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Concentrations of 28 trace elements (Li, Mg, Al, V, Cr, Mn, Fe, Co, Ni, Cu, Zn, Ga, As, Se, Rb, Sr, Mo, Ag, Cd, In, Sn, Sb, Cs, Tl, Hg, Pb, and Bi) in the livers of juvenile and adult American alligators inhabiting two central Florida lakes, Lake Apopka (LA), and Lake Woodruff National Wildlife Refuge (LW) and one lagoon population located in Merritt Island National Wildlife Refuge (MINWR; NASA), were determined. In juveniles from MINWR, concentrations of nine elements (Li, Fe, Ni, Sr, In, Sb, Hg, Pb and Bi) were significantly higher, whereas six elements (V, Fe, As, Sr, Hg and Bi) were elevated in adults (p<0.05) obtained from MINWR. Significant enrichment of some trace elements in adults, relative to juveniles, was observed at all three sampling areas. Specifically, Fe, Pb and Hg were significantly elevated in adults when compared to juveniles, suggesting age-dependent accumulation of these elements. Further, As, Se and Sn showed the same trend but only in animals collected from MINWR. Mean Fe concentrations in the livers of adults from LA, LW and MINWR were 1770 μg g(-1) DW, 3690 μg g(-1) DW and 5250 μg g(-1) DW, respectively. More than half of the adult specimens from LW and MINWR exhibited elevated hepatic Fe concentrations that exceed the threshold value for toxic effects in donkey, red deer and human. These results prompted us to express our concern on possible exposure and health effects in American alligators by some trace elements derived from NASA activities., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Reversed feeding uncouples peripheral and master clock gene rhythms and leads to an increased risk of disease development. The aim of this study was to determine the effects of clock gene uncoupling on sepsis-induced inflammation using a mouse cecal ligation and puncture (CLP) model. C57BL/6N mice were entrained to a 12-h light-dark cycle (lights on at 7 AM). Mice were permitted ad libitum feeding either during the night (7 PM-7 AM) or the nonphysiological light phase (7 AM-7 PM) for a week before CLP. In daytime-fed mice, phase inversion of clock gene expression was observed in the liver, but not in the suprachiasmatic nucleus. Daytime-fed mice also had decreased body weight and food intake. Survival rate was significantly lower in daytime-fed mice (29%) compared with nighttime-fed mice (54%) 72 h after CLP (P = 0.03). Serum levels of interleukin 6 (IL-6), tumor necrosis factor α, high mobility group box 1, IL-1α, IL-9, eotaxin, and granulocyte colony-stimulating factor increased in daytime-fed mice compared with nighttime-fed mice after CLP. Baseline expression levels of sirtuin peroxisome 1 and proliferator-activated receptor γ coactivator 1α in the liver decreased in daytime-fed mice compared with nighttime-fed mice. Thus, daytime feeding induces clock gene uncoupling, which leads to decreased expression of longevity-related and energy metabolism proteins. Daytime feeding may also increase the levels of inflammatory cytokines, thereby increasing mortality in a mouse sepsis model. Our findings suggest that uncoupling of peripheral and master clock gene rhythms by reversed feeding exacerbates inflammatory responses.

Livers from wild pufferfish, Takifugu rubripes, can be described as having a smooth frontal side and an upper-region that is attached to the hepatic portal vein. Based on this description, the liver can be divided into 10 parts (L1-5 and R1-5), and in this work, the lethal potency of each part was determined by mouse bioassay. Among the raw livers from 58 individuals, all 10 parts of 16 individuals, and some parts of 4 individuals showed mouse lethality, but no toxicity was detected in any part of the liver from 22 individuals. In the livers of 4 individuals that were partially toxic, the lethal potency of the toxic parts was less than 4 mouse units (MU)/g, and no part showed especially high toxicity. The remaining 16 individuals were considered non-toxic based on the assay of only one part. Among 13 frozen livers, all parts of 9 individuals were toxic, while all parts of 4 individuals were non-toxic. Liquid chromatography-mass spectrometry analysis revealed that all parts of a weakly toxic raw liver and a strongly toxic frozen liver had tetrodotoxin as the major toxin. Regarding the 16 raw and 9 frozen livers, whose parts were all toxic, the relative lethal potency of each part to the mean lethal potency of the individual (8.9-709 MU/g) was calculated, and subjected to a two-way analysis of variance with 2 factors (left/right and top/bottom) for each group of livers (raw or frozen). The analysis indicated non-significance among factors and interactions at a significance level of 5% in the frozen livers. However, in the raw livers, although no interaction between the 2 factors was detected, the right-side and the 4th-portion from the top were significantly higher than the left-side and the other portions, respectively. Therefore, we concluded that individual inspection using R4, which is the region that lies below the right-center location of the liver, is suitable for toxicity evaluation of liver to ensure the safe consumption of pufferfish.

Background: An important component of postoperative management includes alleviation of hepatic ischemia-reperfusion (I/R) injury, which commonly results from liver surgery. EPC-K1 is a hydroxyl radical scavenger reported to have mitigating effects on I/R injury in many organs. This study evaluates the effects of EPC-K1 on hepatic I/R injury., Materials and Methods: Rats were injected subcutaneously with either EPC-K1 (100 mg/kg) or saline. The hepatic artery and left branch of the portal vein were clamped for 45 min under general anesthesia. Indicators of liver function, including aspartate aminotransferase (AST), alanine aminotransferase (ALT), and lactate dehydrogenase (LDH), and of liver tissue damage were evaluated after 6h and 24h of reperfusion. Serum levels of tumor necrosis factor (TNF)-α, interleukin (IL)-6, and high-mobility group box 1 (HMGB1) protein were measured, and apoptosis was quantified via caspase 3/7 activity and TUNEL assay., Results: AST, ALT, and LDH levels increased significantly as a result of hepatic I/R injury, but were attenuated by EPC-K1 administration. Histologic findings revealed that normal structure of the hepatic parenchyma was maintained in rats pretreated with EPC-K1. TNF-α, IL-6, and HMGB1 levels rose significantly after reperfusion, together with activation of the inflammatory response. However, EPC-K1 administration suppressed levels of inflammatory markers and attenuated the inflammatory response. Moreover, EPC-K1 administration prevented apoptosis as determined by inhibition of caspase 3/7 activity and a decrease in apoptotic cells., Conclusions: Results demonstrate that EPC-K1 inhibits the inflammatory response and suppresses apoptosis during hepatic I/R injury. This suggests that EPC-K1 has hepatoprotective effects, and may be a valuable and novel therapeutic agent in the clinical setting., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Autophagy is a natural process by which a cell maintains homeostasis, usually taking place unnoticed by adjacent cells. Glucose is involved in a negative feedback loop in autophagy. Autophagy is characterized by the induction and secretion of HMGB1, yet the nature of the inflammatory response during and the effect of glucose administration on autophagy are not well understood. Systemic inflammation was induced in experimental animals by LPS injection (7.5 mg/kg) followed by a continuous infusion of either 1%, 5%, or 25% glucose. Autophagy was visualized by immunohistochemistry 12 h after LPS injection. Likewise, protein levels of microtubule-associated protein light chain 3 (LC3)-II, autophagy-related protein 7 (Atg7), and high-mobility group box 1 (HMGB1) were assayed by western blot analysis. We found that autophagy increased in liver tissue in response to LPS-induced systemic inflammation. However, protein levels decreased in rats receiving LPS and a 5% glucose solution. Our results suggest that LPS-induced systemic inflammation increases autophagy in liver cells, potentially involving the upregulation of LC3-II, Atg7, and HMGB1. We also show that a 5% glucose infusion reduces autophagy. We propose that maintaining serum glucose levels with an adequate glucose dose improves systemic inflammation by reducing autophagy.

