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Effects of Insulin on Subcellular Localization of Hexokinase II in Human Skeletal Muscle in Vivo ¹

The Diabetes Division, Department of Medicine (C.V., H.Y-J., P.I., R.Pi., M.P., J.K., R.D., L.M.) and Biochemistry (L.M.), the University of Texas Health Science Center at San Antonio, San Antonio, Texas 78284; The Department of Molecular Physiology (H.A., R.Pr. D.G.), Vanderbilt University School of Medicine, Nashville, Tennessee 373232-0615

Address correspondence and requests for reprints to: Lawrence J. Mandarino, Ph.D., The University of Texas Health Science Center, Department of Medicine/Diabetes Division, 7703 Floyd Curl Drive, San Antonio, Texas 78284-7886.

	Abstract

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The phosphorylation of glucose to glucose-6-phosphate, catalyzed by hexokinase, is the first committed step in glucose uptake into skeletal muscle. Two isoforms of hexokinase, HKI and HKII, are expressed in human skeletal muscle, but only HKII expression is regulated by insulin. HKII messenger RNA, protein, and activity are increased after 4 h of insulin infusion; however, glucose uptake is stimulated much more rapidly, occurring within minutes. Studies in rat muscle suggest that changes in the subcellular distribution of HKII may be an important regulatory factor for glucose uptake. The present studies were undertaken to determine if insulin causes an acute redistribution of HKII activity in human skeletal muscle in vivo. Muscle biopsies (vastus lateralis muscle) were performed before and at the end of 30 min insulin infusion, performed using the euglycemic clamp technique. Muscle biopsies were subfractionated into soluble and particulate fractions to determine if insulin acutely changes the subcellular distribution of HKII. Insulin decreased HKII activity in the soluble fraction from 2.20 ± 0.31 to 1.40 ± 0.18 pmoles/(min[chempt]µg) and increased HKII activity in the particulate fraction from 3.02 ± 0.46 to 3.45 ± 0.46 pmoles/(min[chempt]µg) (P < 0.01 for both). These changes in HKII activity were correlated with changes in HKII protein, as determined by immunoblot analysis (r = 0.53, P = 0.05). Insulin had no effect on the subcellular distribution of HKI activity, which was primarily restricted to the soluble fraction. These studies are consistent with the conclusion that, in vivo in human skeletal muscle, insulin changes the subcellular distribution of HKII within 30 min.

	Introduction

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INSULIN increases glucose transport in skeletal muscle primarily by translocating glucose transporters to the plasma membrane (1). Once glucose is transported into the cell, for its entry to be irreversible it must be phosphorylated by hexokinase to form glucose 6-phosphate. Of the known isoforms of hexokinase, hexokinase I (HKI) and hexokinase II (HKII) are expressed in skeletal muscle (2, 3, 4). Insulin and muscle contraction increase the expression of HKII, but not HKI, in rodents (5, 6) and humans (4), so HKII is considered to be the isoform in skeletal muscle that is subject to regulation. Recent evidence from studies using mathematical modeling of isotopic tracer data indicates that insulin increases glucose transport and phosphorylation independently in forearm muscle of normal volunteers, and that both of these steps are resistant to insulin action in muscle of patients with noninsulin-dependent diabetes mellitus (7, 8).

In normal human volunteers, 4 h of insulin increased the messenger RNA (mRNA) levels of HKII by about 3-fold in skeletal muscle but did not affect HKI mRNA (4). HKII protein content increased comparably, and HKII activity, which is partitioned subcellularly into soluble and particulate fractions, was increased in the soluble fraction. HKI activity was not affected by insulin. In addition to this more long-term regulation of HKII expression, insulin increases the conversion of glucose to glucose 6-phosphate more rapidly in muscle. In studies using the leg or forearm balance technique, insulin increases the arteriovenous difference of glucose across the limb to a nearly maximal degree within 30–60 min in humans (9). It is likely, therefore that insulin exerts an early effect (within minutes) on HKII that increases its activity before the increase in HKII mRNA can be translated into increased HKII protein and activity over several hours. There is evidence from rodent studies that insulin shifts HKII activity from the cytosol to the mitochondria (10), and it has been hypothesized that this results in increased hexokinase activity by increasing proximity of the enzyme to the supply of ATP (11). It is unknown if this occurs in humans. The present studies therefore were undertaken to determine if insulin produces an acute subcellular redistribution of HKII activity in skeletal muscle from healthy volunteers.

	Subjects and Methods

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Subjects

Ten lean, healthy volunteers took part in these studies (3 males, 7 females, age 32 ± 3 yr, body mass index 22.8 ± 0.8 kg/m²). All of the subjects were instructed not to take part in any vigorous exercise for 48 h before the study, and none participated in a regular conditioning program. After obtaining informed written consent, all subjects received a 75 g, 2 h oral glucose tolerance test to confirm the presence of normal glucose tolerance (12). The experimental protocol was approved by the Institutional Review Board of the University of Texas Health Science Center at San Antonio.

