A predominant role of acyl-CoA:monoacylglycerol acyltransferase-2 in dietary fat absorption implicated by tissue distribution, subcellular localization, and up-regulation by high fat diet

Acyl-CoA:monoacylglycerol acyltransferase-2 (MGAT2) catalyzes the synthesis of diacylglycerol and differs from the MGAT1 and MGAT3 in tissue distribution at the mRNA level. In addition to the small intestine, MGAT2 mRNA is also expressed at high levels in human liver, the lower gastrointestinal tract, and the mouse kidney, but the physiological significance of such expression has not yet been studied. Using an affinity-purified antibody, the present study investigated the expression of murine MGAT2 protein along the intestinal tract, determined its subcellular localization, and studied its regulation by diet and in db/db mouse. Results demonstrate a high level of MGAT2 expression in the small intestine in a proximal-to-distal gradient that correlated well with both MGAT enzyme activity and fat absorption pattern. In contrast, MGAT2 protein was not detectable in other sections of the digestive tract, including stomach, cecum, colon, and rectum, or other mouse tissues such as kidney, liver, and adipocytes. Immunohistological studies provided direct evidence that the enzyme is expressed not only in the villi, but also in the crypt regions of the small intestine, which suggests that MGAT2 expression occurs prior to the maturation of enterocytes. MGAT2 is localized in the endoplasmic reticulum (ER) in both MGAT2-transfected COS-7 and Caco-2 cells, indicating that the ER is the primary site for dietary fat re-synthesis. MGAT2 expression appeared not to be affected by diabetes in the db/db mouse, however, the total intestinal MGAT activity was significantly enhanced. Finally, an up-regulation of both MGAT2 protein expression and MGAT activity was observed in mice fed a high fat diet, implicating a role of MGAT2 in diet-induced obesity. Taken together, our data suggest a predominant role of MGAT2 in dietary fat absorption.     

MGAT2, a monoacylglycerol acyltransferase expressed in the small intestine

Acyl CoA:monoacylglycerol acyltransferase (MGAT) catalyzes the synthesis of diacylglycerol, a precursor of triacylglycerol. In the intestine, MGAT plays a major role in the absorption of dietary fat by catalyzing the resynthesis of triacylglycerol in enterocytes. This resynthesis is required for the assembly of lipoproteins that transport absorbed fat to other tissues. Despite intense efforts, a gene encoding an intestinal MGAT has not been found. Previously, we identified a gene encoding MGAT1, which in mice is expressed in the stomach, kidney, adipose tissue, and liver but not in the intestine. We now report the identification of homologous genes in humans and mice encoding MGAT2. Expression of the MGAT2 cDNA in either insect or mammalian cells markedly increased MGAT activity in cell membranes. MGAT activity was proportional to the level of MGAT2 protein expressed, and the amount of diacylglycerol produced depended on the concentration of MGAT substrates (fatty acyl CoA or monoacylglycerol). In humans, the MGAT2 gene is highly expressed in the small intestine, liver, stomach, kidney, colon, and white adipose tissue; in mice, it is expressed predominantly in the small intestine. The discovery of the MGAT2 gene will facilitate studies to determine the functional role of MGAT2 in fat absorption in the intestine and to determine whether blocking MGAT activity in enterocytes is a feasible approach to inhibit fat absorption and treat obesity.     

Identification of acyl coenzyme A:monoacylglycerol acyltransferase 3, an intestinal specific enzyme implicated in dietary fat absorption

Acyl coenzyme A:monoacylglycerol acyltransferase (MGAT) catalyzes the synthesis of diacylglycerol using 2-monoacylglycerol and fatty acyl coenzyme A. This enzymatic reaction is believed to be an essential and rate-limiting step for the absorption of fat in the small intestine. Although the first MGAT-encoding cDNA, designated MGAT1, has been recently isolated, it is not expressed in the small intestine and hence cannot account for the high intestinal MGAT enzyme activity that is important for the physiology of fat absorption. In the current study, we report the identification of a novel MGAT, designated MGAT3, and present evidence that it fulfills the criteria to be the elusive intestinal MGAT. MGAT3 encodes a approximately 36-kDa transmembrane protein that is highly homologous to MGAT1 and -2. In humans, expression of MGAT3 is restricted to gastrointestinal tract with the highest level found in the ileum. At the cellular level, recombinant MGAT3 is localized to the endoplasmic reticulum. Recombinant MGAT3 enzyme activity produced in insect Sf9 cells selectively acylates 2-monoacylglycerol with higher efficiency than other stereoisomers. The molecular identification of MGAT3 will facilitate the evaluation of using intestinal MGAT as a potential point of intervention for antiobesity therapies.     