Background and Aims: Ischemia/reperfusion (I/R) injury is characterized by significant oxidative stress, which induces characteristic changes in the antioxidant system and organ injury leading to significant morbidity and mortality. The aim of this study was to evaluate the protective effect of dihydrolipoyl histidinate zinc complex (DHLHZn) on oxidative damage after severe hepatic I/R injury., Methods: Thirty male Wistar rats were subjected to 45 min of hepatic ischemia by clamping of the hepatic artery and portal vein, followed by a 6-h reperfusion period. DHLHZn (10 mg/kg) (I/R + DHLHZn group) or saline (I/R group) was administered intraperitoneally twice, 30 min before ischemia and at the beginning of the reperfusion. Sham-operated animals (sham group) received equal amounts of saline. The rats were killed at the end of the reperfusion period. Serum levels of aspartate aminotransferase and alanine aminotransferase were determined, and histological examination and oxidative stress were evaluated in liver tissues. In addition, antimycin A-stimulated RAW264.7 cells (murine macrophage-like cells) were treated with DHLHZn to estimate its antioxidant effect., Results: Serum aspartate aminotransferase and alanine aminotransferase levels were increased in the I/R group, but these increases were significantly inhibited in the I/R + DHLHZn group. Similarly, liver tissue damage observed in the I/R group was attenuated in the I/R + DHLHZn group. Cells treated in vitro with both DHLHZn and antimycin A showed reduced reactive oxygen species activity compared to cells treated with antimycin A alone., Conclusion: The new antioxidant DHLHZn may have potential for therapeutic application in liver I/R injury, although this is a limited animal study., (© 2011 Journal of Gastroenterology and Hepatology Foundation and Blackwell Publishing Asia Pty Ltd.)

This study was undertaken to elucidate the effects of dietary protein deprivation on glucose metabolism and hepatic insulin signaling in rats. The results of glucose and pyruvate tolerance tests in rats fed with a 12% casein diet (12C) and a protein-free diet (PF) indicated that protein deprivation enhanced clearance of blood glucose and suppressed gluconeogenesis. Correspondingly, the mRNA level of hepatic phosphoenolpyruvate carboxykinase, a key gluconeogenic enzyme, was suppressed by dietary protein deprivation. In PF-fed rats, total tyrosine phosphorylation of insulin receptor (IR) in the liver induced by insulin injection was enhanced compared with 12C pair-fed rats due to an increase in IR protein level. In addition, protein deprivation caused an increase in protein levels of IR substrate 1 (IRS1) and IRS2, leading to the marked enhancement of insulin-induced tyrosine phosphorylation of IRS2 and its binding to the p85 regulatory subunit of phosphatidylinositol 3-kinase (PI3K). Based on these results, we conclude that protein deprivation suppresses gluconeogenesis by a mechanism primarily mediated by the enhancement of the insulin signals through the IR/IRS/PI3K/mammalian target of rapamycin complex 1 pathway in the liver. Taken together with our previous report, these findings suggest that tissue-specific potentiation of insulin action in the liver and the skeletal muscle plays important roles in maintaining glucose homeostasis even when energy usage is reduced by dietary protein deprivation.

The dysregulated metabolism associated with diabetes mellitus (DM) impairs membrane trafficking events in the liver, including the process of autophagy, which is an essential ongoing cellular process that is highly regulated by nutrients, endocrine factors, and signaling pathways. High-mobility group box 1 (HMGB1) is a nuclear protein with a known role in systemic inflammation and the related various organ injuries. However, its relationship to autophagy is not well understood. The aim of this study was to investigate the effects of inflammation injury on autophagy in the liver in a rat model of DM. DM was induced in animals with streptozotocin, followed four weeks later by induction of inflammation by LPS injection. At 12 h after LPS administration, autophagy was assessed by immunohistochemistry and Western blot analysis of microtubule-associated protein light chain 3 (LC3)-II, as well as transmission electron microscopy. Expression of HMGB1 was also examined by immunohistochemistry and Western blot analysis. Western blot analysis of liver tissue revealed that levels of LC3-II and HMGB1 protein increased in DM rats subjected to LPS-induced inflammation compared with non-DM rats. Autophagy was particularly enhanced in DM rats. Thus, autophagy might be related to progression to organ injury in patients with DM, and inflammation in these patients might be associated with over-induction of autophagy and increased HMGB1 expression.

Attempts were made to examine the effect of paralytic shellfish poisoning toxins (PSP) on hepatic xenobiotic-metabolizing enzymes (XMEs) of tiger puffer (Takifugu rubripes). Two groups of nontoxic tiger fish were analyzed, and one group was fed with a PSP-containing diet (PSP group), and another with a PSP-free diet (control group). After 60 days of feeding, they were compared to each other mainly in terms of the activity of XMEs. Both groups did not differ from each other significantly in body weight gain, hepatosomatic index, and condition factor Hepatic level of cytochrome P450 was lower in PSP group than control group. NADPH-cytochrome P450 reductase, NADH-cytochrome b5 reductase, and ethoxyresorufin-O-deethylase (EROD) exhibited a reduced activity in PSP group than control group. Statistical analysis found that the activity or concentration of those enzymes correlated with the hepatic level of PSR with r2=0.497-0.611.

Dose- and time-dependent effects of 2,3,7,8-tetrabromodibenzo-p-dioxin (TBDD) on the liver were examined by single administration of TBDD by gavage to male and female rats. Fifteen Wistar rats of each sex per group received 0, 10, 30, 100 or 300 microg TBDD/kg body weight. Rats surviving to scheduled necropsy on Day 2, 7 or 36 after the TBDD administration were examined for hepatic histopathology, activities of hepatic microsomal enzymes and serum levels of lipids, total cholesterol and transaminases and hepatic concentrations of TBDD. Tigroid basophilic cytoplasm and hepatocellular hypertrophy were observed at 10 microg/kg on Day 2 or 7 through 36, whereas degenerative and aggressive lesions such as necrosis, fibrosis, multinucleated hepatocytes and disarrangement of hepatocytes occurred later at higher dose levels. Persistently increased activities of hepatic aryl hydrocarbon hydroxylase (AHH), ethoxycoumarin O-deethylase (ECOD) and ethoxyresorufin O-deethylase (EROD), increased serum levels of total cholesterol and phospholipid and increased relative liver weight were observed in all groups dosed 10 mug/kg and above, suggesting that hepatic microsomal monooxygenases and basophilic cytoplasm of hepatocytes were early and sensitive indicators among those TBDD-induced effects. A dose-dependent increase in liver concentrations of TBDD on Day 2 was followed by logarithmic decreases in TBDD concentrations against the days elapsed after the TBDD administration. An elimination half-life (t(1/2)) of TBDD from the liver was estimated to range from 12 to 16 days. It was suggested that females were more susceptible to TBDD than males, and that acute hepatotoxicity of TBDD was as potent as that of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD).

