Altered Hepatic and Muscle Substrate Utilization Provoked by GLUT4 Ablation Free

Female 3- to 4-month-old GLUT4 null mice and sex- and age-matched controls maintained on a C57BL/6J × CBA background were used in this study (8). Animals were fed ad libitum and maintained at a constant temperature (22°C) on a 12-h light and dark cycle. 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.

Blood from fasted animals was collected after an overnight fast (12 h), and blood from ad libitum–fed mice was collected at ∼12:00 a.m. 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 glucose meter (Precision Q.I.D., a gift from Abbott Laboratories, Chicago, IL). Serum insulin concentrations were determined using commercially available radioimmunoassays (Linco Research, St. Charles, MO). Serum NEFA levels were measured using a commercial kit from Wako (Wako Chemicals, Neuss, Germany). Serum TG and glycerol concentrations were analyzed using a colorimetric kit (Sigma, St. Louis, MO.).

Mice were fasted for 4 h and then received an intravenous injection of Triton WR1339 (20 mg/200 μl saline). Blood samples were collected before the injections and then every hour for the next 3 h. Serum TG concentration was determined using a colorimetric kit (Sigma). Hepatic TG production was calculated from the 1-h time point and was expressed as micromoles per kilogram per hour.

Mice were fasted for 5 h, and then olive oil was delivered by a gavage needle at 16.7 μl/g body wt. Blood samples were obtained immediately before and at 30, 60, 120, 180, and 300 min after oil loading. Serum TG levels were analyzed spectrophotometrically using a kit from Sigma.

Absorption studies were performed according to the method reported by Goudriaan et al. (16). The 12- to 14-week-old female control and GLUT4 null mice were fasted overnight for 8–10 h.

Animals were sacrificed by cervical dislocation. Livers from GLUT4 null and control mice were freeze-clamped in liquid nitrogen. Small pieces of liver were homogenized in cold 0.3N perchloric acid and centrifuged at 4°C for 10 min at 5,000g to remove the fat cake. Cleared homogenate (600 μl) was neutralized by adding 25 μl of 5M K₂CO₃ and centrifuged for 1 min. The supernatant was collected and used for determination of glucose-6-phosphate (G-6-P) and glycogen.

Liver G-6-P content was measured spectrophotometrically as described by Lang and Michal (17). Liver glycogen content was determined using a modified version of the method published by Chan and Exton (18). Briefly, aliquots of supernatant were spotted onto small pieces of Whatman paper. Glycogen was precipitated in 66% ice-cold ethanol. The precipitated glycogen was dissolved in 0.2 mol/l sodium acetate (pH 4.5) and hydrolyzed with amyloglucosidase (Boehringer-Mannheim, Indianapolis, IN). The amount of released glucose was determined using a glucose Trinder kit (Sigma). Glycogen content in liver was expressed as micrograms glucose per milligram wet weight of liver.

Liver TG content was determined using a method previously described (19). Final TG concentration was determined using the TG reagent from Roche (Basel, Switzerland; triglyceride/GB 450032).

Acetyl-CoA carboxylase (ACC) activity was assayed in crude liver extracts exactly as previously described (20).

Sample proteins were separated by SDS-PAGE using 5% stacking and 10% resolving gels and then transferred to nitrocellulose filters. Outer mitochondrial membrane proteins were detected using a rabbit polyclonal antibody (1:1,000) (a gift from Dr. P. Scherer, Albert Einstein College of Medicine, Bronx, New York) (21). Relative amounts of serum proteins were quantified using scanning laser densitometry (Molecular Dynamics, Sunnyvale, CA).

