Introduction
5′-AMP-activated protein kinase (AMPK) is a key enzyme in the regulation of energy metabolism. It acts as a cellular energy sensor, and is activated by metabolic stress, such as hypoxia, and muscle contraction [
1]. AMPK controls both fatty acid and carbohydrate metabolism [
2‐
6] by increasing skeletal muscle glucose [
2,
4,
6,
7] and fatty acid [
2,
3] uptake and/or oxidation, suppression of hepatic glucose output [
4] and inhibition of adipose tissue lipolysis [
5,
8]. Importantly, AMPK regulates these processes in an insulin-independent manner. The AMPK pathway appears largely intact in obese and/or type 2 diabetic rodents [
3,
9‐
12] and humans [
5,
13‐
16]. Consequently, AMPK is regarded as a potential target for the treatment of type 2 diabetes.
AMPK is activated by 5-aminoimidazole-4-carboxamide riboside (AICAR) in skeletal muscle [
2‐
4], adipocytes [
8] and hepatocytes [
17]. Although this agent has been widely used to study the metabolic effects of AMPK activation in rodents [
2‐
4,
8,
17], studies investigating the effect of AICAR on AMPK-regulated substrate metabolism in human tissue are scarce [
13,
14,
18]. A recent study investigated the effect of AICAR infusion (10 mg kg
−1 h
−1) on skeletal muscle 2-deoxyglucose uptake in young, healthy males [
19]. However, no studies have examined the impact of AICAR on whole body glucose or fatty acid metabolism in humans. Furthermore, the effects of AICAR administration in type 2 diabetic patients remain to be established.
We hypothesise that i.v. AICAR administration in type 2 diabetic patients reduces both plasma glucose and NEFA concentrations by stimulating blood glucose disposal, lowering hepatic glucose output, and inhibiting adipose tissue lipolysis. In the present study, we combine stable isotope methodology with blood and muscle biopsy sampling to determine the effect of i.v. AICAR administration on blood glucose and fatty acid kinetics in vivo in type 2 diabetic patients.
Methods
Blood sample analysis
Blood samples (8 ml) were collected in EDTA-containing tubes and immediately centrifuged at 1,000 g for 10 min at 4°C. Aliquots of plasma were immediately frozen in liquid nitrogen and stored at −80°C. Plasma glucose (Roche, Basel, Switzerland), lactate (Wako Chemicals, Neuss, Germany), NEFA (Wako Chemicals), glycerol (Roche Diagnostics, Indianapolis, IN, USA) and triacylglycerol (Sigma Diagnostics, St Louis, MO, USA) concentrations were analysed with a COBAS semi-automatic analyser (Roche). Plasma insulin was measured by radioimmunoassay (Linco, St Charles, MO, USA). Blood HbA1c was analysed by HPLC (Variant II; Bio-Rad, Munich, Germany). For determination of plasma palmitate and NEFA kinetics, NEFA were extracted, isolated by thin-layer chromatography and derivatised to their methyl esters. Isotope enrichment of palmitate was analysed by GC-MS (Agilent, Little Falls, DE, USA). Plasma palmitate concentration was determined on an analytical GC with flame ionisation detection using nonadecaenoic acid as the internal standard, and was found to constitute 23.9 ± 0.18% of total NEFA. Following derivatisation, plasma [6,6-2H2]glucose enrichment was determined by electron ionisation GC-MS (Agilent). Palmitate and glucose tracer concentrations in the infusates averaged 2.34 ± 0.03 and 37.3 ± 0.10 mmol/l, respectively, in the AICAR test vs 2.27 ± 0.06 and 37.3 ± 0.06 mmol/l in the control test. Therefore, the exact palmitate and glucose tracer infusion rates averaged 27 ± 1 and 272 ± 7 nmol kg−1 min−1, respectively, in the AICAR test vs 28 ± 1 and 272 ± 8 nmol kg−1 min−1 in the control test. Plasma AICAR concentrations were determined by HPLC, with UV detection set at 260 nm, using a 200 × 4.6 mm 5 μm Hypersil BDS C18 column (ThermoFisher, Waltham, MA, USA). The mobile phase consisted of methanol, 10 mmol/l tetrabutylammonium hydrogen sulphate and 5 mmol/l K2HPO4, pH 8.2 (20:80, vol./vol.).
