1 Department of Human Biology and Nutritional Sciences, University of Guelph, Guelph, Ontario, Canada
2 Department of Medicine, McMaster University, Hamilton, Ontario, Canada
Address correspondence and reprint requests to David J. Dyck, PhD, Department of Human Biology and Nutritional Sciences, 50 Stone Rd. East, University of Guelph, Guelph, ON, N1G 2W1 Canada. E-mail: ddyck{at}uoguelph.ca
AICAR, 5-amino-imidazole carboxamide riboside; AMPK, AMP-activated protein kinase; DAG, diacylglycerol; HAD, hydroxy-acyl-CoA dehydrogenase; TAG, triacylglycerol
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ABSTRACT |
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Adiponectin is a 30-kDa adipose-derived hormone that appears to play an important role in regulating energy homeostasis and insulin sensitivity (1). The mRNA expression of adiponectin is reduced in obese and diabetic mice (2), and plasma levels are suppressed in conditions of obesity, insulin resistance, and type 2 diabetes (3,4). Administration of the globular head group of adiponectin (gAcrp30) reduces plasma glucose levels and ameliorates insulin resistance in mice (57). In addition, mice lacking adiponectin exhibit insulin resistance and diabetes (8,9). Recent studies have also shown that treatment of isolated rodent skeletal muscle with gAcrp30 stimulates glucose uptake (10). The insulin-sensitizing effect of gAcrp30 appears to be mediated by an increase in fatty acid oxidation (1,10), leading to a reduction in muscle lipid content (6).
The molecular mechanism responsible for the stimulation of fatty acid oxidation and glucose uptake appears to be attributable to the activation of AMP-activated protein kinase (AMPK) via specific receptor signaling. Adiponectin receptor-1 is abundantly expressed in skeletal muscle and is a high-affinity receptor for gAcrp30 (11). On binding to its receptor, gAcrp30 activates AMPK, leading to subsequent inhibition of acetyl-CoA carboxylase (1,10). This reduces malonyl-CoA content and promotes mitochondrial fatty acid oxidation by diminishing the inhibitory effect of malonyl CoA on carnitine palmitoyltransferase-1. Adiponectin has no effect on fatty acid oxidation or glucose uptake when AMPK activity is blocked by dominant-negative AMPK expression, demonstrating that the metabolic effects of gAcrp30 occur through activation of AMPK (1).
Despite the growing body of evidence indicating that gAcrp30 is an antidiabetic hormone in rodents, directly regulating fatty acid and glucose metabolism, few studies have examined the role of gAcrp30 in regulating substrate metabolism in human skeletal muscle. Therefore, in the present study, we have used an isolated human skeletal muscle preparation to examine the effect of gAcrp30 on basal and insulin-stimulated glucose uptake, fatty acid oxidation, fatty acid partitioning into intramyocellular lipid pools, and AMPK signaling. Because it has recently been proposed that obesity is associated with adiponectin resistance (12,13), we hypothesized that the metabolic effects of gAcrp30 would be blunted in skeletal muscle from obese individuals because of impaired activation of AMPK.
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RESEARCH DESIGN AND METHODS |
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Muscle viability.
In preliminary experiments, ATP and phosphocreatine were measured spectrophotometrically (16) to ensure muscle viability. Muscle strips were rapidly frozen in liquid N2 immediately after excision (0 min) or after a 120-min incubation.
Glucose uptake.
