Victor A. Zammit

Carnitine, mitochondrial function and therapy

Carnitine is important for cell function and survival primarily because of its involvement in the multiple equilibria between acylcarnitine and acyl-CoA esters established through the enzymatic activities of the family of carnitine acyltransferases. These have different acyl chain-length specificities and intracellular compartment distributions, and act in synchrony to regulate multiple aspects of metabolism, ranging from fuel-selection and -sensing, to the modulation of the signal transduction mechanisms involved in many homeostatic systems. This review aims to rationalise the extensive range of experimental and clinical data that have been obtained through the pharmacological use of L-carnitine and its short-chain acylesters, over the past two decades, in terms of the basic biochemical mechanisms involved in the effects of carnitine on the various cellular acyl-CoA pools in health and disease.

Natrolite: A zeolite with negative Poisson’s ratios

The recently published experimental elastic constants [C. Sanchez-Valle, S. V. Sinogeikin, Z. A. Lethbridge, R. I. Walton, C. W. Smith, K. E. Evans, and J. D. Bass, J. Appl. Phys. 98, 053508 (2005)] for single crystals of the orthorhombic aluminosilicate zeolite NAT (natrolite, Na2(Al2Si3O10)2H2O, Fdd2) throw valuable light on the potential of NAT as a material which exhibits single crystalline negative Poisson’s ratios (auxetic). On performing an off-axis analysis of these elastic constants we confirm that the zeolite natrolite exhibits auxetic behavior in the (001) plane. This supports our preliminary report that NAT-type zeolites exhibit auxetic behavior through a mechanism involving microscopic rotation of semi-rigid structural units.

Diacylglycerol acyltransferase 2 acts upstream of diacylglycerol acyltransferase 1 and utilizes nascent diglycerides and de novo synthesized fatty acids in HepG2 cells

The two diacylglycerol acyltransferases, DGAT1 and DGAT2, are known to have non‐redundant functions, in spite of catalysing the same reaction and being present in the same cell types. The basis for this distinctiveness, which is reflected in the very different phenotypes of Dgat1−/− and Dgat2−/− mice, has not been resolved. Using selective inhibitors of human DGAT1 and DGAT2 on HepG2 cells and gene silencing, we show that, although DGAT2 activity accounts for a modest fraction (< 20%) of overall cellular DGAT activity, inhibition of DGAT2 activity specifically inhibits (and is rate‐limiting for) the incorporation of de novo synthesized fatty acids and of glycerol into cellular and secreted triglyceride to a much greater extent than it affects the incorporation of exogenously added oleate. By contrast, inhibition of DGAT1 affects equally the incorporation of glycerol and exogenous (preformed) oleate into cellular and secreted triacylglycerol (TAG). These data indicate that DGAT2 acts upstream of DGAT1, largely determines the rate of de novo synthesis of triglyceride, and uses nascent diacylglycerol and de novo synthesized fatty acids as substrates. By contrast, the data suggest that DGAT1 functions in the re‐esterification of partial glycerides generated by intracellular lipolysis, using preformed (exogenous) fatty acids. Therefore, we describe distinct but synergistic roles of the two DGATs in an integrated pathway of TAG synthesis and secretion, with DGAT2 acting upstream of DGAT1.

Effects of Insulin Replacements, Inhibitors of Angiotensin, and PKCβ's Actions to Normalize Cardiac Gene Expression and Fuel Metabolism in Diabetic Rats

