Hormonal regulation of fat esterification & oxidation in hepatocytes from fed rats: modulation by 3-mercaptopicolinate

3-Mercaptopicolinate, an inhibitor of phosphoenolpyruvate carboxykinase (PEPCK), decreased esterification of [1-14C] oleate and [1-14C] myristate in hepatocytes from fed rats. In the absence of 3-mercaptopicolinate, adrenaline, noradrenaline, vasopressin or angiotensin II increased esterification to triacylglycerol of [1-14C] oleate but not [1-14C] myristate. Cyclic AMP decreased esterification of both oleate and myristate. In the presence of 3-mercaptopicolinate, stimulation of oleate esterification by the catecholamines, vasopressin or angiotensin II was increased, and stimulatory effects of these hormones on myristate esterification were observed. Adrenaline, noradrenaline, vasopressin or angiotensin II increased 14CO2 production from both [1-14C] oleate and [1-14C] myristate but the degree of stimulation was similar in the absence or presence of 3-mercaptopicolinate. The results indicate a role for the catecholamines and angiotensin II in the regulation of liver fat metabolism and emphasize the potential importance of changes in activity of PEPCK as determinants of hepatic carbon flux.     

Alterations in energy status by menadione metabolism in hepatocytes isolated from fasted and fed rats

The biochemical mechanism of cytotoxicity, induced by the quinoid compound 2-methyl 1,4-naphthoquinone (menadione), was investigated in hepatocytes freshly isolated from fasted and fed rats. Hepatocytes from fasted rats were significantly more vulnerable to the toxicity of menadione than hepatocytes from fed rats. Menadione (150 microM) induced a 50% loss of viability of cells (LT50) from fasted rats after 55 min of incubation, whereas a LT50 of 80 min was observed after exposure of hepatocytes from fed rats to menadione. Glutathione and NADPH levels were rapidly depleted by menadione metabolism. This depletion was sustained during the incubation period. No significant differences were found in the time course and extent of the menadione-induced glutathione and NADPH depletion in hepatocytes of both nutritional states. Menadione also affected the energy status of the hepatocytes. The ATP content of cells from fasted rats decreased to 50% (AT50) within 18 min of exposure to menadione, whereas a 50% loss of ATP content of hepatocytes from fed rats was reached at 65 min. In contrast to depletion of glutathione and NADPH, the time course and extent of menadione-induced ATP depletion correlated well with the time of onset and rate of cell killing. Our results suggest that menadione metabolism may interfere with both mitochondrial and glycolytic ATP production. Depletion of ATP might be a critical step in menadione-induced cytotoxicity.     

Lower fat deposition and energy utilization of growing rats fed diets containing sorbose

1. Growing rats were fed diets containing graded levels (0, 100, 200 and 300 g/kg diet) of sorbose for 6 weeks. Protein, fat and energy deposition were determined by carcass analysis. 2. The values for growth, serum insulin level, digestible energy (DE), metabolizable energy (ME) and fat and energy deposition declined with the increment of dietary sorbose. 3. The efficiency of protein utilization (protein retained/protein consumed) was hardly affected by dietary sorbose. The DE and ME of sorbose per se was calculated as 14.09 and 12.35 kJ/g respectively. The efficiency of energy utilization (energy retained/ME intake) decreased with the increase of dietary sorbose, although sorbose had an ME. 4. The relative weights of gastro-intestinal tract and liver were positively associated with dietary sorbose level, although the reverse was true for the amount of stomach content, being heavier with higher dietary sorbose. 5. It is suggested that dietary sorbose, as a sweetener as well as a bulky agent, seems to be a suitable sugar for the obese and diabetic with special reference to lower body fat and energy deposition without reducing protein utilization.     

