GLUT4 glucose transporter deficiency increases hepatic lipid production and peripheral lipid utilization (original) (raw)
Creation and molecular characterization of adipose- and muscle-specific GLUT4–double KO mice. Mice with KO of GLUT4 simultaneously from both adipose tissue and muscle (aP2-Cre+/––MCK-Cre+/––GLUT4 lox+/+) were obtained by breeding aP2-Cre+/+–GLUT4 lox+/– mice with MCK-Cre+/+–GLUT4 lox+/– mice and identified by PCR using genomic DNA. Homozygous adipose- and muscle -specific GLUT4–double KO (AMG4KO) mice were born with the expected mendelian frequency of 25% and were indistinguishable from control littermates (carrying aP2-Cre+/– and MCK-Cre+/– but not GLUT4 lox) or adipose- and muscle-specific GLUT4–double heterozygous KO (Het KO) mice at birth.
To confirm the effect of the GLUT4 gene targeting on GLUT4 protein expression, we subjected protein extracts from white adipose tissue (WAT), brown adipose tissue (BAT), skeletal muscle, and heart to Western blot analysis. In Het KO mice, GLUT4 protein levels were slightly reduced in WAT and BAT and reduced by 40–50% in skeletal muscle and heart (Figure 1, left panels). In AMG4KO mice, GLUT4 protein levels in WAT and BAT were reduced by 70–99% and those in skeletal muscle and heart were reduced by 98–100%. In Het KO and AMG4KO mice, GLUT1 protein levels were normal in WAT and skeletal muscle (Figure 1, right panels). GLUT1 protein levels were increased in BAT and heart of AMG4KO mice.
Immunoblot analysis of GLUT4 and GLUT1 from control, Het KO, and AMG4KO mice. Tissue extracts from WAT, BAT, gastrocnemius (Gastro), soleus, and heart were prepared and immunoblotted with antibodies for GLUT4 or GLUT1. Shown are representative of 3 different immunoblots performed on 3 separate groups of control, Het KO, and AMG4KO mice. n = 17_22 for control and AMG4KO mice, n = 6 for Het KO mice.
Impact of absence of GLUT4 in adipose tissue and muscle on growth and body composition. AMG4KO mice developed normally from 3 to 25 weeks of age (Figure 2A). Body weight and body fat content determined by dual-energy x-ray absorptiometry (Figure 2B) were also normal in AMG4KO and Het KO mice compared with control littermates at 25 weeks. Body fat assessed as percent of total body weight was normal in AMG4KO mice (data not shown).
Body weight and fat content of AMG4KO mice. (A) Growth curves of female control (open squares), Het KO (open triangles), and AMG4KO (filled circles) littermates. (B) Dual-energy x-ray absorptiometry analysis of body fat content in 5- to 6-month-old female control, Het KO, and AMG4KO mice. n = 5_6 per group.
Impact of absence of GLUT4 in adipose tissue and muscle on glucose tolerance, insulin tolerance, and plasma insulin levels. To determine the physiological consequence of the absence of GLUT4 in AMG4KO mice, we performed glucose tolerance and insulin tolerance tests. Fasting blood glucose was elevated and glucose tolerance was impaired in both female (Figure 3A) and male (not shown) AMG4KO mice as early as 2 months of age. In Het KO mice at this age, fasting glucose and glucose tolerance were normal. AMG4KO mice at 2 months of age were very insulin resistant as evidenced by the fact that blood glucose failed to decrease after insulin injection (0.75 U/kg body weight), whereas in control littermates, glucose decreased about 40% (Figure 3B). Het KO mice showed elevated initial glucose level (4 hours after food was removed) and an intermediate response to insulin (Figure 3B).