Total RNA was isolated using Tri-reagent (Molecular Research Center, Cincinnati, OH) according to the manufacturer’s instructions. Approximately 25–50 μg total RNA was loaded onto a 1.2% formaldehyde-agarose gel, transferred to a Hybond–N nylon membrane (Amersham), and hybridized overnight to random primed ³²P-labeled cDNA probes under high stringency conditions (50% formamide, 42°C). The filter was washed at 42°C in 2 × sodium chloride and sodium citrate, 1% SDS, and 0.2 × sodium chloride and sodium citrate, 0.1% SDS, solutions and subjected to phosphoimage analysis for visualization and quantification. cDNAs for the following probes were kindly provided by the following: fatty acid synthase, Pascal Ferrè; lipoprotein lipase (LPL), Dr. Ira Goldberg; carnitine palmityl transferase 1, Dr. Daniel P. Kelly; peroxisome proliferator–activated receptor (PPAR)-γ, Dr. Ronald M. Evans; glucokinase, Dr. Mark A. Magnuson; GLUT2, Dr. Bernard Thorens; uncoupling protein (UCP)-2, Dr. Daniel Ricquier; and inorganic phosphate carrier (PiC), Dr. Philipp E. Scherer. Fragments for the murine sterol regulatory binding protein were generated using the following primers: 5′-GCCAACTCTCCTGAGAGCTT-3′ and 5′-CTCCTGCTTGAGCTTCTGGTT-3′. UCP-3 and cytochrome C oxidase IV (COX IV) cDNAs were subcloned by RT-PCR using the primer sequences reported by Combatsiaris and Charron (22). Normalization of gel loading was performed by re-probing filters with an oligonucleotide specific for 18S rRNA and end labeled by T4 polynucleotide kinase.

Activity of the pentose phosphate pathway was determined spectrophotometrically by measuring the release of NADPH according to the method described by Cabezas et al. (23). Data are reported as micromoles of NADPH produced per minute per milligram protein.

Fatty acid synthesis rates were determined according to the method described by Lin et al. (24). Mice were tail vein injected with 30 μCi ¹⁴C-acetate (Amersham catalog number CFA13) in the fed state. After 1 h, mice were killed, and liver tissue was harvested. Data are presented as disintegrations per minute per hour per milligram tissue.

Hepatic and peripheral (white adipose and muscle) LPL activity was determined according to the method of Iverius and Ostlund-Lindqvist (25). Mice were tail vein injected with either saline or heparin (200 units/mouse; Sigma), and blood was collected 30 min later from the orbital sinus. Then, 10 μl serum was used with high (1 mol/l) or low (150 mmol/l) NaCl present in the buffer to determine hepatic and total lipase activity, respectively. Data are reported as nanomoles free fatty acid per milligram protein per minute.

LPL activity in quadriceps from control and GLUT4 null mice was determined according to the method of Hocquette et al. (26). Data are reported as counts per minute per milligram tissue.

Individual soleus and extensor digitorum longus (EDL) muscles from 10- to 14-week-old mice were isolated and preincubated for 15 min in Krebs-Ringer buffer supplemented with 4% BSA (fraction V, RIA grade from Sigma), 0.5 mmol/l oleate, and 5 mmol/l glucose. Muscle was 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. Samples were acidified with 15% perchloric acid, and the CO₂ was captured on Whatman paper suspended within the vials. ¹⁴C radioactivity was quantitated after overnight quenching at 4°C by liquid scintillation counting. Data are reported as nanomoles oleate per gram wet muscle weight per 45 min.

Soleus and EDL muscles were isolated and fixed in 2.5% gluteraldehyde in 0.1 mol/l 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 micrographs were taken at 10,000× magnification.

Statistical comparison of control versus GLUT4 null mice was done using the unpaired two-tailed Student’s t test. Data are means ± SE for each group. Significance was accepted at P < 0.05.

