Genes implicated in lipid metabolism
There are several sites of regulation in lipid metabolism in skeletal muscle. The entry of fatty acid into the sarcoplasm is regulated by selective transport of NEFAs across the sarcoplasm (Fig. 1). Fatty acids are transported systemically as triglycerides and are liberated by LPL. Under fasting conditions, Lpl expression in wild-type mice was augmented (81%, p<0.05) (Fig. 2a). This effect was impaired in Prkag3
−/− mice (Fig. 2a).
We then assessed levels of Cd36 mRNA, which is translated into CD36, the putative long-chain fatty acid transporter of skeletal muscle  (Fig. 1). Fasting was associated with an increase in Cd36 expression in the wild-type (214%, p<0.001) and Tg-Prkag3
225Q (183%, p<0.001) mice (Fig. 2b). This fasting-induced increase in Cd36 expression was impaired in the absence of the AMPK γ3 subunit (70%, p<0.05). Overexpression of the mutant γ3225Q subunit was associated with an elevation in Cd36 expression in skeletal muscle during fasting.
Another rate-determining step in lipid metabolism is the transport of the fatty acid into the mitochondria as acyl-CoA. The transport of NEFAs from the sarcoplasm into the mitochondria is facilitated by the carnitine palmitoyl transfer system, which involves CPT-1 (Fig. 1). Cpt1 transcription was increased in response to fasting in wild-type (45%, p<0.01) and Tg-Prkag3
225Q (51%, p<0.01) mice, whereas the fasting-induced response was blunted in Prkag3
−/− mice (Fig. 2c).
In the mitochondrial matrix, acyl-CoA is oxidised in a stepwise manner by a series of enzymes, including HADHSC, leading to the formation of acetyl-CoA (Fig. 1). The transition from the fed to the fasted state was associated with a trend towards an increase in Hadhsc mRNA (41%, p=0.07) in wild-type mice, with essentially similar responses observed among the three groups (Fig. 2d). In the tricarboxylic acid (TCA) cycle, acetyl-CoA derived from oxidation of acyl-CoA or pyruvate is converted to citrate by CS, the first rate-controlling enzyme of the TCA cycle (Fig. 1). Fasting increased the expression of Cs mRNA in Tg-Prkag3
225Q mice (79%, p<0.01). In Prkag3
−/− mice, fasting also increased Cs expression (121%, p<0.001); however, the increase was lower compared with the pronounced effect (240%, p<0.001) noted in wild-type mice.
Electrons derived from the oxidation of substrate in the TCA cycle are transferred to the electron-transport chain to generate a proton gradient across the inner mitochondrial membrane for oxidative phosphorylation. The electron-transport chain is catalysed by a series of enzymes that require CYCS (Cycs) as a co-enzyme for shuttling electrons. Under fed conditions, levels of Cycs mRNA were greater in Tg-Prkag3
225Q mice than in wild-type (37%, p<0.05) or Prkag3
−/− mice (45%, p<0.05) (Fig. 2f). A similar increase in Cycs expression was observed under fasting conditions.
Under fasting conditions, expression of mitochondrial UCP3 is upregulated [4, 30]. One putative function of UCP3 is to export fatty acid anions from the mitochondrial matrix when acyl-CoA flux and lipid oxidation is increased [6, 31, 32] (Fig. 1). Fasting augmented Ucp3 expression (175%, p<0.001) in wild-type mice (Fig. 2g); this increase was more dramatic (328%, p<0.001) in Prkag3
Genes implicated in glucose metabolism
Skeletal muscle glucose uptake is tightly regulated by GLUT4 (which is encoded by Slc2a4), a mammalian facilitative glucose transporter (Fig. 1). Slc2a4 mRNA expression was similar among the three groups. Upon entry into the cytoplasm, glucose is phosphorylated by HK2 to glucose 6-phosphate in the first rate-determining step of glycolysis (Fig. 1). Fasting induced a prominent decrease (77%, p<0.01) in Hk2 expression in Tg-Prkag3
225Q mice (Fig. 3b). Moreover, the level of Hk2 mRNA in Tg-Prkag3
225Q mice was markedly lower than that in wild-type mice (79%, p<0.05).
