Rosalind A. Coleman

Enzymes of triacylglycerol synthesis and their regulation

Since the pathways of glycerolipid biosynthesis were elucidated in the 1950s, considerable knowledge has been gained about the enzymes that catalyze the lipid biosynthetic reactions and the factors that regulate triacylglycerol biosynthesis. In the last few decades, in part due to advances in technology and the wide availability of nucleotide and amino acid sequences, we have made enormous strides in our understanding of these enzymes at the molecular level. In many cases, sequence information obtained from lipid biosynthetic enzymes of prokaryotes and yeast has provided the means to search the genomic and expressed sequence tag databases for mammalian homologs and most of the genes have now been identified. Surprisingly, multiple isoforms appear to catalyze the same chemical reactions, suggesting that each isoform may play a distinct functional role in the pathway of triacylglycerol and phospholipid biosynthesis. This review focuses on the de novo biosynthesis of triacylglycerol in eukaryotic cells, the isoenzymes that are involved, their subcellular locations, how they are regulated, and their putative individual roles in glycerolipid biosynthesis.

Leptin Directly Alters Lipid Partitioning in Skeletal Muscle

Leptin, an adipocyte-derived hormone that directly regulates both adiposity and energy homeostasis, decreases food intake and appears to partition metabolic fuels toward utilization and away from storage. Because skeletal muscle expresses the leptin receptor and plays a major role in determining energy metabolism, we studied leptins effects on glucose and fatty acid (FA) metabolism in isolated mouse soleus and extensor digitorum longus (EDL) muscles. One muscle from each animal served as a basal control. The contralateral muscle was treated with insulin (10 mU/ml), leptin (0.01–10 μg/ml), or insulin plus leptin, and incorporation of [14C]glucose or [14C]oleate into CO2 and into either glycogen or triacylglycerol (TAG) was determined. Leptin increased soleus muscle FA oxidation by 42% (P < 0.001) and decreased incorporation of FA into TAG by 35% (P < 0.01) in a dose-dependent manner. In contrast, insulin decreased soleus muscle FA oxidation by 40% (P < 0.001) and increased incorporation into TAG by 70% (P < 0.001). When both hormones were present, leptin attenuated both the antioxidative and the lipogenic effects of insulin by 50%. Less pronounced hormone effects were observed in EDL muscle. Leptin did not alter insulin-stimulated muscle glucose metabolism. These data demonstrate that leptin has direct and acute effects on skeletal muscle.

AMP-activated kinase reciprocally regulates triacylglycerol synthesis and fatty acid oxidation in liver and muscle: evidence that sn-glycerol-3-phosphate acyltransferase is a novel target

AMP-activated kinase (AMPK) is activated in response to metabolic stresses that deplete cellular ATP, and in both liver and skeletal muscle, activated AMPK stimulates fatty acid oxidation. To determine whether AMPK might reciprocally regulate glycerolipid synthesis, we studied liver and skeletal-muscle lipid metabolism in the presence of 5-amino-4-imidazolecarboxamide (AICA) riboside, a cell-permeable compound whose phosphorylated metabolite activates AMPK. Adding AICA riboside to cultured rat hepatocytes for 3 h decreased [14C]oleate and [3H]glycerol incorporation into triacylglycerol (TAG) by 50% and 38% respectively, and decreased oleate labelling of diacylglycerol by 60%. In isolated mouse soleus, a highly oxidative muscle, incubation with AICA riboside for 90 min decreased [14C]oleate incorporation into TAG by 37% and increased 14CO2 production by 48%. When insulin was present, [14C]oleate oxidation was 49% lower and [14C]oleate incorporation into TAG was 62% higher than under basal conditions. AICA riboside blocked insulins antioxidative and lipogenic effects, increasing fatty acid oxidation by 78% and decreasing labelled TAG 43%. Similar results on fatty acid oxidation and acylglycerol synthesis were observed in C2C12 myoblasts, and in differentiated C2C12 myotubes, AICA riboside also inhibited the hydrolysis of intracellular TAG. These data suggest that AICA riboside might inhibit sn-glycerol-3-phosphate acyltransferase (GPAT), which catalyses the committed step in the pathway of glycerolipid biosynthesis. Incubating rat hepatocytes with AICA riboside for both 15 and 30 min decreased mitochondrial GPAT activity 22-34% without affecting microsomal GPAT, diacylglycerol acyltransferase or acyl-CoA synthetase activities. Finally, purified recombinant AMPKalpha1 and AMPKalpha2 inhibited hepatic mitochondrial GPAT in a time-and ATP-dependent manner. These data show that AMPK reciprocally regulates acyl-CoA channelling towards beta-oxidation and away from glycerolipid biosynthesis, and provide strong evidence that AMPK phosphorylates and inhibits mitochondrial GPAT.