Background/aims: The plasminogen activator (PA)-plasmin system is primarily involved in fibrinolysis, but is also in the patho/physiological events in which breakdown of extracellular matrices is evoked topically. In this present study, we examined the expression of fibrinolytic factors, tissue-type PA (t-PA) and Type 1 PA inhibitor (PAI-1), in acute liver injury., Methods: Acute liver injury was produced in rats by the intraperitoneal administration of carbon tetrachloride (CCl(4)). Plasma aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities were measured to verify the hepatocellular damage. t-PA and PAI-1 gene expressions were measured by Northern blotting, and the cell type(s) expressing these genes was identified by in situ hybridization. t-PA and PAI-1 levels were measured by enzyme-linked immunosorbent assay (ELISA)., Results: A single intraperitoneal administration of CCl(4) caused severe acute parenchymatous liver injury. Both t-PA and PAI-1 gene expressions were induced by the acute liver injury, and plasma t-PA and PAI-1 concentrations were also increased. In situ hybridization studies demonstrated that the hepatocytes were the cells expressing t-PA and PAI-1 genes during the acute liver injury. t-PA was also augmented in the bile, whereas PAI-1 was decreased there., Conclusions: t-PA and PAI-1 gene expressions are induced in the hepatocytes of rats with acute liver injury. These fibrinolytic factors induced by liver injury may play important roles in liver regeneration.

Allantoinase and allantoicase are known to form a complex in amphibian liver. In this study, a new type of allantoinase that did not form a complex with allantoicase was found in the amphibian liver. Purified enzyme had a molecular mass of about 44 kDa both in SDS-PAGE and gel-filtrations. The enzyme cross-reacted with anti-sardine allantoinase polyclonal antibody, and it weakly cross-reacted with anti-bullfrog allantoinase polyclonal antibody.

The effects of dietary restriction of a single essential amino acid (EAA) on insulin-like growth factor-I (IGF-I) and IGF-binding protein (IGFBP)-1 were investigated in rats. Rats were fed experimental diets containing amino acid (AA) mixtures in which the concentrations of all EAA were at levels recommended by the National Research Council (control), in which a single EAA was restricted to 20% of that of the control diets (Leu(-), Lys(-), Met(-) or Thr(-)), or in which the diet was devoid of amino acids (AA(-)). To eliminate the effect of differences in energy intake, rats were fed the mean amount of food as consumed by the AA(-) group on the previous day. Growth was significantly retarded in rats fed diets restricted in just one EAA compared with that of rats fed the control diet, and further growth retardation was observed in rats fed the AA(-) diet. On the other hand, the plasma IGF-I concentrations in the groups with a single EAA restriction or in the AA(-) group were 66% (P: < 0. 05) and 50% (P: < 0.05) of that of the control group, respectively. The effect of any single EAA restriction was not significantly different from that of total AA deprivation. The plasma IGFBP-1 concentration in the control group did not differ from that of rats fed diets with the single EAA restrictions except for methionine restriction, but it was approximately 6-fold greater in the AA(-) group. Differences in plasma IGFBP-1 concentration under these conditions could be explained by differences in hepatic IGFBP-1 mRNA contents. Based on these results, we conclude that restriction of single EAA does not affect IGFBP-1 synthesis in vivo, although the deprivation of a single EAA has been reported to increase IGFBP-1 production in hepatocyte cultures. Our results also indicated that a single EAA restriction decreased IGF-I production but did not affect IGFBP-1 production. The present study suggests that not only plasma IGF-I, but also IGFBP-1, affects the magnitude of growth retardation in vivo.

Protein metabolism of growing animals is greatly affected by quantity and nutritional quality of dietary proteins. When animals are fed diets that contain enough proteins of good nutritional quality, they grow well. However, if they are fed diets deficient in protein or in some essential amino acids, their growth rate is markedly depressed. In this paper, we review the response of plasma insulin-like growth factor-I (IGF-I) to quantity and nutritional quality of dietary proteins. The sound correlation between plasma IGF-I concentration and the gain or loss of body proteins under various nutritional conditions suggests that the plasma IGF-I most possibly regulates the growth rate of animals or the rate of whole body protein synthesis. The quantity and nutritional quality of dietary proteins also regulates plasma concentration of IGF-binding proteins (IGFBPs). The changes in plasma concentration of IGFBPs presumably modifies the activity of IGF-I to regulate whole body protein synthesis. Molecular mechanisms of the changes in plasma concentrations of IGF-I and IGFBPs as affected by dietary proteins are also reviewed.

Disruption of the mouse Hex gene resulted in embryonic lethality around embryonic age (E) 10.5, due to no substantial liver formation. Expression of albumin was detectable in heterozygous (Hex(+/-)) but not in homozygous (Hex(-/-)) [corrected] embryos at E8.5. Instead of liver bud formation at E9.5, a liver-like capsule structure was observed in Hex(-/-) [corrected] embryos. In Hex(-/-) [corrected] mutant liver, we found no hepatocytes but no signs of apoptotic cell death in the area. Expression of transcription factors involved in hepatocyte differentiation, hepatocyte nuclear factor (Hnf)3beta, Hnf6, Hnf4alpha and Hnf1alpha, were restricted to the capsule and internal matrix-like structure in the mutant liver and expression of a subset of these factors were reduced. Hematopoiesis of monocytes was impaired in mutant embryos while erythroid lineage was unaffected. These results indicate that Hex plays an essential role in progenitor cells which commit to the hepatic endoderm and in the hematopoietic differentiation of the monocyte lineage., (Copyright 2000 Academic Press.)

To investigate the molecular mechanisms of increased transcription of the insulin-like growth factor-binding protein-1 (IGFBP-1) gene in dietary protein-deprived animals, the cis-acting sequence that is involved in this regulation was analyzed. We first showed that IGFBP-1 gene transcription was up-regulated by amino acid deprivation in cultured liver cell lines: H4IIE and HuH-7. Since HuH-7 cells showed a greater increase in IGFBP-1 mRNA in response to amino acid deprivation, this cell line was used in further experiments. Using a promoter function assay, we found that up-regulation of promoter activity responding to amino acid deprivation was abolished by deleting the region between -112 and -81 bp from the cap site from the gene construct. This cis-acting region includes the insulin-responsive element (IRE) and glucocorticoid responsive element (GRE) of IGFBP-1. In summary, the present observation suggests that the 32-bp (-112 to -81) in the IGFBP-1 gene 5' promoter region is involved in the induction of the IGFBP-1 gene in response to amino acid deprivation.

