David Hauton

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.

Transcript and metabolite analysis of the effects of tamoxifen in rat liver reveals inhibition of fatty acid synthesis in the presence of hepatic steatosis

Nonalcoholic steatohepatitis (NASH) is a common feature of the metabolic syndrome and toxic reactions to pharmacological drugs. Tamoxifen, (TMX) a widely used anti‐breast cancer drug, can induce NASH and changes in plasma cholesterol levels through mechanisms that are unclear. We studied primary actions of TMX using a short‐term treatment (5 days) that induces microvesicular hepatic steatosis and marked hypercholesterolemia in male rats. Using a combined approach of gene expression profiling and NMR‐based metabolite analysis, we found that TMX‐treated livers have increased saturated fatty acid content despite changes in gene expression, indicating decreased de novo lipogenesis and increased fatty acid oxidation. Our results show that TMX predominantly down‐regulates FAS expression and activity as indicated by the accumulation of malonyl‐CoA, a known inhibitor of mitochondrial β‐oxidation. In the face of a continued supply of exogenous free fatty acids, the blockade of fatty acid oxidation produced by elevated malonyl‐CoA is likely to be the major factor leading to steatosis. Use of a combination of metabolomic and transcriptomic analysis has allowed us to identify mechanisms underlying important metabolic side effects of a widely prescribed drug. Given the broader importance of hepatic steatosis, the novel molecular mechanism revealed in this study should be examined in other forms of steatosis and nonalcoholic steatohepatitis. Lelliott C. J., López M., Curtis R. K., Parker N., Laudes M., Yeo G., Jimenez‐Linan M., Grosse J., Saha A. K., Wiggins D., Hauton D., Brand M. D. ORahilly S., Griffin J. L., Gibbons G. F., Vidal‐Puig A. Transcript and metabolite analysis of the effects of tamoxifen in rat liver reveals inhibition of fatty acid synthesis in the presence of hepatic steatosis. FASEB J. 19, 1–12 (2005)

Utilisation of triacylglycerol and non-esterified fatty acid by the working rat heart: myocardial lipid substrate preference.

Utilisation and subsequent metabolic fate (oxidation; tissue lipid deposition) of non-esterified fatty acid (NEFA), very-low-density lipoprotein-triacylglycerol (VLDL-TAG), and chylomicron-triacylglycerol (CM-TAG) alone or in combination by isolated working rat heart were examined. Cardiac mechanical function was maintained regardless of lipid substrate used. NEFA and CM-TAG were assimilated to a greater extent than VLDL-TAG; CM-TAG utilisation (76+/-10 nmol fatty acid/min per g wet wt.; n=8), but not VLDL-TAG utilisation (16+/-2 nmol fatty acid/min per g wet wt.; n=8), was suppressed in the presence of NEFA, but TAG (CM or VLDL) did not alter NEFA utilisation (57+/-9 nmol fatty acid/min per g wet wt.; n=8). Most (about 75%) of the lipid utilised was oxidised. In the presence of NEFA, CM-TAG deposition as tissue lipid was preserved, despite decreased CM-TAG oxidation; metabolic fate of VLDL-TAG was unaffected by NEFA. TAG (CM or VLDL) in the perfusate tended to decrease lipoprotein lipase (LPL) activity; this may be a reflection of increased LPL turnover in the presence of lipoproteins.

Utilization of triacylglycerol-rich lipoproteins by the working rat heart: routes of uptake and metabolic fates

