Evidence for a Novel Key Regulatory Step in the Triglyceride Synthetic Pathway of Human Adipose Tissue

The triglyceride (TG) synthetic pathway is believed to be regulated at the level of the rate-limiting enzyme acyl-CoA:1,2-diacylglycerol O-acyltransferase (DGAT). Recent reports using rat hepatic tissue suggest a kinase-dependent mechanism for the regulation of DGAT activity. To examine this process further, the present study investigates the regulatory mechanisms involved in the modulation of DGAT in human adipocytes. Adipocytes were fractionated into a microsomal fraction containing DGAT and a cytosolic fraction containing a putative regulatory kinase. DGAT activity was determined bymeasuring the incorporation of 14C-oleoyl-CoA into TG with exogenously supplied 1,2-dioleoyl-sn-glycerol. Kinase activity was assayed by addition of the cytosolic fraction in the presence of Mg2+ and ATP. The results indicate a significant inhibition of human adipose tissue DGAT activity by as much as 43% (avg: 17.5% ± 10.4%, p < 0.01) via a mechanism consistent with a phosphorylation event. Partial purification of the putative cytosolic kinase was achieved by multidimensional chromatography. This study thus provides evidence for a novel and key regulatory step in the human TG biosynthetic pathway. Further research is necessary to determine whether the model outlined here is a physiologic conduit through which extracellular hormones exert a regulatory influence on TG synthesis.

Regulation of direct adipose tissue free fatty acid storage during mixed meal ingestion and high free fatty acid concentration conditions

Direct free fatty acid (FFA) storage into adipocytes relates to body fat distribution. Adipose tissue CD36, acyl-CoA synthetase (ACS), and diacylglycerol acetyl-transferase (DGAT) may account for some of the between-depot and inter-individual variability in FFA storage. These studies were to test whether CD36, ACS or DGAT might be important for direct palmitate storage under meal-ingestion or high FFA conditions. We measured upper (UBSQ) and lower body subcutaneous (LBSQ) adipose tissue FFA storage rates by infusing palmitate tracers intravenously and performing adipose biopsies under hypoinsulinemic (high FFA) and mixed meal conditions. We recruited 5 postmenopausal women, physically active males (5) and females (5), and sedentary males (5) and females (5). We found: 1) the ratio of UBSQ to LBSQ DGAT activity predicted the ratio of palmitate storage (adjusted R = 0.25, F = 8.0, P = 0.01, 95% CI (0.07, 0.48)) under high FFA conditions; 2) the ratio of UBSQ to LBSQ ACS activity predicted the ratio of palmitate storage under meal conditions (adjusted R = 0.18, F = 6.3, P = 0.02, 95% CI (0.12, 1.28); 3) LBSQ direct palmitate storage rates were significantly less in physically active than sedentary adults; 3); 4) adipose tissue CD36 protein content, ACS or DGAT activities did not independently predict palmitate storage rates. We conclude that physically active adults have lesser fatty acid cycling back into adipose tissue and that adipose ACS and DGAT may affect competition between UBSQ and LBSQ adipose for direct palmitate storage.

Hypoxia-inducible lipid droplet-associated interacts with DGAT1 and promotes lipid storage in hepatocytes

ABSTRACTLipid droplets (LD) are dynamic organelles that can expand and shrink, driven by fluctuations in the rate of triglyceride synthesis and degradation. Triglyceride synthesis, storage in LD, and degradation are governed by a complex set of LD-associated proteins. One of these LD-associated proteins, hypoxia-inducible lipid droplet-associated (HILPDA), was found to impair LD breakdown by inhibiting adipose triglyceride lipase. Here we characterized the physiological role and mechanism of action of HILPDA in hepatocytes. Expression of HILPDA was induced by fatty acids in several hepatoma cell lines. Hepatocyte-specific deficiency of HILPDA in mice modestly but significantly reduced hepatic triglycerides in mice with non-alcoholic fatty liver disease. Similarly, deficiency of HILPDA in mouse precision-cut liver slices and primary hepatocytes reduced lipid storage and accumulation of fluorescently-labelled fatty acids in LD, respectively, which was independent of adipose triglyceride lipase. Fluorescence microscopy showed that HILPDA partly colocalizes with LD and with the endoplasmic reticulum, is especially abundant in perinuclear areas, and mainly associates with newly added fatty acids. Real-time fluorescence live-cell imaging further revealed that HILPDA preferentially localizes to LD that are being remodelled. Mechanistically, HILPDA overexpression increased lipid storage in human hepatoma cells concomitant with an increase in DGAT activity and DGAT1 protein levels. Finally, confocal microscopy and Förster resonance energy transfer-fluorescence lifetime imaging microscopy analysis indicated that HILPDA colocalizes and physically interacts with DGAT1. Overall, our data indicate that HILPDA physically interacts with DGAT1 and increases DGAT activity. These findings suggest a novel mechanism in hepatocytes that links elevated fatty acid levels to stimulation of triglyceride synthesis and storage.

