Cardiac hypertrophy with preserved contractile function after selective deletion of GLUT4 from the heart

Glucose enters the heart via GLUT1 and GLUT4 glucose transporters. GLUT4-deficient mice develop striking cardiac hypertrophy and die prematurely. Whether their cardiac changes are caused primarily by GLUT4 deficiency in cardiomyocytes or by metabolic changes resulting from the absence of GLUT4 in skeletal muscle and adipose tissue is unclear. To determine the role of GLUT4 in the heart we used cre-loxP recombination to generate G4H^–/– mice in which GLUT4 expression is abolished in the heart but is present in skeletal muscle and adipose tissue. Life span and serum concentrations of insulin, glucose, FFAs, lactate, and β-hydroxybutyrate were normal. Basal cardiac glucose transport and GLUT1 expression were both increased approximately 3-fold in G4H^–/– mice, but insulin-stimulated glucose uptake was abolished. G4H^–/– mice develop modest cardiac hypertrophy associated with increased myocyte size and induction of atrial natriuretic and brain natriuretic peptide gene expression in the ventricles. Myocardial fibrosis did not occur. Basal and isoproterenol-stimulated isovolumic contractile performance was preserved. Thus, selective ablation of GLUT4 in the heart initiates a series of events that results in compensated cardiac hypertrophy.

J. Clin. Invest.104:1703–1714 (1999).

To meet its substantial energy requirements, the heart is capable of metabolizing a variety of substrates. Cardiac function is therefore very tightly linked to substrate uptake and utilization. Under resting conditions, the heart derives about 70% of its energy from the oxidation of lipids, and the remainder primarily from glycolysis and glucose oxidation (1). Under some physiological conditions such as exercise (2), and in certain pathologic states such as hyperthyroidism (3), ischemia (4, 5), hypertrophy, and congestive heart failure (6, 7), the heart becomes increasingly dependent upon glucose to meet its metabolic demands. Glucose transport is rate limiting for glucose metabolism (8). Glucose enters the heart via the facilitative glucose transporters GLUT1 and GLUT4 (9). The GLUT1 transporter is widely expressed, and is the major mediator of glucose uptake in tissues such as the brain, the blood-brain barrier, erythrocytes, and endothelial cells (9, 10). In contrast, expression of the GLUT4 transporter is limited primarily to muscle (skeletal and cardiac) and adipose tissue, and is the most abundant isoform in these tissues, which also express GLUT1 (9, 10). In the heart, GLUT4 translocates to the plasma membrane in response to insulin (11–13), ischemia (12–14), hypoxia (15), and high-frequency contraction (16). Although these stimuli also result in the translocation of GLUT1, it is likely that GLUT4-mediated uptake represents the major mechanism by which the heart increases glucose uptake under these circumstances (17). In contrast to its role in adipose tissue and skeletal muscle (17–19), GLUT1 is a major mediator of basal cardiac glucose uptake (20, 21).

Glucose transporter expression in the heart is altered in various pathologic states. In untreated diabetes, there is profound downregulation of GLUT4 (22, 23). GLUT1 expression is also downregulated, but the changes are less marked than those described for GLUT4 (24, 25). It has been proposed that these changes in glucose transporter expression contribute to myocardial dysfunction in diabetics (25). The increased myocardial glucose uptake observed in various models of cardiac hypertrophy (6, 7, 26) is uniformly associated with increased expression of GLUT1, with no increase (7), or even a decrease, in GLUT4 expression (26). Agents that induce cardiac myocyte hypertrophy have recently been shown to induce GLUT1 expression at the level of transcription (27). The mechanisms governing the differential regulation of GLUT4 and GLUT1 in this and other pathophysiological states (20, 21, 28), and the relationship between altered glucose transporter expression and cardiac function, are incompletely understood. One approach to address these issues is to alter cardiac glucose transporter expression in transgenic mice.

The phenotype of GLUT4-null mice, in which GLUT4 expression was ablated in all tissues in which it is normally expressed (heart, skeletal muscle, and adipose tissue), provided additional evidence that changes in the cardiac expression of glucose transporters can have profound effects on cardiac structure and function (29). These GLUT4-null mice exhibit significant growth retardation and die prematurely. A striking aspect of this phenotype is the development of marked cardiac hypertrophy (a greater than 2-fold increase in heart weight to body weight ratio), upregulation of cardiac GLUT1 expression, and heart failure. These mice are hyperinsulinemic and have reduced concentrations of cardiac fuels such as β-hydroxybutyrate, FFAs, and lactate, probably because of GLUT4 deficiency in adipose tissue and skeletal muscle. Mice that are heterozygous for the GLUT4-null allele also develop cardiac hypertrophy and histological changes consistent with diabetic cardiomyopathy (30). These mice are hyperinsulinemic, and the majority of these animals develop hypertension and diabetes. Furthermore, normalization of glucose homeostasis by transgenic re-expression of GLUT4 in the skeletal muscle of these mice results in a reversal of the cardiac pathology (31). Thus it is not clear whether all of the cardiac changes are due to the absence of or reduction in GLUT4 expression, or whether the development of cardiac hypertrophy in this interesting model is at least partially due to an altered metabolic milieu that may have independent effects on cardiac structure and function.

Therefore, the aim of this study was to determine the effect of altered cardiac glucose transporter expression in vivo on cardiac contractile performance and the development of cardiac hypertrophy. To achieve this, we used Cre-loxP–mediated gene recombination to generate mice with cardiac-specific inactivation of the GLUT4 gene. With this approach we could study the role of the transporter in the heart in the absence of compensatory metabolic changes that may occur when the gene is also inactivated in skeletal muscle, adipose tissue, or both. Herein we report that mice with cardiac-selective deletion of GLUT4 develop modest cardiac hypertrophy with preserved contractile function.