Very‐low‐density lipoprotein (VLDL) and chylomicrons (CMs) transport triacylglycerol (TAG) to peripheral tissues. Lipoprotein‐TAG may gain access to target cells by lipoprotein lipase (LPL) hydrolysis or via receptor‐mediated uptake; the principal routes of entry of VLDL and CM into heart are unknown, and different routes of entry may result in different metabolic fates. To examine this, isolated working rat hearts were perfused with rat VLDL and CMs, dual‐labelled with [3H]TAG and [14C]cholesterol. Uptake and utilization of CM‐TAG were significantly greater than VLDL‐TAG, but both were decreased significantly (more than halved) by tetrahydrolipstatin (THL, an inhibitor of lipoprotein lipase). By contrast, uptake of VLDL‐cholesterol was much higher than CM‐cholesterol (P < 0.01), and suramin (a lipoprotein receptor antagonist) decreased cholesterol uptake of both forms. CM‐TAG oxidation rate was more than 4‐fold higher than VLDL‐TAG oxidation. However, suramin decreased TAG oxidation from both VLDL and CM without affecting TAG uptake or total utilization, suggesting that the TAG gaining access through receptor‐mediated pathways is preferentially ‘channelled’ towards oxidation. Most (79%) CM‐TAG was oxidized whilst the proportion of VLDL‐TAG oxidized was only about half (49%). In the presence of suramin, there was a significant increase in esterification (incorporation of assimilated [3H]TAG into myocardial tissue [3H]lipids, mainly TAG) of assimilated TAG from both VLDL and CMs, again suggesting that receptor‐mediated TAG uptake is directed towards oxidation rather than esterification. The importance of this relatively small pool of TAG is indicated by the fact that cardiac mechanical function declined markedly when lipoprotein receptors were inhibited. These results suggest that CMs, most fatty acids of which gain access into cardiomyocytes through LPL‐mediated hydrolysis, are the major supplier of TAG for hearts to oxidize; however, the metabolic fate of VLDL was split evenly between oxidation and deposition as myocardial tissue lipid. Most importantly, VLDL may play a regulatory role in heart lipid metabolism through a lipoprotein receptor‐mediated mechanism.

Utilisation of Fatty Acid and Triacylglycerol by Rat Macrophages: the Effect of Endotoxin

Background/Aims: Plasma very-low-density lipoprotein (VLDL) concentrations are increased during sepsis/endotoxaemia and VLDL may be substrates for the activated immune system. Lipid substrate utilisation by quiescent and activated macrophages was therefore examined. Methods: Rat R2 macrophages were incubated with oleate or rat VLDL, with or without lipopolysaccharide (LPS) stimulation. The metabolic fate of the lipids was examined. Results: Macrophages utilised both oleate and (control) VLDL-triacylglycerol (TAG) to a similar extent. Most was deposited as cellular lipid; about 10% of oleate was oxidised compared to 25% of cVLDL-TAG. LPS significantly increased oleate oxidation and its deposition as cellular lipid (mostly as TAG) at 48hrs but abolished both oxidation and storage of VLDL-TAG. VLDL produced during endotoxaemia (eVLDL) was assimilated by the unstimulated macrophage to a greater extent than oleate or cVLDL-TAG and selectively stored as cellular lipids (no oxidation); LPS decreased eVLDL utilisation. VLDL-TAG utilisation was decreased by the lipoprotein lipase (LPL) inhibitor terahydrolipstatin. LPL activity was greater in oleate than in VLDL incubations, but was increased by incubation with eVLDL. LPS had no effect (oleate, cVLDL) or increased (eVLDL) LPL activity. Conclusion: VLDL represented an efficient substrate for the macrophage. However under conditions of sepsis/endotoxaemia eVLDL utilisation was limited and directed away from oxidation, suggesting that the increased plasma TAG (eVLDL) concentrations resulting from sepsis is not a respiratory fuel for the macrophage but may supply cellular lipid.

Preparation of radiolabelled very-low-density lipoprotein in high yield by extended rat liver perfusion

A method is described using an extended (8 h), rat liver perfusion that produces approx. 100 mg of selectively radiolabelled, mature, very-low-density lipoprotein suitable for use in subsequent whole organ perfusion experiments.

