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Abstract Background The active form (T3) of thyroid hormone (TH) controls critical aspects of cerebellar development, such as migration of postmitotic neurons and terminal dendritic differentiation of Purkinje cells. The effects of T3 on early dendritic differentiation are poorly understood. Results In this study, we have analyzed the influence of T3 on the progression of the early steps of Purkinje cell dendritic differentiation in postnatal day 0 organotypic cerebellar cultures. These steps include, successively, regression of immature neuritic processes, a stellate cell stage, and the extension of several long and mature perisomatic protrusions before the growth of the ultimate dendritic tree. We also studied the involvement of RORα, a nuclear receptor controlling early Purkinje cell dendritic differentiation. We show that T3 treatment leads to an accelerated progression of the early steps of dendritic differentiation in culture, together with an increased expression of RORα (mRNA and protein) in both Purkinje cells and interneurons. Finally, we show that T3 failed to promote early dendritic differentiation in staggerer RORα-deficient Purkinje cells. Conclusions Our results demonstrate that T3 action on the early Purkinje cell dendritic differentiation process is mediated by RORα.

Retinoic acid-related Orphan Receptor alpha (RORα; NR1F1) is a widely distributed nuclear receptor involved in several (patho)physiological functions including lipid metabolism, inflammation, angiogenesis, and circadian rhythm. To better understand the role of this nuclear receptor in liver, we aimed at displaying genes controlled by RORα in liver cells by generating HepG2 human hepatoma cells stably over-expressing RORα. Genes whose expression was altered in these cells versus control cells were displayed using micro-arrays followed by qRT-PCR analysis. Expression of these genes was also altered in cells in which RORα was transiently over-expressed after adenoviral infection. A number of the genes found were involved in known pathways controlled by RORα, for instance LPA, NR1D2 and ADIPOQ in lipid metabolism, ADIPOQ and PLG in inflammation, PLG in fibrinolysis and NR1D2 and NR1D1 in circadian rhythm. This study also revealed that genes such as G6PC, involved in glucose homeostasis, and AGRP, involved in the control of body weight, are also controlled by RORα. Lastly, SPARC, involved in cell growth and adhesion, and associated with liver carcinogenesis, was up-regulated by RORα. SPARC was found to be a new putative RORα target gene since it possesses, in its promoter, a functional RORE as evidenced by EMSAs and transfection experiments. Most of the other genes that we found regulated by RORα also contained putative ROREs in their regulatory regions. Chromatin immunoprecipitation (ChIP) confirmed that the ROREs present in the SPARC, PLG, G6PC, NR1D2 and AGRP genes were occupied by RORα in HepG2 cells. Therefore these genes must now be considered as direct RORα targets. Our results open new routes on the roles of RORα in glucose metabolism and carcinogenesis within cells of hepatic origin.

Background: The active form (T3) of thyroid hormone (TH) controls critical aspects of cerebellar development, such as migration of postmitotic neurons and terminal dendritic differentiation of Purkinje cells. The effects of T3 on early dendritic differentiation are poorly understood., Results: In this study, we have analyzed the influence of T3 on the progression of the early steps of Purkinje cell dendritic differentiation in postnatal day 0 organotypic cerebellar cultures. These steps include, successively, regression of immature neuritic processes, a stellate cell stage, and the extension of several long and mature perisomatic protrusions before the growth of the ultimate dendritic tree. We also studied the involvement of RORalpha, a nuclear receptor controlling early Purkinje cell dendritic differentiation. We show that T3 treatment leads to an accelerated progression of the early steps of dendritic differentiation in culture, together with an increased expression of RORalpha (mRNA and protein) in both Purkinje cells and interneurons. Finally, we show that T3 failed to promote early dendritic differentiation in staggerer RORalpha-deficient Purkinje cells., Conclusions: Our results demonstrate that T3 action on the early Purkinje cell dendritic differentiation process is mediated by RORalpha.

Here we show that gene expression of the nuclear receptor RORalpha is induced during adipogenesis, with RORalpha4 being the most abundantly expressed isoform in human and murine adipose tissue. Over-expression of RORalpha4 in 3T3-L1 cells impairs adipogenesis as shown by the decreased expression of adipogenic markers and lipid accumulation, accompanied by decreased free fatty acid and glucose uptake. By contrast, mouse embryonic fibroblasts from staggerer mice, which carry a mutation in the RORalpha gene, differentiate more efficiently into mature adipocytes compared to wild-type cells, a phenotype which is reversed by ectopic RORalpha4 restoration.

Fibrinogen is a plasma protein synthesized by the liver. It is composed of three chains (alpha, beta, gamma). In addition to its main function as a coagulation factor, this acute phase protein is also a risk marker for atherosclerosis. Retinoic acid receptor-related orphan receptor (ROR)alpha is a nuclear receptor modulating physiopathological processes such as cerebellar ataxia, inflammation, atherosclerosis, and angiogenesis. In this study, we identified RORalpha as a regulator of fibrinogen-beta gene expression in human hepatoma cells and in mouse liver. A putative RORalpha response element (RORE) was identified in the human fibrinogen-beta promoter. EMSA showed that RORalpha binds specifically to this RORE, and cotransfection experiments in HepG2 hepatoma cells indicated that this RORE confers RORalpha-dependent transcriptional activation to both the human fibrinogen-beta and the thymidine kinase promoters. Stable transfection experiments in HepG2 and Hep3B hepatoma cells demonstrated that overexpression of RORalpha specifically increases endogenous fibrinogen-beta mRNA levels. Chromatin immunoprecipitation experiments revealed that the fibrinogen-beta RORE is occupied by RORalpha in HepG2 cells. Thus, the human fibrinogen-beta gene is a direct target for RORalpha. Furthermore, fibrinogen-beta mRNA levels in liver and plasma fibrinogen concentrations are specifically decreased in staggerer mice, which are homozygous for a deletion invalidating the Rora gene. Taken together, these data add further evidence for an important role of RORalpha in the control of liver gene expression with potential pathophysiological consequences on coagulation and cardiovascular risk.