Okadaic acid stimulates carnitine palmitoyltransferase I activity and palmitate oxidation in isolated rat hepatocytes

Okadaic acid parallely increased carnitine [corrected] palmitoyltransferase I activity and the rate of palmitate oxidation in isolated rat hepatocytes. Nevertheless, okadaic acid had no significant effect on the rate of octanoate oxidation. Maximal effects of okadaic acid were similar and non-additive to those of dibutyryl-cAMP, forskolin and glucagon. Results indicate that carnitine palmitoyltransferase I activity may be controlled by a mechanism of phosphorylation/dephosphorylation.     

Fine structure of hepatocytes from fasted and fed rats.

The fine structure of hepatocytes from rats maintained on a controlled feeding schedule are described. Liver samples were processed for electron microscopy, histochemistry and chemical determinations of glycogen at precise time-intervals following a 30-hour fast and a 2-hour meal. Hepatocytes from 30-hour-fasted rats with extremely low hepatic glycogen levels were devoid of glycogen particles. Centrilobular cells showed areas of the cytoplasm rich in vesicles of smooth endoplasmic reticulum (SER) while periportal hepatocytes contained less extensive regions of SER. Soon after feeding the fasted rats, glycogen particles appeared in regions of the cell rich in SER. Centrilobular hepatocytes contained numerous glycogen areas which were infiltrated with tubules of SER, while periportal cells showed dense glycogen deposits with SER restricted to the periphery of the masses of glycogen. Throughout glycogen deposition each glycogen particle was closely associated with membranes of SER until maximum glycogen deposition was achieved 12 hours after initiation of feeding. At this point SER was reduced to the lowest amounts of the time-periods studied. During stages of glycogen depletion SER proliferated and reached the highest concentration measured in this study. Tubules of SER were present throughout the glycogen masses of centrilobular hepatocytes, whereas in periportal cells the organelle was restricted to the periphery of the glycogen masses. It is concluded that SER is associated with glycogen particles in rat hepatocytes during both deposition and depletion of glycogen.     

Short and long term effects of ethanol on glycerolipid synthesis from palmitate in fasted rat hepatocytes

Prolonged ethanol administration to rats increased the rates of glycerolipid synthesis from added [U-14C]palmitate in fasted hepatocytes; this increase was more than 2-fold in triglyceride synthesis. Prolonged ethanol administration to rats completely eliminated the acute ethanol-induced increase in triglyceride synthesis from palmitate in hepatocytes from fasted rats. This adaptive change occurred in a short initial period of about 10 days of ethanol feeding. In hepatocytes from fasted control rats, addition of ethanol produced a rapid and strong increase in the concentration of glycerol 3-phosphate. By contrast, this acute effect of ethanol disappeared in hepatocytes from fasted alcoholic rats after a prolonged--5 weeks--administration of ethanol in a liquid diet.     

Influence of malonyl-CoA and palmitate concentration on rate of palmitate oxidation in rat muscle

5-Aminoimidazole-4-carboxamide1--D-ribofuranoside(AICAR) is taken up by perfused skeletal muscle andphosphorylated to form5-aminoimidazole-4-carboxamide-1--D-ribofuraosyl-5'-monophosphate (analog of 5'-AMP) with consequent activation of AMP-activated protein kinase, phosphorylation of acetyl-CoA carboxylase, decrease inmalonyl-CoA, and increase in fatty acid oxidation. Thisstudy was designed to determine the effect of increasing levels ofpalmitate on the rate of fatty acid oxidation. Malonyl-CoAconcentration was manipulated with AICAR at different palmitateconcentrations. Rat hindlimbs were perfused with Krebs-Henseleitbicarbonate containing 4% bovine serum albumin, washed bovine redcells, 200 µU/ml insulin, 10 mM glucose, and different concentrationsof palmitate (0.1-1.0 mM) without or with AICAR (2.0 mM).Perfusion with medium containing AICAR was found to activateAMP-activated protein kinase in skeletal muscle, inactivate acetyl-CoAcarboxylase, and decrease malonyl-CoA at all concentrations ofpalmitate. The rate of palmitate oxidation increased as a function ofpalmitate concentration in both the presence and absence of AICAR butwas always higher in the presence of AICAR. These results provideadditional evidence that malonyl-CoA is an important regulator of therate of fatty acid oxidation at palmitate concentrations in thephysiological range.