The role of lipid droplets in metabolic disease in rodents and humans

Lipid droplets (LDs) are intracellular organelles that store neutral lipids within cells. Over the last two decades there has been a dramatic growth in our understanding of LD biology and, in parallel, our understanding of the role of LDs in health and disease. In its simplest form, the LD regulates the storage and hydrolysis of neutral lipids, including triacylglycerol and/or cholesterol esters. It is becoming increasingly evident that alterations in the regulation of LD physiology and metabolism influence the risk of developing metabolic diseases such as diabetes. In this review we provide an update on the role of LD-associated proteins and LDs in metabolic disease.

Triacylglycerol Synthesis Enzymes Mediate Lipid Droplet Growth by Relocalizing from the ER to Lipid Droplets

Lipid droplets (LDs) store metabolic energy and membrane lipid precursors. With excess metabolic energy, cells synthesize triacylglycerol (TG) and form LDs that grow dramatically. It is unclear how TG synthesis relates to LD formation and growth. Here, we identify two LD subpopulations: smaller LDs of relatively constant size, and LDs that grow larger. The latter population contains isoenzymes for each step of TG synthesis. Glycerol-3-phosphate acyltransferase 4 (GPAT4), which catalyzes the first and rate-limiting step, relocalizes from the endoplasmic reticulum (ER) to a subset of forming LDs, where it becomes stably associated. ER-to-LD targeting of GPAT4 and other LD-localized TG synthesis isozymes is required for LD growth. Key features of GPAT4 ER-to-LD targeting and function in LD growth are conserved between Drosophila and mammalian cells. Our results explain how TG synthesis is coupled with LD growth and identify two distinct LD subpopulations based on their capacity for localized TG synthesis.

Acyl-CoA Synthetase Isoforms 1, 4, and 5 Are Present in Different Subcellular Membranes in Rat Liver and Can Be Inhibited Independently

Inhibition studies have suggested that acyl-CoA synthetase (ACS, EC 6.2.1.3) isoforms might regulate the use of acyl-CoAs by different metabolic pathways. In order to determine whether the subcellular locations differed for each of the three ACSs present in liver and whether these isoforms were regulated independently, non-cross-reacting peptide antibodies were raised against ACS1, ACS4, and ACS5. ACS1 was identified in endoplasmic reticulum, mitochondria-associated membrane (MAM), and cytosol, but not in mitochondria. ACS4 was present primarily in MAM, and the 76-kDa ACS5 protein was located in mitochondrial membrane. Consistent with these locations, N-ethylmaleimide, an inhibitor of ACS4, inhibited ACS activity 47% in MAM and 28% in endoplasmic reticulum. Troglitazone, a second ACS4 inhibitor, inhibited ACS activity <10% in microsomes and mitochondria and 45% in MAM. Triacsin C, a competitive inhibitor of both ACS1 and ACS4, inhibited ACS activity similarly in endoplasmic reticulum, MAM, and mitochondria, suggesting that a hitherto unidentified triacsin-sensitive ACS is present in mitochondria. ACS1, ACS4, and ACS5 were regulated independently by fasting and re-feeding. Fasting rats for 48 h resulted in a decrease in ACS4 protein, and an increase in ACS5. Re-feeding normal chow or a high sucrose diet for 24 h after a 48-h fast increased both ACS1 and ACS4 protein expression 1.5–2.0-fold, consistent with inhibition studies. These results suggest that ACS1 and ACS4 may be linked to triacylglycerol synthesis. Taken together, the data suggest that acyl-CoAs may be functionally channeled to specific metabolic pathways through different ACS isoforms in unique subcellular locations.

