Rosiglitazone Treatment Enhances Acute AMP-Activated Protein Kinase–Me
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《糖尿病学杂志》
1 Diabetes and Obesity Research Program, Garvan Institute of Medical Research, University of New South Wales, Sydney, Australia
2 St. Vincent’s Institute of Medical Research and the Department of Medicine, University of Melbourne, Victoria, Australia
3 Commonwealth Scientific and Industrial Research Organization, Molecular Health Technologies, Victoria, Australia
ACC, acetyl-CoA carboxylase; AICAR, 5-aminoimidazole 4-carboxamide riboside; AMPK, AMP-activated protein kinase; GIR, glucose infusion rate; HGO, hepatic glucose output; LCACoA, long-chain fatty acyl-CoA; pACC, phosphorylated ACC; PPAR, peroxisome proliferator–activated receptor; TZD, thiazolidinedione
ABSTRACT
AMP-activated protein kinase (AMPK) has been implicated in the insulin-sensitizing actions of thiazolidinediones (TZDs), but it is not known whether TZD treatment can enhance tissue glucose uptake in response to AMPK activation. The present study investigated the influence of the TZD rosiglitazone on glucose turnover induced by intravenous infusion of the AMPK activator 5-aminoimidazole 4-carboxamide riboside (AICAR) under euglycemic and iso-insulinemic conditions in insulin-resistant high-fat–fed rats. We found that rosiglitazone treatment significantly enhanced AICAR-stimulated whole-body glucose disposal by 27% in high-fat–fed rats, and a 44% greater glucose infusion rate (both P < 0.01 vs. vehicle control rats) was required to maintain euglycemia. Along with this, both AICAR-stimulated glucose uptake and glucose incorporation into glycogen in muscle and adipose tissue were enhanced (P < 0.05). The enhanced glucose uptake and glycogen synthesis in muscle were associated with increased activity of total AMPK and the AMPK2 subunit. In comparison, these effects were not apparent in rats fed standard rodent diet. Thus, our findings suggest that in addition to ameliorating insulin resistance, TZDs may enhance AMPK-stimulated glucose clearance into peripheral tissues in insulin-resistant states.
It is well recognized that the effect of thiazolidinediones (TZDs) increases the sensitivity of peripheral tissues to insulin in various insulin-resistant states such as type 2 diabetes. Postulated mechanisms involve lessening excessive lipid accumulation in muscle and liver by redistributing circulating lipids to adipose tissue ("lipid steal hypothesis"), a consequence resulting in part from stimulation of peroxisome proliferator–activated receptor (PPAR)-mediated adipocyte differentiation (1,2). In addition to the "lipid steal" mechanism, TZDs may modulate the activity of AMP-activated protein kinase (AMPK) either directly or indirectly via adipokines, particularly adiponectin (3–6). However, the interaction of TZD treatment with AMPK-mediated acute glucose uptake has not been investigated.
AMPK is a metabolic fuel gauge that responds to reductions in cellular energy charge (an increase in AMP-to-ATP ratio) such as during exercise (7). The physiological role of AMPK is to restore the energy charge by stimulating fuel catabolism and inhibiting anabolic processes that are not acutely necessary for survival (8). Activated AMPK mediates an increase in glucose uptake in tissues (e.g., muscle and adipose tissue) by stimulating GLUT4 translocation to the plasma membrane (9). AMPK also decreases malonyl-CoA content by inhibiting acetyl-CoA carboxylase (ACC), thereby promoting fatty acid entry into the mitochondrion for oxidation (8,10). These metabolic effects have been well characterized both in vitro (7,11) and in vivo (12,13) with the use of the AMPK activator 5-aminoimidazole 4-carboxamide riboside (AICAR), which is phosphorylated to ZMP and activates AMPK.
Recent studies have indicated that there is defective AMPK signaling in the presence of excessive lipid accumulation in genetically obese rodents with insulin resistance (14) and in obese patients (15,16). In vitro studies have demonstrated that long-chain fatty acyl-CoAs (LCACoAs; the metabolically active forms of intracellular fatty acids) inhibit AMPK activity (17). High-fat–fed rats also have accumulated tissue LCACoAs, and this increase can be reduced by treatment with TZDs (18). We postulated that TZDs might increase tissue glucose uptake in high-fat–fed rats in response to acute AMPK activation. With the primary aim to test this hypothesis, we report here that the TZD rosiglitazone significantly enhances AICAR-stimulated glucose clearance into muscle associated with increased AMPK activation. As these effects were apparent only in high-fat–fed rats but not in rats fed standard rodent diet, we postulated that treatment with TZDs may be able to enhance AMPK-stimulated glucose metabolism in lipid-induced insulin-resistant states.
RESEARCH DESIGN AND METHODS
Male Wistar rats, supplied from the Animal Resources Centre (Perth, Australia), were conditioned at 22 ± 0.5°C with a 12/12-h light/dark cycle (lights on at 0600) for 1 week and fed standard rodent diet ad libitum. All experimental procedures were approved by the animal experimentation ethics committee (Garvan Institute/St. Vincent’s Hospital) and were in accordance with the National Health and Medical Research Council of Australia guidelines on animal experimentation. Male rats were then fed either a standard rodent chow or a high-fat diet (59% calories as fat) for 4 weeks. During the last week of feeding, animals were given rosiglitazone (4 mg · kg–1 · day–1) or 0.5% methylcellulose as vehicle between 1500 and 1700 once daily by oral gavage. A week before the study, jugular veins were cannulated under halothane anesthesia. Rats were handled daily to minimize stress. Body weight was recorded daily, and only those rats with fully recovered body weight were used for the study.
A preliminary experiment showed that infusion of AICAR alone caused profound hypoglycemia (Fig. 1). The hypoglycemic effect was rapid in onset, sustained, and then gradually reached a steady level after 1 h of infusion. Accordingly, supplement of exogenous glucose was required to maintain euglycemia during AICAR infusion (AICAR-euglycemic clamp). To assess the metabolic response to AMPK activation following treatment with rosiglitazone, an "AICAR-euglycemic clamp" in combination with tracer administration was utilized. The procedures followed those used previously for hyperinsulinemic-euglycemic clamps (18) except that the AMPK activator AICAR was infused in place of insulin. The study was performed in the conscious state 5–7 h after removal of food and at least 12 h after the final dose of rosiglitazone. After collection of baseline blood samples, AICAR was infused at a rate of 5 mg · kg–1 · min–1 via a jugular line, wherein glucose was supplemented to maintain euglycemia. The required glucose infusion rate (GIR) was used as an indicator of the whole-body responsiveness to AICAR (AMPK stimulation). When blood glucose reached the preset steady state (4.5 mmol/l, around 70–90 min), a bolus of mixed [3H]-2-deoxy-glucose and [14C]-glucose was injected via the other jugular line, which was then followed by 45 min of frequent blood sampling to determine glucose disappearance rate (Rd). Hepatic glucose output (HGO) was calculated as the difference between GIR and Rd (18). In separate experiments, the glucose tracers were injected to determine Rd or HGO at the basal state. At the end of the metabolic studies, liver, muscle, retroperitoneal fat, and subcutaneous fat (in the order of tissue collection) were immediately freeze clamped under anesthesia of nembutal. Glucose uptake rate (Rg') in tissues during AICAR infusion was determined by [3H]-2-deoxy-glucose. AICAR-stimulated glycogen synthesis rate was measured by determining [14C]-glucose incorporation into glycogen. Details of these measurements were described in previous publications of this laboratory (13,18,19).
