Carnitine synthesis and uptake into cells are stimulated by fasting in pigs as a model of nonproliferating species

In rodents, fasting increases the carnitine concentration in the liver by an up-regulation of enzymes of hepatic carnitine synthesis and novel organic cation transporter (OCTN) 2, mediated by activation of peroxisome proliferator-activated receptor (PPAR) α. This study was performed to investigate whether such effects occur also in pigs which like humans, as nonproliferating species, have a lower expression of PPARα and are less responsive to treatment with PPARα agonists than rodents. An experiment with 20 pigs was performed, which were either fed a diet ad-libitum or fasted for 24 h. Fasted pigs had higher relative mRNA concentrations of the PPARα target genes carnitine palmitoyltransferase 1 and acyl-CoA oxidase in liver, heart, kidney, and small intestinal mucosa than control pigs, indicative of PPARα activation in these tissues (P<.05). Fasted pigs had a higher activity of γ-butyrobetaine dioxygenase (BBD), enzyme that catalyses the last step of carnitine biosynthesis in liver and kidney, and higher relative mRNA concentrations of OCTN2, the most important carnitine transporter, in liver, kidney, skeletal muscle, and small intestinal mucosa than control pigs (P<.05). Fasted pigs moreover had higher concentrations of free and total carnitine in liver and kidney than control pigs (P<.05). This study shows for the first time that fasting increases the activity of BBD in liver and kidney and up-regulates the expression of OCTN2 in various tissues of pigs, probably mediated by PPARα activation. It is concluded that nonproliferating species are also able to cover their increased demand for carnitine during fasting by an increased carnitine synthesis and uptake into cells.     

Nutritionally induced adipose hypertrophy in young pigs is transient and independent of changes in the expression of the obese and peroxisome proliferator activated receptor genes

Previous studies have shown that piglets weaned to a liquid milk replacer (MR), rather than a typical dry diet (DD) regimen, have improved growth rates and deposit more energy as body fat. In the present study, we used this model to determine whether changes in the expression of genes linked to the regulation of adiposity were related to the accelerated fat accretion. We also determined whether the increase in body fat was sustained throughout a substantial proportion of the growth curve. At weaning (19 plus minus 2 days of age), 96 piglets were placed in 12 replicate pens per diet (4 pigs per pen, 2 barrows and 2 gilts), and fed a liquid MR or conventional DD regimen for 5 weeks. Thereafter, 6 barrows and 6 gilts pigs from each diet were killed for determination of whole body chemical composition (less gastrointestinal contents). The remaining pigs were assigned randomly to weight target groups (60, 85, and 110 kg), placed in individual pens, and fed a conventional dietary regimen until killed at their respective weight targets for tissue sampling and determination of whole body chemical composition. Over the 5-week period in which the MR was fed, the growth rate of the pigs consuming the MR exceeded that of the pigs fed the DD by 36% (P <.05). Fat gain in these pigs was increased to 1.8 times that of the pigs fed the DD, and percentage body fat was 45% greater (P <.05). Acetyl Co-A carboxylase (ACC) activity (per mg of adipose extract protein) was not different between the two diet groups at the conclusion of the 5-week period, or at 110 kg body weight. During the MR period, actual protein gain was increased (P <.05) 22% in the pigs fed the MR as well. By 110 kg of body weight, body fat was reduced (P <.05) by 7.7% (total fat mass) and 8.3% (percentage of body weight basis) in the pigs fed MR vs. the DD group. The expression of the peroxisome proliferator activated receptors (PPAR) alpha and gamma was not influenced by diet or by body weight. Expression of the obese gene was independent of diet, but was greater (P <.09) in pigs at 110 kg body weight than at 60 kg. These data provide additional evidence that piglets weaned to liquid diets have greater rates of growth and deposit more body fat, but that this difference subsides quickly when a typical dry dietary regimen is imposed. Furthermore, the biochemical changes responsible for the increased adiposity are independent of changes in the expression of the obese or PPAR genes, at least at the mRNA level.     

