Yiqing Mao

Resistin and insulin resistance in hepatocytes: Resistin disturbs glycogen metabolism at the protein level

Resistin has been considered to link obesity with type 2 diabetes. Liver glycogen metabolism plays an essential role in maintaining glucose homeostasis, we investigated the effect of resistin on liver glycogen metabolism and attempted to identify its role in initiating insulin resistance and type 2 diabetes. Primary culture of rat hepatocytes was treated by resistin and insulin. Glycogen content was determined by the anthrone-reagent method. Real-time PCR, Western blot and enzymatic activity assay were used to detect key enzymes and genes involved in glucose metabolism. Hepatocytes exposed to resistin, but only in the presence of insulin, show a decrease in insulin-stimulated glycogen content. Decreased insulin receptor expression and GS activity and elevated GP activity was observed after the treatment of hepatocytes with resistin. No significant changes in the expression of the genes for these proteins were observed. These results strongly suggest that resistin effects glycogen metabolism at the protein level, and resistin is highly associated with insulin resistance and type 2 diabetes and is a candidate for the prevention and treatment of type 2 diabetes. Our results should lead to the development of novel strategies for the treatment of type 2 diabetes.

Exposure of tumor-associated macrophages to apoptotic MCF-7 cells promotes breast cancer growth and metastasis.

Tumor-associated macrophages (TAMs) have been found to be associated with the progression and metastasis of breast cancer. To clarify the mechanisms underlying the crosstalk between TAMs and cancer stem cells (CSCs) in breast cancer recurrence and metastasis, we used a co-culture model of macrophages and apoptotic human breast cancer cell line MCF-7 cells to investigate the effects of TAMs on MCF-7 in vitro and in vivo. Macrophages co-cultured with apoptotic MCF-7 had increased tumor growth and metastatic ability in a nude mouse transplantation assay. The macrophages exposed to apoptotic cells also induce an increase in the proportion of CD44+/CD24− cancer stem-like cells, as well as their proliferative ability accompanied with an increase in mucin1 (MUC1) expression. During this process, macrophages secreted increased amounts of interleukin 6 (IL-6) leading to increased phosphorylation of signal transducers and activators of transcription 3 (STAT3), which likely explains the increased transcription of STAT3 target genes such as TGF-β1 and HIF-1α. Our results indicate that when cancer cells endure chemotherapy induced apoptosis, macrophages in their microenvironment can then activate cancer stem cells to promote cancer growth and metastasis by secreting IL-6, which activates STAT3 phosphorylation to regulate the transcription of its downstream target genes.

Differential expression of genes and changes in glucose metabolism in the liver of liver-specific glucokinase gene knockout mice.

To investigate the role of liver-specific expression of glucokinase (GCK) in the pathogenesis of hyperglycemia and to identify candidate genes involved in mechanisms of the onset and progression of maturity onset diabetes of the young, type 2 (MODY-2), we examined changes in biochemical parameters and gene expression in GCK knockout (gck(w/-)) and wild-type (gck(w/w)) mice as they aged. Fasting blood glucose levels were found to be significantly higher in the gck(w/-) mice, compared to age-matched gck(w/w) mice, at all ages (P<0.05), except at 2 weeks. GCK activity of gck(w/-) mice was about 50% of that of wild type (gck(w/w)) mice (P<0.05). Glycogen content at 4 and 40 weeks of age was lower in gck(w/-) mice compared to gck(w/w) mice. Differentially expressed genes in the livers of 2 and 26 week-old liver-specific GCK knockout (gck(w/-)) mice were identified by suppression subtractive hybridization (SSH), which resulted in the identification of phosphoenolpyruvatecarboxykinase (PEPCK, also called PCK1) and Sterol O-acyltransferase 2 (SOAT2) as candidate genes involved in pathogenesis. The expressions of PEPCK and SOAT2 along with glycogen phosphorylase (GP) and glycogen synthase (GS) were then examined in GCK knockout (gck(w/-)) and wild-type (gck(w/w)) mice at different ages. Changes in PEPCK mRNA levels were confirmed by real-time RT-PCR, while no differences in the levels of expression of SOAT2 or GS were observed in age-matched GCK knockout (gck(w/-)) and wild-type (gck(w/w)) mice. GP mRNA levels were decreased in 40-week old gck(w/-) mice compared to age-matched gck(w/w) mice. Changes in gluconeogenesis, delayed development of GCK and impaired hepatic glycogen synthesis in the liver potentially lead to the onset and progression of MODY2.

