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Am J Physiol Endocrinol Metab 293: E1178-E1187, 2007. First published August 21, 2007; doi:10.1152/ajpendo.00628.2006
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Hepatic response to restoration of GLUT4 in skeletal muscle of GLUT4 null mice

Mollie Ranalletta,1 Xiu Quan Du,1 Yoshinori Seki,1 Alan S. Glenn,1 Michael Kruse,1 Ariana Fiallo,1 Irma Estrada,1 Tsu-Shuen Tsao,1 Antine E. Stenbit,1 Ellen B. Katz,1 and Maureen J. Charron1,2

Departments of 1Biochemistry and 2Obstetrics and Gynecology and Women's Health, Albert Einstein College of Medicine, Bronx, New York

Submitted 20 November 2006 ; accepted in final form 3 August 2007


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Expression of GLUT4 in fast-twitch skeletal muscle fibers of GLUT4 null mice (G4-MO) normalized glucose uptake in muscle and restored peripheral insulin sensitivity. GLUT4 null mice exhibit altered carbohydrate and lipid metabolism in liver and skeletal muscle. To test the hypothesis that increased glucose utilization by G4-MO muscle would normalize the changes seen in the GLUT4 null liver, serum metabolites and hepatic metabolism were compared in control, GLUT4 null, and G4-MO mice. The fed serum glucose and triglyceride levels of G4-MO mice were similar to those of control mice. In addition, the alternations in liver metabolism seen in GLUT4 nulls including increased GLUT2 expression and fatty acid synthesis accompanied by an increase in the oxidative arm of the pentose phosphate pathway were absent in G4-MO mice. The transgene used for GLUT4 restoration in muscle was specific for fast-twitch muscle fibers. The mitochondria hypertrophy/hyperplasia in all GLUT4 null skeletal muscles was absent in transgene-positive extensor digitorum longus muscle but present in transgene-negative soleus muscle of G4-MO mice. Results of this study suggest that the level of muscle GLUT4 expression influences mitochondrial biogenesis. These studies also demonstrate that the type and amount of substrate that muscle takes up and metabolizes, determined in part by GLUT4 expression levels, play a major role in directing hepatic carbohydrate and lipid metabolism.

hepatic fatty acid synthesis; muscle mitochondria; substrate utilization

THE MAJORITY OF postprandial glucose is cleared by muscle. Glucose transport through the insulin-responsive glucose transporter, GLUT4, along with phosphorylation by hexokinase constitutes the rate-limiting step in muscle glucose clearance (12). Overexpression of GLUT4 in muscle improves whole body insulin sensitivity and glucose disposal in normal and streptozocin (STZ)-induced diabetic mice (20). The overexpression of GLUT4 specifically in fast-twitch skeletal muscles of mice (MLC-GLUT4) altered muscle substrate utilization by increasing glucose influx and glycolysis (males) or glycogen synthesis (females) and decreasing exogenous free fatty acid (FFA) oxidation (34, 35). MLC-GLUT4 mice are more active, have increased food consumption, and weigh less than controls (35). These results provide insight into the potential therapeutic application of skeletal muscle GLUT4 overexpression in treating complications of peripheral insulin resistance in diabetes and obesity.

The contribution of skeletal muscle GLUT4 to insulin sensitivity and whole body glucose clearance was examined in mice expressing GLUT4 only in fast-twitch muscle on the GLUT4 null background (G4-MO) (36). Interestingly, glucose uptake in extensor digitorum longus (EDL) was completely restored, and peripheral insulin sensitivity was normalized. However, these alterations in skeletal muscle GLUT4 content did not reverse the phenotype of the adipose tissue. G4-MO mice have a reduced adipose tissue phenotype, similar to that of GLUT4 nulls (36).

Alterations in substrate utilization by the GLUT4 null muscle and liver have also been characterized (27). GLUT4 null muscle compensates for decreased glucose uptake by oxidizing more fatty acids to meet its energy demands. This increased oxidation is facilitated by the mitochondria hypertrophy and hyperplasia present in the GLUT4 null muscle (27). GLUT4 null liver supplies the muscle with the needed fatty acids with an increase in fatty acid synthesis and triglyceride (TG) secretion. This process is facilitated by an upregulation of glucose transporter-2 (GLUT2) and the pentose phosphate pathway (PPP) in liver (27). Similar compensatory mechanisms were reported in double knockout mice lacking GLUT4 in their adipocytes and muscle only (AMG4KO) (18). These mice also had increased lipid synthesis in the liver and increased peripheral utilization of these lipids as indicated by the lower respiratory quotient and the greater clearance of lipids from serum after an oil loading test (18).