Muscle strips were preincubated in a shaking water bath at 30°C for 30 min in pregassed (95% O2/5% CO2) Krebs-Henseleit buffer containing 8 mmol/l glucose and 32 mmol/l mannitol. Thereafter, muscle specimens went through three different incubation phases: phase 1 to equilibrate the muscle, phase 2 to wash glucose from the interstitial space, and phase 3 to determine glucose uptake into muscle. During phase 1, muscle strips were incubated for 20 min in the absence (basal) or presence of insulin (120 nmol/l). The concentration of insulin was maintained throughout all remaining incubation steps. In the second phase, muscle specimens underwent two 10-min incubations at 30°C in glucose-free Krebs-Henseleit buffer containing 4 mmol/l pyruvate in the absence or presence of recombinant human gAcrp30 (2.5 µg/ml; Peprotech, Ottawa, ON, Canada). The gAcrp30 concentration was maintained for the remaining incubations. Osmolarity was maintained by the addition of 36 mmol/l mannitol to the media. During phase 3, muscle strips were incubated for 20 min (insulin-stimulated) or 40 min (basal) in Krebs-Henseleit buffer containing 8 mmol/l 3-O-[3H]methyl-D-glucose (800 µCi/mmol) and 32 mmol/l [14C]mannitol (53 µCi/mmol). After incubation, muscles were blotted of excess fluid, frozen in liquid nitrogen, and stored at 80°C until analysis. Glucose uptake was analyzed as the accumulation of intracellular 3-O-[3H]methyl-D-glucose, as described previously (17).
Skeletal muscle fatty acid oxidation.
Muscle strips were placed in a 20-ml glass scintillation vial containing 2 ml of warmed (30°C) pregassed (95% O2/5% CO2, pH 7.4) medium 199 containing 4% fatty acidfree BSA (ICN Biomedicals), 1.0 mmol/l palmitate, and 5.5 mmol/l glucose. Throughout the experiment, vials were gently shaken in a water bath. After a preincubation period of 30 min, muscle samples were incubated with or without gAcrp30 (2.5 µg/ml) for 1 h in the same medium specified above, with the addition of 0.5 µCi/ml of [1-14C]palmitate (Amersham, Oakville, ON, Canada). This permitted the monitoring of exogenous palmitate oxidation and incorporation of palmitate into endogenous lipid pools.
Gaseous 14CO2 produced from the exogenous oxidation of [1-14C]palmitate during incubation was measured by transferring 1.0 ml of the incubation medium to a 20-ml glass scintillation vial containing 1.0 ml of 1 mol/l H2SO4 and a 0.5-ml Fisher microcentrifuge tube containing 1 mol/l benzethonium hydroxide. Liberated 14CO2 was trapped in the benzethonium hydroxide over 60 min, the microcentrifuge tube containing trapped 14CO2 was placed in a scintillation vial, and radioactivity was counted.
Extraction of muscle lipids.
Muscles were placed in 13-ml plastic centrifuge tubes containing 5.0 ml of ice-cold 2:1 chloroform-methanol (vol/vol), and they were homogenized using a polytron (Brinkman Instruments, Mississauga, ON, Canada). After homogenization, samples were centrifuged at 2,000g (4°C) for 10 min. The supernatant was removed with a glass Pasteur pipette and transferred to a clean centrifuge tube. Distilled water (2.0 ml) was added, and samples were shaken for 10 min and centrifuged as before to separate the aqueous and lipophilic phases. Then, 500 µl of the aqueous phase was quantified by liquid scintillation counting to determine the amount of 14C-labeled oxidative intermediates resulting from isotopic fixation. This represented a twofold correction factor for exogenous [14C]palmitate oxidation, as previously described (18). The chloroform phase, which contains the total lipids extracted from muscle, was gently evaporated under a stream of N2 and redissolved in 100 µl of 2:1 chloroform-methanol. Small amounts of dipalmitin and tripalmitin (Sigma Chemical, St. Louis, MO) were added to the 2:1 chloroform-methanol to facilitate the identification of lipid bands on the silica gel plates. We spotted 50 µl of each sample on an oven-dried silica gel plate (Fisher Scientific Canada, Mississauga, ON). Silica gel plates were placed in a sealed tank containing solvent (60:40:3 heptaneisopropyl etheracetic acid) for 40 min. Plates were then dried, sprayed with dichlorofluorescein dye (0.02% wt/vol in ethanol), and visualized under longwave ultraviolet light. The individual lipid bands were marked on the plate with a scalpel and scraped into vials for liquid scintillation counting.