Generation of mice with cardiac-selective GLUT4 ablation (G4H^–/– mice). Three independent targeted clones were obtained after electroporation of the GLUT4 loxP vector into embryonic stem cells. Two clones, each containing 1 wild-type GLUT4 allele and 1 modified loxP allele, were expanded and injected into the blastocysts of C57BL6 mice. Chimeric mice were generated from both clones, and 2 independent lines of mice bearing 1 normal allele and 1 loxP-containing GLUT4 allele (+/lox) were derived from these chimeric mice (Figure 1b). An FVB background was used for α-MHC Cre transgenic mice (Figure 1c). Cre transgenic mice (wild-type for the GLUT4 allele) were crossed with GLUT4 +/lox heterozygotes. Some of the offspring inherited the transgene and were heterozygous for the modified loxP allele (cre+/lox). Mice with cardiac-specific GLUT4 ablation (G4H^–/–) were derived by crossing cre+/lox mice with GLUT4 loxP heterozygotes. This breeding paradigm resulted in 6 genotypes: cre+/+, cre +/lox, and cre lox/lox (all with cre); and +/+, +/lox, and lox/lox (all without cre). G4H^–/– mice have the genotype cre lox/lox and were born with the expected mendelian frequency, indicating that the absence of cardiac GLUT4 did not cause embryonic lethality. G4H^–/– mice exhibited normal life span. Most mice were sacrificed for studies by 12 months of age. In an analysis of a cohort of 78 mice (39 wild-type and 39 G4H^–/–), all animals were alive at 6 months of age. Sixteen wild-type mice and 16 G4H^–/– mice were sacrificed at 7 months. At 9 months, all of the 23 remaining G4H^–/– mice were still alive. Twenty of these mice were sacrificed between 9 and 12 months of age. The remaining 3 mice survived for longer than 18 months, implying that there was no reduction in the longevity of G4H^–/– mice. Growth retardation was not observed in mice of up to 6 months of age. A variable reduction in body weight was seen in older male G4H^–/– mice, but not in female mice (see Table 2b).

Table 2

Heart weights and body weights

Glucose transporter expression. G4H^–/– mice had no GLUT4 immunoreactivity in the heart, in contrast to wild-type or GLUT4 loxP homozygotes (without cre expression) (Figure 2a). We were unable to detect any GLUT4 protein in the hearts of G4H^–/– mice with antibodies to either the carboxyl or amino terminus of GLUT4. Cardiac GLUT1 content was upregulated 2.6-fold (±0.4-fold) in male G4H^–/– mice and 3.7-fold (±0.5-fold) in female G4H^–/– mice (P < 0.001), compared with their respective wild-type controls and with GLUT4 loxP homozygotes (without cre) (Figure 2, b and c). There was no upregulation of GLUT3 in the heart (data not shown). Skeletal muscle GLUT4 content was similar in wild-type mice, loxP homozygotic mice, and G4H^–/– mice (assessed by densitometry of skeletal muscle GLUT4 immunoblots from 5 mice of each genotype), indicating that the modified GLUT4 loxP gene was normally expressed in skeletal muscle and that there was no aberrant cre expression in skeletal muscle (Figure 2d). In loxP homozygous mice, GLUT4 protein content was assessed by laser densitometry of immunoblots of white adipose tissue membrane samples from 17 lox/lox mice and 17 wild-type (+/+) mice of various ages. The mean GLUT4 content of white adipose tissue from lox/lox mice was 52 ± 11% of wild-type control levels. The variable decrease in adipose tissue GLUT4 content indicates that the loxP modification of the GLUT4 gene resulted in lower expression levels in adipose tissue (Figure 2e). Adipose tissue GLUT4 content in G4H^–/– mice was similar to that in loxP homozygotes without cre (Figure 2e). To control for this partial reduction of GLUT4 in adipose tissue, homozygous loxP mice without cre were used as an additional control group for the studies of in vivo metabolism.

Figure 2

Glucose transporter expression. (a) Representative GLUT4 immunoblots of postnuclear membranes of cardiac muscle obtained from female (F) and male (M) wild-type (+/+) and heart-selective GLUT4-ablated (G4H^–/–; cre lox/lox) mice (left) and female mice of other genotypes: loxP homozygotes (lox/lox) and loxP heterozygotes (+/lox) without cre (right). Similar data exist for males. (b) Representative GLUT1 immunoblots of postnuclear membranes of cardiac muscle from female G4H^–/– (cre lox/lox) mice, loxP homozygotic (lox/lox) mice, heterozygotic (+/lox) mice, and wild-type (+/+) mice. Similar data exist for males. (c) Densitometric analysis of GLUT1 immunoblots obtained from male and female G4H^–/– mice and their wild-type controls (aged 18–21 weeks). GLUT1 densities are normalized to 1 for both males and females.Males: wild-type, n = 19; G4H^–/–, n = 7. Females: wild-type, n = 6; G4H^–/–, n = 6. *P < 0.001 vs. wild-type controls (2-tailed t test). (d) Representative GLUT4 immunoblots (from 5 mice of each genotype) demonstrating the preservation of GLUT4 expression in postnuclear membranes of skeletal muscle of G4H^–/– (cre lox/lox) mice and normal expression of GLUT4 in loxP homozygotes without cre (lox/lox). (e) Representative GLUT4 immunoblots (from 17 mice of each genotype) of postnuclear membranes from white adipose tissue of wild-type (+/+), loxP homozygotic (lox/lox), and G4H^–/– (cre lox/lox) male and female mice. Each panel represents a separate immunoblot. The third panel shows that there is no further change in GLUT4 content in G4H^–/– (cre lox/lox) mice compared with loxP homozygous (lox/lox) mice.

