Andrew M. Salter

The inhibition of the oxidation of low density lipoprotein by (+)-Catechin, a naturally occurring flavonoid

(+)-Catechin inhibited the copper-catalysed oxidation of human low density lipoprotein (LDL) in a dose-dependent manner with complete inhibition at 20 micrograms/mL. The flavonoid at a concentration of 50 micrograms/mL also inhibited oxidation of LDL induced by the mouse transformed macrophage J774, human monocyte-derived macrophages and vascular endothelial cells isolated from human umbilical cords. LDL modified by copper-catalysed or cell-induced oxidation was endocytosed and degraded by human macrophages at a much greater rate than native LDL. LDL reisolated from copper or cell incubations in the presence of (+)-catechin was endocytosed and degraded at rates similar to native LDL. (+)-Catechin appeared to inhibit the uptake and degradation by macrophages of cell-modified LDL. The actions of (+)-catechin on cell-induced oxidation of LDL are consistent with the ability of flavonoids of similar structure to inhibit lipoxygenases and with a role for lipoxygenases in cell-induced modification of LDL in vivo.

Stearoyl-CoA desaturase mRNA is transcribed from a single gene in the ovine genome

Clones corresponding to ovine stearoyl-CoA desaturase (SCD) cDNA were isolated from an adipose tissue cDNA library. All of these clones represented a single mRNA species as judged by restriction fragment and DNA sequence analysis. RNase protection analysis demonstrated that this SCD transcript is highly expressed in adipose tissue and liver, and in the mammary gland of lactating animals. A lower level of expression was detectable in a variety of other tissues including brain. Levels of the SCD transcript were decreased in adipose tissue during lactation, and this appears to be related to a marked decline in serum insulin and insulin-responsiveness of the tissue. Southern analysis of ovine and mouse genomic DNA demonstrated that the ovine SCD cDNA hybridised in a manner consistent with a single gene for SCD in ovine DNA; mouse genomic DNA produced a pattern of hybridisation consistent with the previously characterised mouse SCD-1 and SCD-2 genes. Three ovine cosmids were isolated that comprised the restriction fragments predicted by the genomic Southern analysis. The ovine SCD gene was predicted to be encompassed within a 23 kbp region that was present in all three cosmids. These results demonstrate that SCD is transcribed from a single gene in the ovine genome and this gene is insulin-responsive in ovine adipose tissue.

Dietary omega-3 and -6 polyunsaturated fatty acids affect the composition and development of sheep granulosa cells, oocytes and embryos

The evidence that omega-3 (n-3) and -6 (n-6) polyunsaturated fatty acids (PUFAs) have differential effects on ovarian function, oocytes and embryo quality is inconsistent. We report on the effects of n-3 versus n-6 PUFA-enriched diets fed to 36 ewes over a 6-week period, prior to ovarian stimulation and follicular aspiration, on ovarian steroidogenic parameters and embryo quality. Follicle number and size were unaltered by diet, but follicular-fluid progesterone concentrations were greater in n-3 PUFA-fed ewes than in n-6 PUFA-fed ewes. The percentage of saturated FAs (mostly stearic acid) was greater in oocytes than in either granulosa cells or plasma, indicating selective uptake and/or de novo synthesis of saturated FAs at the expense of PUFAs by oocytes. High-density lipoproteins (HDLs) fractionated from sera of these ewes increased granulosa cell proliferation and steroidogenesis relative to the FA-free BSA control during culture, but there was no differential effect of n-3 and n-6 PUFAs on either oestradiol or progesterone production. HDL was ineffective in delivering FAs to embryos during culture, although n-6 PUFA HDL reduced embryo development. All blastocysts, irrespective of the treatment, contained high levels of unsaturated FAs, in particular linoleic acid. Transcripts for HDL and low-density lipoprotein (LDL) receptors (SCARB1 and LDLR) and stearoyl-CoA desaturase (SCD) are reported in sheep embryos. HDL reduced the expression of transcripts for LDLR and SCD relative to the BSA control. The data support a differential effect of n-3 and n-6 PUFAs on ovarian steroidogenesis and pre-implantation development, the latter in the absence of a net uptake of FAs.

