Conditions affecting primary cell cultures of functional adult rat hepatocytes

Summary Primary monolayer cell cultures of adult rat hepatocytes underwent change in morphology and substantial cell loss between 1 and 3 days postinoculation. Dexamethasone-supplementation (1μM) of the culture medium maintained the polygonal epithelial morphology of the hepatocytes and increased longevity such that over 80% of the cells survived for 3 days and at least 30% for 8 or 9 days. This enhancement of survival was obtained up to 48 hr postinoculation, but the earlier the time of dexamethasone supplementation the greater the effect. Removal of dexamethasone resulted in a decrease in longevity. The positive effect of dexamethasone on longevity was observed following dexamethasone replacement of insulin in supplemented cultures, but the combination of insulin and dexamethasone resulted in poorer survival than with dexamethasone alone. The results are interpreted to indicate that dexamethasone provided a requirement of the in vitro environment for survival and suggest that elaboration of a complex medium is required to maintain hepatocytes in culture. This study was supported by an Alexander Ralston Peacock Memorial Grant for Cancer Research (No. BC-133A) from the American Cancer Society.     

Secretory expression of α single-chain insulin precursor in yeast and its conversion into human insulin

A synthetic single-chain porcine insulin precursor (PIP) gene and an α-mating factor leader sequence (αMFL) gene obtained by the PCR method are inserted between the promoter and 3'-terminating sequence of the alcohol dehydrogenase gene ADH1 in plasmid pVT102-U to form plasmid pVT102-U/α MFL-PIP. The single-chain insulin precursor is expressed and secreted to the culture medium by Saccharomyces cererisiae transformed by pVT102-U/αMFL-PIP. The precursor is purified and converted into human insulin by tryptic transpeptidation. The purified human insulin is fully active and can be crystallized. The overall yield of human insulin is 25 mg per liter of culture medium.     

Modulation of response to adenosine in vascular smooth muscle cells cultured in defined medium

Summary Cultured pig aortic smooth muscle cells maintain a viable, quiescent state in a chemically defined medium that contains 10⁻⁶ M insulin, 5μg/ml transferrin, and 0.2 mM ascorbate. DNA synthesis and DNA content were determined by measuring tritiated thymidine incorporation and DNA-binding to the fluorescent probe 4′,6-diamidino-2-phenylindole, respectively. The majority of the population of cells in defined medium cultures were diploid. Tritiated thymidine uptake in cells in defined medium was one-tenth that observed in cells in fetal bovine serum-containing medium. The study of cellular cyclic AMP level in response to extracellular adenosine stimulation in dividing cells and quiescent cells showed that cells in defined medium had a lower extent of response to adenosine compared to cells cultured in serum-containing medium. Both the cell growth index and the response to adenosine of cells cultured in defined medium were reversible after replacing the medium with 10% fetal bovine serum-containing medium, which suggests that the cells in defined medium were healthy and were capable of modulating cellular metabolism depending on culture conditions. This work was supported in part by National Institutes of Health grants HL31854, HL38130, and RR07048.     

Optimization of fermentation medium for xylanase-producing strain Xw2

To improve the fermentation yield of xylanase by optimizing the fermentation conditions for strain Xw2, a Plackett-Burman design was used to evaluate the effects of eight variables on xylanase production by strain Xw2. The steepest ascent （descent） method was used to approach the optimal response surface experimental area. The optimal fermentation conditions were obtained by central composite design and response surface analysis. The results showed that the composition of the optimal fermentation medium was corn cob ＋ 1.5% wheat bran （1：1）, 0.04% MnSO4, 0.04% K2HPO4. 3H2O, and an inoculum size of 6% in 50 mL liquid volume （pH = 6.0）. The optimal culture conditions were 28oc at 150 r/min for 54.23 h. The results of this study can serve as the basis for the industrial production and application of xylanase.     

