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.     

Primary cultures of hepatocytes in serum and hormone-free medium: Identification of conditions which stimulate an in vivo-like induction of G6PD

Summary Recent results from several laboratories suggest that complex interactions between hormones and dietary carbohydrate may be responsible for regulating the induction of several hepatic lipogenic enzymes. Elucidation of these interactions requires the ability to culture hepatocytes for several days in serum-free medium where the hormones or carbohydrate or both present is strictly controlled. The functional response of primary adult rat hepatocytes was examined in a medium without exposure to serum, hormones, or carbohydrates and on three substrata commonly used to culture cells in a defined medium. Hepatocytes cultured on a floating collagen gel in which is embedded a nylon mesh possess cell attachment and morphologic characteristics superior to either cells cultured on a collagen-coated or fibronectin(Fn)-coated substratum. Cells cultured on the gel-mesh system retain insulin responsivity, as measured by protein synthesis rates and glucose-6-phosphate dehydrogenase (G6PD) induction, for at least 6 d in culture. Under these conditions, insulin, dexamethasone, and fructose increase G6PD specific activity to levels comparble to that seen in an induced animal. Hepatocytes cultured on the gel-mesh system tolerate restricted medium conditions better than cells cultured on collagen or Fn-coated substratum, and remain viable for sufficient times to allow, for the first time, full expression and maximal induction (i.e. like in vivo), of G6PD in cultured cells. This system represents a satisfactory model for in vivo liver metabolism and a superior system for studying the effects of hormones and metabolites on G6PD levels, as well as other nutritional-hormonally regulated enzymes.     

Cysteine uptake and taurine biosynthesis in freshly isolated and primary cultured rat hepatocytes

Analysis of the uptake and metabolism of [14C]cysteine in rat liver was undertaken using freshly isolated hepatocytes and hepatocytes maintained in primary culture. The uptake of [14C]cysteine by freshly isolated hepatocytes was by means of both saturable and non-saturable transport systems and the former system was thought to involve facilitated diffusion. The uptake of [14C]cysteine by hepatocytes maintained in primary culture for 24 h also consisted of non-saturated and saturated transport mechanisms. The magnitude of the saturable transport system in cultured hepatocytes was, however, much greater than that found in freshly isolated hepatocytes, and was considered to be operated by active transport. Both freshly isolated and primary cultured hepatocytes had cysteine sulphinic acid decarboxylase activity, but this enzyme activity in the latter cells was noticeably reduced in comparison with that found in freshly isolated hepatocytes. Hepatocytes maintained in primary culture produced not only radiolabelled taurine, but also radiolabelled cysteine sulphinic acid, hypotaurine and alanine when incubated with [14C]cysteine. The present results indicate that cultured hepatocytes actively transport cysteine as well as metabolizing cysteine to taurine via cysteine sulphinic acid and hypotaurine.     

Effect of hydrocortisone and nicotinamide on gamma glutamyltransferase in primary cultures of rat hepatocytes

Isolated rat hepatocytes cultured on collagen coated plates exhibit a gradual fetal phenotypic change during time in culture. The fetal liver marker gamma glutamyltransferase (GGT) was used to follow this change. Inasmuch as a significant overgrowth of nonparenchymal liver derived cells is seen frequently in primary cultures of hepatocytes, a technique was utilized that corrects for the presence of nonparenchymal cells. In media supplemented with either hydrocortisone (10(-5) M) or nicotinamide (25 mM) the original epithelial morphology of hepatocytes was preserved for a longer period of time than in unsupplemented media. Hepatocytes in unsupplemented media exhibited an increase in GGT specific activity over time. Hydrocortisone (10(-5) M) induced an increase in GGT activity compared to controls. Nicotinamide (25 mM) inhibited the increase in GGT activity compared to the unsupplemented hepatocytes. Our results indicate that GGT is regulated by hydrocortisone and nicotinamide.     

