Compensatory increase in lipogenic gene expression in adipose tissue of transgenic mice expressing constitutively active AMP-activated protein kinase-alpha1 in liver

We previously described a line of transgenic mice selectively expressing constitutively active AMPK-α1 under the control of liver-specific human apoE promoter with the hepatic control region sequence. In the short-term activation, the CA-AMPK-α1 transgenic mice at age 10–12 weeks exhibited normal hepatic triglyceride content as compared to wild-type mice due to compensatory increase in mRNA expression of genes in the cholesterol and fatty acid synthesis pathways. But it was not known whether the lipogenic gene expression in white adipose tissue also changed. Here we characterized mRNA expression profile of main lipogenic genes in the cholesterol and fatty acid biosynthesis pathway in white adipose tissue. The data show that short-term chronic activation of AMPK in liver caused marked compensatory increase in lipogenic gene expression both in liver due to induction of Srebp-2 and in white adipose tissue due to upregulation of Srebp-1c. These results support the notion that in addition to its well-recognized function for fat storage adipose tissue can play an adaptive role in fatty acid synthesis when fatty acid synthesis is severely reduced in liver, the main lipogenic organ in mammals.     

Blocking of G1/S transition and cell death in the regenerating liver of Hepatitis B virus X protein transgenic mice

The Hepatitis B virus X (HBx) protein has been strongly implicated in the carcinogenesis of hepatocellular carcinoma (HCC). However, effects of the HBx protein on cell proliferation and cell death are controversial. This study investigates the effects of the HBx protein on liver regeneration in two independent lines of HBx transgenic mice, which developed HCC at around 14 to 16 months of age. High mortality, lower liver mass restoration, and impaired liver regeneration were found in the HBx transgenic mice post-hepatectomy. The levels of alanine aminotransferase and alpha-fetoprotein detected post-hepatectomy increased significantly in the HBx transgenic livers, indicating that they were more susceptible to damage during the regenerative process. Prolonged activation of the immediate-early genes in the HBx transgenic livers suggested that the HBx protein creates a strong effect by promoting the transition of the quiescent hepatocytes from G0 to G1 phase. However, impaired DNA synthesis and mitosis, as well as inhibited activation of G1, S, and G2/M markers, were detected. These results indicated that HBx protein exerted strong growth arrest on hepatocytes and imbalanced cell-cycle progression resulting in the abnormal cell death; this was accompanied by severe fat accumulation and impaired glycogen storage in the HBx transgenic livers. In conclusion, this study provides the first physiological evidence that HBx protein blocks G1/S transition of the hepatocyte cell-cycle progression and causes both a failure of liver functionality and cell death in the regenerating liver of the HBx transgenic mice.     

Decreased GLT-1 and increased SOD1 and HO-1 expression in astrocytes contribute to lumbar spinal cord vulnerability of SOD1-G93A transgenic mice

The SOD1-G93A transgenic mouse is a widely used ALS model, but the death of lower motor neurons is the hallmark. Here, we show that the SOD1-G93A transgene and HO-1 are preferentially over-expressed in the lumbar spinal cord, particularly in the activated astrocytes of the transgenic mice. We also show down-regulation of GLT-1 in spite of the proliferating astrocytes. However, GLT-1, SOD1-G93A transgene and HO-1 expression were not obviously changed in the motor cortex. Our data link spinal cord vulnerability to relatively decreased expression of GLT-1, and high expression of the transgene and HO-1 in astrocytes in SOD1-G93A transgenic mice.     