AMPK activity.
AMPK1 and -
2 activities were examined after the 30-min preincubation (0 min) and after 20 or 60 min of gAcrp30 treatment. Briefly, muscle strips were incubated in medium 199 (5.5 mmol/l glucose) containing 4% fatty acidfree BSA, 5.5 mmol/l glucose, and 1.0 mmol/l palmitate with or without the addition of 2.5 µg/ml gAcrp30. At the end of the incubation period, the muscle strips were quickly freeze-clamped and stored in liquid nitrogen until subsequent analysis.
To determine AMPK activity, muscle strips (20 mg) were homogenized in buffer (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). The homogenates were incubated with AMPK
1 and -
2 (Upstate Scientific, Charlottesville, VA) antibody-bound protein A beads (Sigma, St. Louis, MO) each for 2 h at 4°C. Immunocomplexes were washed with PBS and suspended in 60 µl dilution buffer (50 mmol/l Tris, pH 7.5, 1 mmol/l dithiothreitol, 10% glycerol, and 0.1% Triton-X) for AMPK activity assay (19). Briefly, 20 µl of the sample was combined with 20 µl of reagent mixture (5 mmol/l HEPES, pH 7.5, 1 mmol/l MgCl2, 0.5% glycerol, 1 mmol/l dithiothreitol, and 100 µmol/l SAMS peptide; Upstate Scientific), 250 µmol/l ATP with 32P
-ATP (Amersham Biosciences, QC, Canada) and 100 µmol/l AMP. The reaction proceeded for 15 min, after which 23 µl of reaction mixture was spotted onto p81 filter paper (Upstate Scientific) and washed three times in 1% phosphoric acid. Filter papers were dried and placed in organic scintillant for counting.
Citrate synthase and ß-hydroxy-acyl-CoA dehydrogenase activity.
Citrate synthase and ß-hydroxy-acyl-CoA dehydrogenase (HAD) activity was assayed in a portion of muscle (10 mg) that was rapidly frozen in liquid N2 immediately after excision. Muscle was homogenized in 100 vol/wt of a 100- mmol/l potassium phosphate buffer solution, and citrate synthase activity was assayed spectrophotometrically at 37°C (20). ß-HAD activity was assayed spectrophotometrically at 37°C by measuring the disappearance of NADH, using the whole-muscle homogenate as for citrate synthase (21).
Calculations and statistics.
All data are the means ± SE. Differences within a group were determined using paired t tests. To examine differences between the lean and obese groups, unpaired t tests were used. Statistical significance was accepted at P < 0.05. Total palmitate uptake by the muscle strips was calculated by summing the incorporation of labeled palmitate into lipid pools plus oxidation.
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RESULTS |
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Muscle strip preparation viability.
Muscle viability was preserved during incubations based on maintenance of ATP (0 min, 20.7 ± 0.8; 120 min, 19.1 ± 0.8 µmol/g dry wt) and phosphocreatine content (0 min, 77.5 ± 2.9; 120 min, 70.2 ± 3.2 µmol/g dry wt).
Glucose uptake in isolated skeletal muscle.
Basal glucose uptake was not different between lean and obese subjects (Fig. 1A). Treatment with gAcrp30 increased glucose uptake 37% (P < 0.05) in lean subjects and 33% (P < 0.05) in obese subjects. Insulin increased glucose uptake 110% (P < 0.01) in control subjects and 60% (P < 0.01) in obese subjects (Fig. 1A). The absolute change in glucose uptake in response to insulin was 28% lower in obese subjects (P < 0.05) (Fig. 1B). Combined exposure of insulin and gAcrp30 increased glucose uptake 270% (P < 0.01) in control subjects and 90% (P < 0.01) in obese subjects (Fig. 1A). The additive effect of gAcrp30 and insulin on glucose uptake was 50% lower (P < 0.05) in obese subjects (Fig. 1B).