Regulation of gene expression in adult rat hepatocytes cultured on a basement membrane matrix

Freshly isolated adult rat hepatocytes, when cultured on type I collagen (commercially available as Vitrogen), assume a polygonal shape, form a stable monolayer within 24 hours, but lose the capacity to express some liver-specific functions over time in culture. We incubated hepatocytes in a serum-free medium on a reconstituted basement membrane gel, "matrigel" (prepared from an extract of extracellular matrix of the murine Engelbreth-Holm-Swarm sarcoma), and observed that the cells adhered firmly, remained rounded as single cells or clusters, and maintained liver-specific gene expression for more than 1 week in vitro. Hepatocytes on matrigel secreted substantially higher amounts of albumin, transferrin, haptoglobin, and hemopexin, Northern blot analyses of extracted cellular RNA, expressed increased amounts of mRNA for the liver-specific protein albumin (as compared with cells on vitrogen). In cultures treated with phenobarbital, cytochrome P-450b, and cytochrome P-450e, mRNAs and proteins were barely detectable in cells on Vitrogen but were induced to levels similar to those in the liver in vivo in matrigel cultures. Likewise, the use of matrigel greatly enhanced the induction of mRNA and protein for P-450c by 3-methylcholanthrene and for P-450p by steroidal and nonsteroidal inducers. However, neither substratum permitted induction of P-450d by 3-methylcholanthrene, suggesting that the effects of matrigel are selective even for expression in liver of members of the superfamily of cytochrome P-450 genes. Within 5 days in cultures on Vitrogen, hepatocytes expressed detectable amounts of fetal liver aldolase activity and also mRNA for vimentin and type I collagen, each considered a phenotypic change reflecting hepatocyte "dedifferentiation." None of these was present in cells on matrigel. Responsiveness to mitogenic stimuli, as judged by incorporation of 3H-thymidine into DNA, was also decreased in hepatocytes cultured on matrigel. Finally, there was a remarkable increase in the levels of both matrices during the first 2 days in culture. However, the continuously cytoskeleton mRNA over time in culture than did the rounded cells on matrigel. We conclude that hepatocytes cultured on matrigel, as opposed to the standard collagen, exhibit remarkably enhanced expression of many liver-specific functions.     

Accumulation of pyrroline 5-carboxylic acid in conditioned medium of cultured fibroblast: Stimulatory effects of serum,insulin, and IGF-1

Summary Pyrroline 5-carboxylate, an intermediate of amino acid metabolism, is released into medium by cultured normal human fibroblasts. With cells made quiescent by serum starvation, the addition of 10% fetal bovine serum augmented the release of pyrroline 5-carboxylate into medium by 2.5-fold. Although platelet-derived growth factor was without effect, both insulin and insulinlike growth factor-1 nearly reproduced the serum effect. The dose-dependence of insulin and insulinlike growth factor 1 effects suggested their mediation by their own respective receptors. Although the mechanism for the stimulatory effect remains unknown, these effects of insulin and insulinlike growth factor 1 on pyrroline 5-carboxylate suggest hormonal regulation of pyrroline 5-carboxylate release.     

Insulin-like growth factor-II receptors in cultured rat hepatocytes: regulation by cell density

Insulin-like growth factor-II (IGF-II) receptors in primary cultures of adult rat hepatocytes were characterized and their regulation by cell density examined. In hepatocytes cultured at 5 X 10(5) cells per 3.8 cm2 plate [125I]IGF-II bound to specific, high affinity receptors (Ka = 4.4 +/- 0.5 X 10(9) l/mol). Less than 1% cross-reactivity by IGF-I and no cross-reactivity by insulin were observed. IGF-II binding increased when cells were permeabilized with 0.01% digitonin, suggesting the presence of an intracellular receptor pool. Determined by Scatchard analysis and by polyacrylamide gel electrophoresis after affinity labeling, the higher binding was due solely to an increase in binding sites present on 220 kDa type II IGF receptors. In hepatocytes cultured at low densities, the number of cell surface receptors increased markedly, from 10-20,000 receptors per cell at a culture density of 6 X 10(5) cells/well to 70-80,000 receptors per cell at 0.38 X 10(5) cells/well. The increase was not due simply to the exposure of receptors from the intracellular pool, as a density-related increase in receptors was also seen in cells permeabilized with digitonin. There was no evidence that IGF binding proteins, either secreted by hepatocytes or present in fetal calf serum, had any effect on the measurement of receptor concentration or affinity. We conclude that rat hepatocytes in primary culture contain specific IGF-II receptors and that both cell surface and intracellular receptors are regulated by cell density.     

Co-ordinate induction of hepatic mitochondrial and peroxisomal carnitine acyltransferase synthesis by diet and drugs.

