J. Denis McGarry

Measurement of intracellular triglyceride stores by 1H spectroscopy: validation in vivo

We validate the use of 1H magnetic resonance spectroscopy (MRS) to quantitatively differentiate between adipocyte and intracellular triglyceride (TG) stores by monitoring the TG methylene proton signals at 1.6 and 1.4 ppm, respectively. In two animal models of intracellular TG accumulation, intrahepatic and intramyocellular TG accumulation was confirmed histologically. Consistent with the histological changes, the methylene signal intensity at 1.4 ppm increased in both liver and muscle, whereas the signal at 1.6 ppm was unchanged. In response to induced fat accumulation, the TG concentration in liver derived from 1H MRS increased from 0 to 44.9 ± 13.2 μmol/g, and this was matched by increases measured biochemically (2.1 ± 1.1 to 46.1 ± 10.9 μmol/g). Supportive evidence that the methylene signal at 1.6 ppm in muscle is derived from investing interfascial adipose tissue was the finding that, in four subjects with generalized lipodystrophy, a disease characterized by absence of interfacial fat, no signal was detected at 1.6 ppm; however, a strong signal was seen at 1.4 ppm. An identical methylene chemical shift at 1.4 ppm was obtained in human subjects with fatty liver where the fat is located exclusively within hepatocytes. In experimental animals, there was a close correlation between hepatic TG content measured in vivo by 1H MRS and chemically by liver biopsy [ R = 0.934; P < .0001; slope 0.98, confidence interval (CI) 0.70-1.17; y-intercept 0.26, CI -0.28 to 0.70]. When applied to human calf muscle, the coefficient of variation of the technique in measuring intramyocellular TG content was 11.8% in nonobese subjects and 7.9% in obese subjects and of extramyocellular (adipocyte) fat was 22.6 and 52.5%, respectively. This study demonstrates for the first time that noninvasive in vivo 1H MRS measurement of intracellular TG, including that within myocytes, is feasible at 1.5-T field strengths and is comparable in accuracy to biochemical measurement. In addition, in mixed tissue such as muscle, the method is clearly advantageous in differentiating between TG from contaminating adipose tissue compared with intramyocellular lipids.

Increased lipogenic capacity of the islets of obese rats: A role in the pathogenesis of NIDDM

The onset of NIDDM in obese Zucker diabetic fatty (fa/fa) rats is preceded by a striking increase in the plasma levels of free fatty acids (FFAs) and by a sixfold rise in triglyceride content in the pancreatic islets. The latter finding provides clear evidence of elevated tissue levels of long-chain fatty acyl CoA, which can impair β-cell cell function. To determine if the triglyceride accumulation is entirely the passive consequence of high plasma FFA levels or if prediabetic islets have an increased lipogenic capacity that might predispose to NIDDM, the metabolism of long-chain fatty acids was compared in islets of obese prediabetic and nonprediabetic Zucker diabetic fatty (ZDF) rats and of lean Wistar and lean ZDF rats. When cultured in 1 or 2 mmol/l FFA, islets of both female and male obese rats accumulated, respectively, 7 and 15 times as much triglyceride as islets from lean rats exposed to identical FFA concentrations. The esterification of [14C]palmitate and 9,10-[3H]palmitate was increased in islets of male obese rats and could not be accounted for by defective oxidation of 9,10-[3H]-palmitate. Glycerol-3-PO4 acyltransferase (GPAT) activity was 12 times that of controls. The mRNA of GPAT was increased in islets of obese rats. We conclude that, in the presence of comparable elevations in FFA concentrations, the islets of obese prediabetic rats have a higher lipogenic capacity than controls. This could be a factor in their high risk of diabetes.

