Regulation of mitochondrial sn-glycerol-3-phosphate acyltransferase activity: response to feeding status is unique in various rat tissues and is discordant with protein expression.

Triacylglycerol plays a critical role in an organism's ability to withstand fuel deprivation, and dysregulation of triacylglycerol synthesis is important in the development of diseases such as obesity and diabetes. Glycerol-3-phosphate acyltransferase (GPAT) catalyzes the initial and committed step of glycerolipid synthesis and is therefore a potential site for regulation of triacylglycerol synthesis. Because several studies suggest that triacylglycerol synthesis is linked to the mitochondrial isoform, we studied mitochondrial GPAT expression and the effect of feeding status on the regulation of mitochondrial GPAT in various rat tissues. Liver, adipose, and soleus muscle have high levels of GPAT mRNA, but low protein expression, whereas heart and adrenal, tissues with low GPAT mRNA abundance, have the highest GPAT protein expression. In addition, heart, which has the highest expression of mitochondrial GPAT protein, has low mitochondrial GPAT specific activity (0.02 nmol/min/mg). Liver and adipose have the highest mitochondrial GPAT specific activity (0.17 nmol/min/mg), but very low protein expression. Discrepancies between GPAT protein expression and activity suggest that mitochondrial GPAT may be regulated acutely. In response to a 48-h fast, liver and adipose mitochondrial GPAT protein expression and activity decrease 30-50%. After 24-h refeeding of either chow or high-sucrose diet, mitochondrial GPAT protein expression and activity overshoot normal levels 30-60%. In kidney, mitochondrial GPAT protein and activity increase 65 and 30%, respectively, with refeeding, whereas in the heart, mitochondrial GPAT activity increases 2.3-fold after a fast, with no change in protein expression. We also found that hepatic mitochondrial GPAT activity in the neonatal rat constitutes a lower percentage of the total GPAT activity than in the adult. We postulate that GPAT expression is modulated uniquely in each tissue according to specific needs for triacylglycerol storage.

Expression and characterization of recombinant rat Acyl-CoA synthetases 1, 4, and 5. Selective inhibition by triacsin C and thiazolidinediones.

Inhibition by triacsins and troglitazone of long chain fatty acid incorporation into cellular lipids suggests the existence of inhibitor-sensitive and -resistant acyl-CoA synthetases (ACS, EC ) that are linked to specific metabolic pathways. In order to test this hypothesis, we cloned and purified rat ACS1, ACS4, and ACS5, the isoforms present in liver and fat cells, expressed the isoforms as ACS-Flag fusion proteins in Escherichia coli, and purified them by Flag affinity chromatography. The Flag epitope at the C terminus did not alter the kinetic properties of the enzyme. Purified ACS1-, 4-, and 5-Flag isoforms differed in their apparent K(m) values for ATP, thermolability, pH optima, requirement for Triton X-100, and sensitivity to N-ethylmaleimide and phenylglyoxal. The ACS inhibitor triacsin C strongly inhibited ACS1 and ACS4, but not ACS5. The thiazolidinedione (TZD) insulin-sensitizing drugs and peroxisome proliferator-activated receptor gamma (PPARgamma) ligands, troglitazone, rosiglitazone, and pioglitazone, strongly and specifically inhibited only ACS4, with an IC(50) of less than 1.5 microm. Troglitazone exhibited a mixed type inhibition of ACS4. alpha-Tocopherol, whose ring structure forms the non-TZD portion of troglitazone, did not inhibit ACS4, indicating that the thiazolidine-2,4-dione moiety is the critical component for inhibition. A non-TZD PPARgamma ligand, GW1929, which is 7-fold more potent than rosiglitazone, inhibited ACS1 and ACS4 poorly with an IC(50) of greater than 50 microm, more than 100-fold higher than was required for rosiglitazone, thereby demonstrating the specificity of TZD inhibition. Further, the PPARalpha ligands, clofibrate and GW4647, and various xenobiotic carboxylic acids known to be incorporated into complex lipids had no effect on ACS1, -4, or -5. These results, together with previous data showing that triacsin C and troglitazone strongly inhibit triacylglycerol synthesis compared with other metabolic pathways, suggest that ACS1 and ACS4 catalyze the synthesis of acyl-CoAs used for triacylglycerol synthesis and that lack of inhibition of a metabolic pathway by triacsin C does not prove lack of acyl-CoA involvement. The results further suggest the possibility that the insulin-sensitizing effects of the thiazolidinedione drugs might be achieved, in part, through direct interaction with ACS4 in a PPARgamma-independent manner.