Metabolic profiles. GTTs were performed on age- and sex-matched G4H^–/– mice and control mice between 28 and 30 weeks of age (Figure 3, a and b). Glucose tolerance was similar in male G4H^–/– mice and littermate controls (wild-type for the GLUT4 allele) (Figure 3a). There was a suggestion of mild impairment in glucose tolerance in female G4H^–/– mice at 30 weeks (Figure 3b, right), although the 2 curves were not statistically different by repeated-measures ANOVA. GTTs were then performed on a younger cohort (age 18 weeks), and the resulting curves were completely overlapping (Figure 3b, left). There was no increase in fed or fasted insulin concentrations in G4H^–/– mice of either sex (Table 1). Fasted and fed concentrations of the cardiac metabolic substrates β-hydroxybutyrate, FFAs, and lactate in G4H^–/– mice were similar to levels in controls (Table 1). There were no differences in the metabolic profiles or GTTs of loxP homozygotes (without cre) when compared with wild-type mice (data not shown).

Figure 3

GTTs in awake G4H^–/– mice (squares) and age-matched littermate controls (circles). Blood glucose concentrations at 0, 10, 20, 30, 60, and 120 minutes after intraperitoneal injection of glucose (1 mg/gram body weight). (a) Mean ± SEM for 28-week-old males (6 G4H^–/– and 6 wild-type). (b) Mean ± SEM for 18-week-old females (7 G4H^–/– and 15 wild-type) (left) and 30-week-old females (7 G4H^–/– and 7 wild-type) (right). There are no statistical differences between wild-type and G4H^–/– GTTs by repeated-measures ANOVA.

Table 1

Serum insulin and metabolite concentrations

Cardiac glucose uptake. Basal and insulin-stimulated cardiac glucose uptake were assessed in isolated perfused hearts by measuring the accumulation of 2-DG-P using ³¹P NMR spectroscopy (Figure 4a). The decline in the phosphocreatine peak after insulin stimulation in wild-type mice probably represents transfer of phosphates to 2-DG-P (Figure 4a). There were striking differences in both basal and insulin-stimulated glucose uptake in G4H^–/– mice when compared with wild-type controls (Figure 4b). Insulin-mediated glucose uptake was abolished in G4H^–/– hearts, whereas insulin increased glucose uptake above basal levels by 17-fold (P < 0.0001) in male wild-type mice, and by 8-fold (P < 0.004) in female wild-type mice. Basal glucose uptake in G4H^–/– male hearts was 0.59 ± 0.25 area units/min, which is 4-fold higher than the uptake of 0.142 ± 0.022 area units/min in male wild-type hearts (P < 0.05). The basal uptake in female G4H^–/– mice (0.79 ± 0.12 area units/min) was 2-fold higher than in female controls (0.344 ± 0.090 area units/min) (P < 0.05). The increase in basal glucose uptake in G4H^–/– mice was similar in magnitude to the increase in GLUT1 expression. Interestingly, female wild-type mice tended to have higher basal glucose uptake than did male wild-type mice, and this difference between sexes was lost in G4H^–/– mice.

Figure 4

Evidence for impaired GLUT4-mediated glucose uptake in GLUT4-ablated hearts. (a) Representative ³¹P NMR spectra in wild-type and G4H^–/– hearts, illustrating absence of insulin-mediated augmentation of 2-DG-P accumulation in GLUT4-deficient hearts. The decline in phosphocreatine (PCr) after insulin stimulation in wild-type mice probably represents transfer of phosphates to 2-DG-P. PPA, phenylphosphonic acid standard; Pi, inorganic phosphate. γ, α, and β represent the 3 phosphates on adenosine in ATP. (b) Basal and insulin-stimulated 2-DG uptake in wild-type and G4H^–/– mice. Bars represent mean ± SEM; individual data points are illustrated by the closed circles. Number of animals in each group: Wild-type males: basal, n = 5; insulin-stimulated (Ins), n = 5. G4H^–/– males: basal, n = 4; insulin-stimulated, n = 3. Wild-type females: basal, n = 5; insulin-stimulated, n = 5. G4H^–/– females: basal, n = 5; insulin-stimulated, n = 5. *P < 0.05 vs. wild-type basal of either sex (ANOVA). ‡‡P < 0.0001, **P < 0.004 vs. wild-type basal of the same sex (paired 2-tailed t test).

Cardiac weights and histology. Heart weights were obtained before histological analysis on male and female mice (average age 19 weeks and 21 weeks, respectively) (Table 2a). The hearts of G4H^–/– mice were significantly heavier (by 38% in males and 35% in females) than those of their respective wild-type controls. The heart weight to body weight ratio was significantly higher in G4H^–/– males (43% higher than wild-type; P < 0.01) and tended to be higher in G4H^–/– females (25% higher than wild-type). Wet and dry heart weights were also obtained from mice (average age 35–39 weeks) that underwent glucose uptake measurements (Table 2b). As was observed in the younger cohort, heart weights were higher in the G4H^–/– mice, and the difference achieved statistical significance in both sexes. In absolute terms, heart weights (wet and dry, respectively) were increased by 16% and 25% in G4H^–/– males vs. wild-type males, and by 35% and 42% in G4H^–/– females. When corrected for body weight, the changes were more marked in male G4H^–/– mice (39% and 45% for wet and dry heart weight to body weight ratios, respectively) than in G4H^–/– females (17% and 23%, respectively). Histological examination of hematoxylin and eosin– and trichrome-stained sections was performed on the hearts of mice at 20, 30, and 50 weeks of age. In all age groups, there were no gross morphological abnormalities of myocyte architecture, and analysis of trichrome-stained sections revealed no increase in interstitial collagen compared with controls (Figure 5a). Myocyte cross-sectional areas were measured using quantitative image analysis on sections from 20-week-old mice stained with periodic acid–Schiff. This analysis was performed to address the question of whether increased cardiac size in transgenic mice was due to increased myocyte size. The heavier hearts have greater myocyte cross-sectional areas (Figure 5b), supporting the hypothesis that the observed cardiac hypertrophy (evidenced by increased heart weight) is due to myocyte hypertrophy as opposed to increased interstitial content.

