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.     

Heterozygous disruption of Flk-1 receptor leads to myocardial ischaemia reperfusion injury in mice: application of affymetrix gene chip analysis

This study addresses an important clinical issue by identifying potential candidates of vascular endothelial growth factor (VEGF) signalling through the Flk-1 receptor that trigger cardioprotective signals under ischaemic stress. Isolated working mouse hearts of both wild-type (WT) and Flk-1(+/-) were subjected to global ischaemia (I) for 30 min. followed by 2 hrs of reperfusion (R). Flk-1(+/-) myocardium displayed almost 50% reduction in Flk-1 mRNA as examined by quantitative real-time RT-PCR at the baseline level. Flk-1(+/-) mouse hearts displayed reduction in left ventricular functional recovery throughout reperfusion (dp/dt 605 versus 884), after 2 hrs (P<0.05). Coronary (1.9 versus 2.4 ml) and aortic flow (AF) (0.16 versus 1.2 ml) were reduced in Flk-1(+/-) after 2 hrs of reperfusion. In addition, increased infarct size (38.4%versus 28.41%, P<0.05) and apoptotic cardiomyocytes (495 versus 213) were observed in Flk-1(+/-) knockout (KO) mice. We also examined whether ischaemic preconditioning (PC), a novel method to induce cardioprotection against ischaemia reperfusion injury, through stimulating the VEGF signalling pathway might function in Flk-1(+/-) mice. We found that knocking down Flk-1 resulted in significant reduction in the cardioprotective effect by PC compared to WT. Affymetrix gene chip analysis demonstrated down-regulation of important genes after IR and preconditioning followed by ischaemia reperfusion in Flk-1(+/-) mice compared to WT. To get insight into the underlying molecular pathways involved in ischaemic PC, we determined the distinct and overlapping biological processes using Ingenuity pathway analysis tool. Independent evidence at the mRNA level supporting the Affymetrix results were validated using real-time RT-PCR for selected down-regulated genes, which are thought to play important roles in cardioprotection after ischaemic insult. In summary, our data indicated for the first time that ischaemic PC modifies genomic responses in heterozygous VEGFR-2/Flk-1 KO mice and abolishes its cardioprotective effect on ischaemic myocardium.     

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.     

Generation of Sprn-regulated reporter mice reveals gonadic spatial expression of the prion-like protein Shadoo in mice

The protein Shadoo (Sho) is a paralogue of prion protein, and encoded by the gene Sprn. Like prion protein it is primarily expressed in central nervous system, and has been shown to have a similar expression pattern in certain regions of the brain. We have generated reporter mice carrying a transgene encompassing the Sprn promoter, exon 1, intron 1 and the 5′-end of exon 2 driving expression of either the LacZ or GFP reporter gene to study the expression profile of Shadoo in mice. Expression of the reporter genes was analysed in brains of these transgenic mice and was shown to mimic that of the endogenous gene expression, previously described by Watts et al. [1]. Consequently, the Sprn-LacZ mice were used to study the spatial expression of Sho in other tissues of the adult mouse. Several tissues were collected and stained for β-gal activity, including the thymus, heart, lung, liver, kidney, spleen, intestine, muscle, and gonads. From this array of tissues, the transgene was consistently expressed only in specific cell types of the testicle and ovary, suggesting a role for Shadoo in fertility and reproduction. These mice may serve as a useful tool in deciphering the regulation of the prion-like gene Sprn and thus, indirectly, of the Shadoo protein.     

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     

Deletion of ELOVL5 leads to fatty liver through activation of SREBP-1c in mice

Elongation of very long chain fatty acids (ELOVL)5 is one of seven mammalian fatty acid condensing enzymes involved in microsomal fatty acid elongation. To determine the in vivo substrates and function of ELOVL5, we generated Elovl5(-/-) mice. Studies using liver microsomal protein from wild-type and knockout mice demonstrated that the elongation of gamma-linolenic (C18:3, n-6) to dihomo-gamma-linolenic (C20:3, n-6) and stearidonic (C18:4, n-3) to omega3-arachidonic acid (C20:4, n-3) required ELOVL5 activity. Tissues of Elovl5(-/-) mice accumulated the C18 substrates of ELOVL5 and the levels of the downstream products, arachidonic acid (C20:4, n-6) and docosahexaenoic acid (DHA, C22:6, n-3), were decreased. A consequence of decreased cellular arachidonic acid and DHA concentrations was the activation of sterol regulatory element-binding protein (SREBP)-1c and its target genes involved in fatty acid and triglyceride synthesis, which culminated in the development of hepatic steatosis in Elovl5(-/-) mice. The molecular and metabolic changes in fatty acid metabolism in Elovl5(-/-) mice were reversed by dietary supplementation with arachidonic acid and DHA. These studies demonstrate that reduced ELOVL5 activity leads to hepatic steatosis, and endogenously synthesized PUFAs are key regulators of SREBP-1c activation and fatty acid synthesis in livers of mice.     

