Kimihiko Matsusue

Liver-specific disruption of PPARγ in leptin-deficient mice improves fatty liver but aggravates diabetic phenotypes

To elucidate the function of PPARgamma in leptin-deficient mouse (ob/ob) liver, a PPARgamma liver-null mouse on an ob/ob background, ob/ob-PPARgamma(fl/fl)AlbCre(+), was produced using a floxed PPARgamma allele, PPARgamma(fl/fl), and Cre recombinase under control of the albumin promoter (AlbCre). The liver of ob/ob-PPARgamma(fl/fl)AlbCre(+) mice had a deletion of exon 2 and a corresponding loss of full-length PPARgamma mRNA and protein. The PPARgamma-deficient liver in ob/ob mice was smaller and had a dramatically decreased triglyceride (TG) content compared with equivalent mice lacking the AlbCre transgene (ob/ob-PPARgamma(fl/fl)AlbCre(-)). Messenger RNA levels of the hepatic lipogenic genes, fatty acid synthase, acetyl-CoA carboxylase, and stearoyl-CoA desaturase-1, were reduced in ob/ob-PPARgamma(fl/fl)AlbCre(+) mice, and the levels of serum TG and FFA in ob/ob-PPARgamma(fl/fl)AlbCre(+) mice were significantly higher than in the control ob/ob-PPARgamma(fl/fl)AlbCre(-) mice. Rosiglitazone treatment exacerbated the fatty liver in ob/ob-PPARgamma(fl/fl)AlbCre(-) mice compared with livers from nonobese Cre(-) mice; there was no effect of rosiglitazone in ob/ob-PPARgamma(fl/fl)AlbCre(+) mice. The deficiency of hepatic PPARgamma further aggravated the severity of diabetes in ob/ob mice due to decreased insulin sensitivity in muscle and fat. These data indicate that hepatic PPARgamma plays a critical role in the regulation of TG content and in the homeostasis of blood glucose and insulin resistance in steatotic diabetic mice.

Conditional Disruption of the Peroxisome Proliferator-Activated Receptor γ Gene in Mice Results in Lowered Expression of ABCA1, ABCG1, and apoE in Macrophages and Reduced Cholesterol Efflux

ABSTRACT Disruption of the peroxisome proliferator-activated receptor γ (PPARγ) gene causes embryonic lethality due to placental dysfunction. To circumvent this, a PPARγ conditional gene knockout mouse was produced by using the Cre-loxP system. The targeted allele, containing loxP sites flanking exon 2 of the PPARγ gene, was crossed into a transgenic mouse line expressing Cre recombinase under the control of the alpha/beta interferon-inducible (MX) promoter. Induction of the MX promoter by pIpC resulted in nearly complete deletion of the targeted exon, a corresponding loss of full-length PPARγ mRNA transcript and protein, and marked reductions in basal and troglitazone-stimulated expression of the genes encoding lipoprotein lipase, CD36, LXRα, and ABCG1 in thioglycolate-elicited peritoneal macrophages. Reductions in the basal levels of apolipoprotein E (apoE) mRNA in macrophages and apoE protein in total plasma and high-density lipoprotein (HDL) were also observed in pIpC-treated PPARγ-MXCre+ mice. Basal cholesterol efflux from cholesterol-loaded macrophages to HDL was significantly reduced after disruption of the PPARγ gene. Troglitazone selectively inhibited ABCA1 expression (while rosiglitazone, ciglitazone, and pioglitazone had little effect) and cholesterol efflux in both PPARγ-deficient and control macrophages, indicating that this drug can exert paradoxical effects on cholesterol homeostasis that are independent of PPARγ. Together, these data indicate that PPARγ plays a critical role in the regulation of cholesterol homeostasis by controlling the expression of a network of genes that mediate cholesterol efflux from cells and its transport in plasma.