Downstream of HK2, glucose can be further metabolised through glycolysis or glycogenesis (Fig. 1). We next determined the expression of PFKM, an enzyme that catalyses the second rate-controlling step of glycolysis. Fasting triggered a pronounced decrease (46%, p<0.001) in Pfkm expression in Tg-Prkag3
225Q mice, with no significant change in wild-type or Prkag3
−/− mice (Fig. 3c). Fasting increased (193%, p<0.001) the level of Gys mRNA in wild-type mice (Fig. 3d). This increase was less profound in Prkag3
−/− mice (98%, p<0.01), which expressed a lower level (29%, p<0.05) of this transcript than wild-type mice. Consistent with the reduced Hk2 expression, Gys expression was not elevated in Tg-Prkag3
225Q mice in response to fasting.
The link between glycolysis and the TCA cycle involves the decarboxylation of pyruvate to acetyl-CoA by pyruvate dehydrogenase. The activity of pyruvate dehydrogenase is downregulated when phosphorylated by PDK4  (Fig. 1). Fasting drastically augments the expression of Pdk4 in skeletal muscle [4, 34, 35]. Moreover, the fasting-induced increase in Pdk4 is a proposed mechanism by which entry of pyruvate into the TCA cycle is inhibited to enhance the entry of acetyl-CoA derived from lipid oxidation . Pdk4 mRNA was profoundly increased by fasting in wild-type mice (15-fold, p<0.001) (Fig. 3e), with a similar response noted in Tg-Prkag3
225Q (11-fold, p<0.001) and Prkag3
−/− (17-fold, p<0.001) mice. Thus, Pdk4 expression during fasting is unlikely to be regulated by AMPK heterotrimeric complexes containing the γ3 subunit.
We then evaluated whether fasting influences the expression of Ldh2 mRNA, another enzyme implicated in the pyruvate metabolism (Fig. 1). Fasting induced a two-fold (p<0.05) increase in Ldh2 expression in wild-type mice (Fig. 3f). However, this fasting response was blunted in the Prkag3
−/− mice, suggesting the obligatory role of AMPK γ3 subunit.
Fasting induced a non-significant trend towards increased Ppara expression in wild-type mice. Ppard expression was decreased during fasting in Tg-Prkag3
225Q mice compared with that in wild-type (61%, p<0.01) and Prkag3
−/− (58%, p<0.05) mice (Fig. 4b). Conversely, the expression of Pparg was increased in the skeletal muscle of Tg-Prkag3
225Q mice under fed and fasting conditions (Fig. 4c).
Proteins involved in metabolism
Levels of CD36 were lower in Prkag3
−/− mice than in wild-type (38%, p<0.05) or Tg-Prkag3
225Q mice (40%, p<0.05) under fasting conditions. In contrast, levels of CD36 were increased in Tg-Prkag3
225Q mice under fed conditions (by 30% and 34% relative to those in wild-type and Prkag3
−/− mice, respectively, p<0.05). Levels of UCP3 were also increased in Tg-Prkag3
225Q mice (by 33% compared with those in Prkag3
−/− mice, p<0.05) (Fig. 5b). In contrast to the mRNA results, GLUT4 (Fig. 5c) and HK2 (Fig. 5d) levels were similar for the three groups and were unaltered by fasting. However, fasting increased GYS levels in wild-type (179%, p<0.05) and Prkag3
−/− mice (139%, p<0.05) (Fig. 5e). Consistent with the sustained elevation in glycogen content, fasting had no effect on GYS levels in Tg-Prkag3
Glycogen and triglyceride content
Glycogen content in fed and fasted Tg-Prkag3
225Q mice was increased two-fold (p<0.05) compared with that in wild-type and Prkag3
−/− mice (Fig. 6a), consistent with our previous observations . The increase in glycogen content can be partially attributed to the increase in the expression of genes encoding proteins that regulate lipid and oxidative metabolism (Fig. 2), leading to a glycogen-sparing effect that promotes carbohydrate storage. Fasting leads to significant decrease in skeletal muscle glycogen content in all three groups of mice. In contrast, fed and fasted intramuscular triglyceride content was similar among the three groups (Fig. 6b).