Mouse liver cell culture

126

The proinflammatory cytokine interleukin (IL)-6 has been proposed to be one of the mediators that link obesity-derived chronic inflammation with insulin resistance. Signaling through the mammalian target of rapamycin (mTOR) has been found to impact insulin sensitivity under various pathological conditions, through serine phosphorylation and inhibition of insulin receptor substrate by the downstream effector of mTOR, ribosomal S6 kinase 1 (S6K1). However, an involvement of mTOR in IL-6-induced insulin resistance has not yet been reported. Here we show that rapamycin, the inhibitor of mTOR signaling, rescues insulin signaling and glycogen synthesis from IL-6 inhibition in HepG2 hepatocarcinoma cells as well as in mouse primary hepatocytes. IL-6 activates S6K1 in these cells, but unexpectedly, S6K1 is not involved in IL-6 inhibition of insulin signaling, since the effect of IL-6 persists in cells with drastically reduced S6K1 levels induced by RNA interference, suggesting that the function of mTOR signaling is through a mechanism different from the prevailing model of S6K1 phosphorylation of insulin receptor substrate-1. Interestingly, we find that the phosphorylation of STAT3 on Ser 727 and STAT3 transcriptional activity are regulated by mTOR upon IL-6 stimulation and that STAT3 is required for IL-6 inhibition of insulin signaling. Furthermore, IL-6-induced SOCS3 expression is inhibited by rapamycin, and ectopic expression of SOCS3 blocks the ability of rapamycin to enhance insulin sensitivity in the presence of IL-6. Taken together, we propose that mTOR plays a key role in IL-6-induced hepatic insulin resistance by regulating STAT3 activation and subsequent SOCS3 expression.Insulin resistance, a major risk factor and the principle defect in type II diabetes, is commonly observed with obesity. Chronic production of proinflammatory cytokines is considered a major link between obesity and insulin resistance (1). Elevated local and circulating levels of proinflammatory cytokines, such as tumor necrosis factor-␣ and interleukin (IL) 3 -6, are known to be associated with obesity in both human and rodent models (2-5). Although adipose-derived tumor necrosis factor-␣ may act locally in autocrine and paracrine manners, adipose-derived IL-6 is thought to enter circulation and play a systemic role in modulating insulin actions (6). Depletion of IL-6 improves insulin action in a mouse model of obesity (7), whereas in humans, elevated plasma IL-6 levels positively correlate with obesity and insulin resistance and predict the development of type 2 diabetes (2, 8, 9). Furthermore, IL-6 administration to healthy individuals induces blood glucose increases (10). In vitro, IL-6 has been shown to induce insulin resistance in hepatic cells (11). Although the role of IL-6 within peripheral tissues (adipose and skeletal muscle) is still being debated, it is generally accepted that at least in the liver, IL-6 causes insulin resistance (12).A mechanism of IL-6-induced insulin resistance in the liver has been proposed, which involves the...

Section: Methodsmentioning

confidence: 99%

Regulation of Interleukin-6-induced Hepatic Insulin Resistance by Mammalian Target of Rapamycin through the STAT3-SOCS3 Pathway

Kim

Liu

et al. 2008

126

“…For all experiments, 12-to 20-week-old male Pemt Ϫ/Ϫ and Pemt ϩ/ϩ mice were utilized. Primary hepatocytes were isolated from the livers using the collagenase perfusion technique as described previously (21,22). The cells were isolated and plated in DMEM containing 17% fetal bovine serum and 0.01 mg/ml insulin.…”

Section: Methodsmentioning

confidence: 99%

An Unexpected Requirement for PhosphatidylethanolamineN-Methyltransferase in the Secretion of Very Low Density Lipoproteins

