Berberine Attenuates Development of the Hepatic Gluconeogenesis and Lipid Metabolism Disorder in Type 2 Diabetic Mice and in Palmitate-Incubated HepG2 Cells through Suppression of the HNF-4α miR122 Pathway

Berberine (BBR) has been shown to exhibit protective effects against diabetes and dyslipidemia. Previous studies have indicated that BBR modulates lipid metabolism and inhibits hepatic gluconeogensis by decreasing expression of Hepatocyte Nuclear Factor-4α (HNF-4α). However, the mechanism involved in this process was unknown. In the current study, we examined the mechanism of how BBR attenuates hepatic gluconeogenesis and the lipid metabolism alterations observed in type 2 diabetic (T2D) mice and in palmitate (PA)-incubated HepG2 cells. Treatment with BBR for 4 weeks improve all biochemical parameters compared to T2D mice. Treatment of T2D mice for 4 weeks or treatment of PA-incubated HepG2 cells for 24 h with BBR decreased expression of HNF-4α and the microRNA miR122, the key gluconeogenesis enzymes Phosphoenolpyruvate carboxykinase (PEPCK) and Glucose-6-phosphatase (G6Pase) and the key lipid metabolism proteins Sterol response element binding protein-1 (SREBP-1), Fatty acid synthase-1 (FAS-1) and Acetyl-Coenzyme A carboxylase (ACCα) and increased Carnitine palmitoyltransferase-1(CPT-1) compared to T2D mice or PA-incubated HepG2 cells. Expression of HNF-4α in HepG2 cells increased expression of gluconeogenic and lipid metabolism enzymes and BBR treatment or knock down of miR122 attenuated the effect of HNF-4α expression. In contrast, BBR treatment did not alter expression of gluconeogenic and lipid metabolism enzymes in HepG2 cells with knockdown of HNF-4α. In addition, miR122 mimic increased expression of gluconeogenic and lipid metabolism enzymes in HepG2 cells with knockdown of HNF-4α. These data indicate that miR122 is a critical regulator in the downstream pathway of HNF-4α in the regulation of hepatic gluconeogenesis and lipid metabolism in HepG2 cells. The effect of BBR on hepatic gluconeogenesis and lipid metabolism is mediated through HNF-4α and is regulated downstream of miR122. Our data provide new evidence to support HNF-4α and miR122 regulated hepatic gluconeogenesis and lipid metabolism as promising therapeutic targets for the treatment of T2D.     

The aPKCι blocking agent ATM negatively regulates EMT and invasion of hepatocellular carcinoma

Epithelial-to-mesenchymal transition (EMT) has an important role in invasion and metastasis of hepatocellular carcinoma (HCC). To explore the regulatory mechanism of atypical protein kinase C ι (aPKCι) signaling pathways to HCC development, and find an agent for targeted therapy for HCC, immortalized murine hepatocytes were employed to establish an EMT cell model of HCC, MMH-RT cells. Our study showed that EMT took place in MMH-R cells under the effect of transforming growth factor-β1 (TGF-β1) overexpressing aPKCι. Furthermore, we showed that the aPKCι blocking agent aurothiomalate (ATM) inhibited EMT and decreased invasion of hepatocytes. Moreover, ATM selectively inhibited proliferation of mesenchymal cells and HepG2 cells and induced apoptosis. However, ATM increased proliferation of epithelial cells and had little effect on apoptosis and invasion of epithelial cells. In conclusion, our result suggested that aPKCι could be an important bio-marker of tumor EMT, and used as an indicator of invasion and malignancy. ATM might be a promising agent for targeted treatment of HCC.     