     

Effect of D-galactosamine in vitro on [U-14C]palmitate oxidation, triacylglycerol synthesis and secretion in isolated hepatocytes

Isolated rat hepatocytes were used to study in vitro effects of 10 mM D-galactosamine (GalN) on hepatic fatty acids metabolism. At this concentration, membrane integrity and biochemical competence (i.e., gluconeogenesis and ureogenesis) remained unaffected. Protein synthesis and secretion, as measured by the incorporation of [U-14C]leucine into total and medium protein, was significantly inhibited when incubated for more than 2 h. GalN activated the incorporation of [U-14C]palmitate into triacylglycerols and depressed its utilization in the formation of labelled ketone bodies and 14CO2. Hepatocytes isolated from fasted rats exposed to GalN in vitro did not show any variation in prelabelled triacylglycerol secretion. GalN induced a rapid inhibition of prelabelled triacylglycerol secretion by hepatocytes isolated from fed rats in which this secretion occurred to a larger extent than in hepatocytes isolated from fasted rats. The data reported here suggest that GalN induces a rise of triacylglycerol synthesis by inhibiting the palmitate oxidation pathway and a decrease of triacylglycerol secretion through an early derangement of the secretory pathway.     

A light- and electron-microscope study of hepatocytes of rats fed different diets

Ketogenic diets are used in the treatment of epilepsy in children refractory to drug therapy. This study identifies changes in liver morphology in rats fed four different diets: a normal rodent chow diet, a calorie-restricted high-fat (ketogenic) diet and each diet supplemented with clofibric acid. Hepatocytes of rats fed the ketogenic diet show many lipid droplets and these are reduced to control levels when clofibrate is present in the diet. Mitochondria are enlarged in the livers of rats fed the ketogenic diet and further enlarged if clofibrate is present. Alterations in the appearance or numbers of other organelles are also found.     

Differential effects of acetate on palmitate and octanoate oxidation: Segregation of acetyl CoA pools

In isolated hepatic mitochondria, sodium acetate had little effect on the oxidation of octanoate, but conspicuously inhibited the oxidation of palmitate. This differential effect of acetate on long-chain and short-chain fatty acid oxidation is not due to inhibition of the activation or transfer of long-chain fatty acids into the mitochondria. Both palmitate and octanoate reduced CO₂ production from acetate. Palmitate and octanoate mutually inhibited CO₂ production from each other to the same extent. Acetate stimulated ketogenesis from palmitoyl-1-carnitine to the same extent as it inhibited oxygen uptake and CO₂ production from palmitate. This suggests that acetate causes a redistribution of the end products of palmitate oxidation toward ketogenesis rather than toward total oxidation to CO₂ and H₂O. Acetyl CoA derived from acetate or palmitate may share a common pool or pathway, thus each is mutally inhibitory toward the oxidation of the other. Either because of the existence of separate pools, or because octanoate is the preferred substrate, acetate metabolism does not inhibit O₂ uptake or CO₂ production from octanoate, whereas the oxidation of octanoate dilutes the CO₂ produced from labeled acetate. This may be explained by compartmentation or preferred pathways for the disposition of acetyl CoA derived from different sources.     

Substrate control of glycogen levels in isolated hepatocytes from fed rats.

Isolated parenchymal cells from fed rat liver rapidly lose glycogen when incubated with glucose. The addition of glycerol or fructose but not insulin prevents much of the breakdown. When cells are incubated with glycerol and glucose, more glycogen is retained with the further addition of xylitol than of fructose or pyruvate. Oleate stimulates glycogen breakdown. The results indicate that glycerol may play an important physiological role in the control of glycogen synthesis in the liver, possibly by esterifying fatty acids. Furthermore, the results support the concept that the main effect of insulin on liver glycogen levels $\text{in vivo}$ may be the results of diminished flow of free fatty acids to the liver.     

