Regulation of the branched-chain 2-oxo acid dehydrogenase complex in hepatocytes isolated from rats fed on a low-protein diet.

Hepatocytes isolated from rats fed on a chow diet or a low-protein (8%) diet were used to study the effects of various factors on flux through the branched-chain 2-oxo acid dehydrogenase complex. The activity of this complex was also determined in cell-free extracts of the hepatocytes. Hepatocytes isolated from chow-fed rats had greater flux rates (decarboxylation rates of 3-methyl-2-oxobutanoate and 4-methyl-2-oxopentanoate) than did hepatocytes isolated from rats fed on the low-protein diet. Oxidizable substrates tended to inhibit flux through the branched-chain 2-oxo acid dehydrogenase, but inhibition was greater with hepatocytes isolated from rats fed on the low-protein diet. 2-Chloro-4-methylpentanoate (inhibitor of branched-chain 2-oxo acid dehydrogenase kinase), dichloroacetate (inhibitor of both pyruvate dehydrogenase kinase and branched-chain 2-oxo acid dehydrogenase kinase) and dibutyryl cyclic AMP (inhibitor of glycolysis) were effective stimulators of branched-chain oxo acid decarboxylation with hepatocytes from rats fed on a low-protein diet, but had little effect with hepatocytes from rats fed on chow diet. Activity measurements indicated that the branched-chain 2-oxo acid dehydrogenase complex was mainly (96%) in the active (dephosphorylated) state in hepatocytes from chow-fed rats, but only partially (50%) in the active state in hepatocytes from rats fed on a low-protein diet. Oxidizable substrates markedly decreased the activity state of the enzyme in hepatocytes from rats fed on a low-protein diet, but had much less effect in hepatocytes from chow-fed rats. 2-Chloro-4-methylpentanoate and dichloroacetate increased the activity state of the enzyme in hepatocytes from rats fed on a low-protein diet, but had no effect on the activity state of the enzyme in hepatocytes from chow-fed rats. The results indicate that protein starvation greatly increases the sensitivity of the hepatic branched-chain 2-oxo acid dehydrogenase complex to regulation by covalent modification.     

Up-regulation of ADRP in fatty liver in human and liver steatosis in mice fed with high fat diet

The present study was performed to examine a role of adipose differentiation-related protein (ADRP) in the process of liver steatosis. Immunohistochemical findings indicated that ADRP expression is increased in the hepatocytes in patients with fatty liver when compared with normal liver. ADRP expression is localized in the surface of lipid droplets in the hepatocytes. Increased expression of ADRP mRNA and protein was similarly observed in fatty liver in ob/ob mice and the liver steatosis induced by high fat diet in mice. The up-regulation of ADRP mRNA and protein in the liver by high fat diet was identified in the surface of lipid droplets in a time-dependent manner. Recent studies demonstrated that up-regulation of PPARgamma in the hepatocytes is deeply involved in liver steatosis. To clarify whether ADRP expression is increased by PPARgamma activation in hepatocytes, we examined the effect of a PPARgamma ligand, troglitazone, on ADRP mRNA expression in HepG2 cells. ADRP mRNA expression was increased by troglitazone in dose- and time-dependent manners. All these results suggest that ADRP is up-regulated in liver steatosis in human and mice, and that high fat diet increases expression of ADRP through PPARgamma activation, followed by induction of liver steatosis.     