PPARβ/δ potentiates PPARγ-stimulated adipocyte differentiation

It is well established that peroxisome proliferator‐activated receptor‐γ (PPARγ) has a critical role in modulating adipocyte differentiation based on gain‐of‐function and loss‐of‐function experiments. However, recent gain‐of‐function experiments suggest that PPARβ may also have a role in mediating adipocyte differentiation. Because ligands for PPARs can activate more than one receptor isoform, the specific role of PPARβ in adipocyte differentiation was examined using PPARβ‐null adipocytes. Wild‐type adipocytes accumulate lipids in response to differentiation signaling induced from standard differentiation medium, and this effect is significantly reduced in PPARβ‐null adipocytes. The addition of the PPARβ ligand L165041 to the standard differentiation medium causes enhanced adipocyte differentiation and lipid accumulation, and this effect is diminished in adipocytes lacking expression of PPARβ. Treatment of wild‐type adipocytes with the PPARγ ligand troglitazone causes accelerated adipocyte differentiation and lipid accumulation, and this effect is marginally reduced in PPARβ‐null adipocytes. Expression patterns of mRNA markers of early and late adipocyte differentiation are consistent with the morphological and biochemical differences observed. Results from these studies demonstrate that in the absence of PPARβ expression, adipocyte differentiation is significantly impaired, providing loss‐of‐function evidence supporting a role for this receptor in adipocyte differentiation. These results also demonstrate that L165041‐stimulated adipocyte differentiation and lipid accumulation is mediated by PPARβ. In addition, as the ability of troglitazone to induce adipocyte differentiation is also impaired in PPARβ null adipocytes, this suggests that both PPARβ and PPARγ isoforms are required to facilitate maximal lipid accumulation and differentiation during adipogenesis.

Gene structure of human cholecystokinin (CCK) type-A receptor: body fat content is related to CCK type-A receptor gene promoter polymorphism

The transcriptional start site of the human cholecystokinin (CCK)‐A receptor gene was determined by the Capsite Hunting method. Two sequence changes were detected, a G to T change in nucleotide −128, and an A to G change in nucleotide −81. The homozygote (T/T, G/G) was detected in 25 of 1296 individuals (1.9%) in the cohort study. This polymorphism showed a significantly higher percent body fat and higher levels of serum insulin and leptin, compared with wild type and heterozygotes. Our study provided the possibility that polymorphism in the promoter region of the CCK‐A receptor gene may be one of genetic factors affecting fat deposition.

Fat-Specific Protein 27/CIDEC Promotes Development of Alcoholic Steatohepatitis in Mice and Humans

BACKGROUND & AIMS Alcoholic steatohepatitis (ASH) is the progressive form of alcoholic liver disease and may lead to cirrhosis and hepatocellular carcinoma. We studied mouse models and human tissues to identify molecules associated with ASH progression and focused on the mouse fat-specific protein 27 (FSP-27)/human cell death-inducing DFF45-like effector C (CIDEC) protein, which is expressed in white adipose tissues and promotes formation of fat droplets. METHODS C57BL/6N mice or mice with hepatocyte-specific disruption of Fsp27 (Fsp27(Hep-/-) mice) were fed the Lieber-Decarli ethanol liquid diet (5% ethanol) for 10 days to 12 weeks, followed by 1 or multiple binges of ethanol (5 or 6 g/kg) during the chronic feeding. Some mice were given an inhibitor (GW9662) of peroxisome proliferator-activated receptor γ (PPARG). Adenoviral vectors were used to express transgenes or small hairpin (sh) RNAs in cultured hepatocytes and in mice. Liver tissue samples were collected from ethanol-fed mice or from 31 patients with alcoholic hepatitis (AH) with biopsy-proved ASH and analyzed histologically and immunohistochemically and by transcriptome, immunoblotting, and real-time PCR analyses. RESULTS Chronic-plus-binge ethanol feeding of mice, which mimics the drinking pattern of patients with AH, produced severe ASH and mild fibrosis. Microarray analyses revealed similar alterations in expression of many hepatic genes in ethanol-fed mice and humans with ASH, including up-regulation of mouse Fsp27 (also called Cidec) and human CIDEC. Fsp27(Hep-/-) mice and mice given injections of adenovirus-Fsp27shRNA had markedly reduced ASH following chronic-plus-binge ethanol feeding. Inhibition of PPARG and cyclic AMP-responsive element binding protein H (CREBH) prevented the increases in Fsp27α and FSP27β mRNAs, respectively, and reduced liver injury in this chronic-plus-binge ethanol feeding model. Overexpression of FSP27 and ethanol exposure had synergistic effects in inducing production of mitochondrial reactive oxygen species and damage to hepatocytes in mice. Hepatic CIDEC mRNA expression was increased in patients with AH and correlated with the degree of hepatic steatosis and disease severity including mortality. CONCLUSIONS In mice, chronic-plus-binge ethanol feeding induces ASH that mimics some histological and molecular features observed in patients with AH. Hepatic expression of FSP27/CIDEC is highly up-regulated in mice following chronic-plus-binge ethanol feeding and in patients with AH; this up-regulation contributes to alcohol-induced liver damage.