Activation of STAT3 stimulates AHSP expression in K562 cells

Studies on the chaperone proteinα-hemoglobin stabilizing protein(AHSP)reveal that abundant AHSP in erythroid cells enhance the cells’tolerance to oxidative stress imposed by excessα-hemoglobin in pathological conditions.However,the potential intracellular modulation of AHSP expression itself in response to oxidative stress is still unknown.The present study examined the effect and molecular mechanism of STAT3,an oxidative regulator,on the expression of AHSP.AHSP expression increased in K562 cells upon cytokine IL-6-induced STAT3 activation and decreased in STAT3 knock-down K562 cells.Regulation of AHSP in oxidative circumstance was then examined inα-globin-overloaded K562 cells,and real-time PCR showed strengthened expression of both AHSP and STAT3.ChIP analysis showed binding of STAT3 to AHSP promoter and binding was significantly augmented with IL6 stimulation and uponα-globin overexpression.Dual luciferase reporter assays of the wildtype and mutated SB3 element,an IL-6RE site,in the AHSP promoter in K562 cells highlighted the direct regulatory effect of STAT3 on AHSP gene.Finally,direct binding of STAT3 to SB3 site of AHSP promoter was confirmed with EMSA assays.Our work reveals an adaptive AHSP regulation mediated by the redox-sensitive STAT3 signaling pathway,and provides clues to the therapeutic strategy for AHSP enhancement.     

Disruption effect of Microplitis bicoloratus polydnavirus EGF-like protein, MbCRP, on actin cytoskeleton in lepidopteran insect hemocytes

Microplitis bicoloratus is a braconid endoparasitic wasp associated with the polydnavirus named Microplitis bicoloratus bracovirus （MbBV）. Parasitism of Spodoptera litura larvae leads to an impaired cellular immune response and to the disappearance of the 42 kDa actin in host hemocytes. In this work, we investigated if the absence of actin in blood cells was related to MbBV infection. An MbBV gene similar to egf-like genes identified in another bracovirus was partially cloned and named Mbcrp1. The full-length gene, named Mbcrp, is transcribed throughout the course of parasitism in host hemocytes and the 30 kDa MbCRP protein was detected in hemocytes 6-7 d post-parasitization. The Mbcrp1 gene contains the cysteine-rich trypsin inhibitor-like （TIL） domain coding sequence and the expression of recombinant MbCRP1 inhibited the expression of the 42 kDa actin in Hi5 cells. The 34.1 kDa MbCRPl-green fluorescent protein fusion protein locate specifically in the cytoplasm. These results suggest that expression of MbCRP in lepidopteran insect cells is related to the disruption of the actin cytoskeleton.     

Genetically engineered K cells provide sufficient insulin to correct hyperglycemia in a nude murine model

A gene therapy-based treatment of type 1 diabetes mellitus requires the development of a surrogate β cell that can synthesize and secrete functionally active insulin in response to physiologically relevant changes in ambient glucose levels. In this study, the murine enteroendocrine cell line STC-1 was genetically modified by stable transfection. Two clone cells were selected (STC-1-2 and STC-1-14) that secreted the highest levels of insulin among the 22 clones expressing insulin from 0 to 157.2 μIU/ml/10⁶ cells/d. After glucose concentration in the culture medium was increased from 1 mM to 10 mM, secreted insulin rose from 40.3±0.8 to 56.3±3.2 μIU/ml (STC-1-2), and from 10.8±0.8 to 23.6±2.3 μIU/ml (STC-1-14). After STC-1-14 cells were implanted into diabetic nude mice, their blood glucose levels were reduced to normal. Body weight loss was also ameliorated. Our data suggested that genetically engineered K cells secrete active insulin in a glucose-regulated manner, and in vivo study showed that hyperglycemia could be reversed by implantation of the cells, suggesting that the use of genetically engineered K cells to express human insulin might provide a glucose-regulated approach to treat diabetic hyperglycemia.     

Nitric oxide stimulates insulin release in liver cells expressing human insulin

The establishment of surrogate islet beta cells is important for the treatment of diabetes. Hepatocytes have a similar glucose sensing system as beta cells and have the potential to serve as surrogate beta cells. In this report, we demonstrate that infection of Hepa1-6 liver cells with a lentivirus expressing the human insulin cDNA results in expression and secretion of human insulin. Furthermore, we show that l-arginine at low levels of glucose significantly stimulates the release of insulin from these cells, compared to exposure to high concentration of glucose. The arginine-induced insulin release is via the production of nitric oxide, since treatment with N(G)-nitro-l-arginine, an inhibitor of nitric oxide synthase, blocks insulin secretion induced by l-arginine. These results indicate that nitric oxide plays a role in l-arginine-stimulated insulin release in hepatocytes expressing the human insulin gene, and provides a new strategy to induce insulin secretion from engineered non-beta cells.     

