PURPOSE: Primary mammalian hepatocytes largely retain their liver-specific functions when they are freshly derived from donors. However, long-term cultures of functional hepatocytes are difficult to establish. To increase the longevity and maintain the differentiated functions of hepatocytes in primary culture, cells can be cultured in a sandwich configuration of collagen. In such a configuration, hepatocytes can be cultured for longer periods compared with cultures on single layers of collagen. However, research regarding mouse hepatocytes in sandwich culture is lacking. METHODS: Primary mouse hepatocytes were sandwiched between two layers of collagen to maintain the stability of their liver-specific functions. After gelation, 2 mL of hepatocyte culture medium was applied. RESULTS: After 24 hours, 5, 10 days of culture, the collagen gel sandwich maintained the cellular border and numbers of bile canaliculi more efficiently than a single collagen coating in both high and low density culture dishes. Reverse transcription-polymerase chain reaction analysis of alpha-1-antitrypsin (AAT), hepatocyte nuclear factor 4 alpha (HNF4A), alphafetoprotein, albumin, tryptophan oxygenase (TO), the tyrosine aminotransferase gene, glucose-6-phosphatase, glyceraldehyde-3-phosphate dehydrogenase for mouse primary hepatocytes cultured on collagen coated dishes and collagen gels showed superior hepatocyte-related gene expression in cells grown using the collagen gel sandwich culture system. AAT, HNF4A, albumin, TO were found to be expressed in mouse hepatocytes cultured on collagen gels for 5 and 10 days. In contrast, mouse hepatocytes grown on collagen-coated dishes did not express these genes after 5 and 10 days of culture. CONCLUSION: The collagen gel sandwich method is suitable for primary culture system of adult mouse hepatocytes.
Adult ; Animals ; Bile Canaliculi ; Collagen ; Gels ; Gene Expression ; Glucose-6-Phosphatase ; Hepatocyte Nuclear Factor 4 ; Hepatocytes ; Humans ; Longevity ; Mice ; Oxidoreductases ; Tissue Donors ; Tryptophan Oxygenase ; Tyrosine Transaminase

OBJECTIVETo explore MDOY1 gene mutations in pedigrees of early-onset familial type 2 diabetes.

METHODSWe collected 100 early-onset type 2 diabetes pedigrees in Beijing, in which the probands were diagnosed with type 2 diabetes before the age of 40 years with at least one first-degree relative having such a diagnosis before the age of 45 years. PCR was employed to amplify all the exons and exon/intron splice sites of MDOY1 gene and the PCR products were sequenced to identify the DNA variants.

RESULTSTwo DNA variants in the noncoding region including IVS1C +44A>T and IVS2 -5C>T were identified, and 3 mutations in the coding region we identified M49V, T130I, and S462S were found in these pedigrees.

CONCLUSIONCurrently no sufficient evidence has been obtained to identify the variation in or near MDOY1 genes as the major cause of early-onset type 2 diabetic in Chinese population.

Adult ; Age of Onset ; China ; epidemiology ; DNA Mutational Analysis ; Diabetes Mellitus, Type 2 ; epidemiology ; genetics ; Female ; Genetic Testing ; Hepatocyte Nuclear Factor 4 ; genetics ; Humans ; Male ; Mutation ; Pedigree

OBJECTIVETo observe the expression of hepatocyte nuclear factor 4alpha (HNF4alpha) and the activity of key enzyme glucokinase (GK) in glucose metabolism, and further to investigate the possible mechanism of berberine in treating type 2 diabetes.

METHODMouse primary hepatocytes were isolated by an improved single two-step perfusion method. The murine hepatocytes were cultured and incubated with berberine (0, 1, 3, 10, 30, 100 micromol x L(-1)) and 1 mmol x L(-1) metformin for 24 h respectively. The mRNA expression of HNF4alpha were quantified by RT-PCR and the protein expression of HNF4alpha were quantified by Western-blot. And the activity of GK were detected with enzyme kinetics method.

RESULTAs compared with the negative control group, at a certain concentration range, the expression of HNF4alpha mRNA and protein and the activity of GK were promoted by berberine. Both of them reached the top at the concentration of 30 micromol x L(-1) (P<0.01). But the metformin made no difference with the negative control group on the expression of HNF4alpha and the activity of GK.