Isocaloric maternal low-protein diet alters IGF-I,IGFBPs, and hepatocyte proliferation in the fetal rat

We investigated the effect of an isocaloric maternal low-protein diet during pregnancy in rats on the proliferative capacity of cultured fetal hepatocytes. The potential roles of these changes on the IGF-IGF-binding protein (IGFBP) axis, and the role of insulin and glucocorticoids in liver growth retardation, were also evaluated. Pregnant Wistar rats were fed a control (C) diet (20% protein) or a low-protein (LP) diet (8%) throughout gestation. In primary culture, the DNA synthesis of hepatocytes derived from LP fetuses was decreased by approximately 30% compared with control hepatocytes (P < 0.05). In parallel, in vivo moderate protein restriction in the dam reduced the fetal liver weight and IGF-I level in fetal plasma (P < 0.01) and augmented the abundance of 29- to 32-kDa IGFBPs in fetal plasma (P < 0.01) and fetal liver (P < 0.01). By contrast, the abundance of IGF-II mRNA in liver of LP fetuses was unaffected by the LP diet. In vitro, the LP-derived hepatocytes produced less IGF-I (P < 0.01) and more 29- to 32-kDa IGFBPs (P < 0.01) than hepatocytes derived from control fetuses. These alterations still appeared after 3-4 days of culture, indicating some persistence in programming. Dexamethasone treatment of control-derived hepatocytes decreased cell proliferation (54 +/- 2.3%, P < 0.01) and stimulated 29- to 32-kDa IGFBPs, whereas insulin promoted fetal hepatocyte growth (127 +/- 5.5%, P < 0.01) and inhibited 29- to 32-kDa IGFBPs. These results show that liver growth and cell proliferation in association with IGF-I and IGFBP levels are affected in utero by fetal undernutrition. It also suggests that glucocorticoids and insulin may modulate these effects.     

The regulation of IGFs and IGFBPs by prolactin in primary culture of fetal rat hepatocytes is influenced by maternal malnutrition

During perinatal development, the regulation of IGF system appears to be growth hormone (GH) independent. By using highly purified primary fetal hepatocytes, we investigated the role of prolactin (PRL) in the regulation of IGF system and hepatocyte proliferation. We also analyzed the consequence of a maternal low-protein (LP) diet on the regulation of IGF, IGF-binding protein (IGFBP), and hepatocyte proliferation by prolactin. Pregnant Wistar rats were fed a control (C) diet (20% protein) or isocaloric (LP; 8%) diet throughout gestation. On day 21.5, fetal hepatocytes were cultured for 4 days and incubated with rat prolactin. In the C hepatocytes, PRL at 100 ng/ml decreased the abundance of IGFBP-1 and IGFBP-2 by 50 (P < 0.05) and 60% (P < 0.01), respectively. It also reduced by 70% the level of IGF-II mRNA (P < 0.01). By contrast, PRL failed to modulate IGFBP-1 and IGFBP-2 production by LP hepatocytes, and this was associated with reduced abundance of the short form of PRL receptor (P < 0.05). PRL had no effect on either the proliferation or the IGF-I production by C and LP hepatocytes, although it reduced the expression of IGF-II. These results suggest that prolactin influences hepatocyte proliferation in vitro by inhibiting IGFBP-1, IGFBP-2, and IGF-II levels, which may coincide with the decline of IGF-II observed in rodents during late gestation in vivo. On the other hand, maternal LP diet induces a resistance of fetal hepatocytes to PRL.     

Nutritional control of branched chain alpha-ketoacid dehydrogenase in rat hepatocytes

Branched chain alpha-ketoacid dehydrogenase (EC 1.2.4.4) complex, the rate-limiting enzyme of branched chain amino acid catabolism in most tissues, is subject to regulation by covalent modification, with phosphorylation inactivating and dephosphorylation activating the complex. The enzyme complex from liver of chow-fed rats is mainly in the active form but that from liver of rats fed a low-protein diet is mainly in the inactive form. Isolated hepatocytes were used to identify factors that affect interconversion of branched chain alpha-ketoacid dehydrogenase. The enzyme present in hepatocytes of rats fed a low-protein diet appears much more responsive to regulation by covalent modification than the branched chain alpha-ketoacid dehydrogenase present in hepatocytes of normal chow-fed rats. alpha-Chloroisocaproate, a specific inhibitor of the kinase responsible for phosphorylation and inactivation of the complex, greatly stimulates oxidation of alpha-keto[1-14C]isovalerate by hepatocytes prepared from rats fed a low-protein diet but not from normal chow-fed rats. Oxidizable substrates are also much more effective inhibitors of branched chain alpha-ketoacid oxidation with hepatocytes from rats fed a low-protein diet than from normal chow-fed rats. Activity measurements with cell-free extracts suggest that changes in flux through the dehydrogenase with intact hepatocytes prepared from rats fed a low-protein diet are explained in large part by changes in the proportion of the enzyme in the active, dephosphorylated form. Regulation of liver branched chain alpha-ketoacid dehydrogenase by covalent modification functions to conserve branched chain amino acids for protein synthesis during periods of restricted dietary protein intake.     