Acylation-stimulating protein deficiency and altered adipose tissue in alternative complement pathway knockout mice

Acylation-stimulating protein (C3adesArg/ASP) is an adipokine that acts on its receptor C5L2 to stimulate triglyceride (TG) synthesis in adipose tissue. The present study investigated ASP levels in mouse models of obesity and leanness and the effect of ASP deficiency in C3 knockout (C3KO) mice on adipose tissue morphology. Plasma ASP levels in wild-type (WT) mice correlated positively with plasma nonesterified fatty acids (NEFA) ( R = 0.664, P < 0.001) and total cholesterol ( R = 0.515, P < 0.001). Plasma ASP was increased by 85% in obese ob/ob leptin-deficient mice and decreased in lean diacylglycerol acyltransferase 1 (DGAT1) KO mice (−54%) and C/EBPαβ/βtransgenic mice (−70%) compared with WT. Mice lacking alternative complement factor B or adipsin (FBKO or ADKO), required for ASP production, were also ASP deficient. Both FBKO and C3KO mice had delayed postprandial TG and NEFA clearance on low-fat (LF) and high-fat (HF) diets, suggesting that lack of ASP, not C3, drives the metabolic phenotype. Adipocyte size distribution in C3KO mice was polarized (increased number of both small and large cells), with decreased adipsin expression (−33% gonadal HF), DGAT1 expression (−31% to −50%) and DGAT activity (−41%). Overall, a reduction/deficiency in ASP is associated with an antiadipogenic state and ASP may provide a target for controlling fat storage.

Diacylglycerol acyltransferase (DGAT) activity in relation to muscle fat content and <i>DGAT1</i> genotype in two different breeds of <i>Bos Taurus</i> (short communication)

To circumvent the obvious waste of resources when producing good marbled beef, research is needed to clarify mechanisms which are involved in intramuscular fat storage in cattle. The possible association between the activity of diacylglycerol acyltransferase (DGAT) and muscle fat content was examined in samples of longissimus dorsi (LD) and semitendinosus (ST) muscle from Holstein and Charolais bulls. The Holstein bulls exhibited higher fat content in both muscles and higher marbling score. In Holstein, DGAT activity was enhanced in the LD muscle, and there was a tentative positive relationship between DGAT activity and the fat content in ST muscle. When muscle DGAT activity was examined as a function of DGAT1 genotype for all animals, regardless of breed, the DGAT activity of LD muscle of the K/K genotype was about five-fold greater than for either the K/A or A/A genotypes. Further investigation on the relationship between DGAT1 genotype and i.m. fat is required before this may be developed as a selection tool for marbling.

Downregulation of hepatic acyl-CoA:diglycerol acyltransferase in chronic renal failure

Chronic renal failure (CRF) is associated with hypertriglyceridemia and elevated plasma VLDL and IDL concentrations. These events can be due to either increased production or depressed catabolism of triglyceride-rich lipoproteins. Several studies have documented downregulation of lipoprotein lipase, hepatic triglyceride lipase, and the VLDL receptor, leading to depressed clearance and elevated plasma concentration of triglyceride-rich lipoproteins and their remnants in CRF. However, the effect of CRF on the triglyceride biosynthetic pathway has not been explored. Diglycerol acyltransferase (DGAT) is a microsomal enzyme that joins acyl-CoA to 1,2 diacylglycerol and, as such, constitutes the final step in triglyceride biosynthesis. Two distinct forms of DGAT (DGAT-1 and -2) have thus far been identified. The present study was undertaken to examine the effect of CRF on DGAT gene expression and activity in the liver, which is the source of endogenous triglycerides in the circulation. Male Sprague-Dawley rats were studied 8 wk after [Formula: see text]nephrectomy (CRF) or sham operation. DGAT-1 and DGAT-2 mRNA abundance and DGAT activity were quantified. The CRF group showed reduced creatinine clearance, elevated plasma triglycerides, and VLDL concentrations. This was accompanied by significant reductions in hepatic DGAT-2 mRNA abundance ( P < 0.01) and total DGAT activity ( P < 0.1), pointing to diminished hepatic triglyceride production capacity in CRF animals. In conclusion, CRF results in significant downregulation of hepatic DGAT gene expression and activity. Given the critical role of DGAT in triglyceride biosynthesis, the present study points to diminished, not increased, hepatic triglyceride synthetic capacity in CRF rats.