The assembly of triacylglycerol-rich lipoproteins: an essential role for the microsomal triacylglycerol transfer protein

Raised plasma triacylglycerol is an independent risk factor for cardiovascular disease, and an understanding of factors which regulate the synthesis and degradation of lipoproteins which carry triacylglycerol in the blood may lead to novel approaches to the treatment of hypertriacylglycerolaemia. An active microsomal triacylglycerol transfer protein (MTP) is essential for the assembly of particles which transport triacylglycerol through the circulation. After absorption in the intestine, dietary fat and fat-soluble vitamins are incorporated into chylomicrons in the intestinal epithelial cells, and these lipoproteins reach the bloodstream via the lymphatic system. Patients with the rare genetic disorder, abetalipoproteinaemia, in which MTP activity is absent, present clinically with fat-soluble vitamin and essential fatty acid deficiency, indicating a key role for MTP in the movement of fat into the body. The triacylglycerol-rich lipoprotein found in fasting blood, VLDL, is assembled in the liver by an MTP-dependent process similar to chylomicron assembly, and transports triacylglycerol to extra-hepatic tissues such as adipose tissue and heart. In the absence of MTP activity, VLDL are not synthesized and only extremely low levels of triacylglycerol are present in the blood. Dietary components, including fat, cholesterol and ethanol, can modify the expression of the MTP gene and, hence, MTP activity. The present review summarizes current knowledge of the role of MTP in the assembly and secretion of triacylglycerol-rich lipoproteins, and the regulation of its activity in both animal and cell systems.

Dietary fatty acids and cardiovascular disease

In 1991, the Committee on Medical Aspects of Food Policy produced a report on the dietary reference values for food energy and nutrients for groups of people in the United Kingdom. The resulting recommendations, which included specific limits for intakes of total, saturated, trans- and cis-polyunsaturated fatty acids (PUFA) have remained a cornerstone of public health policy ever since, and similar recommendations have been adopted by the World Health Organization. These recommendations were made largely on the basis of specific effects of these fatty acids on the risk of developing atherosclerotic cardiovascular disease (CVD). The intervening years have seen a plethora of human epidemiological and intervention trials to further elucidate the specific relationship between dietary fatty acid intake, plasma lipids and lipoproteins and cardiovascular morbidity and mortality. A number of recent meta-analyses and systematic reviews have revisited the role of specific dietary fatty acid classes and CVD risk. In general, these continue to support a link between saturated fatty acids (SFA) and CVD morbidity/mortality. They also highlight the potent adverse effects of trans fatty acids derived from partially hydrogenated vegetable oil. The most recent data suggest that replacing SFA with cis-PUFA (primarily linoleic acid) has the greatest impact on reducing CVD risk. Evidence of specific beneficial effects of n-3 PUFA is generally stronger for secondary, rather than primary, CVD risk, and it is restricted to very long chain fatty acids of marine origin as opposed to alpha-linolenic acid. Taken together, these data suggest that recent focus on dietary n-6-to-n-3 PUFA ratios may have been misguided, and that future strategies should focus on replacing dietary SFA with total PUFA, rather than concentrating on n-6 : n-3 PUFA ratio.

The effect of different dietary fatty acids on lipoprotein metabolism: concentration-dependent effects of diets enriched in oleic, myristic, palmitic and stearic acids

While it is well established that the fatty acid composition of dietary fat is important in determining plasma lipoprotein cholesterol concentrations, the effects of changing the absolute quantities of the individual fatty acids are less clear. In the present study Golden Syrian hamsters were fed on isoenergetic, low cholesterol (0.05 g/kg) diets containing 100, 150 or 200 g added fat/kg. This consisted of triolein (TO) alone, or equal proportions of TO and either trimyristin (TM), tripalmitin (TP) or tristearin (TS). Each trial also included a control group fed on a diet containing 50 g TO/kg. As the mass of TO in the diet increased, plasma VLDL-cholesterol concentrations rose. The TM-rich diets produced a concentration-dependent increase in total plasma cholesterol which was a result of significant increases in both VLDL and HDL levels. The TP-rich diets increased plasma LDL- and HDL-cholesterol levels in a concentration-dependent manner. TS-containing diets did not increase the cholesterol content of any of the major lipoprotein fractions. Hepatic LDL-receptor mRNA concentrations were significantly decreased in animals fed on TP, while apolipoprotein B mRNA concentrations were significantly increased. Thus, on a low-cholesterol diet, increasing the absolute amount of dietary palmitic acid increases LDL-cholesterol more than either myristic or stearic acid. These effects on lipoprotein metabolism may be exerted through specific modulation of the expression of the LDL receptor and apolipoprotein B genes.

Regulation of hepatic gene expression by saturated fatty acids

Diets rich in saturated fatty acids have long been associated with increased plasma cholesterol concentrations and hence increased risk of cardiovascular disease. More recently, they have also been suggested to promote the development of non-alcoholic fatty liver disease. While there is now considerable evidence to suggest that polyunsaturated fatty acids exert many of their effects through regulating the activity of transcription factors, including peroxisome proliferator activated receptors, sterol regulatory binding proteins (SREBPs) and liver X receptor, our understanding of how saturated fatty acids act is still limited. Here we review the potential mechanisms whereby saturated fatty acids modulate hepatic lipid metabolism thereby impacting on the synthesis, storage and secretion of lipids. Evidence is presented that their effects are, at least partly, mediated through modulation of the activity of the SREBP family of transcription factors.