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.     

Enzyme activities of intestinal triacylglycerol and phosphatidylcholine biosynthesis in Atlantic salmon (Salmo salar L.)

The substitution of fish oil with plant-derived oil in diets for carnivorous fish, such as Atlantic salmon, has previously revealed the potentially deleterious supranuclear accumulation of lipid droplets in intestinal cells (enterocytes) which may compromise gut integrity, and consequently, fish health. This suggests that unfamiliar dietary lipid sources may have a significant impact on intestinal lipid metabolism, however, the mode of lipid resynthesis is largely unknown in teleost fish intestine. The present study aimed at characterising three key lipogenic enzymes involved in the biosynthesis of triacylglycerol (TAG) and phosphatidylcholine (PC) in Atlantic salmon enterocytes: monoacylglycerol acyltransferase (MGAT), diacylglycerol acyltransferase (DGAT), and diacylglycerol cholinephosphotransferase (CPT). Furthermore, to investigate the dietary effect of plant oils on these enzymes, two experimental groups of fish were fed a diet with either capelin (fish oil) or vegetable oil (rapeseed oil:palm oil:linseed oil, 55:30:15 w/w) as the lipid source. The monoacylglycerol (MAG) pathway was highly active in the intestinal mucosa of Atlantic salmon as demonstrated by MGAT activity (7 nmol [1-(14)C]palmitoyl-CoA incorporated min(-1) mg protein(-1)) and DGAT activity (4 nmol [1-(14)C]palmitoyl-CoA incorporated min(-1) mg protein(-1)), with MGAT appearing to also provide adequate production of sn-1,2-diacylglycerol for potential utilisation in PC synthesis via CPT activity (0.4 nmol CDP-[(14)C]choline incorporated min(-1) mg protein(-1)). Both DGAT and CPT specific activity values were comparable to reported mammalian equivalents, although MGAT activity was lower. Nevertheless, MGAT appeared not to be the rate-limiting step in salmon intestinal TAG synthesis. The homology between piscine and mammalian enzymes was established by similar stimulation and inhibition profiles by a variety of tested cofactors and isomeric substrates. The low dietary n-3/n-6 PUFA ratio presented in the vegetable oil diet did not significantly affect the activities of MGAT, DGAT, or CPT under optimised assay conditions, or in vivo intestinal mucosa lipid class composition, when compared to a standard fish oil diet.     

Cloning and functional characterization of a mouse intestinal acyl-CoA:monoacylglycerol acyltransferase,MGAT2

Acyl-CoA:monoacylglycerol transferase (MGAT) plays a predominant role in dietary fat absorption in the small intestine, where it catalyzes the first step of triacylglycerol resynthesis in enterocytes for chylomicron formation and secretion. Although the mouse small intestine exhibits the highest MGAT enzyme activity among all of the tissues studied, the gene encoding the enzyme has not been identified so far. In the present studies, we report the identification and characterization of a mouse intestinal MGAT, MGAT2. Transient expression of MGAT2 in AV-12, COS-7, and Caco-2 cells led to a more than 70-, 30-, and 35-fold increase in the synthesis of diacylglycerol, respectively. MGAT2 expressed in mammalian cells can catalyze the acylation of rac-1-, sn-2-, and sn-3-monoacylglycerols, and the enzyme prefers monoacylglycerols containing unsaturated fatty acyls as substrates. MGAT2 also demonstrates weak DGAT activity, which can be distinguished from its MGAT activity by detergent treatment that abolishes DGAT but not MGAT activity. We also analyzed the biochemical features of MGAT2 and demonstrated homogenate protein-, time-, and substrate concentration-dependent MGAT enzyme activity in transiently transfected COS-7 cells. Northern blot analysis indicates that the mouse MGAT2 is most abundantly expressed in the small intestine, suggesting that MGAT2 may play an important role in dietary fat absorption.     