When two copies of the sequences spanning -57 to -35 of the fatty acid synthase (FAS) or -64 to -41 of the ATP citrate-lyase (ACL) gene linked to a reporter gene were transfected into primary cultured hepatocytes, the reporter activities significantly increased in response to insulin/glucose treatment. In cotransfection experiments of the FAS(-57/-35) with the Sp1 or Sp3 expression vector, the reporter activities of transcription were suppressed by Sp1 and stimulated by Sp3. In the cotransfection experiments of ACL(-64/-41), the activities were suppressed by Sp1 but were unchanged by Sp3. A similar effect of Sp1 and Sp3 on transcription was seen in mRNA concentrations and enzyme activities of endogenous FAS and ACL. Moreover, the mRNA concentrations and enzyme activities of endogenous acetyl-CoA carboxylase were suppressed by Sp1 and greatly increased by Sp3. Gel mobility super shift assays using antibodies against Sp1 or Sp3 revealed the binding of the transcription factors Sp1 and Sp3 with the GC rich regions located within FAS(-57/-35) and ACL(-64/-41) genes. The formation of DNA-protein complexes was decreased in rats fed a high-carbohydrate diet in comparison with that in fasted rats, but feeding the corn oil diet inhibited this decrease. In Western immunoblotting assay, however, the amount of Sp1 and Sp3 remained unchanged in the dietary conditions. Therefore, the binding of DNA-protein complexes was not due to changes in the amount of Sp1 and Sp3 but to changes in the binding activity, suggesting that these transcription factors may be an important determinant of lipogenic enzyme expression.

Type 1 plasminogen activator inhibitor (PAI-1), a major physiological inhibitor of plasminogen activation, is an important component of the hepatic acute phase response. We studied the acute phase regulation of murine hepatic PAI-1 in response to systemic toxicity and local tissue injury in both wild-type mice and in mice in which the interleukin (IL)-1beta gene had been inactivated by gene targeting. Endotoxin induced plasma PAI-1 antigen levels and PAI-1 mRNA accumulation in liver to the same extent in both wild-type and IL-1beta-deficient mice. In contrast, turpentine increased plasma PAI-1 and hepatic PAI-1 mRNA accumulation in wild-type mice but not in IL-1beta-deficient mice. Intraperitoneal injection of murine IL-1beta rapidly increased plasma PAI-1 and hepatic PAI-1 mRNA in both wild-type and IL-1beta-deficient mice. These results suggest that IL-1beta is a critical inducer of hepatic PAI-1 gene expression during the acute phase response to local tissue injury. In situ hybridization studies revealed that hepatocytes are the cells primarily responsible for the hepatic expression of the PAI-1 gene induced by lipopolysaccharide and turpentine.

We have shown that dexamethasone stimulates insulin-like growth factor binding protein-1 (IGFBP-1) production due to an increase in IGFBP-1 mRNA content in primary cultures of rat hepatocytes. In the present study, we investigated the effect of dexamethasone on the stabilization of IGFBP-1 mRNA in this system. We found that dexamethasone stabilized IGFBP-1 mRNA from the dot blot analysis with actinomycin D in the culture medium. It is suggested that the stabilization of IGFBP-1 mRNA is one of the mechanisms in which IGFBP-1 mRNA increases during dexamethasone treatment in primary cultures of rat hepatocytes.

Effects of dietary protein restriction and ovariectomy on plasma concentrations and hepatic messenger RNA (mRNA) of insulin-like growth factor-I (IGF-I) and insulin-like growth factor binding proteins (IGFBP) were investigated in young female rats. Ovariectomy increased plasma IGF-I concentration in rats fed on either a 50 g casein/kg diet (protein-restricted diet) or a 200 g casein/kg diet (control diet), but it increased IGF-I mRNA in liver only in the rats fed on the control diet. On the other hand, by Western ligand blot analysis, we observed that ovariectomy increased plasma IGFBP-3 concentration, and decreased plasma IGFBP-4 concentration. Ovariectomy did not affect IGFBP-1 and IGFBP-2 mRNA in liver, but dietary protein restriction significantly increased them, which may correspond to their plasma concentrations. The present results show that ovarian hormones and dietary protein content affect the plasma concentrations of IGF-I, IGFBP-3 and IGFBP-4 and hepatic mRNA of IGF-I, IGFBP-1, IGFBP-2, IGFBP-3 and IGFBP-4 in different manners.

Insulin stimulates tyrosine phosphorylation of 175-195 kDa proteins including insulin receptor substrate-1 (IRS-1) in various tissues and cell types. In intact rat livers, starvation increased the insulin-dependent tyrosine phosphorylation of the insulin receptor and IRS-1 as has been described by others. Surprisingly, starvation greatly increased the tyrosine phosphorylation of the 195-kDa protein induced by insulin, indicating that this protein may be a new substrate of the insulin receptor kinase. The marked increase in tyrosine phosphorylation of the 195-kDa protein may have a physiological role in signal transmission in response to insulin under starvation conditions.

The binding of insulin to its receptor rapidly induces intrinsic insulin receptor tyrosine kinase activity, resulting in tyrosine phosphorylation of various cytosolic substrates, such as insulin receptor substrate-1 (IRS-1) which, in turn, associates with a p85 subunit of phosphatidylinositol 3-kinase (PI 3-kinase) followed by activation of this enzyme. In the present study, we have examined these early steps of insulin signalling in rat liver in vivo after food ingestion. After fasting for 22 h, a 12% casein diet was available ad libitum throughout the 8-h experimental period. Plasma insulin concentrations increased within 45 min after feeding, reached a maximum at 1.5 h and gradually decreased until 8 h. Autophosphorylation of the insulin receptor beta-subunit in liver was detected even during fasting and increased about 1.5-fold at 1.5 h after feeding. Basal tyrosine phosphorylation of IRS-1 was detectable during starvation, increased about twofold at 3 h after feeding and levels were maintained until 8 h. The content of the p85 subunit of PI 3-kinase associated with IRS-1 also increased after feeding in parallel with the changes in tyrosine phosphorylation of IRS-1. Because tyrosine phosphorylation of the insulin receptor beta-subunit and IRS-1 and the association of the p85 subunit of PI 3-kinase with IRS-1 in liver were closely correlated with the changes in the plasma concentration of insulin, we concluded that endogenous insulin secreted in response to eating caused these insulin-dependent intracellular changes in the liver.