The present study was conducted to examine the effect of restoration of GLUT4 expression in fast-twitch muscle on the altered hepatic metabolism observed in GLUT4 null mice. Comparison of liver function and muscle substrate utilization in control, GLUT4 null, and G4-MO mice provides insight into how muscles differing in GLUT4 content and consequently in the type of substrate utilized for energy production play a role in the regulation of liver metabolism.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals. Using homologous recombination and embryonic stem cell technology, Katz et al. (16) generated global GLUT4 knockout mice (GLUT4 null). Mice with transgenic overexpression of GLUT4 in fast-twitch skeletal muscle (MLC-GLUT4) were generated by placing the GLUT4 gene under the myosin light chain promoter (MLC) as previously described by Tsao et al. (34). MLC-GLUT4 mice were mated with the GLUT4 null mice to generate GLUT4 null mice with muscle-specific overexpression of GLUT4 (G4-MO) (36). General characteristics of G4-MO mice were previously described by Tsao et al. (36). Mice that had formerly been bred onto a C57Bl/6J/CBA background were bred onto a C57BL/6J background (10 generations). Male and female 3- to 4-mo-old mice were used in this study. Animals were fed ad libitum and maintained at a constant temperature (22°C) on a 12:12-h light-dark cycle. Mice were killed between 9:00 and 10:00 AM in the postabsorptive state unless otherwise noted. The Animal Care and Use Committee of Albert Einstein College of Medicine, in accordance with the Public Health Service Animal Welfare Policy, approved all protocols.

Determination of serum hormones, metabolites, and adipokines. Blood from ad libitum-fed mice was collected at ~12:00 AM. Whole blood was drawn from the orbital sinus and centrifuged. Serum was frozen on dry ice and kept at –70°C until further use. Blood glucose was determined using a glucometer and strips (Precision QID, kind gifts from Andrew Adler, Abbott Laboratories, Chicago, IL.). Serum insulin concentration was determined using a commercially available radioimmunoassay (Linco Research, St. Charles, MO). Serum TG and lactate were determined using colorimetric kits from ThermoDMA (Louisville, KY) and Sigma Diagnostics (St. Louis, MO), respectively. Adiponectin and resistin were measured with commercially available RIAs (Linco Research).

Northern blot analysis. Total RNA was isolated using TRIzol (Molecular Research Center, Cincinnati, OH) according to the manufacturer's instructions. Approximately 25 µg of total RNA were loaded onto a 1.2% formaldehyde-agarose gel, transferred to a Hybond-N nylon membrane (Amersham, Piscataway, NJ), and hybridized overnight to a random primed 32P-labeled probe under high-stringency conditions (50% formamide, 42°C). The filter was washed at 42°C in 2x SSC, 1% SDS, and 0.2x SSC, 0.1% SDS, solutions and subjected to phosphoimage analysis for visualization and quantification. cDNA for the fatty acid synthase (FAS) probe was kindly provided by Pascal Ferre (INSERM, Paris, France).

Quantitative RT-PCR analysis of candidate gene expression. Total RNA was prepared using TRIzol reagent and reverse-transcribed with SuperScript III RT (Invitrogen, Carlsbad, CA), and cDNA was amplified using SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA). Primer pairs for the genes studied were designed using Primer Express software (Applied Biosystems), and their sequences are shown in Table 1. Normalization across samples will be performed using the average of the constitutive gene ubiquitin or cyclophilin b. Relative expression is determined using the comparative threshold cycle (CT) method.


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  		Table 1. Primers and annealing temperatures for real-time RT-PCR

