Insulin-mediated increases in renal plasma flow are impaired in insulin-resistant normal subjects.

BACKGROUND: Impaired vasodilatation in skeletal muscle is a possible mechanism linking insulin resistance to blood pressure regulation. Increased renal vascular resistance has been demonstrated in the offspring of essential hypertensives. We assessed whether insulin-mediated renal vasodilatation is impaired in insulin-resistant normal subjects. DESIGN: In two groups of 10 insulin-resistant and 10 insulin-sensitive normal subjects, we compared the effects of sequential physiological and supraphysiological insulin dosages (50 and 150 mU kg(-1) h(-1)) on renal plasma flow (RPF) and leg blood flow using the euglycaemic clamp technique, 131I-labelled Hippuran clearances and venous occlusion plethysmography. Time-control experiments were performed in the same subjects. RESULTS: Whole-body glucose uptake amounted to 4.9 +/- 2.1 and 11.0 +/- 2.4 mg kg(-1) min(-1) in the insulin-resistant and to 12.7 +/- 2.3 and 17.4 +/- 2.6 mg kg(-1) min(-1) in the insulin-sensitive subjects during physiological and supraphysiological hyperinsulinaemia, respectively. RPF increased more in insulin-sensitive compared to insulin-resistant subjects during physiological hyperinsulinaemia (13.7 vs. 6.8%, P < 0.05). RPF increased to comparable levels during supraphysiological hyperinsulinaemia. Insulin-mediated changes in leg blood flow did not differ between groups. In the combined group, we found a positive correlation between insulin-mediated glucose uptake and changes in RPF during physiological hyperinsulinaemia (r = 0.57, P = 0.009), whereas insulin-mediated glucose uptake correlated with changes in leg blood flow during supraphysiological hyperinsulinaemia (r = 0.54. P = 0.017). CONCLUSIONS: Our results suggest that the sensitivities of the skeletal muscle and renal vascular bed differ for insulin's vasodilatory action. Insulin-mediated increases in RPF are impaired in insulin-resistant but otherwise normal subjects during physiological hyperinsulinaemia.

Identification of human liver carboxylesterase as one of the proteins involved in Plasmodium falciparum malaria sporozoite invasion in primary cultures of human hepatocytes.

BACKGROUND/AIMS: In a previous study, we have demonstrated that primary human hepatocytes in culture are susceptible for Plasmodium falciparum sporozoite invasion and for development of parasites into exo-erythrocytic forms. In a separate study we demonstrated the involvement of two human liver plasma membrane proteins (55 kD and 20 kD) in the invasion of P. falciparum sporozoites in vitro. In this study, we have unravelled the nature of the 55 kD protein. METHODS: For the identification of this protein, a 53-58 kD membrane protein fraction from human liver was isolated, radioactively labelled, incubated with sporozoites and cross-linked. After reduction of the cross-linker, the released proteins were mixed with unlabelled 53-58 kD protein fraction and separated on two-dimensional SDS-PAGE. Autoradiography showed a single spot corresponding to a protein of 55 kD and pI of 5.7-5.8. RESULTS: Amino acid sequencing revealed the 55 kD protein as carboxylesterase. The biological activity of purified human liver carboxylesterase and of an antiserum against carboxylesterase on sporozoite invasion in vitro was evaluated. Human carboxylesterase as well as a rabbit antiserum against carboxylesterase inhibited the invasion of P. falciparum sporozoites into primary human hepatocytes in culture. A number of carboxylesterase cDNA clones were isolated from a human liver cDNA library. Sequence analysis revealed two different iso-types. Immunoaffinity purified recombinant human carboxylesterase was shown also to inhibit the invasion of sporozoites into primary human hepatocytes. Immunocytochemical analysis of the localisation of carboxylesterase in primary cultures of human hepatocytes using specific antibodies, showed its presence inside the hepatocytes and on the membrane. CONCLUSIONS: Carboxylesterase plays a role in the invasion process of P. falciparum sporozoites into human hepatocytes in vitro. The implications of these findings are further discussed.

Inflammation-induced changes in expression and glycosylation of genetic variants of alpha 1-acid glycoprotein. Studies with human sera, primary cultures of human hepatocytes and transgenic mice.