Histomorphologic hepatic features and growth performances of juvenile Senegalese sole (Solea senegalensis) fed isogenertic practical diets with variable protein/lipid levels

Histological and morphometric techniques were employed in juvenile Senegalese sole (Solea senegalensis) livers to evaluate diet‐related adaptive changes and to establish how an integrated morphofunctional approach could improve the understanding of rearing strategies. Four isoenergetic diets with variable protein/lipid content (44/22, 49/20, 54/18 and 59/16) were formulated. Data compared statistically showed that an increase in protein and decrease of lipid in the diet was associated with a significantly higher specific growth rate (SGR) and reduced feed conversion rate (FCR). Histologic evaluation (visual scoring of lipid storage) and morphometry (area and perimeter) of hepatocytes showed that a high lipid diet resulted in increased lipid storage. The data confirmed that a concomitant decrease of protein with an increase in lipids in the diet led to a massive storage of unused lipid within hepatocytes associated with a reduced growth performance.     

Effect of long-term administration of a high protein or low protein diet on rat liver. Morphological and biochemical findings

An increase in relative liver weight, the total liver DNA content, hepatocyte volume and the total surface area of the membranes of mitochondria and the granular and smooth endoplasmic reticulum of hepatocytes, but a decrease in the size of the nuclei, were found in adult male rats fed three weeks on a high protein diet compared with animals given a standard laboratory diet. Serum transaminase (ALT, AST) and alkaline phosphatase activity was practically the same as the control values. Rats fed three weeks on a low protein diet showed a decrease in relative liver weight, in the total liver DNA content, in hepatocyte and nuclear volume and in ploidy, and also in the surface area of the membranes of the mitochondria and the smooth and granular endoplasmic reticulum; conversely, the number of binucleate hepatocytes rose. Serum ALT, AST and alkaline phosphatase activity was mildly, but statistically significantly elevated.     

Metabolic studies on isolated hepatocytes of the rat. Examples of application to various physico-pathological problems

Isolated hepatocytes offer many advantages. Many experiments can be realized with homogeneous cells. Furthermore, if a pathological state or a nutritional deficiency are induced in the whole animals, freshly isolated hepatocytes can be used to evaluate the consequences of such treatment. Gluconeogenesis was studied, using isolated hepatocytes of high protein diet fed rats, partial magnesium deficient rats and streptozotocin diabetic rats Asialoglycoprotein uptake by isolated hepatocytes of normal. streptozotocin diabetic rats with and without insulin treatment was also measured.     

PKCδ is activated in the liver of obese Zucker rats and mediates diet-induced whole body insulin resistance and hepatocyte cellular insulin resistance