Retinoic acid-receptor-related orphan receptor (ROR) alpha is a nuclear receptor involved in many pathophysiological processes such as cerebellar ataxia, inflammation, atherosclerosis and angiogenesis. In the present study we first demonstrate that hypoxia increases the amount of Rora transcripts in a wide panel of cell lines derived from diverse tissues. In addition, we identified a functional promoter sequence upstream of the first exon of the human Rora gene, spanning -487 and -45 from the translation initiation site of RORalpha1. When cloned in a luciferase reporter vector, this sequence allowed the efficient transcription of the luciferase gene in several cell lines. Interestingly, the activity of the Rora promoter was enhanced by hypoxia in HepG2 human hepatoma cells, and this effect was dependent on an HRE (hypoxia response element) spanning from -229 to -225. Using electrophoretic-mobility-shift assays, we showed that HIF-1 (hypoxia-inducible factor 1), which plays a key role in the transcriptional response to hypoxia, bound to this HRE. Overexpression of HIF-1alpha increased the activity of the Rora promoter through the HRE. Overexpression of a dominant-negative form of HIF-1alpha producing transcriptionally inactive HIF-1alpha/HIF-1beta dimers abolished hypoxic activation of the Rora promoter. This indicated that HIF-1 is involved in the response of RORalpha to hypoxia. Taken together, our data reveal Rora as a new HIF-1 target gene. This illustrates, at the molecular level, the existence of cross-talk between signalling pathways mediated by HIF-1 and those mediated by nuclear receptors.

The rat alpha-fetoprotein ( afp ) gene is controlled by three enhancers whose function depends on their interaction with liver-enriched transcription factors. The afp enhancer III, located at -6 kb, is composed of three regions that act in synergy. Two of these regions, called s1 and s2, contain a putative binding site for hepatocyte nuclear factor-6 (HNF-6). This factor is the prototype of the ONECUT family of cut-homoeodomain proteins and is a known regulator of liver gene expression in adults and during development. We show here that the two splicing isoforms of HNF-6 bind to a site in the s1 region and in the s2 region. The core sequence of the s1 site corresponds to none of the known HNF-6 binding sites. Nevertheless, the binding properties of the s1 site are identical with those of the s2 site and of previously characterized HNF-6 binding sequences. The HNF-6 consensus should therefore be rewritten as DRRTCVATND. Binding of HNF-6 to the s1 and s2 sites requires both the cut and the homoeo domains, is co-operative and induces DNA bending. HNF-6 strongly stimulates the activity of the afp enhancer III in transient transfection experiments. This effect requires the stereo-specific alignment of the two HNF-6 sites. Moreover, HNF-6 stimulates the enhancer in synergy with the retinoic-acid-receptor-related orphan receptor alpha (RORalpha), which binds to a neighbouring site in the s1 region. Thus expression of the afp gene requires functional interactions between HNF-6 molecules and between HNF-6 and RORalpha.

The retinoic acid receptor-related orphan receptor alpha (RORalpha) is critically involved in many physiological functions in several organs. We find that the main RORalpha isoform in the mouse liver is the RORalpha4 isoform, in terms of both mRNA and protein levels, while the RORalpha1 isoform is less abundant. Because hypoxia is a major feature of liver physiology and pathology, we examined the effect of this stress on Rora gene expression and RORalpha transcriptional activity. HepG2 human hepatoma cells were cultured for 24 h under normoxia (20% O2) or hypoxia (10, 2, and 0.1% O2) and the abundance of the Rora transcripts measured by Northern blot and semi-quantitative RT-PCR. Hypoxic HepG2 cells contained more Rora mRNA than controls. This was also observed in rat hepatocytes in primary culture. Cobalt chloride and desferrioxamine also increased the amount of Rora mRNA in HepG2 cells. It is likely that these treatments increase the amount of the RORalpha4 protein in HepG2 cells as evidenced by Western blotting in the case of desferrioxamine. Transient transfection experiments indicated that hypoxia, cobalt chloride, and desferrioxamine all stimulate RORalpha transcriptional activity in HepG2 cells. Hence, we believe that RORalpha participates in the control of gene transcription in hepatic cells and modulates gene expression in response to hypoxic stress.

Hypoxia is an important component of many pathological processes including cancerogenesis and cirrhosis. We have attempted to identify additional hepatic genes sensitive to hypoxia by postulating that genes with possible binding sites for hypoxia inducible factor-1 (HIF-1) are regulated by hypoxia. A computer analysis identified the oncodevelopmental alpha-fetoprotein gene (afp) as one of them. The amounts of both alpha-fetoprotein mRNA and protein were decreased under hypoxic conditions in HepG2 hepatoma cells. Stability of afp mRNA was not altered, and de novo synthesis of proteins was required. Transfection experiments in HepG2 cells showed that both hypoxia and overproduction of HIF-1alpha specifically repressed the transcriptional activity of the rat afp regulatory region through the sequence 5'-CACGTGGG-3' located at -3625 to -3619. Mutation in this sequence strongly impaired these repressions. Interestingly, this sequence was a functional stimulatory target for c-Myc, suggesting that c-Myc regulates afp gene expression. Lastly, the amounts of c-myc mRNA and protein were reduced when these cells were grown under hypoxic conditions. Taken together, these results suggest the existence of a possible competition between HIF-1 and c-Myc that could modulate the transcriptional activity of the afp gene in response to hypoxia.