Figure 5

Cardiac histology. (a) Representative transverse sections of left ventricle stained with hematoxylin and eosin (H&E) (top row) and trichrome (middle row) of a wild-type and a G4H^–/– female mouse (aged 30 weeks) illustrating the absence of gross morphological abnormalities of myocyte architecture and the absence of any increase in interstitial collagen. ×600 (original magnification). Representative periodic acid–Schiff (PAS) cross-sections (bottom row) taken from the left ventricle of a female G4H^–/– mouse and an age-matched control (aged 20 weeks), illustrating the sections used for quantitative histomorphometry. Data are similar for males (not shown). (b) Quantitative histomorphometry of male and female G4H^–/– and wild-type hearts (a single heart is represented for each). Heart weight to body weight ratios (HW/BW) are shown beneath each bar. Males: n = 28 cross-sections for wild-type mice and 27 cross-sections for G4H^–/– mice. Females: n = 37 cross-sections for wild-type and 43 cross-sections for G4H^–/–. **P < 0.0001 vs. wild-type of the same sex (unpaired 2-tailed t test).

Expression of ANP and BNP in the heart. Because increased cardiac expression of the natriuretic peptides ANP and BNP are universal features of cardiac hypertrophy of diverse etiologies (38, 39), we examined the expression of the ANP and BNP genes in the ventricles of G4H^–/– mice and littermate controls. There was a striking 43-fold increase in BNP gene expression in G4H^–/– mice (P = 0.016). Although more variable, ANP expression on average was induced 7-fold (Figure 6, a and b).

Figure 6

ANP and BNP gene expression. (a) Northern blot showing changes in the expression of ANP and BNP mRNA in wild-type (first 5 lanes) and G4H^–/– mice (last 4 lanes). Data shown are from male mice 13–14 weeks of age. Thirty micrograms of total RNA was loaded in each lane. (b) Densitometric analysis of Northern blot data after correcting for loading with cyclophyllin. Note differences in the scales (y axis) of the ANP and BNP plots. P = 0.016 for wild-type BNP vs. G4H^–/– BNP expression. P = 0.112 for wild-type ANP vs. G4H^–/– ANP expression (Mann-Whitney U test 2-tailed probabilities). Wild-type: n = 5. G4H^–/–: n = 4.

Cardiac contractile function. Table 3 shows baseline measurements in isolated perfused hearts immediately before the 2-DG uptake assay. Heart rate and coronary flow per gram heart weight were similar in all hearts. When LV end-diastolic pressure was set to 5–10 mmHg, LV systolic pressure and RPP were indistinguishable between wild-type and G4H^–/– hearts. Figure 7 shows contractile responses to isoproterenol infusion in G4H^–/– and control hearts. The responses in LV developed pressure, LV end-diastolic pressure, heart rate, and rates of contraction and relaxation were similar in both groups.

Figure 7

Isovolumic contractile performance in wild-type mice (open circles) and G4H^–/– mice (closed circles) in response to 2 doses of isoproterenol as described in Methods. There are 5 animals (3 males and 2 females) in each group (32–40 weeks of age). Each G4H^–/– mouse was paired with an age-matched wild-type control. LVDP, left ventricular developed pressure; EDP, end diastolic pressure; RPP, rate pressure product; +dP/dt, rate of contraction; –dP/dt, rate of relaxation.

Table 3

Contractile performance of isolated perfused hearts

We used cre-loxP gene targeting to inactivate the GLUT4 gene selectively in the heart. GLUT4 expression was preserved in skeletal muscle, and although the expression of GLUT4 was variably reduced in the adipose tissue of loxP homozygous (lox/lox) mice (without cre expression), in extensive metabolic analyses comparing lox/lox mice with wild-type mice, we found no differences in glucose tolerance, concentration of insulin or metabolic substrates, body weight, or heart weight. We have also measured contractile performance and glucose uptake in a small number of lox/lox hearts and found no difference from wild-type mice. For this reason we compared G4H^–/– mice with wild-type controls. These G4H^–/– mice allow assessment of the physiological role of GLUT4 in the heart in vivo in the absence of metabolic perturbations such as altered substrate supply and hyperinsulinemia, which could potentially confound the interpretation of the cardiac phenotype.

The major finding of this study is that the absence of GLUT4 in the heart results in the development of cardiac hypertrophy with preserved contractile function, and does not impair longevity. Hypertrophy is evidenced by increased cardiac weight, myocyte cross-sectional area, and the induction of ANP and BNP gene expression, and occurs in the absence of changes in the circulating concentrations of insulin and cardiac metabolic substrates. The observed hypertrophy is intermediate in degree between the physiological hypertrophy observed after exercise training in rodents (40, 41) and the more severe changes observed in pathological conditions such as hypertension or pressure overload (26, 42). In G4H^–/– mice, there was no histological evidence of myocyte disarray and no increase in myocardial fibrosis — both common features of pathological hypertrophy (30, 43). Furthermore, the mild induction of ANP gene expression is similar to that reported with chronic exercise in the rat, but less than that observed in hypertensive rats (42). The induction of natriuretic peptide gene expression is one of the most conserved features of ventricular hypertrophy (irrespective of the etiology of the hypertrophy; 44) and is observed in multiple species (38). BNP is expressed predominantly in ventricular myocytes, and its expression and secretion are more closely correlated with changes in ventricular mass than are the expression and secretion of ANP (44). Hence the significant increase in BNP expression provides strong evidence for the presence of myocyte hypertrophy in G4H^–/– mice. The molecular mechanisms inducing hypertrophy in this model are not currently understood. Our study raises the possibility that the inability to increase cardiac glucose uptake by GLUT4 results in the activation of signaling pathways that ultimately lead to the development of cardiac hypertrophy.