Insulin resistance can arise when pathological levels of free fatty acids (FFAs) and proinflammatory cytokines disrupt insulin signaling. Protein kinase C delta (PKCδ) is a FFA- and a proinflammatory cytokine-regulated protein kinase that is associated with inhibition of insulin signaling and action. To gain insight into the role of PKCδ in insulin resistance, PKCδ activation was studied in a genetic model of obesity-linked insulin resistance. PKCδ was found to be activated in the liver of obese insulin-resistant Zucker rats and in isolated cultured hepatocytes. PKCδ was further studied in PKCδ-null mice and their wild-type littermates fed a high-fat or control diet for 10 weeks. PKCδ-null mice on a high-fat diet had improved insulin sensitivity and hepatic insulin signaling compared to wild-type littermates. Additionally, the deleterious effect of a high-fat diet on glucose tolerance in wild-type mice was completely blocked in PKCδ-null mice. To directly test the role of PKCδ in cellular insulin resistance, primary hepatocytes from the high-fat diet mice were isolated and stimulated with insulin. Primary hepatocytes from PKCδ-null mice had improved insulin-stimulated Akt and FOXO phosphorylation compared to hepatocytes from wild-type littermates. Consistent with this result, tumor necrosis factor alpha-mediated inhibition of insulin signaling was blocked in PKCδ knockdown primary hepatocytes. These results indicate that PKCδ plays a role in insulin resistance and is consistent with the hypothesis that PKCδ is a negative regulator of insulin signaling and thus may be a therapeutic target for the treatment of type 2 diabetes.     

Diet and diabetic state modify glycogen synthase activity and expression in rat hepatocytes

Glycogen synthase (GS), a key regulatory enzyme in glycogen synthesis, is controlled by multisite phosphorylation and allosteric regulation and is activated by insulin. This study investigated changes in GS activity and expression in hepatocytes isolated from rats under altered nutritional and diabetic conditions. Experiments were carried out in healthy rats fed a chow diet, rats on high simple sugar (60% of energy from fructose and sucrose) or high fat (46% of energy from fat) diet, and in rats with streptozotocin induced diabetes. In the presence of insulin, activated GS activity (GS(I) form) was increased by 89% in hepatocytes isolated from healthy rats. The stimulatory effect of insulin on GS activity and expression was blunted by cycloheximide and actinomycin treatment. In rats fed a high simple sugar or high fat diet, insulin stimulation of GS(I) in isolated hepatocytes was impaired and GS expression was significantly lower in rats fed the high fat diet in comparison to controls. GLUT-2 protein expression was significantly lowered by both the high fat and high simple sugar diets. In hepatocytes isolated from diabetic rats, total GS activity (GS(T)) was lower than in hepatocytes from healthy animals. Insulin added to the incubation medium did not stimulate GS activity, demonstrating impaired sensitivity to insulin in diabetic rats. However, insulin administration significantly increased GS expression indicating that a defect in synthase phosphorylation may be responsible for impaired GS activity in the diabetic state. The results presented in this study further confirm that GS activity is affected by both dietary and hormonal factors which can be measured in a rat hepatocyte model.     

Structural and functional changes after the administration of tetrachlormethane in the liver of rats fed on diets with different protein contents

Morphological and biochemical changes characterizing the degree of liver damage and the development of liver repair were studied in rats fed 21 days on a low protein diet (LPD), a standard diet (SLD) and a high protein diet (HPD) and then given a single i.p. injection of tetrachlormethane (CCl4) in a dose of 0.75 ml/kg body weight. The HPD was found to increase sensitivity to CCl4, but it also promoted the liver repair process, as seen from the increment in liver DNA synthesis and the total DNA content of the liver, increased ploidy of the hepatocytes and growth of the size of their nuclei and of the hepatocytes themselves. An increase in the total surface area of the membranes of the granular endoplasmic reticulum and the inner and outer membrane of the mitochondria, but a decrease in the surface area of the membranes of the smooth endoplasmic reticulum, were also observed after the administration of CCl4. The LPD raised liver resistance to CCl4, but the development of liver repair activity differed from the process after the SLD and HPD, since polyploidy of the hepatocytes (especially the growth of octaploid cells) predominated and there was also an increase in the number of binuclear hepatocytes. Cell hypertrophy was expressed less in rats fed on the LPD than in animals given the HPD. As far as liver repair was concerned, the HPD showed no explicit advantages over the SLD.     

