Increased oxidative capacity in skeletal muscles from cold-acclimated ducklings: a comparison with rats

1. The effects of prolonged cold exposure on cytochrome oxidase activity were investigated in skeletal muscles, liver and adipose tissues from cold-acclimated (CA) and control (TN) ducklings and rats. 2. Cold acclimation increased the oxidative capacity of skeletal muscles (+33% in gastrocnemius and +195% in pectoral) and liver (+47%) from CA ducklings, but decreased the oxidative capacity of gastrocnemius muscle (-22%) from CA rats. On the other hand, in these CA rats it increased the oxidative capacity of liver by 88% and, above all, brown adipose tissue by 544%. 3. The significance of these changes due to acclimation to cold in ducklings and rats is discussed. Such an increase in oxidative capacity of CA duckling muscles may explain the non-shivering thermogenesis observed in these birds.     

Increased respiration in skeletal muscle mitochondria from cold-acclimated ducklings: uncoupling effects of free fatty acids

Intermyofibrillar mitochondria from skeletal muscle (m. gastrocnemius) and liver mitochondria were isolated from cold-acclimated (4 degrees C) or control (30 degrees C) 4-week old ducklings. The respiratory rate of isolated mitochondria, with Na-succinate as substrate, was followed polarographically at 25 degrees C in order to determine the basal respiratory rate, the rate of respiration in the presence of free fatty acids (FFA) (Na-palmitate), and the fully uncoupled rate, after addition of FCCP. The basal respiration (which in liver mitochondria was unaffected by acclimation to cold) was higher (+53%) in intermyofibrillar mitochondria from cold-acclimated ducklings than from controls, and the maximal FCCP-stimulated respiration was also increased (+98%) by acclimation to cold. FFA-stimulated respiration increased as a function of FFA concentration in both types of mitochondria. The increase in respiration due to FFA was about double in intermyofibrillar mitochondria from cold-acclimated ducklings than that of controls, but in liver mitochondria there was no increase due to cold. The membrane potential was estimated by the dye safranine in the absence or in the presence of FFA in the incubation medium. There were no significant differences in the basal membrane potential in the two groups and the addition of FFA led to the same depolarization in both groups. The significance of these alterations for acclimation to cold is discussed.     

Regulation of lipogenesis in isolated hepatocytes by triglyceride-rich lipoproteins.

Very low density lipoproteins, chylomicrons, and remnants caused, within an hour, significant inhibition of fatty acid synthesis but not cholesterol synthesis in hepatocytes isolated from meal-fed rats. In contrast, low density lipoproteins, high density lipoproteins, and the serum fraction of density greater than 1.21 failed to significantly inhibit either fatty acid or cholesterol synthesis within 1 h. The Scatchard plots of specific binding showed that rat and human very low density lipoproteins interact with the high affinity sites on the hepatocytes with the apparent dissociation constants of 64 and 106 nM, respectively. These data also indicated that each hepatocyte was capable of binding 6 X 10(5) molecules of very low density lipoproteins.     

Temperature dependence of lipogenesis in isolated hepatocytes from rainbow trout (Salmo gairdneri)

Temperature dependence of lipogenesis in trout liver cells was investigated in the presence of 5 mM lactate using either [14C]lactate or [3H]water. A ratio of 3H/14C-incorporation greater than one is found, irrespective of temperature. Acclimation of fish to 4, 10 or 16 degrees C affects neither the height of lipid synthesis nor its temperature sensitivity. The distribution of [14C]lactate between the main lipid classes and the capacities for cholesterol- and triacylglycerol-synthesis are correlated to the glycogen stores of the hepatocytes. A comparison of fatty acid synthesis and cholesterogenesis in livers of normal fed rat and of trout suggests a capability for lipogenesis in trout somewhat similar to that in mammals.     

Tissue variation of mitochondrial oxidative phosphorylation efficiency in cold-acclimated ducklings

We investigated the oxidative phosphorylation efficiency of liver and gastrocnemius muscle mitochondria in thermoneutral and cold-acclimated ducklings. The yield of oxidative phosphorylation was lower in muscle than in liver mitochondria, a difference that was associated with a higher proton conductance in muscle mitochondria. Cold exposure did not affect oxidative phosphorylation efficiency or basal proton leak in mitochondria. We conclude that the basal proton conductance of mitochondria may regulate mitochondrial oxidative phosphorylation efficiency, but is not an important contributor to thermogenic processes in cold-acclimated ducklings.     