Enhanced production of fructose palmitate by lipase-catalyzed esterification in ionic liquids

For the enhancement of enzyme activity, application of ultrasound irradiation on lipase-catalyzed esterification of fructose with palmitic acid in ionic liquids (ILs) mixture containing supersaturated fructose solution was investigated. In the mixture of [Bmim][TfO] and [Omim][Tf₂N] (1:1, v/v), 1.44 times higher enzyme activity (29.2 μmoL/min/g) was achieved under ultrasound irradiation. Besides, ultrasound irradiation enhanced enzyme stability in viscous ILs mixture. After 5 times reuse of Novozym 435 and ILs mixture, 84.4% of initial enzyme activity was remained under ultrasound irradiation, while the residual activity using magnetic stirring only method was 76.2%. These results show that enzymatic reaction in viscous ILs mixture under ultrasound irradiation is an effective method for enzyme activity, as well as, enzyme stability resulting in economic competitiveness of green process.     

Fractional oxidation of chylomicron-derived oleate is greater than that of palmitate in healthy adults fed frequent small meals

Differences in oxidation of individual dietary fatty acids could contribute to the effect of dietary fat composition on risk factors for non-insulin-dependent diabetes mellitus and cardiovascular disease. Using a novel stable isotope technique, we compared fractional oxidation of chylomicron-derived oleate and palmitate in 10 healthy adults in a crossover study. 1-(13)C-labeled oleate or palmitate was emulsified into a eucaloric formula diet administered each 20 min for 7 h to produce a plateau in excretion of (13)C label in breath CO(2). Unlabeled oleate and palmitate each provided 16% of dietary energy, and other fatty acids provided 8% of energy. Total dietary fat was 40% of energy, carbohydrate was 46%, and protein was 14%. Diet without tracer was fed for 2 h before beginning tracer administration to establish a baseline fed state. Relative oxidation of oleate versus palmitate was defined as fractional oxidation of oleate divided by fractional oxidation of palmitate. Relative oxidation averaged 1.21 (99.5% confidence interval = 1.03;-1.39), indicating that fractional oxidation of oleate was significantly greater than that of palmitate.     

Metabolic regulation of fatty acid esterification and effects of conjugated linoleic acid on glucose homeostasis in pig hepatocytes

Conjugated linoleic acids (CLAs) are geometric and positional isomers of linoleic acid (LA) that promote growth, alter glucose metabolism and decrease body fat in growing animals, although the mechanisms are poorly understood. A study was conducted to elucidate the effects of CLA on glucose metabolism, triglyceride (TG) synthesis and IGF-1 synthesis in primary culture of porcine hepatocytes. In addition, hormonal regulation of TG and IGF-1 synthesis was addressed. Hepatocytes were isolated from piglets (n = 5, 16.0 ± 1.98 kg average body weight) by collagenase perfusion and seeded into collagen-coated T-25 flasks. Hepatocytes were cultured in William's E containing dexamethasone (10-8 and 10-7 M), insulin (10 and 100 ng/ml), glucagon (0 and 100 ng/ml) and CLA (1 : 1 mixture of cis-9, trans-11 and trans-10, cis-12 CLA, 0.05 and 0.10 mM) or LA (0.05 and 0.10 mM). Addition of CLA decreased gluconeogenesis (P < 0.05), whereas glycogen synthesis and degradation, TG synthesis and IGF-1 synthesis were not affected compared with LA. Increased concentration of fatty acids in the media decreased IGF-1 production (P < 0.001) and glycogen synthesis (P < 0.01), and increased gluconeogenesis (P < 0.001) and TG synthesis (P < 0.001). IGF-1 synthesis increased (P < 0.001) and TG synthesis decreased (P < 0.001) as dexamethasone concentration in the media rose. High insulin/glucagon increased TG synthesis. These results indicate that TG synthesis in porcine hepatocytes is hormonally regulated so that dexamethasone decreases and insulin/glucagon increases it. In addition, CLA decreases hepatic glucose production through decreased gluconeogenesis.