Effects of troglitazone and insulin on glucose production in cultured hepatocytes isolated from rats on high fat diet

Increased dietary fat intake in general, and saturated fat specifically, will lead to the impairment of insulin action. The aim of this study was to find out the changes in hepatic glucose output in dependence of fat diet and a possible direct action of insulin and trogitazone in hepatocytes. Hepatocytes were isolated by a collagenase perfusion technique and cultured for 24 h in M 199 serum-free medium. The glucose production in hepatocytes isolated from rats on high fat diet (unsaturated fat) was 79% higher compared to control and even 139% higher than in rats on high-fat diet (saturated fat). Troglitazone significantly decreased the glucose production in hepatocytes obtained from rats on unsaturated fat diet. The troglitazone in presence of insulin totally normalized glucose production but also only in hepatocytes obtained from rats on unsaturated-fat diet. The troglitazone showed an insulinomimetic as well as insulin-sensitizing effect but only in rats on unsaturated-fat diet.     

Immunocytochemical localization of liver-specific proteins in pancreatic hepatocytes of rat

Hepatocytes are induced in the pancreas of rats maintained first on a copper-deficient diet for 8 weeks and then on normal rat chow. These cells are morphologically identical to parenchymal cells of the liver. These hepatocytes contain two liver-specific proteins: carbamyl phosphate synthetase I, a mitochondrial matrix protein that participates in the conversion of ammonia to carbamyl phosphate; and urate oxidase, an enzyme that catalyzes the oxidation of uric acid to allantoin. In addition, we also present evidence indicating that dietary administration of ciprofibrate induces peroxisomal beta-oxidation pathway enzymes, while the levels of catalase are unaltered in pancreatic hepatocytes. These observations along with the previously published results further establish the identity of pancreatic hepatocytes to parenchymal cells of liver and clearly indicate that transdifferentiation of pancreatic cells to hepatocytes is associated with activation of several liver-specific genes.     

Role of hepatic lipase and scavenger receptor BI in clearing phospholipid/free cholesterol-rich lipoproteins in PLTP-deficient mice

Phospholipid transfer protein knock-out (PLTP0) mice have defective transfer of phospholipids (PL) from triglyceride-rich lipoproteins (TRL) into high-density lipoproteins (HDL). In this study, we examined the role of diet, hepatic lipase (HL) and scavenger receptor BI (SRBI) in determining the accumulation of excess PL and free cholesterol (FC, "surface remnants") in plasma of PLTP0 mice. PL and FC accumulated in the very low-density lipoprotein (VLDL)-LDL region of PLTP0 mice on a highly saturated, coconut oil-based diet, but not on chow or milk-fat based Western diets. Accumulation of PL and FC was dramatically increased in PLTP0/HL0 mice, compared to PLTP0 mice, but only on the coconut oil diet. Turnover studies indicated that the coconut oil diet was associated with delayed catabolism of PL of PL/FC-rich particles. Incubation of these particles with primary hepatocytes in the presence of SRBI neutralizing antibody indicated that SRBI was primarily responsible for removal of FC and PL on the Western diet. In hepatocytes of coconut oil-fed mice, removal of FC and PL from these particles by SRBI was markedly reduced, even though SRBI protein expression levels were unchanged. These studies indicate that HL and SRBI both have major role in the clearance of PL and FC of surface remnants in PLTP0 mice. SRBI appears to be dysfunctional in coconut oil diet-fed animals, possibly related to changes in hepatocyte membrane fatty acid composition.     