Irreversible inhibition of hepatic glutathione S-transferase by ciprofibrate, a peroxisome proliferator

Ciprofibrate (2-[4-(2,2-dichlorocyclopropyl) phenoxy]2-methyl propionic acid) which is a hypolipidemic agent and has been shown to cause peroxisome proliferation, non-competitively inhibits glutathione S-transferase activity of rat liver, both in vivo and in vitro. Among all the glutathione S-transferases of rat liver, ligandin is maximally inhibited by ciprofibrate. Studies with the purified glutathione S-transferases of rat liver indicate that the affinities of different subunits of liver enzymes for ciprofibrate are in the order Ya greater than Yb, Yb' greater than Yc.     

Suppression of mouse hepatocyte apoptosis by peroxisome proliferators: role of PPARalpha and TNFalpha

Peroxisome proliferators (PPs) are a diverse group of nongenotoxic chemicals that in rodents cause hepatic peroxisome proliferation, liver enlargement, increased replicative DNA synthesis and suppression of apoptosis. The effects of PPs in vivo can be reproduced in vitro where PPs can induce mouse hepatocyte DNA synthesis and suppress both spontaneous apoptosis and that induced by transforming growth factor beta (TGFbeta). In vitro, high concentrations (>500 U/ml) of exogenous tumour necrosis factor (TNFalpha) [M. Rolfe, N.H. James, R.A. Roberts, TNF suppresses apoptosis and induces S-phase in rodent hepatocytes: a mediator of the hepatocarcinogenicity of peroxisome proliferators?, Carcinogenesis 18 (1997) 2277-2280] are also able to stimulate hepatocyte DNA synthesis and suppress apoptosis, implicating TNFalpha in mediating or permitting the liver growth response to PPs. Here, using cultured mouse hepatocytes isolated from PPARalpha null mice, we have examined the role of the peroxisome proliferator activated receptor alpha (PPARalpha) in mediating the suppression of apoptosis caused by PPs. In addition we have investigated further the role of TNFalpha in mediating the rodent response to PPs. The PP nafenopin (50 microM) was unable to stimulate DNA synthesis measured by bromodeoxyuridine incorporation in these PPARalpha null mouse hepatocytes (96% of control), unlike epidermal growth factor, a growth factor used as a positive control. In assays of apoptosis using H33258 staining of chromatin condensation, nafenopin was unable to suppress either spontaneous or TGFbeta1-induced apoptosis. In contrast, high concentrations of TNFalpha (>500 U/ml) were able to both stimulate DNA synthesis (204% of control) and suppress apoptosis in PPARalpha null hepatocytes (40% and 38% of control for spontaneous and TGFbeta1-induced apoptosis respectively). However, TNFalpha could not stimulate beta-oxidation of palmitoyl CoA in either PPARalpha null mouse or B6C3F1 (PPARalpha wild type) mouse hepatocytes. These data confirm the dependence of the response to PPs on PPARalpha by demonstrating that PPARalpha mediates the suppression of hepatocyte apoptosis in response to PPs. In addition, the data provide evidence that high concentrations of TNFalpha can modulate DNA synthesis and apoptosis in the absence of PPs and PPARalpha. Thus, in vivo, physiological levels of TNFalpha may be permissive for a PPARalpha-dependent growth response to PPs.     

Tissue-selective, bidirectional regulation of PEX11 alpha and perilipin genes through a common peroxisome proliferator response element

Most cis-acting regulatory elements have generally been assumed to activate a single nearby gene. However, many genes are clustered together, raising the possibility that they are regulated through a common element. We show here that a single peroxisome proliferator response element (PPRE), located between the mouse PEX11 alpha and perilipin genes, confers on both genes activation by peroxisome proliferator-activated receptor alpha (PPAR alpha) and PPAR gamma. A functional PPRE 8.4 kb downstream of the promoter of PEX11 alpha, a PPAR alpha target gene, was identified by a gene transfection study. This PPRE was positioned 1.9 kb upstream of the perilipin gene and also functioned with the perilipin promoter. In addition, this PPRE, when combined with the natural promoters of the PEX11 alpha and perilipin genes, conferred subtype-selective activation by PPAR alpha and PPAR gamma 2. The PPRE sequence specifically bound to the heterodimer of RXR alpha and PPAR alpha or PPAR gamma 2, as assessed by electrophoretic gel mobility shift assays. Furthermore, tissue-selective binding of PPAR alpha and PPAR gamma to the PPRE was demonstrated in hepatocytes and adipocytes, respectively, by chromatin immunoprecipitation assay. Hence, the expression of these genes is induced through the same PPRE in the liver and adipose tissue, where the two PPAR subtypes are specifically expressed.     