Stimulation of glycogen synthesis and lipogenesis by glutamine in isolated rat hepatocytes.

Glutamine stimulated glycogen synthesis and lactate production in hepatocytes from overnight-fasted normal and diabetic rats. The effect, which was half-maximal with about 3 mM-glutamine, depended on glucose concentration and was maximal below 10 mM-glucose. beta-2-Aminobicyclo[2.2.1.]heptane-2-carboxylic acid, an analogue of leucine, stimulated glutaminase flux, but inhibited the stimulation of glycogen synthesis by glutamine. Various purine analogues and inhibitors of purine synthesis were found to inhibit glycogen synthesis from glucose, but they did not abolish the stimulatory effect of glutamine on glycogen synthesis. The correlation between the rate of glycogen synthesis and synthase activity suggested that the stimulation of glycogen synthesis by glutamine depended solely on the activation of glycogen synthase. This activation of synthase was not due to a change in total synthase, nor was it caused by a faster inactivation of glycogen phosphorylase, as was the case after glucose. It could, however, result from a stimulation of synthase phosphatase, since, after the addition of 1 nM-glucagon or 10 nM-vasopressin, glutamine did not interfere with the inactivation of synthase, but did promote its subsequent re-activation. Glutamine was also found to inhibit ketone-body production and to stimulate lipogenesis.     

Increased ketogenesis related to insulin deficiency in isolated hepatocytes from NIDDM model rats.

To investigate the hepatic ketone body metabolism in NIDDM, we studied the ketone body production rates in hepatocytes from newly developed non-obese NIDDM model rats. NIDDM model rats were prepared by intraperitoneal injection of streptozotocin at 2 or 5 days of age (STZ2, STZ5 respectively). After 10-15 weeks, ketone body production rates in hepatocytes isolated from these rats were compared with those from control rats as well as ketotic rats made by intravenous injection of streptozotocin into adult rats. Basal ketone body production rates from 0.3 mM [U-14C] palmitate in hepatocytes from control, STZ 2, STZ 5 and ketotic rats were 11.7 +/- 0.98, 14.9 +/- 0.72, 16.0 +/- 0.45, 22.8 +/- 2.32 nmole.palmitate/mg.prot/hr, respectively. These rates were stimulated by 1 microgram/ml of glucagon in control, STZ 2 and STZ 5 rats (14.1 +/- 0.99, 18.6 +/- 1.36, 18.7 +/- 0.69 nmole.palmitate/mg.prot/hr, respectively), but not in ketotic rats (22.8 +/- 2.07 nmole.palmitate/mg.prot/hr). The similar effects were observed by 1 microgram/ml of epinephrine. The basal ketone body production rates were negatively correlated to both hepatic glycogen contents and plasma IRI levels. Considering these parameters together, the extent of metabolic derangement in STZ 2 and STZ 5 rats was between that in control and ketotic rats. These results indicate that the derangements of hepatic ketone body production are related to the severity of insulin deficiency and suggest that the enhanced hepatic ketogenesis contributes in part to the elevated plasma ketone body levels in non-obese NIDDM.     

Increased oxidation of p-nitrophenol and aniline by intact hepatocytes isolated from pyrazole-treated rats