Study design

All studies began at 0800 in the morning after a 10–12 h overnight fast. Euglycemic, hyperinsulinemic clamp experiments were performed as described previously (13). An antecubital vein was catheterized for infusion of insulin and 20% glucose, and a hand vein was canulated in a retrograde fashion and placed in a heated box (60 C) for sampling of arterialized blood. Percutaneous needle biopsies of the vastus lateralis muscle were obtained as previously described (4) before the start of the insulin infusion. After the subject rested 30 min, insulin was infused in a primed, continuous manner at a rate of 40 mU/m²[chempt]min, while the plasma glucose was maintained at euglycemia. Plasma glucose was measured with a Beckman glucose analyzer (Beckman Instruments, Fullerton, CA), every 5 min in arterialized blood, and a variable infusion of 20% glucose was adjusted to maintain euglycemia. The second percutaneous muscle biopsy was obtained from the other leg 30 min after starting the insulin infusion. Muscle biopsy specimens were immediately blotted free of blood, frozen in liquid nitrogen within 15–20 sec, and stored in liquid nitrogen until assay.

Muscle biopsies

Biopsies were homogenized while still frozen using a Polytron Homogenizer (Brinkman Instruments, Westbury, CT) in a buffer consisting of 50 mM potassium phosphate, pH 7.4, 2 mM dithiothreitol; 2 mM EDTA, 20 mM sodium fluoride; 10 µg/mL soybean trypsin inhibitor; 20 µg/mL p-aminobenzamidine; 70 µg/mL phenylmethylsulfonyl fluoride. Homogenates were centrifuged at 14,000g. The supernatant (soluble fraction) was removed and kept on ice, and the pellet (particulate fraction) was resuspended in the extraction buffer containing 0.1% Triton X-100. The soluble fraction contains greater than 95% of the activity of cytosolic enzymes such as glycogen synthase, and the particulate fraction contains greater than 98% of mitochondrial enzymes such as pyruvate dehydrogenase (14).

Enzyme activity assays

The different temperature sensitivities of HKII and HKI were used to separate the activities of the two enzymes (4, 16). Aliquots of soluble and particulate fractions were either heated at 45 C for 1 h or kept on ice. Because HKII activity is denatured at 45 C (16), HK activity assayed on the heated sample represents HKI activity, whereas activity assayed on the samples kept at 4 C represents total HK (HKI plus HKII) activity. HKII activity was determined as the difference between these values. HK activity was determined using a modification (4) of an enzyme-linked fluorometric assay (2). Previous studies using this technique have shown that HKI and HKII activities determined in this manner correspond in the subcellular fractions with HKI or HKII protein content determined by immunoblot analysis using specific antibodies (4). The HK assay buffer consisted of 40 mM Tris-HCl, pH 7.5; 100 mM KCl; 20 mM/MgCl₂; 2 mM EDTA; 10 mM glucose; 2 mM ATP; 0.25 mM NADP⁺; and 0.01 U/mL glucose 9-phosphate dehydrogenase (Sigma Chemical, St. Louis, MO). Total assay volume was 1.0 mL, and the assay was started by adding 10 µL of muscle extract fractions (30–60 µg protein). The increase in fluorescence was monitored for 15 min using a fluorometer (Turner model 112, Sequoia-Turner, Mountain View, CA) calibrated to 5 nmol nicotinamide adenine dinucleotide phosphate (NADPH) full scale. This assay was linear for greater than 20 min during which time less than 1% of substrates and cofactors were consumed. Multiple assays performed on separate portions of muscle biopsies from the same individuals (n = 5) revealed the interassay coefficient of variation to be 11.5 ± 1.6% over a range of activity of 1–4 pmol/min^-1µg protein^-1. Glycogen synthase (CS) fractional velocity was defined as the ratio of GS activities determined at 0.1 and 10 mM G-6-P. This ratio is increased by insulin infusion in humans and represents dephosphorylation and activation of GS. All enzyme activities are expressed per protein content and therefore are specific activities.