Isovolumic contractile performance is preserved in G4H^–/– hearts under the experimental conditions described here. In response to the inotropic stimulant isoproterenol, wild-type and G4H^–/– hearts achieved heart rates similar to those observed in vivo, and experienced increased contractile performance to a similar extent. However, in preliminary experiments, we observed that isolated perfused G4H^–/– hearts have increased susceptibility to ischemic injury, and develop irreversible diastolic dysfunction after low-flow ischemia (45, 46). In preliminary in vivo experiments performed under ketamine/xylazine anesthesia, blood pressures and basal in vivo contractility were similar in G4H^–/– mice and control mice (47). However, diastolic dysfunction developed in G4H^–/– mice after isoproterenol administration. In these experiments, the anesthesia resulted in reduced baseline heart rate and diminished cardiac contractility in both groups. Therefore the basis of the diastolic dysfunction in response to isoproterenol under these in vivo conditions requires further investigation. Taken together, these observations suggest that under basal conditions, the GLUT4-deficient heart is capable of maintaining normal function, and that dysfunction occurs only in the context of significant stress, such as ischemia.

Another major finding is that deletion of GLUT4 expression in the heart is associated with induction of GLUT1 expression. The degree of GLUT1 upregulation in G4H^–/– mice is similar to that reported in GLUT4-null mice (29). Cardiac GLUT1 expression has been reported to increase during euglycemic hyperinsulinemic clamps in rats (21). The upregulation was blunted by increasing glucose or FFA concentration, suggesting that both insulin exposure and the energy status of cardiac muscle could be important determinants of cardiac GLUT1 expression. GLUT4-null mice develop substantial hyperinsulinemia and reduced concentrations of FFAs, which could account in part for the upregulation of GLUT1. However, our mice with cardiac-selective loss of GLUT4 are not hyperinsulinemic and have normal serum concentrations of FFAs and glucose. Therefore it is more likely that GLUT1 induction results directly from the effects of GLUT4 deficiency in cardiomyocytes, and from altered signal transduction pathways that ultimately lead to hypertrophy.

The degree of upregulation of basal transport and GLUT1 expression observed in G4H^–/– hearts is similar to that reported in aortically banded rats with compensated hypertrophy (7), and in the hearts of spontaneously hypertensive rats (26). Recent evidence suggests that activation of the Ras, Raf, MEK, and ERK pathways, which may play a role in the induction of cardiac hypertrophy (48, 49), is associated with induction of GLUT1 expression (27). These pathways also activate the expression of ANP (50). Furthermore, additional pathways involving members of the stress-activated protein kinase family such as JNK have also been shown to be important mediators of the hypertrophic response in vivo (51). Our model of cardiac GLUT4 deficiency will therefore be a useful model system with which to dissect the interactions between changes in cardiac glucose utilization and activation of signal transduction pathways that lead to hypertrophy.

Our studies support the hypothesis that GLUT1 is an important mediator of basal glucose uptake in the heart. The absence of GLUT4 was not associated with a reduction in basal glucose uptake. On the contrary, basal glucose uptake was increased in the hearts of G4H^–/– mice; this correlated with the increase in GLUT1 expression. This contrasts with adipose tissue and skeletal muscle, in which GLUT4 is the major mediator of basal glucose uptake (17–19). Insulin induces translocation of the GLUT1 transporter in the heart in vivo (13, 52), in primary rat adipocytes (17), and in 3T3L1 adipocytes (53). GLUT1 also translocates to the plasma membrane in the heart in response to ischemia (14). The lack of augmentation of glucose uptake in GLUT4-deficient hearts in response to a maximally stimulating dose of insulin confirms that GLUT4 is the major mediator of insulin-mediated glucose uptake in the heart.

Our findings support those of Katz et al., who initially reported the development of cardiac hypertrophy in mice that lacked GLUT4 in all tissues (GLUT4-null) (29). There are, however, important differences between GLUT4-null mice and our mice, which lack the transporter selectively in the heart. Of note, G4H^–/– mice have normal longevity in contrast to GLUT4-null mice, the majority of which died between 5 and 7 months of age (29). G4H^–/– mice have a normal metabolic profile (29), the degree of cardiac hypertrophy is modest, there is no myocardial fibrosis (54), and ventricular function is preserved (55). The milder cardiac phenotype in our mice is consistent with the hypothesis that reduced substrate supply and hyperinsulinemia impaired cardiac function and induced further cardiac hypertrophy in GLUT4-null mice. Diminished oxidation of long-chain fatty acids is associated with the development of cardiac hypertrophy in rodents (56). Chronic hyperinsulinemia is associated with ventricular hypertrophy in rats (57), and reversal of hyperinsulinemia in GLUT4 heterozygous-null mice with transgenic complementation of GLUT4 in skeletal muscle leads to regression of cardiac hypertrophy (31). The absence of multiple metabolic derangements in our animals provides a plausible explanation for the difference in the cardiac phenotypes of G4H^–/– mice vs. GLUT4-null mice. It is also possible that differences in the genetic background of these 2 models could account for some of the differences in the magnitude of cardiac hypertrophy and in contractile function. Importantly, both models define a critical role for GLUT4 in cardiac physiology. The absence of GLUT4 results in compensated hypertrophy in G4H^–/– mice, and in overt cardiac dysfunction when the heart is subjected to additional metabolic stresses as occurs in GLUT4-null animals.

