GLUCOSE TRANSPORTER CONTENT AND GLUCOSE UPTAKE IN SKELETAL MUSCLE CONSTRUCTS ENGINEERED IN VITRO

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1 In Vitro Cell. Dev. Biol. Animal 39: , November December Society for In Vitro Biology /03 $ GLUCOSE TRANSPORTER CONTENT AND GLUCOSE UPTAKE IN SKELETAL MUSCLE CONSTRUCTS ENGINEERED IN VITRO ERIN L. BAKER, ROBERT G. DENNIS, AND LISA M. LARKIN 1 Department of Biomedical Engineering (E. L. B., R. G. D.), Division of Geriatric Medicine (L. M. L.), Muscle Mechanics Laboratory (R. G. D., L. M. L.), Department of Mechanical Engineering (R. G. D.), University of Michigan, Ann Arbor, Michigan (Received 18 November 2003; accepted 14 January 2004) SUMMARY Engineered muscle may eventually be used as a treatment option for patients suffering from loss of muscle function. The metabolic and contractile function of engineered muscle has not been well described; therefore, the purpose of this experiment was to study glucose transporter content and glucose uptake in engineered skeletal muscle constructs called myooids. Glucose uptake by way of 2-deoxyglucose and GLUT-1 and GLUT-4 transporter protein content was measured in basal and insulin-stimulated myooids that were engineered from soleus muscles of female Sprague Dawley rats. There was a significant increase in the basal 2-deoxyglucose uptake of myooids compared with adult control (fivefold), contraction-stimulated (3.4-fold), and insulin-stimulated (threefold) soleus muscles (P , , and , respectively). In addition, there was a significant increase in the insulin-stimulated 2-deoxyglucose uptake of myooids compared with adult control soleus muscles in basal conditions (6.5-fold) and adult contraction-stimulated (4.5-fold) and insulinstimulated (3.9-fold) soleus muscles (P , , and , respectively). There was a significant 30% increase in insulin-stimulated compared with basal 2-deoxyglucose uptake in the myooids. The myooid GLUT-1 protein content was 820% of the adult control soleus muscle, whereas the GLUT-4 protein content was 130% of the control soleus muscle. Myooid GLUT-1 protein content was 6.3-fold greater than GLUT-4 protein content, suggesting that the glucose transport of the engineered myooids is similar in several respects to that observed in both fetal and denervated skeletal muscle tissue. Key words: skeletal muscle; myooid; glucose uptake; glucose transporter. INTRODUCTION The production and use of engineered muscle from cells of muscle biopsy samples is potentially a powerful tool for the restoration of muscle function after acute injury, surgery, or disease. The current clinical method for restoration of massive muscle tissue loss is whole-muscle grafting. However, the clinical success of whole-muscle grafting has been tempered by the incomplete and variable return of muscle function, especially in elderly patients (Prendergast et al., 1977; Young et al., 1984, 1985; Guelinckx et al., 1988). As a result, the use of whole-muscle grafting has been used mainly in young individuals and is reserved for a limited set of conditions. The ability to engineer muscle from cells of young and senescent muscle donors may lead to the eventual use of engineered muscle as a treatment option for patients suffering from loss of muscle function. Although some previously engineered in vitro skeletal muscle constructs have been described mechanically (Vandenburgh, 1982; Vandenburgh and Karlisch, 1989; Vandenburgh et al., 1991a, 1991b), to date, no studies have examined the relationship between their contractile function and metabolic capabilities. Therefore, the goal of this experiment was to study glucose transporter content and 1 To whom correspondence should be addressed at Institute of Gerontology, NIB, Room 964, 300 North Ingalls Street, University of Michigan, Ann Arbor, Michigan llarkin@umich.edu glucose uptake in these functional engineered skeletal muscle constructs, termed myooids (Dennis and Kosnik, 2000; Dennis et al., 2001; Kosnik et al., 2001). Glucose enters skeletal muscle by way of a facilitated transport system that involves two isoforms of glucose transporters: GLUT-1, which is responsible for uptake during basal conditions (Klip and Paquet, 1990; Ren et al., 1993), and GLUT-4, which is responsible for uptake in the presence of insulin and contraction