Mitigation of isolation-associated adipocyte interleukin-6 secretion following rapid dissociation of adipose tissue.

Primary adipocyte isolation by collagenase digestion is a widely used technique to study metabolic regulation and insulin action in adipocytes. However, induction of a proinflammatory response characterized by enhanced secretion of interleukin (IL)-6 has been tightly linked to the isolation process itself. To test the hypothesis that the shaking mechanical force exerted on adipocytes stimulates inflammation during isolation, rat primary adipocytes were prepared by collagenase digestion in orbital shaking incubators maintained at varying speeds. Contrary to expectation, the isolation-induced release of IL-6 was attenuated by increasing the rotational speed of digestion and the concentration of collagenase, both of which resulted in rapid dissociation of adipocytes from the vasculature. In addition, the attenuation of IL-6 secretion was associated with decreased phosphorylation of the stress-related p38 mitogen-activated protein kinase (p38 MAPK) and preserved insulin action. The data suggest that optimization of parameters including, but not limited to, mincing technique, time of digestion, and collagenase concentration will make it possible to isolate primary adipocytes without activation of a proinflammatory response leading to elevated secretion of IL-6.

Zinc enhances adiponectin oligomerization to octadecamers but decreases the rate of disulfide bond formation.

Adiponectin, a hormone secreted from adipocytes, has been shown to protect against development of insulin resistance, ischemia-reperfusion injury, and inflammation. Adiponectin assembles into multiple oligomeric isoforms: trimers, hexamers and several higher molecular weight (HMW) species. Of these, the HMW species are selectively decreased during the onset of type 2 diabetes. Despite the critical role of HMW adiponectin in insulin responsiveness, its assembly process is poorly understood. In this report, we investigated the role of divalent cations in adiponectin assembly. Purified adiponectin 18mers, the largest HMW species, did not collapse to smaller oligomers after treatment with high concentrations of EDTA. However, treatment with EDTA or another chelator DTPA inhibited the oligomerization of 18mers from trimers in vitro. Zn(2+) specifically increased the formation of 18mers when compared with Cu(2+), Mg(2+), and Ca(2+). Distribution of adiponectin oligomers secreted from zinc chelator TPEN-treated rat adipocytes skewed toward increased proportions of hexamers and trimers. While we observed presence of zinc in adiponectin purified from calf serum, the role of zinc in disulfide bonding between oligomers was examined because the process is critical for 18mer assembly. Surprisingly, Zn(2+) inhibited disulfide bond formation early in the oligomerization process. We hypothesize that initial decreases in disulfide formation rates could allow adiponectin subunits to associate before becoming locked in fully oxidized conformations incapable of further oligomerization. These data demonstrate that zinc stimulates oligomerization of HMW adiponectin and possibly other disulfide-dependent protein assembly processes.