The effects of pancreatic hormones and cyclic AMP on the induction of ketogenesis and long-chain fatty acid oxidation were studied in primary cultures of hepatocytes from fetal and newborn rabbits. Hepatocytes were cultivated during 4 days in the presence of glucagon (10(-6) M), forskolin (2 x 10(-5) M), dibutyryl cyclic AMP (10(-4) M), 8-bromo cyclic AMP (10(-4) M) or insulin (10(-7) M). Ketogenesis and fatty acid metabolism were measured using [1-14C]oleate (0.5 mM). In hepatocytes from fetuses at term, the rate of ketogenesis remained very low during the 4 days of culture. In hepatocytes from 24-h-old newborn, the rate of ketogenesis was high during the first 48 h of culture and then rapidly decreased to reach a low value similar to that measured in cultured hepatocytes from term fetuses. A 48 h exposure to glucagon, forskolin or cyclic AMP derivatives is necessary to induce ketone body production in cultured fetal hepatocytes at a rate similar to that found in cultured hepatocytes from newborn rabbits. In fetal liver cells, the induction of ketogenesis by glucagon or cyclic AMP results from changes in the partitioning of long-chain fatty acid from esterification towards oxidation. Indeed, glucagon, forskolin and cyclic AMP enhance oleate oxidation (basal, 12.7 +/- 1.6; glucagon, 50.0 +/- 5.5; forskolin, 70.6 +/- 5.4; cyclic AMP, 77.5 +/- 3.4% of oleate metabolized) at the expense of oleate esterification. In cultured fetal hepatocytes, the rate of fatty acid oxidation in the presence of cyclic AMP is similar to the rate of oleate oxidation present at the time of plating (85.1 +/- 2.6% of oleate metabolized) in newborn rabbit hepatocytes. In hepatocytes from term fetuses, the presence of insulin antagonizes in a dose-dependent fashion the glucagon-induced oleate oxidation. Neither glucagon nor cyclic AMP affect the activity of carnitine palmitoyltransferase I (CPT I). The malonyl-CoA concentration inducing 50% inhibition of CPT I (IC50) is 14-fold higher in mitochondria isolated from cultured newborn hepatocytes (0.95 microM) compared with fetal hepatocytes (0.07 microM), indicating that the sensitivity of CPT I decreases markedly in the first 24 h after birth. The addition of glucagon or cyclic AMP into cultured fetal hepatocytes decreased by 80% and 90% respectively the sensitivity of CPT I to malonyl-CoA inhibition. In the presence of cyclic AMP, the sensitivity of CPT I to malonyl-CoA inhibition in cultured fetal hepatocytes is very similar to that measured in cultured hepatocytes from 24-h-old newborns.     

Growth- and development-dependent expression of gangliosides in rat hepatocytes and liver tissues.

Expression of gangliosides in the liver was examined in primary cultures of hepatocytes from adult rats and liver tissues from rats of different ages. Hepatocytes were isolated from 7-week-old rat liver and cultured in L-15 medium containing insulin, dexamethasone and 10% fetal bovine serum. Hepatocytes proliferated only on the first day, and then ceased proliferation. The content of GD3 and GD1a increased during the period of active proliferation and reached a nearly constant level, whereas GM1, GD1b, GT1b, and GQ1b gradually increased throughout culture. Addition of EGF to the culture medium caused significant increases in the content of GD3, and to a lesser degree of GM3, but exhibited little effect on the expression of other ganglioside species. The specific induction of GD3 and GM3 expression by EGF was reproduced under serum-free conditions, despite the lack of hepatocyte proliferation. Expression of gangliosides in cultured hepatocytes was also modulated by cell density; higher cell density brought about increased content of GM1, GD1a, GD1b, GT1b, and GQ1b with concomitant reduction of GM3 in cells. The composition of gangliosides in liver tissues demonstrated a unique developmental pattern. GD3 and GD1a were strongly expressed in E-16 embryonic tissue and rapidly decreased with increasing age. GD1b, GT1b, and GQ1b were found only in postnatal liver tissues. These findings suggest that the expression of gangliosides in rat hepatocytes and liver tissues are regulated by growth- and development-dependent factors.     