Specific repression of transthyretin gene expression in rat liver by a peroxisome proliferator clofibrate.

The effect of clofibrate, a hypolipidemic drug and a potent peroxisome proliferator, on expression of a nonperoxisomal transthyretin (prealbumin) gene was studied using rats fed clofibrate for various periods. Upon feeding a diet containing clofibrate, the level of transthyretin mRNA was down-regulated, reaching 20% of the control level, in an almost reciprocal time course to that of increases in the levels of peroxisomal mRNAs. Studies on expression of other steroid hormone regulated genes suggest that clofibrate may down-regulate several but not all types of steroid hormone regulated mRNA expression.     

Suppression of liver cell apoptosis in vitro by the non-genotoxic hepatocarcinogen and peroxisome proliferator nafenopin

Suppression of apoptosis has been implicated as a mechanism for the hepatocarcinogenicity of the peroxisome proliferator class of non- genotoxic carcinogens. The ability of the peroxisome proliferator nafenopin to suppress or delay the onset of liver apoptosis was investigated using primary cultures of rat hepatocytes and the Reuber hepatoma cell line FaO. 50 microM nafenopin reversibly maintained the viability of primary rat hepatocyte cultures which otherwise degenerated within 8 d of establishment. The maintenance of viability of hepatocyte monolayers was associated with a significant decrease in the number of cells exhibiting chromatin condensation patterns typical of apoptosis. Apoptosis could be induced in hepatocytes by administration of 5 ng/ml TGF beta 1. Co-addition of 50 microM nafenopin significantly reduced TGF beta 1-induced apoptosis by 50-60%. TGF beta 1 (1-5 ng/ml) also induced apoptosis in the FaO rat hepatoma cell line. Cell death was accompanied by detachment of FaO cells from the monolayer and detached cells exhibited chromatin condensation and non-random DNA fragmentation patterns typical of apoptosis. Co-addition of 50 microM nafenopin to TGF beta 1-treated FaO cultures significantly reduced the number of apoptotic cells detaching from the monolayer at 24 h. In contrast, nafenopin had no significant effect on FaO apoptosis induced by the DNA damaging agents etoposide and hydroxyurea. We conclude that suppression of liver cell death by apoptosis may play a role in the hepatocarcinogenicity of the peroxisome proliferators, although the extent of this protection is dependent on the nature of the apoptotic stimulus.     

Comitogenicity of eicosanoids and the peroxisome proliferator ciprofibrate in cultured rat hepatocytes

Several hypolipidemic drugs and environmental contaminants induce hepatic peroxisome proliferation and hepatic tumors when administered to rodents. These chemicals increase the expression of the peroxisomal β-oxidation pathway and the cytochrome P-450 4A family, which metabolize lipids, including eicosanoids and their precursor fatty acids. We previously found that the peroxisome proliferator ciprofibrate decreases the level of eicosanoids in the liver and in cultured hepatocytes. In this study, we examined the effect of prostaglandins E₂ and F_2α (PGE₂ and PGF_2α), leukotriene C₄ (LTC₄) and the peroxisome proliferator ciprofibrate on DNA synthesis in cultured hepatocytes. Primary rat hepatocytes were cultured on collagen gels in serum-free L-15 medium with varying concentrations of eicosanoids and ciprofibrate, and the absence or presence of growth factors. Ciprofibrate lowered hepatocyte eicosanoid concentrations; the addition of eicosanoids restored their levels. After a 48-h exposure with [³H]-thymidine, DNA synthesis was determined by measuring [³H]-thymidine incorporation into DNA. The addition of PGE₂, PGF_2α, and LTC₄ to cultures along with ciprofibrate increased DNA synthesis, whereas treatment with ciprofibrate or eicosanoids alone resulted in a much smaller increase. The addition of epidermal growth factor (EGF) to the eicosanoid-ciprofibrate combination increased DNA synthesis more than EGF or the eicosanoid-ciprofibrate combination alone. The PGF_2α-ciprofibrate combination also was comitogenic with transforming growth factor-α and hepatocyte growth factor. The addition of both ciprofibrate and prostaglandins also blocked the growth inhibitory effect of transforming growth factor-β on DNA synthesis induced by EGF. These results show that the eicosanoids PGE₂, PGF_2α, and LTC₄ are comitogenic with the peroxisome proliferator ciprofibrate in cultured rat hepatocytes. © 1996 Wiley-Liss, Inc.     