FATTY ACIDS RAPIDLY INDUCE THE CARNITINE PALMITOYLTRANSFERASE I GENE IN THE PANCREATIC BETA -CELL LINE INS-1

Fatty acids are important metabolic substrates for the pancreatic β-cell, and long term exposure of pancreatic islets to elevated concentrations of fatty acids results in an alteration of glucose-induced insulin secretion. Previous work suggested that exaggerated fatty acid oxidation may be implicated in this process by a mechanism requiring changes in metabolic enzyme expression. We have therefore studied the regulation of carnitine palmitoyltransferase I (CPT I) gene expression by fatty acids in the pancreatic β-cell line INS-1 since this enzyme catalyzes the limiting step of fatty acid oxidation in various tissues. Palmitate, oleate, and linoleate (0.35 mM) elicited a 4-6-fold increase in CPT I mRNA. The effect was dose-dependent and was similar for saturated and unsaturated fatty acids. It was detectable after 1 h and reached a maximum after 3 h. The induction of CPT I mRNA by fatty acids did not require their oxidation, and 2-bromopalmitate, a nonoxidizable fatty acid, increased CPT I mRNA to the same extent as palmitate. The induction was not prevented by cycloheximide treatment of cells indicating that it was mediated by pre-existing transcription factors. Neither glucose nor pyruvate and various secretagogues had a significant effect except glutamine (7 mM) which slightly induced CPT I mRNA. The half-life of the CPT I transcript was unchanged by fatty acids, and nuclear run-on analysis showed a rapid (less than 45 min) and pronounced transcriptional activation of the CPT I gene by fatty acids. The increase in CPT I mRNA was followed by a 2-3-fold increase in CPT I enzymatic activity measured in isolated mitochondria. The increase in activity was time-dependent, detectable after 4 h, and close to maximal after 24 h. Fatty acid oxidation by INS-1 cells, measured at low glucose, was also 2-3-fold higher in cells cultured with fatty acid in comparison with control cells. Long term exposure of INS-1 cells to fatty acid was associated with elevated secretion of insulin at a low (5 mM) concentration of glucose and a decreased effect of higher glucose concentrations. It also resulted in a decreased oxidation of glucose. The results indicate that the CPT I gene is an early response gene induced by fatty acids at the transcriptional level in β- (INS-1) cells. It is suggested that exaggerated fatty acid oxidation caused by CPT-1 induction is implicated in the process whereby fatty acids alter glucose-induced insulin secretion.

Regulation of the activity of caspases by L-carnitine and palmitoylcarnitine

L‐Carnitine facilitates the transport of fatty acids into the mitochondrial matrix where they are used for energy production. Recent studies have shown that L‐carnitine is capable of protecting the heart against ischemia/reperfusion injury and has beneficial effects against Alzheimers disease and AIDS. The mechanism of action, however, is not yet understood. In the present study, we found that in Jurkat cells, L‐carnitine inhibited apoptosis induced by Fas ligation. In addition, 5 mM carnitine potently inhibited the activity of recombinant caspases 3, 7 and 8, whereas its long‐chain fatty acid derivative palmitoylcarnitine stimulated the activity of all the caspases. Palmitoylcarnitine reversed the inhibition mediated by carnitine. Levels of carnitine and palmitoyl‐CoA decreased significantly during Fas‐mediated apoptosis, while palmitoylcarnitine formation increased. These alterations may be due to inactivation of β‐oxidation or to an increase in the activity of the enzyme that converts carnitine to palmitoylcarnitine, carnitine palmitoyltransferase I (CPT I). In support of the latter possibility, fibroblasts deficient in CPT I activity were relatively resistant to staurosporine‐induced apoptosis. These observations suggest that caspase activity may be regulated in part by the balance of carnitine and palmitoylcarnitine.

Regulation of ketogenesis and clinical aspects of the ketotic state

Abstract Recent studies of the regulation of ketogenesis are reviewed. Under circumstances of relative or absolute insulin deficiency there is a mobilization of free fatty acids from adipose tissue to the liver. While an increased delivery of fatty acids to this organ is important in providing substrate for ketone body formation, it is emphasized that enhanced uptake of fatty acids by the liver is not sufficient in itself to initiate maximal ketogenesis. It appears likely that a major determinant of the rate of ketogenesis is competition for the fatty acid substrate between the β-oxidative and triglyceride synthesizing pathways. While it is widely held that the rate of triglyceride synthesis is primary and that only those fatty acids not utilized for esterification become available for oxidation, evidence for the reverse sequence is presented. It is considered likely that fatty acids are utilized for triglyceride synthesis only insofar as they escape uptake and oxidation in the mitochondria. Regardless of the mechanism, fatty acid oxidation is increased in the ketotic state with the consequence that acetyl-CoA production is accelerated. Since the utilization of acetyl-CoA for fatty acid synthesis and, to a much lesser extent, its oxidation in the Krebs cycle is impaired, the synthesis of acetoacetate and β-hydroxybutyrate is stimulated to a remarkable degree. The hepatic overproduction of ketones appears to be coupled to a limited capacity for their utilization by peripheral tissues, the combined effect of which accounts for the life-threatening acidosis seen in diabetic coma. From a clinical standpoint, newer studies relating to the treatment of diabetic ketoacidosis have been covered, with particular attention paid to the problems of late cerebral edema, paradoxical acidification of the cerebrospinal fluid during treatment, shifts of the oxygen dissociation curve due to 2,3-diphosphoglycerate depletion and initial hypokalemia. Recommendations for therapy designed to minmize complications are presented.