Metabolites and hormone measurements.
Plasma glucose was determined using a glucose analyzer (YSI 2300, Yellow Springs, OH). Plasma fatty acids were determined spectrophotometrically using an acyl-CoA oxidase–based colorimetric kit (NEFA-C; Wako Pure Chemical Industries, Osaka, Japan). Plasma triglyceride concentrations were measured using enzymatic colorimetric methods (Triglyceride INT procedure 336 and GPO Trinder; Sigma). Plasma insulin and adiponectin concentrations were determined using commercially available radioimmunoassay kits according to the manufacturer’s instructions (Linco Research). Tissue LCACoAs and triglyceride content was determined using enzymatic methods as described in our previous publications (13).
AMPK and ACC assays.
The activity of total AMPK in the tissue homogenate was assayed in a buffer that contained 40 mmol/l HEPES (pH 7.0), 200 μmol/l AMP, 200 μmol/l ATP, 80 mmol/l NaCl, 8% glycerol, 0.8 mmol/l NaCl, 0.8 mmol/l dithiothreitol, and 200 μmol/l [-32P] ATP for 8 min at 37°C (13). The AMPK-specific SAMS peptide was synthesized and used as a substrate (20). For the determination of the AMPK activity associated with 1 and 2 subunits, 50 mg muscle (200 mg fat) was homogenized in buffer A (50 mmol/l Tris HCl, pH 7.5, 1 mmol/l EDTA, 1 mmol/l EGTA, 1 mmol/l dithiothreitol, 50 mmol/l NaF, 5 mmol/l Na pyrophosphate, 10% glycerol, 1% Triton X-100, 10 μg/ml trypsin inhibitor, 2 μg/ml aprotinin, 1 mmol/l benzamidine, and 1 mmol/l phenylmethylsulfonyl fluoride). Homogenates were centrifuged at 14,000g for 20 min and supernatants incubated with AMPK2 or -1 antibody–bound protein A beads for 2 h at 4°C. Immunocomplexes were washed with PBS and PBS plus 1% Triton X-100 before being resuspended in 50 mmol/l HEPES buffer (pH 7.5). The AMPK activities in the immunocomplexes were measured in assay buffer (50 mmol/l HEPES, pH 7.5, 10 mmol/l MgCl2, 1 mmol/l dithiothreitol, 250 mmol/l ATP, [-32P]ATP ([1,000 cpm/pmol], 100 μmol/l SAMS peptide, 5% glycerol, and 0.05% Triton-X-100) containing 200 μmol/l AMP as described previously (20). Activities were calculated as picomole of Pi transferred to the SAMS peptide per minute per milligram total protein. Protein concentration was determined using the bicinchoninic acid protein assay kit (Pierce, Rockford, IL). The polyclonal antipeptide antibodies to AMPK1 and -2 were raised to nonconserved regions of the AMPK isoforms 1 (373–390) and 2 (490–516). For the measurement of phosphorylated ACC (pACC), protein fractions were resolved by SDS-PAGE, transferred to nitrocellulose, and probed using an ACC-pSer79 phosphospecific antibody as described previously (21). Total ACC was measured using a conjugated streptavidin antibody (Rockland Immunochemicals), and immunoblots were analyzed using the Odyssey infrared imaging system (LI-COR Biosciences).
Statistical analysis.
All results are presented as means ± SE. A Student’s t test was used for comparisons between two individual groups. The Macintosh Statview SE + Graphic program (Abacus Concepts-Brain Power) was used to perform the statistics.
RESULTS
Whole-body metabolic parameters in the basal state and during AICAR infusion.
Table 1 summarizes whole-body metabolic parameters in the basal state and during AICAR infusion. Treatment with rosiglitazone did not significantly alter any measured metabolic parameters in chow-fed control (CH-CON) rats. Basal plasma levels of insulin and triglyceride were higher in high-fat–fed control (HF-CON) rats, while rosiglitazone reduced their values to the levels of CH-CON rats. AICAR infusion suppressed plasma levels of triglyceride and fatty acids in all four groups (P < 0.01 vs. basal values) despite maintenance of similar levels of plasma glucose and insulin. The AICAR-stimulated suppression of both triglyceride and fatty acids was greater in rosiglitazone-treated groups compared with their corresponding control groups.
Figure 2 shows whole-body glucose turnover rate in the absence and presence of AICAR infusion. In chow-fed rats, whole-body glucose disappearance rate (Rd) or HGO was similar between the CH-CON and rosiglitazone-treated chow-fed (CH-RSG) groups without AICAR infusion, whereas in high-fat–fed rats, this parameter was slightly lower in rosiglitazone-treated high-fat–fed (HF-RSG) rats (P < 0.05 vs. HF-CON). During the AICAR-euglycemic clamp, there was no significant difference in GIR required for euglycemia, Rd, and HGO between CH-CON and CH-RSG groups. However, the GIR during AICAR infusion was 25% lower in HF-CON rats compared with CH-CON rats (P < 0.05). This reduction was completely corrected in HF-RSG rats with a significant increase (25%) in AICAR-induced Rd. Compared with the HF-CON group, the increment in AICAR-induced Rd was 69% higher in the HF-RSG group (15.7 vs. 9.3 mg · kg–1 · min–1). HGO was almost completely suppressed in both HF-CON and HF-RSG groups, and there was no detectable difference in this parameter between them. This suggests that the increase in GIR in the HF-RSG was due to an enhancement of AICAR-stimulated glucose disposal (Rd) in peripheral tissues.
Changes in AICAR-stimulated glucose metabolism in tissues.