Properties of the mouse intestinal acyl-CoA:monoacylglycerol acyltransferase,MGAT2

Acyl-CoA:monoacylglycerol acyltransferase (MGAT) plays an important role in dietary fat absorption by catalyzing a rate-limiting step in the re-synthesis of diacylglycerols in enterocytes. The present study reports further characterization of MGAT2, a newly identified intestinal MGAT (Cao, J., Lockwood, J., Burn, P., and Shi, Y. (2003) J. Biol. Chem. 278, 13860-13866) for its substrate specificity, requirement for lipid cofactors, optimum pH and Mg2+, and other intrinsic properties. MGAT2 enzyme expressed in COS-7 cells displayed a broad range of substrate specificity toward fatty acyl-CoA derivatives and monoacylglycerols, among which the highest activities were observed with oleoyl-CoA and rac-1-monolauroylglycerol, respectively. MGAT2 appeared to acylate monoacylglycerols containing unsaturated fatty acyls in preference to saturated ones. Lipid cofactors that play roles in signal transduction were shown to modulate MGAT2 activities. In contrast to oleic acid and sphingosine that exhibited inhibitory effects, phosphatidylcholine, phosphatidylserine, and phosphatidic acid stimulated MGAT2 activities. Using recombinant murine MGAT2 expressed in Escherichia coli, we demonstrated conclusively that MGAT2 also possessed an intrinsic acyl-CoA:diacylglycerol acyltransferase (DGAT) activity, which could provide an alternative pathway for triacylglycerol synthesis in the absence of DGAT. In contrast to the inhibitory effect on MGAT2 activities, nonionic and zwitterionic detergents led to a striking activation of DGAT activity of the human DGAT1 expressed in mammalian cells, which further distinguished the behaviors of the two enzymes. The elucidation of properties of MGAT2 will facilitate future development of compounds that inhibit dietary fat absorption as a means to treat obesity.     

Proteolysis of bovine beta-lactoglobulin during thermal treatment in subdenaturing conditions highlights some structural features of the temperature-modified protein and yields fragments with low immunoreactivity.

Bovine beta-lactoglobulin was hydrolyzed with trypsin or chymotrypsin in the course of heat treatment at 55, 60 and 65 degrees C at neutral pH. At these temperatures beta-lactoglobulin undergoes significant but reversible structural changes. In the conditions used in the present study, beta-lactoglobulin was virtually insensitive to proteolysis by either enzyme at room temperature, but underwent extensive proteolysis when either protease was present during the heat treatment. High-temperature proteolysis occurs in a progressive manner. Mass spectrometry analysis of some large-sized breakdown intermediates formed in the early steps of hydrolysis indicated that both enzymes effectively hydrolyzed some regions of beta-lactoglobulin that were transiently exposed during the physical treatments and that were not accessible in the native protein. The immunochemical properties of the products of beta-lactoglobulin hydrolysis were assessed by using various beta-lactoglobulin-specific antibodies, and most epitopic sites were no longer present after attack of the partially unfolded protein by the two proteases.     

Protease-activated protein kinase C in rat liver

In regenerating rat liver, an elevated protein kinase activity was detected which phosphorylated ribosomal protein S6 and histones. The properties of this enzyme were closely similar with those of protease-activated protein kinase C with Mr 45,000. During the study of the mechanism of proteolytic activation, type III protein kinase C (encoding alpha-sequence) was shown to be subjected to limited proteolysis by trypsin-like protease and converted to protein kinase M in ionic strength- and pH-dependent manner. This reaction was stimulated in the presence of Ca2+ and phospholipid under slightly higher ionic strength condition than physiological level (greater than 140 mM NaCl) and alkaline pH (7.5-8.0). These results suggest that activation of Na+/H+ exchanger in plasma membrane may trigger this type of proteolytic activation of protein kinase C. In addition to protein kinase M, another type of protease-activated kinase with Mr 80,000 was detected when limited proteolysis of protein kinase C was performed on inactive form of this enzyme (in the absence of either Ca2+ or phospholipid or both activators) under lower ionic strength condition. The molecular mass of this active enzyme was slightly smaller (approximately 200) than that of native protein kinase C. However, it is not clear at this time whether this small fragment was released from amino-terminal or carboxy-terminal domain to make protein kinase C partially active in the absence of Ca2+ and phospholipid. Although it has been proposed that proteolytic degradation of protein kinase C is involved in down regulation of this enzyme, the physiological significance of these two types of protease-activated forms of protein kinases in liver has remained obscure.     