Immunoblot analysis

For seven of the subjects who had sufficient protein remaining after enzyme activity assays, HKII protein content was determined using 50 µg of each soluble fraction and 50 µg of the particulate fractions. Extracts were subjected to sodium dodecyl sulfate (SDS)-PAGE under reducing conditions (7% running gel, 5% stacking gel) with a Mini-Protean electrophoresis apparatus (Bio-Rad, Richmond, CA) and were transferred to nitrocellulose electrophoretically. The blots were washed twice in tris-(hydroxymethyl) amino-methane-buffered saline (TBS) and then blocked at room temperature for 1 h by use of TBS containing 2% nonfat dried milk and 0.1% Tween 20. An antibody specific for HKII was produced by synthesizing peptides corresponding to the COOH-terminal 18 amino acids of rat HKII (GAALITAVAC-RIREAGQR). This sequence is distinct from the COOH-terminal 16 amino acids of HKI (LITAVGVRLRGDPTNA), and competition experiments with the carboxy-terminal half of HKII confirmed the specificity of the HKII antibody (17). The amino acid sequences of rat and human HKII show 94% identity, and the peptide used to produce the antibody is identical in rat and human HKII (2, 3). The peptides were conjugated to rabbit serum albumin and injected into rabbits for production of polyclonal antibodies. Each of these antibodies was added directly to the blocking mixture at a concentration of 1:1,000 and incubated overnight at 4 C. Immunoblots were developed using the ECL detection system (Amersham Life Science, Little Chalfont, Buckinghamshire, UK), and signals were quantified using NIH Image software (National Institutes of Health, Bethesda, MD).

Statistics

HKI and HKII activities and HKII protein content in the soluble and particulate fractions after insulin stimulation were compared with basal values by two factor repeated measures analysis of variance (BMDP Statistical Software, Los Angeles, CA) where the two factors were treatment (basal vs. insulin) and fraction (soluble vs. particulate). Post hoc t tests were used to compare cell means where the F-test for interaction (treatment x fraction) was significant. Hexokinase activities were also analyzed as insulin-stimulated changes from basal values and compared by paired t test. Correlation analysis was performed by the Pearson product-moment method.

	Results

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Euglycemic clamp

The plasma insulin concentration was 7 ± 1 µU/mL in the basal state and was 73 ± 5 µU/mL during the last 10 min of the insulin infusion. The glucose infusion rate required to maintain euglycemia during the last 10 min (time t = 20 to 30 min) of the hyperinsulinemic euglycemic clamp was 3.56 ± 0.35 mg/kg fat free mass.

Enzyme activities

HKI and HKII activities are given in Table 1. HKII activity was divided between soluble and particulate fractions under basal conditions. Insulin infusion decreased HKII activity in the soluble fraction from a basal value of 2.20 ± 0.31 to 1.40 ± 0.18 and increased HKII activity in the particulate fraction from 3.02 ± 0.46 to 3.45 ± 0.46 pmol/(min[chempt]µg) protein (F₁,₃₆ = 21.96, P < 0.0001, for interaction effect of insulin and subcellular fraction, by repeated measures analysis of variance). Both of these changes were statistically significant by post hoc t tests (P < 0.01). HKII activities were also expressed as changes from basal values. HKII activity in the soluble fraction decreased by 0.80 ± 0.23 pmoles/(min[chempt]µg) protein (P < 0.01 compared with a value of 0) and increased by 0.43 ± 0.13 pmoles/(min[chempt]µg) protein in the particulate fraction (P < 0.01 vs. 0). These changes in the two fractions were not statistically different from one another (paired t = 1.60, P = 0.14). Insulin had no effect on total HKII activity (soluble plus particulate), which was 5.22 ± 0.70 pmoles/(min[chempt]µg) protein in the basal state compared with 4.85 ± 0.53 during insulin infusion (paired t = 1.59, P = 0.15).

To determine whether the redistribution of HKII activity after 30 min insulin infusion was accompanied by a shift in HKII protein, soluble and particulate fractions were subjected to immunoblot analysis. In the soluble fraction, the relative density of the HKII band tended to decrease from 4.15 ± 0.77 to 2.99 ± 0.91, and in the particulate fraction it tended to increase from 2.19 ± 0.31 to 3.75 ± 1.24 (F₁,₃₆ = 3.45, P = 0.07 by repeated measures analysis of variance). Insulin had no effect on total HKII protein (6.33 ± 0.93 vs. 6.74 ± 1.94 units). Insulin-induced changes in HKII were significantly correlated (r = 0.53, P = 0.05) with changes in HKII protein (Fig. 1).

View larger version (12K): [in this window] [in a new window]   Figure 1. The relationship between insulin-stimulated changes in HKII protein and activity in soluble (circles) and particulate (squares) fractions of muscle biopsies after 30 minutes of insulin infusion is illustrated in this figure. The correlation coefficient relating these variables was 0.53 (P = 0.05).

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

The precise intracellular sites of localization were not determined in the present study. Previous studies have shown that, using the same extraction procedure used in the current study, glycogen synthase and phosphofructokinase activity were located in the soluble fraction, while pyruvate dehydrogenase, a mitochondrial inner membrane enzyme, was located in the particulate fraction (14). In addition, immunoblot analysis has shown that HKI is located entirely in the soluble fractions, and HKII is partitioned between soluble and particulate fractions (4). It is likely that freezing and homogenization disrupt organelle structure to some extent; however, since detergent was not used in the extraction and centrifugation steps, proteins that were bound to membrane structures were likely to have remained bound. Such is the case for pyruvate dehydrogenase (14). Therefore, although it cannot be said with certainty to which membranous structure HKII was bound, this fractionation method can clearly differentiate between soluble and bound fractions.