Overexpression of Nd1, a novel Kelch family protein, in the heart of transgenic mice protects against doxorubicin-induced cardiomyopathy

Doxorubicin is one of the most effective drugs available for cancer chemotherapy. However, the clinical use of doxorubicin has been greatly limited because of severe side effects on cardiomyocytes. Since Nd1-L, a novel actin-binding protein, is expressed most abundantly in the heart of adult mice, we examined a role of Nd1-L in doxorubicin-induced cardiomyopathy. When doxorubicin (5 mg/kg × 4 times) was injected into adult mice at a 3-day-interval, approximately 50% of injected mice died within 4 weeks of the first injection. Nd1-L mRNA expression in the heart decreased within 3 weeks after the first injection and many cardiomyocytes of injected mice died by apoptosis. Overexpression of Nd1-L in the heart of transgenic mice protected the cardiomyocytes from apoptosis and improved survival rate after doxorubicin injection. Furthermore, activation of Erk1/2 was observed in cultured cells overexpressing Nd1-L. Thus, Nd1-L plays a critical role in protecting the heart from doxorubicin-induced cardiomyopathy.     

Overexpression of TIMP-1 under the MMP-9 promoter interferes with wound healing in transgenic mice

We have generated transgenic mice harboring the murine matrix metalloproteinase 9 (MMP-9) promoter cloned in front of human TIMP-1 cDNA. The transgenic mice were viable and fertile and exhibited normal growth and general development. During wound healing the mice were shown to express human TIMP-1 in keratinocytes that normally express MMP-9. However, the healing of skin wounds was significantly retarded with slow migration of keratinocytes over the wound in transgenic mice. In situ zymography carried out on wound tissues revealed total blockage of gelatinolytic activity (i.e., MMP-9 and MMP-2). The results confirm studies with MMP-9 knockout mice showing that MMP-9 is not essential for general development, but they also demonstrate an important role of keratinocyte MMP-9, as well that of other keratinocyte MMPs that are inhibited by TIMP-1, in wound healing. The transgenic mice generated in this study provide a model for the role of MMPs in MMP-9-producing cells in other challenging situations such as bone fracture recovery and cancer invasion.The expert technical assistance of M. Jarva, L. Ollitervo, S. Kangas, and R. Jokisalo is gratefully acknowledged. This work was supported in part by grants from the Finnish Academy of Science, the Swedish Cancer Foundation, the Novo Nordisk Foundation and EC contract QLG1-CT-2000-01131 (K.T.), the Finnish Dental Society Apollonia and the Northern Finland Cancer Foundation (M.P.), as well as the K. Albin Johansson Foundation and the Einar and Karin Stroems Foundation (E.P.)     

口蹄疫病毒VP1基因转化马铃薯及其块茎专一性表达

报道了FMDV VP1基因与马铃薯块茎专一性表达class Ipatatin基因5′区融合,经农杆菌介导导入马铃薯植株,PCR、RT-PCR证实了其整合及转录表达。ELISA结果进一步表明,VP1在转基因马铃薯块茎中具有免疫活性。为探讨在马铃薯块茎中高表达VP1蛋白及进一步开发其作为FMDV口服疫苗生物反应器奠定基础。     

Expression of humanized anti-Her2/neu single-chain IgG1-like antibody in mammary glands of transgenic mice

A system for production of single-chain antibody in mammary glands of mice was developed on the basis of a hybrid gene constructed from the coding sequence of anti-Her2/neu single-chain antibody inserted into the first exon of the sheep beta-lactoglobulin gene. Lines of transgenic mice were obtained that expressed humanized single-chain anti-Her2/neu IgG1-like antibody in their milk. These antibodies interact with Her2/neu antigen with high affinity (K_d = 0.4 nM). The expression level of the transgene depended on its integration site in the genome but not on the copy number. The transgene had no toxic effect on the mice and was stably inherited, at least for two generations. The results reveal new opportunities of producing single-chain antibodies in the milk of animals.     