In conclusion, we have shown that cardiac-selective GLUT4 deficiency leads to compensated ventricular hypertrophy with preserved cardiac contractile function. These changes do not impair longevity. The upregulation of GLUT1 and the increase in basal cardiac glucose uptake is similar to that observed in other models of cardiac hypertrophy. Our observations raise the important possibility that primary alterations in cardiac glucose transporter expression may play a direct role in the pathogenesis of cardiac hypertrophy.

This work was supported by National Institutes of Health (NIH) grants HL-62886 and DK-02495 (to E.D. Abel), HL-59246 and AG-00837 (to R. Tian), HL-52320 (to J. Ingwall), and DK-43051 (to B.B. Kahn) and the transgenic core of the Boston Obesity Nutrition Research Center (P30DK 46200). E.D. Abel was the recipient of a Faculty Development Award from the Robert Wood Johnson Foundation, recipient of the Eleanor and Miles Shore 50th Anniversary Scholars in Medicine Fellowship from Harvard Medical School, and recipient of a pilot and feasibility grant from the Boston Obesity and Nutrition Research Center (P30 DK46200). H. Kaulbach was supported by NIH training grant T32-DK07561. J. Duffy and T. Doetschmann wished to express their thanks to Mark Lewandowski and Gail Martin for the gift of the cre recombinase plasmid. We also thank Odile Peroni, Mark Pham, Dionne Rudder, Sandrine Betuing, and Mikhail Yeframashvili for technical assistance. One of the authors, John Duffy, passed away during the preparation of this manuscript. We therefore dedicate this work to his memory.

Helen C. Kaulbach and Rong Tian contributed equally to this work.