The relative occurrence of genetic variants of human alpha 1-acid glycoprotein (AGP) in relation to changes in glycosylation was studied in sera of patients with burn injury, media of cytokine-treated primary cultures of human hepatocytes and Hep 3B cells, and sera of transgenic mice expressing the human AGP-A gene. It is concluded (i) that the glycosylation of AGP was not dependent on its genetic expression and (ii) that both the variants determined by the AGP-A gene as well as by the AGP-B/B' genes are increased after inflammation or treatment with interleukins 1 and 6.

Tumor necrosis factor (TNF) inhibits interleukin (IL)-1 and/or IL-6 stimulated synthesis of C-reactive protein (CRP) and serum amyloid A (SAA) in primary cultures of human hepatocytes.

Interleukin (IL)-1, IL-6 and tumor necrosis factor (TNF) are considered as important mediators for the modulation of liver synthesis of acute phase proteins. However, studies of the direct effect of individual or a combination of these cytokines on the synthesis of acute phase proteins in human hepatocytes are still very limited. In this study, we have examined the synthesis of C-reactive protein (CRP) and serum amyloid A (SAA) in primary cultures of human hepatocytes exposed to recombinant(r)IL-1 alpha (100 U/ml), rIL-6 (2000 U/ml), rTNF alpha (30 U/ml) and to various combinations of these cytokines in the presence of 1 microM dexamethasone. Monoclonal antibodies to rTNF alpha and monospecific anti-rIL-6 sheep antiserum were also used to investigate the possible endogenous production of TNF or IL-6. The findings indicate: (1) IL-1 and IL-6 are stimulatory cytokines for the liver synthesis of CRP and SAA. Anti IL-6 abolishes the stimulatory effect of IL-1. These findings support the previous observation and indicate that IL-1 exerts its action on the enhanced synthesis of CRP and SAA at least in part via IL-6 production in the liver cell. (2) TNF is an inhibitory cytokine for the liver synthesis of CRP. It inhibits also the stimulatory effect of IL-1 and IL-6 on the synthesis of CRP and SAA. (3) Since anti-TNF enhances the stimulatory effect of IL-6 on the synthesis of CRP and SAA, it seems likely that TNF is also produced by the human hepatocytes. However, further studies for more direct evidence of the liver cell production of TNF, such as the detection of TNF messenger RNA are required.

Differential effects of endotoxin and fibrinogen degradation products (FDPS) on liver synthesis of fibrinogen and albumin: evidence for the involvement of a novel monokine in the stimulation of fibrinogen synthesis induced by FDPS.

1. Administration of endotoxin or fibrinogen degradation products (FDPs) in rats increase fibrinogen synthesis comparable to that found during the acute phase response. 2. An increased fibrinogen synthesis is also found in co-cultures of hepatocytes with peripheral blood mononuclear cells upon administration of endotoxin or FDPs, but not in primary cultures of hepatocytes alone. 3. However, the increased synthesis of fibrinogen by FDPs is not accompanied by a decreased albumin synthesis, as in the case of stimulated fibrinogen synthesis induced by endotoxin in vivo and in co-cultures of hepatocytes with peripheral blood mononuclear cells, or induced by monocytic products in vivo and in primary cultures of hepatocytes alone. 4. Since IL-1 and/or IL-6 could not be accounted for the stimulation of fibrinogen synthesis without a decreased albumin synthesis, a novel monokine produced by mononuclear cells upon FDP administration might be involved.

Effects of monocytic products, recombinant interleukin-1, and recombinant interleukin-6 on glycosylation of alpha 1-acid glycoprotein: studies with primary human hepatocyte cultures and rats.

Changes in the carbohydrate moieties of acute-phase glycoproteins (APGPs) often accompany the increase in their secretion by the liver during inflammation. In this study, we investigated whether factors known to regulate APGP gene expression are also involved in the altered glycosylation. For this purpose, the glycosylation pattern of alpha 1-acid glycoprotein (AGP) as secreted by human hepatocytes, cultured in the presence and absence of dexamethasone and monokines, was studied by crossed affino- (concanavalin A) immunoelectrophoresis (CAIE). The monokines rIL-1 and rIL-6, in the presence of dexamethasone, both stimulated AGP secretion and caused a change in glycosylation towards an increased Con A reactivity, including the appearance of two strongly reactive forms (D and E) normally not present. Dexamethasone alone did not influence either process. When tested in vivo in rats, rIL-6 also induced an increased presence of Con A-reactive forms of AGP in serum. In conclusion, the changes in secretion and glycosylation of AGP as seen during inflammation seem to be mediated by the same factor(s).

Transgenic expression of the muscle-specific intermediate filament protein desmin in nonmuscle cells.