Overexpression of ABIN-2, a negative regulator of NF-kappaB, delays liver regeneration in the ABIN-2 transgenic mice

Activation of NF-kappaB is one of the earliest responses at the start of liver regeneration, and is required for hepatocyte cell cycle progression. The A20-binding inhibitor of NF-kappaB activation-2, ABIN-2, is an inhibitor of NF-kappaB. However, its effects on hepatocyte cell cycle progression are not known and its involvement in liver regeneration has not been explored. In this study, the temporal expression pattern of the mouse ABIN-2 was studied during liver regeneration induced by partial hepatectomy. We demonstrate that ABIN-2 is rapidly and transiently induced, and expression peaked at around 8h post-hepatectomy. To test that the inducible expression of ABIN-2 serves to regulate NF-kappaB during liver regeneration, transgenic mice overexpressing human ABIN-2 protein in the liver were generated. Our transgenic data demonstrated that overexpression of ABIN-2 inhibited NF-kappaB nuclear translocation, which peaked at around 2-4h post-hepatectomy, and this led to an impairment of the G1/S transition as well as a delay in hepatocyte cell cycle progression of the regenerating liver. In addition, overexpression of ABIN-2 specifically inhibited endogenous ABIN-2 mRNA induction, suggesting a negative feedback mechanism for ABIN-2 expression. In conclusion, ABIN-2 may function as a negative regulator that downregulates NF-kappaB activation during liver regeneration.     

MUC1-specific immune responses in human MUC1 transgenic mice immunized with various human MUC1 vaccines

Analyses of MUC1-specific cytotoxic T cell precursor (CTLp) frequencies were performed in mice immunized with three different MUC1 vaccine immunotherapeutic agents. Mice were immunized with either a fusion protein comprising MUC1 and glutathione S-transferase (MUC1-GST), MUC1-GST fusion protein coupled to mannan (MFP) or with a recombinant vaccinia virus expressing both MUC1 and interleukin-2. Mouse strain variations in immune responsiveness have been observed with these vaccines. We have constructed mice transgenic for the human MUC1 gene to study MUC1-specific immune responses and the risk of auto-immunity following MUC1 immunization. Transgenic mice immunized with MUC1 were observed to be partially tolerant in that the MUC1-specific antibody response is lower than that observed in syngeneic but non-transgenic mice. However, a significant MUC1-specific CTLp response to all three vaccines was observed, indicating the ability to overcome T cell, but to a lesser extent B cell, tolerance to MUC1 in these mice. Histological analysis indicates no evidence of auto-immunity to the cells expressing the human MUC1 molecule. These results suggest that it is possible to generate an immune response to a cancer-related antigen without damage to normal tissues expressing the antigen. Received: 7 July 1999 / Accepted: 26 August 1999     

Genetic model for the chronic activation of skeletal muscle AMP-activated protein kinase leads to glycogen accumulation

The AMP-activated protein kinase (AMPK) is an important metabolic sensor/effector that coordinates many of the changes in mammalian tissues during variations in energy availability. We have sought to create an in vivo genetic model of chronic AMPK activation, selecting murine skeletal muscle as a representative tissue where AMPK plays important roles. Muscle-selective expression of a mutant noncatalytic gamma1 subunit (R70Qgamma) of AMPK activates AMPK and increases muscle glycogen content. The increase in glycogen content requires the presence of the endogenous AMPK catalytic alpha-subunit, since the offspring of cross-breeding of these mice with mice expressing a dominant negative AMPKalpha subunit have normal glycogen content. In R70Qgamma1-expressing mice, there is a small, but significant, increase in muscle glycogen synthase (GSY) activity associated with an increase in the muscle expression of the liver isoform GSY2. The increase in glycogen content is accompanied, as might be expected, by an increase in exercise capacity. Transgene expression of this mutant AMPKgamma1 subunit may provide a useful model for the chronic activation of AMPK in other tissues to clarify its multiple roles in the regulation of metabolism and other physiological processes.     