Resistin disrupts glycogen synthesis under high insulin and high glucose levels by down-regulating the hepatic levels of GSK3β

The effect of mouse resistin on hepatic insulin resistance in vivo and in vitro, and its possible molecular mechanism were examined. Focusing on liver glycogen metabolism and gluconeogenesis, which are important parts of glucose metabolism, in primary cultures of rat hepatocytes we found that glycogen content was significantly lower (P<0.05) after treatment with recombinant murine resistin only in the presence of insulin plus glucose stimulation. Protein levels of factors in the insulin signaling pathway involved in glycogen synthesis were examined by Western blot analysis, with the only significant change observed being the level of phosphorylated (at Ser 9) glycogen synthase kinase-3β (GSK-3β) (P<0.001). No differences in the protein levels for the insulin receptor β (IRβ), insulin receptor substrates (IRS1 and IRS2), phosphatidylinositol 3-kinase (PI3K), protein kinase B (Akt) or their phosphorylated forms were observed between control and resistin treated primary rat hepatocytes. In a mouse model with high liver-specific expression of resistin, fasting blood glucose levels and liver glycogen content changed. Fasting blood glucose levels were significantly higher (P<0.001) in the model mice, compared to the control mice, while the glycogen content of the liver tissue was about 60% of that of the control mice (P<0.05). The gluconeogenic response was not altered between the experimental and control mice. The level of phosphorylated GSK-3β in the liver tissue was also decreased (P<0.05) in the model mice, consistent with the results from the primary rat hepatocytes. Our results suggest that resistin reduces the levels of GSK-3β phosphorylated at Ser 9 leading to impaired hepatic insulin action in primary rat hepatocytes and in a mouse model with high liver-specific expression of resistin.

Expression of the Human Glucokinase Gene: Important Roles of the 5′ Flanking and Intron 1 Sequences

Background Glucokinase plays important tissue-specific roles in human physiology, where it acts as a sensor of blood glucose levels in the pancreas, and a few other cells of the gut and brain, and as the rate-limiting step in glucose metabolism in the liver. Liver-specific expression is driven by one of the two tissue-specific promoters, and has an absolute requirement for insulin. The sequences that mediate regulation by insulin are incompletely understood. Methodology/Principal Findings To better understand the liver-specific expression of the human glucokinase gene we compared the structures of this gene from diverse mammals. Much of the sequence located between the 5′ pancreatic beta-cell-specific and downstream liver-specific promoters of the glucokinase genes is composed of repetitive DNA elements that were inserted in parallel on different mammalian lineages. The transcriptional activity of the liver-specific promoter 5′ flanking sequences were tested with and without downstream intronic sequences in two human liver cells lines, HepG2 and L-02. While glucokinase liver-specific 5′ flanking sequences support expression in liver cell lines, a sequence located about 2000 bases 3′ to the liver-specific mRNA start site represses gene expression. Enhanced reporter gene expression was observed in both cell lines when cells were treated with fetal calf serum, but only in the L-02 cells was expression enhanced by insulin. Conclusions/Significance Our results suggest that the normal liver L-02 cell line may be a better model to understand the regulation of the liver-specific expression of the human glucokinase gene. Our results also suggest that sequences downstream of the liver-specific mRNA start site have important roles in the regulation of liver-specific glucokinase gene expression.