Induction of cytochrome P-450 IIE1 by pyrazole has been shown in a variety of studies with isolated microsomes or reconstituted systems containing the purified P-450 isozyme. Experiments were conducted to document induction by pyrazole in intact hepatocytes by studying the oxidation of p-nitrophenol to 4-nitrocatechol or of aniline to p-aminophenol. Hepatocytes prepared from rats treated with pyrazole for 2 days oxidized p-nitrophenol or aniline at rates which were 3- to 4-fold higher than saline controls. To observe maximal induction in hepatocytes, it was necessary to add metabolic substrates such as pyruvate, sorbitol or xylitol, which suggests that availability of the NADPH cofactor may be rate-limiting in the hepatocytes from the pyrazole-treated rats. Carbon monoxide inhibited the oxidation of p-nitrophenol and aniline by hepatocytes from the pyrazole-treated rats and controls, demonstrating the requirement for cytochrome P-450. The oxidation of both substrates by the hepatocyte preparations was inhibited by a variety of agents that interact with and are effective substrates for oxidation by P-450 IIE1 such as ethanol, dimethylnitrosamine, pyrazole and 4-methylpyrazole. Microsomes isolated from pyrazole-treated rats oxidized aniline and p-nitrophenol at elevated rats compared to saline controls. These results indicate that induction by pyrazole of the oxidation of drugs which are effective substrates for P-450 IIE1 can be observed in intact hepatocytes. The extent of induction and many of the characteristics of aniline or p-nitrophenol oxidation observed with isolated microsomes from pyrazole-treated rats can also be found in the intact hepatocytes.     

Iron mobilization from isolated hepatocytes

It is not known which message and mechanism triggers the cell to mobilize iron from ferritin. In this paper we present the results of incubation experiments with 59Fe-labelled hepatocytes. Anemic serum gives a significant higher rate of iron mobilization than normal serum. The involvement of apo-transferrin is ruled out because it did not increase iron mobilization. Citrate increased iron mobilization which is not the result of an increase in NADH/NAD+-ratio because addition of ethanol did not stimulate iron mobilization. Desferrioxamine is used clinically in iron overloaded patients and it is known that iron removal is a very slow process. Although desferrioxamine can mobilize iron from ferritin in hepatocytes, a considerable amount remains inside the cell as a low molecular weight fraction. This fraction represents chelator bound iron and is slowly released into the circulation.     

Increased catalytic activity of cytochrome P-450IIE1 in pericentral hepatocytes compared to periportal hepatocytes isolated from pyrazole-treated rats.

Cytochrome P-450IIE1 is induced by a variety of agents, including acetone, ethanol and pyrazole. Recent studies employing immunohistochemical methods have shown that P-450IIE1 was expressed primarily in the pericentral zone of the liver. In order to evaluate whether catalytic activity of P-450IIE1 is preferentially localized in the pericentral zone of the liver acinus, the oxidation of aniline and p-nitrophenol, two effective substrates for P-450IIE1, by periportal and pericentral hepatocytes isolated from pyrazole-treated rats was determined. Periportal and pericentral hepatocytes were prepared by a digitonin-collagenase procedure; the marker enzymes glutamine synthetase and gamma-glutamyl transpeptidase indicated reasonable separation of the two cell populations. Viability, yield and total cytochrome P-450 content were similar for the periportal and pericentral hepatocytes. Pericentral hepatocytes oxidized aniline and p-nitrophenol at rates that were 2-4-fold greater than periportal hepatocytes under a variety of conditions. Carbon monoxide inhibited the oxidation of the substrates with both preparations and abolished the increased oxidation found with the pericentral hepatocytes. Pyrazole or 4-methylpyrazole, added in vitro, effectively inhibited the oxidation of aniline and p-nitrophenol and prevented the augmented rate of oxidation by the pericentral hepatocytes. Western blots carried out using isolated microsomes revealed a more than 2-fold increase in immunochemical staining with microsomes isolated from the pericentral hepatocytes, which correlated to the 2-4-fold increase in the rate of oxidation of aniline or p-nitrophenol by the pericentral hepatocytes. These results suggest that functional catalytic activity of cytochrome P-450IIE1 is preferentially localized in the pericentral zone of the liver acinus, and that most of the induction by pyrazole of P-450IIE1 appears to occur within the pericentral zone.     