1. Taegtmeyer, H. Energy metabolism of the heart: from basic concepts to clinical applications. Curr Probl Cardiol 1994. 19:57-116.
2. Gertz, EW, Wisneski, JA, Stanley, WC, Neese, RA. Myocardial substrate utilization during exercise in humans. Dual carbon-labeled carbohydrate isotope experiments. J Clin Invest 1988. 82:2017-2025.
   View this article via: JCI PubMed Google Scholar
3. Seymour, AM, Eldar, H, Radda, GK. Hyperthyroidism results in increased glycolytic capacity in the rat heart. A 31P-NMR study. Biochim Biophys Acta 1990. 1055:107-116.
   View this article via: PubMed CrossRef Google Scholar
4. Opie, LH. Myocardial ischemia—metabolic pathways and implications of increased glycolysis. Cardiovasc Drugs Ther 1990. 4:777-790.
   View this article via: PubMed CrossRef Google Scholar
5. Owen, P, Dennis, S, Opie, LH. Glucose flux rate regulates onset of ischemic contracture in globally underperfused rat hearts. Circ Res 1990. 66:344-354.
   View this article via: PubMed Google Scholar
6. Nascimben, L, et al. Rates of insulin-independent glucose entry and glycolysis are increased in hypertrophied hearts. Circulation 1995. 92(Suppl. I):I–770-(Abstr.).
7. Weinberg, EO, Thienelt, CD, Lorell, BH. Pretranslational regulation of glucose transporter isoform expression in hearts with pressure-overload left ventricular hypertrophy. Circulation 1995. 92(Suppl. I):I–385-(Abstr.).
8. Manchester, J, Kong, X, Nerbonne, J, Lowry, OH, Lawrence (Jr), JC. Glucose transport and phosphorylation in single cardiac myocytes: rate-limiting steps in glucose metabolism. Am J Physiol 1994. 266:E326-E333.
   View this article via: PubMed Google Scholar
9. Mueckler, M. Family of glucose transporter genes. Implications for glucose homeostasis and diabetes. Diabetes 1990. 39:6-11.
   View this article via: PubMed CrossRef Google Scholar
10. Bell, GI, et al. Molecular biology of mammalian glucose transporters. Diabetes Care 1990. 13:198-208.
    View this article via: PubMed CrossRef Google Scholar
11. Slot, JW, Geuze, HJ, Gigengack, S, James, DE, Lienhard, GE. Translocation of the glucose transporter GLUT4 in cardiac myocytes of the rat. Proc Natl Acad Sci USA 1991. 88:7815-7819.
    View this article via: PubMed CrossRef Google Scholar
12. Egert, S, Nguyen, N, Brosius (III), FC, Schwaiger, M. Effects of wortmannin on insulin- and ischemia-induced stimulation of GLUT4 translocation and FDG uptake in perfused rat hearts. Cardiovasc Res 1997. 35:283-293.
    View this article via: PubMed CrossRef Google Scholar
13. Russell (III), RR, et al. Additive effects of hyperinsulinemia and ischemia on myocardial GLUT1 and GLUT4 translocation in vivo. Circulation 1998. 98:2180-2186.
    View this article via: PubMed Google Scholar
14. Young, LH, et al. Low-flow ischemia leads to translocation of canine heart GLUT-4 and GLUT-1 glucose transporters to the sarcolemma in vivo. Circulation 1997. 95:415-422.
    View this article via: PubMed Google Scholar
15. Sun, D, Nguyen, N, Degrado, TR, Schwaiger, M, Brosius, FC. Ischemia induces translocation of the insulin-responsive glucose transporter GLUT4 to the plasma membrane of cardiac myocytes. Circulation 1994. 89:793-798.
    View this article via: PubMed Google Scholar
16. Till, M, Kolter, T, Eckel, J. Molecular mechanisms of contraction-induced translocation of GLUT4 in isolated cardiomyocytes. Am J Cardiol 1997. 80:85A-89A.
    View this article via: PubMed CrossRef Google Scholar
17. Holman, GD, et al. Cell surface labeling of glucose transporter isoform GLUT4 by bis-mannose photolabel. Correlation with stimulation of glucose transport in rat adipose cells by insulin and phorbol ester. J Biol Chem 1990. 265:18172-18179.
    View this article via: PubMed Google Scholar
18. Dudek, RW, Dohm, GL, Holman, GD, Cushman, SW, Wilson, CM. Glucose transporter localization in rat skeletal muscle. Autoradiographic study using ATB-[2-3H]BMPA photolabel. FEBS Lett 1994. 339:205-208.
    View this article via: PubMed CrossRef Google Scholar
19. Kahn, BB, Rossetti, L, Lodish, HF, Charron, MJ. Decreased in vivo glucose uptake but normal expression of GLUT1 and GLUT4 in skeletal muscle of diabetic rats. J Clin Invest 1991. 87:2197-2206.
    View this article via: JCI PubMed Google Scholar
20. Kraegen, EW, et al. Glucose transporters and in vivo glucose uptake in skeletal and cardiac muscle: fasting, insulin stimulation and immunoisolation studies of GLUT1 and GLUT4. Biochem J 1993. 295:287-293.
    View this article via: PubMed Google Scholar
21. Laybutt, DR, Thompson, AL, Cooney, GJ, Kraegen, EW. Selective chronic regulation of GLUT1 and GLUT4 content by insulin, glucose, and lipid in rat cardiac muscle in vivo. Am J Physiol 1997. 273:H1309-H1316.
    View this article via: PubMed Google Scholar
22. Burcelin, R, et al. Regulation of glucose transporter and hexokinase II expression in tissues of diabetic rats. Am J Physiol 1993. 265:E392-E401.
    View this article via: PubMed Google Scholar
23. Slieker, LJ, et al. Glucose transporter levels in tissues of spontaneously diabetic Zucker fa/fa rat (ZDF/drt) and viable yellow mouse (A^vy/a). Diabetes 1992. 41:187-193.
    View this article via: PubMed CrossRef Google Scholar
24. Kainulainen, H, et al. In vivo glucose uptake and glucose transporter proteins GLUT1 and GLUT4 in heart and various types of skeletal muscle from streptozotocin-diabetic rats. Biochim Biophys Acta 1994. 1225:275-282.
    View this article via: PubMed Google Scholar
25. Garvey, WT, Hardin, D, Juhaszova, M, Dominguez, JH. Effects of diabetes on myocardial glucose transport system in rats: implications for diabetic cardiomyopathy. Am J Physiol 1993. 264:H837-H844.
    View this article via: PubMed Google Scholar
26. Paternostro, G, Clarke, K, Heath, J, Seymour, AM, Radda, GK. Decreased GLUT-4 mRNA content and insulin-sensitive deoxyglucose uptake show insulin resistance in the hypertensive rat heart. Cardiovasc Res 1995. 30:205-211.
    View this article via: PubMed CrossRef Google Scholar
27. Montessuit, C, Thorburn, A. Transcriptional activation of the glucose transporter GLUT1 in ventricular cardiac myocytes by hypertrophic agonists. J Biol Chem 1999. 274:9006-9012.
    View this article via: PubMed CrossRef Google Scholar
28. Brosius (III), FC, et al. Persistent myocardial ischemia increases GLUT1 glucose transporter expression in both ischemic and non-ischemic heart regions. J Mol Cell Cardiol 1997. 29:1675-1685.
    View this article via: PubMed CrossRef Google Scholar