Northern blot analysis of liver revealed a 3.3-fold (P < 0.002) increase in fatty acid synthase mRNA in fed but not fasted GLUT4 null mice compared with control mice (Fig. 1A). The overall rates of fatty acid synthesis, as measured by incorporation of ¹⁴C-acetate into liver fatty acids, revealed a sixfold increase in fatty acid synthesis in GLUT4 null compared with control mice (Fig. 1B). Regulators of lipid metabolism were also measured. An elevated level of PPAR-γ is associated with upregulation of ACC and fatty acid synthase leading to TG accumulation. GLUT4 null mice have significantly higher (1.8-fold, P < 0.02) expression levels of PPAR-γ mRNA compared with control mice in the fed state (Table 1). As expected, ACC activity is also increased 1.8- and 1.2-fold in GLUT4 null mice compared with control mice in the fed and fasted states under stimulated conditions (10 mmol/l citrate), respectively (Figs. 2A and B). The overall TG content in liver was not different in GLUT4 null mice compared with control mice in the fed and fasted states (Table 1). Genes involved in hepatic fatty acid utilization were examined. There were no differences in expression of LPL (8.20 ± 0.62 vs. 8.25 ± 1.31 arbitrary units, n = 5 mice per group) in liver from fed GLUT4 null and control mice, respectively. The transport of long-chain fatty acyl-CoA into the mitochondria for oxidation is regulated by carnitine palmityl transferase 1. There was no difference in expression levels of carnitine palmityl transferase 1 (5.14 ± 0.59 vs. 4.47 ± 0.47 arbitrary units, n = 5 mice per group) in liver of fed GLUT4 null or control mice, respectively. These data suggest that fatty acid oxidation is not altered in GLUT4 null liver because of normal levels of fatty acid transport into liver and mitochondria in the fed state. Hepatic TG secretion rates, on the other hand, were increased 2.5-fold (P < 0.001) in GLUT4 null mice compared with control mice (Fig. 2C). Collectively these data demonstrate increased hepatic fatty acid synthesis and secretion in GLUT4 null mice.

Fatty acid synthesis requires the generation of NADPH by the pentose phosphate pathway. Levels of the oxidative and nonoxidative branches were measured by analyzing NADPH release from liver homogenates. The oxidative and nonoxidative branches were significantly increased in GLUT4 nulls, 1.6-fold (P < 0.001) and 1.4-fold (P < 0.05), respectively (Fig. 3A). The ratio of oxidative to nonoxidative activity was not significantly different between GLUT4 null and control mice, indicating that they equally recycle ribose for NADPH production (Fig. 3B). However, the difference between the oxidative and nonoxidative branches was significantly increased—1.8-fold (P < 0.001) in GLUT4 nulls, indicating a high rate of ribose production that is consistent with tissues undergoing increased fatty acid synthesis (Fig. 3B). These data support the observed increase in fatty acid synthesis rates observed in GLUT4 null liver.

While GLUT4 null mice have small elevations in circulating glucose and insulin, they do not develop diabetes on normal pellet diet (Table 1). As previously reported (8), GLUT4 null mice have a 1.7-fold increase in the mRNA expression of GLUT2 in liver when compared with age- and sexed-matched control mice. Hepatic GLUT2 mRNA expression was increased 2.1-fold (P < 0.001) in fed GLUT4 null mice (Table 1). The initial step of glycolysis, glucose phosphorylation, is regulated by glucokinase in liver. During the fed state, glucokinase expression was increased twofold (P < 0.001) in GLUT4 null mice compared with control mice, but showed no difference in the fasted state (data not shown; Table 1). The product of glucokinase, G-6-P, was also elevated 1.3-fold (P < 0.05) in fed GLUT4 null mice, but decreased fourfold (P < 0.001) with fasting (Table 1). The transcription factor sterol regulatory binding protein, which has been shown to activate glucokinase transcription and inhibit PEPCK transcription (27), was significantly upregulated 1.5-fold (P < 0.01) in fed GLUT4 null liver (Table 1). Overall glycogen content was not significantly different in GLUT4 null liver compared with control mice in the fed state (Table 1). GLUT4 null liver also showed normal mRNA expression of PEPCK (7.17 ± 0.34 vs. 6.34 ± 0.23 arbitrary units, n = 5 per group) and glucose-6-phosphatase (9.46 ± 1.65 vs. 7.43 ± 1.20 arbitrary units, n = 5 per group) in the fed state when compared with control mice. Together these data suggest that during feeding, when circulating glucose and insulin become elevated, the GLUT4 null liver significantly increases glucose uptake and glycolysis compared with control liver, but maintain normal hepatic glycogen content.