 
Immunoblot analysis. Livers were homogenized in a buffer containing 50 mM HEPES, 5 mM EDTA, 15 mM Na-pyro-phosphate, 25 mM NaF, 100 mM beta-glycerol-phosphate, 2 mM Na3VO4, 10% Triton X-100, and Complete Protease Inhibitor Cocktail Tablets (Roche, Mannheim, Germany). Homogenates (40 µg of protein) were separated by 10% SDS-PAGE and transferred to Hybond enhanced chemiluminescence (ECL) nitrocellulose (Amersham Pharmacia Biotech, Piscataway, NJ). Rabbit polyclonal GLUT2 antibodies (1:2,000) generated to a GLUT2-specific carboxy-terminus peptide were used to detect GLUT2 protein in total liver homogenates. Blots were normalized by incubation with mouse monoclonal {alpha}-tubulin antibodies (1:4,000) (Sigma, St. Louis, MO). ECL (Amersham Pharmacia Biotech) detection was used in combination with scanning laser densitometry for quantitation. Gastrocnemius muscles were homogenized in a buffer containing 50 mM HEPES, 5 mM EDTA, 15 mM Na-pyro-phosphate, 25 mM NaF, 100 mM beta-glycerol-phosphate, 2 mM Na3VO4, 10% Triton X-100, and Complete Protease Inhibitor Cocktail Tablets. Homogenates (100 µg protein) were boiled for 5 min and then separated by 7% SDS-PAGE and transferred to Hybond ECL nitrocellulose. Rabbit polyclonal phosphorylated AMP-activated protein kinase (phospho-AMPK) antibodies (1:1,000) (Cell Signaling, Danvers, MA) were used to detect endogenous phospho-AMPK protein in total muscle homogenates. Blots were normalized by incubation with rabbit polyclonal antibodies that detect total AMPK (1:1,000) (Cell Signaling). Signals were visualized and quantified using the infrared Odyssey Western Blotting System (Li-Cor, Lincoln, NE).

PPP. Activity of the PPP in male and female mouse livers was determined as previously described (14). NADPH release was measured spectrophotometrically to assess PPP activity (6). Data are reported as micromoles of NADPH produced per minute per milligram of protein.

Fatty acid synthesis rates. Fatty acid synthesis rates were determined as previously described (20) according to the method described by Lin et al. (21). Three- to four-month-old female GLUT4 null and control mice were tail vein injected with 30 µCi of [14C]acetate (Amersham Cat. No. CFA13) in the fed state. After 1 h, mice were killed and liver was harvested. Samples were immediately frozen in liquid nitrogen and stored at –70°C until analysis; 250 mg of liver were digested in 500 µl of 30% KOH solution for 30 min at 90–95°C; and 500 µl of 100% ethanol were added, and samples were heated for an additional 3 h at 90–95°C. Cholesterol was extracted with petroleum ether. Once all the cholesterol was removed, the samples were acidified with 18 N H2SO4 and washed several times with petroleum ether to extract all the TG. Each wash was collected and counted for 14C content. Data are presented as disintegrations per minute per hour (time after injection of [14C]acetate) per milligram of tissue.

Transmission electron microscopy. Soleus and EDL muscles were isolated and fixed in 2.5% gluteraldehyde in 0.1 M cacodylate buffer, postfixed with 1% osmium tetroxide followed by 1% uranyl acetate, dehydrated through a graded series of ethanol, and embedded in LX112 resin (LADD Research Industries, Burlington, VT). Ultrathin sections were cut on a Reichaert Ultracut E microtome, stained with uranyl acetate followed by lead citrate, and viewed on a JEOL 1200EX transmission electron microscope at 80 kV. Representative images were acquired at x10,000 magnification.

Oleate oxidation in EDL and soleus. Oleate oxidation rates were determined as previously described by Tsao et al. (35). Briefly, individual EDL and soleus muscles from 10- to 14-wk-old female mice were isolated and preincubated for 15 min in Krebs-Ringer buffer supplemented with 4% bovine serum albumin (fraction V, RIA grade from Sigma), 0.5 mM oleate, and 5 mM glucose. Muscles were then transferred to new vials containing the same buffer plus 0.8 µCi/ml [1-14C]oleic acid (60 mCi/mmol, Amersham) and incubated for 45 min. Before the addition of perchloric acid, 200 µl of solvable were injected onto the filter paper. Samples were acidified with 15% perchloric acid, and the CO2 was captured on Whatman paper soaked in 200 µl of Solvable suspended within the vials. 14C radioactivity was quantitated after overnight quenching at 4°C by liquid scintillation counting. Data are reported as nanomoles of oleate per gram wet muscle weight per 45 minutes.

Statistical analysis. Statistical comparison of control, GLUT4 null, and G4-MO mice within each gender was done using ANOVA and Bonferroni post hoc analysis. Values are means ± SE for each group. Significance was accepted at P < 0.05.