AMG4KO mice have glucose intolerance and insulin resistance. (A) Glucose tolerance tests were performed after 16-hours fasting on 2-month-old female control (open squares), Het KO(open triangles), and AMG4KO(filled circles) mice. Animals were injected i.p. with 1 g D-glucose/kg body weight. Blood glucose was measured immediately before injection and 15, 30, 60, and 120 minutes after injection. n = 9_17 per group. (B) Random-fed, 2-month-old mice were injected intraperitoneally with 0.75 U/kg body weight of human regular insulin. Blood glucose was determined in the tail vein at 0, 15, 30, 45, 60, and 90 minutes. n = 5_9 per group. Results are expressed as mean blood glucose concentration ± SEM; *P < 0.05 vs. control mice; #P < 0.05 vs. Het KO mice assessed by repeated-measures ANOVA.
To determine whether glucose intolerance and insulin resistance worsens with age, we measured glucose tolerance in 6-month-old AMG4KO mice and compared the results to those from mice lacking GLUT4 in either tissue (Figure 4A). Fasting glucose was elevated and glucose tolerance was impaired in all 3 lines of GLUT4-KO mice. Surprisingly, glucose intolerance was not more marked in AMG4KO mice than in either AG4KO or MG4KO mice (Figure 4A). However, AMG4KO mice had a higher risk for insulin resistance than MG4KO mice, as evidenced by higher insulin levels in the fed state (Figure 4B). Fed insulin was increased 2.3-fold in AG4KO mice (left panel), 1.7-fold in MG4KO mice (middle panel), and 3.4-fold in AMG4KO mice (right panel) compared with their respective controls. Furthermore, in control littermates of AMG4KO, 55% of the insulin values were less than 120 pM, whereas in Het KO, AMG4KO, and AG4KO mice, none of the values were below this level. A range of fed insulin levels was observed in each of the genotypes studied, possibly due to different genetic modifiers in these outbred populations. However, these results suggest that the higher level of insulin sensitivity (lower plasma insulin levels) seen in half the control group was eliminated in Het KO, AMG4KO, and AG4KO mice. In addition, 56% of AMG4KO mice and 30% of Het KO mice had insulin values greater than 300 pM, whereas none of the control littermates had values this high. Thus, heterozygous or homozygous disruption of GLUT4 from muscle and adipose tissue simultaneously is associated with an increased risk for elevated insulin levels (Figure 4B) and insulin resistance (Figure 3B).
Effect of GLUT4 KO in both adipose tissue and skeletal muscle compared with single-tissue GLUT4 knockout mice. (A) Glucose tolerance tests were performed in female AG4KO, MG4KO, AMG4KO, and Het KO mice compared with control littermates at the age of 5_6 months. n = 9_17 per group. (B) Fed serum insulin levels were measured from female AG4KO, MG4KO, AMG4KO, and Het KO mice compared with control littermates at 4_8 months of age. Bars indicate mean values. *P < 0.05 vs. control littermates; #P < 0.05 vs. MG4KO mice; –P < 0.05 vs. AMG4KO mice.
Fed insulin levels were 2.6-fold higher in AMG4KO mice compared with MG4KO mice (Figure 4B). Interestingly, insulin levels in AMG4KO mice were not significantly higher than in AG4KO mice, which underscores the importance of GLUT4 in adipocytes in the regulation of whole-body insulin sensitivity. In Het KO mice, mean fed insulin was not elevated at 2 months (not shown) but became elevated by 4–8 months (Figure 4B), which indicates an age-related effect on insulin resistance as is seen in humans with type 2 diabetes and in mice that are heterozygous for GLUT4 targeting in all tissues (13). Fasting insulin levels in AMG4KO mice were also elevated 2-fold compared with control mice (Table 1). For all metabolic studies (Figures 5–8), AMG4KO mice with insulin values spanning the range shown in Figure 4B were used.
In vivo glucose metabolism during hyperinsulinemic-euglycemic clamp studies in awake female AMG4KO mice at 6 months of age. (A) GINF, insulin-stimulated whole-body glucose uptake, glycolysis, and glycogen synthesis in control (white bars) and AMG4KO (black bars) mice. (B) Insulin-stimulated glucose uptake in cerebellum (Cerebel), gastrocnemius, extensor digitorum longus (EDL), soleus, heart, BAT, and WAT of control and AMG4KO mice in vivo. Values are means ± SEM; *P < 0.05 vs. control littermates. n = 8 per group.