High-density oligonucleotide arrays were used to compare gene expression of rat hearts from control, untreated diabetic, and diabetic groups treated with islet cell transplantation (ICT), protein kinase C (PKC)β inhibitor ruboxistaurin, or ACE inhibitor captopril. Among the 376 genes that were differentially expressed between untreated diabetic and control hearts included key metabolic enzymes that account for the decreased glucose and increased free fatty acid utilization in the diabetic heart. ICT or insulin replacements reversed these gene changes with normalization of hyperglycemia, dyslipidemia, and cardiac PKC activation in diabetic rats. Surprisingly, both ruboxistaurin and ACE inhibitors improved the metabolic gene profile (confirmed by real-time RT-PCR and protein analysis) and ameliorated PKC activity in diabetic hearts without altering circulating metabolites. Functional assessments using Langendorff preparations and 13C nuclear magnetic resonance spectroscopy showed a 36% decrease in glucose utilization and an impairment in diastolic function in diabetic rat hearts, which were normalized by all three treatments. In cardiomyocytes, PKC inhibition attenuated fatty acid–induced increases in the metabolic genes PDK4 and UCP3 and also prevented fatty acid–mediated inhibition of basal and insulin-stimulated glucose oxidation. Thus, PKCβ or ACE inhibitors may ameliorate cardiac metabolism and function in diabetes partly by normalization of fuel metabolic gene expression directly in the myocardium.

Hepatic triacylglycerol synthesis and secretion : DGAT2 as the link between glycaemia and triglyceridaemia

lThe liver regulates both glycaemia and triglyceridaemia. Hyperglycaemia and hypertriglyceridaemia are both characteristic of (pre)diabetes. Recent observations on the specialised role of DGAT2 (diacylglycerol acyltransferase 2) in catalysing the de novo synthesis of triacylglycerols from newly synthesized fatty acids and nascent diacylglycerols identifies this enzyme as the link between the two. This places DGAT2 at the centre of carbohydrate-induced hypertriglyceridaemia and hepatic steatosis. This function is complemented, but not substituted for, by the ability of DGAT1 to rescue partial glycerides from complete hydrolysis. In peripheral tissues not normally considered to be lipogenic, synthesis of triacylglycerols may largely bypass DGAT2 except in hyperglycaemic/hyperinsulinaemic conditions, when induction of de novo fatty acid synthesis in these tissues may contribute towards increased triacylglycerol secretion (intestine) or insulin resistance (adipose tissue, and cardiac and skeletal muscle).

Carnitine palmitoyltransferase 1: Central to cell function

INTRODUCTION A sentinel paper in 1977 described the inhibition of the oxidation of fatty acids by isolated mitochondria by malonyl-CoA in vitro and its correlation with the inhibition of the ‘‘outer’’ CPT activity that could be measured in intact, well-coupled mitochondria (1, 2). This explained the ability of some tissues to perform high rates of both fatty acid synthesis or oxidation under separate physiological conditions. Malonyl-CoA, an intermediate of fatty acid synthesis, through inhibition of the ‘‘ratelimiting’’ step of fatty acid oxidation, ensures that the two processes would not occur simultaneously. The inhibition of CPT 1 by malonyl-CoA is crucial because it is the point at which metabolism of fatty acids and glucose come into the most direct ‘‘contact’’ to influence each other’s metabolism. Other interactions, such as the inhibition of phosphofructokinase by citrate, the provision of glucose-derived glycerol for acylglyceride synthesis, the regulation of pyruvate dehydrogenase activity by fatty acid-derived acetyl-CoA, do not have the same ability to affect the continuous substrate selection that tissues have to make primarily between glucose and fatty acid oxidation.

Evidence That Diacylglycerol Acyltransferase 1 (DGAT1) Has Dual Membrane Topology in the Endoplasmic Reticulum of HepG2 Cells

Triacylglycerol (TAG) synthesis and secretion are important functions of the liver that have major impacts on health, as overaccumulation of TAG within the liver (steatosis) or hypersecretion of TAG within very low density lipoproteins (VLDL) both have deleterious metabolic consequences. Two diacylglycerol acyltransferases (DGATs 1 and 2) can catalyze the final step in the synthesis of TAG from diacylglycerol, which has been suggested to play an important role in the transfer of the glyceride moiety across the endoplasmic reticular membrane for (re)synthesis of TAG on the lumenal aspect of the endoplasmic reticular (ER) membrane (Owen, M., Corstorphine, C. C., and Zammit, V. A. (1997) Biochem. J. 323, 17–21). Recent topographical studies suggested that the oligomeric enzyme DGAT1 is exclusively lumen facing (latent) in the ER membrane. By contrast, in the present study, using two specific inhibitors of human DGAT1, we present evidence that DGAT1 has a dual topology within the ER of HepG2 cells, with approximately equal DGAT1 activities exposed on the cytosolic and lumenal aspects of the ER membrane. This was confirmed by the observation of the loss of both overt (partial) and latent (total) DGAT activity in microsomes prepared from livers of Dgat1−/− mice. Conformational differences between DGAT1 molecules having the different topologies were indicated by the markedly disparate sensitivities of the overt DGAT1 to one of the inhibitors. These data suggest that DGAT1 belongs to the family of oligomeric membrane proteins that adopt a dual membrane topology.