We previously mapped the sequences responsive to insulin/glucose stimulation and polyunsaturated fatty-acid suppression in the proximal promoter region between positions -104 and -20 of the ATP citrate-lyase (ACL) gene [Fukuda, H., Iritani, N., Katsurada, A. & Noguchi, T. (1996) FEBS Lett. 380, 204-207]. To investigate further the regulatory DNA sequences required for stimulation and suppression of this gene, primary cultured hepatocytes were transfected with plasmids containing the 5'-flanking sequences of the rat ACL gene fused to the chloramphenicol acetyltransferase (CAT) gene. When two copies of the sequences spanning -64 to -41 (linked to ACLcat20) were used for transfection, CAT activity significantly increased in response to insulin/glucose treatment. This increase was inhibited by addition of polyunsaturated fatty acid. Mutational analysis of this region showed that sequences between -55 and -51 are essential for recognition and interaction with trans-acting factors. Gel mobility shift assays using the sequence from -64 to -41 as a probe revealed nuclear factor(s) from rat liver that specifically complexed with the sequences. In addition, by antibody supershift assays, we have detected the binding of the transcriptional factor Sp1 at the G+C-rich region located within -64 to -41 of the ACL promoter. On the other hand, the formations of DNA-protein complexes with Sp1 binding site or ACL(-64 to -41) were decreased in rats fed a high-carbohydrate diet in comparison with those in rats fasted or fed a polyunsaturated fatty-acid-rich diet. Cotransfection studies in rat hepatocytes, with the Sp1 expression vector and ACLcat constructs, showed the inactivation of the promoter. These results demonstrated that the region from -64 to -41 of the ACL gene was responsible for stimulation due to insulin/glucose, the stimulation was suppressed by polyunsaturated fatty acid, and Sp1 may be involved in the regulation.

The present studies were undertaken to determine whether the interaction between cAMP-dependent and insulin-dependent pathways in primary cultures of rat hepatocytes affects biological functions and tyrosine phosphorylation. Quiescent hepatocytes were pretreated with dibutyryl cAMP or cAMP-generating agents such as glucagon, and then treated or not with insulin. Preincubation for 6 h with dibutyryl cAMP or glucagon enhanced the effect of insulin on DNA synthesis, but not the effect of insulin on amino acid transport or glycogen and protein synthesis. Tyrosine phosphorylation of intracellular proteins was determined by immunoblot analysis using an anti-phosphotyrosine antibody. Maximum tyrosine phosphorylation of a 195 kDa protein, which may be a substrate of insulin receptor kinase, of 175-180 kDa proteins, including insulin receptor substrate (IRS)-1, and of 90-95 kDa proteins, including the insulin receptor beta-subunit, was reached within 30 s of incubation with insulin. Pretreatment for about 3 h with dibutyryl cAMP or cAMP-generating agents clearly increased insulin-dependent tyrosine phosphorylation of the 195 kDa protein, but not IRS-1, IRS-2 or the insulin receptor beta-subunit. Because dibutyryl cAMP and cAMP-generating agents did not increase insulin receptor number or its kinase activity, the effect of cAMP on this potentiation of tyrosine phosphorylation is assumed to be exerted at a step distal to insulin receptor kinase activation. The potentiation by cAMP pretreatment of insulin-stimulated tyrosine phosphorylation may in part be secondary to inhibition of phosphotyrosine phosphatase activity, because cAMP pretreatment blunted the effect of Na3VO4 on the net tyrosine phosphorylation of the 195 kDa protein as compared with cells pretreated with no additive. In summary, the interactions between cAMP-dependent and insulin-dependent pathways that lead to augmentation of DNA synthesis appear to parallel the changes in tyrosine phosphorylation. Further studies will be required to determine whether there is a causal relationship between these phenomena.

We previously mapped the sequences responsive to insulin/glucose stimulation and polyunsaturated fatty acid (PUFA) suppression in proximal promoter region from -57 to -35 of fatty acid synthase (FAS) gene of rat liver [Fukuda et al. (1996) Biochem. Mol. Biol. Int. 38, 987-9961. When two copies of the sequences spanning -57 to -35 were linked to a reporter gene containing heterologous promoter and were used for transfection, the reporter activity significantly increased in response to insulin/glucose treatment in hepetocytes. This increase was inhibited by addition of PUFA. Gel mobility shift assays using the sequence from -57 to -35 as a probe revealed nuclear factor(s) from rat liver that specifically complexed with the sequences. In addition, by antibody supershift assays, we have detected the binding of the transcriptional factor Sp1 at the GC-rich region located within -57 to -35 of the FAS promoter. Cotransfection studies in rat hepatocytes, with the Sp1 expression vector and FAScat constructs, showed the inactivation of the promoter. These results were similar to those for the region from -68 to -52 of FAS gene (an insulin response element). The region from -68 to -52 of FAS gene competed for the formation of DNA-protein complexes to the region from -57 to -35 in the gel shift assay. Mutational analysis showed that the overlapping region of these two sequences was essential for the binding of Sp1. It has been demonstrated that both the regions from -57 to -35 and from -68 to -52 of the FAS gene are responsible for regulation due to insulin/glucose and PUFA, and Sp1 may be involved in the regulation.

In marine fish liver, degradative enzymes able to convert purines to urate have been shown to be located in the cytosol and degradative enzymes able to convert urate to urea and glyoxylate in the peroxisomes. The end products of purine degradation are urea and glyoxylate in fish. Glyoxylate may be converted to glycine by alanine:glyoxylate aminotransferase for the reutilization of purine carbons. The present report describes that hepatic alanine:glyoxylate aminotransferase is located both in the peroxisomes and in the mitochondria in fresh water fish, showing that the intracellular localization of the enzyme differs between fresh water fish and marine fish. This is the first report on the presence of alanine:glyoxylate aminotransferase in fish peroxisomes.

Hepatic cystathionine beta-synthase activity was increased in rats fed a high-protein diet for three or five days as compared to rats given laboratory chow (day 0). In the low-protein diet group, the enzyme activity decreased after both feeding periods. Similar changes in the cystathionine beta-synthase mRNA levels were observed in the rats fed these diets. The changes in cystathionine beta-synthase activity caused by alteration of the dietary protein content were mainly due to changes in the mRNA levels.

Background: Few studies have made direct comparisons of the metabolic effects of growth hormone (GH) and insulin-like growth factor 1 (IGF-1). We have assessed the dose-dependent effects of GH and IGF-1 treatments on nitrogen metabolism, intestinal structure, and hepatic IGF-1-messenger RNA (mRNA) expression in postoperative parenterally fed rats., Methods: Rats were maintained on total parenteral nutrition (TPN) for 3 days after gastrectomy. GH (0.4 or 0.8 IU/kg/d) or IGF-1 (1,2, or 4 mg/kg/d) was infused throughout the experimental period. Anabolic effects of GH and IGF-1 were assessed by body weight change, nitrogen excretion, and whole-body protein turnover. Organ weights, intestinal structure, plasma IGF-1 levels and hepatic IGF-1-mRNA contents were also determined., Results: Both GH and IGF-1 attenuated body weight loss and nitrogen excretion and increased whole-body protein synthesis and spleen weight. These observations suggest that the anabolic effects of 1 mg/kg/d of IGF-1 were equivalent to those of 0.66 IU/kg/d of GH. IGF-1, but not GH, reduced atrophy of the intestinal mucosa. GH treatment increased hepatic IGF-1-mRNA and the plasma IGF-1 level, whereas IGF-1 treatment increased the plasma IGF-1 level with no change in the hepatic IGF-1-mRNA content., Conclusions: Administration of GH or IGF-1 attenuates catabolism after surgery. The anabolic effects of 1 mg/kg/d of IGF-1 are equivalent to those of 0.66 IU/kg/d of GH. IGF-1 reduces intestinal mucosal atrophy. GH increases hepatic IGF-1-mRNA and the plasma IGF-1 level.