Increased translatable mRNA and decreased lipogenesis are responsible for the augmented secretion of lipid-deficient apolipoprotein E by hepatocytes from fasted rats

We examined the mechanism through which fasting selectively increases the secretion of apoE while it decreases the secretion of all lipoprotein lipids (Davis, R. A., Boogaerts, J. R., Borchardt, R. A., Malone-McNeal, M., and Archambault-Schexnayder, J. (1985) J. Biol. Chem. 260, 14137-14144). Livers were obtained from rats that were fed chow plus drinking water (control) and drinking water only (fasted) for three days. Livers were extracted for both total and poly(A) RNA. Using full length, nick-translated 32P-labeled cDNA probes for both apoE and beta-actin, the relative abundance was determined by slot blot hybridization assays. There was 2-fold more apoE mRNA in the livers of fasted rats. Furthermore, translation of poly(A) RNA using a reticulocyte lysate showed a similar 2.3-fold increase in the synthesis of immunoprecipitable [35S]methionine-labeled apoE. The 2-fold increase in translatable apoE mRNA correlates with a similar increase in apoE secretion. We also characterized the form of apoE secreted by hepatocytes from fasted cells. Cells were labeled with [35S]methionine, and the medium was separated by agarose 0.5m column chromatography. The majority of the apoE secreted cells from both control and fasted rats eluted in fractions that contained no detectable lipid. Furthermore, almost all of the increased apoE secreted by fasted cells was in these lipid-deficient fractions. The isoform distribution of apoE secreted by cells from both groups consisted of six major apoE isoforms. Consistent with previous results, treatment with neuraminidase transformed the acidic forms into the three most basic, suggesting that the three most acidic isoforms contain varying amounts of sialic acid. The isoform pattern of apoE secreted by cells from fasted rats was significantly enriched in two acidic isoforms, while it was significantly decreased in the major basic isoform. Moreover, when oleic acid (1 mM) was added to the culture medium to stimulate lipogenesis, the amount of apoE secreted with lipid increased as did the more basic isoforms. These data suggest that the secretion of lipid-deficient apoE by cells from fasted rats is the result of increased mRNA and a concomitant reduction in lipogenesis. Furthermore, the parallel shift of both the amount of apoE secreted associated with lipid as well as its isoform pattern to a more basic one by oleic acid suggests that the lipid availability plays a role in determining the lipid complement and sialic acid content of apoE secreted by the hepatocyte.     

Increased lipoprotein lipase activity of skeletal muscle in cold-acclimated rats

The activity of lipoprotein lipase (LPL) in the heart, diaphragm, and soleus muscles was markedly increased in cold-acclimated rats and it was even greater in rats treated with oxytetracycline (OTC) while exposed to cold. Other skeletal muscles studied had low and variable activities which were not significantly increased by cold acclimation or by cold plus OTC treatment. It appears therefore that, apart from the heart and the muscles involved in respiratory movements, LPL activity is primarily associated with those muscles which contain a predominance of slow-twitch oxidative fibers, and that the enzyme in muscle, heart, and diaphragm responds to cold acclimation and cold plus OTC treatment in a parallel fashion in these tissues.     

The effects of oxalate and glucose on lipogenesis by isolated hepatocytes from normal and biotin-deficient chicks (Gallus domesticus)

• 1.1. Isolated hepatocytes synthesize fatty acids and cholesterol from lactate and acetate with lactate being the more effective substrate.
• 2.2. Biotin deficiency decreased fatty add synthesis from both substrates but stimulated cholesterogenesis.
• 3.3. Exposure of intact hepatocytes to oxalate inhibited fatty acid and cholesterol synthesis from lactate, this effect was enhanced in biotin-deficient chicks. A similar effect was not observed when acetate was the substrate.
• 4.4. Synthesis of fatty acids from lactate and acetate was stimulated by glucose, biotin deficiency increased this response. Cholesterogenesis was reduced in control but not biotin-deficient chicks.

     