Hepatic triacylglycerol accumulation and insulin resistance

The association of hepatic steatosis with hepatic insulin resistance and type 2 diabetes has prompted investigators to elucidate the underlying mechanism. In this review we focus on pathways of lipid metabolism, and we review recent data, primarily from mouse models, that link lipid intermediates with insulin resistance. Most of the studies that implicate acyl-CoA, lysophosphatidic acid, phosphatidic acid, diacylglycerol, or ceramide rely on indirect associations. Convincing data to support the hypothesis that specific lipid intermediates initiate pathways that alter insulin signaling will require studies in which the concentration of each purported signaling molecule can be manipulated independently.

Glycogen Storage Disease in Adults

Table 1 The glycogen storage diseases (GSD) include more than ten separate genetic defects that impair glycogen breakdown, primarily in liver or muscle or both. Even the types most frequently encountered (GSD-Ia and GSD-III) are uncommon, each with an incidence of approximately 1 in 100 000 births. Thus, no single institution has followed and reported on a large series of patients. The importance of several major complications was recognized only recently because only single cases were initially reported. Our study represents the largest number of adults with GSD-Ia and GSD-Ib to be included in one investigation and is the first to focus on clinical and social outcomes. Although two groups of investigators recently described the clinical course of patients with GSD in Europe and Israel, most of the patients studied were children [1, 2]. Relatively little information is available about adults with these diseases. We collected information on adults with GSD-Ia, GSD-Ib, and GSD-III in the United States and Canada in order to identify long-term complications that may be amenable to prevention and to determine the effect of the disease on education, employment, and family life. Table 1. SI Units Glycogen Storage Disease Types Ia, Ib, and III Glycogen storage disease type Ia results from deficient glucose-6-phosphatase activity in liver, kidney, and intestine [3]. Glucose-6-phosphatase is a single 35-kd protein [4]. When glucose-6-phosphatase activity is deficient, the liver is unable to hydrolyze glucose from glucose-6-phosphate that has been derived either from the metabolism of stored glycogen or from gluconeogenesis. Patients must depend on dietary carbohydrate to maintain euglycemia; during a fast of more than a few hours, the serum glucose concentration may decrease profoundly, and seizures are common in children. Mental retardation is uncommon, however, because the brain is protected by its ability to metabolize lactate that is present at high concentrations in the serum. Chronic hypoglycemia causes a sustained increase of counter-regulatory hormones, such as cortisol. In childhood, GSD-Ia typically results in poor growth and delayed puberty. Hyperuricemia occurs probably because ATP synthesis from ADP is driven by deamination of the AMP product to inosine that is subsequently metabolized to uric acid. Renal excretion of uric acid may also be decreased because lactate competes for the renal anion transporter. Fatty liver and hyperlipidemia result from the large influx of adipose-derived fatty acids into the liver in response to low insulin and high glucagon and cortisol concentrations. Anemia that is refractory to iron supplementation is believed to occur because of chronic disease. In untreated adults with GSD-Ia, the blood glucose decreases only to about 2.8 mmol/L (50 mg/dL) after an overnight fast. Symptomatic hypoglycemia is uncommon in untreated adults, but increases of counter-regulatory hormones probably persist. Adults with GSD-Ia have a high incidence of hepatic adenomas and focal segmental glomerulosclerosis [3, 5, 6]. The continuing abnormalities in counter-regulatory hormones, together with the hyperuricemia and hyperlipidemia, may be responsible for many of the complications observed in adult patients. Glycogen storage disease type Ib results from a deficiency of the glucose-6-phosphate translocase that transports glucose-6-phosphate into the lumen of the endoplasmic reticulum where it is hydrolyzed by glucose-6-phosphatase [3]. The translocase has not been purified. Without the translocase, glucose-6-phosphate cannot reach the hydrolytic enzyme; thus, patients with GSD-Ib are also unable to maintain euglycemia. The resulting metabolic consequences are identical in both forms of GSD-I. Because patients with GSD-Ib also have neutropenia and recurrent bacterial infections [3, 7], it seems likely that the glucose-6-phosphate translocase plays a role in normal neutrophil function. In GSD-III, glycogen debranching enzyme is deficient [3]. This enzyme is a 165-kd protein that contains two catalytic sites that are required for activity. The enzyme has been cloned and sequenced [8]. Normally, successive glucose residues are released from glycogen by glycogen phosphorylase until the glycogen chains are within four glucose residues of a branch point. The first catalytic activity of the debranching enzyme (oligo-1,4,-1,4-glucantransferase) transfers three of the remaining glucose residues to the terminus of another glucose chain. The second catalytic activity (amylo-1,6-glucosidase) then hydrolyzes the