A series of experiments were conducted to characterize the autocrine role of adiponectin in modulating fatty acid metabolism and inflammation in the pig. In the first study, we cloned and sequenced the porcine adiponectin open reading frame and evaluated the regulation of adiponectin, in vitro and in vivo. The porcine sequence shares approximately 88, 86, 85 and 83% homology with the dog, human, cow and mouse adiponectin, respectively, and 79–83% similarity with dog, human, cow and mouse proteins at the amino acid level, based on the translated porcine sequence and GenBank submissions for the other species. Analysis of serum from very lean vs. a substantially fatter line of pigs indicated that relative circulating adiponectin concentrations are higher (P \u3c 0.01) in lean pigs than in the fatter line, and that the difference is established relatively early in the growth curve. Incubating pig adipocytes for 6 hours with recombinant pig adiponectin also resulted in an approximate 30% reduction (P \u3c 0.05) in lipogenesis compared with adipocytes under basal conditions and with those incubated in the presence of insulin.^ Based on in vivo and in vitro data showing adiponectin is positively correlated with leanness and reduces lipogenesis in porcine adipocytes, experiment investigated adipocyte lipid metabolism. In a series of cell culture studies for experiment two, we further investigated the role of adiponectin in modulating lipid metabolism by measuring mRNA abundance of genes associated with fatty acid synthesis and oxidation in porcine adipocytes. Adiponectin transiently increased (2–6.5 fold, P \u3c 0.05) the expression of acetyl Co-A carboxylase (ACC), AMP activated kinase (AMPK), acyl Co-A oxidase (ACO) and peroxisome proliferator activated receptor-&agr; (PPAR&agr;). After the initial increase in mRNA abundance, there was either a return to initial levels of expression (PPAR&agr;), a significant reduction (AMPK), or a cyclic pattern of expression (ACC, ACO). The abundance of the uncoupling protein-3 (UCP3) transcript was not influenced by adiponectin until the final measure at 360 minutes, at which time it was increased 3.8-fold (P \u3c 0.05). ^ Experiment three was designed to examine anti-inflammatory properties of adiponectin contributing to its insulin sensitizing effects through regulation of the adiponectin receptors, adipoR1 and adipoR2. Cells were incubated ± TNF&agr; or IL-6 (30 ng/mL), with or without pretreatment of 10 &mgr;M AG490, in hyper-glycemic or normo-glycemic conditions for 6 h. There was no effect of cytokines at 5.5 mM glucose for R1 expression. However, there was a trend for a down-regulation of R2 by AG490-TNF&agr; (P \u3c 0.07). At 25 mM glucose, there was an increase in R1 by AG490-TNF&agr; treatment (P = 0.06). R2 was marginally reduced by IL-6, but there was a reduction (P \u3c .01) with AG490 plus IL-6. High glucose caused a reduction of both receptors, whereas TNF&agr; increased both in the high glucose media. R1 (P \u3c 0.1) and R2 (P \u3c 0.05) were further elevated by AG490 plus TNF&agr; in high glucose. Interestingly, IL-6 and AG490 plus IL-6 (P \u3c 0.05) reduced R1 and R2 expression, but only in the high glucose media. Collectively, these data indicate that the effects of TNF&agr; and IL-6 on adiponectin receptor expression are influenced by glucose concentration, and that the JAK-STAT pathway may be a determinant of adiponectin receptor expression.^ In the final experiment, the effects of hyperglycemia on insulin sensitivity in pig adipocytes, and 3T3-L1 adipocytes were examined. In pig adipocytes, concentrations of 25 and 40 mM glucose inhibited insulin stimulated glucose uptake (P \u3c 0.05), that could not be reversed by addition of adiponectin to the culture media. To examine if hyperglycemic conditions were associated with impaired insulin signaling pig adipocytes were cultured in euglycemic and hyperglycemic conditions and insulin receptor and Akt phosphorylation were measured by semiquantitative Western blots. However, insulin resistance in primary pig adipocytes could not be attributed to changes in protein phosphosphorlation of the insulin receptor or its down-stream target Akt (P \u3e 0.05). To examine glycemic regulation of insulin-induced glucose uptake for longer duration the 3T3-L1 mouse derived adipocytes were used. Similar to the effects seen in acute porcine adipocyte cultures hyperglycemia inhibited insulin stimulated 2-deoxyglucose uptake in 3T3-L1 adipocytes (P \u3c 0.05), and 24 hour treatment with adiponectin could not reverse the insulin resistance. Again, difference in phosphorylation of the insulin receptor and Akt could not be detected in hyperglycemic culture cells compared with control cells. Interestingly, although adiponectin did not appear to impact insulin signaling pathways in under hyperglycemic conditions it did have an effect on inflammatory pathways in the adipocyte. Adiponectin reduced the production of intracellular ROS in 3T3-L1 adipocytes cultured in hyperglycemic conditions (P \u3c 0.05). (Abstract shortened by UMI.