A quantitative study of the effect of carnitine deficiency on expression of glycolytic and gluconeogenic enzymes was performed using juvenile visceral steatosis mice which are systemically deficient in carnitine. The amounts of glucokinase and L-type pyruvate kinase mRNA were reduced in homozygotes, compared to heterozygotes and normal controls at 2 and 8 weeks. Liver-type phosphofructokinase, however, did not differ significantly. The abundance of fructose 1,6-bisphosphatase mRNA was unchanged at 2 and 8 weeks. The level of phosphoenolpyruvate carboxykinase mRNA was increased slightly at 2 weeks, but not at 8 weeks. A part of these changes could not be explained by the plasma glucose or insulin level. Carnitine administration restored the mRNA of these enzymes to normal levels. These results suggest that carnitine deficiency affects the expression of these liver enzymes.

Expression of key regulatory enzymes involved in glucose metabolism was studied in the livers of Otsuka Long-Evans Tokushima fatty (OLETF) rats, a model of non-insulin dependent diabetes mellitus. The activity and mRNA levels of glucokinase and L-type pyruvate kinase was increased in the liver of OLETF rats compared with control rats. There was no such remarkable change in liver-type phosphofructokinase. The activities of glucose-6-phosphatase and fructose-1,6-biphosphatase also increase despite high plasma levels of glucose and insulin. The activity of phosphoenolpyruvate carboxykinase did not show any significant change. The mRNA levels for fructose-1,6-biphosphatase, and phosphoenolpyruvate carboxykinase exhibited no marked changes. These results suggest that the expression of glucose-6-phosphatase and fructose-1,6-biphosphatase is disordered in OLETF rats.

The regulation of gene expression of glucokinase (GK) and L-type pyruvate kinase (L-PK) in rat liver was investigated and compared with the previously reported regulation of lipogenic enzymes. Experiments were conducted in which the time courses and responses to diet quantity of mRNA concentrations and enzyme activities after refeeding a carbohydrate/protein diet (CP) to food-deprived rats were measured. The effects of dietary nutrients on the gene expression were investigated in rats refed either the CP diet, a carbohydrate diet without protein (C), a protein diet without carbohydrate (P), or a carbohydrate/protein/corn oil diet (CPF). The effects of the CPF diet on the gene expression after insulin treatment to diabetic rats were also investigated. After refeeding the CP diet, GK mRNA concentration and enzyme activity reached maximum levels in 2 h and 16-24 h, respectively, whereas those of L-PK peaked in 16 h and 48 h, respectively, similar timecourse to lipogenic enzymes. Moreover, GK mRNA concentrations were maximal in rats fed 20% of the ad libitum diet intake, and L-PK mRNA concentrations, like lipogenic enzyme mRNA, were maximal in rats fed approximately 50% of ad libitum intake. GK mRNA concentrations were significantly increased in parallel with an increase in plasma insulin and glucose concentrations. GK and L-PK mRNA and enzyme levels in rats fed the C diet were comparably induced to the levels in those fed the CP diet. L-PK mRNA induction by the CP diet was significantly reduced by dietary polyunsaturated fatty acids (CPF diet), whereas the GK mRNA induction was not significantly reduced.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of the production of insulin-like growth factor (IGF)-I, IGF-II, IGF binding proteins (IGFBPs), and their related proteins by various hormones was investigated in primary cultures of rat liver parenchymal and nonparenchymal cells. Freshly isolated parenchymal cells contained mRNAs of IGF-I, IGF-II, IGFBP-1, IGFBP-4, growth hormone (GH) receptor, and the acid-labile subunit (ALS), which forms a ternary complex with IGF-I and IGFBP-3; however, parenchymal cells did not express the IGFBP-3 gene. In contrast, nonparenchymal cells contained IGFBP-3 mRNA exclusively, as we reported previously [Takenaka et al. Agric. Biol. Chem., 55, 1191-1193 (1991)]. Cultured rat parenchymal cells produced IGF-I, IGFBP-1, and IGFBP-4 prominently. In these cells, secretion of IGF-I and the content of IGF-I mRNA was greatly increased in the presence of GH in the medium. Insulin also increased the production of IGF-I. Secretion of IGFBP-1 into the medium was enhanced by treatment with glucagon, dibutyrylcyclic AMP (Bu2cAMP), and dexamethasone (Dex) and these enhancements with glucagon and Dex reflected the increase in its mRNA content. Insulin depressed the secretion of IGFBP-1. The content of IGFBP-4 in the parenchymal cells was increased by insulin, Bu2cAMP, and triiodothyronine (T3), thereby enhancing the production of IGFBP-4 and secretion into the medium. Cultured liver nonparenchymal cells of rats produced IGFBP-1, IGFBP-3, and IGFBP-4. Secretion of IGFBP-1 was increased by Bu2cAMP in the medium, that of IGFBP-3 by IGF-I, and that of IGFBP-4 by both IGF-I and Bu2cAMP. Regulation of the production of IGFBP-3 by IGF-I was demonstrated in these investigations. These results suggest that GH increases production of IGF-I in the parenchymal cells and this IGF-I, in turn, increases the production of IGFBP-3 in nonparenchymal cells. As we found GH also increases ALS production in parenchymal cells, by these mechanisms, GH increases the formation of the ternary complex of IGF-I, IGFBP-3, and ALS. This study clearly demonstrates the interrelationship between parenchymal and nonparenchymal cells in the production of IGF-I and IGFBPs in the liver.

Hepatic cystathionine beta-synthase activity is decreased by the addition of cysteine to the diet. This effect of cysteine was slightly greater in diets containing 0.25% methionine than in those containing 1% methionine, and was reduced during aging. Similar changes were observed in the level of the mRNA of this enzyme, although the changes in the transcript levels were slightly greater than the changes in enzyme activity. Thus, we conclude that the addition of cysteine to a methionine-containing diet causes a decrease in cystathionine beta-synthase activity mainly by diminishing its mRNA level.

Novel erythrocyte pyruvate kinase gene defects were found in a patient without a family history of consanguinity. The polymerase chain reaction products of the R-type pyruvate kinase cDNA from the propositus contained two point mutations of Ser80 (TCC)-->Pro (CCC) and Arg490 (CGG)-->Trp (TGG). Allele-specific polymerase chain reaction of the genomic DNA revealed that this patient was a compound heterozygote. The mobilities of the patient's L- and R-type pyruvate kinase by thin-layer polyacrylamide gel electrophoresis were abnormal. The results are consistent with the fact that these mutations are within exons common to the hepatic and erythrocyte isozymes.