Expression of N-Acetylglucosaminyltransferase III Suppresses α2,3-Sialylation,and Its Distinctive Functions in Cell Migration Are Attributed to α2,6-Sialylation Levels

N-Acetylglucosaminyltransferase III (GnT-III), which catalyzes the addition of the bisecting GlcNAc branch on N-glycans, is usually described as a metastasis suppressor. Overexpression of GnT-III inhibited migration in multiple types of tumor cells. However, these results seem controversial to the clinical observations for the increased expression of GnT-III in human hepatomas, glioma, and ovarian cancers. Here, we present evidence that these inconsistencies are mainly attributed to the different expression pattern of cell sialylation. In detail, we show that overexpression of GnT-III significantly inhibits α2,3-sialylation but not α2,6-sialylation. The migratory ability of cells without or with a low level of α2,6-sialylation is consistently suppressed after GnT-III overexpression. In contrast, the effects of GnT-III overexpression are variable in tumor cells that are highly α2,6-sialylated. Overexpression of GnT-III promotes the cell migration in glioma cells U-251 and hepatoma cells HepG2, although it has little influence in human breast cancer cell MDA-MB-231 and gastric cancer cell MKN-45. Interestingly, up-regulation of α2,6-sialylation by overexpressing β-galactoside α2,6-sialyltranferase 1 in the α2,6-hyposialylated HeLa-S3 cells abolishes the anti-migratory effects of GnT-III. Conversely, depletion of α2,6-sialylation by knock-out of β-galactoside α2,6-sialyltranferase 1 in α2,6-hypersialylated HepG2 cells endows GnT-III with the anti-migratory ability. Taken together, our data clearly demonstrate that high expression of α2,6-sialylation on the cell surface could affect the anti-migratory role of GnT-III, which provides an insight into the mechanistic roles of GnT-III in tumor metastasis.     

Formation of a large, complex domain of histone hyperacetylation at human 14q32.1 requires the serpin locus control region

The human serine protease inhibitor (serpin) gene cluster at 14q32.1 is a useful model system to study cell-type-specific gene expression and chromatin structure. Activation of the serpin locus can be induced in vitro by transferring human chromosome 14 from non-expressing to expressing cells. Serpin gene activation in expressing cells is correlated with locus-wide alterations in chromatin structure, including the de novo formation of 17 expression-associated DNase I-hypersensitive sites (DHSs). In this study, we investigated histone acetylation throughout the proximal serpin subcluster. We report that gene activation is correlated with high levels of histone H3 and H4 acetylation at serpin gene promoters and other regulatory regions. However, the locus is not uniformly hyperacetylated, as there are regions of hypoacetylation between genes. Furthermore, genetic tests indicate that locus-wide controls regulate both gene expression and chromatin structure. For example, deletion of a previously identified serpin locus control region (LCR) upstream of the proximal subcluster reduces both gene expression and histone acetylation throughout the ~130 kb region. A similar down regulation phenotype is displayed by transactivator-deficient cell variants, but this phenotype can be rescued by transfecting the cells with expression cassettes encoding hepatocyte nuclear factor-1α (HNF-1α) or HNF-4. Taken together, these results suggest that histone acetylation depends on interactions between the HNF-1α/HNF-4 signaling cascade and the serpin LCR.     

HCV+ Hepatocytes Induce Human Regulatory CD4+ T Cells through the Production of TGF-β

Background

Hepatitis C Virus (HCV) is remarkably efficient at establishing persistent infection and is associated with the development of chronic liver disease. Impaired T cell responses facilitate and maintain persistent HCV infection. Importantly, CD4⁺ regulatory T cells (Tregs) act by dampening antiviral T cell responses in HCV infection. The mechanism for induction and/or expansion of Tregs in HCV is unknown.

Methodology/Principal Findings

HCV-expressing hepatocytes were used to determine if hepatocytes are able to induce Tregs. The infected liver environment was modeled by establishing the co-culture of the human hepatoma cell line, Huh7.5, containing the full-length genome of HCV genotype 1a (Huh7.5-FL) with activated CD4⁺ T cells. The production of IFN-γ was diminished following co-culture with Huh7.5-FL as compared to controls. Notably, CD4⁺ T cells in contact with Huh7.5-FL expressed an increased level of the Treg markers, CD25, Foxp3, CTLA-4 and LAP, and were able to suppress the proliferation of effector T cells. Importantly, HCV⁺ hepatocytes upregulated the production of TGF-β and blockade of TGF-β abrogated Treg phenotype and function.