CONCLUSIONIt is suggested that the effects of berberine on improving glucose metabolism can be mechanically associated with its up-regulating the HNF4a expression and inducing the activity of hepatic glucokinase.

Animals ; Berberine ; pharmacology ; Cell Survival ; drug effects ; Cells, Cultured ; Gene Expression Regulation ; drug effects ; Glucokinase ; genetics ; metabolism ; Hepatocyte Nuclear Factor 4 ; genetics ; metabolism ; Hepatocytes ; cytology ; drug effects ; metabolism ; Male ; Mice ; Plant Extracts ; pharmacology

OBJECTIVETo examine dietary zinc supplementation could alleviate the damage of alcoholic liver disease and the relationship with the expression of hepatocyte nuclear factor 4alpha (HNF-4alpha).

METHODS40 adult C57 BL/6 mice were randomly divided into four groups (n = 10): control, zinc, ethanol and zinc plus ethanol, which were sacrificed after fed four different diets for 6 months. Zinc sulfate was added in the drinking water of the Zinc and Zinc Plus Ethanol group and the content was 75 mg/L. Liver regeneration was assessed by immunohistochemical staining of proliferating cell nuclear antigen (PCNA), and the expression of HNF-4alpha was determined by RT-PCR and Western blot. And as to assess the status of oxidative stress of the mice, malondialdehyde (MDA) and superoxide dismutase (SOD) were detected.

RESULTSCompared with the control group, the expression level of HNF-4alpha decreased significantly in the ethanol group (P < 0.05), and the content of MDA increased significantly in this group, while the content of SOD declined significantly (P < 0.05). Compared with the ethanol group, the number of PCNA-positive hepatocytes increased significantly, and the expression level of HNF-4alpha also increased in the zinc plus ethanol group (P < 0.05), and the content of SOD increased in this group, while MDA decreased significantly (P < 0.05).

CONCLUSIONLong term ethanol exposure can lead to oxidoreduction imbalances which can be reversed by zinc supplementation. We suppose that zinc-enhanced liver regeneration is associated with an increase in HNF-4alpha, suggesting that dietary zinc supplementation may have beneficial effects in alcoholic liver disease.

Animals ; Dietary Supplements ; Hepatocyte Nuclear Factor 4 ; metabolism ; Liver ; metabolism ; Liver Diseases, Alcoholic ; metabolism ; therapy ; Male ; Malondialdehyde ; metabolism ; Mice ; Mice, Inbred C57BL ; Superoxide Dismutase ; metabolism ; Zinc Sulfate ; pharmacology ; therapeutic use

OBJECTIVETo screen the mutation of hepatocyte nuclear factor 4 alpha gene (HNF4A) in Chinese pedigrees with early and/or multiplex-onset diabetes in Shanghai and nearby area.

METHODSBy PCR-single strand conformation polymorphism (PCR-SSCP) and direct sequencing, the mutation screen of HNF4A gene was performed in 93 normal controls and 154 unrelated probands from early- and/or multiplex-onset diabetes. The PCR-RFLP was used to analyze the frequencies of the discovered mutations and variants.

RESULTSTwo synonymous mutations (N153N, A158A) were found in two families, of which the N153N was co-segregated with early-onset diabetes. These two synonymous mutations were not detected in the 93 normal controls. Three variants, IVS1+308(A to G)(rs2071197), IVS1+357(A to T)(rs2071198), IVS1-5(C to T)(rs745975), were also identified in this study. The genotype and allele frequencies of the three variants had no difference between the probands and normal controls.

CONCLUSIONHNF4A gene mutation is rare in Chinese pedigrees with early and/or multiplex-onset diabetes.