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 ) 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.

Acyl-CoAs are functionally channeled in liver: potential role of acyl-CoA synthetase.

Acyl-CoA synthetase (ACS) catalyzes the activation of long-chain fatty acids to acyl-CoAs, which can be metabolized to form CO(2), triacylglycerol (TAG), phospholipids (PL), and cholesteryl esters (CE). To determine whether inhibiting ACS affects these pathways differently, we incubated rat hepatocytes with [(14)C]oleate and the ACS inhibitor triacsin C. Triacsin inhibited TAG synthesis 70% in hepatocytes from fed rats and 40% in starved rats, but it had little effect on oleate incorporation into CE, PL, or beta-oxidation end products. Triacsin blocked [(3)H]glycerol incorporation into TAG and PL 33 and 25% more than it blocked [(14)C]oleate incorporation, suggesting greater inhibition of de novo TAG synthesis than reacylation. Triacsin did not affect oxidation of prelabeled intracellular lipid. ACS1 protein was abundant in liver microsomes but virtually undetectable in mitochondria. Refeeding increased microsomal ACS1 protein 89% but did not affect specific activity. Triacsin inhibited ACS specific activity in microsomes more from fed than from starved rats. These data suggest that ACS isozymes may be functionally linked to specific metabolic pathways and that ACS1 is not associated with beta-oxidation in liver.

Physiological and nutritional regulation of enzymes of triacylglycerol synthesis.

Although triacylglycerol stores play the critical role in an organism's ability to withstand fuel deprivation and are strongly associated with such disorders as diabetes, obesity, and atherosclerotic heart disease, information concerning the enzymes of triacylglycerol synthesis, their regulation by hormones, nutrients, and physiological conditions, their mechanisms of action, and the roles of specific isoforms has been limited by a lack of cloned cDNAs and purified proteins. Fortunately, molecular tools for several key enzymes in the synthetic pathway are becoming available. This review summarizes recent studies of these enzymes, their regulation under varying physiological conditions, their purported roles in synthesis of triacylglycerol and related glycerolipids, the possible functions of different isoenzymes, and the evidence for specialized cellular pools of triacylglycerol and glycerolipid intermediates.

Rat sn-glycerol-3-phosphate acyltransferase: molecular cloning and characterization of the cDNA and expressed protein.

Rat mitochondrial glycerol-3-phosphate acyltransferase (GPAT) cDNA was cloned and characterized. We identified a cDNA containing an open reading frame of 828 amino acids that had an 89% homology with the coding region of the previously characterized mouse mitochondrial GPAT and a predicted amino acid sequence that was 96% identical. The rat 5' UTR was only 159 nucleotides, in contrast to the 926 nucleotide 5' UTR of the mouse cDNA and had an internal deletion of 167 nucleotides. GPAT was expressed in Sf21 insect cells, and specific inhibitors strongly suggest that, like the Escherichia coli GPAT, the recombinant mitochondrial GPAT and the mitochondrial GPAT isoform in rat liver contain critical serine, histidine, and arginine residues.

Analysis of amino acid motifs diagnostic for the sn-glycerol-3-phosphate acyltransferase reaction.

