Geoffrey F. Gibbons

Mobilisation of triacylglycerol stores.

Triacylglycerol (TAG) is an energy dense substance which is stored by several body tissues, principally adipose tissue and the liver. Utilisation of stored TAG as an energy source requires its mobilisation from these depots and transfer into the blood plasma. The means by which TAG is mobilised differs in adipose tissue and liver although the regulation of lipid metabolism in each of these organs is interdependent and synchronised in an integrated manner. This review deals principally with the mechanism of hepatic TAG mobilisation since this is a rapidly expanding area of research and may have important implications for the regulation of plasma very-low-density lipoprotein metabolism. TAG mobilisation plays an important role in fuel selection in non-hepatic tissues such as cardiac muscle and pancreatic islets and these aspects are also reviewed briefly. Finally, studies of certain rare inherited disorders of neutral lipid storage and mobilisation may provide useful information about the normal enzymology of TAG mobilisation in healthy tissues.

Adipose tissue metabolism in obesity: lipase action in vivo before and after a mixed meal.

Physiological actions of insulin include suppression of fat mobilization from adipose tissue and activation of adipose tissue lipoprotein lipase. Here, we report measurements of adipose tissue hormone-sensitive lipase (HSL) and lipoprotein lipase (LPL) action in vivo in 10 normal and eight obese subjects, with the latter group having varying degrees of glucose intolerance. HSL and LPL actions (per gram of adipose tissue) were similar in the two groups, after an overnight fast. In the normal subjects, HSL action was suppressed after a meal (by 75% +/- 6% between 60 to 300 minutes, P less than .01), and the action of LPL was increased (clearance of circulating triacylglycerol [TAG] increased by 140% +/- 57% at 300 minutes, P less than .05). Despite hyperinsulinemia, these responses were blunted in the obese subjects (P less than .05 for each change being less than in normal group). The adipose tissue of the obese subjects showed continued nonesterified fatty acid (NEFA) release at a time when NEFA mobilization was completely suppressed in the normal group. Both impaired suppression of HSL and low fractional retention of fatty acids for reesterification within the adipose tissue contributed to this abnormal NEFA release. Impaired activation of LPL was associated with a greater absolute increase in plasma TAG concentration postprandially in the obese. In obese subjects, adipose tissue HSL and LPL fail to respond to immunoreactive insulin postprandially, which may be an important maladaptation in terms of lipoprotein metabolism and risk of coronary heart disease.

A role for PPARα in the control of SREBP activity and lipid synthesis in the liver

Inclusion of the PPARalpha (peroxisome-proliferator-activated receptor alpha) activator WY 14,643 in the diet of normal mice stimulated the hepatic expression of not only genes of the fatty acid oxidation pathway, but also those of the de novo lipid synthetic pathways. Induction of fatty acid synthase mRNA by WY 14,643 was greater during the light phase of the diurnal cycle, when food intake was low and PPARalpha expression was high. Hepatic fatty acid pathway flux in vivo showed a similar pattern of increases. The abundance of mRNAs for genes involved in hepatic cholesterol synthesis was also increased by WY 14,643, but was associated with a decrease in cholesterogenic carbon flux. None of these changes were apparent in PPARalpha-null mice. Mice of both genotypes showed the expected decreases in 3-hydroxy-3-methylglutaryl-CoA reductase mRNA levels and cholesterol synthesis in response to an increase in dietary cholesterol. The increase in fatty acid synthesis due to WY 14,643 was not mediated by increased expression of SREBP-1c (sterol regulatory element binding protein-1c) mRNA, but by an increase in cleavage of the protein to the active form. An accompanying rise in stearoyl-CoA desaturase mRNA expression suggested that the increase in lipogenesis could have resulted from an alteration in membrane fatty acid composition that influenced SREBP activation.

