Dietary control and exercise prevent metabolic disorders, but they are not usually successful interventions. Drugs that mimic calorie restriction and exercise are being developed to combat metabolic disorders. One target for treating metabolic disorders is Sirt1 (8
), an NAD+
-dependent protein deacetylase (48
). Resveratrol, which was discovered in a small-molecule screen to be a Sirt1 activator (1
), has drawn a great deal of interest for its therapeutic potential in treating metabolic disorders such as type 2 diabetes. By increasing mitochondrial biogenesis and metabolic rate, resveratrol reduced fat and increased glucose tolerance and insulin sensitivity and physical endurance.
If Sirt1 is the target of resveratrol, transgenic mice overexpressing Sirt1 should have phenotypes very similar to those induced by resveratrol. Thus far, three models of whole-body Sirt1 gain of function have been reported. In Sirt1 knockin mice (36
), the Sirt1 transgene, which was expressed from the β-actin locus, is overexpressed in fat but not in muscle or liver, key organs affected in metabolic disorders. These mice have increased metabolic rate, lower fat mass, and are more insulin sensitive than control mice, resembling mice treated with resveratrol. More recently, transgenic mice in which the Sirt1 transgene is expressed from its own promoter have been reported by two independent groups (35
). These transgenic mice, which have a more physiological expression pattern of the Sirt1 transgene than the knockin mice (36
), are more insulin sensitive partly due to increased adiponectin levels and decreased hepatic steatosis. Surprisingly, the transgenic mice developed by Banks et al. (35
), which are congenic in C57BL6/J background, have reduced metabolic rate and body temperature and have the same amount of fat despite eating less. It should be noted that unlike the other Sirt1 gain-of-function studies, this study measured the metabolic rate using regular diet not high-fat diet. Nevertheless, this study shows that the function of Sirt1 on energy balance may be opposite of what was previously thought (7
) and would be predicted if the central target of resveratrol was Sirt1. One possible explanation for the reduced fat mass in the knockin mice (36
) is that expression of the Sirt1 transgene from the β-actin locus impaired adipocyte differentiation during development (49
). Therefore, the Sirt1 knockin model (36
) cannot distinguish between a direct effect of Sirt1 and an indirect one caused by reduced fat.
Resveratrol has been reported to affect the activities of many enzymes (11
) including AMPK (12
). To evaluate the possibility that the effects of resveratrol are mediated by AMPK, we studied the effects of resveratrol in mice deficient in AMPKα1 or -α2. Our findings indicate that all of the salient effects induced by resveratrol are abolished in AMPKα1- and/or AMPKα2-deficient mice, suggesting that the metabolic changes induced by resveratrol are largely mediated through AMPK rather than Sirt1. Because the wild-type mice used in our study were not littermates of AMPK-null mice, it is possible that some differences between wild-type mice and AMPK-null mice may be due to differences in the genetic background. However, we feel that this difference is minimal because the AMPK-null mice used in this study have been backcrossed to C57BL6/J mice, which we used as wild-type control, for at least six generations. Therefore, we expect our AMPK-null mice to be at least 98% congenic to the wild-type controls. Moreover, the dominant role of AMPK in the metabolic effects of resveratrol is supported by our studies using Sirt1-deficient mefs. Resveratrol-induced transcription of PGC-1α and PGC-1α–dependent genes was shorter in duration but was not abolished in Sirt1-deficient mefs (), whereas it was abolished in AMPK-deficient mefs (). The ability of AMPK to increase NAD and the NAD-to-NADH ratio (45
) may also explain how resveratrol treatment can lead to Sirt1 activation without directly activating it. Moreover, AMPK can activate PGC-1α, one of Sirt1 substrates, by directly phosphorylating it (50
), indicating that activation of AMPK can affect the Sirt1-dependent pathways in multiple ways. Thus, our findings suggest that the direct target of resveratrol in vivo may not be Sirt1 and supports the possibility that Sirt1 plays a modulatory role, rather than a central role, in resveratrol response. Whether it is the direct target of resveratrol or not, it appears that Sirt1 can also function upstream of AMPK in HepG2 hepatocytes and HEK293T cell line (26
). Since Sirt1 is not required for resveratrol-mediated activation of AMPK activation in mefs (A
), it is possible that Sirt1 is upstream of AMPK only in certain cell types.
Resveratrol-induced physical endurance has been largely attributed to increased mitochondrial function (7
). Certainly, converting muscle fiber to mitochondria-rich, slow-twitch fibers increases physical endurance (52
). However, it is also likely that the glycogen content in skeletal muscle, which is known to be increased by AMPK activity (34
), also plays a role in resveratrol-induced physical endurance.
Although the expression levels of UCPs in WAT and BAT increased with resveratrol in wild-type mice, the body temperature of wild-type or AMPK−/−
mice did not increase with resveratrol. It is possible that the stress associated with the rectal temperature measurement may have masked any subtle differences in body temperature. It should also be noted that unlike AMPKα1−/−
mice gained less weight on resveratrol even though the expression levels of UCPs were not induced by resveratrol in the adipose tissue. We do not have a clear explanation for this, but one possibility is that the antiadipogenic function of Sirt1 (49
) is being driven by the resveratrol-AMPKα1-Sirt1 pathway in AMPKα2−/−
The complex nature of the resveratrol effect is also demonstrated by our observation that even though resveratrol induced weight loss in AMPKα2−/−
mice, it did not improve their insulin sensitivity (B
). One reason resveratrol failed to improve insulin sensitivity in AMPKα2−/−
mice may be that the failure to increase mitochondrial biogenesis and fat oxidation in skeletal muscle led to a build up of lipids that are known to inhibit insulin action. Indeed, resveratrol failed to increase mitochondrial content and decrease DAG and ceramide in both AMPKα1−/−
mice. In skeletal muscle, the AMPKα1 isoform makes up only ~20% of the total AMPK activity (17
), and, yet, resveratrol-induced mitochondrial biogenesis (D
) or reduction in ROS (C
), DAG (D
), and ceramide (E
) did not occur in the skeletal muscle of AMPKα1−/−
mice. One possible explanation is that in addition to the AMPKα2 activity, a crosstalk between the skeletal muscle and either the nervous system or fat, where the AMPKα1 isoform is predominant (17
), is required for resveratrol-induced mitochondrial biogenesis or reduction of ROS in skeletal muscle. For example, the AMPKα2 activity in skeletal muscle and the low-energy signal from resveratrol-induced weight loss, which requires AMPKα1, may both be required for the full resveratrol effect. It is also likely that in skeletal muscle, AMPKα1 and AMPKα2 have nonoverlapping functions, both of which are required for responding to resveratrol.