Yuguang Shi

Lipid metabolic enzymes: emerging drug targets for the treatment of obesity

Obesity is a complex disease that is regulated by both central and peripheral mechanisms. New medicines for the treatment and prevention of obesity will need to overcome the powerful compensatory responses of the overlapping, and often redundant, networks that have evolved to safeguard efficient energy use. Despite great progress in the identification of central signals that regulate satiety, and considerable investment in the development of appetite-controlling medications, success has been modest so far. Here we review the main enzymes that are involved in different stages of lipid metabolism — from digestion and absorption through synthesis and storage to mobilization and oxidation — which might be successfully targeted by new pharmacotherapies.

Identification and Characterization of a Gene Encoding Human LPGAT1, an Endoplasmic Reticulum-associated Lysophosphatidylglycerol Acyltransferase

Phosphatidylglycerol (PG) is an important membrane polyglycerolphospholipid required for the activity of a variety of enzymes and is a precursor for synthesis of cardiolipin and bis(monoacylglycerol) phosphate. PG is subjected to remodeling subsequent to its de novo biosynthesis to incorporate appropriate acyl content for its biological functions and to prevent the harmful effect of lysophosphatidylglycerol (LPG) accumulation. The enzymes involved in the remodeling process have not yet been identified. We report here the identification and characterization of a human gene encoding an acyl-CoA: lysophosphatidylglycerol acyltransferase (LPGAT1). Expression of the LPGAT1 cDNA in Sf9 insect and COS-7 cells led to a significant increase in LPG acyltransferase activity. In contrast, no significant acyltransferase activities were detected against glycerol 3-phosphate or a variety of lysophospholipids, including lysophosphatidylcholine, lysophosphatidylethanolamine, lysophosphatidylinositol, and lysophosphatidylserine. The recombinant human LPGAT1 enzyme recognized various acyl-CoAs and LPGs as substrates but demonstrated clear preference to long chain saturated fatty acyl-CoAs and oleoyl-CoA as acyl donors, which is consistent with the lipid composition of endogenous PGs identified from different tissues. Kinetic analyses of LPGAT1 expressed in COS-7 cells showed that oleoyl-LPG was preferred over palmitoyl-LPG as an acyl receptor, whereas oleoyl-CoA was preferred over lauroyl-CoA as an acyl donor. Consistent with its proposed microsomal origin, LPGAT1 was localized to the endoplasmic reticulum by subcellular fractionation and immunohistochemical analyses. Northern blot analysis indicated that the human LPGAT1 was widely distributed, suggesting a dynamic functional role of the enzyme in different tissues.

Catalytic properties of MGAT3, a putative triacylgycerol synthase

Acyl-coenzyme A:monoacylglycerol acyltransferase 3 (MGAT3) is a member of the MGAT family of enzymes that catalyze the synthesis of diacylglycerol (DAG) from monoacylglycerol (MAG), a committed step in dietary fat absorption. Although named after the initial identification of its MGAT activity, MGAT3 shares higher sequence homology with acyl-coenzyme A:diacylglycerol acyltransferase 2 (DGAT2) than with other MGAT enzymes, suggesting that MGAT3 may also possess significant DGAT activity. This study compared the catalytic properties of MGAT3 with those of MGAT1 and MGAT2 enzymes using both MAG and DAG as substrates. Our results showed that in addition to the expected MGAT activity, the recombinant MGAT3 enzyme expressed in Sf-9 insect cells displayed a strong DGAT activity relative to that of MGAT1 and MGAT2 enzymes in the order MGAT3 > MGAT1 > MGAT2. In contrast, none of the three MGAT enzymes recognized biotinylated acyl-CoA or MAG as a substrate. Although MGAT3 possesses full DGAT activity, it differs from DGAT1 in catalytic properties and subcellular localization. The MGAT3 activity was sensitive to inhibition by the presence of 1% CHAPS, whereas DGAT1 activity was stimulated by the detergent. Consistent with high sequence homology with DGAT2, the MGAT3 enzyme demonstrated a similar subcellular distribution pattern to that of DGAT2, but not DGAT1, when expressed in COS-7 cells. Our data suggest that MGAT3 functions as a novel triacylglycerol (TAG) synthase that catalyzes efficiently the two consecutive acylation steps in TAG synthesis.