Lipid molecular order in liver mitochondrial outer membranes, and sensitivity of carnitine palmitoyltransferase I to malonyl-CoA

Mitochondrial outer membranes were prepared from livers of rats that were in the normal fed state, starved for 48 h, or made diabetic by injection of streptozotocin. Membranes were also prepared from starved late-pregnant rats. The latter three conditions have previously been shown to induce varying degrees of desensitization of mitochondrial overt carnitine palmitoyltransferase (CPT I) to malonyl-CoA inhibition. We measured the fluorescence polarization anisotropy of two probes, 1,6-diphenyl-1,3,5-hexatriene (DPH) and 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene-p-toluenesulfonate (TMA-DPH) which, when incorporated into membranes, report on the hydrophobic core and on the peripheral regions of the bilayer, respectively. The corresponding polarization indices (rDPH and rTMA-DPH) were calculated. In membranes of all three conditions characterized by CPT I desensitization to malonyl-CoA, rDPH was decreased, whereas there was no change in rTMA-DPH, indicating that CPT I is sensitive to changes in membrane core, rather than peripheral, lipid order. The major lipid components of the membranes were analyzed. Although significant changes with physiological state were observed, there was no consistent pattern of changes in gross lipid composition accompanying the changes to membrane fluidity and CPT I sensitivity to malonyl-CoA. We conclude that CPT I kinetic characteristics are sensitive to changes in lipid composition that are localized to specific membrane microdomains.

Submitochondrial and subcellular distributions of the carnitine-acylcarnitine carrier

The submitochondrial and subcellular distributions of the carnitine‐acylcarnitine translocase (CAC) have been studied. CAC is enriched to a much lesser extent than the carnitine palmitoyltransferases within the contact sites of mitochondria. A high‐abundance protein of identical molecular size as the mitochondrial CAC that is immunoreactive with an anti‐peptide antibody raised against a linear epitope of mitochondrial CAC is present in peroxisomes but not in microsomes. This suggests that CAC is targeted to at least two different locations within the liver cell and that acylcarnitine transport into peroxisomes is CAC mediated.

Regulation of mitochondrial outer-membrane carnitine palmitoyltransferase (CPT I): Role of membrane-topology

The topology of the outer membrane carnitine palmitoyltransferase (CPT I) of rat liver mitochondria was studied systematically using several experimental approaches. Studies with immobilized malonyl-CoA and octanoyl-CoA showed that functionally the active and regulatory sites of CPT I are exposed on the outer (cytosolic) surface of the mitochondrial outer membrane. Anti-peptide antibodies generated against three linear peptide sequences that occur in between and on either side of two hydrophobic, putative transmembrane domains were used to (a) ascertain which were bound by intact mitochondria and mitochondria in which the outer membrane was permeabilized to proteins; and (b) to determine the size of fragments generated by limited proteolysis (by trypsin or proteinase K) of CPT I in intact or outer membrane-ruptured mitochondria. The sizes and immunoreactivity of the proteolytic fragments generated were correlated with the effects of the proteases on CPT I activity and malonyl-CoA sensitivity. The results of all the different approaches suggested the following: (i) CPT I has two transmembrane domains; (ii) both the N- and C-termini are exposed on the cytosolic side of the membrane; (iii) the linker region between the two transmembrane domains protrudes into the intermembrane space; (iv) both the active site and the malonyl-CoA-binding site are exposed on the cytosolic side of the membrane; (v) the amino-terminus of the protein interacts with the C-terminal domain of the protein to maintain the optimal conformation required for activity of the enzyme and its sensitivity to malonyl-CoA.