Figure 3 shows glucose uptake, as indicated by the glucose uptake rate Rg' in major peripheral tissues in response to AICAR infusion. In chow-fed rats, rosiglitazone treatment did not significantly influence AICAR-stimulated Rg'. The average Rg' from white and red muscles was similar between CH-CON and CH-RSG groups (27 ± 1 vs. 29 ± 3 μmol · 100 mg–1 · min–1). In high-fat–fed rats, there was a trend of increased Rg' in both white and red muscle with rosiglitazone treatment. Taking both muscles together, the average Rg' was 20% greater in HF-RSG rats compared with HF-CON rats (30 ± 1 and 25 ± 2 μmol · 100 mg–1 · min–1, P < 0.05). Additionally, rosiglitazone enhanced AICAR-stimulated Rg' in adipose tissue. Consistent with changes in Rg', there was no difference in AICAR-stimulated glycogen synthesis in all measured tissues between CH-CON and CH-RSG groups (Fig. 4). In contrast, compared with HF-CON rats, there was a significant increase in red muscle (by 1.2-fold) and visceral fat (by 2.7-fold) glucose incorporation into glycogen in HF-RSG rats.
Tissue lipid levels post-AICAR clamp.
Tissue LCACoA levels reflect a metabolically active form of fatty acids within cells (22). In red muscle, LCACoA levels were 48% lower in the HF-RSG group compared with HF-CON rats, (5.2 ± 0.5 vs. 7.9 ± 0.7 nmol/g, P < 0.01) but not between CH-CON and CH-RSG groups (3.4 ± 0.5 vs. 3.7 ± 1.0 nmol/g, respectively, n = 6 per group). There was no difference in this parameter in either white muscle or liver between rosiglitazone and vehicle treatments in either chow-fed or high-fat–fed rats (data not shown). Compared with CH-CON rats, triglyceride levels were high in both red muscle (1.0 ± 0.1 vs. 1.4 ± 0.1 μmol/g, P < 0.05) and liver (8.6 ± 1.0 vs. 15.3 ± 0.8 μmol/g, P < 0.01) in HF-CON rats. However, its levels were similar between HF-CON and HF-RSG groups in muscle (1.4 ± 0.1 vs. 1.6 ± 0.2 μmol/g in red muscle; 2.6 ± 0.6 vs. 3.6 ± 0.6 μmol/g in white muscle) and liver (15.3 ± 0.8 vs. 14.8 ± 2.5 μmol/g, n = 6 per group).
Activity of AMPK and ACC.
As shown in Fig. 5A, rosiglitazone treatment had no significant influence on AICAR-stimulated activity of total AMPK in either white or red muscle in chow-fed rats. While there appeared to be a trend toward increased AMPK1 activity in white muscle of CH-RSG rats in response to AICAR stimulation, its value was similar in red muscle compared with CH-RSG rats. There was no difference in AICAR-stimulated AMPK2 activity between the CH-CON and CH-RSG groups. In contrast, there was a substantial increase (47%) in the activity of total AMPK in red muscle in response to AICAR stimulation in HF-RSG rats compared with HF-CON rats (Fig. 5B). Associated with the increased total AMPK activity, AMPK2 activity in red muscle was almost twice as high in HF-RSG rats as in HF-CON rats, whereas AICAR-stimulated AMPK2 activity was not different between the CH-CON and CH-RSG groups. In the absence of AICAR, AMPK2 activity was similar between vehicle- and rosiglitazone-treated groups (data not shown). In comparison, there was no difference in AMPK1 activity in either white or red muscle between the HF-CON and HF-RSG groups following AICAR infusion. While AICAR-stimulated AMPK1 activity in fat tissue was not different between CH-CON and CH-RSG rats (78 ± 14 vs. 87 ± 16 pmol · mg protein–1 · min–1), it tended to be higher in HF-RSG rats compared with HF-CON rats (138 ± 17 vs. 95 ± 17 pmol · mg protein–1 · min–1, P = 0.09). No AMPK2 activity was detected in adipose tissue of chow-fed or high-fat–fed rats.
In liver, the activity of total AMPK after the AICAR clamp was 36% lower in the HF-CON group compared with the CH-CON group (313 ± 21 vs. 464 ± 61 pmol · mg protein–1 · min–1, P < 0.05, n = 6 per group). Rosiglitazone treatment did not show any significant effect on AICAR-stimulated liver AMPK activity in either chow-fed rats (469 ± 60 pmol · mg protein–1 · min–1) or high-fat–fed rats (362 ± 35 pmol · mg protein–1 · min–1) compared with their corresponding control groups.
Figure 6 shows the ratio of pACC to total values obtained from Western blots as an indication of ACC activity. The overall pattern in muscles and adipose tissue was similar in chow-fed and high-fat–fed rats. We did not detect a significant difference in ACC content and activity between rosiglitazone and control groups in either chow-fed or high-fat–fed rats after AICAR infusion. There was also no difference in muscle pACC–to–total ACC ratio in the absence of AICAR between vehicle and rosiglitazone treatments (data not shown). Total levels of ACC were unchanged with rosiglitazone treatment in both chow-fed and high-fat–fed rats in all tissues measured. In liver, the ratio of pACC to total ACC was 29% lower in HF-CON (1.58 ± 0.21 vs. 2.04 ± 0.10 in CH-CON, P < 0.05, n = 6 per group). However, this ratio was not altered by rosiglitazone treatment in either HF-RSG (1.79 ± 0.15) or CH-RSG (1.67 ± 0.41) group compared with their corresponding control groups.
DISCUSSION
While TZDs have been shown to modulate AMPK activity (3–5), it is not clear whether TZD treatment contributes to AMPK-mediated metabolic effects. The purpose of this study was to investigate whether TZD treatment increases peripheral glucose uptake in response to acute AMPK activation. The study demonstrates that treatment with rosiglitazone significantly enhances glucose uptake into muscle and adipose tissue (as indicated by Rd, Rg', and glycogen synthesis rate) in response to acute stimulation by the AMPK activator AICAR in insulin-resistant high-fat–fed rats. This was also indicated by a greater infusion rate of exogenous glucose (GIR) required to maintain euglycemia. Interestingly, we found that this potentiation was only present in high-fat–fed rats and did not occur in normal chow-fed rats. At least part of the mechanism of the enhanced peripheral glucose clearance involves potentiation of AMPK activation by AICAR in high-fat–fed rats.
To identify the target sites responsible for the enhanced glucose disposal, we determined AICAR-stimulated Rg' in individual muscle types and adipose tissue. The obtained data showed significantly greater increases in AICAR-stimulated Rg' in muscle (as indicated by the average value from combined red and white muscle) and adipose tissue in HF-RSG rats (not in CH-RSG rats). Since these effects appeared to be more apparent in red muscle and visceral fat, we further examined AICAR-stimulated glycogen synthesis and found that changes in AICAR-stimulated glycogen synthesis were in accord with changes in Rg' in muscle and fat. These findings, taken together, indicate that both muscle and adipose tissues contributed to AICAR-stimulated whole-body glucose disposal in high-fat–fed rats.
