucose phosphorylation.

In soleus muscle from both control and HF rats, hyperinsulinemia resulted in an increase in [G]im above zero. If the only defect in glucose uptake in HF rats occurred at the glucose transport step, one would predict that [G]im would be lower in HF than in control rats. Instead, it appears that glucose phosphorylation becomes limiting at a lower rate of glucose uptake in HF rats. An impairment in hexokinase II activity has been reported in muscle from insulin-resistant mice (3) and humans (25), but this has not been observed in high-fat diet-induced insulin resistance (17, 44) or muscle from obese Zucker rats (38). Even if hexokinase activity is normal in insulin-resistant muscle, the high sensitivity of hexokinase I and hexokinase II to inhibition by G-6-P means that a defect in utilization of G-6-P (either by incorporation into glycogen or by metabolism in glycolysis) may also explain the inability of phosphorylation in HF muscle to match pace with the rate of glucose transport. In support of this, there are some data suggesting that muscle G-6-P levels are higher in HF rats after a hyperinsulinemic clamp (18), and it has also been reported that hexokinase is more sensitive to inhibition by G-6-P in insulin-resistant muscle (38). This is clearly an area in which more work needs to be performed.

One interesting result from these experiments is that the fall in plasma NEFA concentration that normally occurs in response to hyperinsulinemia was not observed in HF rats. We view this as both a consequence of insulin resistance (i.e., insulin resistance at the adipocyte) and a possible contributing factor to the development of muscle insulin resistance in these rats. An acute infusion of a triglyceride emulsion designed to increase the plasma NEFA concentration is able to cause insulin resistance within ~2-4 h in humans (2, 36) and rats (14, 19). Acute NEFA elevation is also able to prevent endothelium-dependent increases in leg blood flow in humans (39). This could be a mechanism for the fall in the muscle glucose-to-plasma glucose ratio observed during hyperinsulinemia in HF rats in soleus, vastus, and gastroc. In addition, an acute elevation of NEFA concentration during a hyperinsulinemic clamp results in a rise in the muscle G-6-P concentration in rats (14), and may therefore relate to the impairment observed in glucose phosphorylation in soleus muscle from HF rats.

In conclusion, we have demonstrated that the impairment in muscle glucose uptake induced by consumption of a high-fat diet for ~4 wk is due not only to an impairment in glucose transport but also to defects in glucose delivery and glucose phosphorylation. In soleus muscle from HF rats, glucose phosphorylation is impaired in both the basal and insulin-stimulated state, whereas glucose delivery becomes more limiting to glucose uptake in soleus during hyperinsulinemia. In two muscles comprised of fast-twitch fibers, it appears that glucose delivery is a more important site of resistance to insulin-stimulated glucose uptake in HF rats than in control rats. Taken in total, these data and data from the literature suggest that the consumption of a high-fat diet affects each of the steps that comprise muscle glucose uptake. The phenotype of "skeletal muscle insulin resistance" is in fact due to defects at both muscle and the vasculature, which impair both the delivery and extraction of glucose.


    ACKNOWLEDGEMENTS

The authors are grateful to Dr. Andrea Mari for insight in the preparation of the manuscript, and Dr. George Reed for assistance with statistical analyses.


    FOOTNOTES

This work was supported by NIH Grant RO1 DK-50277. A. Halseth was supported by Training Grant 5 T32 DK-07563-08.

Address for reprint requests and other correspondence: A. Halseth, Pharmacia Corporation, 800 N. Lindbergh Blvd., Mail Zone T1G, St. Louis, MO 63117.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 24 August 1999; accepted in final form 12 June 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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