Multivesicular bodies isolated from rat hepatocytes

Summary Plama lipoproteins (and other ligands) are endocytosed by hepatocytes and appear in multivesicular bodies (MVBs) in the Golgi-lysosome region of the cell prior to their degradation. We have isolated MVB fractions from livers of estradiol-treated rats, permitting studies of their properties (Hornick et al. 1985). Here we report our cytochemical studies of lysosomal enzyme activity in partially and highly purified MVB fractions and in MVBs in hepatocytes in situ.Only about 15% of partially or highly purified MVBs were positive for acid phosphatase and arylsulfatase, consistent with the prelysosomal nature of this compartment. Partially purified MVB fractions contained small round vesicles, 70–120 nm in diameter, which, stained intensely for these enzymes; occasionally these vesicles appeared to fuse with MVBs, suggesting that these structures are primary lysosomes. Such stained vesicles were rarely seen in highly purified MVB preparations. Acid phosphatase reaction product with cerium as capture reagent appeared as uniform precipitates surrounding endocytosed plasma lipoproteins in positively stained MVBs. Arylsulfatase reaction product, however, appeared as distinctive are or plaque-like deposits just inside the MVB-limiting membrane, often in continuity with intense reaction product contained in a fusing primary lysosome. Similar putative primary lysosomes were occasionally observed in isolated, intact Golgi fractions from the same livers. Similar histochemical reactivities of MVBs and putative primary lysosomes were observed in thin sections of hepatoyctes in situ.These observations support the conclusion that, in hepatocytes, MVBs represent the immediate prelysosomal compartment in the endocytic pathway of macromolecular catabolism, and suggest that MVBs are converted to secondary lysosomes by direct fusion with primary lysosomes arising from closely adjacent Golgi compartment.A portion of this work was published in abstract form in the Journal of Histochemistry and Cytochemistry, Vol. 34, page 120, 1986.This work was supported by U.S. Public Health Service Grant HL 14237 (Arteriosclerosis SCOR)     

Increased mitochondrial fusion and autophagy help isolated hepatocytes repolarize in collagen sandwich cultures

Freshly isolated, depolarized rat hepatocytes can repolarize into bile canalicular networks when plated in collagen sandwich cultures. We studied the events underlying this repolarization process, focusing on how hepatocytes restore ATP synthesis and resupply biosynthetic precursors after the stress of being isolated from liver. We found that soon after being plated in collagen sandwich cultures, hepatocytes converted their mitochondria into highly fused networks. This occurred through a combination of upregulation of mitochondrial fusion proteins and downregulation of a mitochondrial fission protein. Mitochondria also became more active for oxidative phosphorylation, leading to overall increased ATP levels within cells. We further observed that autophagy was upregulated in the repolarizing hepatocytes. Boosted autophagy levels likely served to recycle cellular precursors, supplying building blocks for repolarization. Repolarizing hepatocytes also extensively degraded lipid droplets, whose fatty acids provide precursors for β-oxidation to fuel oxidative phosphorylation in mitochondria. Thus, through coordination of mitochondrial fusion, autophagy, and lipid droplet consumption, depolarized hepatocytes are able to boost ATP synthesis and biosynthetic precursors to efficiently repolarize in collagen sandwich cultures.     

PPARgamma2 regulates lipogenesis and lipid accumulation in steatotic hepatocytes

Peroxisome proliferator-activated receptor-gamma (PPARgamma) is considered to be one of the master regulators of adipocyte differentiation. PPARgamma2 is abundantly expressed in mature adipocytes and is elevated in the livers of animals that develop fatty livers. The aim of this study was to determine the ability of PPARgamma2 to induce lipid accumulation in hepatocytes and to delineate molecular mechanisms driving this process. The hepatic cell line AML-12 was used to generate a cell line stably expressing PPARgamma2. Oil Red O staining revealed that PPARgamma2 induces lipid accumulation in hepatocytes. This phenotype is accompanied by a selective upregulation of several adipogenic and lipogenic genes including adipose differentiation-related protein (ADRP), adipocyte fatty acid-binding protein 4, sterol regulatory element-binding protein-1 (SREBP-1), fatty acid synthase (FAS), and acetyl-CoA carboxylase, genes whose expression levels are known to increase in steatotic livers of ob/ob mice. Furthermore, the PPARgamma2-regulated induction of both SREBP-1 and FAS parallels an increase in de novo triacylglycerol synthesis in hepatocytes. Triacylglycerol synthesis and lipid accumulation are further enhanced by culturing hepatocytes with troglitazone in the absence of exogenous lipids. These results correspond with an increase in the lipid droplet protein, ADRP, and the data demonstrate that ADRP functions to coat lipid droplets in hepatocytes as observed by confocal microscopy. Taken together, these observations propose a role for PPARgamma2 as an inducer of steatosis in hepatocytes and suggest that this phenomenon occurs through an induction of pathways regulating de novo lipid synthesis.