Studies of in vivo mutations in rpsL transgene in UVB-irradiated epidermis of XPA-deficient mice

We have established xeroderma pigmentosum group A (XPA) gene-knockout mice with nucleotide excision repair (NER) deficiency, which rapidly developed skin tumors when exposed to a low dose of chronic UV like XP-A patients, confirming that the NER process plays an important role in preventing UVB-induced skin cancer. To examine the in vivo mutation in the UVB-irradiated epidermis, we established XPA (−/−), (+/−) and (+/+) mice carrying the Escherichia coli rpsL transgene with which the mutation frequencies and spectra in the UVB-irradiated epidermal tissue can be examined conveniently. The XPA (−/−) mice showed a higher frequency of UVB-induced mutation in the rpsL transgene with a low dose (150 J/m²) of UVB-irradiation than the XPA (+/−) and (+/+) mice, while, at a high dose (900 J/m²) they showed almost the same frequency of mutation as the XPA (+/−) and (+/+) mice, probably because of cell death in the epidermis of the XPA (−/−) mice. However, CC→TT tandem transition, a hallmark of UV-induced mutation, was detected at higher frequency in the XPA (−/−) mice than the XPA (+/−) and (+/+) mice at both doses of UVB. This rpsL/XPA mouse system will be useful for further analyzing the role of NER in the mutagenesis and carcinogenesis induced by various carcinogens.     

Chromatin breakdown by deoxyribonuclease1 promotes acetaminophen-induced liver necrosis: an ultrastructural and histochemical study on male CD-1 mice

We analyzed in male wild-type (WT) and Dnase1 knockout (KO) CD-1 mice after acetaminophen (APAP)-intoxication the hepatolobular distribution of APAP-adducts in relation to DNA-damage by terminal deoxyribonucleotidyl-transferase dUTP nick end-labeling (TUNEL), the ultrastructural alterations of hepatocellular morphology and the intracellular localization of Dnase1. Treatment of WT-mice with 600 mg/kg APAP led to extensive pericentral necrosis. Electron microscopy (EM) demonstrated vesiculation of the rough endoplasmatic reticulum and swelling of mitochondria. Pericentral WT-hepatocyte nuclei exhibited pyknosis, karyorrhexis and karyolysis. In contrast, livers from treated KO-mice exhibited almost normal light microscopical structure and EM revealed only mild signs of hepatocellular damage. In WT-mice several layers of pericentral hepatocytes displayed APAP-adduct formation and subsequent DNA-damage, whereas in KO-animals only few cells were affected. Serum aminotransferases rose similarly in both mouse strains up to 12 h, thereafter increased only in WT-mice. Immunogold-EM revealed the translocation of Dnase1 from the rER into the nuclei of treated WT-mice. In KO-mice, APAP-adduct formation was retarded and less extensive suggesting that detoxification of APAP must have been more effective in KO-mice possibly due to the lack of energy depletion otherwise caused by Dnase1-induced DNA-damage in WT-mice.     

Augmenting energy expenditure by mitochondrial uncoupling: a role of AMP-activated protein kinase

Strategies to prevent and treat obesity aim to decrease energy intake and/or increase energy expenditure. Regarding the increase of energy expenditure, two key intracellular targets may be considered (1) mitochondrial oxidative phosphorylation, the major site of ATP production, and (2) AMP-activated protein kinase (AMPK), the master regulator of cellular energy homeostasis. Experiments performed mainly in transgenic mice revealed a possibility to ameliorate obesity and associated disorders by mitochondrial uncoupling in metabolically relevant tissues, especially in white adipose tissue (WAT), skeletal muscle (SM), and liver. Thus, ectopic expression of brown fat-specific mitochondrial uncoupling protein 1 (UCP1) elicited major metabolic effects both at the cellular/tissue level and at the whole-body level. In addition to expected increases in energy expenditure, surprisingly complex phenotypic effects were detected. The consequences of mitochondrial uncoupling in WAT and SM are not identical, showing robust and stable obesity resistance accompanied by improvement of lipid metabolism in the case of ectopic UCP1 in WAT, while preservation of insulin sensitivity in the context of high-fat feeding represents the major outcome of muscle UCP1 expression. These complex responses could be largely explained by tissue-specific activation of AMPK, triggered by a depression of cellular energy charge. Experimental data support the idea that (1) while being always activated in response to mitochondrial uncoupling and compromised intracellular energy status in general, AMPK could augment energy expenditure and mediate local as well as whole-body effects; and (2) activation of AMPK alone does not lead to induction of energy expenditure and weight reduction.     