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DISCUSSION |
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Effect of gAcrp30 on skeletal muscle glucose uptake.
Studies in rodents have reported that gAcrp30 increases glucose uptake in skeletal muscle (1,10). However, no studies have examined the effect of gAcrp30 on glucose uptake in human muscle. Therefore, a major finding of the present study was that gAcrp30 increased glucose uptake in the absence of insulin in skeletal muscle from both lean and obese individuals. Importantly, we also demonstrate that gAcrp30 had an additive effect on insulin-stimulated glucose uptake in skeletal muscle from both lean and obese subjects. Similar observations have recently been reported in L6 myotubes. Ceddia et al. (25) found that gAcrp30 increased basal rates of glucose uptake via translocation of GLUT4 to the cell surface. Furthermore, when used in combination with insulin, gAcrp30 exerted an additive effect on glucose uptake and GLUT4 translocation (25). It should be noted that the pharmacological insulin concentration used in the present study was chosen because this is known to maximally stimulate glucose uptake in an incubation model. Thus, it must be acknowledged that the high level of insulin, particularly in the absence of other hormones and peptides normally present in the circulation, makes it impossible to directly extrapolate our finidngs to the in vivo condition. Taken together, the results of the current study and those of Ceddia et al. (25) suggest that like muscle contraction and 5-amino-imidazole carboxamide riboside (AICAR), gAcrp30 increases glucose uptake via an insulin-independent mechanism, possibly involving AMPK. It is also noteworthy that in the current study, the stimulation of glucose uptake in the presence of gAcrp30 and insulin was actually greater than that which would be predicted by adding the individual responses to these hormones, i.e., a greater than additive effect. This may imply that adiponectin also increases insulin sensitivity in muscle.
A novel finding of the current study was that the additive effect of gAcrp30 and insulin on glucose uptake was blunted in the muscle from obese patients. Recently, it has been proposed that in animal models of obesity and human type 2 diabetes, adiponectin resistance may develop, resulting in a decrease in the adiponectin effects on fatty acid oxidation and AMPK activation (12,13). Interestingly, in muscle from the obese individuals, the increase in basal glucose uptake in response to gAcrp30 was not impaired, despite reduced activation of AMPK compared with lean muscle. Therefore, these results suggest that the reduction in insulin-stimulated glucose uptake in the muscle from obese individuals may be responsible for the impairment in glucose uptake in response to a combination of gAcrp30 and insulin. Surprisingly, the fasting levels of blood glucose and insulin were not different between the lean and obese groups (Table 1), suggesting that whole-body insulin sensitivity was not actually impaired in the obese subjects. However, it should be noted that these measurements were made after a prolonged fast (1218 h) before surgery, and that a more dynamic test, such as a euglycemic-hyperinsulinemic clamp or an oral glucose tolerance test, may have revealed a difference in insulin sensitivity between the groups.
Effect of gAcrp30 on skeletal muscle fatty acid metabolism.
The insulin-sensitizing effect of gAcrp30 has been suggested to be mediated by an increase in fatty acid oxidation (1,10), leading to a reduction in muscle lipid content (6). In the current study, we demonstrated that gAcrp30 increases fatty acid oxidation in skeletal muscle from both lean and obese subjects and that this is associated with enhanced insulin sensitivity. However, similar to the effect on insulin-stimulated glucose uptake, the stimulatory effect of gAcrp30 on fatty acid oxidation is blunted in skeletal muscle from obese individuals. This is consistent with the finding of impaired activation of AMPK in muscle from obese subjects. Our data are in agreement with the recent study of Chen et al. (12), who report that gAcrp30 stimulates fatty acid oxidation in cultured human myotubes and that this response is impaired in obese and obese type 2 diabetic myotubes.