Expression of the oncodevelopmental alpha-fetoprotein (AFP) gene is tightly regulated and occurs in the yolk sac, fetal liver and intestine, and cancerous liver cells. Transcription of the AFP gene is under the control of three enhancers that are very tissue specific. We have shown that the most upstream of these enhancers, located at -6 kb, works through the combined action of liver-enriched factors and nuclear receptors that bind to three regions of this DNA regulatory element. This study showed that orphan nuclear receptors of the ROR alpha, Re-verb alpha, and Rev-erb beta groups can bind as monomers with high affinity and specificity to an evolutionarily conserved AGGTCA motif in the functionally important region 1 of this AFP enhancer. Transient transfection experiments performed with human HepG2 hepatoma cells showed that overproduction of ROR alpha 4 stimulated the activity of the AFP enhancer in a dose-dependent manner, while that of Rev-erb alpha and Rev-erb beta had the opposite effect. These effects were highly specific and required the integrity of the AGGTCA motif. The action of these nuclear receptors also occurred in the context of the entire 7-kb regulatory region of the rat AFP gene. These results suggest that altering the amounts or activities of these orphan receptors in cells of hepatic or endodermal origin could modulate AFP gene expression in response to a variety of developmental or carcinogenic stimuli.

The alpha-fetoprotein (AFP) gene is expressed mainly in the yolk sac, liver and intestine during embryonic and fetal life. We have analyzed the activities of some of the rat AFP regulatory elements in vivo, using transgenic mice bearing the LacZ gene with a nuclear localization signal (nls-lacZ) placed under the control of the rat AFP promoter and the most proximal enhancer regions (from -3,127 to +102). Four of the six transgenic lines, with two genetic backgrounds, had highly specific reproducible patterns of transgene expression on embryonic days E10.5, E12 and E15. Analyses were performed on the whole embryo and histologically. There was nuclear staining in the yolk sac endodermal cells and in the epithelial cells of the intestine, indicating that the proximal enhancer and promoter drive expression in these cells where the AFP gene is actively transcribed. The pharyngo-tympanic canal was also stained in the transgenic embryos. But there was no expression of the lacZ transgene in the embryonic liver, indicating that additional sequences of rat genomic DNA are required for correct expression in the liver., (Copyright 2000 S. Karger AG, Basel.)

The visceral yolk sac is a fetal membrane with essential placental functions. It is the major site of synthesis of alpha-fetoprotein (AFP), the most abundant plasma protein in the fetus. We developed a system of rat yolk sac explants in serum-free culture medium to study the regulation of endodermal gene expression in yolk sac. The explanted yolk sac tissues retained their double-sided morphology for up to 48 hours. The epithelial cells of both layers remained tightly joined on a basement membrane as seen by light and electron microscopy. This probably accounts for the continued expression of several endodermal cell-specific markers. The levels of mRNA encoding AFP, vitamin D-binding protein (DBP), hepatocyte nuclear factor 1alpha and beta transcription factors did not change during the 48-hour culture period. This reflects the stability of the differentiation state of the yolk sac endodermal cells. Dexamethasone and phorbol ester (TPA) specifically reduced the AFP mRNA level without affecting that of DBP. This suggests that these transduction pathways are functional in the yolk sac during this period of gestation and could be involved in the physiological down-regulation of AFP gene expression before birth. All these results show that this serum-free culture of rat yolk sac explants is a valuable system for further investigating the action of natural compounds and pharmacological drugs on endodermal gene expression during the embryonic and fetal periods.

The effects of phorbol esters (phorbol-12,13-dibutyrate, PDB) on alpha-fetoprotein expression and cell growth were assayed by using fetal hepatocytes in primary culture. PDB acts synergistically with epidermal growth factor (EGF) to specifically decrease alpha-fetoprotein (AFP) mRNA levels, without affecting the expression of other genes of the same family, such as albumin and Vitamin D-binding protein (DBP). This effect is PDB-dose dependent, maximal effects being at 10 ng/ml. The implication of protein kinase C (PKC) in this effect seems clear since bisindolylmaleimide (BIS), a specific PKC inhibitor, completely blocks the PDB effect on AFP expression. Nuclear run-on experiments show that the decrease in AFP mRNA levels is mainly due to an inhibition in the transcription rate of the gene. Determination of PKC activities shows that fetal hepatocytes contain mainly Ca(2+)-independent isoenzymes, which patterns of activation was not modified by EGF plus PDB treatment with respect to PDB treatment. We have found that MAPK and JNK activities, c-jun and c-fos mRNA levels and AP-1 binding activity are notably increased when cells are incubated with both EGF and PDB, PDB does not stimulate growth of fetal hepatocytes, measured either as [3H]-thymidine incorporation into DNA or by cell cycle analysis using flow cytometry. All these results suggest that activation of PKC may affect liver gene expression rather than cell growth in fetal hepatocytes.

We have further characterized the most distal of the three alpha-fetoprotein (AFP) enhancers required for expression of the AFP gene in fetal hepatocytes and yolk sac endodermal cells. Almost total rat AFP enhancer 3 (E3) activity is driven by a 160-bp fragment at -6 kb containing three target regions for nuclear proteins that cooperate to stimulate transcription from the AFP and the thymidine kinase promoters in HepG2 hepatoma cells. Region 1, recently shown to be crucial for correct function of the enhancer in liver of transgenic mice, is recognized by two sets of transcription factors that bind to partly overlapping sites, 1a and 1b, in a noncooperative and nonexclusive manner. Site 1a contains a motif, AGGTCA, which is recognized by chicken ovalbumin upstream promoter transcription factors (COUP-TFs), but not by hepatocyte nuclear factor 4. Hepatocyte nuclear factor 3 (HNF3) and CCAAT/enhancer binding protein (C/EBP), which bind to regions 2 and 3, respectively, are likely responsible for the liver-specific E3 action. They play a key role by acting in synergy. The participation of nuclear receptors such as COUP-TFs, with C/EBP and HNF3, in the tight control of the distal AFP enhancer is a new, and perhaps key, step toward understanding the regulation and function of this enhancer, which may remain active throughout development.