Characterization of a mutant pancreatic eIF-2α kinase, PEK, and co- localization with somatostatin in islet delta cells

Phosphorylation of eukaryotic translation initiation factor-2α (eIF-2α) is one of the key steps where protein synthesis is regulated in response to changes in environmental conditions. The phosphorylation is carried out in part by three distinct eIF-2α kinases including mammalian double-stranded RNA-dependent eIF-2α kinase (PKR) and heme-regulated inhibitor kinase (HRI), and yeast GCN2. We report the identification and characterization of a related kinase, PEK, which shares common features with other eIF-2α kinases including phosphorylation of eIF-2α in vitro. We show that human PEK is regulated by different mechanisms than PKR or HRI. In contrast to PKR or HRI, which are dependent on autophosphorylation for their kinase activity, a point mutation that replaced the conserved Lys-614 with an alanine completely abolished the eIF-2α kinase activity, whereas the mutant PEK was still autophosphorylated when expressed in Sf-9 cells. Northern blot analysis indicates that PEK mRNA was predominantly expressed in pancreas, though low expression was also present in several tissues. Consistent with the high levels of mRNA in pancreas, the PEK protein was only detected in human pancreatic islets, and the kinase co-localized with somatostatin, a pancreatic delta cell-specific hormone. Thus PEK is believed to play an important role in regulating protein synthesis in the pancreatic islet, especially in islet delta cells.

ALCAT1 is a polyglycerophospholipid acyltransferase potently regulated by adenine nucleotide and thyroid status

Acyl-CoA:lysocardiolipin acyltransferase-1 (ALCAT1) catalyzes acylation of lysocardiolipin back to cardiolipin, an important step in cardiolipin remodeling. The present study reports the catalytic properties of ALCAT1 in vitro and its regulation by thyroid hormone status in mouse liver and heart. Recombinant ALCAT1 expressed in Sf9 cells preferred basic pH conditions and did not require divalent cations or integrity of the subcellular membrane for its enzymatic activity. Recombinant ALCAT1 was potently inhibited by ADP and ATP, but not by adenosine nucleotide analogs or other nucleotides, such as UTP and GTP, suggesting that ALCAT1 does not require ATP hydrolysis for its enzyme activity. In addition to cardiolipin, ALCAT1 also catalyzed acylation of other members of the polyglycerophospholipid family, including phosphatidylglycerol, a precursor for cardiolipin synthesis, and bis(monoacylglycero)phosphate, a structural isomer of lysophosphatidylglycerol and a metabolic intermediate of cardiolipin. These findings suggest that ALCAT1 plays a role in the remodeling of other polyglycerophospholipids. In support of a regulatory role of ALCAT1 in cardiolipin remodeling in response to oxidative stress, ALCAT1 expression in liver and heart was significantly downregulated in mice with hypothyroidism and upregulated in mice treated with thyroid hormone, which is known to stimulate mitochondrial activity, oxidative stress, and cardiolipin remodeling.

Assignment1 of pancreatic eIF-2α kinase (EIF2AK3) to human chromosome band 2p12 by radiation hybrid mapping and in situ hybridization

Pancreatic eIF-2· kinase (EIF2AK3 alias PEK) is a new member of a family of eIF-2· kinases, which include hemeregulated inhibitor kinase (HRI), the double stranded RNA dependent protein kinase (PKR), and the yeast GCN2 (Shi et al., 1998). EIF2AK3 activity has also been linked to responsiveness to endoplasmic reticulum stress (Harding et al., 1999). Overexpression of one member of the eIF-2· kinase family has been implicated in apoptosis while overexpression of a dominant negative form of the same protein leads to cell proliferation (for review Williams et al., 1997). To determine whether altered EIF2AK3 expression may be linked to a possible disease state, we have used radiation hybrid (RH) mapping and fluorescent in situ hybridization (FISH) analysis to localize the EIF2AK3 gene.

Threonine phosphorylations induced by RX-871024 and insulin secretagogues in βTC6-F7 cells

Treatment of the pancreatic β-cell line βTC6-F7 with an imidazoline compound, RX-871024, KCl, or tolbutamide resulted in increased threonine phosphorylation of a 220-kDa protein (p220) concurrent with enhanced insulin secretion, which can be partially antagonized by diazoxide, an ATP-sensitive potassium (KATP) channel activator. Although phosphorylation of p220 was regulated by cytoplasmic free calcium concentration ([Ca2+]i), membrane depolarization alone was not sufficient to induce phosphorylation. Phosphorylation of p220 also was not directly mediated by protein kinase A, protein kinase C, or insulin exocytosis. Analysis of subcellular fractions indicated that p220 is a hydrophilic protein localized exclusively in the cytosol. Subsequently, p220 was purified to homogeneity, sequenced, and identified as nonmuscle myosin heavy chain-A (MHC-A). Stimulation of threonine phosphorylation of nonmuscle MHC-A by KCl treatment also resulted in increased phosphorylation of a 40-kDa protein, which was coimmunoprecipitated by antibody to MHC-A. Our results suggest that both nonmuscle MHC-A and the 40-kDa protein may play roles in regulating signal transduction, leading to insulin secretion.