Obesity and insulin resistance are considered chronic inflammatory states, in part because circulating IL-6 is elevated. Exogenous IL-6 can induce hepatic insulin resistance in vitro and in vivo. The importance of endogenous IL-6, however, to insulin resistance of obesity is unresolved. To test the hypothesis that IL-6 contributes to the inflammation and insulin resistance of obesity, IL-6 was depleted in Lep(ob) mice by injection of IL-6-neutralizing antibody. In untreated Lep(ob) mice, signal transducer and activator of transcription-3 (STAT3) activation was increased compared with that in lean controls, consistent with an inflammatory state. With IL-6 depletion, activation of STAT3 in liver and adipose tissue and expression of haptoglobin were reduced. Expression of the IL-6-dependent, hepatic acute phase protein fibrinogen was also decreased. Using the hyperinsulinemic-euglycemic clamp technique, insulin-dependent suppression of endogenous glucose production was 89% in IL-6-depleted Lep(ob) mice, in contrast to only 32% in Lep(ob) controls, indicating a marked increase in hepatic insulin sensitivity. A significant change in glucose uptake in skeletal muscle after IL-6 neutralization was not observed. In a direct comparison of hepatic insulin signaling in Lep(ob) mice treated with anti-IL-6 vs. IgG-treated controls, insulin-dependent insulin receptor autophosphorylation and activation of Akt (pSer473) were increased by nearly 50% with IL-6 depletion. In adipose tissue, insulin receptor signaling showed no significant change despite major reductions in STAT3 phosphorylation and haptoglobin expression. In diet-induced obese mice, depletion of IL-6 improved insulin responsiveness in 2-h insulin tolerance tests. In conclusion, these results indicate that IL-6 plays an important and selective role in hepatic insulin resistance of obesity.

Summary While adipose tissue-associated macrophages contribute to development of chronic inflammation and insulin resistance of obesity, little is known about the role of hepatic Kupffer cells in this environment. Here we address the impact of Kupffer cell ablation using clodronate-encapsulated liposome depletion in a diet-induced obese (DIO) and insulin resistant mouse model. Hepatic expression of macrophage markers measured by realtime RT-PCR remained unaltered in DIO mice despite characteristic expansion of adipose tissue-associated macrophages. DIO mouse livers displayed increased expression of alternative activation markers but unaltered proinflammatory cytokine expression when compared to lean mice. Kupffer cell ablation reduced hepatic anti-inflammatory cytokine IL-10 mRNA expression in lean and DIO mice by 95% and 84%, respectively. Despite decreased hepatic IL-6 gene expression after ablation in lean and DIO mice, hepatic STAT3 phosphorylation, Socs3 and acute phase protein mRNA expression increased. Kupffer cell ablation in DIO mice resulted in additional hepatic triglyceride accumulation and a 30-40% reduction in hepatic insulin receptor autophosphorylation and Akt activation. Implicating systemic loss of IL-10, high-fat-fed IL-10 knockout mice also displayed increased hepatic STAT3 signaling and hepatic triglyceride accumulation. Insulin signaling was not altered, however. In conclusion, Kupffer cells are a major source of hepatic IL-10 expression, the loss of which is associated with increased STAT3-dependent signaling and steatosis. One or more additional factors appear to be required, however, for the Kupffer cell-dependent protective effect on insulin receptor signaling in DIO mice.