Inhibition of endogenous BMP in the glomerulus leads to mesangial matrix expansion

Bone morphogenetic protein7 (BMP7) attenuates renal tubular and interstitial damage in a variety of experimental models. The function of BMP in the glomerulus is, however, not well understood. In the present study, we generated transgenic mice carrying cDNA for noggin, an endogenous inhibitor of BMPs, driven by the podocyte-specific promoter nephrin. Transgenic founder mice could be divided into two groups based on gross histological analyses at 2 months of age. One group was characterized by the presence of cystic glomeruli with collapsed capillary tufts and a decrease in mesangial cell number, representing a developmental defect during glomerular morphogenesis ("cystic" Tg mice). In contrast, the kidneys appeared to be normal in the other group ("non-cystic" Tg mice). In both groups, however, massive mesangial expansion developed at 10 months of age. The lesion was characterized by the accumulation of fibronectin, but not type I collagen, type IV collagen or laminin. This phenotype is similar to the fibronectin nephropathy. These results suggest that endogenous BMP can have an important role in regulating glomerular structural homeostasis.     

Hepatic monocyte chemoattractant protein-1 is upregulated by dietary cholesterol and contributes to liver steatosis

To assess the role of monocyte chemoattractant protein-1 (MCP-1/CCL2) in the development of fatty liver, we have used LDLr^−/− mice as an animal model of high-fat, high-cholesterol diet-induced liver steatosis. The rapid dietary induction of hepatic mRNA MCP-1 expression was paralleled by a concomitant increase in plasma MCP-1 that was strongly associated with the degree of liver steatosis. Hepatocytes showed an intense immunoreactivity for MCP-1 that was mainly located surrounding the hepatic lipid droplets. The intake of cholesterol also increased the concentration of MCP-1 in liver homogenates. This was accompanied by a differential expression of members of the PPAR family. Additionally, complete MCP-1 deficiency prevents the development of liver steatosis in LDLr^−/− mice and partial deficiency is accompanied by a certain protective effect. Our data also suggest that MCP-1 may be important in the regulation of hepatic insulin resistance and may represent a link between inflammation and metabolic diseases. We conclude that dietary cholesterol upregulation of hepatic MCP-1 may help to understand the role of circulating MCP-1 in conditions where liver derangements are clinically important and in the association of liver steatosis with the metabolic syndrome.     

Role of deleted in breast cancer 1 (DBC1) protein in SIRT1 deacetylase activation induced by protein kinase A and AMP-activated protein kinase

The NAD(+)-dependent deacetylase SIRT1 is a key regulator of several aspects of metabolism and aging. SIRT1 activation is beneficial for several human diseases, including metabolic syndrome, diabetes, obesity, liver steatosis, and Alzheimer disease. We have recently shown that the protein deleted in breast cancer 1 (DBC1) is a key regulator of SIRT1 activity in vivo. Furthermore, SIRT1 and DBC1 form a dynamic complex that is regulated by the energetic state of the organism. Understanding how the interaction between SIRT1 and DBC1 is regulated is therefore essential to design strategies aimed to activate SIRT1. Here, we investigated which pathways can lead to the dissociation of SIRT1 and DBC1 and consequently to SIRT1 activation. We observed that PKA activation leads to a fast and transient activation of SIRT1 that is DBC1-dependent. In fact, an increase in cAMP/PKA activity resulted in the dissociation of SIRT1 and DBC1 in an AMP-activated protein kinase (AMPK)-dependent manner. Pharmacological AMPK activation led to SIRT1 activation by a DBC1-dependent mechanism. Indeed, we found that AMPK activators promote SIRT1-DBC1 dissociation in cells, resulting in an increase in SIRT1 activity. In addition, we observed that the SIRT1 activation promoted by PKA and AMPK occurs without changes in the intracellular levels of NAD(+). We propose that PKA and AMPK can acutely activate SIRT1 by inducing dissociation of SIRT1 from its endogenous inhibitor DBC1. Our experiments provide new insight on the in vivo mechanism of SIRT1 regulation and a new avenue for the development of pharmacological SIRT1 activators targeted at the dissociation of the SIRT1-DBC1 complex.     