The ability of GLUT4 null mice to use lipids was assessed by oral administration of olive oil. GLUT4 null mice showed an enhanced ability to clear orally administered lipids (Fig. 4A). However, GLUT4 null mice show no difference compared with control mice in their ability to absorb TGs or fatty acids (Figs. 4B and C). Serum levels of NEFAs were reduced 1.3-fold (P < 0.001) and 1.4-fold (P < 0.001) in the fed and fasted states, respectively, in the GLUT4 null mice compared with control mice (Table 1). Fed and fasted serum TGs were reduced 2.2-fold (P < 0.001) and 1.9-fold (P < 0.05), respectively, in GLUT4 null mice compared with control mice (Table 1). Fatty acid oxidation by muscle and adipose tissue requires the breakdown of serum TGs into NEFAs through the action of LPL. Serum levels of total LPL (63.0 ± 2.4 and 66.8 ± 2.8 nmol free fatty acid · mg⁻¹ protein · min⁻¹, n = 5 per group) and hepatic LPL (11.3 ± 0.39 and 11.0 ± 0.82 nmol free fatty acid · mg⁻¹ protein · min⁻¹, n = 5 per group) were not significantly different (P = 0.2 and P = 0.7) between control and GLUT4 null mice, respectively, in the fed state. Northern blot analysis from gastrocnemius muscle revealed a 1.6-fold (P < 0.01) increase in LPL mRNA in fed GLUT4 null mice compared with controls (Fig. 5A). Muscle LPL activity was increased 2.1-fold (P < 0.001) in GLUT4 null mice compared with control mice (Fig. 5B).

Based on the findings of increased hepatic TG production and increased muscle LPL activity, we next examined the ability of GLUT4 null muscle to oxidize TGs. Oleate oxidation rates in the GLUT4 null soleus and EDL muscles were increased 1.5-fold (P < 0.05) and 1.3-fold (P < 0.05), respectively (Figs. 5C and D). This coincides with mitochondrial hypertrophy/hyperplasia noted in transmission electron micrographs of GLUT4 null soleus and EDL muscle (Fig. 6A). Mitochondrial hypertrophy was accompanied by increased expression of mitochondrial outer membrane proteins (Fig. 6B). The average increase was ∼1.8-fold in soleus and 1.4-fold in EDL of GLUT4 null mice relative to control mice (P < 0.01). Expression of genes encoding the inner mitochondrial membrane proteins COX IV and PiC were increased 30 and 50%, respectively, in GLUT4 null skeletal muscle compared with control mice (P < 0.01) (Fig. 6C). UCP-2 mRNA levels were not altered in GLUT4 null skeletal muscle relative to controls, but UCP-3 mRNA levels were reduced 50% in GLUT4 null muscle compared with control (P < 0.01) (Fig. 6C).

GLUT4 null mice have invoked compensatory mechanisms in the absence of GLUT4 that allow them to resist diabetic hyperglycemia in the absence of GLUT4. The liver senses hepatoportal glucose concentrations in a GLUT2-dependent manner and adjusts glucose influx and efflux accordingly to maintain euglycemia (14). In GLUT4 null mice, GLUT2 is increased to promote increased influx and thus clearance of glucose from serum. Glucose influx correlates with increased levels of glucokinase mRNA, the first step in glycolysis, and increased generation of G-6-P. Transgenic mice overexpressing glucokinase have reduced fasting glucose levels, improved glucose tolerance, and increased hepatic glycogen (28). Chronic glucokinase overexpression (>6 months) results in glucose intolerance, hyperglycemia, hyperinsulinemia, and hypertriglyceridemia (29). Therefore, although glucokinase appears to be beneficial in lowering blood glucose concentrations, prolonged overexpression leads to lipid storage problems due to continual hepatic lipogenesis. Enhanced TG utilization by muscle protects GLUT4 null mice from the detrimental effects of chronic hepatic lipogenesis seen in older glucokinase transgenic mice.