   RESULTS
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 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Serum metabolites of GLUT4 null mice with restoration of GLUT4 in skeletal muscle. As previously reported (16), GLUT4 null mice maintain euglycemia. The elevation in fed glucose often seen in GLUT4 null males was normalized to control levels in G4-MO mice (Table 2). Both GLUT4 null and G4-MO females had slightly higher glucose levels compared with control mice. Overall, fed insulin levels were low in all mice. The increased insulin levels in the GLUT4 null female mice (1.9-fold, P < 0.05) were normalized in the G4-MO. The decreased lactate levels seen in the GLUT4 null mice were restored to control levels in the G4-MO mice (Table 2). Serum TG concentrations were not significantly different among the genotypes in both males and females. These data show that reexpression of GLUT4 in muscle of GLUT4 null mice results in nearly normal fed glucose, insulin, and lactate levels in the G4-MO mouse.


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  		Table 2. Serum metabolites

 
Serum adipokines. The adipose depots of GLUT4 null and G4-MO mice are smaller than for controls (24). The adipocyte-secreted factors, adiponectin and resistin, were measured in the serum to assess the endocrine function of the fat pad. The serum of male GLUT4 null and G4-MO mice had similar significantly reduced levels of adiponectin compared with controls (Table 3). However, surprisingly, the serum of female GLUT4 null and G4-MO mice had the same levels of adiponectin as did controls. The resistin levels in both males and females were the same among the genotypes. These results show that, in the male GLUT4 null and G4-MO mice, the endocrine function of the reduced adipose tissue is similar to that of each other and different from that of control. However, in the female GLUT4 null and G4-MO mice, the reduced mass of the adipose tissue does not result in a decrease in circulating adiponectin and resistin levels.


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  		Table 3. Serum adipokines

 
Liver metabolism in G4-MO mice. As previously reported (27), GLUT4 null mice utilize glucose for hepatic fatty acid synthesis. GLUT2 protein expression levels were restored to control values in G4-MO mice (Fig. 1A). FAS mRNA expression was partially normalized to control values in female G4-MO and completely normalized in male G4-MO liver (Fig. 1B). Rates of hepatic fatty acid synthesis were reduced in both male (1.4-fold, P < 0.07) and female (2.4-fold, P < 0.01) G4-MO compared with GLUT4 null mice (Fig. 2). Generation of NADPH by the PPP is used to support fatty acid synthesis. The 1.7-fold (P < 0.01) increase in the oxidative phase of the PPP of GLUT4 null compared with control mice was absent in both male and female G4-MO mice (Fig. 3, A and B). Collectively, these data suggest that, when GLUT4 was restored in fast-twitch skeletal muscle fibers, the G4-MO mice did not have increased glucose uptake through GLUT2 or the increased fatty acid synthesis seen in GLUT4 null liver.


Figure 1
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  		Fig. 1. GLUT2 protein (A) and fatty acid synthase (FAS) mRNA expression (B) in the liver. A: liver homogenates from fed control mice, GLUT4 null mice, and mice expressing GLUT4 only in fast-twitch muscle on the GLUT4 null background (G4-MO) were analyzed by immunoblot for GLUT2 protein expression (n = 3–5). B: Northern blot of FAS mRNA expression in fed liver of control, GLUT4 null, and G4-MO mice was quantified by phosphoimage analysis (n = 5). Statistical analysis was performed using ANOVA. Values for both analyses are represented as means ± SE. Bars lacking a common letter differ, P < 0.05.

 

Figure 2
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  		Fig. 2. Liver fatty acid synthesis rates in female and male mice. Values are means ± SE for n = 4–10 per group. Data are reported as the incorporation rate of [14C]acetate into fatty acids per hour per milligram of liver. Statistical analysis was performed using ANOVA. Values are means ± SE. Bars lacking a common letter differ, P < 0.05.

 

Figure 3
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  		Fig. 3. Liver pentose phosphate pathway activity in male (A) and female mice (B). Liver homogenates from fed mice were analyzed for whole pathway, oxidative, and nonoxidative pentose phosphate pathway activity. Statistical analysis was performed using ANOVA. Values are means ± SE for n = 5–6 mice per group. Bars lacking a common letter differ, P < 0.05.

 
Expression of genes of hepatic lipid and glucose metabolism was measured by quantitative RT-PCR (qRT-PCR) (Table 4). The expression of peroxisome proliferator-activated receptor-{gamma}2 (PPAR{gamma}2), a transcription factor that controls expression of genes of glucose metabolism and lipogenesis (26, 38), and its coactivator, PGC1{alpha}, are increased significantly in the GLUT4 null compared with G4-MO and control mice. This result is in agreement with the increases in rates of the fatty acid synthesis and PPP pathway discussed above. In addition, stearoyl-CoA desaturase [SCD-1; a target of PPAR{gamma}2 (38)] and transketolase [part of the PPP (39)] gene expression is significantly increased in GLUT4 null compared with control mice. However, the G4-MO mouse shows an intermediate level of expression. These results support the conclusion that the GLUT4 null liver is more lipogenic than the G4-MO and control livers.