stimuli (Douen et al., 1990; Marette et al., 1992). GLUT-4 is the primary isoform in adult skeletal muscle and is known to translocate to the cell surface in response not only to insulin (Cushman and Wardzala, 1980; Klip et al., 1987; Lund et al., 1993) but also to muscle contraction (Holloszy and Narahara, 1965; Ploug et al., 1984; Brozinick et al., 1994) and hypoxia (Cartee et al., 1991). GLUT-1 is located primarily in the plasma membrane, whereas GLUT-4 is found predominantly in intracellular membranes (Zorzano et al., 1996). Because skeletal muscle is the principal target tissue for glucose disposal and is the main tissue involved in insulin-stimulated glucose uptake, glucose transport is considered to be the ratelimiting step in glucose utilization, and GLUT-4 protein content of skeletal muscle is of the utmost importance. Decreased expression of either the GLUT-1 or GLUT-4 transporter may result in a diminished fuel availability to muscle during endurance contraction (Larkin et al., 1997). Because engineered myooids are cultured from satellite cells and 434

2 GLUCOSE UPTAKE IN ENGINEERED MUSCLE 435 lack innervation, we hypothesize that myooid glucose transporter content and glucose uptake will exhibit characteristics similar to those observed in fetal or denervated skeletal muscle tissue. Fetal skeletal muscle tissue has been shown to express a high number of GLUT-1 transporters, and significantly less GLUT-1 expression is observed during postnatal life (Santalucia et al., 1992; Castello et al., 1994; Gaster et al., 2000a). GLUT-4 transporters are present in fetal tissue, although to a much lesser extent than GLUT-1 transporters (Wang and Hu, 1991; Santalucia et al., 1992; Castello et al., 1994; Gaster et al., 2000a). Accordingly, other studies of human adult skeletal muscle have shown increased GLUT-1 expression during tissue regeneration after damage (Gaster et al., 2000b). Alterations in the expression of GLUT-1 and GLUT-4 proteins in fetal tissue are associated with the process of muscle innervation (Gaster et al., 2000). Denervation is associated with elevated basal glucose transport and diminished insulin-stimulated glucose transport (Coderre et al., 1992; Handberg et al., 1996). Accordingly, denervation is associated with upregulation of GLUT-1 protein and messenger ribonucleic acid (mrna) content and downregulation of GLUT-4 protein and mrna content (Block et al., 1991; Coderre et al., 1992; Handberg et al., 1996). Unlike GLUT-1 normally found in innervated tissue, it has been shown that the denervation-induced upregulation of GLUT-1 is associated with GLUT-1 localization to intracellular vesicles that respond to insulin by translocating to the cell surface (Zhou et al., 2000). In fact, the formation of insulin-sensitive GLUT- 1 containing vesicles in denervated muscle may act as a compensatory mechanism for diminished GLUT-4 protein content (Zhou et al., 2000). To examine the glucose transporter content and glucose uptake in engineered muscle, we first produced myooids by harvesting and culturing myogenic cells from the soleus muscles of adult female rats as described by Dennis and Kosnik (2000). The myooid is a three-dimensional skeletal muscle construct developed from the coculture of mammalian skeletal muscle satellite cells and fibroblasts that does not use an artificial scaffold for the contractile region of the structure. The myooid includes anchor materials that are designed to serve as artificial tendons and to allow manual manipulation (Dennis and Kosnik, 2000). In this study, the basal and insulin-stimulated myooids were examined for glucose uptake by way of 2-deoxyglucose and GLUT-1 and GLUT-4 transporter protein content and were compared with basal, insulin-stimulated, and contraction-stimulated adult soleus muscles. MATERIALS AND METHODS Animal model and animal care. Tissue engineering studies were carried out using muscle tissue from female Sprague Dawley retired breeder rats obtained from the Harlan Sprague Dawley Laboratory (Indianapolis, IN). The glucose-uptake studies in whole adult mouse muscle were performed on female mice obtained from a genetically heterogeneous mouse population, designated the UM-HET3 strain, purchased from Jackson Laboratories (Bar Harbor, ME). All animals underwent a surgical