Alignment of amino acid sequences from various acyltransferases [sn-glycerol-3-phosphate acyltransferase (GPAT), lysophosphatidic acid acyltransferase (LPAAT), acyl-CoA:dihydroxyacetone-phosphate acyltransferase (DHAPAT), 2-acylglycerophosphatidylethanolamine acyltransferase (LPEAT)] reveals four regions of strong homology, which we have labeled blocks I-IV. The consensus sequence for each conserved region is as follows: block I, [NX]-H-[RQ]-S-X-[LYIM]-D; block II, G-X-[IF]-F-I-[RD]-R; block III, F-[PLI]-E-G-[TG]-R-[SX]-[RX]; and block IV, [VI]-[PX]-[IVL]-[IV]-P-[VI]. We hypothesize that blocks I-IV and, in particular, the invariant amino acids contained within these regions form a catalytically important site in this family of acyltransferases. Using Escherichia coli GPAT (PlsB) as a model acyltransferase, we examined the role of the highly conserved amino acid residues in blocks I-IV in GPAT activity through chemical modification and site-directed mutagenesis experiments. We found that the histidine and aspartate in block I, the glycine in block III, and the proline in block IV all play a role in E. coli GPAT catalysis. The phenylalanine and arginine in block II and the glutamate and serine in block III appear to be important in binding the glycerol 3-phosphate substrate. Since blocks I-IV are also found in LPAAT, DHAPAT, and LPEAT, we believe that these conserved amino acid motifs are diagnostic for the acyltransferase reaction involving glycerol 3-phosphate, 1-acylglycerol 3-phosphate, and dihydroxyacetone phosphate substrates.

Membrane insertion characteristics of the various transmembrane domains of the Escherichia coli TolQ protein.

The Escherichia coli TolQ protein is a 230-amino acid integral cytoplasmic membrane protein required for the import of group A colicins, for infection by the filamentous phage, and for maintenance of the integrity of the bacterial envelope. TolQ is a polytopic protein with three membrane-spanning regions. The first membrane-spanning region has a 19-residue periplasmic NH2-terminal tail, while the second and third membrane-spanning segments are separated by a short 17-amino acid periplasmic loop. To study the membrane assembly of TolQ, fusions of different membrane-spanning regions were examined for their ability to insert in the absence of functional SecA or the membrane potential. Fusions containing the first membrane-spanning region plus the adjacent cytoplasmic domain and a construct containing the "hairpin loop," formed by the second and third membrane-spanning regions, insert in the absence of functional SecA. The fusion containing the second and third membrane-spanning regions required the membrane potential for insertion while the first membrane-spanning region was able to insert even in the absence of a membrane potential. Taken together, these results suggest that insertion of intact TolQ is not dependent on the Sec system, but does require the membrane potential.

Membrane topology and mutational analysis of the TolQ protein of Escherichia coli required for the uptake of macromolecules and cell envelope integrity.

TolQ is a 230-amino-acid protein required to maintain the integrity of the bacterial envelope and to facilitate the import of both filamentous bacteriophage and group A colicins. Cellular fractionation experiments showed TolQ to be localized to the cytoplasmic membrane. Bacteria expressing a series of TolQ-beta-galactosidase and TolQ-alkaline phosphatase fusion proteins were analyzed for the appropriate enzyme activity, membrane location, and sensitivity to exogenously added protease. The results are consistent with TolQ being an integral cytoplasmic membrane protein with three membrane-spanning regions. The amino-terminal 19 residues as well as a small loop in the 155 to 170 residue region appear exposed in the periplasm, while the carboxy terminus and a large loop after the first transmembrane region are cytoplasmic. Amino-terminal sequence analysis of TolQ purified from the membrane revealed the presence of the initiating formyl methionine group, suggesting a rapid translocation of the amino-terminal region across the cytoplasmic membrane. Analysis of various tolQ mutant strains suggests that the third transmembrane region as well as parts of the large cytoplasmic loop are necessary for activity.

Expression of insulin-like growth factor I stimulates normal somatic growth in growth hormone-deficient transgenic mice.

A line of transgenic mice expressing insulin-like growth factor-I (IGF-I) under the control of the mouse metallothionien-1 promoter was crossed to a line of dwarf transgenic mice lacking GH expressing cells that were genetically ablated by diphtheria toxin expression. Mice generated from this cross that carry both transgenes express IGF-I in the absence of GH. These mice grew larger than their GH-deficient transgenic littermates and exhibited weight and linear growth indistinguishable from that of their nontransgenic siblings. These results confirm the suspected role of IGF-I in mediating GH's stimulation of somatic growth, including that of long bones, and illustrates the essential role of GH and IGF-I in the modulation of postnatal growth. Analysis of differences in organ growth among these mice, however, suggests that GH and IGF-I also have growth promoting actions that are independent of one another; GH appears to be necessary for the attainment of normal liver size, while IGF-I can stimulate brain growth.