The oncodevelopmentally regulated alpha-fetoprotein (AFP) gene offers a very good model system to better understand the molecular mechanisms which dictate the specificity of gene expression in liver and control its tight modulation in the course of development and carcinogenesis. Transcription factors of the CCAAT/enhance-binding protein (C/EBP), hepatocyte nuclear factor-1 (HNF-1), and nuclear factor-1 (NF-1) families can bind in vitro to the promoter of the rat AFP gene, which makes the expression of the AFP gene specific to the liver. We have evaluated the influence of some of these factors on the activity of the AFP promoter by transfection of HepG2 hepatoma cells with the appropriate expression vector plus a CAT plasmid under the control of the AFP promoter. A similar plasmid bearing the rat albumin promoter was used as a control. C/EBP alpha, and C/EBP beta acted as transactivators on the AFP promoter, while LIP, a truncated form of C/EBP beta, was a potent negative regulator of the promoter. Interestingly, HNF-1 beta was found to be more potent than HNF-1 alpha in activating the AFP promoter in the HepG2 cells. This effect was highly promoter and cell specific since it did not occur with the rat albumin promoter or in Chinese hamster ovary cells. HNF-1 beta, which is produced earlier than HNF-1 alpha during liver development, would thus have the greater influence on the AFP promoter in early development. Our results pointed to a key role that NF1 might play in the functioning of the AFP promoter. Indeed, overexpression of NF1 induced a specific decrease in the activity of the AFP promoter. Competition between NF1 and HNF-1 for binding to their overlapping binding sites on the AFP promoter would be critical for modulating its activity.

The effects of a phorbol ester (TPA) and of members of the Jun and Fos oncoprotein family on the activity of the rat alpha-fetoprotein (AFP) promoter were checked by using transient expression experiments in HepG2 hepatoma cells. TPA blocked the activity of the rat AFP promoter in a dose-dependent manner. Overexpression of c-Jun specifically repressed the rat AFP promoter but not the albumin promoter. JunB and JunD were poorer inhibitors. c-Fos expression did not potentiate the negative effect of Jun. The Jun-induced repression does not require binding of c-Jun to the AFP promoter. DNase 1 footprinting experiments did not display any high affinity binding site for Jun on the AFP promoter. Integrity of the c-Jun DNA binding domain is not required for the c-Jun protein to block the AFP promoter. The N-terminal part of Jun, which contains the activating domain, is responsible for the repression as shown by using Jun-Gal4 chimera. Jun likely exerts its negative control on the AFP promoter via protein-protein interactions with a not yet identified trans-activating factor within the -134 to +6 region or with a component of the general machinery of transcription. Jun proteins can thus be key intermediates in regulatory cascades which result in the differential modulation of the AFP and albumin gene expression in the course of liver development and carcinogenesis.

The promoter of the rat alpha-fetoprotein (AFP) gene, which makes the expression of the developmentally regulated AFP gene specific to the liver, is a putative target for transcription factors of the CAAT/enhancer-binding protein (C/EBP), hepatocyte nuclear factor-1 (HNF-1) and nuclear factor-1 (NF-1) families. We have evaluated the influence of these factors on the activity of the AFP promoter by transfection of HepG2 hepatoma cells with the appropriate expression vector plus a CAT plasmid under the control of the AFP promoter. A similar plasmid bearing the rat albumin promoter was used as a control. C/EBP alpha, C/EBP beta and D-binding protein (DBP) acted as trans-activators on the AFP promoter, whereas liver inhibitory protein (LIP), a truncated form of C/EBP beta, was a potent negative regulator of the promoter. C/EBP alpha also bound to and stimulated the activity of the AFP enhancer at -2.5 kb. Interestingly, HNF-1 beta was found to be more potent than HNF-1 alpha in activating the AFP promoter. This effect was specific, as it did not occur with the rat albumin promoter. HNF-1 beta, which is produced earlier than HNF-1 alpha during liver development, would thus have the greater influence on the AFP promoter in early development. Both HNF-1s allowed expression of the AFP promoter in cells of nonhepatic origin. Overexpression of NF-1 induced a specific decrease in the activity of the AFP promoter. This strongly suggests that competition between NF-1 and HNF-1 for binding to their overlapping binding sites on the AFP promoter is critical for modulating its activity. Thus changing combinations of these trans-acting factors may tightly modulate the AFP promoter activity in the course of liver development and carcinogenesis.

Energy intake, weight gain, carcass composition, plasma fuels, hepatic metabolites and lipogenic enzyme activities were studied in adult rats fed either a low fat, high carbohydrate (LF) diet or one of two fat-containing diets in which 32% of the metabolizable energy was constituted by long-chain triglycerides (LCT) or medium-chain triglycerides (MCT). Compared with the LF diet, the MCT diet did not depress food and energy intake, weight gain, energy and nitrogen retention or lipid deposition and did not produce ketogenesis. The weight gain of rats fed LCT was 25% higher, and increased lipid deposition was observed. Lower lipogenic enzyme activities were observed in rats fed the LF diet containing 4% corn oil than in rats fed the MCT diet containing 1% corn oil. This effect disappeared when rats previously adapted to the LCT diet were fed LF or MCT diets containing 1% corn oil for 21 d. By d 21, in both groups, hepatic malic enzyme, ATP-citrate lyase, acetyl CoA carboxylase and fatty acid synthase activities were 2.2-, 2.0-, 2.3- and 1.8-fold higher than those of rats fed LCT. Intermediate hepatic glucose-6-phosphate dehydrogenase activities were observed in rats fed the MCT diet, compared with LCT (40% lower) and LF (1.6-fold higher) diets. These data show that in rats fed a diet in which MCT supplies 32% of metabolizable energy, a high activity of lipogenic enzymes is observed, suggesting that MCT had no inhibitory effect on the activity of these enzymes.