procedure for the removal of the right and left soleus muscles. All animals were acclimated to our colony conditions, i.e., light cycle and temperature, for 1 wk before the removal procedure. Rats were housed individually and mice were housed in groups of four in hanging plastic cages (28 56 cm) and kept on a 12:12 light dark light cycle at a temperature of C. The animals were fed Purina Rodent Chow 5001 laboratory chow and water ad libitum. All surgical procedures were performed in an aseptic environment with animals in a deep plane of anesthesia induced by intraperitonial injections of sodium pentobarbital (65 mg/kg). Supplemental doses of pentobarbital were administered as required to maintain an adequate depth of anesthesia. All animal care and animal surgery were in accordance with The Guide for Care and Use of Laboratory Animals (Public Health Service, 1996, NIH Publication No ); the experimental protocol was approved by the University Committee for the Use and Care of Animals. Preparation of solutions and media. Unless otherwise indicated, all solutions and media were prepared and stored at 4 C before isolation and culture of muscle cells and warmed to 37 C in a heated water bath immediately before use. The media, with slight modifications from Dennis and Kosnik (2000), were as follows: (1) Growth medium (GMA) consisted of 400 ml of HAM F-12 nutrient mixture (GIBCO-BRL, Catalog No ), 100 ml fetal bovine serum (GIBCO-BRL, Catalog No ), and 5 ml A9909 (Sigma Chemical Co., St. Louis, MO, Catalog No. A9909). (2) Differentiation medium (DMA) consisted of 465 ml of Dulbecco modified Eagle medium (DMEM, GIBCO-BRL, Carlsbad, CA, Catalog No ), 35 ml of 100% horse serum albumin (GIBCO-BRL, Catalog No ), and 5 ml A9909. (3) The tissue was dissociated in a dispase and collagenase solution (D&C). Ten milliliters of the solution was prepared per two muscle strips and consisted of 4 units of dispase (Sigma, Catalog No. P-3417; 0.4 units/mg) and 100 units of type 4 collagenase (GIBCO-BRL, Catalog No ; 239 units/mg) per milliliter of DMEM. (4) Five milliliters of transport medium (TM) was prepared per muscle dissected at the concentration of 2% A9909 in Dulbecco phosphate-buffered saline (DPBS). (5) Three milliliters of preincubation medium (PIM) was prepared per plate and consisted of 2.5 ml of 0.05% sodium azide (NaN 3, Sigma, Catalog No. S-8032) in DPBS solution, 22.5 ml DMA, and 0.25 ml A9909 per muscle dissected. Preparation of culture dishes and anchors. Myooids were engineered in individual 35-mm plates as described earlier (Dennis and Kosnik, 2000). In brief, individual plates allowed for functional evaluation of individual myooids and facilitated the removal of any contaminated plate. Each 35-mm plate was coated with 1.5 ml of Sylgard (Dow Chemical Corporation, Midland, MI; Type 184 silicon elastomer) and allowed to cure for 3 wk before use. One week before use, Sylgard-coated plates were then coated with laminin at 1.0 g/cm 2 per plate (10 g of natural mouse laminin [GIBCO-BRL, Catalog No ] and 3 ml of DPBS, ph 7.2, [GIBCO-BRL, Catalog No ] per plate) and left to dry for 48 h. Salt crystals were dissolved and removed by rinsing the plates with 3 ml of DPBS. Forty-eight hours after laminin coating, sutures (Ethicon braided silk size 0 nonsterile, Catalog No. 116-S) were cut to a length of 6 mm, one end of each suture was frayed, and the entire suture was soaked in the laminin solution consisting of 50 g of the natural mouse laminin and 1 ml DPBS. Two sutures were then pinned to each plate (with the frayed ends facing each other) 12 mm apart with four pins (0.1-mm stainless steel minutien pins Fine Science Tools, Foster City, CA, Catalog No ) to serve as artificial tendons for the myooids (Dennis and Kosnik, 2000). The sutures were left to dry for 2 h, and plates were filled with 2 ml of GMA and capped. The plates were then decontaminated with UV light (wavelength, nm) for 90 min and placed in a 37 C5%CO 2 incubator for 1 wk before plating muscle cells. Preparation of muscle and isolation of satellite cells. Both soleus muscles were surgically removed under aseptic conditions, weighed, sterilized in 70% ethanol (ETOH), and incubated for 5 min in TM. A single-edged razor blade and a pair of No. 5 forceps (Fine Science Tools) were then used to slice the