Peroxisome-proliferator-activated receptor-α (PPARα) deficiency leads to dysregulation of hepatic lipid and carbohydrate metabolism by fatty acids and insulin

The aim of the present study was to determine whether peroxisome-proliferator-activated receptor-alpha (PPARalpha) deficiency disrupts the normal regulation of triacylglycerol (TAG) accumulation, hepatic lipogenesis and glycogenesis by fatty acids and insulin using PPARalpha-null mice. In wild-type mice, hepatic TAG concentrations increased (P<0.01) with fasting (24 h), with substantial reversal after refeeding (6 h). Hepatic TAG levels in fed PPARalpha-null mice were 2.4-fold higher than in the wild-type (P<0.05), increased with fasting, but remained elevated after refeeding. PPARalpha deficiency also impaired hepatic glycogen repletion (P<0.001), despite normal insulin and glucose levels after refeeding. Higher levels of plasma insulin were required to support similar levels of hepatic lipogenesis de novo ((3)H(2)O incorporation) in the PPARalpha-null mice compared with the wild-type. This difference was reflected by corresponding changes in the relationship between plasma insulin and the mRNA expression of the lipogenic transcription factor sterol-regulatory-element-binding protein-1c, and that of one of its known targets, fatty acid synthase. In wild-type mice, hepatic pyruvate dehydrogenase kinase (PDK) 4 protein expression (a downstream marker of altered fatty acid catabolism) increased (P<0.01) in response to fasting, with suppression (P<0.001) by refeeding. Although PDK4 up-regulation after fasting was halved by PPARalpha deficiency, PDK4 suppression after refeeding was attenuated. In summary, PPARalpha deficiency leads to accumulation of hepatic TAG and elicits dysregulation of hepatic lipid and carbohydrate metabolism, emphasizing the importance of precise control of lipid oxidation for hepatic fuel homoeostasis.

The effect of peroxisome-proliferator-activated receptor-alpha on the activity of the cholesterol 7 alpha-hydroxylase gene.

Cholesterol 7 alpha-hydroxylase (Cyp7a1) plays a central role in the regulation of bile acid and cholesterol metabolism, and transcription of the gene is controlled by bile acids and hormones acting through a complex interaction with a number of potential steroid-hormone-binding sites. Transcriptional activity of the human CYP7A1 gene promoter transfected into HepG2 cells was decreased in a concentration-dependent manner by co-transfection with an expression vector for peroxisome-proliferator-activated receptor-alpha (PPAR alpha). This effect was augmented by 9-cis-retinoic acid receptor-alpha (RXR alpha) and activators of PPAR alpha to give a maximum inhibition of approx. 80%. The region responsible for this inhibition contained a site known to bind hepatocyte nuclear factor 4 (HNF4), and mutation of this site greatly decreased the effect. Co-expression of HNF4 increased promoter activity and decreased the effect of PPAR alpha. Gel-mobility-shift assays failed to detect any binding of PPAR alpha/RXR alpha dimers to any regions of the promoter containing potential binding sites. Also the hepatic abundance of Cyp7a1 mRNA in mice in which the PPAR alpha gene was disrupted was the same as in normal mice, both during the dark phase, when the animals were feeding, and during the light phase, when mRNA abundance was greatly increased. Cholesterol feeding produced the same increase in hepatic Cyp7a1 mRNA abundance in PPAR alpha-null animals as in normals. It is concluded that, whereas PPAR alpha can affect CYP7A1 gene transcription in vitro through an indirect action, probably by competing for co-factors, this is unlikely to be a major influence on Cyp7a1 activity under normal physiological conditions.

Inhibition of cholesterol absorption associated with a PPARα-dependent increase in ABC binding cassette transporter A1 in mice