Effects of dietary fat on cholesterol metabolism : Regulation of plasma LDL concentrations

INTRODUCTION . . 241 EFFECTS O F DIETARY FATTY ACIDS O N PLASMA LIPOPROTEIN CONCENTRATIONS I N H U M A N S . . 242 B A C K G R O U N D . . . 242 RECENT M E T A A N A L Y S E S . . 242 C O M P A R A T I V E EFFECTS OF D I F F E R E N T S A T U R A T E D FATTY A C I D S . . 244 T R A N S F A T T Y A C I D S . . 245 N-3 P O L Y U N S A T U R A T E D F A T T Y A C I D S . . 245 REGULATION O F LOW DENSITY LIPOPROTEIN METABOLISM . . 246 MECHANISMS WHEREBY DIETARY FATTY ACIDS MAY I N F L U E N C E PLASMA L D L CONCENTRATIONS . . 249

Effect of dietary triacylglycerol structure on lipoprotein metabolism: A comparison of the effects of dioleoylpalmitoylglycerol in which palmitate is esterified to the 2-or 1(3)-position of the glycerol

The effect on lipoprotein metabolism of diets enriched in different isomers of dioleoylpalmitoylglycerol was studied. One diet contained fat in which palmitate was esterified to the two outer positions of the glycerol (OOP) and the other in which it was esterified to the middle carbon (OPO). The lipid composition of chylomicrons was similar in rats fed either fat blend. However, triacylglycerol (TAG) in chylomicrons from OPO fed animals was relatively enriched in palmitic acid, at the expense of stearic, oleic and linoleic acids. Silver phase HPLC and 2-positional analysis clearly demonstrated that the identity of the fatty acid in the 2-position was similar in both dietary and chylomicron TAG. No significant differences could be seen in the in vitro hydrolysis of chylomicron TAG from animals fed the two fats labelled with [14C]palmitate. As expected, following hydrolysis, palmitate was released as free fatty acid from chylomicrons isolated from OOP-fed animals but within 2-monoacylglycerol from those fed OPO. The enrichment of chylomicrons with palmitate in animals fed O[14C]PO resulted in increased delivery of [14C]palmitate to the liver. In a further series of experiments Golden Syrian hamsters were fed diets containing the fat blends and either 0.005% or 0.12% (w/w) cholesterol, for 28 days. No differences in fasting plasma lipoprotein concentrations were seen in response to the dietary fats. We conclude that, while these isometric triacylglycerols had transient effects on chylomicron metabolism, no significant longer term effect on plasma concentrations of endogenous lipoproteins could be found.

Effects of omega-3 and -6 polyunsaturated fatty acids on ovine follicular cell steroidogenesis, embryo development and molecular markers of fatty acid metabolism

We previously reported increased follicular fluid progesterone (P(4)) concentrations in ewes fed an n-3 compared to an n-6 polyunsaturated fatty acid (PUFA)-enriched diet, but detected no differential effect of n-3 and n-6 PUFA-enriched high-density lipoproteins (HDL) on granulosa cell (GC) steroidogenesis in vitro. Moreover, net n-6 PUFA-enriched HDL reduced early embryo development, but in the absence of a net uptake of FA. Consequently, we hypothesised that a) effects of n-3 PUFA on ovarian steroidogenesis are mediated by theca rather than GCs and b) during embryo culture lipids are acquired solely from the albumin fraction of serum, so that albumin-delivered n-3 and n-6 PUFA exert a greater differential effect on embryo development than either low-density lipoprotein (LDL)- or HDL-delivered PUFA. Data confirmed that n-3 PUFA increases P(4) production solely in theca cells and that this is associated with an increase in STAR transcript expression. Furthermore, LDL- and HDL-delivered n-3 PUFA are equally efficacious in this regard during the first 96 h of culture, but thereafter only HDL-delivered n-3 PUFA induces this effect in partially luteinised theca cells. We also demonstrate that albumin is the sole serum fraction that leads to a net uptake of FA during embryo culture. PUFA-enriched serum and albumin increased the yield of morphologically poorer quality blastocysts with increased transcript expression for the antioxidant enzyme SOD1. Important differential effects of n-3 and n-6 PUFA on ovarian steroidogenesis acting solely on theca cells are identified, but differential effects of PUFA on embryo development are less apparent.