Effects of dexamethasone on insulin receptor in aging

The aim of this study was to examine the effects of dexamethasone (Dex) on functional properties of the rat insulin receptor (IR). Male Mill Hill hooded rats, 3, 6, 12, 18 and 21 months old, were injected with Dex (4 mg/kg) and rat liver and erythrocytes were used for experiments 18 h after Dex administration. Treatment with Dex lowered the specific binding (SB) of insulin (INS) in the liver of 3- and 18-month-old rats and concentration of INS binding sites (N1, N2) and the dissociation constant of low-affinity binding sites (Kd2) in the liver of 6- and 18-month-old rats. In addition, Dex treatment lowered the liver IR protein level in all analyzed groups, except 21-month-old rats where it remained unchanged, but raised the IR mRNA level in 18-month-old rats. In erythrocytes, treatment with Dex decreased SB and Kd2 (in animals 3 and 6 months old) and N1 (in ones 3 and 18 months old). Following Dex treatment, the INS plasma level increased (in rats 3, 18 and 21 months old), while glucose (Glu) concentration increased in 3 and 12 months old, but decreased in 6- and 21-month-old rats. In summary, Dex exerts the strongest effect on the erythrocyte IR of 3- and 6-month-old rats and the hepatic IR of 18-month-old rats. IR in both tissues is almost insensitive to Dex in 12- and 21-month-old rats. The pattern of age-related changes of IR induced by Dex does not correlate with changes of plasma Glu and INS.     

Simultaneous expression of salivary and pancreatic amylase genes in cultured mouse hepatoma cells.

The tissue-specific expression of two types of mouse amylase genes does not overlap in vivo; the Amy-1 locus is transcribed in the parotid gland and the liver, while expression of Amy-2 is limited to the pancreas. We identified a mouse hepatoma cell line, Hepa 1-6, in which both amylase genes can be simultaneously expressed. Amy-1 is constitutively active in these cells and is inducible by dexamethasone at the level of mRNA. We demonstrated that the liver-specific promoter of Amy-1 is utilized by the dexamethasone-treated hepatoma cells, and that glucocorticoid consensus sequences are present upstream of this promoter. Amy-2 is not detectable constitutively, but can be activated if the cells are cultured in serum-free medium containing dexamethasone. Expression of Amy-2 in a nonpancreatic cell type has not previously been observed. We speculate that induction of Amy-1 and activation of Amy-2 may involve different regulatory mechanisms. Hepa 1-6 cells provide an experimental system for molecular analysis of these events.     

ENPP1 affects insulin action and secretion: evidences from in vitro studies

The aim of this study was to deeper investigate the mechanisms through which ENPP1, a negative modulator of insulin receptor (IR) activation, plays a role on insulin signaling, insulin secretion and eventually glucose metabolism. ENPP1 cDNA (carrying either K121 or Q121 variant) was transfected in HepG2 liver-, L6 skeletal muscle- and INS1E beta-cells. Insulin-induced IR-autophosphorylation (HepG2, L6, INS1E), Akt-Ser(473), ERK1/2-Thr(202)/Tyr(204) and GSK3-beta Ser(9) phosphorylation (HepG2, L6), PEPCK mRNA levels (HepG2) and 2-deoxy-D-glucose uptake (L6) was studied. GLUT 4 mRNA (L6), insulin secretion and caspase-3 activation (INS1E) were also investigated. Insulin-induced IR-autophosphorylation was decreased in HepG2-K, L6-K, INS1E-K (20%, 52% and 11% reduction vs. untransfected cells) and twice as much in HepG2-Q, L6-Q, INS1E-Q (44%, 92% and 30%). Similar data were obtained with Akt-Ser(473), ERK1/2-Thr(202)/Tyr(204) and GSK3-beta Ser(9) in HepG2 and L6. Insulin-induced reduction of PEPCK mRNA was progressively lower in untransfected, HepG2-K and HepG2-Q cells (65%, 54%, 23%). Insulin-induced glucose uptake in untransfected L6 (60% increase over basal), was totally abolished in L6-K and L6-Q cells. GLUT 4 mRNA was slightly reduced in L6-K and twice as much in L6-Q (13% and 25% reduction vs. untransfected cells). Glucose-induced insulin secretion was 60% reduced in INS1E-K and almost abolished in INS1E-Q. Serum deficiency activated caspase-3 by two, three and four folds in untransfected INS1E, INS1E-K and INS1E-Q. Glyburide-induced insulin secretion was reduced by 50% in isolated human islets from homozygous QQ donors as compared to those from KK and KQ individuals. Our data clearly indicate that ENPP1, especially when the Q121 variant is operating, affects insulin signaling and glucose metabolism in skeletal muscle- and liver-cells and both function and survival of insulin secreting beta-cells, thus representing a strong pathogenic factor predisposing to insulin resistance, defective insulin secretion and glucose metabolism abnormalities.