Dietary supplementation with the peroxisome proliferator-activated receptor α (PPARα) ligand WY 14,643 gave rise to a 4- to 5-fold increase in the expression of mRNA for the ATP binding cassette transporter A1 (ABCA1) in the intestine of normal mice. There was no effect in the intestine of PPARα-null mice. Consumption of a high-cholesterol diet also increased intestinal ABCA1 expression. The effects of WY 14,643 and the high-cholesterol diet were not additive. WY 14,643 feeding reduced intestinal absorption of cholesterol in the normal mice, irrespective of the dietary cholesterol concentration, and this resulted in lower diet-derived cholesterol and cholesteryl ester concentrations in plasma and liver. At each concentration of dietary cholesterol, there was a similar significant inverse correlation between intestinal ABCA1 mRNA content and the amount of cholesterol absorbed. The fibrate-induced changes in the intestines of the normal mice were accompanied by an increased concentration of the mRNA encoding the sterol-regulatory element binding protein-1c gene (SREBP-1c), a known target gene for the oxysterol receptor liver X receptor α (LXRα). There was a correlation between intestinal ABCA1 mRNA and SREBP-1c mRNA contents, but not between SREBP-1c mRNA content and cholesterol absorption. These results suggest that PPARα influences cholesterol absorption through modulating ABCA1 activity in the intestine by a mechanism involving LXRα.

Human triacylglycerol-rich lipoprotein subfractions as substrates for lipoprotein lipase

In order to test the hypothesis that lipoprotein lipase (LPL) acts preferentially on larger lipoprotein particles, we determined the susceptibility of triacylglycerol-rich lipoprotein (TRL) subfractions to hydrolysis by LPL in vitro. Chylomicrons (Sf > 400), very low density lipoproteins (VLDL)1 (Sf 60-400) and VLDL2 (Sf 20-60) were isolated from six subjects with a range of plasma-triacylglycerol (TAG) concentrations following an overnight fast and for up to 6 h after the consumption of a mixed meal (41% fat). The percent of TRL-TAG hydrolysed by LPL in subfractions isolated following overnight fast was VLDL1 > VLDL2 (46.8 +/- 10.2 vs. 25.9 +/- 7.4%, P = 0.006) and 3 h after the meal it was chylomicrons > VLDL1 > VLDL2 (81.0 +/- 12.6 vs. 52.8 +/- 10.2 vs. 27.7 +/- 6.2%, chylomicrons vs. VLDL1 and VLDL1 vs. VLDL2, both P < or = 0.005). The percent of VLDL1-TAG hydrolysed increased both within and between subjects as VLDL1-TAG concentrations increased. This relationship could be explained by the positive correlation observed between VLDL1-TAG and VLDL1-TAG:apolipoprotein B. In conclusion, increasing the size and TAG content of a lipoprotein particle increases its susceptibility to hydrolysis by LPL.

Evaluation of the role of peroxisome-proliferator-activated receptor α in the regulation of cardiac pyruvate dehydrogenase kinase 4 protein expression in response to starvation, high-fat feeding and hyperthyroidism

Inactivation of cardiac pyruvate dehydrogenase complex (PDC) after prolonged starvation and in response to hyperthyroidism is associated with enhanced protein expression of pyruvate dehydrogenase kinase (PDK) isoform 4. The present study examined the potential role of peroxisome-proliferator-activated receptor alpha (PPARalpha) in adaptive modification of cardiac PDK4 protein expression after starvation and in hyperthyroidism. PDK4 protein expression was analysed by immunoblotting in homogenates of hearts from fed or 48 h-starved rats, rats rendered hyperthyroid by subcutaneous injection of tri-iodothyronine and a subgroup of euthyroid rats maintained on a high-fat/low-carbohydrate diet, with or without treatment with the PPARalpha agonist WY14,643. In addition, PDK4 protein expression was analysed in hearts from fed, 24 h-starved or 6 h-refed wild-type or PPARalpha-null mice. PPARalpha activation by WY14,643 in vivo over the timescale of the response to starvation failed to up-regulate cardiac PDK4 protein expression in rats maintained on standard diet (WY14,643, 1.1-fold increase; starvation, 1.8-fold increase) or influence the cardiac PDK4 response to starvation. By contrast, PPARalpha activation by WY14,643 in vivo significantly enhanced cardiac PDK4 protein expression in rats maintained on a high-fat diet, which itself increased cardiac PDK4 protein expression. PPARalpha deficiency did not abolish up-regulation of cardiac PDK4 protein expression in response to starvation (2.9-fold increases in both wild-type and PPARalpha-null mice). Starvation and hyperthyroidism exerted additive effects on cardiac PDK4 protein expression, but PPARalpha activation by WY14,643 did not influence the response of cardiac PDK4 protein expression to hyperthyroidism in either the fed or starved state. Our data support the hypothesis that cardiac PDK4 protein expression is regulated, at least in part, by a fatty acid-dependent, PPARalpha-independent mechanism and strongly implicate a fall in insulin in either initiating or facilitating the response of cardiac PDK4 protein expression to starvation.