Food intake, plasma glucose, insulin (I) and glucagon (G), hepatic glycogen and fructose 2,6-bisphosphate (F-2, 6-P2) and liver glucokinase, glucose 6-phosphatase (G6-Pase), 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (6-PF-2 kinase/F-2, 6-P2ase), pyruvate kinase (PK-L) and phosphoenolpyruvate carboxykinase (PEPCK) activities were measured in 2 and 22-month-old rats before 3 d starvation and after 2, 4, 6, 24 and 48 h refeeding a high carbohydrate (HC, 74% w/w) diet. Expressed per 100 g of body weight, the food intake of old rats was 55% lower than that of young rats and the amount of carbohydrate absorbed hourly during the first 6 h of refeeding was 2.4-fold higher in young than in old rats. During the first 6 h of refeeding plasma glucose increased 2-fold and returned to normal values after 24 h in young rats, while plasma glucose did not change during refeeding in old rats. In young rats [I] fell by 85% after starvation and returned to normal values 2 h after refeeding. [I] was higher in old than in young rats; it decreased by 40% after starvation and returned to the basal value 4 h after refeeding. No marked changes were observed in plasma [G] in both groups. No difference was observed in hepatic glycogen in the two groups, while F-2, 6-P2 was higher in old than in young rats. In young rats, the opposite changes in liver glucokinase and G6-Pase activities occurring after starvation and during refeeding were

Food intake, plasma glucose, insulin (I) and triiodothyronine (T3) and liver glucose 6-phosphate dehydrogenase (G6P-DH), malic enzyme (ME). ATP-citrate lyase, acetyl-CoA carboxylase (AcCoACx) and fatty acid synthase (FAS) activities were measured in 2 and 22 months old rats before, after 3 d starvation and 2,4,6. 24 and 48 h refeeding a high carbohydrate (74% w/w) diet. Expressed per 100 g of body weight, the carbohydrate intake of old rats was 55% lower than that of young rats. Plasma insulin was higher in old than in young rats and decreased (-40%) after starvation and returned to control values 4 h after refeeding. In young rats plasma insulin fell after starvation (-85%) and returned to normal values 2 h after refeeding. No significant differences were observed in plasma [T3] between the two groups. During the first 6 h of refeeding, plasma glucose increased 2-fold and returned to control values after 24 h in young rats. In old rats, plasma glucose returned to its control value after 2 h. Compared to the starved level, 48 h after refeeding, G6P-DH, ME, ATP-citrate lyase, AcCoACx and FAS activities increased 5- to 6-fold in young rats, while in old rats the increase was much smaller and represented 35% of that observed in young rats. These results suggest, that the age-related reduction in inducibility of hepatic lipogenic enzymes of rats refed a high carbohydrate diet after starvation may be due to a spontaneous decrease in the carbohydrate intake and to a decrease effectiveness of insulin (insulin resistance).

Gluconeogenic enzymes and substrates were measured in the livers of fasted and suckled newborn pigs in the first 48 h postpartum. The activities at birth of glucose-6-phosphatase, fructose-1,6-diphosphatase, pyruvate carboxylase and phosphoenolpyruvate carboxykinase were, respectively, 70%, 45%, 117% and 35% of adult values. At birth, cytosolic phosphoenolpyruvate carboxykinase represented 35% of total activity, a similar distribution to that in the adult. In suckled piglets, all activities were greater at 24 and 48 h that at birth. In starved piglets, the increases were greater in all cases; the increase in cytosolic phosphoenolpyruvate carboxykinase was much more pronounced than for that for the particulate enzyme, with the former representing more than 50% of total at 48 h. The levels of gluconeogenic enzymes in the piglets in the early neonatal period would appear to be adequate for their needs and do not provide an explanation for their fasting hypoglycaemia. Hepatic levels of lactate, pyruvate, phosphoenolpyruvate, ketone bodies, and amino acids were determined in these piglets. No significant differences were observed in these metabolites between fasted and suckled animals except that glutamine was doubled in fed piglets, Evidence for the metabolic block in the livers of fasted animals was lacking and ketone bodies did not accumulate. These observations suggest that the limitations to gluconeogenesis result from unavailability of energy substrates and/or carbon precursors to the liver or the deficiency in their uptake.

Changes in hepatic levels of lactate, pyruvate, phosphoenolpyruvate (PEP), alpha-ketoglutarate, malate, oxaloacetate, ketone bodies, alanine, serine, glycine, aspartate, glutamate, glutamine, valine, urea, adenine nucleotides and inorganic phosphate were examined in rats consuming a high protein, carbohydrate-free diet for up to 40 days. While some components showed transient changes, others (pyruvate, malate, oxaloacetate, PEP, ketone bodies, alanine, glycine, glutamine, valine, urea, adenine nucleotides and inorganic phosphate) were permanently altered. The cytoplasmic and mitochondrial redox states were only transiently affected and by day 24 were not different from control values. In contrast, the cytoplasmic phosphorylation state was affected from day 1 on; this suggests a role for the latter in permanently reorienting metabolism toward gluconeogenesis and ureogenesis.

The effects of dietary protein on the metabolism of proteins, carbohydrates, and especially, lipids were investigated in genetically obese Zucker rats and their lean siblings. For 40 days the rats received diets containing 15%, 64%, or 82% protein, included at the expense of cornstarch. In the obese animals, the high-protein diets led to decreased food intake and weight gain. While these diets decreased the activities of lipogenic enzymes along with the lipid gain, they did not decrease the final body-fat content. The increase protein intake stimulated hepatic ureogenesis and gluconeogenesis. Lipolysis was stimulated, as demonstrated by an accumulation of ketone bodies in the liver. Blood levels of triacylglycerols, free glycerol, and nonesterified fatty acids were concomitantly decreased, which suggests an accelerated turnover of lipids. Whatever the composition of the diet, total energy retention of the lean rats was always less than that of the obese rats. The changes observed on high-protein diets were essentially the same for the two groups, except that the final body-content of lipids in the lean rats was significantly lower. In the absence of exogenous carbohydrate, the lean rats were barely able to retain nitrogen and to maintain hepatic lipogenesis. Unlike the rats from other strains, the lean Zucker rats could not adapt to a low-carbohydrate diet; this failure may be due to a metabolic disorder.

