Genetic ablation or chemical inhibition of phosphatidylcholine transfer protein attenuates diet-induced hepatic glucose production.

UNLABELLED: Phosphatidylcholine transfer protein (PC-TP, synonym StARD2) is a highly specific intracellular lipid binding protein that is enriched in liver. Coding region polymorphisms in both humans and mice appear to confer protection against measures of insulin resistance. The current study was designed to test the hypotheses that Pctp-/- mice are protected against diet-induced increases in hepatic glucose production and that small molecule inhibition of PC-TP recapitulates this phenotype. Pctp-/- and wildtype mice were subjected to high-fat feeding and rates of hepatic glucose production and glucose clearance were quantified by hyperinsulinemic euglycemic clamp studies and pyruvate tolerance tests. These studies revealed that high-fat diet-induced increases in hepatic glucose production were markedly attenuated in Pctp-/- mice. Small molecule inhibitors of PC-TP were synthesized and their potencies, as well as mechanism of inhibition, were characterized in vitro. An optimized inhibitor was administered to high-fat-fed mice and used to explore effects on insulin signaling in cell culture systems. Small molecule inhibitors bound PC-TP, displaced phosphatidylcholines from the lipid binding site, and increased the thermal stability of the protein. Administration of the optimized inhibitor to wildtype mice attenuated hepatic glucose production associated with high-fat feeding, but had no activity in Pctp-/- mice. Indicative of a mechanism for reducing glucose intolerance that is distinct from commonly utilized insulin-sensitizing agents, the inhibitor promoted insulin-independent phosphorylation of key insulin signaling molecules. CONCLUSION: These findings suggest PC-TP inhibition as a novel therapeutic strategy in the management of hepatic insulin resistance.

Regulatory role for phosphatidylcholine transfer protein/StarD2 in the metabolic response to peroxisome proliferator activated receptor alpha (PPARalpha).

Phosphatidylcholine transfer protein (PC-TP, a.k.a. StarD2) is abundantly expressed in liver and is regulated by PPARalpha. When fed the synthetic PPARalpha ligand fenofibrate, Pctp(-/-) mice exhibited altered lipid and glucose metabolism. Microarray profiling of livers from fenofibrate fed wild type and Pctp(-/-) mice revealed differential expression of a broad array of metabolic genes, as well as their regulatory transcription factors. PC-TP expression in cell culture controlled the activities of both PPARalpha and HNF4alpha, suggesting that the mechanism by which it modulates hepatic metabolism is at least in part via activation of transcription factors that govern nutrient homeostasis.

Regulation of hepatic lipogenesis by the transcription factor XBP1.

Dietary carbohydrates regulate hepatic lipogenesis by controlling the expression of critical enzymes in glycolytic and lipogenic pathways. We found that the transcription factor XBP1, a key regulator of the unfolded protein response, is required for the unrelated function of normal fatty acid synthesis in the liver. XBP1 protein expression in mice was elevated after feeding carbohydrates and corresponded with the induction of critical genes involved in fatty acid synthesis. Inducible, selective deletion of XBP1 in the liver resulted in marked hypocholesterolemia and hypotriglyceridemia, secondary to a decreased production of lipids from the liver. This phenotype was not accompanied by hepatic steatosis or compromise in protein secretory function. The identification of XBP1 as a regulator of lipogenesis has important implications for human dyslipidemias.

Regulation of energy substrate utilization and hepatic insulin sensitivity by phosphatidylcholine transfer protein/StarD2.

Phosphatidylcholine transfer protein (PC-TP, also known as StarD2) is a highly specific intracellular lipid binding protein with accentuated expression in oxidative tissues. Here we show that decreased plasma concentrations of glucose and free fatty acids in fasting PC-TP-deficient (Pctp(-/-)) mice are attributable to increased hepatic insulin sensitivity. In hyperinsulinemic-euglycemic clamp studies, Pctp(-/-) mice exhibited profound reductions in hepatic glucose production, gluconeogenesis, glycogenolysis, and glucose cycling. These changes were explained in part by the lack of PC-TP expression in liver per se and in part by marked alterations in body fat composition. Reduced respiratory quotients in Pctp(-/-) mice were indicative of preferential fatty acid utilization for energy production in oxidative tissues. In the setting of decreased hepatic fatty acid synthesis, increased clearance rates of dietary triglycerides and increased hepatic triglyceride production rates reflected higher turnover in Pctp(-/-) mice. Collectively, these data support a key biological role for PC-TP in the regulation of energy substrate utilization.