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  		Table 4. Quantitative RT-PCR of liver tissue

 
Muscle-specific loss of mitochondria hypertrophy/hyperplasia in G4-MO mice. GLUT4 nulls exhibit increased oleate oxidation and mitochondria hypertrophy/hyperplasia in both soleus and EDL muscles (27). The transgene (MLC-GLUT4) was used to restore GLUT4 expression to GLUT4 null skeletal muscle (34). As expected, GLUT4 expression in a mixed fiber muscle such as gastrocnemius is significantly elevated G4-MO mice (Table 5). MLC1 is expressed in fast-twitch fiber types making up most of the skeletal muscle mass in mice (34). EDL is a fast-twitch muscle. Expression of MLC1 in slow-twitch muscles such as soleus is negligible (34). Restoration of GLUT4 expression in EDL muscle of G4-MO mice abolished the mitochondria hypertrophy/hyperplasia previously described in GLUT4 null EDL (Fig. 4). Consistent with this observation, PGC1{alpha} and mitochondrial transcription factor A are significantly elevated in GLUT4 null compared with control mice and expressed at either intermediate or lower levels, respectively, in G4-MO gastrocnemius (Table 5). G4-MO soleus muscle, however, maintained the mitochondria hypertrophy/hyperplasia characteristic of GLUT4 null muscle (Fig. 4). Transgenic overexpression of GLUT4 in fast-twitch muscle (MLC-GLUT4 mice) of wild-type mice did not alter mitochondrial morphology (Fig. 4). These data suggest that restoration of GLUT4, and thus glucose transport, in fast-twitch EDL muscle of GLUT4 null mice was sufficient to eliminate the mitochondria hypertrophy/hyperplasia in that muscle.


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  		Table 5. Quantitative RT-PCR of gastrocnemius muscle tissue

 

Figure 4
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  		Fig. 4. Transmission electron micrographs of mitochondria from soleus and extensor digitorum longus (EDL) muscle of male mice. Representative micrographs are shown at an original magnification of x10,000. Mice were 18 wk old. MLC-GLUT4, overexpression of GLUT4 specifically in fast-twitch skeletal muscles of mice; MLC, myosin light chain promoter. *Mitochondria.

 
Muscle-specific normalization of oleate oxidation in fast-twitch muscle. Since previous and present studies show that glucose uptake and mitochondrial morphology and number are normalized in G4-MO EDL, studies were conducted to determine whether the oleate oxidation levels in G4-MO EDL would be returned to normal as well. G4-MO EDL showed oleate oxidation levels similar to control EDL (Fig. 5A.). These results demonstrate that restoration of GLUT4-mediated glucose uptake in G4-MO fast-twitch muscle returns lipid utilization levels to near normal in the G4-MO. Reflecting the mitochondria hypertrophy/hyperplasia discussed above, oleate oxidation rates were significantly increased in GLUT4 null and G4-MO soleus compared with controls (Fig. 5B). The lack of GLUT4 expression and continued mitochondrial hypertrophy/hyperplasia contribute to the high rate of oleate oxidation seen in the G4-MO soleus. Consistent with these results is the significantly increased expression of genes involved in fatty acid uptake and oxidation, lipoprotein lipase (LPL) and PGC1{alpha}, seen in the gastrocnemius of the GLUT4 nulls compared with G4-MO and controls (Table 5). Additionally, the phosphorylated form of AMPK, a kinase that regulates fatty acid oxidation (33), is increased in GLUT4 nulls compared with controls (P = 0.07) and G4-MO (P ≤ 0.05) (Fig. 6).


Figure 5
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  		Fig. 5. Oleate oxidation rates in isolated EDL (A) and soleus muscles (B). Muscles were isolated from female control, GLUT4 null, and G4-MO mice, and oxidation was measured as described in the METHODS. Values are means ± SE for n = 4 per group. Bars lacking a common letter differ, P < 0.005.

