We previously showed that Cidea À/À mice are resistant to diet-induced obesity through the upregulation of energy expenditure. The AMP-activated protein kinase (AMPK), consisting of catalytic a subunit and regulatory subunits b and c, has a pivotal function in energy homoeostasis. We show here that AMPK protein levels and enzymatic activity were significantly increased in the brown adipose tissue of Cidea À/À mice. We also found that Cidea is colocalized with AMPK in the endoplasmic reticulum and forms a complex with AMPK in vivo through specific interaction with the b subunit of AMPK, but not with the a or c subunit. When co-expressed with Cidea, the stability of AMPK-b subunit was dramatically reduced due to increased ubiquitinationmediated degradation, which depends on a physical interaction between Cidea and AMPK. Furthermore, AMPK stability and enzymatic activity were increased in Cidea À/À adipocytes differentiated from mouse embryonic fibroblasts or preadipocytes. Our data strongly suggest that AMPK can be regulated by Cidea-mediated ubiquitindependent proteosome degradation, and provide a molecular explanation for the increased energy expenditure and lean phenotype in Cidea-null mice.

Cu,Zn-superoxide dismutase (SOD) was chemically modified with low molecular weight heparin (LMWH). To characterize the conjugate, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and native polyacrylamide gel electrophoresis (native PAGE) with protein staining and polysaccharide staining were employed. The stabilities of the modified enzyme to heat, acid, alkali, and trypsin treatment were also investigated. SDS-PAGE of the conjugate presented two major bands, and native PAGE of the conjugate showed similar banding position with protein staining and polysaccharide staining, which was different from that of the unmodified SOD and LMWH/SOD mixture. Moreover, the conjugate migrated faster with increasing extent of the modification. Enhanced heat stability, acid resistance, alkali resistance, and anti-trypsin stability of the modified enzyme were observed compared with those of the unmodified enzyme. Results of the study suggest that covalent linkage in LMWH-SOD can be effectively characterized by electrophoretic techniques and the chemical modification of SOD with LMWH can enhance the stabilities of the enzyme. In addition, native PAGE with protein staining can be used to evaluate the extent of the modification.

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