Induction of fatty liver by fasting in suncus

We found that a fatty liver was easily induced in a novel experimental animal, Suncus murinus (suncus), by withholding food. Hepatic triglyceride content increased linearly for up to 24 h after fasting in these animals. Serum levels of neutral lipids are very low in the fed state compared with those in rats, and decreased significantly after 24 h fasting. On the other hand, serum free fatty acids, which are at the same level in fed animals as in rats, increased threefold in the fasting suncus. In order to learn whether the fatty liver induced by fasting is an unusual physiological state or a pathological on-going state in suncus, they were refed after 24 h fasting. Refeeding resulted in a decrease in hepatic triglyceride content to the level of fed animals. Serum lipid levels, which decreased with fasting, returned to those of fed animals. This evidence indicates that hepatic lipid secretion is impaired even in a physiological state to some extent and that starvation causes increasing influx of free fatty acid to the liver, which might be followed by esterification and result in triglyceride accumulation in the liver. In conclusion, hepatic lipid and lipoprotein metabolism is unique to the suncus, which is a useful animal model for the study of intra-hepatic lipid transport.     

Induction of rat liver mitochondrial fatty acid elongation by the administration of peroxisome proliferator di-(2-ethylhexyl)phthalate: absence of elongation activity in peroxisomes

The administration of di-(2-ethylhexyl)phthalate (DEHP)3 to male Sprague-Dawley rats resulted in more than a threefold increase in activity of acetyl CoA-dependent hepatic mitochondrial fatty acid elongation. Peroxisomes obtained either from control or DEHP-treated rats were not capable of elongating any of the fatty acyl CoAs tested. Furthermore, the peroxisomes possessed no trans-2-enoyl CoA reductase activity. Therefore, the elongation activity in the 7500g fraction from both control and DEHP-fed animals can be attributed totally to the mitochondria. Maximal incorporation of acetyl CoA occurred in the presence of both NADH and NADPH, and octanoyl CoA (8:0) and decanoyl CoA (10:0) were found to be optimal primers for fatty acid elongation in both control and DEHP-treated animals. The apparent Km for 8:0 CoA was 17 microM in both animal groups while the Vmax was increased from 4.5 to 12.5 nmol/min/mg following treatment. The apparent Km for 10:0 CoA was 10 microM in both control and DEHP-treated groups while the apparent Vmax increased from 2.5 to 10 nmol/min/mg; palmitoyl-CoA (16:0) was a very poor primer for chain elongation. Although the acetyl CoA-dependent fatty acid elongation was stimulated by DEHP treatment, the mitochondrial trans-2-enoyl CoA reductase activity was unaffected. The mitochondrial total elongation activity following DEHP-treatment using 8:0 CoA as primer was about two times higher than enoyl CoA reductase activity using trans-2-decenoyl CoA (10:1). This was the result of accumulation of intermediates, which were identified as trans-2-10:1 (35%), beta-hydroxy 10:0 (25%), unidentified (15%), and elongated saturated product 10:0 (24%). Elongation by one acetate unit was found in both the control and DEHP-treated animals. The results are discussed in terms of physiological significance.     

Impact of peroxisome proliferator activated receptor agonist drugs in a model of nephrotoxicity in rats

Doxorubicin (DOX) is one of the basic anticancer drugs, nonetheless its use is restricted due to noxious side effects. Kidney failure is one of the main side effects that restrict its medical use. The current study assessed the nephroprotective effects of fenofibrate and pioglitazone against the renal injury induced by doxorubicin in rats and illustrated the probable mechanisms underlying these protective effects. For this purpose, Male Sprague–Dawley rats weighing (200–230 g) were allocated into seven groups treated for 15 days as following: control (50% corn oil + 50% DMSO p.o), fenofibrate (100 mg/kg p.o) and pioglitazone (10 mg/kg p.o) as well as four groups of DOX (15 mg/kg i.p on 11th day). DOX groups included DOX alone and DOX with protective drugs fenofibrate, pioglitazone or both of them. As a result of doxorubicin nephrotoxicity; serum creatinine and blood urea nitrogen were remarkably elevated. Moreover, renal glutathione was significantly reduced while tissue lipid peroxidation malondialdehyde, tumor necrosis factor-α, nuclear factor-kappa B p65 (NF-κB p65), interleukin-1β, p38 mitogen activated protein kinase (p38-MAPK) and caspase-3 (Casp-3) were significantly augmented. Treatment with fenofibrate and pioglitazone either alone or in combination markedly attenuated DOX-induced injury by suppression of oxidative stress, inflammation and apoptosis. The above-mentioned biochemical markers were affirmed by histological assessment. In conclusion, fenofibrate, pioglitazone, and their combination possess potential prophylactic effects against doxorubicin-induced renal injury through modulation of p38-MAPK/NF-κB p65 pathway with superiority to the combination.     