Differential Expression of Genes Associated with the Progression of Renal Disease in the Kidneys of Liver-Specific Glucokinase Gene Knockout Mice

Liver glucokinase (GCK) deficient mice possess mild renal complications associated with diabetes. To investigate the progression of kidney disease and identify candidate genes involved in the pathogenesis of renal damage, we examined changes in tissue structure and gene expression in the kidneys of liver-specific GCK knockout (gckw/−) mice and age-matched normal wild-type control (gckw/w) mice as they aged. Suppression subtractive hybridization (SSH) was used to identify candidate genes that showed a pattern of differential expression between kidneys of gckw/− and gckw/w mice at 60 weeks of age. Differential expression of the candidate genes was examined by real-time qPCR in liver-specific gckw/− and gckw/w mice at 16, 26, 40, 60, and 85 weeks of age. Among the candidate genes, only glutathione peroxidase-3 (GPX3) was confirmed to show differential expression by qPCR in the 60-week old mice, however two others genes, MALAT1 and KEG, showed significant changes at other ages. This study shows that liver-specific glucokinase deficient mice display changes in kidney morphology by 40 weeks of age, and that renal complication may be correlated with a reduction in GPX3 levels. Since decreased GPX3 mRNA expression was observed at 26 weeks, which is younger than the age when pathological changes can be seen in kidney biopsies, GPX3 may serve as an early marker for kidney damage.

Long-term renal changes in the liver-specific glucokinase knockout mouse: implications for renal disease in maturity-onset diabetes of the young 2

To investigate the functional and structural renal changes in a long-term liver-specific glucokinase (gck) knockout mouse, a model was developed of maturity-onset diabetes of the young (MODY2). Hemizygous gck knockout mice, gck(w/-) groups, were compared at 6, 10, and 14 months with their age-matched normal littermates, gck(w/w) groups. To examine changes, we compared body weight, fasting blood glucose, serum insulin, and creatinine levels, as well as 24-h urine samples that were collected for urine volume and protein analysis between the 2 groups. Renal tissues were collected and stained with hemotoxylin-eosin and periodic-acid Schiff for light microscopic observation. The expression of renal transforming growth factor β1 (TGF-β1) was determined by Western blot. Our results show that fasting blood glucose levels were significantly higher in gck(w/-) mice compared with gck(w/w) mice (P < 0.01) for all age groups. Compared with age-matched gck(w/w) mice, 10-month old gck(w/-) mice have significantly elevated body weights (P < 0.01) and protein contents (P < 0.001). A gradual increase in mesangial matrix and a thickening of the glomerular basement membrane was observed in gck(w/-) mice at 10 and 14 months. The levels of renal TGF-β1 expression are increasing in both gck(w/-) and gck(w/w) mice. Our results indicate that renal changes occur in the liver-specific gck knockout mouse model of MODY2 and suggest that TGF-β1 may play a key role in pathogenesis of these renal changes.

An over expression APP model for anti-Alzheimer disease drug screening created by zinc finger nuclease technology.

Zinc Finger Nucleases (ZFNs), famous for their ability to precisely and efficiently modify specific genomic loci, have been employed in numerous transgenic model organism and cell constructions. Here we employ the ZFNs technology, with homologous recombination (HR), to construct sequence-specific Amyloid Precursor Protein (APP) knock-in cells. With the use of ZFNs, we established APP knock in cell lines with gene-modification efficiencies of about 7%. We electroporated DNA fragment containing the promoter and the protein coding regions of the zinc finger nucleases into cells, instead of the plasmids, to avoid problems associated with off target homologous recombination, and adopted a pair of mutated FokI cleavage domains to reduce the toxic effects of the ZFNs on cell growth. Since over-expression of APP, or a subdomain of it, might lead to an immediately lethal effect, we used the Cre-LoxP System to regulate APP expression. Our genetically transformed cell lines, w5c1 and s12c8, showed detectable APP and Amyloid β (Aβ) production. The Swedish double mutation in the APP coding sequence enhanced APP and Aβ abundance. What is more, the activity of the three key secretases in Aβ formation could be modulated, indicating that these transgenic cells have potential for drug screening to modify amyloid metabolism in cells. Our transformed cells could readily be propagated in culture and should provide an excellent experimental medium for elucidating aspects of the molecular pathogenesis of Alzheimer’s disease, especially those concerning the amyloidogenic pathways involving mutations in the APP coding sequence. The cellular models may also serve as a tool for deriving potentially useful therapeutic agents.