soleus muscle longitudinally into six strips. Next, 35-mm Sylgard-treated plates were sterilized in 70% ETOH, and muscle slices were pinned to length, with two muscle strips per plate. Then 3 ml of PIM was added to each plate, and the plates were UV treated for 90 min. The plates were then placed in a 37 C5%CO 2 incubator for 50 h. After a 48- to 50-h incubation period, muscle slices were inspected for contamination, and infected plates were discarded. The remaining muscle strips were then removed from the plates and incubated in D&C (two muscle strips per 10 ml) in a 37 C shaking water bath for 3 4 h. The dissociation was aided by occasionally shaking each vial slightly by hand. Once the muscle was fully digested by 3 4 h, the vials were centrifuged at 1000 g for 15 min at 25 C. Last, the supernatant was aspirated from the vials, and the pellet was resuspended in GMA to obtain a concentration of 10 mg of dissociated muscle per 2 ml of GMA. Cell culture and myooid formation. The previously prepared laminin-coated plates were examined for contamination after storage in an incubator for 1 wk, and the GMA was aspirated from the plates. Two milliliters of the cell suspension was plated in each culture dish and placed in a 37 C5%CO 2 incubator for 5 d. Culture plates were not disturbed for at least 72 h to allow

3 436 BAKER ET AL. cell adherence to the plates. After 5 d, the cells were fed with GMA every 48 h until the cells became confluent (approximately 7 d). Once the cells achieved confluence, they were fed with DMA every 48 h until the myocytes fused to form multinucleated myotubes that began to contract spontaneously. The contractions led to delamination of the monolayer, which detached from the Sylgard coat while remaining attached to the artificial tendons (Dennis and Kosnik, 2000). This detachment began peripherally and progressed radially inward until the entire sheet of myotubes rolled up between the tendons and lifted off the substrate (Dennis and Kosnik, 2000). The tension generated by the cells within the construct enabled the sheet to be suspended above the substrate between the tendons. At this point, the cells rapidly remodeled in response to the new mechanical environment, and all the cells eventually aligned themselves along the axis of the construct, now called a myooid (Dennis and Kosnik, 2000). In vitro stimulation of adult soleus muscle for contraction-induced glucoseuptake studies. The soleus muscles from the adult mice were activated by field stimulation between two platinum plate electrodes placed longitudinally on either side of the muscle. Square wave pulses of 0.2 ms in duration were produced by a stimulator (model S88, Grass Instruments, Quincy, MA) and amplified (model DC-300A Series II, Crown International Inc., Elkhart, IN). Stimulation voltage and subsequently muscle length (L o ) were adjusted to give maximum twitch force (Brooks and Faulkner, 1988). Optimum fiber length (L f ) was determined by multiplying the value for L o by a previously determined fiber length to muscle length ratio of 0.45 (Brooks and Faulkner, 1988). The use of protocols of repeated shortening contractions to assess maximum sustained power (Brooks and Faulkner, 1991) was used to assess contraction-stimulated glucose uptake. In brief, the soleus muscle was subjected to a 15-min contraction protocol during which shortening contractions were initiated at a low-intensity duty cycle of 0.01 that was increased to 0.02 after 5 min and to 0.04 after another 5 min, which accomplished the goals stated above. The duty cycle of contractions or the fraction of the work rest cycle during which the muscles produce work was increased by increasing the number of contractions per unit time. The average contraction duration of 56 ms combined with duty cycles of 0.01, 0.02, and 0.04 resulted in contractions occurring at a rate of approximately one each 5.6 s during the first 5 min, one each 2.8 s during the next 5 min, and one each 1.4 s during the last 5 min. During the contraction protocol, the muscles were exposed to a buffer containing radiolabeled 2-deoxyglucose for determination of contraction-induced glucose uptake. The protocol is explained in detail below. Determination of 2-deoxyglucose uptake in myooids and adult soleus muscle. A total of 21 myooids