Induction of pigmentation in nonproliferating cells of Serratia marcescens by addition of single amino acids

Addition of casein hydrolysate to suspensions of washed, nonpigmented, nonproliferating Serratia marcescens incubating at 27 C induced biosynthesis of prodigiosin. Four amino acids of casein hydrolysate, dl-aspartic acid, l-glutamic acid, l-proline, and l-alanine caused formation of pigment when added individually. dl-Ornithine also was effective. Optimal concentrations for maximal pigmentation were 5 to 10 mg/ml; at these high concentrations, d-serine also induced biosynthesis of some prodigiosin. dl-Alanine and -ornithine were as effective as the l-iosomers, but l-glutamic acid and l-proline gave better responses than their racemic mixtures. Kinetics of prodigiosin biosynthesis after addition of dl-alanine (20 mg/ml) were similar to those of cells suspended in 0.2% casein hydrolysate. The other amino acids were less effective. Addition of 5 mg of dl-alanine or casein hydrolysate per ml to minimal medium increased by 30% the amount of prodigiosin formed by growing cells after incubation for 7 days at 27 C. Cultures grown for 7 days at 27 C in 0.2% casein hydrolsate formed more prodigiosin than did suspensions of nonproliferating cells containing individual amino acids or casein hydrolysate. However, more pigment was produced by cells suspended in l-alanine (5 mg/ml) or l-proline (10 mg/ml) than when suspended in 0.4% natural or synthetic casein hydrolysate. Filtrates from suspensions of nonproliferating cells forming pigment in l-proline induced more rapid formation of prodigiosin, but filtrates from suspensions in dl-alanine did not. The data supported the hypothesis that pyrrole groups of prodigiosin may be synthesized from 5-carbon amino acids such as proline, ornithine, aspartic, and glutamic acids, but the role of alanine is unknown.     

Induction of control genes in intestinal gluconeogenesis is sequential during fasting and maximal in diabetes

We studied in rats the expression of genes involved in gluconeogenesis from glutamine and glycerol in the small intestine (SI) during fasting and diabetes. From Northern blot and enzymatic studies, we report that only phosphoenolpyruvate carboxykinase (PEPCK) activity is induced at 24 h of fasting, whereas glucose-6-phosphatase (G-6-Pase) activity is induced only from 48 h. Both genes then plateau, whereas glutaminase and glycerokinase strikingly rebound between 48 and 72 h. The two latter genes are fully expressed in streptozotocin-diabetic rats. From arteriovenous balance and isotopic techniques, we show that the SI does not release glucose at 24 h of fasting and that SI gluconeogenesis contributes to 35% of total glucose production in 72-h-fasted rats. The new findings are that 1) the SI can quantitatively account for up to one-third of glucose production in prolonged fasting; 2) the induction of PEPCK is not sufficient by itself to trigger SI gluconeogenesis; 3) G-6-Pase likely plays a crucial role in this process; and 4) glutaminase and glycerokinase may play a key potentiating role in the latest times of fasting and in diabetes.     

Sulfate and thiosulfate transport in Escherichia coli K-12: evidence for a functional overlapping of sulfate- and thiosulfate-binding proteins.

In Escherichia coli, sulfate and thiosulfate ions are transported by an ABC-type transporter consisting of both the membrane components (the products of cysT, cysW, and cysA genes) and the periplasmic binders (the products of cysP and sbp genes). The single cysP and sbp mutants are able to utilize both sulfate and thiosulfate as a sole sulfur source, while the inactivation of both genes leads to cysteine auxotrophy resulting from the block in the transport of both ions.