Intestine-specific Deletion of Acyl-CoA:Monoacylglycerol Acyltransferase (MGAT) 2 Protects Mice from Diet-induced Obesity and Glucose Intolerance

The absorption of dietary fat involves the re-esterification of digested triacylglycerol in the enterocytes, a process catalyzed by acyl-CoA:monoacylglycerol acyltransferase (MGAT) 2. Mice without a functional gene encoding MGAT2 (Mogat2^−/−) are protected from diet-induced obesity. Surprisingly, these mice absorb normal amounts of dietary fat but increase their energy expenditure. MGAT2 is expressed in tissues besides intestine, including adipose tissue in both mice and humans. To test the hypothesis that intestinal MGAT2 regulates systemic energy balance, we generated and characterized mice deficient in MGAT2 specifically in the small intestine (Mogat2^IKO). We found that, like Mogat2^−/− mice, Mogat2^IKO mice also showed a delay in fat absorption, a decrease in food intake, and a propensity to use fatty acids as fuel when first exposed to a high fat diet. Mogat2^IKO mice increased energy expenditure although to a lesser degree than Mogat2^−/− mice and were protected against diet-induced weight gain and associated comorbidities, including hepatic steatosis, hypercholesterolemia, and glucose intolerance. These findings illustrate that intestinal lipid metabolism plays a crucial role in the regulation of systemic energy balance and may be a feasible intervention target. In addition, they suggest that MGAT activity in extraintestinal tissues may also modulate energy metabolism.     

Tissue expression of three structurally different fatty acid binding proteins from rat heart muscle, liver, and intestine

Three structurally different 14-15 kDa fatty acid binding proteins have have been purified from rat liver, small intestinal epithelium, and heart muscle, and were quantitated using specific antisera in rat tissues. Heart muscle fatty acid binding protein comprised 5% of heart muscle cytosol protein and was also expressed in stomach, muscle, testis, ovary, kidney, brain, and adipose tissue, a pattern distinct from both liver protein (expressed in liver, small and large intestinal epithelium, and adipose tissue) and intestinal protein (expressed in small and large intestinal epithelium and stomach). Distinctive patterns of tissue expression of the three different fatty acid binding proteins suggest that they may perform different specific functions in fatty acid transport and metabolism.     

Some molecular properties of asparagine synthetase from rat liver

Asparagine synthetase purified from rat liver reveals two species (slower migrating band I and faster migrating band II) when subjected to polyacrylamide gel electrophoresis under nondenaturing conditions (S. Hongo and T. Sato (1981) Anal. Biochem. 114, 163-166). We have investigated some molecular properties of these species. Elution of band I from the gel and re-electrophoresis showed that band I yielded band II similar to that of the initial run. Peptide maps by limited proteolysis were very similar and amino acid compositions were also alike in the two species. L-Lysine was identified as the sole NH2-terminal amino acid in both the species. By cross-linking experiments the enzyme was shown to be a dimeric protein. When the purified enzyme was subjected to isoelectric focusing the enzyme activity and protein focused at pH 6.0 in a single peak. These results demonstrate that rat liver asparagine synthetase is composed of two identical subunits. The enzyme, inactivated by storage at -20 degrees C for about 3 months, showed aggregated forms in polyacrylamide gel electrophoresis, and was reactivated markedly by the addition of dithiothreitol.     

Monoacylglycerol Acyltransferase-2 Is a Tetrameric Enzyme That Selectively Heterodimerizes with Diacylglycerol Acyltransferase-1