SOCS3 is a cytokine-inducible negative regulator of cytokine receptor signaling. Recently, SOCS3 was shown to be induced by a cAMP-dependent pathway involving exchange protein directly activated by cAMP (Epac). We observed in livers of fasted mice that Socs3 mRNA was increased 4-fold compared with refed mice, suggesting a physiologic role for SOCS3 in the fasted state that may involve glucagon and Epac. Treating primary hepatocytes with glucagon resulted in a 4-fold increase in Socs3 mRNA levels. The Epac-selective cAMP analog 8 -4-(chlorophenylthio)-2-O-methyladenosine-3,5-monophosphate, acetoxymethyl ester (cpTOME) increased Socs3 expression comparably. In gain-of-function studies, adenoviral expression of SOCS3 in primary hepatocytes caused a 50% decrease in 8-br-cAMP-dependent PKA phosphorylation of the transcription factor CREB. Induction of the gluconeogenic genes Ppargc1a, Pck1, and G6pc by glucagon or 8-br-cAMP was suppressed nearly 50%. In loss-of-function studies, hepatocytes from liver-specific SOCS3 knock-out mice responded to 8-br-cAMP with a 200% greater increase in Ppargc1a and Pck1 expression, and a 30% increase in G6pc expression, relative to wild-type cells. Suppression of SOCS3 by shRNA in hepatocytes resulted in a 60% increase in cAMP-dependent G6pc and Pck1 expression relative to control cells. SOCS3 expression also inhibited cAMP-dependent phosphorylation of the IP3 receptor but did not inhibit nuclear localization of the catalytic subunit of PKA. Using an in vitro kinase assay, cAMP-dependent PKA activity was reduced by 80% in hepatocytes expressing ectopic SOCS3. These data indicate that cAMP activates both the PKA and Epac pathways with induction of SOCS3 by the Epac pathway negatively regulating the PKA pathway.Glucagon is a catabolic hormone that is synthesized and released by ␣ cells in the pancreatic islets in response to hypoglycemia (1). Glucagon maintains normoglycemia by increasing hepatic glucose output through mobilization of glucose from glycogen (glycogenolysis) and increasing de novo glucose production (gluconeogenesis) (1, 2). Glucagon receptor expression has been found in the pancreas, small intestine, and kidney, but is minimal compared with levels in the liver (3). The glucagon receptor is a classic G s ␣-coupled receptor. Receptor-ligand coupling results in activation of adenylate cyclase, cAMP production, and protein kinase A (PKA) activation. In a reaction that is critical to gluconeogenesis, activated PKA phosphorylates cAMP response element-binding protein (CREB) 2 leading to its transcriptional activation at the cAMPresponsive element (CRE) in the promoter region of key genes, including peroxisome proliferator-activated receptor-␥ coactivator 1␣ (Ppargc1a), phosphoenolpyruvate carboxykinase (Pck1), and glucose-6-phosphatase (G6pc) (1, 4). PEPCK catalyzes the rate-limiting step in gluconeogenesis, while G-6-Pase hydrolyzes glucose-6-phosphate to generate free glucose that can be released from the hepatocyte into the circulation. PGC-1␣ coactivates hepatic nuclear fa...

Objective Obesity is associated with increased circulating interleukin-6, which may contribute to hepatic insulin resistance by impairing insulin receptor signaling. This study was designed to assess the impact of the systemic absence of IL-6 on the development of insulin resistance and glucose intolerance in an obese mouse model. Materials/Methods Systemic insulin, glucose, and pyruvate tolerance tests were performed in IL-6 knockout (IL6KO) mice that had been crossed with a genetically obese (Lepdb) mouse model. Real-time RT-PCR and Western blot analysis assessed cellular and molecular markers of insulin signaling, inflammation, and metabolism. Results Absence of IL-6 did not improve systemic glucose or insulin tolerance, but Lepdb × IL6KO mice displayed a smaller blood glucose increase following a pyruvate challenge. These results suggest that loss of IL-6 in the context of obesity may locally reduce hepatic glucose production from a gluconeogenic precursor. Hepatic insulin-dependent insulin receptor autophosphorylation, Akt activation, and FoxO1 phosphorylation were similar between Lepdb × IL6KO mice and Lepdb controls. Basal gene expression of the gluconeogenic enzyme, PEPCK, was reduced in male Lepdb × IL6KO mice relative to Lepdb controls, but gene expression of another regulatory enzyme, G6Pase, remained unaltered. Absence of IL-6 reduced gene expression of serum amyloid A and RelA in female Lepdb mice, but did not alter hepatic triglyceride accumulation or lipogenic gene expression. Conclusion Overall, our results suggest that IL-6 may be detrimental in obesity by contributing to elevated hepatic glucose output.

As models of chronic liver injury in the mouse are limited, we have developed such a model in the C57B6 mouse that utilizes Adeno-associated viral vectors (AAV) engineered to express both enhanced Green Fluorescent Protein and the CD8+ epitope SIINFEKL. After injection of this vector directly into the liver, 1x104 SIINFEKL-specific OT-1 cells are administered. Injection of these cells results in an increase in OT-1 cell numbers in the liver until Day 20 correlating with liver damage from Days 10-25. Addition of these cells does not cause decreased vector DNA but significantly decreases vector mRNA expression. To define the mechanisms by which OT-1 cells mediate their effect, we focused on both IFNγ and Fas. In looking at the role of these two molecules, we have found that IFNγ is important in the activation and/or recruitment of the OT-1 cells as IFNγR KO hosts have a significant decrease in the number of OT-1 in the liver at Day 20 compared to their WT controls. The same has been found when comparing WT and Faslpr hosts indicating a role of FasL in the same activation or recruitment processes. FasL has been shown by Suzuki et al to play a role in costimulation in a peptide model, but this phenomenon has not been seen in other viral or vector models1. The role of these two molecules in our AAV model may give clues to the molecular events that occur in other types of hepatitis. Suzuki I JI 2000 Nov 15; 165(10):5537-43. Support: NIH RO1 AI064463