Changes in TGF-β receptors of rat hepatocytes during primary culture and liver regeneration: Increased expression of TGF-β receptors associated with increased sensitivity to TGF-β-mediated growth inhibition

To clarify the role of transforming growth factor-β (TGF-β) and its receptors in hepatocyte growth, we studied the expression of TGF-β1 and its receptors and the sensitivity to growth inhibition by TGF-β1 protein in rat hepatocytes derived from resting and regenerating livers. In hepatocytes derived from resting livers, mRNAs for TGF-β type II receptor (TβR-II), insulin-like growth factor-II/mannose 6-phosphate receptor (IGF-II/M-6-PR), and TGF-β1 increased with time in primary culture. The cell surface TGF-β receptor proteins (TβR-I, II, and III), examined by the receptor affinity-labeling assay using ¹²⁵I-TGF-β1, also increased, especially after 48 hr of culture. Hepatocytes were more sensitive to inhibition of DNA synthesis, when the TGF-β1 protein was added at later times in culture, corresponding to the presence of increased TGF-β receptors. In hepatocytes from regenerating livers after a partial hepatectomy (PH), an increase of TβR-I, TβR-II, TβR-III, IGF-II/M-6-PR, and TGF-β1 mRNAs was found, compared with hepatocytes from resting livers. Similarly, using TGF-β receptor affinity-labeling assay, hepatocytes from PH livers were found to have an increase in TβR-I, II, and III proteins, with a peak at 4 days post-PH, compared with hepatocytes from resting livers. When TGF-β1 protein was added for a short period (6 or 24 hr) after cell attachment to hepatocyte cultures, it inhibited DNA synthesis more effectively in hepatocytes from regenerating compared with resting livers. Our results show that hepatocyte TGF-β receptors and sensitivity to growth inhibition by TGF-β1 protein change together and are modulated during liver regeneration, as well as during the conditions of primary culture. J. Cell. Physiol. 176:612–623, 1998. © 1998 Wiley-Liss, Inc.     

Gene transfection of multicellular spheroid of hepatocytes on an artificial substrate

The handling of hepatocytes, a major cell population in the liver, is an important technique in both liver tissue engineering and hepatology. However, these cells are so fragile that it has been impossible to harvest hepatocytes with high viability from tissue culture dishes after a period of culture in vitro. In this study, we employed an artificial substrate for transfection of multilayer hepatocytes and harvested these cells with high viability after transfection. Hepatocytes cultured on an amphiphilic artificial substrate form multilayer aggregates (spheroids) in the presence of growth factors during gene transfection with cation liposomes. Compared to cells cultured on a collagen-coated plate, these spheroids are easily harvested with high viability by pipetting in EDTA solution. In addition, these spheroids rapidly spread on collagen after transfer from the artificial substrate, demonstrating that hepatocytes in the center of the spheroids were viable. Epidermal growth factor (EGF) increased the transfection efficiency into hepatocytes while hepatocyte growth factor (HGF) alone did not increase the efficiency. However, HGF synergestically increased the effect of EGF on transfection. Interestingly, this transfection required the process of spheroid formation because the gene was not transfected once the spheroid formation completed or under conditions where hepatocytes did not form spheroids. This method using spheroidal hepatocytes for in vitro transfection is promising for the development of ex vivo gene therapy.     

Primary rat hepatocyte culture on 3D nanofibrous polymer scaffolds for toxicology and pharmaceutical research

Primary rat hepatocytes are a widely used experimental model to estimate drug metabolism and toxicity. In currently used two‐dimensional (2D) cell culture systems, typical problems like morphological changes and the loss of liver cell‐specific functions occur. We hypothesize that the use of polymer scaffolds could overcome these problems and support the establishment of three‐dimensional (3D) culture systems in pharmaceutical research. Isolated primary rat hepatocytes were cultured on collagen‐coated nanofibrous scaffolds for 7 days. Cell loading efficiency was quantified via DNA content measurement. Cell viability and presence of liver‐cell‐specific functions (albumin secretion, glycogen storage capacity) were evaluated. The activity of liver‐specific factors was analyzed by immunofluorescent staining. RNA was isolated to establish quantitative real‐time PCR. Our results indicate that primary rat hepatocytes cultured on nanofibrous scaffolds revealed high viability and well‐preserved glycogen storage. Albumin secretion was existent during the entire culture period. Hepatocytes remain HNF‐4 positive, indicating highly preserved cell differentiation. Aggregated hepatocytes re‐established positive signaling for Connexin 32, a marker for differentiated hepatocyte interaction. ZO‐1‐positive hepatocytes were detected indicating formation of tight junctions. Expression of cytochrome isoenzymes was inducible. Altogether the data suggest that nanofibrous scaffolds provide a good in vitro microenvironment for neo tissue regeneration of primary rat hepatocytes. Biotechnol. Bioeng. 2011; 108:141–150. © 2010 Wiley Periodicals, Inc.     