Acyl-CoA:monoacylglycerol acyltransferases (MGATs) and diacylglycerol acyltransferases (DGATs) catalyze the two consecutive steps in the synthesis of triacylglycerol, a key process required for dietary fat absorption into the enterocytes of the small intestine. In this report, we investigated the tendency of MGAT2 to form an enzyme complex with DGAT1 and DGAT2 in intact cells. We demonstrated that in addition to the 38-kDa monomer of the MGAT2 enzyme predicted by its peptide sequence, a 76-kDa moiety was detected in SDS-PAGE without reducing agent and heat inactivation. The 76-kDa MGAT2 moiety was greatly enhanced by treatment with a cross-linking reagent in intact cells. Additionally, the cross-linking reagent dose-dependently yielded a band corresponding to the tetramer (152 kDa) in SDS-PAGE, suggesting that the MGAT2 enzyme primarily functions as a homotetrameric protein and as a tetrameric protein. Likewise, DGAT1 also forms a homodimer under nondenaturing conditions. When co-expressed in COS-7 cells, MGAT2 heterodimerized with DGAT1 without treatment with a cross-linking reagent. MGAT2 also co-eluted with DGAT1 on a gel filtration column, suggesting that the two enzymes form a complex in intact cells. In contrast, MGAT2 did not heterodimerize with DGAT2 when co-expressed in COS-7 cells, despite high sequence homology between the two enzymes. Furthermore, systematic deletion analysis demonstrates that N-terminal amino acids 35–80 of DGAT1, but not a signal peptide at the N terminus of MGAT2, is required for the heterodimerization. Finally, co-expression of MGAT2 with DGAT1 significantly increased lipogenesis in COS-7 cells, indicating the functional importance of the dimerization.     

Monoacylglycerol and diacylglycerol acyltransferases and the synthesis of neutral glycerides in Manduca sexta

The insect fat body and the adipose tissue of vertebrates store fatty acids (FA) as triacylglycerols (TG). However, the fat body of most insects has the unique ability to rapidly produce and secrete large amounts of diacylglycerol (DG). Monoacylglycerol acyltransferase (MGAT), which catalyzes the synthesis of DG from MG, and a diacylglycerol acyltransferase (DGAT), which catalyzes the synthesis of TG from DG, are key enzymes in the metabolism of neutral glycerides. However, very little is known about these acyltransferases in insects. In the present study we have cloned two predicted MGATs and a DGAT from Manduca sexta and compared their sequences with predicted MGAT and DGAT homologs from a number of insect species. The comparison suggested that insects may only have a single DGAT gene, DGAT1. The apparent absence of a DGAT2 gene in insects would represent a major difference with vertebrates, which contain DGAT1 and DGAT2 genes. Insects seem to have a single MGAT gene which is similar to the MGAT2 of vertebrates. A number of conserved phosphorylation sites of potential physiological significance were identified among insect proteins and among insect and vertebrate proteins. DGAT1 and MGAT are expressed in fat body, midgut and ovaries. The relative rates of utilization of FAs for the synthesis of DG and TG correlated with the relative expression levels of MGAT and DGAT suggesting that regulation of the expression levels of these acyltransferases could be determining whether the fat body secretes DG or stores fatty acids as TG. The expression patterns of the acyltransferases suggest a role of the monoacylglycerol pathway in the production and mobilization of DG in M. sexta fat body.     

Seasonal changes in critical enzymes of lipogenesis and triacylglycerol synthesis in the marmot (Marmota flaviventris)

Fatty acid metabolism and triacylglycerol synthesis are critical processes for the survival of hibernating mammals that undergo a prolonged fasting period. Fatty acid synthase, fatty-acid-CoA ligase, diacylglycerol acyltransferase, and monoacylglycerol acyltransferase activities were measured in liver and in white and brown adipose tissue, in order to determine whether enzymes of lipogenesis and triacylglycerol synthesis vary seasonally during hibernation in the yellow-bellied marmot (Marmota flaviventris). Compared with mid-winter hibernation, fatty acid synthase activity was higher in all three tissues during early spring when marmots emerged from hibernation and in mid-summer when they were feeding, consistent with the synthesis of fatty acids from the carbohydrate-rich summer diet. Fatty-acid-CoA ligase and diacylglycerol acyltransferase activities were highest in summer in white adipose tissue when triacylglycerol synthesis would be expected to be high; diacylglycerol acyltransferase activity was also high in brown adipose tissue during spring and summer. In liver, however, diacylglycerol acyltransferase specific activity was highest during hibernation, suggesting that triacylglycerol synthesis may be prominent in liver in winter. Monoacylglycerol acyltransferase activity, which may aid in the retention of essential fatty-acids, was 80-fold higher in liver than in white or brown adipose tissue, but did not vary seasonally. Its dependence on palmitoyl-CoA suggests that a divalent cation might play a role in enzyme activation. The high hepatic diacylglycerol acyltransferase activity during hibernation suggests that the metabolism of very low density lipoprotein may be important in the movement of adipose fatty acids to brown adipose tissue and muscle during the rewarming that occurs periodically during hibernation. These studies suggest that enzymes of lipid metabolism vary seasonally in the marmot, consistent with requirements of this hibernator for triacylglycerol synthesis and metabolism.Abbreviations BAT brown adipose tissue - DGAT diacylglycerol acyltransferase - FAS fatty acid synthase - K _m Michaelis constant - MGAT monoacylglycerol acyltransferase - RQ respiratory quotiant - VLDL very low density lipoprotein - WAT white adipose tissue     