Substituting dietary polyunsaturated fat with monounsaturated fat increases insulin sensitivity in cultured rat hepatocytes

Increased dietary fat intake generally, and saturated fat specifically, will lead to impairment of insulin action and to development of metabolic syndrome. The aim of this study was to find out changing of hepatic glucose output in dependence of high monounsaturated and high polyunsaturated fat diet and possible direct action of insulin in hepatocytes. Hepatocytes were isolated by a collagenase perfusion technique and cultured for 24 h in M 199 serum-free medium. The glucose production in hepatocytes isolated from rats on high polyunsaturated fat diet, as well as those isolated from rats on high monounsaturated fat diet was significantly higher than in standard controls. Insulin significantly decreased glucose production but only in hepatocytes obtained from rats on high monounsaturated fat diet. On the other side energy expenditure was significantly higher in rats on high monounsaturated fat diet and significantly lower in rats on high polyunsaturated fat diet comparable with animals on standard diet. Monounsaturated fat in Mediterranean diet could have beneficial effect on the development of the metabolic syndrome by increasing insulin sensitivity and energy expenditure.     

CCN1 expression in hepatocytes contributes to macrophage infiltration in nonalcoholic fatty liver disease in mice

Our objective was to investigate the potential roles of CCN1 in the inflammation and macrophage infiltration of nonalcoholic fatty liver disease (NAFLD). The regulation of hepatic CCN1 expression was investigated in vitro with murine primary hepatocytes treated with free fatty acids or lipopolysaccharide (LPS) and in vivo with high-fat (HF) diet-fed mice or ob/ob mice. CCN1 protein and a liver-specific CCN1 expression plasmid were administered to mice fed a normal diet (ND) or HF diet. Myeloid-derived macrophages and RAW264.7 cells were also treated with CCN1 in vitro to determine the chemotactic effects of CCN1 on macrophages. LPS treatment significantly increased hepatic CCN1 expression in HF diet-fed mice and ob/ob mice. LPS and FFAs induced CCN1 expression in primary murine hepatocytes in vitro through the TLR4/MyD88/AP-1 pathway. CCN1 protein and overexpression of CCN1 in the liver induced more severe hepatic inflammation and macrophage infiltrates in HF mice than in ND mice. CCN1 recruited macrophages through activation of the Mek/Erk signaling pathway in myeloid-derived macrophages and RAW264.7 cells in vitro. Endotoxin and FFA-induced CCN1 expression in hepatocytes is involved in the hepatic proinflammatory response and macrophage infiltration in murine NAFLD.     

Effects of vanadate on glucose production in cultured hepatocytes isolated from rats on high saturated fat diet

Insulin resistance is a common phenomenon in obesity and Type 2 diabetes. Common factor important for development of diabetes and insulin resistance is intake of saturated fat. Vanadate treatment improves glucose homeostasis in vivo. The aim of this study was to find out changing of hepatic glucose output in dependence of saturated fat diet and possible direct action of vanadate in cultured hepatocytes. Hepatocytes were isolated by a collagenase perfusion technique and cultured for 24 h in M 199 serum-free medium. The glucose production in hepatocytes isolated from rats on high saturated fat diet was significantly 139% higher comparable to standard controls. Glucagon 100% increased glucose production in hepatocytes from rats on standard diet and 200% in hepatocytes on saturated high fat diet. The addition vanadate significantly decreased basic glucose production and did not influence glucagon stimulated glucose production. Presence of insulin did not influence either glucagon or vanadate effect. High saturated fat diet not only increases insulin resistance but also decreases chances of successful therapy of diabetes.     

Acetoacetate: a major substrate for the synthesis of cholesterol and fatty acids by isolated rat hepatocytes

The diuretic drug amiloride antagonises the insulin-dependent increase in phosphorylation of ATP-citrate lyase in hepatocytes isolated from rats that had been starved and refed a fat-free diet. Studies with a range of protein kinases and protein phosphatases that have been shown to phosphorylate or dephosphorylate purified ATP-citrate lyase in vitro revealed that amiloride was a non-specific inhibitor of all protein kinases tested, but did not significantly affect any of the protein phosphatases. These results cast doubt on previous claims that growth factors stimulate phosphorylation of ribosomal protein S6 by activating an amiloride-sensitive Na+/H+ exchange system, and that insulin inhibits a protein phosphatase that is activated by amiloride.