29. Katz, EB, Stenbit, AE, Hatton, K, Depinho, R, Charron, MJ. Cardiac and adipose tissue abnormalities but not diabetes in mice deficient in GLUT4. Nature 1995. 377:151-155.
    View this article via: PubMed CrossRef Google Scholar
30. Stenbit, AE, et al. GLUT4 heterozygous knockout mice develop muscle insulin resistance and diabetes. Nat Med 1997. 3:1096-1101.
    View this article via: PubMed CrossRef Google Scholar
31. Tsao, TS, et al. Prevention of insulin resistance and diabetes in mice heterozygous for GLUT4 ablation by transgenic complementation of GLUT4 in skeletal muscle. Diabetes 1999. 48:775-782.
    View this article via: PubMed CrossRef Google Scholar
32. Rajewsky, K, et al. Conditional gene targeting. J Clin Invest 1996. 98:600-603.
    View this article via: JCI PubMed Google Scholar
33. Li, E, Bestor, TH, Jaenisch, R. Targeted mutation of the DNA methyltransferase gene results in embryonic lethality. Cell 1992. 69:915-926.
    View this article via: PubMed CrossRef Google Scholar
34. Papaioannou, V., and Johnson, R. 1993. Production of chimeras and genetically defined offspring from targeted ES cells. In Gene targeting: a practical approach. A.L. Joyner, editor. Oxford University Press. Oxford, United Kingdom. 107–146.
    View this article via: PubMed Google Scholar
35. Subramaniam, A, et al. Tissue-specific regulation of the alpha-myosin heavy chain gene promoter in transgenic mice. J Biol Chem 1991. 266:24613-24620.
    View this article via: PubMed Google Scholar
36. Frevert, EU, Kahn, BB. Protein kinase C isoforms ε, η, δ, and ζ, in murine adipocytes: expression, subcellular localization and tissue-specific regulation in insulin-resistant states. Biochem J 1996. 316:865-871.
    View this article via: PubMed Google Scholar
37. Armstrong, AT, Binkley, PF, Baker, PB, Myerowitz, PD, Leier, CV. Quantitative investigation of cardiomyocyte hypertrophy and myocardial fibrosis over 6 years after cardiac transplantation. J Am Coll Cardiol 1998. 32:704-710.
    View this article via: PubMed CrossRef Google Scholar
38. Chien, KR, Knowlton, KU, Zhu, H, Chien, S. Regulation of cardiac gene expression during myocardial growth and hypertrophy: molecular studies of an adaptive physiologic response. FASEB J 1991. 5:3037-3046.
    View this article via: PubMed Google Scholar
39. Fujiwara, H, Nishigaki, K. Brain natriuretic peptide in hypertrophic obstructive cardiomyopathy. Cardiologia 1998. 43:901-908.
    View this article via: PubMed Google Scholar
40. Kaplan, ML, et al. Cardiac adaptations to chronic exercise in mice. Am J Physiol 1994. 267:H1167-H1173.
    View this article via: PubMed Google Scholar
41. Hickson, RC, Hammons, GT, Holoszy, JO. Development and regression of exercise-induced cardiac hypertrophy in rats. Am J Physiol 1979. 236:H268-H272.
    View this article via: PubMed Google Scholar
42. Buttrick, PM, Kaplan, M, Leinwand, LA, Scheuer, J. Alterations in gene expression in the rat heart after chronic pathological and physiological loads. J Mol Cell Cardiol 1994. 26:61-67.
    View this article via: PubMed CrossRef Google Scholar
43. Factor, SM, Minase, T, Sonnenblick, EH. Clinical and morphological features of human hypertensive-diabetic cardiomyopathy. Am Heart J 1980. 99:446-458.
    View this article via: PubMed CrossRef Google Scholar
44. Sagnella, GA. Measurement and significance of circulating natriuretic peptides in cardiovascular disease. Clin Sci (Colch) 1998. 95:519-529.
    View this article via: CrossRef Google Scholar
45. Tian, R, Pham, M, Abel, ED. Cardiac glucose transporter deficiency increases the susceptibility of the heart to ischemic injury. Diabetes 1999. 48(Suppl. 1):A127-(Abstr.).
46. Tian, R, Pham, M, Abel, ED. Cardiac selective GLUT4 ablation exacerbates ATP depletion and contractile dysfunction during low flow ischemia. Circulation 1999. 100(Suppl. I):I-344-(Abstr.).
47. Hampton, T, Wang, J-F, Deangelis, J, Abel, ED. Preserved basal contractility but impaired responses to isoproterenol in vivo in mice with cardiac selective GLUT4 ablation. Circulation 1999. 100(Suppl. I):I-118-(Abstr.).
48. Bogoyevitch, MA, et al. Endothelin-1 and fibroblast growth factors stimulate the mitogen-activated protein kinase signaling cascade in cardiac myocytes. The potential role of the cascade in the integration of two signaling pathways leading to myocyte hypertrophy. J Biol Chem 1994. 269:1110-1119.
    View this article via: PubMed Google Scholar
49. Sadoshima, J, Izumo, S. Mechanical stretch rapidly activates multiple signal transduction pathways in cardiac myocytes: potential involvement of an autocrine/paracrine mechanism. EMBO J 1993. 12:1681-1692.
    View this article via: PubMed Google Scholar
50. Bogoyevitch, MA, et al. Adrenergic receptor stimulation of the mitogen-activated protein kinase cascade and cardiac hypertrophy. Biochem J 1996. 314:115-121.
    View this article via: PubMed Google Scholar
51. Sugden, PH, Clerk, A. “Stress-responsive” mitogen-activated protein kinases (c-Jun N-terminal kinases and p38 mitogen-activated protein kinases) in the myocardium. Circ Res 1998. 83:345-352.
    View this article via: PubMed Google Scholar
52. Fischer, Y, et al. Insulin-induced recruitment of glucose transporter 4 (GLUT4) and GLUT1 in isolated rat cardiac myocytes. Evidence of the existence of different intracellular GLUT4 vesicle populations. J Biol Chem 1997. 272:7085-7092.
    View this article via: PubMed CrossRef Google Scholar
53. Clarke, JF, Young, PW, Yonezawa, K, Kasuga, M, Holman, GD. Inhibition of the translocation of GLUT1 and GLUT4 in 3T3-L1 cells by the phosphatidylinositol 3-kinase inhibitor, wortmannin. Biochem J 1994. 300:631-635.
    View this article via: PubMed Google Scholar
54. Laidlaw, JS, Jelicks, LA, Charron, MJ. Metabolically altered and hypertrophied GLUT4-null hearts resist loss of function following ischemia/reperfusion. Diabetes 1999. 48(Suppl. 1):A75-(Abstr.).
55. Weiss, RG, et al. In vivo cardiac energetics in GLUT4-null mice. Circulation 1998. 98(Suppl. I):I–627-(Abstr.).
56. Bressler, R, Goldman, S. A role of fatty acid oxidation in cardiac hypertrophy. Cardioscience 1993. 4:133-142.
    View this article via: PubMed Google Scholar
57. Holmang, A, Yoshida, N, Jennische, E, Waldenstrom, A, Bjorntorp, P. The effects of hyperinsulinaemia on myocardial mass, blood pressure regulation and central haemodynamics in rats. Eur J Clin Invest 1996. 26:973-978.
    View this article via: PubMed CrossRef Google Scholar