 

Figure 6
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  		Fig. 6. Phosphorylated AMP-activated protein kinase (phospho-AMPK) expression in gastrocnemius muscle. Gastrocnemius homogenates from fed control, GLUT4 null, and G4-MO mice were analyzed by immunoblot for total and phospho-AMPK protein expression (n = 5) using the Li-Cor Odyssey System. Statistical analysis was performed using ANOVA. Values shown are means ± SE. Bars lacking a common letter differ, P < 0.005; GLUT4 null vs. control, P < 0.07.

 
Expression of muscle-secreted factors. Since fed glucose and insulin levels that in part direct hepatic gene expression (10) are similar in GLUT4 null and G4-MO mice, the possibility exists that skeletal muscle may secrete factors that affect liver metabolism. To address this question, expression of the muscle-secreted factors IL-6 (29), TNF{alpha} (25), and musclin (1, 23) was quantified by qRT-PCR in gastrocnemius muscle (Table 5). There was no significant difference in TNF{alpha} expression among genotypes. IL-6 expression was significantly elevated in the GLUT4 nulls compared with controls (2.5-fold, P < 0.01). G4-MO muscle IL-6 expression levels were between those of GLUT4 nulls and controls. Interestingly, musclin expression levels were significantly increased, threefold, in GLUT4 nulls compared with controls and G4-MO.


   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The goal of this study was to test the hypothesis that restoration of GLUT4 in GLUT4 null muscle would normalize the metabolic changes seen in the GLUT4 null liver. GLUT4 null mice have alterations in both carbohydrate and lipid metabolism (16, 27, 36). As previously reported, GLUT4 null mice have greatly reduced adiposity and altered serum lipid profiles (16). Restoration of GLUT4 in skeletal muscle of GLUT4 null mice restores whole body glucose utilization but does not normalize adipose mass (36). Serum TG concentrations are reduced in GLUT4 null mice in large part because of increased skeletal muscle LPL activity and increased beta-oxidation (27). Here we report normalization of serum TG concentrations when GLUT4 expression is restored in fast-twitch skeletal muscle of GLUT4 null mice. The GLUT4-compensated muscles of the G4-MO no longer need to use mostly fatty acids to meet energy needs. These data further support the idea that the reduction in serum TG concentration in GLUT4 null mice results from increased lipid uptake and oxidation in skeletal muscle (16, 27).

The mitochondria hypertrophy/hyperplasia present in GLUT4 null skeletal muscle is believed to be part of the compensatory mechanism for maintaining euglycemia (27). The increased oxidation seen in the GLUT4 null muscle utilizes the fatty acids made in the liver from glucose not taken up by the GLUT4 null muscle. Restoration of GLUT4 in skeletal muscle of GLUT4 null mice was specific to muscles containing fast-twitch muscle fibers such as EDL muscle (36). G4-MO mice have normal glucose uptake into transgene-expressing muscle (EDL) but not in transgene-negative muscle (soleus) (36). In agreement with the transgene expression and glucose uptake data, mitochondria hypertrophy/hyperplasia was absent in EDL and present in soleus muscle of G4-MO mice. In addition, the oleate oxidation rate was reduced to control levels in G4-MO EDL but not in the G4-MO soleus.

The mitochondria hypertrophy/hyperplasia is seen in the GLUT4 EDL but not in the G4-MO EDL. Changes in mitochondria number and morphology have been seen in other transgenic mouse models that disrupt the normal energy metabolism of the muscle. When creatine kinase, an enzyme that catalyzes the phosphocreatine-to-creatine + ATP reaction, is knocked out in both the mitochondria (ScCKmit) and the cytosolic isoform (M-CK), muscle loses contractile force under strenuous ex vivo exercise conditions (31). In both models, mitochondria volume is expanded in the muscle (31). In addition, in the M-CK–/– muscle, a large number of mitochondria contained glycogen, lipofuscin, and other lysosomal structures (31). In the heart type fatty acid-binding protein knockout mouse (H–/–), fatty acid uptake and oxidation are significantly reduced in soleus muscle, whereas glucose uptake is almost twofold elevated (2, 12). The mitochondria structure does not change in the H–/– muscle, but the number of mitochondria concentrated at the sarcolemma increased while the concentration of mitochondria between fibers did not change. The fatty acid uptake and utilization in skeletal muscle of the fatty acid transporter CD36 knockout mouse are significantly decreased (3). Although mitochondria hyperplasia/hypertrophy was not examined in these mice, several proteins in the respiratory chain were examined, and it was concluded that the fatty acid oxidation capacity of the muscle was not diminished (3).