Fatty Acid and Triacylglycerol Utilisation by Perfused Rat Spleen: Differential Metabolic Fate and the Effect of Endotoxin

Background/Aims: The spleen uses significant amounts of non-esterified fatty acid (NEFA) and chylomicron (CM)-derived triacylglycerol (TAG); however, its utilisation of very-low-density lipoprotein triacylglycerol (VLDL-TAG), and the effect of endotoxin on lipid assimilation and fate are unknown. This study aims to define spleen utilisation of esterified and non-esterified fatty acid in quiescent and endotoxin-stimulated states.Methods: Rat spleens were perfused for 2h with glucose (11mM) and either NEFA (oleate; 0.4mM fatty acid), rat CM (0.4mM TAG), or rat VLDL (0.4mM TAG). Lipid oxidation and tissue lipid deposition were measured.Results: Total utilisation of oleate and VLDL-TAG were not significantly different. Utilisation of CM was 2-3 fold greater; however oxidation of CM-TAG (5.5nmols FA/min/gram spleen) was not significantly different from either NEFA or VLDL-TAG. More TAG (CM and VLDL) was deposited as tissue lipid compared to oxidation than NEFA; the pattern of intracellular lipid accumulation of TAG and NEFA also differed. Endotoxin (LPS) did not affect the proportion of lipid oxidised and accumulated or on TAG tissue lipid distribution but it increased the proportion of oleate recovered as tissue TAG and cholesterol ester. Perfusion with CM-TAG significantly decreased heparin-releasable lipoprotein lipase (LPL) activity compared with oleate or VLDL-perfused spleens. LPS increased LPL activity 5-fold in oleate-perfused spleens, but had no effect on VLDL-perfused spleens.Conclusion: VLDL-TAG is utilised by spleen to a similar extent as NEFA, but its pattern of metabolic fate resembles that of CM-TAG. Endotoxin affects the pattern of NEFA deposition in tissue lipids but has no significant effect on VLDL utilisation by the spleen.

Modulation of the orthostatic blood pressure response by acute nitrate consumption is dependent upon ethnic origin.

Orthostatic stress triggers a response to maintain cerebral perfusion and prevent syncope. Given the hypotensive effects of inorganic nitrate this response to orthostasis may be altered by acute supplementation with inorganic nitrate and modified by ethnic origin. Caucasian and SE Asian (n = 30 for both), were recruited and subjected to an ‘active stand test’ and brachial artery blood pressure (BP), digit blood flow and ECG were recorded. Following inorganic nitrate supplementation, (10 mg/kg body mass) the tests were repeated. For both Caucasian and SE Asians transition to standing increased diastolic pressure (DP) and heart rate (HR) (P < 0.001 for both) and by calculation increased rate‐pressure product (P < 0.001) and decreased pulse pressure (P < 0.01 for both) indicative of decreased ventricular filling. Nitrate supplementation decreased both DP (P < 0.001) and HR (P < 0.001). Assessment of HR variability suggested sympathetic nerve activity, was higher throughout in Caucasians (P < 0.05) coupled with higher parasympathetic tone (P < 0.01). Nitrate had no effect on cardiac autonomic nerve activity, as estimated using HR variability, for supine or standing subjects. The tachycardia and hypertension associated with orthostatic stress were preserved in both Caucasian and SE Asian subjects, however, we highlight possible differences in autonomic nervous system activity between Caucasians and SE Asians. SE Asians are resistant to the hypotensive effects of inorganic nitrate supplementation suggesting the absence of a crucial mechanism for activation of the nitrate‐nitrite‐nitric oxide system.

Caffeine, gravity, and baroreceptor function: the integration of diet and cardiovascular control

We describe a simple, cost-effective experiment to demonstrate cardiovascular integration of heart rate and blood pressure to accommodate the environmental and dietary factors of gravity and caffeine. Specific learning objectives associated with this include understanding the effects of posture on blood pressure and heart rate, coupled with the role of caffeine in modifying this response. Inclusion of ECG measurements, coupled with heart rate variability analysis, added a demonstration of the contribution made by the autonomic nervous system under these conditions. We clearly demonstrate that the cardiac work, estimated as rate-pressure product, necessary to undertake the transition from supine to standing, is fixed for a given group of subjects. However, the individual contribution of heart rate and systolic pressure to the cardiac workload is subject to the external factors of gravity and caffeine. Such an activity also demonstrates additional benefits, including unstructured teaching opportunities to augment classroom learning associated with integrative physiology and also the discussion of ethical issues with regard to human experimentation.