VLDL-triglycerides production and hepatic de novo lipogenesis in female AMG4KO mice. (A) Determination of VLDL-triglyceride production rates. After a 4-hour fast Triton WR 1339 was injected i.v. and serum samples were taken for a period of 4 h and assayed for triglycerides in AMG4KO (filled circles) and control (open squares) littermates. *P < 0.05 vs. control littermates. n = 6_7 per genotype. (B) Rates of 3H2O (left panel) and 14C-glucose (right panel) incorporation into liver fatty acids in fed state. n = 7_8 per genotype. (C) ACC expression in female control and AMG4KO mice after the hyperinsulinemic-euglycemic clamp study. Tissue extracts from liver, WAT, BAT, gastrocnemius, and heart were prepared and immunoblotted with peroxidase-labeled streptavidin to detect the relative content of ACC isoforms. (D) RNase protection assay for SREBP-1c mRNA in livers of control and AMG4KO mice. Total RNA (30 μg) isolated from liver was subjected to RNase protection assay. The data were normalized relative to β-actin mRNA. (E) A representative Western blot analysis of glucokinase in liver of female control and AMG4KO mice. n = 8 per genotype.
Metabolic parameters before and during hyperinsulinemic-euglycemic clamp study in control and AMG4KO mice
We found no significant differences between groups in fed plasma levels of adipokines. Leptin levels were 6.35 ± 1.88 ng/ml in controls and 9.76 ± 3.10 ng/ml in AMG4KO mice (P = NS). When data were expressed per gram of body weight for each mouse, there were still no differences between genotypes. Total adiponectin was 8.93 ± 1.61 μg/ml in controls and 8.90 ± 1.37 μg/ml in AMG4KO mice (P = NS). The proportion of high-molecular-weight adiponectin was low and did not appear to differ between groups (1.5% ± 0.7% of total adiponectin for controls, 0.2% ± 0.2% for AMG4KO; P = NS). Resistin levels were not statistically different but tended to be increased 25–30% in AMG4KO females (control, 6.88 ± 1.04 ng/ml; AMG4KO, 8.60 ± 0.95 ng/ml; P = NS). It appears that AMG4KO mice may have a higher risk for elevated plasma resistin levels. However, fed insulin levels did not correlate with resistin levels. There were no differences in levels of plasminogen activator inhibitor–1 (PAI-1) or adipose-secreted cytokines, IL-1β, IL-6, IL-8, TNF-α, or monocyte chemoattractant protein-1 (MCP-1).
Impact of decreasing GLUT4 in adipose tissue and muscle on in vivo glucose metabolism. To gain insight into glucose metabolism in different tissues, we performed 2-hour hyperinsulinemic-euglycemic clamp studies in awake 5-month-old female mice. Basal plasma glucose concentration was increased by approximately 40% in AMG4KO mice compared with control littermates (P < 0.01; Table 1). During the clamp, plasma insulin concentrations were raised to approximately 700 pM, and plasma glucose was clamped at 6.3–6.5 mM. The rate of glucose infusion needed to maintain euglycemia increased rapidly in the control mice. In contrast, glucose infusion rate (GINF) in response to insulin was markedly reduced (by approximately 75%) in AMG4KO mice (Figure 5A). Insulin-stimulated whole-body glucose uptake and whole-body glycolysis were reduced by 60–65% in AMG4KO mice, and glycogen synthesis tended to be reduced (Figure 5A).
To determine which tissues are responsible for the decrease in insulin-stimulated whole-body glucose utilization in AMG4KO mice, we measured the rate of insulin-stimulated 2-deoxyglucose (2-DG) uptake in individual tissues during the last 40 minutes of the clamp (Figure 5B). There was no difference in 2-DG uptake rate in cerebellum between AMG4KO and control mice. In extensor digitorum longus and gastrocnemius muscle, 2-DG uptake rates were markedly depressed by 85–90% in AMG4KO mice. In soleus muscle and heart, insulin-stimulated 2-DG uptake in AMG4KO was decreased by 75%. In AMG4KO WAT and BAT, 2-DG uptake was decreased approximately 65% and 50%, respectively (Figure 5B). The induction of GLUT1 expression in BAT and heart (Figure 1) may have contributed to the smaller defects in these tissues.