Staurosporine Induced Apoptosis May Activate Cancer Stem-Like Cells (CD44+/CD24-) in MCF-7 by Upregulating Mucin1 and EpCAM

Malignant tumors recur after chemotherapy. A small population of cancer stem-like cells within tumors is now generally considered the prime source of the recurrence. To better understand how cancer stem-like cells induce relapse after fractionated chemotherapy, we examined changes in the CD44+/CD24- cancer stem-like cells population and behavior using the breast cancer cell line MCF-7. Our results show that apart from an increase in the CD44+/CD24- population, proliferation and clone formation, but not migration, were enhanced after recovery from apoptosis induced by two pulses of staurosporine (STS). The distribution of cells in the cell cycle differed between acutely induced apoptosis and fractionated chemotherapy. Sorted CD44+/CD24- stem-like cells from MCF-7 cells recovered from STS treatment possessed greater proliferation abilities. We also observed that mucin1 (MUC1) and Epithelial cell adhesion molecule (EpCAM) were up-regulated in abundance coincidently with proliferation and clone formation enhancement. Our findings suggest that fractionated chemotherapy induced apoptosis could stimulate cancer stem-like cell to behave with a stronger malignant property than cancer cells themselves and MUC1 and EpCAM are important factors involving in this process. By demonstrating changes in cancer stem cell during chemotherapy and identifying the crucial factors, we potentially can target them, to eradicate tumors and overcome cancer relapse.

Xinmailong mitigated epirubicin-induced cardiotoxicity via inhibiting autophagy.

ETHNOPHARMACOLOGICAL RELEVANCE Using insects, such as the cockroach, for the treatment of disease has a long history in traditional Chinese medicine. Xinmailong (XML) Injection, a bioactive composite extracted from Periplaneta americana (a species of cockroach), shows reasonable protective effects against cardiovascular injury and was approved for the use in the treatment of cardiac dysfunction in 2006, yet its cardio protective mechanisms remain unclear. AIM The present study aims to examine the protective effects of XML against epirubicin-induced cardiotoxicity in vivo and determine its underlying mechanisms. MATERIALS AND METHODS The chemical characteristics of XML were identified using high performance liquid chromatography (HPLC). Rats were intraperitoneally injected with epirubicin and then treated with XML for 14 days. Survival rate, echocardiography, electrocardiographic recordings and Masson staining were used to evaluate the cardioprotective activity of XML. Western blot and quantitative real time reverse transcriptase polymerase chain reaction (RT-PCR) analyses were used to investigate the molecular mechanisms underlying the actions of XML. RESULTS XML treatment significantly enhanced the survival rate of rats from epirubicin-induced heart failure. XML prevented left ventricle dilatation, improved cardiac function. Furthermore, treatment with XML also significantly inhibited the accumulation of collagen, reduced the levels of mRNA for matrix metalloproteinases-9 (Mmp9) and transforming growth factor-β 1(Tgfb1). This action of XML therefore might be responsible, at least in part, for the attenuation of cardiac fibrotic remodeling. XML inhibited autophagy as evidenced by the decreased accumulation of Beclin1 and autophagy related 7 (Atg7), which are necessary to form autophagosome structures. Protein kinase B (PKB/Akt), phosphatidylinositol 3 kinase (PI3K) and B cell lymphoma2 (Bcl2) levels were up-regulated, while significantly decreased protein levels for phosphorylated P38 and extracellular regulated protein kinases 1/2 (Erk1/2) were observed in the XML treated rats. The autophagy related results suggested that the increase in PI3K/Akt levels and inhibition of the phosphorylation of P38 MAPK and Erk1/2 contributed to the anti-autophagic activity of XML. CONCLUSIONS Our data suggest that XML may be effective for mitigating epirubicin-induced cardiomyopathy and inhibits autophagy via activating the PI3K/Akt signaling pathway and inhibiting the Erk1/2 and P38 MAPK signaling pathways.