The mitochondria hypertrophy/hyperplasia seen in the GLUT4 EDL but not in the G4-MO EDL is characteristic of endurance-trained athletes (4, 14, 9, 15). During endurance training, both fatty acid oxidation and GLUT4 levels are increased in muscle (11, 13, 28). When PGC1{alpha}, a regulator of nuclear respiratory factors 1 and 2, involved in mitochondrial biogenesis (24, 25), was deleted from the mouse, mitochondrial number and respiratory capacity were diminished in slow-twitch muscle (19). When PGC1{alpha} was overexpressed in murine muscle, as expected, mitochondrial hyperplasia was accompanied by an increase in fatty acid oxidation (22). Indeed, PGC1{alpha} expression is significantly higher GLUT4 null gastrocnemius muscle than in control, while the G4-MO muscle has an intermediate expression level. Additionally, when PGC1{alpha} was overexpressed in murine muscle, GLUT4 mRNA expression was decreased, resulting in insulin resistance. Conversely, the overexpression of GLUT4 in skeletal muscle (MLC-GLUT4) of wild-type mice increased physical activity, food consumption, and insulin sensitivity but decreased oleate oxidation rates in EDL muscle (35). Furthermore, as seen in the present study, MLC-GLUT4 transgenic mice also lack the mitochondria hypertrophy/hyperplasia associated with increased lipid oxidation in the GLUT4 null and PGC1{alpha} overexpressors.

These data, and the studies discussed above, demonstrate the impact of the type and amount of substrate available intracellularly for oxidation on mitochondrial biogenesis in skeletal muscle. The GLUT4 null mice with dramatically decreased skeletal muscle glucose uptake but increased fatty acid uptake and utilization exhibit mitochondria hypertrophy/hyperplasia. This increased reliance on fatty acids for energy was also seen in the AMG4KO mice, with a 98% reduction of GLUT4 in skeletal muscle (18). Mitochondria hypertrophy/hyperplasia was not reported. In contrast, the MLC-GLUT4 mice, with overexpression of GLUT4 in muscle, showed increased glucose uptake and utilization with decreased oleate oxidation (34) and no mitochondria hypertrophy/hyperplasia. Likewise, the overexpression of LPL in the muscle to increase fatty acid uptake, utilization, and storage did not result in mitochondria hypertrophy/hyperplasia (17, 37). In one study, LPL overexpression resulted in TG accumulation and decreased glucose uptake in muscle under clamp conditions (17), whereas in the other study, no insulin resistance was found (37). The increased LPL gene expression in the GLUT4 null gastrocnemius returns to normal levels in the G4-MO gastrocnemius. Interestingly, the mitochondrial hypertrophy/hyperplasia seen in GLUT4 null soleus seems to be slightly diminished in the G4-MO soleus, even though fatty acid oxidation levels remain as high as in GLUT4 nulls. This decreased level of mitochondrial compensation could be the result of increased serum lactate levels available for oxidation in the G4-MO. The results of the present study and the LPL studies show that increased myocyte intracellular glucose (MLC-GLUT4) or lipid (LPL overexpressor) alone did not result in increased mitochondrial biogenesis. However, making the muscle metabolically inflexible by completely deleting GLUT4 to severely restrict the amount of intracellular glucose, necessitating a reliance on fatty acid oxidation for energy, initiated muscle mitochondria hypertrophy/hyperplasia.

GLUT4 null livers utilize glucose that is not able to be taken up by GLUT4 null muscle to fuel fatty acid synthesis (27). The present studies were performed to determine whether nearly normal glucose uptake in skeletal muscle, and subsequent decreased reliance on lipids for energy, would alter fatty acid metabolism in liver. Indeed, we observed normalization of GLUT2 protein and FAS mRNA expression in G4-MO mice. Hepatic fatty acid synthesis rates were completely normalized to control rates in female G4-MO mice and were near normal in males. More striking, however, was the complete reversal of the 1.7-fold increase in the oxidative phase of the PPP in G4-MO mice. To support these findings, RT-PCR studies showed significantly increased expression levels of PPAR{gamma}2 and its coactivator PGC1{alpha} in GLUT4 null livers that returned to control levels in the G4-MO. PPAR{gamma}2 is a transcription factor that controls expression of genes of glucose metabolism and lipogenesis (26, 38). The expression of two lipogenic genes activated by PPAR{gamma}2, SCD-1 and transketolase, is also significantly increased in the GLUT4 null liver compared with control. The expression levels of these two genes are only slightly elevated in the G4-MO compared with controls. Collectively, these data demonstrate that restoration of GLUT4 only in fast-twitch skeletal muscle fibers of GLUT4 null mice is sufficient to reduce to near normal the rate at which the liver utilizes glucose for fatty acid synthesis. These results also demonstrate that the liver of the GLUT4 null mouse is metabolically flexible and able to respond to peripheral signals to synthesize appropriate substrates for energy production in the GLUT4 null muscle with impaired glucose uptake.