Impact of impaired muscle and adipose tissue glucose utilization on hepatic insulin sensitivity. Hepatic glucose production (HGP) was normal in the basal state (Table 1), but the ability of insulin to suppress HGP was impaired by 50% in AMG4KO mice (Figure 6A). To understand the mechanism, we measured the expression of the gluconeogenic enzymes, phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase), in liver samples after the insulin clamp. These enzymes are important determinants of HGP. Their expression is increased by fasting and is rapidly inhibited at the transcriptional level by insulin. Northern blot analysis revealed that after insulin infusion in fasted mice, PEPCK and G6Pase mRNA levels normalized to β-actin mRNA levels were 50% and 120% higher, respectively, in liver of AMG4KO compared with controls (Figure 6B). Under non-clamp conditions as well, liver PEPCK and G6Pase mRNA levels tended to be higher in AMG4KO mice than in controls (data not shown).
Insulin action in liver during hyperinsulinemic-euglycemic clamp study in awake female AMG4KO mice. (A) Percent suppression of basal hepatic glucose production in control and AMG4KO mice. (B) Insulin inhibition of PEPCK and G6Pase gene expression in liver of control and AMG4KO mice. RNA was prepared from liver after the hyperinsulinemic-euglycemic clamp study and was subjected to Northern blot analysis. PEPCK and G6Pase mRNA levels were normalized to β-actin mRNA levels and expressed as % of control levels. *P < 0.05 vs. control littermates. n = 6_8 per group.
Impact of impaired muscle and adipose tissue glucose utilization on energy metabolism. We expected AMG4KO mice to be more severely diabetic since glucose uptake into muscle and adipose tissue was so markedly impaired (Figure 5B). We wanted to understand more about fuel utilization in these mice, and we hypothesized that they might have increased lipid oxidation, which could enhance their insulin sensitivity (14). To test this, we performed indirect calorimetry and calculated the respiratory quotient (RQ), which is a ratio of carbohydrate oxidation to lipid oxidation. During the light period, the RQ was lower in AMG4KO mice compared with control littermates (Figure 7A). This indicates that AMG4KO mice have relatively lower carbohydrate utilization and higher fat utilization than control littermates, even though control mice are also primarily metabolizing lipid stores due to low levels of food intake during the light period. During the dark period, the RQ rose from 0.72 to 0.92 in control mice as they ate chow that is high in carbohydrate. In AMG4KO mice, the RQ rose from 0.66 to only 0.76, which reflected their continued dependence on lipid metabolism, most likely due to the impairment of glucose uptake in muscle and adipocytes.
Energy metabolism in AMG4KO mice. (A) Twenty-four-hour respiratory quotient was measured by indirect calorimetry in ad libitum_fed female mice. Light period data were collected for 5 hours starting from 13:00, and dark period data were collected for 5 hours starting from 22:00. *P < 0.05 vs. same period in control littermates. (B) Serum triglyceride and FFA levels during a fat-loading test. After a 4-hour fast, control (open symbols) and AMG4KO (closed symbols) mice were gavaged with olive oil (16.7 μl/g body weight; circles) or saline (rectangles). Before and after fat loading, blood was collected serially, and serum triglyceride and FFA levels were measured. *P < 0.05 vs. control littermates given lipid. n = 6 per group given lipids, n = 1 per group given saline.