Apparent 'glucokinase' activity in non-hepatic tissues due to N-acetyl-D-glucosamine kinase

1. Electrophoretic examination of tissue extracts from rat intestinal mucosa, kidney, lung, spleen, mammary gland, adipose tissue, heart muscle and placenta in agarose gels did not reveal the presence of any glucokinase (ATP:D-glucose 6-phosphotransferase, EC 2.7.1.2) activity corresponding to that present in rat liver. 2. All these tissues do contain an enzyme that possesses very high-Km glucose-phosphorylating activity but which has a slightly lower electrophoretic mobility than glucokinase and can be separated from it by various means. 3. This phosphotransferase activity is due to N-acetyl-D-glucosamine kinase (ATP:2-acetamido-2-deoxy-D-glucose 6-phosphotransferase, EC 2.7.1.59), which has been partialyy purified from intestinal mucosa tissue and shown to have similar kinetic properties to the same enzyme previously purified more extensively from liver and kidney. 4. It is suggested that many of the effects reported in the literature of 'glucokinase' activity in non-hepatic tissues are probably due to N-acetyl-D-glucosamine kinase.     

Low Concentration of Inducer Favors Production of Active Form of 6-Phosphofructo-2-kinase/Fructose-2,6-bisphosphatase inEscherichia coli

Expression of chicken and rat liver bifunctional enzyme, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, inEscherichia coliencountered two common problems: the chicken enzyme was liable to proteolysis and the rat enzyme was prone to form inclusion bodies. Reducing the rate of protein synthesis by lowering either growth temperature or isopropyl-β- -thiogalactopyranoside (IPTG) concentration alleviated these two problems. Growth at 22°C was optimum for expression of both enzymes. The optimum range of IPTG concentration for expression was 0.1–1 μ for the chicken liver bifunctional enzyme and 10 μ for rat liver enzyme. The components of growth medium also influenced the production. Compared with Luria–Bertani medium, an enriched medium—tryptone–phosphate medium—tripled the production of the active enzymes. Addition of glucose (0.2%) doubled the expression level of active chicken liver enzyme, but reduced the production of active rat liver enzyme to half the maximal level, while the phosphate in tryptone–phosphate medium had no effect on the production of the two enzymes.     

Biochemical properties of porcine white adipose tissue mitochondria and relevance to fatty acid oxidation

The capacity of white adipose tissue mitochondria to support a high beta-oxidative flux was investigated by comparison to liver mitochondria. Based on marker enzyme activities and electron microscopy, the relative purity of the isolated mitochondria was similar thus allowing a direct comparison on a protein basis. The results confirm the comparable capacity of adipose tissue and liver mitochondria for palmitoyl-carnitine oxidation. Relative to liver, both citrate synthase and α-ketoglutarate dehydrogenase were increased 7.87- and 10.38-fold, respectively. In contrast, adipose tissue NAD-isocitrate dehydrogenase was decreased (2.85-fold). Such modifications in the citric acid cycle are expected to severely restrict citrate oxidation in porcine adipose tissue. Except for cytochrome c oxidase, activities of the enzyme complexes comprising the electron transport chain were not significantly different. The decrease in adipose cytochrome c oxidase activity could partly be attributed to a decreased inner membrane as suggested by lipid and enzyme analysis. In addition, Western blotting indicated that adipose and liver mitochondria possess similar quantities of cytochrome c oxidase protein. Taken together these results indicate that not only is the white adipose tissue protoplasm relatively rich in mitochondria, but that these mitochondria contain comparable enzymatic machinery to support a relatively high beta-oxidative rate.