Noga¹,

Zhao

Vance

2002

212

164

Phosphatidylethanolamine N-methyltransferase (PEMT) catalyzes the conversion of phosphatidylethanolamine to phosphatidylcholine (PC). We investigated whether there was diminished secretion of lipoproteins from hepatocytes derived from mice that lacked PEMT (Pemt ؊/؊ ) compared with Pemt ؉/؉ mice. Hepatocytes were incubated with 0.75 mM oleate, the media were harvested, and triacylglycerol (TG), PC, apolipoprotein (apo) B100, and apoB48 were isolated and quantified. Compared with hepatocytes from Pemt ؉/؉ mice, hepatocytes from Pemt ؊/؊ mice secreted 50% less TG, whereas secretion of PC was unaffected. Fractionation of the secreted lipoproteins on density gradients demonstrated that the decrease in TG was in the very low density lipoprotein (VLDL)/low density lipoprotein fractions. The secretion of apoB100 was decreased by ϳ70% in VLDLs/ low density lipoproteins, whereas there was no significant decrease in apoB48 secretion in any fraction. Transfection of McArdle hepatoma cells (that lack PEMT) with PEMT cDNA enhanced secretion of TG in the VLDLs. Because the levels of PC in the hepatocytes and hepatoma cells were unaffected by the lack of PEMT expression, there appears to be an unexpected requirement for PEMT in the secretion of apoB100-containing VLDLs.

“…In Vitro Measurement of VLDL-triglyceride Secretion-Mouse hepatocyte isolation and culturing was done as described previously (6,29). In short, the portal vein was cannulated with a 22-gauge plastic cannula.…”

Section: Methodsmentioning

confidence: 99%

Apolipoprotein E Participates in the Regulation of Very Low Density Lipoprotein-Triglyceride Secretion by the Liver

Mensenkamp¹,

Jong²,

Goor³

et al. 1999

121

ApoE-deficient mice on low fat diet show hepatic triglyceride accumulation and a reduced very low density lipoprotein (VLDL) triglyceride production rate. To establish the role of apoE in the regulation of hepatic VLDL production, the human APOE3 gene was introduced into apoE-deficient mice by cross-breeding with APOE3 transgenics (APOE3/apoe؊/؊ mice) or by adenoviral transduction. APOE3 was expressed in the liver and, to a lesser extent, in brain, spleen, and lung of transgenic APOE3/apoe؊/؊ mice similar to endogenous apoe. Plasma cholesterol levels in APOE/apoe؊/؊ mice (3.4 ؎ 0.5 mM) were reduced when compared with apoe؊/؊ mice (12.6 ؎ 1.4 mM) but still elevated when compared with wild type control values (1.9 ؎ 0.1 mM). Hepatic triglyceride accumulation in apoE-deficient mice was completely reversed by introduction of the APOE3 transgene. The in vivo hepatic VLDL-triglyceride production rate was reduced to 36% of control values in apoE-deficient mice but normalized in APOE3/ apoe؊/؊ mice. Hepatic secretion of apoB was not affected in either of the strains. Secretion of 3 H-labeled triglycerides synthesized from [ 3 H]glycerol by cultured hepatocytes from apoE-deficient mice was four times lower than by APOE3/apoe؊/؊ or control hepatocytes. The average size of secreted VLDL particles produced by cultured apoE-deficient hepatocytes was significantly reduced when compared with those of APOE3/ apoe؊/؊ and wild type mice. Hepatic expression of human APOE3 cDNA via adenovirus-mediated gene transfer in apoE-deficient mice resulted in a reduction of plasma cholesterol depending on plasma apoE3 levels. The in vivo VLDL-triglyceride production rate in these mice was increased up to 500% compared with LacZ-injected controls and correlated with the amount of apoE3 per particle. These findings indicate a regulatory role of apoE in hepatic VLDL-triglyceride secretion, independent from its role in lipoprotein clearance.Apolipoprotein E is an important constituent of triglyceriderich lipoproteins such as VLDL 1 and chylomicrons and is essential for effective receptor-mediated uptake of their remnants (1). High levels of apoE delay lipoprotein lipase-mediated lipolysis of these lipoproteins (2, 3). ApoE deficiency in mice leads to elevated plasma cholesterol concentrations because of the accumulation of VLDL-and chylomicron-remnants (␤-VLDL), which results from impaired hepatic uptake of these particles (4 -6). As a consequence, atherosclerotic lesions rapidly develop in apoE-deficient mice (Refs. 5 and 7; for review see Ref. 8). A secretion-recapture role for apoE has been proposed in which the apoprotein is secreted by hepatocytes into the space of Disse to interact with heparan sulfate proteoglycans, followed by binding and internalization of circulating lipoproteins (9, 10). Data from in vitro studies indicate that apoE may also serve a function in intracellular metabolism and distribution of lipids after their uptake by macrophages (11) and hepatoma cells (12). Recent studies from our laboratory have shown that apoE defi...