The coding region of the hamster desmin gene was fused to the 5' flanking sequences of the hamster vimentin gene and introduced into the germ line of mice. The expression of this intermediate filament gene construct (pVDes) was analyzed at the RNA and protein level in transgenic mice as well as in fibroblast cell lines and primary hepatocyte cultures derived from these mice. In all transgenic mice, the pVDes-encoded protein was coexpressed with mouse vimentin in a tissue-specific fashion and was indistinguishable from normal hamster desmin. Culturing of transgenic hepatocytes induced desmin expression indicating that 3.2 kbp of the vimentin gene 5' region regulates both tissue-specific and tissue culture-induced intermediate filament protein expression. Immunohistochemical staining and double-label immunoelectron microscopy of cultured transgenic fibroblasts showed that the pVDes protein assembled into intermediate filaments which colocalized with the mouse vimentin filaments. Endogenous vimentin RNA levels were not influenced by high-level pVDes expression. The coexpression of desmin and vimentin in nonmuscle cells did not result in detectable developmental, morphological, or physiological abnormalities.

Fibrinogen and albumin synthesis are regulated at the transcriptional level during the acute phase response.

During acute inflammation or after administration of monocytic products, an enhanced transcription of the fibrinogen polypeptide genes and a reduced transcription of the albumin gene were observed. The changes in the fibrinogen polypeptide transcriptional rate were found to precede the change in albumin gene transcription. These findings indicate that the altered synthesis of fibrinogen and albumin during inflammation are regulated at the transcriptional level and are most probably mediated by monocytic products (including interleukin-1).

The effect of interleukin-1, interleukin-6 and its interrelationship on the synthesis of serum amyloid A and C-reactive protein in primary cultures of adult human hepatocytes.

During the acute phase response, synthesis of C-reactive protein and serum amyloid A is increased. To investigate whether the enhanced synthesis of these proteins are due to stimulatory effect of inflammatory mediators such as interleukin-1 (IL-1) and interleukin-6 (IL-6) produced by macrophages and monocytes, primary cultures of adult human hepatocytes were exposed to recombinant (r)IL-1, rIL-6 or rIL-1 and monospecific anti rIL-6 antibodies in the presence of 1 microM dexamethasone. The findings indicate that rIL-1 and rIL-6 both stimulate the liver synthesis of C-reactive protein and serum amyloid A, however monospecific anti rIL-6 antibodies reduce the stimulatory effect of rIL-1 on the synthesis of these proteins. These findings suggest that IL-6 plays a key role in the stimulation of synthesis of serum amyloid A and C-reactive protein by the human liver cells.

Primary culture of cryopreserved adult human hepatocytes on homologous extracellular matrix and the influence of monocytic products on albumin synthesis.

Cultured human hepatocytes provide a useful method for studies of specific metabolic functions, such as plasma protein synthesis or drug metabolism in man and specific interaction of microorganisms with the human liver cell. In vitro studies using human hepatocytes are seriously hampered by the limited availability of viable tissue and the lack of suitable methods for preservation of hepatocytes. In addition, normal adult human hepatocytes, under classical culture conditions, do not proliferate in vitro. Recently, we have reported a method for long-term storage of human hepatocytes, using a cryopreservation technique. However, after thawing, the efficiency of cell seeding onto tissue culture plastic and, accordingly, the survival of hepatocytes in primary cultures were decreased as compared to freshly prepared and cultured hepatocytes. In the present study, we report the effects of extracellular matrix (ECM) obtained from normal liver of kidney donors on the attachment efficiency, survival, some metabolic functions and fine structure of human hepatocytes. It was found that the deleterious effect of deep-freeze storage on attachment efficiency and survival of hepatocytes could be significantly reduced by using tissue culture plastic precoated with human liver ECM. Hepatocytes survived for more than 4-6 weeks, without evidence of fibroblast overgrowth. Using this in vitro experimental system, we have also shown that these hepatocytes synthesize several liver-specific acute phase proteins, and monocytic products were able to decrease the hepatocytic synthesis of albumin while total protein synthesis remained unchanged. These results support our previous observations in cultured rodent hepatocytes, indicating the important role of monocytic products in the regulation of liver synthesis of albumin in inflammatory diseases in man.

Prostaglandins E2 and F2 alpha are not involved in the monocytic-product (interleukin-1)-induced stimulation of hepatic fibrinogen synthesis in rats.