AMPK activity assays were performed as previously described [
23]. Briefly, AMPKα1 and -α2 were immunoprecipitated from 100 μg of protein using isoform-specific antibodies (2 μg) coupled to 15 μl of protein A beads (Pierce, Rockford, IL, USA). Immune complexes were washed twice with 1 ml of lysis buffer containing 0.5 mol/l NaCl, and once with 1 ml of Buffer A (50 mmol/l Tris pH 7.5, 0.1 mmol/l EGTA, 0.1% [wt/vol.] 2-mercaptoethanol). Assays were performed in a total volume of 50 μl (50 mmol/l Tris pH 7.5, 0.1 mmol/l EGTA, 0.1% 2-mercaptoethanol, 10 mmol/l MgCl
2, 0.1 mmol/l [
32P]ATP (∼200 cpm/pmol) and 30 μmol/l AMARA peptide (AMARAASAAALARRR). The assays were carried out for 30 min at 30°C and terminated by applying 40 μl of the reaction mixture onto P81 papers. Phosphotransferase activity was measured by scintillation counting.
Discussion
The present study shows that i.v. AICAR infusion in vivo in type 2 diabetic patients inhibits hepatic glucose output while maintaining whole body glucose uptake, thereby lowering plasma glucose concentrations. Furthermore, AICAR infusion is shown to suppress whole body lipolysis, resulting in a decline in plasma NEFA concentration.
The effects of AICAR on glucose metabolism have been studied extensively in rodent models, both in vitro and in vivo. These studies demonstrate that AMPK activation by AICAR stimulates glucose uptake [
2‐
4,
6,
7] and inhibits hepatic glucose output [
5] in an insulin-independent manner. In obese and/or insulin-resistant rodent models, glucose tolerance is improved after long-term AICAR administration [
10‐
12]. Ex vivo studies in human skeletal muscle tissue samples have yielded similar results, demonstrating that AICAR increases glucose transport [
13] and fatty acid oxidation [
14], which is accompanied by an increase in AMPK phosphorylation and/or activity [
13,
14] and ACC phosphorylation [
13,
14]. It is evident that it would be of great interest to determine the effects of in vivo AICAR administration in humans. Cuthbertson et al. [
19] recently described a twofold increase in 2-deoxyglucose uptake in skeletal muscle after 3 h of AICAR infusion (10 mg kg
−1 h
−1) in young men. However, this was not accompanied by changes in plasma glucose concentration [
19]. During a euglycaemic–hyperinsulinaemic clamp in combination with AICAR infusion, whole body glucose uptake (i.e.
M value) was slightly increased (7%) [
19].
To date, no study has investigated the effects of AICAR administration on plasma glucose and fatty acid kinetics in vivo in type 2 diabetic patients. In the present study, we demonstrate a strong decline in the rate of appearance of plasma glucose following AICAR infusion (0.75 mg kg
−1 min
−1, or 45 mg kg
−1 h
−1), suggesting that AICAR infusion strongly suppresses hepatic glucose output in type 2 diabetic patients. This is in accordance with previous results in obese Zucker rats, in which AICAR infusion was shown to suppress hepatic glucose output [
5]. Even though plasma glucose appearance rates declined during AICAR infusion, whole body glucose uptake remained unchanged (Figs.
1,
2). Consequently, glucose disposal (when defined as the percentage of glucose
R
a that is taken up from the circulation) was significantly greater during AICAR infusion (Figs.