To investigate associated changes in AMPK signaling, we compared the activity of total AMPK in the tissue lysate from muscle at the end of AICAR infusion. As predicted, the activity of total AMPK was higher in red muscle in HF-RSG rats compared with HF-CON rats, consistent with AICAR-stimulated glucose uptake in this group. Further analyses showed that while AICAR stimulated AMPK1 activity to a similar extent in control and rosiglitazone-treated rats, the activity of AMPK2 in red muscle was almost twofold higher in HF-RSG rats when compared with HF-CON rats. As AMPK2 is the main subunit mediating glucose metabolism (23), the enhancement of AICAR-stimulated glycogen synthesis (as well as glucose uptake) in red muscle was likely to be, at least in part, due to an increased AMPK2 activation. Our results are consistent with a very recent report showing that the activity of muscle AMPK2, but not -1, is reduced in obese Zucker rats and chronic rosiglitazone administration can restore its activity (24). While AMPK2 activity was not detectable in adipose tissue, AMPK1 activity appeared to be increased in this tissue. It remains to be determined whether AMPK1 was involved in the AICAR-stimulated glucose uptake in adipose tissue.
The mechanism by which rosiglitazone enhances AMPK-stimulated glucose uptake in muscle is not clear. Several research groups (3,4) have shown that TZDs acutely activate AMPK in rat muscle in vitro and in vivo by a PPAR-independent mechanism, which appears to be associated with a decrease in cellular energy state. Recently, Lebrasseur et al. (25) extended these earlier studies by showing increases in muscle AMPK activity in intact rats 15 min after an intraperitoneal injection of troglitazone at a dose of 8 mg/kg. One possibility for the increase in AMPK in muscle of the fat-fed rats treated with rosiglitazone in the present study could be due to such a mechanism. However, time-course studies in isolated muscle suggest that the direct activation of AMPK by TZDs and associated metabolic changes subside within 60 min (25). Consistent with this, we found no difference between the HF-CON and HF-RSG groups in muscle AMPK2 activity or pACC in the absence of AICAR stimulation 12 h after the last dose of rosiglitazone (4 mg/kg by oral gavage). Thus, the enhanced AICAR-stimulated AMPK activity with rosiglitazone treatment may be more likely to result from PPAR-induced changes in gene expression, as indicated by increases in plasma adiponectin levels. It has been reported that TZDs can upregulate adiponectin expression and plasma adiponectin levels, which could potentially activate AMPK activity. Although plasma levels of adiponectin in rosiglitazone-treated chow-fed and high-fat–fed rats were substantially increased, this is unlikely to be a major mechanism because there was no increase in basal Rd (or HGO) in rosiglitazone-treated rats. Moreover, the enhancement of AMPK-mediated glucose metabolism was specific to AICAR stimulation in high-fat–fed rats.
Several lines of evidence appear to point to a possible link between the enhanced AMPK stimulation of glucose uptake in muscle to rosiglitazone’s effects on lipid me-tabolism. First, we found the enhancement of AICAR-stimulated glucose uptake and glycogen synthesis by rosiglitazone only apparent in high-fat–fed rats but not in chow-fed rats. Second, AMPK signaling has been shown to be defective in the presence of excessive lipid accumulation in genetically obese rodents (14,24) and obese patients (15,16). Furthermore, it has been recently shown that LCACoAs can inhibit AMPK activation by LKB1 (17). We have previously reported that TZDs lessen an excessive accumulation of LCACoAs in red muscle of high-fat–fed rats (18,26) in the absence of insulin or AICAR stimulation. Based on these studies, we speculate that the enhancement of AICAR-stimulated glucose uptake by rosiglitazone in high-fat–fed rats may be related to the lipid- lowering effects of rosiglitazone. This interpretation is indeed supported by the fact that a lower LCACoA level was preserved in red muscle during AICAR clamp in rosiglitazone-treated high-fat–fed rats in association with the improved AMPK activation and glucose uptake in this tissue. However, further studies are required to establish whether this is a causal relationship.
Although pACC was increased at the end of the AICAR infusion (data not shown), we did not find an enhancement of its phosphorylation in either muscle or adipose tissue in rosiglitazone-treated high-fat–fed rats. This was somewhat unexpected because the extent of ACC phosphorylation is believed to be closely associated with AMPK activity. However, a recent study has also shown that acute ischemia in the kidney increases AMPK activity up to threefold without detected increases in ACC phosphorylation (27). There is new evidence to suggest that lipids affects AMPK phosphorylation and ACC phosphorylation separately in skeletal muscle cells (28). Regardless of this disparity, our data support the notion that AMPK-stimulated glucose uptake is mediated by different downstream signaling molecules rather than via ACC (29).
AICAR has been shown to exert a strong effect to suppress hepatic glucose output involving both AMPK activation and AMPK-independent pathways (30,31). In an earlier study in normal rats during AICAR infusion, hepatic glucose output was close to zero (12). In the present study, hepatic glucose production was almost completely suppressed by AICAR infusion at the dose used, and no significant difference in hepatic glucose output was found between the HF-CON and HF-RSG groups. Consistent with this, rosiglitazone did not show a significant effect to enhance AICAR-stimulated AMPK activity in the liver. These data suggest that the liver is unlikely to be a major contributor to the improvement of AMPK-induced glucose metabolism in high-fat–fed rats following rosiglitazone treatment.
In summary, the present study demonstrates for the first time that treatment with the TZD rosiglitazone for 1 week enhances acute AMPK-mediated glucose disposal in insulin-resistant high-fat–fed rats. Muscle and adipose tissue were the major sites for the enhanced disposal of circulating glucose. The enhanced glucose uptake in muscle was associated with increased activity of the AMPK2 subunit, suggesting that rosiglitazone potentiates AICAR-stimulated glucose uptake in peripheral tissues via enhancing AMPK2. Thus, we conclude that in addition to being insulin sensitizers, TZDs can also sensitize AMPK-mediated glucose disposal in peripheral tissues in lipid-induced insulin-resistant states. Since AMPK is activated during physical activity, our findings also imply that TZDs may increase effectiveness of exercise for the control of hyperglycemia in insulin-resistant subjects such as obese patients with type 2 diabetes.
ACKNOWLEDGMENTS
This study was supported by grants from the Diabetes Australian Research Trust (DART) (to J.-M.Y. and E.K.) and the National Health and Medical Research Council (NHMRC) (to E.K.). The Australian Research Council and NHMRC support B.K. N.Z. is currently supported by a National Heart Foundation postgraduate scholarship.