Increased glucose flux into GLUT4 null liver leads to increased G-6-P concentrations. G-6-P is a potent stimulator of fatty acid synthesis, as demonstrated by in vitro experiments with cultured rat hepatocytes (30). Both the oxidative branch of the pentose phosphate pathway and fatty acid synthesis are increased in GLUT4 null mice in the fed state. PPAR-γ promotes transcription of genes associated with lipid storage and is upregulated in GLUT4 null liver. Increased fatty acid synthesis is often associated with activation of the transcription factor PPAR-γ in response to thiazolidinediones treatment. In general, levels of PPAR-γ are low in liver (10–30% of what is seen in adipose tissue) (31) but are increased in several mouse models of hepatic steatosis (32). Liver-specific inactivation of PPAR-γ reduced hepatic steatosis by 30% in lipoatrophic A-ZIP/FI (AZIP) fatless mice but impaired TG clearance and increased muscle TG storage (33). AZIP mice without PPAR-γ were more diabetic, presumably because of increased peripheral insulin resistance. Similarly, disruption of PPAR-γ in liver of leptin-deficient mice results in reduced hepatic steatosis but increased serum TGs and free fatty acids and the severity of diabetes (34). In models of reduced PPAR-γ, liver steatosis was improved, but peripheral insulin sensitivity was worsened because of increased storage of TGs in muscle and adipose tissue. GLUT4 null mice resist the deleterious effects of increased hepatic fatty acid synthesis through enhanced TG clearance and oxidation in skeletal muscle. Muscle-specific inactivation of PPAR-γ results in increased adiposity with normal insulin sensitivity in muscle but whole-body insulin resistance due to hepatic insulin resistance (35). Collectively, these mouse models demonstrate that alteration of PPAR-γ in liver or muscle has systemic effects on both glucose and lipid metabolism. GLUT4 null mice overcome severe peripheral insulin resistance by altering liver metabolism in a way that complements alterations in skeletal muscle substrate utilization.

Despite increased hepatic fatty acid synthesis and low adipose tissue mass, GLUT4 null mice have reduced serum TG levels. The clearance of serum TGs is greatly accelerated in GLUT4 null mice and is suggestive of enhanced fatty acid clearance and oxidation. Based on the phenotype of GLUT4 null mice, excess hepatic-derived TGs are not stored in adipose tissue (GLUT4 null mice have an 80% reduction in adipose tissue mass). Proper storage of TGs is essential for the maintenance of insulin sensitivity. Accumulation of TGs in nonadipose tissues such as skeletal muscle, liver, and/or pancreas can result in the development of insulin resistance and diabetes (36). Serum TGs are cleared through the activation of LPL. Serum LPL and hepatic lipase activity were not altered in GLUT4 null mice, but skeletal muscle LPL mRNA levels and activity were significantly increased. Transgenic mice overexpressing human LPL have reduced serum TGs and are protected from diet-induced hypertriglyceridemia (37). Overexpression of LPL specifically in skeletal muscle increased muscle TG content, decreased insulin-stimulated glucose uptake into muscle, and caused skeletal muscle mitochondrial hyperplasia/hypertrophy (38,39). In combination with increased LPL activity, GLUT4 null skeletal muscles also exhibit enhanced rates of oleate oxidation, which may protect them from the adverse effects of intramyocellular TGs. Future studies will be conducted to determine the direct contribution of hepatic-derived fatty acid particles to the increased β-oxidation observed in GLUT4 null mice during the fed state.

GLUT4 null mice display skeletal muscle mitochondrial hyperplasia/hypertrophy associated with increased oleate oxidation, although the direct contribution of lipid uptake was not measured. The observed mitochondrial hypertrophy is confirmed by increased expression of outer mitochondrial membrane proteins. The hypertrophied mitochondria most likely embody an increased capability to undergo oxidative phosphorylation. This is strongly supported by increased expression of inorganic phosphate carrier and COX IV mRNA in muscle (40). COX IV is one of the major rate-limiting factors in the mitochondrial respiratory chain, and its activity has been positively correlated with pyruvate and palmitate oxidation rates (41,42). PiC is an inner mitochondrial membrane protein that transports inorganic phosphate into the mitochondrial matrix, where it serves as substrate for ATP synthesis (43). Decreased expression of UCP-3 should complement the increased capacity for substrate oxidation to preserve a normal energetic state in GLUT4 null skeletal muscle. This is supported by results from Tsuboyama-Kasaoka et al. (44) demonstrating that increased glucose flux in skeletal muscle after GLUT4 overexpression led to increased UCP-3 expression and oxygen consumption. UCP-3 knockout mice have a fourfold increase in skeletal muscle ATP synthesis with no change in the tricarboxylic acid cycle, demonstrating increased mitochondrial energy coupling (45).