and 15 adult soleus muscles were analyzed for 2- deoxyglucose uptake. Of the myooids, 12 were examined in basal conditions and nine were examined after insulin stimulation. Of the adult muscle, five were examined in basal conditions, five were examined during contractions, and five were examined after insulin stimulation. The myooids were compared with five adult mouse soleus muscles in basal, insulin-stimulated, and contraction-stimulated conditions. Mouse soleus muscles were used for comparison in these in vitro glucose-uptake studies instead of rat soleus muscles because of the fact that unlike rat soleus muscle the mouse soleus muscle is approximately the size of a myooid in length and width, and it is hypothesized that the diffusion of glucose into the mouse muscle is similar to that of myooids. Using in vivo methods, previous investigators have observed a similar glucose uptake in mouse and rat soleus muscles, making the mouse soleus muscle an appropriate control for the myooids derived from rat soleus muscle (Gaudreanult et al., 2001; Fueger et al., 2003). Eight additional myooids were analyzed for GLUT-1 and GLUT-4 transporter protein content. The glucose-uptake assay media consisted of 2-deoxyglucose transport medium (2DG-TM), incubation medium (IM), and wash medium (WM), all of which were based on Krebs Henselit buffer (KHB). The 2DG-TM basal solution (BAS 2DG-TM) consisted of 1.5 ml KHB consisting of 1 mm 2-deoxy- [1,2-3 H] glucose (1.5 Ci/mmol), 39 mm [1-14 C] mannitol (8 Ci/mmol) (New England Nuclear, Boston, MA), and 13.3 nm insulin. 2-Deoxyglucose transport medium, l 3 H-2-DG, and 2.34 l 14 C-mannitol. The IM consisted of 2 ml of KHB consisting of 0.1% BSA, 40 mm mannitol, and 2 mm pyruvate. The WM consisted of 2 ml KHB consisting of 0.1% BSA, 32 mm mannitol, and 8 mm glucose. All media was warmed to 37 C, and each myooid was rinsed with IM. The IM was then applied to the myooids and incubated at 37 C for 10 min in a CO 2 incubator. During the incubation period, BAS 2DG-TM and insulin-stimulated 2DG-TM (INS 2DG-TM) solution was prepared. The INS 2DG-TM consisted of BAS 2DG-TM with an additional 0.54 g of insulin (at 27.8 units/mg). Next, a 20- l sample of both FIG Deoxyglucose uptake in myooids and adult mouse soleus muscles; n, no. of samples. Values are means standard error expressed in micromoles per gram. BAS 2DG-TM and INS 2DG-TM were taken for total counts and added to a scintillation vial. After incubation, one-half of the plates were filled with 2 ml of BAS 2DG-TM and the other half were filled with 2 ml INS 2DG-TM; they were then placed in a CO 2 incubator at 37 C for 20 min. At the end of the incubation period, the myooids were thoroughly rinsed in WM, removed from the sutures, blotted, weighed, freeze clamped, and stored at 80 C. Individual myooids were dissolved in a 1:10 (wt/vol) dilution of 1 M KOH solution, placed in boiling water for 2 min, placed on ice for 5 min, and then centrifuged for 10 s at 10,000 g. After this, samples were neutralized by adding 1 M HCl in a 1:10 (wt/vol) dilution of myooid wet weight, and 20 l of each sample was placed in a scintillation vial and counted on a scintillation counter. GLUT-1 and GLUT-4 Western blotting. Western analysis of GLUT-1 and GLUT-4 was conducted as described previously (Larkin et al., 1997). In brief, myooids and adult soleus (n 5) were homogenized in phosphate buffer in a 1:100 (wt/vol) dilution. Twenty microliters of homogenate was used to determine proteins by way of Bradford protein assay (BioRad, Richmond, CA). Western analysis was performed by using vertical slab polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and aliquots of muscle membranes (30 g protein/lane). An internal control sample of adult rat soleus muscle was run on all gels, and duplicate samples from each myooid were separated on a 10% polyacrylamide resolving gel and then eletrophoretically transferred onto Immobilon polyvinyldifluoride membrane (Millipore, Mildford, MA). These membranes were then blocked for 60 min with tris(hydroxymethyl)aminomethane-buffered saline with Tween-20 (100 mm tris(hydroxymethyl)aminomethane, 0.9% NaCl, 2.0 ml Tween-20, ph 7.5) and 5% Carnation nonfat dry milk. Immunoblotting was performed by using a rabbit polyclonal antibody against rat brain GLUT-1 or