Pyruvate kinase activity and rates of gluconeogenesis and glycolysis in rat hepatocytes were evaluated by production of glucose and lactate + pyruvate from dihydroxyacetone during the first 48 hours after the shift from a low protein, high carbohydrate diet to a high protein, carbohydrate-free diet. The effect of glucagon was also studied. In the absence of glucagon, 11-17 hours after the dietary shift when glycogen was lowest, gluconeogenesis was maximal and glycolysis minimal. The concentration of fructose 1,6-biphosphate was high and did not change during the experiment. The activity ratio of pyruvate kinase measured with phosphoenolpyruvate (PEP) (V0.5 mM PEP/V4 mM PEP) was high in crude extracts and low in (NH4)2SO4-treated extracts, but remained unchanged during the whole experiment. There was no correlation of the rates of gluconeogenesis or glycolysis from dihydroxyacetone with the activity ratio of pyruvate kinase. With glucagon, gluconeogenesis from dihydroxyacetone was increased and a concurrent decrease in glycolysis was paralleled with a decrease in the fructose 1,6-bisphosphate concentration and in the activity ratio of pyruvate kinase. The activity ratio of pyruvate kinase in (NH4)2SO4-treated cells represented about 50% of that in the absence of the hormone. This difference may be related to glucagon-induced phosphorylation of pyruvate kinase.

Energy intake, weight gain, carcass composition, plasma hormones and fuels, hepatic metabolites and the activities of phosphoenolpyruvate carboxykinase (PEPCK), malic enzyme, and glucose 6-phosphate dehydrogenase (G6P-DH) were examined in adult rats during a 44-day period of low fat, high carbohydrate (LF) feeding or of consumption of one or two high (70% metabolizable energy) fat diets composed of 63% (metabolizable energy) long-chain (LCT) or medium-chain (MCT) triglycerides. Energy intake was similar in the LCT and MCT groups but was less than that of LF group. The weight gain of rats fed MCT diet was 30% less than that of rats fed LF or LCT diets. Energy retention was less when the diet provided MCT than LCT or LF, and that resulted in a 60% decrease in the daily lipids deposition. Plasma glucose, free fatty acids, glycerol, and insulin/glucagon ratio were similar in the three groups. Blood ketone body (KB) concentrations in rats fed the high fat diets were extremely elevated, particularly in the MCT group, but declined throughout the experiment and by the 44th day hyperketonemia decreased by 50% but remained higher than in the LF diet. The blood beta-hydroxybutyrate/acetoacetate (B/A) ratio remained slightly elevated in rats fed the high fat diets. Similar changes were observed in liver KB concentration and in the B/A ratio. Liver lactate/pyruvate ratio elevated in the LCT and MCT groups at the initiation of the diets decreased by 50% at the end of the experiment. The consumption of high fat diets led to a 1.5-fold increase in liver PEPCK activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Gluconeogenesis from dihydroxyacetone (DHA), glycerol, lactate, pyruvate or alanine was studied in the absence or in the presence of glucagon in hepatocytes isolated from starved rats or from rats fed a high protein diet for 2-48 h. In both groups, gluconeogenesis from DHA, glycerol, lactate and pyruvate exhibited similar changes over 48 h; the rates of glucose production increased progressively until 24 h and then plateaued. During the early phase (2-11 h), gluconeogenesis from DHA and glycerol were higher than gluconeogenesis from lactate and pyruvate. During the first 24 h of the experiment, gluconeogenesis from alanine displays a kinetic similar to that from lactate or pyruvate. After feeding a high protein diet for 24 to 48 h, gluconeogenesis from alanine was slightly higher than that in starved rats and paralleled the increase in alanine aminotransferase activity. Glucagon stimulated gluconeogenesis from DHA up to 48 h, but with glycerol this effect occurred only during the early phase (2-11 h). Glucagon stimulated gluconeogenesis from lactate, pyruvate or alanine by 1.35-fold throughout the experimental period. These findings suggest that the development of gluconeogenesis during starvation or after feeding a high protein diet displays different kinetics, depending on the substrate used and on the level of entry in the gluconeogenic pathway: triose phosphates or pyruvate.

Diurnal variation in hepatic levels of lactate, pyruvate, phosphoenolpyruvate (PEP), alpha-ketoglutarate, malate, oxaloacetate, ketone bodies, alanine, serine, glycine, aspartate, glutamate, glutamine, valine, urea, adenine nucleotides and inorganic phosphate were studied in rats adapted to a high protein, carbohydrate-free diet for 24 days. Most circadian rhythms differed in relation to controls (10% protein diet); many merely had different amplitudes, some were inverted, and some exhibited drastically altered patterns. Cytoplasmic redox state exhibited nearly similar variations and phosphorylation state differed primarily in amplitude whereas mitochondrial redox state was highly depressed in the absorptive phase. The metabolic regulation implied by the results is discussed in relation to both circadian variations of plasma insulin and glucagon concentrations, and pyruvate kinase and phosphoenolpyruvate carboxykinase activities previously reported.

The activities of glucose 6-phosphate dehydrogenase (EC 1.1.1.49), malic enzyme (EC 1.1.1.40), ATP-citrate lyase (EC 4.1.3.8), acetyl-CoA carboxylase (EC 6.4.1.2) and fatty acid synthetase were lower (-25 to -60%) in liver of rats fed during 45 days with a moderate long-chain triglycerides (LCT) content diet (32% of metabolizable energy, ME), than in control rats fed with a low fat diet (LCT, 10% of ME). However, the fall in malic enzyme activity was not significant. In contrast, these activities were higher (+40 to +160%) in rats fed with a diet with a moderate medium-chain triglycerides (MCT) content (32% of ME), than in control rats. Nevertheless, the increase in activity of malic enzyme and ATP-citrate lyase was more important. Contrary to LCTs, MCTs had no inhibitory effect on the activity of enzymes involved in hepatic lipogenesis.