1. Monocytic products, especially interleukin-1 (IL-1), play an important role in the acute-phase response. Prostaglandins have been shown to act as second messengers in several physiological alterations of the acute-phase response, such as fever, muscle wasting and immunoregulation. The present study was undertaken to determine the role of prostaglandins in the monocytic-product-induced stimulation of the hepatic synthesis of fibrinogen, a well-known acute-phase protein. 2. Prostaglandin (PG) E2, PGF2 alpha and 16,16-dimethyl-PGE2 did not stimulate fibrinogen synthesis and fibrinogen polypeptide mRNA content when administered intraperitoneally to rats or when added to monolayer cultures of rat hepatocytes. 3. Cyclo-oxygenase inhibitors did not abolish the stimulation of fibrinogen synthesis and its mRNA content induced by monocytic products in vivo or in vitro. 4. These findings indicate that the enhanced synthesis of fibrinogen induced by monocytic products (including IL-1) during the acute-phase response is not mediated by prostaglandins or other products of the cyclo-oxygenase pathway of arachidonic acid.

In vitro infection of primary cultures of cryopreserved adult human hepatocytes with hepatitis B virus.

Infection with HBeAg and HBV DNA positive serum in primary cultures of cryopreserved human hepatocytes in the presence of human whole blood in the medium was performed in 8 consecutive experiments. HBsAg and HBV DNA release into the medium was increased in the second week after infection. Via immunostaining, HBcAg was first observed in the nucleus of hepatocytes approximately 3 days after infection. A maximal percentage of HBcAg positive cells in 0.1% of cultured hepatocytes was detected on the 7th day. HBsAg was also first demonstrated on the 3rd day, and predominantly localized in the cytoplasm. About 5% of hepatocytes were HBsAg positive on the 12th day after infection. The percentage of positive cells did not appear to increase after this time. Using in situ cytohybridization and agarose gel electrophoresis and Southern blot analysis, HBV DNA was first detected on the 4th day. In addition, electron microscopic studies revealed the presence of 42 nm virus-like particles in the cytoplasm of infected cells in the second week after infection. This in vitro system provides a model for studying the mechanism of HBV infection, viral replication and maturation. However, further improvement of culture systems is needed, to increase the number of infected cells and for active HBV replication.

Study of the molecular mechanism of decreased liver synthesis of albumin in inflammation.

Hypoalbuminemia in inflammatory disorders is not an infrequent finding. However, little is known about albumin synthesis in these patients. In the present study we have measured the albumin synthesis in four patients with inflammatory diseases using the [14C]carbonate technique. Because inflammation causes a decreased albumin synthesis and this decreased synthesis could not be related to a reduced amino acid supply, we have also examined the possible molecular mechanisms of reduced albumin synthesis during inflammation using in vivo and in vitro experiments in rats. In rats with turpentine-induced inflammation, serum albumin concentration and liver albumin mRNa level were markedly decreased. These changes could not be reproduced by administration of fibrinogen-, or fibrin-degradation products, or several hormones, such as corticosteroids, growth hormone, and adrenaline. However, monocytic products, especially interleukin 1, postulated to be important mediators of the inflammatory response, reduced albumin synthesis and liver albumin messenger RNA content but not total protein synthesis in rats in vivo and in primary cultures of rat hepatocytes. These findings suggest that monocytic products play an important role in reduced albumin synthesis during inflammation.

Cryopreservation of adult human hepatocytes. The influence of deep freezing storage on the viability, cell seeding, survival, fine structures and albumin synthesis in primary cultures.

Isolated and cultured human hepatocytes provide a useful model for studies of the liver cell function in man. In vitro studies using human hepatocytes are scarce, due to the limited availability and the lack of suitable methods for storage. In this study, we report the effect of deep freezing storage on the viability, fine structures and albumin synthesis of human adult hepatocytes in classical culture conditions. Hepatocytes were isolated using collagenase perfusion (9 isolations). The cell yield was 4-37 X 10(8) with a viability of 60-87%. Cryopreservation was performed in medium containing 10% DMSO and 20% fetal calf serum using a Cryoson BV-4 programmable freezer (0 degree C for 5 min, followed by a freezing rate of 1.5 degrees C/min for 20 min and 7 degrees C/min for 10 min). The cells were stored for 25-275 days in the liquid nitrogen vapor phase (-150 degrees C). Within 16 h about 80% of viable cells from freshly isolated hepatocytes whereas after cryopreservation, 55% of viable cells as determined by Trypan Blue exclusion before the cryopreservation attached to plastic and survived. Electron microscopy showed well developed tight junctions, structures similar to bile canaliculi. Cell polarity was evident. However, 'bleb' formation, more lipid droplets and lysosomes were found in cryopreserved hepatocytes during a short period after thawing. At the 3rd week, cells detached and died. These changes were associated with increased secretion of lactate dehydrogenase, whereas the albumin secretion dropped (from 10 to 4 micrograms/micrograms DNA), regardless of whether hepatocytes were cultured from fresh preparations or after cryopreservation. These findings suggest the cryopreservation is a useful technique to preserve hepatocytes for in vitro studies. Nevertheless, an improved method is necessary to increase the efficiency of cell seeding after cryopreservation.