FOOTNOTES
The costs of publication of this article were defrayed in part by the payment of page charges. This 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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2 St. Vincent’s Institute of Medical Research and the Department of Medicine, University of Melbourne, Victoria, Australia
3 Commonwealth Scientific and Industrial Research Organization, Molecular Health Technologies, Victoria, Australia
ACC, acetyl-CoA carboxylase; AICAR, 5-aminoimidazole 4-carboxamide riboside; AMPK, AMP-activated protein kinase; GIR, glucose infusion rate; HGO, hepatic glucose output; LCACoA, long-chain fatty acyl-CoA; pACC, phosphorylated ACC; PPAR, peroxisome proliferator–activated receptor; TZD, thiazolidinedione
ABSTRACT
AMP-activated protein kinase (AMPK) has been implicated in the insulin-sensitizing actions of thiazolidinediones (TZDs), but it is not known whether TZD treatment can enhance tissue glucose uptake in response to AMPK activation. The present study investigated the influence of the TZD rosiglitazone on glucose turnover induced by intravenous infusion of the AMPK activator 5-aminoimidazole 4-carboxamide riboside (AICAR) under euglycemic and iso-insulinemic conditions in insulin-resistant high-fat–fed rats. We found that rosiglitazone treatment significantly enhanced AICAR-stimulated whole-body glucose disposal by 27% in high-fat–fed rats, and a 44% greater glucose infusion rate (both P < 0.01 vs. vehicle control rats) was required to maintain euglycemia. Along with this, both AICAR-stimulated glucose uptake and glucose incorporation into glycogen in muscle and adipose tissue were enhanced (P < 0.05). The enhanced glucose uptake and glycogen synthesis in muscle were associated with increased activity of total AMPK and the AMPK2 subunit. In comparison, these effects were not apparent in rats fed standard rodent diet. Thus, our findings suggest that in addition to ameliorating insulin resistance, TZDs may enhance AMPK-stimulated glucose clearance into peripheral tissues in insulin-resistant states.
It is well recognized that the effect of thiazolidinediones (TZDs) increases the sensitivity of peripheral tissues to insulin in various insulin-resistant states such as type 2 diabetes. Postulated mechanisms involve lessening excessive lipid accumulation in muscle and liver by redistributing circulating lipids to adipose tissue ("lipid steal hypothesis"), a consequence resulting in part from stimulation of peroxisome proliferator–activated receptor (PPAR)-mediated adipocyte differentiation (1,2). In addition to the "lipid steal" mechanism, TZDs may modulate the activity of AMP-activated protein kinase (AMPK) either directly or indirectly via adipokines, particularly adiponectin (3–6). However, the interaction of TZD treatment with AMPK-mediated acute glucose uptake has not been investigated.
AMPK is a metabolic fuel gauge that responds to reductions in cellular energy charge (an increase in AMP-to-ATP ratio) such as during exercise (7). The physiological role of AMPK is to restore the energy charge by stimulating fuel catabolism and inhibiting anabolic processes that are not acutely necessary for survival (8). Activated AMPK mediates an increase in glucose uptake in tissues (e.g., muscle and adipose tissue) by stimulating GLUT4 translocation to the plasma membrane (9). AMPK also decreases malonyl-CoA content by inhibiting acetyl-CoA carboxylase (ACC), thereby promoting fatty acid entry into the mitochondrion for oxidation (8,10). These metabolic effects have been well characterized both in vitro (7,11) and in vivo (12,13) with the use of the AMPK activator 5-aminoimidazole 4-carboxamide riboside (AICAR), which is phosphorylated to ZMP and activates AMPK.
Recent studies have indicated that there is defective AMPK signaling in the presence of excessive lipid accumulation in genetically obese rodents with insulin resistance (14) and in obese patients (15,16). In vitro studies have demonstrated that long-chain fatty acyl-CoAs (LCACoAs; the metabolically active forms of intracellular fatty acids) inhibit AMPK activity (17). High-fat–fed rats also have accumulated tissue LCACoAs, and this increase can be reduced by treatment with TZDs (18). We postulated that TZDs might increase tissue glucose uptake in high-fat–fed rats in response to acute AMPK activation. With the primary aim to test this hypothesis, we report here that the TZD rosiglitazone significantly enhances AICAR-stimulated glucose clearance into muscle associated with increased AMPK activation. As these effects were apparent only in high-fat–fed rats but not in rats fed standard rodent diet, we postulated that treatment with TZDs may be able to enhance AMPK-stimulated glucose metabolism in lipid-induced insulin-resistant states.
RESEARCH DESIGN AND METHODS
Male Wistar rats, supplied from the Animal Resources Centre (Perth, Australia), were conditioned at 22 ± 0.5°C with a 12/12-h light/dark cycle (lights on at 0600) for 1 week and fed standard rodent diet ad libitum. All experimental procedures were approved by the animal experimentation ethics committee (Garvan Institute/St. Vincent’s Hospital) and were in accordance with the National Health and Medical Research Council of Australia guidelines on animal experimentation. Male rats were then fed either a standard rodent chow or a high-fat diet (59% calories as fat) for 4 weeks. During the last week of feeding, animals were given rosiglitazone (4 mg · kg–1 · day–1) or 0.5% methylcellulose as vehicle between 1500 and 1700 once daily by oral gavage. A week before the study, jugular veins were cannulated under halothane anesthesia. Rats were handled daily to minimize stress. Body weight was recorded daily, and only those rats with fully recovered body weight were used for the study.
A preliminary experiment showed that infusion of AICAR alone caused profound hypoglycemia (Fig. 1). The hypoglycemic effect was rapid in onset, sustained, and then gradually reached a steady level after 1 h of infusion. Accordingly, supplement of exogenous glucose was required to maintain euglycemia during AICAR infusion (AICAR-euglycemic clamp). To assess the metabolic response to AMPK activation following treatment with rosiglitazone, an "AICAR-euglycemic clamp" in combination with tracer administration was utilized. The procedures followed those used previously for hyperinsulinemic-euglycemic clamps (18) except that the AMPK activator AICAR was infused in place of insulin. The study was performed in the conscious state 5–7 h after removal of food and at least 12 h after the final dose of rosiglitazone. After collection of baseline blood samples, AICAR was infused at a rate of 5 mg · kg–1 · min–1 via a jugular line, wherein glucose was supplemented to maintain euglycemia. The required glucose infusion rate (GIR) was used as an indicator of the whole-body responsiveness to AICAR (AMPK stimulation). When blood glucose reached the preset steady state (4.5 mmol/l, around 70–90 min), a bolus of mixed [3H]-2-deoxy-glucose and [14C]-glucose was injected via the other jugular line, which was then followed by 45 min of frequent blood sampling to determine glucose disappearance rate (Rd). Hepatic glucose output (HGO) was calculated as the difference between GIR and Rd (18). In separate experiments, the glucose tracers were injected to determine Rd or HGO at the basal state. At the end of the metabolic studies, liver, muscle, retroperitoneal fat, and subcutaneous fat (in the order of tissue collection) were immediately freeze clamped under anesthesia of nembutal. Glucose uptake rate (Rg') in tissues during AICAR infusion was determined by [3H]-2-deoxy-glucose. AICAR-stimulated glycogen synthesis rate was measured by determining [14C]-glucose incorporation into glycogen. Details of these measurements were described in previous publications of this laboratory (13,18,19).