Deficiency of sterol regulatory element‐binding protein‐1c induces schizophrenia‐like behavior in mice

Schizophrenia is a hereditary disease that approximately 1% of the worldwide population develops. Many studies have investigated possible underlying genes related to schizophrenia. Recently, clinical studies suggested sterol regulatory element‐binding protein (SREBP) as a susceptibility gene in patients with schizophrenia. SREBP controls cellular lipid homeostasis by three isoforms: SREBP‐1a, SREBP‐1c and SREBP‐2. This study used SREBP‐1c knockout (KO) mice to examine whether a deficiency in SREBP‐1c would affect their emotional and psychiatric behaviors. Altered mRNA expression in genes downstream from SREBP‐1c was confirmed in the brains of SREBP‐1c KO mice. Schizophrenia‐like behavior, including hyperactivity during the dark phase, depressive‐like behavior, aggressive behavior and deficits in social interaction and prepulse inhibition, was observed in SREBP‐1c KO mice. Furthermore, increased volume of the lateral ventricle was detected in SREBP‐1c KO mice. The mRNA levels of several γ‐aminobutyric acid (GABA)‐receptor subtypes and/or glutamic acid decarboxylase 65/67 decreased in the hippocampus and medial prefrontal cortex of SREBP‐1c KO mice. Thus, SREBP‐1c deficiency may contribute to enlargement of the lateral ventricle and development of schizophrenia‐like behaviors and be associated with altered GABAergic transmission.     

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.     

Fasting and sampling time affect liver gene expression of high-fat diet-fed mice

Several physiological and biological variables are known to affect peroxisome proliferator-activated receptor (PPAR)-α-dependent signaling pathway and plasma biochemical profiles. However, less is known about the effect of these variables on high-fat diet-fed mice. In a 5-week study, C57BL/6 mice were divided into control (C) and high-fat diet-fed (H) groups, whereby before dissection, each group was subdivided into non-fasted (nC and nH) and a 15-h fasted mice (fC and fH) killed in the early light cycle, and a 15-h fasted mice (eC and eH) killed in the late phase of the light cycle. Liver and blood from the vena cava were collected. Non-fasted nC and nH mice have a marginal difference in their body weight gain, whereas significant differences were found for fasted mice. In nH mice, PPAR-α, acyl-CoA oxidase and insulin-like growth factor-binding protein expressions were significantly elevated, in contrast to fatty acid synthase (Fasn), stearoyl CoA-desaturase (SCD)-1, and elongase (ELOVL)-6 expressions. Fasn was profoundly induced in fH mice, while decreased sterol regulatory-binding protein-1 and SCD-1 were found only in eH mice. Different from the gene expression profiles, plasma total cholesterol level of the eH mice was higher than controls, whereas nH mice have increased plasma non-esterified fatty acids. Only glucose level of the fH mice was higher than that observed for controls. Results showed that fasting and sampling time have significantly affected liver gene expression and plasma biochemical indices of the high-fat diet-treated mice. An overlook in these aspects can cause serious discrepancies in the experimental data and their interpretations.     

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.     

Nephropathy and defective spermatogenesis in mice transgenic for a single isoform of the Wilms' tumour suppressor protein, WT1-KTS, together with one disrupted Wt1 allele

The Wilms' tumour suppressor protein, WT1, is a zinc finger protein essential for the development of several organs, including the kidney and gonads. In each of these tissues WT1 is required at multiple stages of development and its persistent expression in podocytes and Sertoli cells suggests WT1 may also have a role in the maintenance of kidney and testis function throughout adult life. Naturally occurring isoforms of WT1 are generated by alternative mRNA splicing. An altered ratio of the splice isoforms WT1-KTS and WT1 + KTS appears to be sufficient to account for the developmental abnormalities (pseudohermaphroditism and nephropathy) characteristic of Frasier syndrome. We show that mice with a transgene encoding WT1-KTS do not differ from their wild-type littermates unless they are also heterozygous for a null mutation at the endogenous Wt1 locus. Animals with both genetic modifications develop proteinuria, together with multiple glomerular cysts, and male infertility. These pathologic changes may be explained as a consequence of altering the WT1 isoform ratio in tissues that express WT1 during adulthood. The results suggest WT1 misexpression could contribute to human glomerulocystic kidney disease.     

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.     

Fluoxetine suppresses AMP-activated protein kinase signaling pathway to promote hepatic lipid accumulation in primary mouse hepatocytes

In the previous study, we demonstrated that fluoxetine (FLX) regulated lipogenic and lipolytic genes to promote hepatic lipid accumulation. On this basis, underlying mechanisms were investigated by focusing on the intracellular signaling transduction in the present study using primary mouse hepatocytes. The expression of lipogenesis- and lipolysis-related genes was evaluated with the application of specific activators and inhibitors. Activation status of respective signaling pathway and the lipid accumulation in hepatocytes were analyzed. We provided evidence that AMP-activated protein kinase (AMPK) activator AICAR (5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside) significantly suppressed the increased expression of representative lipogenesis-related genes, acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS) by FLX, while increased the repressed expression of lipolysis-related genes, carboxylesterases. In the meanwhile, FLX regulated the above genes in the same way as AMPK inhibitor Compound C did. Furthermore, AICAR inhibited the proteolytic activation of SREBP1c induced by FLX, resulting in the decreased level of nuclear SREBP1c. Further studies demonstrated that FLX significantly suppressed the phosphorylation of AMPK and subsequent phosphorylation of ACC, following the inhibited phosphorylation and nuclear export of liver kinase B1 (LKB1). As a functional analysis, FLX-induced lipid accumulation in hepatocytes was repeatedly abolished by AICAR. In conclusion, FLX-induced hepatic lipid accumulation is mediated by the suppression of AMPK signaling pathway. The findings not only provide new insight into the understanding of the mechanisms for selective serotonin reuptake inhibitors-mediated dyslipidemia effects, but also suggest a novel therapeutic target to interfere.