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Adiponectin is an adipocyte-derived hormone with antidiabetic activities that include increasing the sensitivity of cells to insulin. Adaptor protein containing pleckstrin homology domain, phosphotyrosine-binding domain, and leucine zipper motif (APPL1) stimulates adiponectin signaling and promotes adiponectin's insulin-sensitizing effects by binding to two adiponectin receptors, AdipoR1 and AdipoR2, and the insulin receptor. In this study, we report an alternative splicing variant of APPL1 (APPL1sv) that is highly expressed in mouse liver, pancreas, and spleen tissues. The expression levels of APPL1sv in liver tissues were enhanced in a mouse model of obesity and diabetic dyslipidemia (i.e. db/db mice) and reduced in calorierestricted mice compared with ad libitum--fed mice. APPL1sv overexpression or suppression inhibited or enhanced, respectively, adiponectin-stimulated phosphorylation ofAMPprotein kinase (AMPK) in mouse hepatocytes. We also found that APPL1sv binds to AdipoR1 and AdipoR2 under basal conditions and that adiponectin treatment reduces this binding. Overexpression of APPL1sv blocked adiponectin-induced interactions of APPL1 with the adiponectin receptors. Moreover, adenovirus-mediated and short hairpin RNA--based suppression of APPL1sv greatly reduced high fat diet--induced insulin resistance and hepatic glucose production in mice. Our study identifies a key suppressor of hepatic adiponectin signaling and insulin sensitivity, a finding that may shed light on identifying effective therapeutic targets for treating insulin resistance and type 2 diabetes. [ABSTRACT FROM AUTHOR]

Adiponectin is an adipokine with insulin-sensitizing and anti-inflammatory functions. We previously reported that adiponectin multimerization and stability are promoted by the disulfide bond A oxidoreductase-like protein (DsbA-L) in cells and in vivo. However, the precise mechanism by which DsbA-L regulates adiponectin biosynthesis remains elusive. Here we show that DsbA-L is co-localized with the endoplasmic reticulum (ER) marker protein disulfide isomerase and the mitochondrial marker Mito Tracker. In addition, DsbA-L interacts with the ER chaperone protein Ero1-Lα in 3T3-L1 adipocytes. In silico analysis and truncation mapping studies revealed that DsbA-L contains an ER targeting signal at its N terminus. Deletion of the first 6 residues at theNterminus greatly impaired DsbA-L localization in the ER. Overexpression of the wild type but not the ER localization-defective mutant of DsbA-L protects against thapsigargin-induced ER stress and adiponectin down-regulation in 3T3-L1 adipocytes. In addition, overexpression of the wild type but not the ER localization-defective mutant of DsbA-L promotes adiponectin multimerization. Together, our results reveal that DsbA-L is localized in both the mitochondria and the ER in adipocytes and that its ER localization plays a critical role in suppressing ER stress and promoting adiponectin biosynthesis and secretion. [ABSTRACT FROM AUTHOR]

Disulfide-bond A oxidoreductase-like protein (DsbA-L) possesses beneficial effects such as promoting adiponectin multimerization and stability, increasing insulin sensitivity, and enhancing energy metabolism. The expression level of DsbA-L is negatively correlated with obesity in mice and humans, but the underlying mechanisms remain unknown. To address this question, we generated reporter gene constructs containing the promoter sequence of the mouse DsbA-L gene. Deletion analysis showed that the proximal promoter of mouse DsbA-L is located between -186 and -34 bp relative to the transcription start site. In silico analysis identified a putative Sp1 transcription factor binding site in the first intron of the DsbA-L gene. Electrophoretic mobility shift assay and chromatin immunoprecipitation analysis indicated that Sp1 bound to this intron region in vitro and in intact cells. Overexpression of Sp1 or suppressing Sp1 expression by siRNA reduced or increased DsbA-L promoter activity, respectively. The binding activity of Sp1 was gradually decreased during 3T3-L1 cell differentiation and was significantly increased in adipose tissues of obese mice. Our results identify Sp1 as an inhibitor of DsbA-L gene transcription, and the Sp1-mediated inhibition of DsbA-L gene expression may provide a mechanism underlying obesity-induced adiponectin downregulation and insulin resistance. [ABSTRACT FROM AUTHOR]