insulin-regulatable GLUT-4 protein (Chemicon, Temecula, CA), followed by fluorescent-labeled IgG, and the fluorescent signal was monitored, digitized, and analyzed using a Molecular Dynamics Storm Imager (Molecular Dynamics, Piscataway, NJ). Myooid signals were corrected for background, and signals were compared with the adult soleus muscle signal. A single band resulted for both GLUT- 1 and GLUT-4 proteins at a molecular mass of 45 kda. Statistics. Values are presented as means standard error. Statistical analysis was performed by using Statview 4.01 (Abacus Concepts, Berkeley, CA). A one-way analysis of variance was conducted to compare the differences between myooids and adult mouse soleus muscles. Differences were considered significant at P RESULTS Glucose uptake. There was a significant increase in the basal 2- deoxyglucose uptake of myooids compared with adult unstimulated (fivefold), contraction-stimulated (3.4-fold), and insulin-stimulated (threefold) soleus muscles (P , , and , respectively). In addition, there was a significant increase in the insulin-stimulated 2-deoxyglucose uptake of myooids compared with adult unstimulated control soleus muscles in basal conditions (6.5- fold) and adult contraction-stimulated (4.5-fold) and insulin-stimulated (3.9-fold) soleus muscles (P , , and , respectively). There was a significant 30% increase in insulin-stimulated 2-deoxyglucose uptake compared with basal 2-deoxyglucose uptake in the myooids (P ; Fig. 1).

4 GLUCOSE UPTAKE IN ENGINEERED MUSCLE 437 FIG. 2. Western blot of GLUT-1 and GLUT-4 protein content (single band at 45 kda). Each myooid lane represents protein isolated from an individual myooid. Each control lane represents a homogenate of five adult soleus muscles. FIG. 3. GLUT-1 and GLUT-4 glucose transporter protein content in myooids and adult mouse soleus muscles; n, no. of samples. Values are means standard error expressed as percentage of adult soleus muscle. GLUT-1 and GLUT-4 glucose transporters. Myooid GLUT-1 and GLUT-4 protein content showed a significant increase compared with the sedentary adult mouse soleus muscle (Fig. 2). The average myooid GLUT-1 content was 820% of the control, and the average GLUT-4 content was 130% of the control (Fig. 3). Myooid GLUT-1 protein content was 6.3-fold greater than GLUT-4 protein content. DISCUSSION To examine the glucose transporter content and glucose uptake in engineered muscle, we first produced myooids by harvesting and culturing satellite cells from the soleus muscles of female rats. To date, the metabolic function of engineered muscle has not been well described. The myooids were examined for glucose uptake by way of 2-deoxyglucose, and GLUT-1 and GLUT-4 transporter protein content was measured in basal and insulin-stimulated myooids. The basal 2-deoxyglucose uptake of myooids was significantly higher than that of unstimulated control (fivefold) and contraction-stimulated (2.9-fold) adult soleus muscles, and the insulin-stimulated 2- deoxyglucose uptake of myooids was significantly higher than that of unstimulated control (6.5-fold) and contraction-stimulated (3.8- fold) adult soleus muscles. However, there was a significant increase in insulin-stimulated 2-deoxyglucose uptake compared with basal 2-deoxyglucose uptake in the myooids. Myooid GLUT-1 protein content was 6.3-fold greater than GLUT-4 protein content, and the protein content of both glucose transporters was significantly greater than that observed in adult soleus muscle. These results suggest that the glucose transporter content and glucose uptake of the engineered myooids is similar in several respects to that observed in both fetal and denervated skeletal muscle tissue and is in these respects unlike adult skeletal muscle. Previous studies indicate that during fetal and early-postnatal life, the GLUT-1 transporter is a predominant isoform expressed in skeletal muscle, with significantly reduced GLUT-1 expression during postnatal life (Santalucia et al., 1992; Castello et al., 1994; Gaster et al., 2000a). GLUT-4 transporters have been observed in fetal tissue, although to a much lesser extent than GLUT-1 transporters (Santalucia et al., 1992; Castello et al., 1994; Gaster et al., 2000a). GLUT-4 transporters are sparse in fetal tissue, but after birth, the GLUT-4 transporter