In chicken liver, alanine:2-oxoglutarate aminotransferase was located only in the mitochondria. In 40-day-old chickens, starvation resulted in a dramatic increase of liver mitochondrial alanine:2-oxoglutarate aminotransferase activity, reaching about a 100-fold increase in the activity (units/g of liver) on Day 7 of starvation. The mitochondrial alanine:2-oxoglutarate aminotransferase was purified to homogeneity and characterized from the mitochondrial extract of 7-day-starved chicken liver. The enzyme possessed alanine:glyoxylate aminotransferase activity and was also present in control chicken liver. The enzyme was found in the present study for the first time and named alanine:2-oxoglutarate (glyoxylate) aminotransferase. Alanine:2-oxoglutarate aminotransferase specific for alanine and 2-oxoglutarate as substrates was not detected in both control and starved chicken livers. In contrast, liver mitochondrial alanine:2-oxoglutarate aminotransferase from mammals did not possess alanine:glyoxylate aminotransferase activity. The increase in mitochondrial alanine:2-oxoglutarate aminotransferase activity during starvation was found to be attributed to an increase in enzyme protein.

The chain of enzymes necessary to convert uric acid to its metabolic products urea and glyoxylic acid in vertebrates is truncated through the successive loss of allantoicase, allantoinase, and urate oxidase during phylogenetic evolution. Previous studies have assigned the localization of both urate oxidase and allantinase to the peroxisome in the amphibian liver. This study reports the cloning of a cDNA encoding bullfrog (Rana catesbeiana) allantoinase, an enzyme that converts allantoin to allantoic acid. The cDNA is 2112 base pairs in length containing a 1449-base pair open reading frame which corresponds to a 483-residue protein (53,296 Da). Structural analysis of the deduced protein suggested two potential transmembrane segments and the presence of a putative mitochondrial localization sequence in the amino terminus. Immunocytochemical analysis revealed that allantoinase is localized to mitochondria and not to peroxisomes. On Northern blotting, a single mRNA species was detected in the liver and kidney of frog but not in other tissues; this distribution was confirmed by immunoblotting. The hepatic- and renal-specific expression of allantoinase coincides with the distribution of urate oxidase in these tissues in the frog. The allantoinase expressed in Saccharomyces cerevisiae and in Spodoptera frugiperda (Sf9) insect cells exhibits catalytic activity and is antigenically identical to the native frog enzyme.

On the basis of differential and density gradient centrifugation studies, the site of the uric acid degrading enzymes, urate oxidase and allantoinase, in amphibia was previously assigned to the hepatic peroxisomes. Using specific antibodies against frog urate oxidase and allantoinase, we have undertaken an immunocytochemical study of the localization of these two proteins in frog liver and kidney, and demonstrate that whereas urate oxidase is present in peroxisomes, allantoinase is localized in mitochondria. Urate oxidase and allantoinase were detected by immunoblot analysis in both frog liver and kidney. The subcellular localization of these two enzymes was ascertained by Protein A-gold immunocytochemical staining of Lowicryl K4M-embedded tissue. Peroxisomes in frog liver parenchymal cells and kidney proximal tubular epithelium contained a semi-dense subcrystalloid core, which was found to be the exclusive site of urate oxidase localization. Allantoinase was detected within mitochondria, but not in peroxisomes of hepatocytes or proximal tubular epithelium. No allantoinase was detected in the mitochondria of nonhepatic parenchymal cells in liver and of the cells lining the distal convoluted tubules of the kidney. These results demonstrate that, unlike rat kidney peroxisomes which lack urate oxidase, peroxisomes of frog kidney contain this enzyme. Contrary to previous assumptions, these studies also clearly establish that urate oxidase and allantoinase, the first two enzymes involved in uric acid degradation, are localized in different subcellular organelles in frog liver and kidney.

To study the regulatory mechanism of pyruvate kinase M gene transcription, we analyzed its chromatin structure and cis-acting DNA regions. Two DNase-I-hypersensitive sites were detected in dRLh-84 hepatoma cells, but not in hepatocytes, which coincides with expression of the M gene in the two types of cells. These sites, designated HS2 and HS1, were located around the major transcription start site and about 2.9 kb downstream from this site, respectively. A transient chloramphenicol acetyltransferase expression assay indicated that the region around HS1 did not show any activity, whereas the upstream region up to -457 had promoter activity in hepatoma cells. Most of this activity was lost by a 5'-deletion from -286 to -225. Further analysis identified a cluster of three cis-acting regions from -279 to -216, which are named boxes A, B and C. These regions did not have any independent effect, but the inclusion of all regions were synergistic. These regions were not active in hepatocytes, suggesting that they have cell-type specificity. A gel mobility shift assay indicated that unidentified, but distinct, nuclear proteins bound to the three boxes. These results suggest that transcriptional regulation of the M gene involves alteration of chromatin structure and binding of proteins to three cis-acting elements.

The regulation by insulin and carbohydrates of the gene expression of three key enzymes involved in glucose metabolism was studied in the liver of the Wistar fatty rat, a model of obese non-insulin-dependent diabetes mellitus. A high glucose or fructose diet, or insulin administration caused a similar magnitude of increase in the level of L-type pyruvate kinase mRNA in the liver of Wistar fatty rats and their lean littermates. However, the induction of glucokinase mRNA and repression of phosphoenolpyruvate carboxykinase mRNA by dietary glucose or insulin were impaired in the fatty rats, whereas fructose caused a similar decrease in phosphoenolpyruvate carboxykinase mRNA in both types of rats. These results indicate that the regulation of gene expression of glucokinase and phosphoenolpyruvate carboxykinase, but not of L-type pyruvate kinase, by insulin is impaired in the liver of the Wistar fatty rat.

Effect of quantity and nutritional quality of dietary proteins on the content of mRNA of insulin-like growth factor-binding protein-1 (IGFBP-1) was studied in rat liver and kidney. IGFBP-1 mRNA content per unit RNA increased in liver and kidney of rats fed on a protein-free diet and in those of fasted rats compared with that in the rats fed on a casein diet. When rats were given a gluten diet for 7 d, IGFBP-1 mRNA content in liver did not change significantly but that in kidney increased considerably compared with that in those organs of the rats fed on the casein diet. Because IGFBP-1 mRNA has been demonstrated both in liver parenchymal and non-parenchymal cells (Takenaka et al. 1991), the effect of the protein-free diet on these two types of cells has been studied. An increase in IGFBP-1 mRNA content under protein deprivation was observed in both liver parenchymal and non-parenchymal cells, suggesting that these two types of cells are regulated in a similar mode as far as IGFBP-1 mRNA content is concerned. The physiological and nutritional significance of the previously stated results on protein anabolism are discussed when considered together with our previous observations on the plasma concentrations of IGF-1 (Takahashi et al. 1990) and IGFBP (Umezawa et al. 1991) and insulin-like growth factor-1 mRNA content in liver (Miura et al. 1991).