Metabolites and hormone measurements.
Plasma glucose was determined using a glucose analyzer (YSI 2300, Yellow Springs, OH). Plasma fatty acids were determined spectrophotometrically using an acyl-CoA oxidase–based colorimetric kit (NEFA-C; Wako Pure Chemical Industries, Osaka, Japan). Plasma triglyceride concentrations were measured using enzymatic colorimetric methods (Triglyceride INT procedure 336 and GPO Trinder; Sigma). Plasma insulin and adiponectin concentrations were determined using commercially available radioimmunoassay kits according to the manufacturer’s instructions (Linco Research). Tissue LCACoAs and triglyceride content was determined using enzymatic methods as described in our previous publications (13).
AMPK and ACC assays.
The activity of total AMPK in the tissue homogenate was assayed in a buffer that contained 40 mmol/l HEPES (pH 7.0), 200 μmol/l AMP, 200 μmol/l ATP, 80 mmol/l NaCl, 8% glycerol, 0.8 mmol/l NaCl, 0.8 mmol/l dithiothreitol, and 200 μmol/l [-32P] ATP for 8 min at 37°C (13). The AMPK-specific SAMS peptide was synthesized and used as a substrate (20). For the determination of the AMPK activity associated with 1 and 2 subunits, 50 mg muscle (200 mg fat) was homogenized in buffer A (50 mmol/l Tris HCl, pH 7.5, 1 mmol/l EDTA, 1 mmol/l EGTA, 1 mmol/l dithiothreitol, 50 mmol/l NaF, 5 mmol/l Na pyrophosphate, 10% glycerol, 1% Triton X-100, 10 μg/ml trypsin inhibitor, 2 μg/ml aprotinin, 1 mmol/l benzamidine, and 1 mmol/l phenylmethylsulfonyl fluoride). Homogenates were centrifuged at 14,000g for 20 min and supernatants incubated with AMPK2 or -1 antibody–bound protein A beads for 2 h at 4°C. Immunocomplexes were washed with PBS and PBS plus 1% Triton X-100 before being resuspended in 50 mmol/l HEPES buffer (pH 7.5). The AMPK activities in the immunocomplexes were measured in assay buffer (50 mmol/l HEPES, pH 7.5, 10 mmol/l MgCl2, 1 mmol/l dithiothreitol, 250 mmol/l ATP, [-32P]ATP ([1,000 cpm/pmol], 100 μmol/l SAMS peptide, 5% glycerol, and 0.05% Triton-X-100) containing 200 μmol/l AMP as described previously (20). Activities were calculated as picomole of Pi transferred to the SAMS peptide per minute per milligram total protein. Protein concentration was determined using the bicinchoninic acid protein assay kit (Pierce, Rockford, IL). The polyclonal antipeptide antibodies to AMPK1 and -2 were raised to nonconserved regions of the AMPK isoforms 1 (373–390) and 2 (490–516). For the measurement of phosphorylated ACC (pACC), protein fractions were resolved by SDS-PAGE, transferred to nitrocellulose, and probed using an ACC-pSer79 phosphospecific antibody as described previously (21). Total ACC was measured using a conjugated streptavidin antibody (Rockland Immunochemicals), and immunoblots were analyzed using the Odyssey infrared imaging system (LI-COR Biosciences).
Statistical analysis.
All results are presented as means ± SE. A Student’s t test was used for comparisons between two individual groups. The Macintosh Statview SE + Graphic program (Abacus Concepts-Brain Power) was used to perform the statistics.
RESULTS
Whole-body metabolic parameters in the basal state and during AICAR infusion.
Table 1 summarizes whole-body metabolic parameters in the basal state and during AICAR infusion. Treatment with rosiglitazone did not significantly alter any measured metabolic parameters in chow-fed control (CH-CON) rats. Basal plasma levels of insulin and triglyceride were higher in high-fat–fed control (HF-CON) rats, while rosiglitazone reduced their values to the levels of CH-CON rats. AICAR infusion suppressed plasma levels of triglyceride and fatty acids in all four groups (P < 0.01 vs. basal values) despite maintenance of similar levels of plasma glucose and insulin. The AICAR-stimulated suppression of both triglyceride and fatty acids was greater in rosiglitazone-treated groups compared with their corresponding control groups.
Figure 2 shows whole-body glucose turnover rate in the absence and presence of AICAR infusion. In chow-fed rats, whole-body glucose disappearance rate (Rd) or HGO was similar between the CH-CON and rosiglitazone-treated chow-fed (CH-RSG) groups without AICAR infusion, whereas in high-fat–fed rats, this parameter was slightly lower in rosiglitazone-treated high-fat–fed (HF-RSG) rats (P < 0.05 vs. HF-CON). During the AICAR-euglycemic clamp, there was no significant difference in GIR required for euglycemia, Rd, and HGO between CH-CON and CH-RSG groups. However, the GIR during AICAR infusion was 25% lower in HF-CON rats compared with CH-CON rats (P < 0.05). This reduction was completely corrected in HF-RSG rats with a significant increase (25%) in AICAR-induced Rd. Compared with the HF-CON group, the increment in AICAR-induced Rd was 69% higher in the HF-RSG group (15.7 vs. 9.3 mg · kg–1 · min–1). HGO was almost completely suppressed in both HF-CON and HF-RSG groups, and there was no detectable difference in this parameter between them. This suggests that the increase in GIR in the HF-RSG was due to an enhancement of AICAR-stimulated glucose disposal (Rd) in peripheral tissues.
Changes in AICAR-stimulated glucose metabolism in tissues.