NtSKP1基因的反义载体构建及转基因烟草的产生

根据枯斑三生烟SKP1基因(NtSKP1)的序列,设计一对分别含有特定酶切位点的特异引物,以重组质粒pMD18-SKP1为模板,扩增目的基因(约473bp)片段。将反向目的片段插入中间载体pHANNIBAL的内含子右侧,再经NotⅠ酶切回收约3443bp的目的片段,插入到双元载体质粒pART27中,成功构建了含NtSKP1基因片段反向序列的植物表达载体pART27-skp1a,其转录产物能减弱目的基因的表达。将pART27-skp1a质粒导人根癌农杆菌LBA4404中并转化烟草叶片细胞,经选择分化培养,获得转基因烟草。     

Elucidation of the role of COX-2 in liver fibrogenesis using transgenic mice

Hepatic COX-2 overexpression is sufficient to induce hepatitis, but its role on liver fibrosis remains unknown. We aim to elucidate possible biological effects of COX-2 in liver fibrosis using both gain-of-function and loss-of-function mouse models. COX-2 transgenic (TG) mice that specifically overexpress the human COX-2 cDNA in the liver, knockout (KO), and wild type (WT) mice were studied in two different murine fibrosis models induced by carbon tetrachloride (CCl₄) injection or methionine and choline-deficient (MCD) diet. Liver injury was assessed by serum ALT and bilirubin levels and histological examination. Hepatic collagen content was determined by picrosirius red stain morphometry assay and quantitation of hydroxyproline. Hepatic stellate cell (HSC) activation was determined by immunohistochemical analysis of α-smooth muscle actin (α-SMA). mRNA expression of fibrogenic genes was assayed by real-time quantitative PCR. COX-2 protein was overexpressed in the liver of TG mice compared with WT littermates. CCl₄ or MCD-induced liver fibrotic injury was equally severe in TG and WT mice, as demonstrated by similar elevated levels of hepatic collagen contents. Enhanced COX-2 expression in TG liver did not affect HSC activation and fibrogenic gene expression upon CCl₄ or MCD treatment. Importantly, CCl₄-treated KO mice did not show significant difference in liver fibrotic damage and fibrogenic gene expression compared with the WT counterparts. This is the first report on the effect of COX-2 in liver fibrosis based on genetic mouse models. The results suggest that COX-2 does not appear to mediate the development of liver fibrosis.     

转基因小鼠和基因突变小鼠微卫星不稳定性分析

目的探讨微卫星在转基因和基因突变小鼠中的变化,为基因修饰和遗传突变动物的遗传检测和表型分析提供理论依据和技术手段。方法根据文献报道,从GenBank中选取198个等位基因数量多、富含多态性的微卫星位点,以野生型动物为对照,对6种近交系遗传背景的转基因小鼠和5种自然基因突变的近交系小鼠进行微卫星多态性检测,选用1.5%琼脂糖凝胶电泳和STR扫描技术,比较分析微卫星不稳定性。结果共有40个微卫星位点在转基因和基因突变小鼠中表现出多态性。在基因突变小鼠中,微卫星不稳定性有55.6%(10/18)是由纯合变为杂合(Ⅰ型),有3个位点(16.6%,3/18)是纯合突变(Ⅱ型),有5个位点同时存在2种类型的突变。但是在转基因动物中,大多数的微卫星多态性为Ⅰ型突变(87.5%,28/32),只有2个位点(6.2%,2/32)是Ⅱ型突变。另外有2个位点同时存在2种类型的突变。结论基因修饰或基因突变可引起小鼠相关微卫星发生不稳定性,而且某些微卫星位点对基因改变敏感性较高。