The molecular mechanism(s) responsible for mitochondrial alterations seen in the absence of insulin-stimulated flux of glucose through GLUT4 are not fully understood. Mitochondrial proliferation and increased fatty acid oxidation are also characteristics of endurance-trained athletes (46). PPAR-γ coactivator 1 (PGC-1) regulates transcription factors involved in mitochondrial biogenesis (47). Skeletal muscle transgenic overexpression of PGC-1 caused conversion of fast-twitch glycolytic fibers to slow-twitch oxidative fibers and, in the highest transgenic expressing line, had decreased body weight (48). Consistent with increased oleate oxidation, GLUT4 null tibialis anterior and EDL muscles have increased fast-twitch oxidative type IIA fibers and reduced fast-twitch glycolytic type IIB fibers (12). Mice with transgenic overexpression of a related protein, PGC-1b, were lean, hyperphagic, and resistant to diet-induced obesity because of increased energy expenditure (49). A key regulator of fatty acid synthesis and oxidation is ACC. ACC2-deficient mice have increased fatty acid oxidation rates and accumulate less fat than control mice (50). Interestingly, these mice, like GLUT4 nulls, have increased fatty acid oxidation in soleus muscle. However, unlike GLUT4 nulls, the liver of ACC2-deficient mice exhibits increased fatty acid oxidation. When challenged with a high-fat diet, ACC2-deficient mice gained less weight than controls and were protected from development of diabetes (50). Malonyl-CoA at high concentrations promotes fatty acid synthesis and inhibits β-oxidation. Malonyl-CoA concentrations in GLUT4 null gastrocnemius muscle were not different from control mice (data not shown). In GLUT4 null mice, increased skeletal muscle fatty acid oxidation complements increased liver fatty acid synthesis and prevents the accumulation of intramyocellular TGs. This alteration in substrate utilization also provides fuel for high-energy phosphate formation in the absence of significant insulin-stimulated glucose utilization. Whether the alterations in substrate utilization noted in GLUT4 null mice modulate overall energy expenditure require additional studies.

Data presented here clearly demonstrate the significance of coordinated alterations in substrate utilization as a major compensatory mechanism invoked by GLUT4 null mice for maintenance of euglycemia. The liver plays a critical role in glucose clearance and in providing alternative energy substrates (TGs) for use by peripheral tissues such as skeletal muscle. Skeletal muscle, through increased mitochondrial hypertrophy/hyperplasia and oxidative capacity, is able to rapidly clear and use serum TGs and prevent their accumulation in nonadipose depots. A schematic representation of the adaptations in GLUT4 null liver and skeletal muscle substrate utilization as a means to manage the potential diabetic effects of global GLUT4 ablation are presented in Fig. 7. New therapeutic approaches for the treatment of diabetes should consider the interactions of lipid and glucose metabolism and cross-talk that occurs among tissues.

This work was supported by grants from the National Institutes of Health (NIH) (DK47425 and HL58119), American Diabetes Association, Comprehensive Cancer Center of the Albert Einstein College of Medicine, and the Diabetes Research and Training Center. M.R., T.S.T., and A.E.S. were supported by NIH (5T32DK07513, 5T32HL07675, and 5T32GM07491, respectively).

We wish to acknowledge the following individuals: Dr. Juleen Zierath for helpful advice with the oleate oxidation studies; Dr. Clay Semenkovich, Trey Coleman, and Dr. Ira Goldberg for providing technical guidance for LPL measurements; Dr. Peter Voshol for advice on intestinal lipid absorption measurements and manuscript preparation; Leslie Gunter for transmission electron microscopy; Drs. Sally Du and Lingguang Cui for mouse assistance; and Drs. Ann Louise Olson, Luciano Rossetti, and Philipp Scherer for valuable discussions and critical reading of the manuscript.

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