THE CIRCUMFUSION SYSTEM FOR MULTIPURPOSE CULTURE CHAMBERS : II. The Protracted Maintenance of Differentiation of Fetal and Newborn Mouse Liver in Vitro

The circumfusion system is a complex in vitro pumping unit incorporating 12 multipurpose culture chambers through which a serum-supplemented fluid nutrient is recirculated at a rate of 4.5 ml/min per chamber. This system was used to study the differentiative responses of fetal and newborn mouse liver explants placed in the serum-free environment formed between the sheets of unperforated cellophane and cover glasses of the chambers. Hepatocytes (parenchymal cells) were discernible in 3–5 days. They retained many of their features of differentiation in the circumfusion system for more than 120 days of cultivation. The living morphological characteristics of the hepatocytes were studied by phase-contrast microscopy (direct viewing and time-lapse cinemicrography) and by special cytochemical staining. Electron micrographs were made of both fresh liver specimens and the cultured cells. Comparisons of the cultured parenchymal cells with their in vivo progenitors showed a remarkable preservation of their differentiated state.     

Effects of medium composition on the morphology and function of rat hepatocytes cultured as spheroids and monolayers

Primary hepatocytes cultured as monolayers or as spheroids were studied to compare the effects of four different culture media (Williams' E, Chee's, Sigma Hepatocyte, and HepatoZYME medium). Rat hepatocytes were cultured as conventional monolayers for 3 d or as spheroids for 2 wk. For spheroid formation a method was emplOyed that combined the use of a nonadherent substratum with rotation of cultures. Hepatocyte integrity and morphology were assessed by light and electron microscopy and by reduced glutathione content. Hepatocyte function was measured by albumin secretion and 7-ethoxycoumarin metabolism. Chee's medium was found to be optimal for maintenance of hepatocyte viability and function in monolayers, but it failed to support spheroid formation. For spheroid formation and for the maintenance of spheroid morphology and function, Sigma HM was found to be optimal. These results demonstrate that the medium requirements of hepatocytes differ markedly depending on the culture model employed. Spheroid culture allowed better preservation of morphology and function of hepatocytes compared with conventional monolayer culture. Hepatocytes in spheroids formed bile canaliculi. and expressed an actin distribution resembling that found in hepatocytes in vivo. Albumin secretion was maintained at the same level as that found during the first d in primary culture, and 7-ethoxycoumarin metabolism was maintained over 2 wk in culture at approximately 30% of the levels found in freshly isolated hepatocytes. The improved morphology and function of hepatocyte cultures as spheroids may provide a more appropriate in vitro model for certain applications where the maintenance of liver-specific functions in long-term culture is crucial.     

Induction of unscheduled DNA synthesis in primary rat hepatocytes by benzidine-congener-derived azo dyes in the in vitro and in vivo/in vitro assays

The genotoxicity of the benzidine-congener-derived azo dyes. Direct Blue 1 ( DB1 ), Direct Blue 14 ( DB14 ), Direct Brown 95 ( DB95 ), and Direct Red 46 ( DR46 ) was studied in the in vitro and in vivo/in vitro unscheduled DNA synthesis (UDS) assays in primary rat hepatocytes to determine if in vivo metabolism of these compounds was required for induction of UDS. Hepatocytes were isolated, cultured, and treated with the azo dyes and [3H]thymidine (in vitro assay); alternatively, in the in vivo/in vitro assay, rats were intubated with the azo dyes, the hepatocytes isolated at 17 h after dosing and incubated in a medium containing [3H]thymidine. UDS was quantified by an autoradiographic method. None of the azo dyes induced UDS in the in vitro assay. However, DR46 did induce marginal, but significant UDS in 1 experiment (1.2 net grains at 500 micrograms/ml media). No significant UDS was observed when DR46 was tested in a subsequent in vitro assay. In the in vivo/in vitro assay, DB95 (100 mg/kg), DB14 (125 mg/kg), and DR46 (100 mg/kg) induced significant UDS (12, 2.1, and 3.5 net grains, respectively). None of the azo dyes tested was mutagenic in the Salmonella/microsome assay in the presence and absence of rat liver enzymes. Therefore, in vivo reduction of azo dyes, presumably by the gut microflora, is a requirement for the genotoxicity of these azo dyes in the primary rat hepatocyte UDS assay.