Figure 3 shows glucose uptake, as indicated by the glucose uptake rate Rg' in major peripheral tissues in response to AICAR infusion. In chow-fed rats, rosiglitazone treatment did not significantly influence AICAR-stimulated Rg'. The average Rg' from white and red muscles was similar between CH-CON and CH-RSG groups (27 ± 1 vs. 29 ± 3 μmol · 100 mg–1 · min–1). In high-fat–fed rats, there was a trend of increased Rg' in both white and red muscle with rosiglitazone treatment. Taking both muscles together, the average Rg' was 20% greater in HF-RSG rats compared with HF-CON rats (30 ± 1 and 25 ± 2 μmol · 100 mg–1 · min–1, P < 0.05). Additionally, rosiglitazone enhanced AICAR-stimulated Rg' in adipose tissue. Consistent with changes in Rg', there was no difference in AICAR-stimulated glycogen synthesis in all measured tissues between CH-CON and CH-RSG groups (Fig. 4). In contrast, compared with HF-CON rats, there was a significant increase in red muscle (by 1.2-fold) and visceral fat (by 2.7-fold) glucose incorporation into glycogen in HF-RSG rats.
Tissue lipid levels post-AICAR clamp.
Tissue LCACoA levels reflect a metabolically active form of fatty acids within cells (22). In red muscle, LCACoA levels were 48% lower in the HF-RSG group compared with HF-CON rats, (5.2 ± 0.5 vs. 7.9 ± 0.7 nmol/g, P < 0.01) but not between CH-CON and CH-RSG groups (3.4 ± 0.5 vs. 3.7 ± 1.0 nmol/g, respectively, n = 6 per group). There was no difference in this parameter in either white muscle or liver between rosiglitazone and vehicle treatments in either chow-fed or high-fat–fed rats (data not shown). Compared with CH-CON rats, triglyceride levels were high in both red muscle (1.0 ± 0.1 vs. 1.4 ± 0.1 μmol/g, P < 0.05) and liver (8.6 ± 1.0 vs. 15.3 ± 0.8 μmol/g, P < 0.01) in HF-CON rats. However, its levels were similar between HF-CON and HF-RSG groups in muscle (1.4 ± 0.1 vs. 1.6 ± 0.2 μmol/g in red muscle; 2.6 ± 0.6 vs. 3.6 ± 0.6 μmol/g in white muscle) and liver (15.3 ± 0.8 vs. 14.8 ± 2.5 μmol/g, n = 6 per group).
Activity of AMPK and ACC.
As shown in Fig. 5A, rosiglitazone treatment had no significant influence on AICAR-stimulated activity of total AMPK in either white or red muscle in chow-fed rats. While there appeared to be a trend toward increased AMPK1 activity in white muscle of CH-RSG rats in response to AICAR stimulation, its value was similar in red muscle compared with CH-RSG rats. There was no difference in AICAR-stimulated AMPK2 activity between the CH-CON and CH-RSG groups. In contrast, there was a substantial increase (47%) in the activity of total AMPK in red muscle in response to AICAR stimulation in HF-RSG rats compared with HF-CON rats (Fig. 5B). Associated with the increased total AMPK activity, AMPK2 activity in red muscle was almost twice as high in HF-RSG rats as in HF-CON rats, whereas AICAR-stimulated AMPK2 activity was not different between the CH-CON and CH-RSG groups. In the absence of AICAR, AMPK2 activity was similar between vehicle- and rosiglitazone-treated groups (data not shown). In comparison, there was no difference in AMPK1 activity in either white or red muscle between the HF-CON and HF-RSG groups following AICAR infusion. While AICAR-stimulated AMPK1 activity in fat tissue was not different between CH-CON and CH-RSG rats (78 ± 14 vs. 87 ± 16 pmol · mg protein–1 · min–1), it tended to be higher in HF-RSG rats compared with HF-CON rats (138 ± 17 vs. 95 ± 17 pmol · mg protein–1 · min–1, P = 0.09). No AMPK2 activity was detected in adipose tissue of chow-fed or high-fat–fed rats.
In liver, the activity of total AMPK after the AICAR clamp was 36% lower in the HF-CON group compared with the CH-CON group (313 ± 21 vs. 464 ± 61 pmol · mg protein–1 · min–1, P < 0.05, n = 6 per group). Rosiglitazone treatment did not show any significant effect on AICAR-stimulated liver AMPK activity in either chow-fed rats (469 ± 60 pmol · mg protein–1 · min–1) or high-fat–fed rats (362 ± 35 pmol · mg protein–1 · min–1) compared with their corresponding control groups.
Figure 6 shows the ratio of pACC to total values obtained from Western blots as an indication of ACC activity. The overall pattern in muscles and adipose tissue was similar in chow-fed and high-fat–fed rats. We did not detect a significant difference in ACC content and activity between rosiglitazone and control groups in either chow-fed or high-fat–fed rats after AICAR infusion. There was also no difference in muscle pACC–to–total ACC ratio in the absence of AICAR between vehicle and rosiglitazone treatments (data not shown). Total levels of ACC were unchanged with rosiglitazone treatment in both chow-fed and high-fat–fed rats in all tissues measured. In liver, the ratio of pACC to total ACC was 29% lower in HF-CON (1.58 ± 0.21 vs. 2.04 ± 0.10 in CH-CON, P < 0.05, n = 6 per group). However, this ratio was not altered by rosiglitazone treatment in either HF-RSG (1.79 ± 0.15) or CH-RSG (1.67 ± 0.41) group compared with their corresponding control groups.
DISCUSSION
While TZDs have been shown to modulate AMPK activity (3–5), it is not clear whether TZD treatment contributes to AMPK-mediated metabolic effects. The purpose of this study was to investigate whether TZD treatment increases peripheral glucose uptake in response to acute AMPK activation. The study demonstrates that treatment with rosiglitazone significantly enhances glucose uptake into muscle and adipose tissue (as indicated by Rd, Rg', and glycogen synthesis rate) in response to acute stimulation by the AMPK activator AICAR in insulin-resistant high-fat–fed rats. This was also indicated by a greater infusion rate of exogenous glucose (GIR) required to maintain euglycemia. Interestingly, we found that this potentiation was only present in high-fat–fed rats and did not occur in normal chow-fed rats. At least part of the mechanism of the enhanced peripheral glucose clearance involves potentiation of AMPK activation by AICAR in high-fat–fed rats.
To identify the target sites responsible for the enhanced glucose disposal, we determined AICAR-stimulated Rg' in individual muscle types and adipose tissue. The obtained data showed significantly greater increases in AICAR-stimulated Rg' in muscle (as indicated by the average value from combined red and white muscle) and adipose tissue in HF-RSG rats (not in CH-RSG rats). Since these effects appeared to be more apparent in red muscle and visceral fat, we further examined AICAR-stimulated glycogen synthesis and found that changes in AICAR-stimulated glycogen synthesis were in accord with changes in Rg' in muscle and fat. These findings, taken together, indicate that both muscle and adipose tissues contributed to AICAR-stimulated whole-body glucose disposal in high-fat–fed rats.