Hepatic fatty acid oxidation and ketogenesis after clofibrate treatment

The effect of clofibrate treatment on hepatic ketogenic capacity was studied in rats. Ketogenesis from octanoate and oleate was increased 2- and 4,5-fold, respectively, in hepatocytes from fed, treated rats. In contrast to controls ketogenic rates did not increase upon starvation. While ketogenesis from oleate was higher in fed, treated animals than in fasted controls, endogenous ketogenesis was lower and increased upon starvation. Ketogenesis from octanoate and oleate was stimulated approx. 2-fold in homogenates from treated animals. Labeled pyruvate and succinate oxidation was unaltered. [1-14C]Oleate oxidation was severely inhibited by cyanide, both in homogenates from controls and treated animals. Clofibrate caused a 3-fold increase in hepatic carnitine levels. Catalase and glutamate dehydrogenase activities were also increased by the drug. Cytochrome c oxidase did not change. Despite their increased ketogenic capacity hepatocytes from treated rats esterified as much oleate as controls. The increased oxidation was matched by an increased oleate uptake. Plasma ketones were increased 2-fold in fasted, treated animals. Plasma free fatty acids were unaffected. It is concluded that the enhanced ketogenic capacity induced by clofibrate is the result of an increase in mitochondrial beta-oxidation, an increase in the activity of carnitine palmitoyltransferase and possibly of the observed increases in hepatic carnitine content and fatty acid uptake.

Bulk magnetic susceptibility effects on the assessment of intra- and extramyocellular lipids in vivo

Localized proton spectroscopy provides a novel method for noninvasive measurement of lipid content in skeletal muscle. It has been suggested that the chemical shift difference between lipid signals from distinct compartments in skeletal muscle might be caused by bulk magnetic susceptibility (BMS) differences from lipids stored in intra‐ (IMCL) and extramyocellular (EMCL) compartments. Direct evidence is provided to confirm the theoretical prediction that compartment symmetry is responsible for discrimination between resonances of IMCL and EMCL. Phantoms imitating lipids in skeletal muscle were constructed using soybean oil to represent EMCL, and Intralipid™, an intravenous fat emulsion of fine droplets, to represent IMCL. It was found that the chemical shift of Intralipid™ is independent of the BMS effects, while the resonance of soybean oil shifts in a predictable manner determined by the geometry of the compartment. Magn Reson Med 47:607–610, 2002.

The glucose paradox: new perspectives on hepatic carbohydrate metabolism

Abstract In the past few years the question of how the body handles a glucose load when a fast is terminated has taken on renewed interest. As a result, longstanding concepts have been challenged and new formulations are emerging. The mechanism by which liver glycogen is repleted during refeeding has come under particular scrutiny and is the primary focus of this article.

Ketogenesis and its regulation

Abstract The ketotic state has been of major interest to biochemists, physiologists and clinicians for many years. It is our purpose here to review recent advances that provide new insights into the mechanisms by which ketogenesis is regulated. To this end, we shall briefly discuss current understanding of the biochemical physiology of ketogenesis, the hormonal induction of ketosis and clinical implications.

Topological and functional analysis of the rat liver carnitine palmitoyltransferase 1 expressed in Saccharomyces cerevisiae.

The rat liver carnitine palmitoyltransferase 1 (L‐CPT 1) expressed in Saccharomyces cerevisiae was correctly inserted into the outer mitochondrial membrane and shared the same folded conformation as the native enzyme found in rat liver mitochondria. Comparison of the biochemical properties of the yeast‐expressed L‐CPT 1 with those of the native protein revealed the same detergent lability and similar sensitivity to malonyl‐CoA inhibition and affinity for carnitine. Normal Michaelis‐Menten kinetics towards palmitoyl‐CoA were observed when careful experimental conditions were used for the CPT assay. Thus, the expression in S. cerevisiae is a valid model to study the structure‐function relationships of L‐CPT 1.