branch-point glucose residue. Three molecular subgroups of GSD-III have been well defined [9]; each is associated with enzyme deficiency in the liver and with childhood hypoglycemia. In adults with GSD-III, hypoglycemia is uncommon. As in GSD-I, poor growth may be prominent, but the growth rate increases before puberty, and adult height is normal [10]. Additionally, increases in transaminase levels provide evidence of hepatocellular damage, and liver biopsies show periportal fibrosis [10], perhaps related to the abnormal short-branched glycogen structure. In patients with subtype GSD-IIIb, enzyme activity and immunoreactive material are absent in liver but are present in muscle; these patients do not have a myopathy. Patients with GSD-IIIa (78% of cases) lack enzyme activity and lack immunoreactive material in liver and muscle. Patients with GSD-IIId (7% of cases) lack only the transferase activity but have normal immunoreactive material in liver and muscle. In patients with GSD-IIIa and IIId, muscle weakness may occur either in childhood or after the third decade. Cardiomyopathy is apparent only after age 30 years [9]. Treatment of Glycogen Storage Disease For only the past 10 to 15 years, children with GSD-Ia and GSD-Ib were treated with either intermittent uncooked cornstarch or a nocturnal glucose infusion given by intragastric tube. When euglycemia is maintained in this manner, growth and pubertal development are normal, and it is hoped that the late complications of GSD-I will be prevented. A high-protein diet was recommended for patients with GSD-III. Diet supplementation can increase the growth rate in children with GSD-III [11], but beneficial results on the myopathy have been less well documented. In this retrospective study of adults with GSD types Ia, Ib, and III, we found, in addition to complications frequently recognized, a high incidence of osteopenia and fractures and of nephrocalcinosis, kidney stones, and pyelonephritis. We describe the long-term outlook for adult patients with GSD who have not had optimal lifelong dietary glucose therapy. Methods Information on patients 18 years of age or older was obtained by contacting specialists in pediatric metabolism, endocrinology, gastroenterology, and genetics throughout the United States and Canada and by advertising through the Association for Glycogen Storage Diseases and The New England Journal of Medicine. No registries of patients with GSD are available. Information was included on living adult patients with GSD and patients who had died since 1967. Diagnosis of GSD had been confirmed by enzyme assay of each patient or of an affected sibling. Fifty-six physicians were individually contacted. Nineteen stated that they were not treating any adult patients with GSD. Thirteen physicians in private practice or at 1 of 12 medical centers filled out a detailed questionnaire or sent copies of clinic and hospital records that were reviewed by two of us. To obtain an estimate of how many patients might be missed by this survey, we reviewed records from a reference laboratory (Washington University) of 21 patients with GSD-Ia and of 21 patients with GSD-III who were diagnosed between 1955 and 1972. If still alive, these patients would now range in age from 18 to 64 years. Our study includes only 5 of these patients with GSD-I and 1 with GSD-III. Thus, this report incompletely represents North American patients with GSD who are currently older than 18 years of age. Clinical, radiographic, and laboratory findings at the latest visit were obtained, but data were not universally available for every item on the questionnaire. In analyzing each response, information was considered to be available only if specifically recorded; omission of information was not recorded as either a negative or a positive response. The presence of liver adenomas, nephrocalcinosis, or kidney stones was based on data from ultrasound or radiographic studies. The diagnosis of osteopenia was based on data from radiographic studies. The normal values for height were taken from the National Center for Health Statistics [12]. Normal values for serum chemistry tests [13] were used. Results Glycogen Storage Disease Type Ia Case Report Patient 1, a 43-year-old divorced father of one child, is a poultry farmer. A liver biopsy and enzymatic assay were obtained at 4 years of age because of poor growth, hypoglycemia without seizures, hepatomegaly, and frequent nosebleeds. Despite frequent meals, growth continued to be poor, puberty was delayed, and the final adult height of 168 cm was achieved after 20 years of age. Allopurinol was taken inconsistently after one of many gouty attacks beginning from 18 years of age. The patient did not complete high school. As an adult, he has smoked 2 to 4 packs of cigarettes per day. After divorcing in his 20s, he frequently skipped breakfast and failed to follow a recommended diet. Instead, his diet was high in fat and consisted primarily of foods that required little preparation, such as candy and sandwiches. He has always denied symptomatic hypoglycemia, although his serum glucose concentration after an overnight fast is about 2.8 mmol/L (50 mg/dL). Beginning in his mid-20s, he had recurrent episodes of flank pain and hematuria that were treated with antibiotics, and he passed kidney stones. At age 24, an intravenous pyelogram showed punctate calcificati