To investigate associated changes in AMPK signaling, we compared the activity of total AMPK in the tissue lysate from muscle at the end of AICAR infusion. As predicted, the activity of total AMPK was higher in red muscle in HF-RSG rats compared with HF-CON rats, consistent with AICAR-stimulated glucose uptake in this group. Further analyses showed that while AICAR stimulated AMPK1 activity to a similar extent in control and rosiglitazone-treated rats, the activity of AMPK2 in red muscle was almost twofold higher in HF-RSG rats when compared with HF-CON rats. As AMPK2 is the main subunit mediating glucose metabolism (23), the enhancement of AICAR-stimulated glycogen synthesis (as well as glucose uptake) in red muscle was likely to be, at least in part, due to an increased AMPK2 activation. Our results are consistent with a very recent report showing that the activity of muscle AMPK2, but not -1, is reduced in obese Zucker rats and chronic rosiglitazone administration can restore its activity (24). While AMPK2 activity was not detectable in adipose tissue, AMPK1 activity appeared to be increased in this tissue. It remains to be determined whether AMPK1 was involved in the AICAR-stimulated glucose uptake in adipose tissue.
The mechanism by which rosiglitazone enhances AMPK-stimulated glucose uptake in muscle is not clear. Several research groups (3,4) have shown that TZDs acutely activate AMPK in rat muscle in vitro and in vivo by a PPAR-independent mechanism, which appears to be associated with a decrease in cellular energy state. Recently, Lebrasseur et al. (25) extended these earlier studies by showing increases in muscle AMPK activity in intact rats 15 min after an intraperitoneal injection of troglitazone at a dose of 8 mg/kg. One possibility for the increase in AMPK in muscle of the fat-fed rats treated with rosiglitazone in the present study could be due to such a mechanism. However, time-course studies in isolated muscle suggest that the direct activation of AMPK by TZDs and associated metabolic changes subside within 60 min (25). Consistent with this, we found no difference between the HF-CON and HF-RSG groups in muscle AMPK2 activity or pACC in the absence of AICAR stimulation 12 h after the last dose of rosiglitazone (4 mg/kg by oral gavage). Thus, the enhanced AICAR-stimulated AMPK activity with rosiglitazone treatment may be more likely to result from PPAR-induced changes in gene expression, as indicated by increases in plasma adiponectin levels. It has been reported that TZDs can upregulate adiponectin expression and plasma adiponectin levels, which could potentially activate AMPK activity. Although plasma levels of adiponectin in rosiglitazone-treated chow-fed and high-fat–fed rats were substantially increased, this is unlikely to be a major mechanism because there was no increase in basal Rd (or HGO) in rosiglitazone-treated rats. Moreover, the enhancement of AMPK-mediated glucose metabolism was specific to AICAR stimulation in high-fat–fed rats.
Several lines of evidence appear to point to a possible link between the enhanced AMPK stimulation of glucose uptake in muscle to rosiglitazone’s effects on lipid me-tabolism. First, we found the enhancement of AICAR-stimulated glucose uptake and glycogen synthesis by rosiglitazone only apparent in high-fat–fed rats but not in chow-fed rats. Second, AMPK signaling has been shown to be defective in the presence of excessive lipid accumulation in genetically obese rodents (14,24) and obese patients (15,16). Furthermore, it has been recently shown that LCACoAs can inhibit AMPK activation by LKB1 (17). We have previously reported that TZDs lessen an excessive accumulation of LCACoAs in red muscle of high-fat–fed rats (18,26) in the absence of insulin or AICAR stimulation. Based on these studies, we speculate that the enhancement of AICAR-stimulated glucose uptake by rosiglitazone in high-fat–fed rats may be related to the lipid- lowering effects of rosiglitazone. This interpretation is indeed supported by the fact that a lower LCACoA level was preserved in red muscle during AICAR clamp in rosiglitazone-treated high-fat–fed rats in association with the improved AMPK activation and glucose uptake in this tissue. However, further studies are required to establish whether this is a causal relationship.
Although pACC was increased at the end of the AICAR infusion (data not shown), we did not find an enhancement of its phosphorylation in either muscle or adipose tissue in rosiglitazone-treated high-fat–fed rats. This was somewhat unexpected because the extent of ACC phosphorylation is believed to be closely associated with AMPK activity. However, a recent study has also shown that acute ischemia in the kidney increases AMPK activity up to threefold without detected increases in ACC phosphorylation (27). There is new evidence to suggest that lipids affects AMPK phosphorylation and ACC phosphorylation separately in skeletal muscle cells (28). Regardless of this disparity, our data support the notion that AMPK-stimulated glucose uptake is mediated by different downstream signaling molecules rather than via ACC (29).
AICAR has been shown to exert a strong effect to suppress hepatic glucose output involving both AMPK activation and AMPK-independent pathways (30,31). In an earlier study in normal rats during AICAR infusion, hepatic glucose output was close to zero (12). In the present study, hepatic glucose production was almost completely suppressed by AICAR infusion at the dose used, and no significant difference in hepatic glucose output was found between the HF-CON and HF-RSG groups. Consistent with this, rosiglitazone did not show a significant effect to enhance AICAR-stimulated AMPK activity in the liver. These data suggest that the liver is unlikely to be a major contributor to the improvement of AMPK-induced glucose metabolism in high-fat–fed rats following rosiglitazone treatment.
In summary, the present study demonstrates for the first time that treatment with the TZD rosiglitazone for 1 week enhances acute AMPK-mediated glucose disposal in insulin-resistant high-fat–fed rats. Muscle and adipose tissue were the major sites for the enhanced disposal of circulating glucose. The enhanced glucose uptake in muscle was associated with increased activity of the AMPK2 subunit, suggesting that rosiglitazone potentiates AICAR-stimulated glucose uptake in peripheral tissues via enhancing AMPK2. Thus, we conclude that in addition to being insulin sensitizers, TZDs can also sensitize AMPK-mediated glucose disposal in peripheral tissues in lipid-induced insulin-resistant states. Since AMPK is activated during physical activity, our findings also imply that TZDs may increase effectiveness of exercise for the control of hyperglycemia in insulin-resistant subjects such as obese patients with type 2 diabetes.
ACKNOWLEDGMENTS
This study was supported by grants from the Diabetes Australian Research Trust (DART) (to J.-M.Y. and E.K.) and the National Health and Medical Research Council (NHMRC) (to E.K.). The Australian Research Council and NHMRC support B.K. N.Z. is currently supported by a National Heart Foundation postgraduate scholarship.
FOOTNOTES
The costs of publication of this article were defrayed in part by the payment of page charges. This 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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