Conclusions/Significance

These results demonstrate that HCV infected hepatocytes are capable of directly inducing Tregs development and may contribute to impaired host T cell responses.     

Laron Dwarfism and Non-Insulin-Dependent Diabetes Mellitus in the Hnf-1α Knockout Mouse

Mice deficient in hepatocyte nuclear factor 1 alpha (HNF-1α) were produced by use of the Cre-loxP recombination system. HNF-1α-null mice are viable but sterile and exhibit a phenotype reminiscent of both Laron-type dwarfism and non-insulin-dependent diabetes mellitus (NIDDM). In contrast to an earlier HNF-1α-null mouse line that had been produced by use of standard gene disruption methodology (M. Pontoglio, J. Barra, M. Hadchouel, A. Doyen, C. Kress, J. P. Bach, C. Babinet, and M. Yaniv, Cell 84:575–585, 1996), these mice exhibited no increased mortality and only minimal renal dysfunction during the first 6 months of development. Both dwarfism and NIDDM are most likely due to the loss of expression of insulin-like growth factor I (IGF-I) and lower levels of insulin, resulting in stunted growth and elevated serum glucose levels, respectively. These results confirm the functional significance of the HNF-1α regulatory elements that had previously been shown to reside in the promoter regions of both the IGF-I and the insulin genes.     

Fish Oil and Fenofibrate Prevented Phosphorylation-dependent Hepatic Sortilin 1 Degradation in Western Diet-fed Mice

Obesity and diabetes are associated with hepatic triglyceride overproduction and hypertriglyceridemia. Recent studies have found that the cellular trafficking receptor sortilin 1 (Sort1) inhibits hepatic apolipoprotein B secretion and reduces plasma lipid levels in mice, and its hepatic expression was negatively associated with plasma lipids in humans. This study investigated the regulation of hepatic Sort1 under diabetic conditions and by lipid-lowering fish oil and fenofibrate. Results showed that hepatic Sort1 protein, but not mRNA, was markedly lower in Western diet-fed mice. Knockdown of hepatic Sort1 increased plasma triglyceride in mice. Feeding mice a fish oil-enriched diet completely restored hepatic Sort1 levels in Western diet-fed mice. Fenofibrate also restored hepatic Sort1 protein levels in Western diet-fed wild type mice, but not in peroxisome proliferator-activated receptor α (PPARα) knock-out mice. PPARα ligands did not induce Sort1 in hepatocytes in vitro. Instead, fish oil and fenofibrate reduced circulating and hepatic fatty acids in mice, and n-3 polyunsaturated fatty acids prevented palmitate inhibition of Sort1 protein in HepG2 cells. LC/MS/MS analysis revealed that Sort1 phosphorylation at serine 793 was increased in obese mice and in palmitate-treated HepG2 cells. Mutations that abolished phosphorylation at Ser-793 increased Sort1 stability and prevented palmitate inhibition of Sort1 ubiquitination and degradation in HepG2 cells. In summary, therapeutic strategies that prevent posttranslational hepatic Sort1 down-regulation in obesity and diabetes may be beneficial in improving dyslipidemia.     

βKlotho Suppresses Tumor Growth in Hepatocellular Carcinoma by Regulating Akt/GSK-3β/Cyclin D1 Signaling Pathway

βKlotho is a regulator in multiple metabolic processes, while its role in cancer remains unclear. We found the expression of βKlotho was down-regulated in human hepatocellular carcinoma tissues compared with that in paired adjacent non-tumourous liver tissues. Hepatoma cells also showed decreased expression of βKlotho compared with normal hepatocyte cells. Reintroduction of βKlotho into hepatoma cells inhibited their proliferation. The anti-proliferative effect of βKlotho might be linked with G1 to S phase arrest, which was mediated by Akt/GSK-3β/cyclin D1 signaling, since forced expression βKlotho reduced the phosphorylation level of Akt and GSK-3β and induced down-regulation of cyclin D1. Furthermore, βKlotho overexpression could inhibit tumorgenesis, while constitutively activated Akt could override the suppressive effects of βKlotho in vivo. These data suggest βKlotho suppresses tumor growth in hepatocellular carcinoma.