content increases and localizes to subcellular vesicles. The dependence of transporter expression on developmental stage suggested that there are differences in the metabolic conditions of developing and mature muscles (Gaster et al., 2000b). In accordance with these studies, this study suggests that the engineered myooids have metabolic characteristics similar to those of fetal skeletal muscle. These fetal-like metabolic characteristics are evidenced by the eightfold increase in myooid GLUT-1 transporter protein content compared with sedentary adult mouse soleus muscle and the 6.3-fold increase of myooid GLUT-1 compared with GLUT-4 transporter protein content. The GLUT-1 and GLUT-4 transporter protein content data also suggest that the engineered myooids exhibit characteristics of denervated skeletal muscle. Similar to fetal muscle, denervated muscle shows increased synthesis of GLUT-1 transporter protein and decreased GLUT-4 transporter content (Suarez et al., 2001). Several studies have demonstrated the characteristic trait of denervated skeletal muscle as upregulation of GLUT-1 protein and mrna content and downregulation of GLUT-4 protein and mrna content (Block et al., 1991; Coderre et al., 1992; Handberg et al., 1996). Basal 2-deoxyglucose uptake has been shown to increase after denervation, whereas insulin-stimulated 2-deoxyglucose uptake has been observed to decrease concomitantly as a result of the marked GLUT-4 protein downregulation (Suarez et al., 2001). In normal muscle, all GLUT-1 is located at the cell surface (Cushman and Wardzala, 1980), whereas in denervated muscle a significant portion of GLUT-1 protein is found in intracellular vesicles (Goodyear et al., 1991). On insulin stimulation, these vesicles translocate to the cell surface. Zhou et al. (2000) suggest that in denervated rat extensor digitorum longus (EDL) muscle the formation of insulin-sensitive GLUT-1 containing vesicles may compensate for the diminished action of GLUT-4 transporters. In accordance with previous studies of denervated muscle, myooids (which are cultured in the absence of motorneurons) exhibit higher basal glucose uptake compared with glucose uptake of the innervated adult rat soleus muscle. Note that, during culture, the myooids are not actively stimulated

5 438 BAKER ET AL. (mechanically or electrically), but they do exhibit submaximal spontaneous contractions intermittently for the duration of the culture period (Dennis and Kosnik, 2000). It should also be noted, however, that insulin-stimulated glucose uptake of the myooid significantly increased compared with that of the adult muscle control. Overall, our results suggest that the metabolic characteristics of myooids are similar to those of the denervated muscle. However, whether the shifts in glucose transporter content and glucose uptake are due to a lack of innervation, the high energy demand of regenerating muscle, or both, still needs to be investigated. Although our experiments have yielded insight into the metabolic properties of in vitro engineered muscle, additional studies of myooid glucose uptake and glucose transporter protein content are necessary to further characterize the metabolic capabilities as compared with human and rodent adult skeletal muscle. In particular, future studies may define more clearly the localization and activity of glucose transporters in the myooid. In addition, previous studies have shown that myooids can be stimulated to generate contractile force, work, and power (Dennis and Kosnik, 2000). To date, no detailed study of contraction-stimulated glucose metabolism has been carried out. Overall, the results suggest that myooid glucose transporter content and glucose uptake is comparable with that of both fetal and denervated skeletal muscle tissue, both of which are in a state of rapid growth or high energy demand. ACKNOWLEDGMENTS This research was supported by DARPA/Navy contract N C-8034 and the U.S. Department of Defense (Navy) Grant N G20G. We thank Reena Narula and Rukmini Vasupuram for their technical assistance in conducting experiments. REFERENCES Block, N. E.; Menick, D. R.; Buse, M. G. Effect of denervation on the expression of two glucose transporter isoforms in rat hindlimb muscle. J. Clin. 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