Since fed glucose and insulin levels that in part direct hepatic gene expression (10) are similar in GLUT4 null and G4-MO mice, the possibility exists that skeletal muscle may secrete factors that also affect liver metabolism. Another mouse model supporting this concept is the muscle-specific insulin receptor knockout mouse (MIRKO) (5, 7). These mice have increased adiposity, due to increased adipocyte hyperplasia, and elevated serum lipids (TG and FFA) but normal blood glucose, serum insulin, and glucose tolerance. Like GLUT4 null mice, MIRKO mice have normal hepatic insulin sensitivity and normal hepatic glycogen and TG content. The anti-diabetic phenotype in MIRKO mice results from increased white adipose tissue (WAT) glucose uptake associated with increased adipose hyperplasia. Like GLUT4 nulls, altered skeletal muscle insulin sensitivity of MIRKO mice provoked a compensatory response in peripheral tissues (WAT and perhaps liver) to compensate for the defect in muscle. These results suggest that altering skeletal muscle metabolism to utilize predominately one fuel can affect substrate metabolism in other tissues. To address this issue, expression levels of several myokines were measured in the gastrocnemius muscle. Indeed, while the expression levels of TNF{alpha}, a proinflammatory myokine (25), are the same among the genotypes, the expression levels of muscle-secreted factors IL-6 and musclin are significantly increased in GLUT4 nulls compared with controls. Muscle IL-6 expression increases with exercise and promotes fatty acid oxidation, while an increase in musclin secretion is indicative of muscle insulin resistance (23, 25). These results are consistent with the insulin resistance and increased fatty acid oxidation seen in the GLUT4 null muscle (16, 27). As expected, the musclin expression levels returned to control levels in the non-insulin-resistant G4-MO muscle. However, the IL-6 expression level was intermediate in G4-MO. Further studies are needed to determine whether these secreted factors contribute to the ability of the GLUT4 null mouse to avoid diabetes.

In summary, the ability of GLUT4 null mice to maintain euglycemia requires the muscle and liver to make and metabolize fat at levels not seen in normal mice (27). Expression of GLUT4 in skeletal muscle of GLUT4 null mice restores whole body insulin sensitivity (36). GLUT4 null mice rely on hepatic fatty acid synthesis to provide lipids for oxidative fuel for skeletal muscle. This study clearly demonstrates that GLUT4 restoration in fast-twitch skeletal muscle only is able to diminish the compensatory increase in hepatic lipid synthesis seen in GLUT4 null liver. Likewise, no mitochondria hypertrophy/hyperplasia is evident when glucose flux and fatty acid oxidation are restored to normal by the restoration of GLUT4 expression in the EDL. Restoration of GLUT4 in skeletal muscle in G4-MO mice abolishes the need for the compensatory mechanisms for maintenance of euglycemia observed in GLUT4 null liver (20). Whether skeletal muscle is communicating with liver directly (secreted factors) or indirectly (decreased clearance of serum TG, increased lactate) remains to be determined.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by grants to M. J. Charron from the National Institutes of Health (NIH; nos. DK-47425, HL-58119, and HL-073163), the American Diabetes Association, the Comprehensive Cancer Center of Albert Einstein College of Medicine, and the Diabetes Research and Training Center. M. Ranalletta was supported by NIH Grant 5T32-DK-07513.


   ACKNOWLEDGMENTS
 
We thank Dr. Patricia Vuguin for critical reading of the manuscript. We also thank Andrew Adler of Abbott Laboratories for the glucose strips and glucometer.

This work was submitted in partial fulfillment of the PhD degree requirements for the Albert Einstein College of Medicine (M. Ranalletta).

Present addresses: M. Ranalletta, Merck and Co., Inc., Whitehouse Sta., NJ 08889; T.-S. Tsao, Dept. of Biochemistry and Molecular Biophysics, Univ. of Arizona, Tucson, AZ 83521; and A. E. Stenbit, Dept. of Medicine, Medical University of South Carolina, Charleston, SC 29125.


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. J. Charron, Dept. of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461 (e-mail: charron{at}aecom.yu.edu)

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.


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