To examine whether the AMG4KO mice could clear lipids more efficiently, we administered exogenous triglycerides intragastrically 4 hours after food removal and monitored serum triglyceride and FFA concentrations. Initial triglyceride and FFA levels were normal in AMG4KO mice (Figure 7B). Serum triglyceride levels in mice of both genotypes gavaged with saline decreased by more than 90% during the first 2 hours (Figure 7B, squares). In contrast, the serum triglyceride levels in mice of both genotypes gavaged with lipids increased to 240 mg/dl over the first 2 hours. After that, the clearance of triglycerides in AMG4KO mice was faster than in control littermates (Figure 7B, left panel). Similarly, serum FFA levels were normal in AMG4KO mice before the gavage and rose comparably in control and AMG4KO mice gavaged with lipid (Figure 7B, right panel). However, the clearance of FFA after 2 hours was faster in AMG4KO mice than in the control littermates. The comparable rise in triglycerides and FFA in the 2 genotypes suggests that the absorption of lipid from the gut was normal. There was no excess lipid in the feces of the AMG4KO mice. Thus, the enhanced lipid clearance in AMG4KO mice reflects an increased ability to metabolize triglycerides.
Hepatic lipid metabolism and glucose flux. There was no significant difference in serum triglyceride concentrations in the fed state between AMG4KO and control mice (76 ± 11 mg/dl vs. 104 ± 14 mg/dl, respectively; P = 0.79). To determine whether there was an enhanced supply and production of lipids, we injected Triton WR1339 in awake mice to block serum triglyceride clearance and measured the increase in serum triglycerides. The injection of Triton WR1339 resulted in a linear increase in serum triglyceride concentrations for 4 hours after injection (Figure 8A). Linear regression analysis of the increase in serum triglyceride concentrations revealed that the rate of hepatic triglyceride production in AMG4KO mice was increased 1.5-fold compared with control littermates (6.3 ± 0.8 mg/dl vs. 4.3 ± 0.5 mg/dl per hour; P < 0.05), which suggests increased hepatic VLDL synthesis in AMG4KO mice.
To investigate whether increased VLDL-triglyceride secretion could be caused by increased de novo lipogenesis, we injected mice with both tritiated water and [U-14C]-glucose and assessed de novo lipogenesis. The total rate of hepatic fatty acid synthesis (measured by 3H2O incorporation into fatty acids) was 2-fold higher in AMG4KO mice compared with control littermates (P < 0.05) (Figure 8B left panel). The incorporation of [U-14C]-glucose into de novo fatty acids was increased more than 3-fold in AMG4KO mice compared with control littermates (P < 0.05) (Figure 8B, right panel).
Expression of the key lipogenic enzyme, acetyl-CoA carboxylase (ACC), was increased 1.6-fold in liver of AMG4KO mice compared with control littermates (Figure 8C). This effect was specific to liver, since ACC levels were decreased in WAT and not changed in BAT, muscle, or heart of AMG4KO mice (Figure 8C). SREBP-1c is a membrane-bound transcription factor that regulates enzymes responsible for fatty acid metabolism (15). SREBP-1c mRNA levels normalized to β-actin mRNA were increased by 1.9-fold in AMG4KO mice (Figure 8D). In spite of these changes, hepatic triglyceride content did not differ between AMG4KO and control littermates (18.1 ± 1.8 mg/g vs. 16.5 ± 2.1 mg/g of liver, respectively). Muscle triglyceride content was also not altered in AMG4KO compared with control mice (13 ± 4 mg/g in AMG4KO vs. 16 ± 3 mg/g of gastrocnemius in control). Thus, increased production of triglycerides is offset by increased utilization, so there is no increase in tissue storage of triglycerides. The in vivo lipogenesis data (Figure 8B) demonstrate that part of the adaptation to the impairment of glucose transport in muscle and adipose tissue is increased glucose incorporation into fatty acids in the liver.
Glucokinase is a key enzyme in determining glucose utilization by the liver, and in some aspects it is a marker for glucose uptake in the liver. Glucokinase protein levels were increased 2.3-fold in the liver of AMG4KO mice (Figure 8E), which suggests increased glucose uptake. This is confirmed by the measured increase in glucose incorporation into lipids in the liver of AMG4KO mice compared with controls (Figure 8B). Thus, the lack of a more marked elevation of blood glucose levels in AMG4KO mice could be because the liver is compensating by taking up more glucose.