Tubes were vortexed and centrifuged, and the supernatants were transferred to a fresh set of tubes containing 200 mg mixed-bed ion-exchange resins, AG-501 × 8 resins (BioRad). Cells were preincubated (in absence of substrate) for 30 min with insulin (10 nmol/L) or glucagon (10 ng/mL), or a combination, followed by 3 h incubation with the substrate. The next morning, cultures were washed in HEPES-phosphate–salt–bicarbonate buffer containing 0.2% BSA and incubated in the same buffer containing insulin or glucagon and 14C-pyruvate (2 mmol/L, 0.5 ♜i pyruvate/incubation, PerkinElmer) as substrate. In control wells without CM, RPMI was used to dilute the culture medium. During this incubation, if condition medium (CM) was used for an experiment, CM was diluted 1:1 with the culture medium and incubated overnight. After attachment, medium was switched to Williams E without serum and dexamethasone (culture medium) and incubated overnight in a 5% CO 2 incubator. Hepatocyte suspension was filtered and centrifuged at 50 g for 6 min, and the pellet was resuspended in Williams Medium E (Life Technologies, Inc.) fortified with glutamax, antibiotics, 10% FBS, and dexamethasone (10 nmol/L) and allowed to attach for 6 h on collagen-coated plates. The digested livers were excised, and cells were teased out in Earle balanced salt solution without collagenase. Mice were infused through the inferior vena cava with a calcium-free HEPES-phosphate buffer, followed by Liberase TM collagenase solution (Roche Diagnostics) for another 5 min. In support of these ideas, chemical deletion or genetic impairment of hepatic macrophages in obese mice leads to an improvement in insulin sensitivity ( 13– 15). In addition, there is strong evidence that macrophages play a significant role in hepatic inflammation and insulin resistance. Thus, obesity promotes hepatic inflammation in humans and rodents, leading to increased production of proinflammatory cytokines and acute-phase reactants. Other cardinal features of obesity are nonalcoholic fatty liver disease and hepatic inflammation ( 11, 12). In obesity and type 2 diabetes, the liver is insulin resistant, resulting in increased gluconeogenesis and glycogenolysis leading to increased overall hepatic glucose production ( 9, 10). Insulin stimulates storage of glucose as liver glycogen and inhibits hepatic glucose production by restricting glycogenolysis and gluconeogenesis ( 8). The liver is responsible for the metabolism, synthesis, storage, and distribution of nutrients and is a key organ with central importance in the maintenance of glucose homeostasis. These RHMs then enhance the severity of obesity-induced inflammation and hepatic insulin resistance. We conclude that KCs can participate in obesity-induced inflammation by causing the recruitment of RHMs, which are distinct from KCs and are not precursors to KCs. A comparison of the MCP-1/C-C chemokine receptor type 2 (CCR2) chemokine system between the two cell types showed that the ligand (MCP-1) is more highly expressed in KCs than in RHMs, whereas CCR2 expression is approximately fivefold greater in RHMs. Furthermore, RHMs from obese mice were more inflamed and expressed higher levels of tumor necrosis factor-α and interleukin-6 than RHMs from lean mice. In addition, RHMs comprised smaller size and immature, monocyte-derived cells compared with KCs. In vivo macrophage tracking revealed an approximately sixfold higher number of RHMs in obese mice than in lean mice, whereas the number of KCs was comparable. With these approaches, the inflammatory phenotype of these distinct macrophage populations was determined under lean and obese conditions. We used a combination of in vivo macrophage tracking methodologies and adoptive transfer techniques in which KCs and RHMs are differentially labeled with fluorescent markers. Therefore, we assessed whether KCs and RHMs, or both, represent the major liver inflammatory cell type in obesity. However, recruited hepatic macrophages (RHMs) were recently shown to represent a sizable liver macrophage population in the context of obesity. The current dogma is that obesity-associated hepatic inflammation is due to increased Kupffer cell (KC) activation.
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