Insulin resistance is associated with accumulation of cytosolic lipids within muscle (26). It has been hypothesized that interventions that increase fatty acid oxidation will improve insulin sensitivity by reducing the accumulation of muscle lipids. Indeed, adiponectin treatment has been shown to improve insulin sensitivity by decreasing muscle triglyceride content (6). Therefore, we examined whether gAcrp30 treatment would affect fatty acid esterification into DAG and TAG pools. Interestingly, fatty acid esterification into DAG and TAG was unaffected by gAcrp30 treatment, despite the observed increase fatty acid oxidation. It is possible that our incubation period was too short in duration to see an effect of gAcrp30 on fatty acid esterification into the endogenous lipid pools. Furthermore, the effect of gAcrp30 on fatty acid esterification into the lipid pools may only become evident when in the presence of other hormones that stimulate lipogenesis, such as insulin. However, despite no changes in the esterification of fatty acids into the lipid pools, we were able to detect a significant reduction in the esterification-to-oxidation ratio in both lean and obese muscle, indicating a partitioning of fatty acid toward oxidation rather than storage.
Effect of gAcrp30 on AMPK activation.
The signaling pathways regulated by adiponectin are just beginning to be characterized. On binding to its receptor, adiponectin receptor-1, gAcrp30 activates AMPK, which appears to mediate the metabolic effects of gAcrp30 (11). Previous studies using cell culture and isolated rodent muscle have shown that AMPK activity is rapidly and transiently activated by gAcrp30 (1,10). However, here we show that AMPK1 activity is only activated after 60 min of exposure to gAcrp30 in both lean and obese skeletal muscle. In contrast, AMPK
2 activity is increased after 20 min of incubation with gAcrp30 in muscle from lean and obese individuals. This effect is maintained in muscle from lean subjects after 60 min of gAcrp30, whereas there was a tendency for AMPK
2 to remain elevated at this time point in muscle from obese subjects. Although we do not know the precise mechanisms underlying these differences, it is possible that species differences exist such that a more prolonged AMPK response is observed in human skeletal muscle after acute treatment with gAcrp30.
Interestingly, we found impaired stimulation of AMPK1 and -
2 by gAcrp30 in skeletal muscle of obese subjects. In fact, we found that the basal activity of AMPK
1 is reduced in obese skeletal muscle. The metabolic significance of this reduction in AMPK
1 activity is unknown because currently it appears that the
2 isoform plays a more important role in regulating substrate metabolism (27,28). Indeed, AMPK
2 knockout mice are resistant to AICAR-induced glucose uptake (27) and develop whole-body insulin resistance (28), whereas AMPK
1 knockout mice appear normal (25). In this regard, the reduction in AMPK
2 activity in the obese subjects may explain the impaired metabolic responses to gAcrp30. Furthermore, it has recently been reported that the mRNA expression of adiponectin receptor-1 is reduced in insulin-resistant rodents (13) and humans (29), which may result in decreased activation of AMPK by gAcrp30.
In conclusion, this is the first study to show that gAcrp30 acutely increases glucose uptake in human skeletal muscle. Furthermore, we show an additive effect of gAcrp30 on insulin-stimulated glucose transport in lean and obese skeletal muscle. These effects may be mediated by a shift in fatty acid partitioning toward oxidation and away from storage, which is likely due to activation of AMPK. Importantly, our data also show that the effects of gAcrp30 on insulin-stimulated glucose uptake and fatty acid oxidation are blunted in skeletal muscle from obese subjects because of impaired activation of AMPK. These results suggest that adiponectin resistance develops in obesity.
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ACKNOWLEDGMENTS |
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We acknowledge the excellent technical assistance of Brianne Thrush and Angela Smith. We also gratefully acknowledge the invaluable contribution of the surgeons, Drs. Sibley, Loopstra, and Lightheart.
Received for publication May 30, 2005 and accepted in revised form August 15, 2005
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REFERENCES |
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