Changes in hepatic levels of lactate, pyruvate, phosphoenolpyruvate, alpha-ketoglutarate, malate, oxaloacetate, adenine nucleotides, inorganic phosphate, ketone bodies, alanine, serine, glycine, aspartate, glutamate, valine and urea were examined in adult rats during the first 24 h of either starvation or consumption of a high protein (HP) diet. No differences were found between these two conditions in the concentration of metabolites studied or the cytosolic redox state. Under both conditions, the cytosolic phosphorylation state decreased to a low 15 h into the experiment but the changes were more pronounced on the HP diet. Hepatic ketone bodies rose sharply after 12 h, with the increase 2.5 times greater for starved rats. In starvation, hepatic aspartate, valine, and urea were low and glycine was high, whereas the opposite was seen for the HP diet. In both groups, alanine fell within 9 h and remained low thereafter. These findings suggest that, in the first 24 h of starvation, the energy necessary for gluconeogenesis is obtained from fatty acid oxidation, while during HP feeding the energy for both gluconeogenesis and ureagenesis are derived from fatty acid oxidation and amino acid oxidation.

Plasma insulin (I), glucagon (G) and glucose, hepatic glycogen, fructose 2, 6-bisphosphate (F2, 6-P2), fructose 1, 6-bisphosphate, phosphoenolpyruvate, and some liver key enzymes involved in glycolysis (6-phosphofructo-2-kinase/fructose-2, 6-bisphosphatase (6-PF-2kinase/F-2,6-P2ase), activity ratio (velocity at suboptimal substrate concentration/maximum velocity) of pyruvate kinase (PK-L] and in gluconeogenesis (phosphoenolpyruvate carboxykinase activity) have been compared in young (2 months) and old (16 months) rats upon starvation or transition to a high protein (HP) diet. In the 10 and 24 hours after the dietary switch, plasma glucose decreased less and hepatic glycogen was less depleted in the old rats. The ratios of plasma I/G and of hepatic 6-PF-2kinase/F-2,6-P2ase were higher in the old rats and their decrease delayed at both time points, as was the concentration of hepatic F-2,6-P2 and the activity ratio of PK-L (before and after removal of endogenous noncovalent factors). The consistency of these differences indicate that the mechanisms for control of glycolysis/gluconeogenesis are similar in young and old rats, but it appears that in old rats starved or fed HP diet, the switch from glycolysis to gluconeogenesis is delayed. This suggests that as a result of the slowness of the hormonal changes the process of phosphorylation/dephosphorylation, which is so important in the short-term regulation of the glycolysis/gluconeogenesis pathway, may be impaired with age.

Pertinent hepatic metabolites and enzymes were examined in rats fed a high carbohydrate (HC) diet and during the first 24 h of either starvation or feeding a high protein (HP) diet. Consumption of the HC diet induced slight but definite 24-h oscillations in hepatic concentrations of cyclic AMP, glycogen, glucose 6-phosphate, fructose 2,6-bisphosphate, fructose 1,6-bisphosphate and phosphoenolpyruvate, as well as the activities of 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase and phosphoenolpyruvate carboxykinase. The transition to starvation or the HP diet induced, within 12 h, concurrent increases in cyclic AMP and phosphoenolpyruvate and decreases in glycogen, glucose 6-phosphate, fructose 6-phosphate, fructose 2,6-bisphosphate and fructose 1,6-bisphosphate. These changes were associated with a decrease in the ratio of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase and an increase in phosphoenolpyruvate carboxykinase. These results suggest that the activity of the fructose 6-phosphate/fructose 1,6-bisphosphate cycle is similar during the first 24 h of starvation or HP consumption.

Plasma hormones, glucose and free fatty acids, liver glycogen and two key enzymes of glycolysis and gluconeogenesis were examined in adult rats during a 40-day period of high protein feeding. Plasma insulin fell within 1 day but returned to normal after 4 days. Glucagon changed more slowly, reaching a maximum on day 4 and declined to near the control value within 24 days. Consequently, the insulin to glucagon ratio was lower on days 1, 4 and 8 and was nearly normal on day 24. With respect to hepatic enzymes, phosphoenolpyruvate carboxykinase activity rose sharply on the 1st day and remained elevated for 40-day period; the L-isozyme of pyruvate kinase, although unchanged on the 1st day, decreased thereafter and from day 8 on represented 15--20% of control. Circadian variations in these parameters were also measured in rats adapted to the high protein diet. In such animals, the diurnal change in plasma hormones was less marked but tended to be inverted with respect to controls; the insulin/glucagon ratio was highest during daylight on high protein and in late night on the control diet. Over 24 hours, pyruvate kinase activity was related directly and phosphoenolpyruvate carboxykinase inversely to the hormone ratio. We concluded that in rats adapted to high protein, as in controls, the diurnal balance between glycolysis and gluconeogenesis is probably regulated by the same factor, namely the insulin/glucagon ratio.

The rates of glucose production from various substrates entering gluconeogenesis at different steps were investigated in hepatocytes isolated from term-fetus and newborn rabbits fasted during the first 2 days of life. The data were compared to the rate of glucose production measured in hepatocytes from young rabbits (50-60 days) starved for 48 h. The net production of glucose from substrates (lactate, pyruvate, propionate, alanine) entering gluconeogenesis below phosphoenolpyruvate was very low at birth and increased during the first day of life, in relation with an increased cytosolic phosphoenolpyruvate carboxykinase activity. The net production of glucose from precursors entering gluconeogenesis at the level of triose phosphates (dihydroxyacetone, fructose) was low at birth but a maximal capacity for gluconeogenesis was reached within 6 h after birth. This enhanced gluconeogenic capacity was associated with a fall in hepatic fructose 2,6-bisphosphate concentration and a reduced glycolytic flux. In contrast, a high glucose production from galactose was already present at birth and did not rise at 24 or 48 h after delivery. These results suggest that the development of gluconeogenic capacity in hepatocytes isolated from newborn rabbit is dependent upon two factors, a decrease in the F2,6-P2 concentration which reduces the glycolytic flux and an increase in the activity of cytosolic phosphoenolpyruvate carboxykinase.