Hepatic insulin resistance is sufficient to produce dyslipidemia and susceptibility to atherosclerosis.

Insulin resistance plays a central role in the development of the metabolic syndrome, but how it relates to cardiovascular disease remains controversial. Liver insulin receptor knockout (LIRKO) mice have pure hepatic insulin resistance. On a standard chow diet, LIRKO mice have a proatherogenic lipoprotein profile with reduced high-density lipoprotein (HDL) cholesterol and very low-density lipoprotein (VLDL) particles that are markedly enriched in cholesterol. This is due to increased secretion and decreased clearance of apolipoprotein B-containing lipoproteins, coupled with decreased triglyceride secretion secondary to increased expression of Pgc-1 beta (Ppargc-1b), which promotes VLDL secretion, but decreased expression of Srebp-1c (Srebf1), Srebp-2 (Srebf2), and their targets, the lipogenic enzymes and the LDL receptor. Within 12 weeks on an atherogenic diet, LIRKO mice show marked hypercholesterolemia, and 100% of LIRKO mice, but 0% of controls, develop severe atherosclerosis. Thus, insulin resistance at the level of the liver is sufficient to produce the dyslipidemia and increased risk of atherosclerosis associated with the metabolic syndrome.

Interacting proteins dictate function of the minimal START domain phosphatidylcholine transfer protein/StarD2.

The Star (steroidogenic acute regulatory protein)-related transfer (START) domain superfamily is characterized by a distinctive lipid-binding motif. START domains typically reside in multidomain proteins, suggesting their function as lipid sensors that trigger biological activities. Phosphatidylcholine transfer protein (PC-TP, also known as StarD2) is an example of a START domain minimal protein that consists only of the lipid-binding motif. PC-TP, which binds phosphatidylcholine exclusively, is expressed during embryonic development and in several tissues of the adult mouse, including liver. Although it catalyzes the intermembrane exchange of phosphatidylcholines in vitro, this activity does not appear to explain the various metabolic alterations observed in mice lacking PC-TP. Here we demonstrate that PC-TP function may be mediated via interacting proteins. Yeast two-hybrid screening using libraries prepared from mouse liver and embryo identified Them2 (thioesterase superfamily member 2) and the homeodomain transcription factor Pax3 (paired box gene 3), respectively, as PC-TP-interacting proteins. These were notable because the START domain superfamily contains multidomain proteins in which the START domain coexists with thioesterase domains in mammals and with homeodomain transcription factors in plants. Interactions were verified in pulldown assays, and colocalization with PC-TP was confirmed within tissues and intracellularly. The acyl-CoA thioesterase activity of purified recombinant Them2 was markedly enhanced by recombinant PC-TP. In tissue culture, PC-TP coactivated the transcriptional activity of Pax3. These findings suggest that PC-TP functions as a phosphatidylcholine-sensing molecule that engages in diverse regulatory activities that depend upon the cellular expression of distinct interacting proteins.

Structure and function of phosphatidylcholine transfer protein (PC-TP)/StarD2.

Phosphatidylcholine transfer protein (PC-TP) is a highly specific soluble lipid binding protein that transfers phosphatidylcholine between membranes in vitro. PC-TP is a member of the steroidogenic acute regulatory protein-related transfer (START) domain superfamily. Although its biochemical properties and structure are well characterized, the functions of PC-TP in vivo remain incompletely understood. Studies of mice with homozygous disruption of the Pctp gene have largely refuted the hypothesis that this protein participates in the hepatocellular selection and transport of biliary phospholipids, in the production of lung surfactant, in leukotriene biosynthesis and in cellular phosphatidylcholine metabolism. Nevertheless, Pctp(-/-) mice exhibit interesting defects in lipid homeostasis, the understanding of which should elucidate the biological functions of PC-TP.