The effects of dexamethasone (Dex) on insulin-like growth factor (IGF)-I and IGF binding protein (IGFBP)-1 production were investigated in primary cultures of rat hepatocytes. Dex enhanced the secretion of IGFBP-1 as measured by ligand blot analysis but did not show any prominent effect on immunoreactive IGF-I secretion. EC50 of Dex on IGFBP-1 secretion was calculated to be 3 x 10(-8) M. The content of IGFBP-1 mRNA in the cells increased greatly in the presence of Dex but the IGF-I mRNA content did not change significantly under the same conditions. Insulin showed the opposite effect of Dex by decreasing the production of IGFBP-1 and the cellular content of IGFBP-1 mRNA. This effect of insulin was observed also with Dex in the medium. These results show that the gene expression of IGF-I and IGFBP-1 is differently regulated by glucocorticoids and insulin in primary cultures of rat hepatocytes. The results most possibly explain the in vivo effects of glucocorticoids and insulin in regulation of IGF-I and IGFBP-1 production by liver.

Key enzymes related to lipogenesis in the liver are induced by a high glucose diet or insulin and suppressed by starvation, diabetes, or glucagon. Most of these enzymes are also induced by dietary fructose, even in diabetic liver. This regulation occurs at the posttranscriptional level as well as at the transcriptional level. We studied extensively the molecular mechanism of induction of L-type pyruvate kinase (LPK). The transcription of the LPK gene in the liver was stimulated by insulin and inhibited by glucagon. This insulin action required ongoing protein synthesis and metabolism of glucose and was enhanced by glucocorticoid. On the other hand, the mechanism of induction of the LPK by dietary fructose depended on plasma insulin levels. Dietary fructose stimulated transcription of the LPK gene in normal rats, whereas it acted mainly at the posttranscriptional level in diabetic rats. These fructose effects were attributable to a common metabolite of fructose and glycerol. The induction of LPK mRNA by dietary glucose was impaired in the liver of Wistar fatty rats, a model of obese non-insulin-dependent diabetes mellitus, but fructose-induced accumulation of the mRNA was not. Studies on transgenic mice indicated that the 5'-flanking region up to -3 kb of the LPK gene contained all cis-acting elements necessary for tissue-specific expression of LPK and its stimulation by diets and insulin. Further analysis using a transient expression assay revealed the presence of three cis-acting elements necessary for expression of LPK in hepatocytes in the region up to -170 kb. However, these elements alone were not sufficient for dietary and hormonal regulation of this enzyme when analyzed in transgenic mice.

Effects of quantity and quality of dietary proteins on plasma immunoreactive insulin-like growth factor-1 (IGF-1) concentration, and content of IGF-1 mRNA in rat liver were investigated in rats. Plasma immunoreactive IGF-1 concentration in rats given a casein diet was higher than that in rats given a soya-bean-protein or protein-free diet. The IGF-1 mRNA content in liver was estimated by the Northern blot hybridization technique employing 32P-labelled rat IGF-1 complementary DNA (cDNA). At least four molecular species of IGF-1 mRNA of different molecular weight were found in rat liver. The sizes were 0.8-1.2, 2.0, 3.6-4.0 and 7.4 kb. Most of the mRNA species decreased in the livers of rats given a gluten diet (120 g gluten/kg diet) compared with rats given the casein diet. In particular, mRNA of 7.4 kb decreased markedly. When rats were fed on the protein-free diet, mRNA of all species decreased significantly. The estimated IGF-1 mRNA in the livers of rats fed on the gluten or protein-free diet was almost 0.4 of that of the rats given the casein diet. Feeding the soya-bean-protein diet did not result in a marked effect on the hepatic content of mRNA species of IGF-1. The results showed that liver IGF-1 mRNA content is sensitively regulated by quantity and nutritional quality of dietary proteins.

We have cloned a full-length cDNA for rat-liver-type phosphofructokinase. The similarities of the rat liver-type phosphofructokinase mRNA to the human and mouse counterparts were 94% and 99% in their amino acid sequences and 88% and 94% in the nucleotide sequences of their coding regions, respectively. Rat liver-type phosphofructokinase mRNA was expressed in all tissues examined, but its level was regulated tissue-specifically. The nutritional and hormonal regulations of the mRNA in the liver were examined in comparison with those of two other key glycolytic enzymes, glucokinase and L-type pyruvate kinase. The level of liver-type phosphofructokinase mRNA was essentially unchanged by starvation (72 h) or diabetes. The mRNA level also did not change significantly on refeeding starved rats on a high carbohydrate diet, or treating diabetic ones with insulin. These results suggested that rat liver-type phosphofructokinase mRNA in the liver was not under control of diet or insulin, in contrast to glucokinase and L-type pyruvate kinase.

The messages for LF-B1, which interacts with the cis-acting element of PKL-I to play an essential role in expression of L-type pyruvate kinase (PK) in the liver, and L-type PK were found to be present in RIN-m5F insulinoma cells as well as the liver, kidney and small intestine, although the levels of the two mRNAs in these tissues were not correlated. Gel retardation assay suggested that similar nuclear proteins bound to two other cis-acting elements, PKL-II and PKL-III, were expressed in both liver and insulinoma cells, and that additional PKL-III-binding proteins were present only in RIN-m5F cells. Thus, we suggest that the mechanism of L-type PK expression in pancreatic B cells is similar to that in the liver.

A 36-year-old woman was admitted to our hospital because of general fatigue. The physical and laboratory findings on admission revealed splenomegaly, pancytopenia, hypocoagulopathy, liver hypofunction with a hepaplastin test of 55% and ICG Rmax of 0.6 mg/kg/min. Diagnostic imaging showed a hypoechoic mass 1.5 in diameter a low density area on the CT scan and a faint tumor stain on the AAG in the posterior inferior area of the liver. On a diagnosis of hepatocellular carcinoma with liver cirrhosis and hypersplenism, partial hepatectomy and splenectomy were performed. The resected hepatic specimen revealed a small liver cancer of 1.9 x 1.5 x 1.3 cm with liver cirrhosis. The specimen consisted of a firm rubbery mass. Macroscopically, the tumor appeared oval and was lobulated with a thin capsule. A fibrous scar was observed in the central area. Microscopically, malignant hepatocytes showed various shapes, ranging from polygonal to spindle form, with eosinophilic granular cytoplasm and were surrounded by abundant fibrous stroma. Orcein stain, revealed that these malignant hepatocytes contained many black granules of copper-binding protein. Immunoperoxidase staining for alpha 1-antitrypsin was also positive in the malignant hepatocytes. However, within this lamellar fibrous regions, there were many cords of tumor cells in which nucleoli were absent and abortive biliary differentiation was suggested. Consequently this tumor was diagnosed as an atypical fibrolamellar hepatocellular carcinoma. We think that this case is the 3rd case reported in Japan and the 2nd case in a Japanese person.

The relation of expression of the LF-B1 gene with the L-type pyruvate kinase (L-PK) mRNA level in rat liver and hepatoma cells was investigated. The L-PK mRNA level in rat liver changed after partial hepatectomy, during development and on intake of a high carbohydrate diet, while the level of LF-B1 mRNA remained unchanged or altered reciprocally. Dedifferentiated AH-130 cells, which did not express L-PK mRNA, expressed LF-B1 mRNA. These results suggest that transcription of the pyruvate kinase L gene is not simply regulated by the level of LF-B1 mRNA.