Glycerol-3-phosphate acyltransferases: Rate limiting enzymes of triacylglycerol biosynthesis

Four homologous isoforms of glycerol-3-phosphate acyltransferase (GPAT), each the product of a separate gene, catalyze the synthesis of lysophosphatidic acid from glycerol-3-phosphate and long-chain acyl-CoA. This step initiates the synthesis of all the glycerolipids and evidence from gain-of-function and loss-of-function studies in mice and in cell culture strongly suggests that each isoform contributes to the synthesis of triacylglycerol. Much work remains to fully delineate the regulation of each GPAT isoform and its individual role in triacylglycerol synthesis.

Mitochondrial Glycerol-3-Phosphate Acyltransferase-Deficient Mice Have Reduced Weight and Liver Triacylglycerol Content and Altered Glycerolipid Fatty Acid Composition

ABSTRACT Microsomal and mitochondrial isoforms of glycerol-3-phosphate acyltransferase (GPAT; E.C. 2.3.1.15) catalyze the committed step in glycerolipid synthesis. The mitochondrial isoform, mtGPAT, was believed to control the positioning of saturated fatty acids at the sn-1 position of phospholipids, and nutritional, hormonal, and overexpression studies suggested that mtGPAT activity is important for the synthesis of triacylglycerol. To determine whether these purported functions were true, we constructed mice deficient in mtGPAT. mtGPAT−/− mice weighed less than controls and had reduced gonadal fat pad weights and lower hepatic triacylglycerol content, plasma triacylglycerol, and very low density lipoprotein triacylglycerol secretion. As predicted, in mtGPAT−/− liver, the palmitate content was lower in triacylglycerol, phosphatidylcholine, and phosphatidylethanolamine. Positional analysis revealed that mtGPAT−/− liver phosphatidylethanolamine and phosphatidylcholine had about 21% less palmitate in the sn-1 position and 36 and 40%, respectively, more arachidonate in the sn-2 position. These data confirm the important role of mtGPAT in the synthesis of triacylglycerol, in the fatty acid content of triacylglycerol and cholesterol esters, and in the positioning of specific fatty acids, particularly palmitate and arachidonate, in phospholipids. The increase in arachidonate may be functionally significant in terms of eicosanoid production.