The effects of protein levels and types of fat in the diet on the metabolism of lean and obese Zucker rats were studied. For 40 days the rats were fed ad libitum one of four diets: two "usual protein" diets (19% protein by weight) with 19.4% triacylglycerols, either long chain (UP-LCT diet) or medium chain (UP-MCT diet); and two high protein (64% protein), carbohydrate-free diets, again with 19.4% triacylglycerols (HP-LCT and HP-MCT diets, respectively). The energy intakes of the obese rats decreased about equally on the HP-LCT, UP-MCT, and HP-MCT diets. The daily weight gain, which was high in the UP-LCT rats, was lower when carbohydrates were replaced by proteins, or when LCTs were replaced by MCTs; furthermore, when these two changes were made together, their beneficial effects on body weight were additive. The lipid gain, too, was high with the UP-LCT diet and lower both with the high protein diets and with the MCT diets; again combining the two amplified the two individual effects, so much that the final lipid concentration in the body was lowered, whereas the concentration of water increased. Hepatic acetyl CoA carboxylase activity was low when the diet supplied plenty of LCTs, but replacing carbohydrates with proteins in such a diet produced an additional decrease in this enzymatic activity. When either a normal protein or a high protein diet supplied MCTs in place of LCTs, acetyl CoA carboxylase activity was high and similar to that found with a high carbohydrate diet.(ABSTRACT TRUNCATED AT 250 WORDS)

Newborn guinea-pigs delivered by Caesarean section at term were fasted for 48h at 35 degrees C and compared with suckling neonates. For the first 18h of fasting, blood glucose concentration remains at a normal level: a rapid liver glycogenolysis contributes to glucose production but indirect evidence suggests also that active gluconeogenesis from lactate, alanine and glycerol occurs at this time. Despite high liver triacylglycerol levels at birth and increased plasma free fatty acids the blood ketone body concentration remains very low which suggests that free fatty acid availability is not the only factor controlling the onset of ketogenesis at birth. After 24h of fasting, blood glucose concentration decreases whereas suckling newborns remain normoglycaemic. A mild hypoglycaemia is observed in fasting newborn which is not related to an unavailability of fat-derived substrates since mobilization of fat stores induces an increase of plasma free fatty acid levels and high blood concentration of ketone bodies. This relative hypoglycaemia is probably related to the lack of glucose and/or gluconeogenic substrate intake strengthened by an unavailability of some endogenous gluconeogenic precursors since a lack of a mobilization of protein stores characterizes this period. The maintenance of glucose homeostasis in suckling neonates reflects an active gluconeogenesis; a theoretical calculation shows an important contribution of each milk constituent including amino acids to sustain an endogenous glucose production.

Changes in plasma glucose, hepatic cyclic AMP, glycogen and fructose 2,6-bisphosphate (F-2,6-P2), and liver 6-phosphofructo-2-kinase (6-PF-2kinase), fructose 2,6-bisphosphatase (F-2,6-P2ase) and phosphoenolpyruvate carboxykinase (PEPCK) activities were examined in rats fed a low protein, high carbohydrate (HC) diet during 3 d of either starvation or feeding a high protein, carbohydrate-free (HP) diet. Under both HP feeding or starvation, liver cyclic AMP increased after 1 d and remained constant thereafter. Whereas plasma glucose was low during starvation, it was unaffected by HP feeding. In both experimental groups, liver glycogen fell after 1 d; thereafter it remained low on starvation, but increased progressively on HP diet reaching 70% of the HC-fed rats value on day 3. Under both experimental conditions, F-2,6-P2 fell 85% after day 1 and was unchanged thereafter. One day after the start of starvation or consumption of the HP diet, 6-PF-2kinase decreased, F-2,6-P2ase increased and 6-PF-2kinase/F-2,6-P2ase ratio decreased, but changes were significantly more important with the HP diet than with starvation. PEPCK activity increased in both experimental conditions, but the increase was greater on the HP diet than on starvation. These findings suggest that during the first 3 d the adaptative response of hepatic gluconeogenesis is higher with a HP diet than upon starvation.

Adult male rats were force-fed either a single dose of 200 mg of DL-methionine or an amino acid mixture with the pattern of casein. The animals were anesthetized with pentobarbital at 0, 1.5, 3 and 4 hours after intubation, and samples of liver were removed by a freeze-clamping technique at 0, 15 and 30 seconds of ischemia. Hepatic ATP, ADP, AMP, Pi, glucose-6-phosphate, glucose and certain nitrogenous compounds as well as plasma glucose were determined. After methionine was given by intubation, hepatic methionine and glutathione increased severalfold within 1.5 hours; cystathionine showed a transient rise at this time but returned to normal at 3 and 4 hours, when taurine was progressively increasing. Several nonessential amino acids decreased, suggesting that they may be utilized for energy. Methionine force-feeding did not modify the concentration of hepatic adenine nucleotides and probably did not change their turnover, as estimated from changes during ischemia. The level and production of Pi during ischemia was increased however. After force-feeding the amino acid mixture, hepatic methionine, cystathionine and taurine were unaffected, and glutathione increased only at hours 3 and 4; glycine and threonine were elevated by 1.5 hours. Hepatic adenine nucleotides, inorganic phosphate and glucose were not significantly affected by the feeding of amino acids by intubation.