The site and regulation of the subcellular distribution of hexokinase is a controversial and complex issue that may depend on the hexokinase isoform and the tissue studied. In brain, where HKI predominates, the majority of the enzyme is bound to membrane (18). Most of the enzyme appears to be associated with porin, a mitochondrial membrane anion channel (19), and this may serve to bring hexokinase into close proximity with the site of ATP (adenosine triphosphate) production. Regeneration of ADP (adenosine diphosphate) by hexokinase can then promote increased oxidative phosphorylation, and glucose can serve as an "acceptor" of phosphate (11). The biological significance and net energy gain of such a system is not clear, but ischemia or hypoglycemia increases hexokinase binding to brain mitochondria (20), suggesting that this is an effort to protect energy supply. Other investigators have found that HKI in brain can be associated with membrane structures other than mitochondria (21). In macrophages, phorbol esters translocate hexokinase to microfilaments in a zone adjacent to the plasma membrane (22). This could place hexokinase in closer apposition to the supply of glucose from glucose transporters in the membrane and could potentially increase the rate of glucose phosphorylation and uptake. In heart muscle, which primarily expresses HKII, insulin has been reported to redistribute hexokinase activity to the mitochondria (23). These investigators propose that this redistribution is important in insulin stimulation of glucose uptake.

In rat skeletal muscle, both contraction and insulin have been reported to increase the proportion of HKII bound to mitochondria (10, 24). Increased muscle contraction produced by electrical stimulation caused an increase in the ratio of bound to free hexokinase in rats (24). However, this effect apparently required two days to be seen and seemed to depend upon increased HKII synthesis, so it may be a different phenomenon than acute subcellular redistribution. In the isolated rat diaphragm (10), and in rat adipocytes (25), another insulin-sensitive tissue, insulin produced a rapid increase in the activity of hexokinase bound to mitochondria.

Glycogen synthase fractional velocity was also assayed in the soluble fraction of the muscle biopsies. The fractional velocity of glycogen synthase reflects the phosphorylation state of the enzyme (26). Insulin activated glycogen synthase within minutes, and the extent of activation was similar to that observed with 2–4 h insulin infusion (14). The time course of insulin’s activation of glycogen synthase has previously been reported (27), although to our knowledge this is the earliest demonstration of activation of glycogen synthase by insulin in human muscle. The current study demonstrates that, after 30 min, at a time when insulin had altered the subcellular distribution of HKII, glycogen synthase had been maximally activated. These observations show that the enzyme responsible for glycogen deposition is maximally stimulated before the time of maximal activation of glycogen synthesis in vivo (28), and they illustrate the utility of percutaneous muscle biopsies in humans for examining early steps in insulin action.

The ability of insulin to activate glycogen synthase and translocate HKII within 30 min is consistent with its effects on skeletal muscle glucose uptake. In human studies, insulin increases forearm muscle glucose uptake rapidly, and this effect reaches near maximal levels within 60 min insulin infusion (9). This rapid activation of the metabolic effects of insulin is comparable to the ability of insulin to activate its known intracellular signaling pathways within minutes. These rapid effects on glucose uptake and metabolism are likely to reflect the acute changes brought about by insulin secreted following a meal and therefore may have direct relevance to normal physiology. Moreover, the plasma insulin concentrations achieved during the insulin infusion are within the normal physiologic range, so these findings are likely to bear directly on normal physiology.

In summary, the present study demonstrates that insulin alters the subcellular distribution of HKII, but not HKI, in normal human skeletal muscle. At the same time, insulin has activated glycogen synthase. This acute effect of insulin is to shift HKII from a soluble, most likely cytosolic, to a particulate, possibly mitochondrial, fraction of muscle extract. These efforts are consistent with the rapid activation of glucose uptake by muscle.

	Acknowledgments

 
The authors gratefully acknowledge the nursing assistance of Magda Ortiz and Diana Frantz, the technical assistance of Cindy Munoz, Sheila Taylor, Gilbert Gomez, Andrea Barrentine, and Jean Finlayson, and the editorial aid of Ms. Jerry Lynn Beesley.

	Footnotes

 
¹ This study was supported by NIH Grants DK-47936(LM), DK-35107, DK-46867(DG), and DK-20593 (Vanderbilt Diabetes Research and Training Center), DK-24092(RD), and General Clinical Research Center Grant RR MO1RR01346.

Received June 20, 1997.

Revised August 19, 1997.

Accepted September 24, 1997.

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