1,
2). These findings extend the previous observations by Cuthbertson et al. [
19] and indicate that AICAR infusion in patients with type 2 diabetes has only a modest impact on plasma glucose uptake but strongly inhibits hepatic glucose output. In contrast to Cuthbertson et al. [
19], we observed a significant decline in plasma glucose levels during AICAR infusion. This discrepancy between studies may be attributed to the 4.5-fold higher AICAR dose that was administered and the selection of insulin-resistant type 2 diabetic patients as opposed to healthy, young men.
In the present study we also assessed the effect of AICAR infusion on plasma NEFA kinetics. Activation of AMPK by AICAR has been shown to inhibit lipolysis and lipogenesis in vitro in adipocytes [
8,
24,
25] and in vivo in both lean and insulin-resistant obese rat models [
5]. This study is the first to demonstrate that i.v. AICAR infusion (0.75 mg kg
−1 min
−1) inhibits the whole body lipolytic rate in type 2 diabetic patients, resulting in a significant decline in circulating plasma NEFA concentrations (Fig.
3). Furthermore, as AMPK activation in the liver also stimulates hepatic fatty acid oxidation [
26], it might be assumed that the decline in the rate of appearance of plasma NEFA is also partly due to a greater hepatic extraction and oxidation rate of fatty acids released from the splanchnic area. Altogether, it appears that the effects of AICAR infusion on plasma glucose and NEFA levels in type 2 diabetic patients are mainly exerted through its effects on the liver and adipose tissue, by inhibition of endogenous glucose production, stimulation of hepatic fatty acid oxidation and/or a reduction in whole body lipolysis. Contrary to our expectations, the effect of AICAR infusion on whole body and/or skeletal muscle glucose and NEFA uptake seems to be of less quantitative importance in type 2 diabetic patients.
To investigate the effects of AICAR on AMPK activation in skeletal muscle tissue, we measured potential changes in the phosphorylation state of AMPK and its downstream target, ACC, as a more sensitive marker of AMPK activation [
27]. Although we failed to detect a significant increase in AMPK phosphorylation (Fig.
4), we did observe a substantial increase in ACC phosphorylation in muscle biopsy samples collected after 2 h of AICAR infusion (185 ± 28%). As the ACC phosphorylation state can be used as a more sensitive measure of in vivo AMPK activity, our findings suggest that modest allosteric activation of AMPK had occurred without substantial phosphorylation of AMPK by its kinase. However, this was not supported by the AMPK activity assays, which showed no changes in either AMPKα1 or -α2 activity. Alternatively, it is possible that an unknown kinase that is responsive to AICAR was responsible for the increase in ACC phosphorylation. We did not detect any effect of AICAR infusion on the phosphorylation of other known AMPK substrates, such as AS160, GSK3α, GSK3β and HDAC5 (on S259 and S498). Cuthbertson et al. [
19] showed an increase in ERK1/2 phosphorylation with AICAR infusion in humans. However, no change was detected in the present study. As such, our data indicate that AICAR infusion in vivo in type 2 diabetic patients only modestly activates AMPK in skeletal muscle tissue. The impact of AICAR infusion on AMPK activation seems to be much greater in adipose and/or liver tissue. It should be noted that a small but significant increase in circulating plasma insulin concentrations during the AICAR test might have contributed to the observed effects on hepatic glucose output and whole body lipolysis [
28]. Future studies using AICAR administration in vivo in humans might consider the use of octreotide to suppress insulin secretion. Furthermore, it should be mentioned that some effects of AICAR may not be AMPK-mediated, especially the effects on hepatic glucose output [
29]. As adipose and/or liver tissue samples were not collected, we can only speculate on the impact of AICAR infusion on AMPK activation in hepatic and/or adipose tissue.
In conclusion, i.v. AICAR infusion (0.75 mg kg−1 min−1) in type 2 diabetic patients inhibits hepatic glucose output, stimulates hepatic fatty acid oxidation and/or reduces whole body lipolysis in vivo, thereby lowering plasma glucose and NEFA concentrations.