Adult ; Age of Onset ; Base Sequence ; China ; epidemiology ; Diabetes Mellitus ; epidemiology ; genetics ; Female ; Gene Frequency ; Genotype ; Hepatocyte Nuclear Factor 4 ; genetics ; Humans ; Male ; Mutation ; Pedigree ; Polymerase Chain Reaction ; Polymorphism, Single-Stranded Conformational ; Sequence Analysis, DNA

Phenylketonuria is an autosomal recessive disorder caused by a deficiency of phenylalanine hydroxylase. Transthyretin has been implicated as an indicator of nutritional status in phenylketonuria patients. In this study, we report that phenylalanine and its metabolite, phenylpyruvic acid, affect MAPK, changing transthyretin expression in a cell- and tissue-specific manner. Treatment of HepG2 cells with phenylalanine or phenylpyruvic acid decreased transcription of the TTR gene and decreased the transcriptional activity of the TTR promoter site, which was partly mediated through HNF4alpha. Decreased levels of p38 MAPK were detected in the liver of phenylketonuria-affected mice compared with wild-type mice. In contrast, treatment with phenylalanine increased transthyretin expression and induced ERK1/2 activation in PC-12 cells; ERK1/2 activation was also elevated in the brainstem of phenylketonuria-affected mice. These findings may explain between-tissue differences in gene expression, including Ttr gene expression, in the phenylketonuria mouse model.
Animals ; Brain Stem/metabolism/pathology ; Disease Models, Animal ; Gene Expression Regulation ; Hep G2 Cells ; Hepatocyte Nuclear Factor 4/metabolism ; Humans ; Liver/*metabolism/pathology ; Mice ; Mice, Mutant Strains ; Mitogen-Activated Protein Kinase 3/genetics/*metabolism ; Organ Specificity ; Phenylalanine/metabolism ; Phenylalanine Hydroxylase/deficiency ; Phenylketonurias/*genetics/metabolism/pathology/physiopathology ; Phenylpyruvic Acids/metabolism ; Prealbumin/*biosynthesis/genetics ; p38 Mitogen-Activated Protein Kinases/genetics/*metabolism

BACKGROUNDKCNJ11, ABCC8, PPARG, and HNF4A have been found to be associated with type 2 diabetes in populations with different genetic backgrounds. The aim of this study was to test, in a Chinese Han population from Beijing, whether the genetic variants in these four genes were associated with genetic predisposition to type 2 diabetes.

METHODSWe studied the association of four representative SNPs in KCNJ11, ABCC8, PPARG, and HNF4A by genotyping them using ABI SNaPshot Multiplex System in 400 unrelated type 2 diabetic patients and 400 unrelated normoglycaemic subjects.

RESULTSrs5219 (E23K) in KCNJ11 was associated with genetic susceptibility to type 2 diabetes (OR = 1.400 with 95% CI 1.117 1.755, P = 0.004 under an additive model, OR = 1.652 with 95% CI 1.086 2.513, P = 0.019 under a recessive model, and OR = 1.521 with 95% CI 1.089 2.123, P = 0.014 under a dominant model) after adjusting for sex and body mass index (BMI). We did not find evidence of association for ABCC8 rs1799854, PPARG rs1801282 (Pro12Ala) and HNF4A rs2144908. Genotype-phenotype correlation analysis revealed that rs1799854 in ABCC8 was associated with 2-hour postprandial insulin secretion (P = 0.005) after adjusting for sex, age and BMI. Although no interactions between the four variants on the risk of type 2 diabetes were detected, the multiplicative interaction between PPARG Pro12Ala and HNF4A rs2144908 was found to be associated with 2-hour postprandial insulin (P = 0.004 under an additive model for rs2144908; and P = 0.001 under a dominant model for rs2144908) after adjusting for age, sex and BMI, assuming a dominant model for PPARG Pro12Ala.

CONCLUSIONSOur study replicated the association of rs5219 in KCNJ11 with type 2 diabetes in Chinese Han population in Beijing. And we also observed that ABCC8 as well as the interaction between PPARG and HNF4A may contribute to post-challenge insulin secretion.

ATP-Binding Cassette Transporters ; genetics ; Adult ; Body Mass Index ; Diabetes Mellitus, Type 2 ; genetics ; Female ; Genetic Predisposition to Disease ; genetics ; Genotype ; Hepatocyte Nuclear Factor 4 ; genetics ; Humans ; Male ; Middle Aged ; PPAR gamma ; genetics ; Polymorphism, Single Nucleotide ; genetics ; Potassium Channels, Inwardly Rectifying ; genetics ; Receptors, Drug ; genetics ; Sulfonylurea Receptors