Fibrinogen fragments X, Y, D and E increase levels of plasma fibrinogen and liver mRNAs coding for fibrinogen polypeptides in rats.

Previously, we demonstrated that in vivo regulation of liver fibrinogen synthesis occurs via the fibrinogen mRNA level. However, the molecular regulatory mechanism of fibrinogen synthesis is still not well understood. Fibrinogen or fibrin degradation products might play an important role in regulating fibrinogen synthesis. In our present study, we have injected rats intraperitoneally with purified homologous fragments and measured the liver content of mRNA specific coding for fibrinogen. Increased levels of fibrinogen mRNA and elevated plasma fibrinogen concentrations were observed in rats after administration of fibrinogen degradation products X, Y, DEGTA, Dcate or E. Fragment E or E' has a less stimulatory effect than X, Y or Dcate, whereas cross-linked fibrin degradation product D dimer does not increase fibrinogen synthesis. This article reports for the first time a stimulatory effect of the high molecular weight fibrinogen degradation products on fibrinogen synthesis.

The influence of glucocorticoid on albumin synthesis and its messenger RNA in rat in vivo and in hepatocyte suspension culture.

Corticosteroids are known to stimulate the synthesis of a number of liver-specific proteins. The reports regarding the effect of glucocorticoid on albumin synthesis in vivo and in vitro are controversial. In an attempt to determine the mechanism by which glucocorticoid exerts its influence on hepatic albumin synthesis and to find an explanation for the conflicting data, we have studied the effect of dexamethasone disodium phosphate on albumin synthesis and albumin messenger RNA as determined by the molecular hybridization technique in hepatocytes in rat in vivo and in suspension culture. In hepatocyte suspension culture, addition of 0.48 microM dexamethasone in medium at zero time led to a significant increase (20%) in incorporation of labeled precursor into albumin as compared to control experiments; this was accompanied by a maintainance of the initial level of full-length albumin mRNA for a 9 h period. In hepatocytes cultured without dexamethasone in the medium there was a progressive loss of albumin mRNA content. Despite this finding, dexamethasone was not able to increase the albumin mRNA content in hepatocyte to a level higher than the initial value. Moreover, administration of this hormone either intraperitoneally or intravenously into rats did not lead to enhanced cell-free albumin synthesis or to an increased level of albumin mRNA. These findings suggest that glucocorticoid does not play an essential role in the regulation of albumin synthesis in vivo. In vitro, however, glucocorticoid leads to a preservation of the initial level of albumin mRNA and thus plays a role in the control of spontaneous dedifferentiation of liver cells in culture.

The influence of glucocorticoid on the fibrinogen messenger RNA content of rat liver in vivo and in hepatocyte suspension culture.

The plasma concentration of fibrinogen, one of the major acute-phase proteins produced by the liver, increases during the acute-phase response as a result of enhanced synthesis in liver. Since adrenal-cortical hormones have been thought to have a key role in the regulation of the fibrinogen synthesis, fibrinogen-polypeptide mRNA sequences were determined in the present study, by using a specific complementary-DNA probe, in RNA fractions obtained from rat hepatocytes exposed to glucocorticoids in vitro (hepatocyte suspension cultures) and in vivo. Maximal induction of the fibrinogen-polypeptide mRNA (to 400% of the control value) was found in vitro at 0.1 microM-dexamethasone after 9 h of incubation. The same magnitude of induction was obtained with 20 microM-cortisol or 60 microM-corticosterone. In contrast with the findings in vitro, no induction of the fibrinogen-polypeptide mRNA was observed in the liver at various times after injection of different doses of glucocorticoids into rats. These results suggest that more complex regulatory mechanisms are involved and that glucocorticoids are not the sole regulatory factors in vivo in the enhanced synthesis of fibrinogen during the acute-phase response.