Intracellular triacylglycerol lipase: Its role in the assembly of hepatic very-low-density lipoprotein (VLDL)

Extracellular fatty acids entering the hepatocyte are either esterified to cytosolic TAG or oxidized to ketone bodies. Very little is esterified and secreted directly in association with VLDL. Thus, even when extracellular fatty acids are available, the major, direct source of VLDL TAG is the cytosolic pool. The recruitment of cytosolic TAG for VLDL assembly involves lipolysis followed by re-esterification. At least 70% of the secreted TAG is derived via this route. Fatty acids released at this lipolytic step are utilized exclusively for VLDL TAG synthesis and are not available for ketogenesis. Substantially more cytosolic TAG undergoes lipolysis than is required to meet the needs of VLDL assembly. The remaining fatty acids are re-esterified and re-cycled to the cell cytosol. From a physiological viewpoint, the presence of this indirect route for VLDL TAG recruitment would provide a means of regulation of VLDL secretion which is independent of the plasma fatty acid concentration. In this respect, several pathophysiological conditions are known in which there is a negative association between plasma fatty acid concentration and the rate of VLDL secretion. These are: (a) insulin-dependent diabetes, (b) starvation, (c) fat-feeding. Lipolysis of cytosolic TAG and transfer of fatty acids into the ER lumen may provide a regulatory focus for the control of hepatic VLDL output.

Up-regulation of pyruvate dehydrogenase kinase isoform 4 (PDK4) protein expression in oxidative skeletal muscle does not require the obligatory participation of peroxisome-proliferator-activated receptor alpha (PPARalpha).

In insulin deficiency, increased lipid delivery and oxidation suppress skeletal-muscle glucose oxidation by inhibiting pyruvate dehydrogenase complex (PDC) activity via enhanced protein expression of pyruvate dehydrogenase kinase (PDK) isoform 4, which phosphorylates (and inactivates) PDC. Signalling via peroxisome-proliferator-activated receptor alpha (PPARalpha) is an important component of the mechanism enhancing hepatic and renal PDK4 protein expression. Activation of PPARalpha in gastrocnemius, a predominantly fast glycolytic (FG) muscle, also increases PDK4 expression, an effect that, if extended to all muscles, would be predicted to drastically restrict whole-body glucose disposal. Paradoxically, chronic activation of PPARalpha by WY14,643 treatment improves glucose utilization by muscles of insulin-resistant high-fat-fed rats. In the resting state, oxidative skeletal muscles are quantitatively more important for glucose disposal than FG muscles. We evaluated the participation of PPARalpha in regulating PDK4 protein expression in slow oxidative (SO) skeletal muscle (soleus) and fast oxidative-glycolytic (FOG) skeletal muscle (anterior tibialis) containing a high proportion of oxidative fibres. In the fed state, acute (24 h) activation of PPARalpha by WY14,643 in vivo failed to modify PDK4 protein expression in soleus, but modestly enhanced PDK4 protein expression in anterior tibialis. Starvation enhanced PDK4 protein expression in both muscles, with the greater response in anterior tibialis. WY14,643 treatment in vivo during starvation did not further enhance upregulation of PDK4 protein expression in either muscle type. Enhanced PDK4 protein expression after starvation was retained in SO and FOG skeletal muscles of PPARalpha-deficient mice. Our data indicate that PDK4 protein expression in oxidative skeletal muscle is regulated by a lipid-dependent mechanism that is not obligatorily dependent on signalling via PPARalpha.