Direct interaction between metastasis-associated protein 1 and endophilin 3.

The yeast two‐hybrid system was used to search for partners of mouse metastasis‐associated protein 1 (Mta1). Screening of a cDNA library prepared from mouse embryo yielded positive clones coding for endophilin 3. The site of interaction was suggested to be the SH‐3‐binding domain of Mta1 and SH‐3 domain of endophilin 3. This interaction was confirmed by GST pull‐down assay in vitro and immunoprecipitation in vivo. The Mta1 and endophilin 3 transcripts were highly expressed in testis and brain. But, Mta1 localized mainly in nucleus and to a lesser extent in cytoplasm while endophilin 3 localized mainly in cytoplasm. If Mta1 functions in cytoplasm, it might be involved in the regulation of endocytosis mediated by endophilin 3.

Hepatic PPARγ and LXRα independently regulate lipid accumulation in the livers of genetically obese mice

The nuclear hormone receptors liver X receptor α (LXRα) and peroxisome proliferator‐activated receptor γ (PPARγ) play key roles in the development of fatty liver. To determine the link between hepatic PPARγ and LXRα signaling and the development of fatty liver, a LXRα‐specific ligand, T0901317, was administered to normal OB/OB and genetically obese (ob/ob) mice lacking hepatic PPARγ (Pparγ ΔH). In ob/ob‐Pparγ ΔH and OB/OB‐Pparγ ΔH mice, as well as ob/ob‐Pparγ WT and OB/OB‐Pparγ WT mice, the liver weights and hepatic triglyceride levels were markedly increased in response to T0901317 treatment. These results suggest that hepatic PPARγ and LXRα signals independently contribute to the development of fatty liver.

Hepatic peroxisome proliferator-activated receptor-γ–fat-specific protein 27 pathway contributes to obesity-related hypertension via afferent vagal signals

AIMS Obesity is commonly associated with hypertension. Increased sympathetic tonus in obese subjects contributes to the underlying mechanism. However, the precise mechanisms whereby obesity induces this sympathetic activation remain unclear. Hepatic peroxisome proliferator-activated receptor (PPAR)-γ2 expression, which is reportedly upregulated during obesity development, affects sympathetic activation via hepatic vagal afferents. Herein, we report involvement of this neuronal relay in obesity-related hypertension. METHODS AND RESULTS Peroxisome proliferator-activated receptor-γ and a direct PPARγ target, fat-specific protein 27 (Fsp27), were adenovirally overexpressed or knocked down in the liver, in combination with surgical dissection or pharmacological deafferentation of the hepatic vagus. Adenoviral PPARγ2 expression in the liver raised blood pressure (BP) in wild-type but not in β1/β2/β3 adrenergic receptor-deficient mice. In addition, knockdown of endogenous PPARγ in the liver lowered BP in murine obesity models. Either surgical dissection or pharmacological deafferentation of the hepatic vagus markedly blunted BP elevation in mice with diet-induced and genetically-induced obesity. In contrast, BP was not elevated in other models of hepatic steatosis, DGAT1 and DGAT2 overexpressions, in which PPARγ is not upregulated in the liver. Thus, hepatic PPARγ upregulation associated with obesity is involved in BP elevation during obesity development. Furthermore, hepatic expression of Fsp27 raised BP and the effect was blocked by hepatic vagotomy. Hepatic Fsp27 is actually upregulated in murine obesity models and its knockdown reversed BP elevation. CONCLUSION The hepatic PPARγ-Fsp27 pathway plays important roles in the development of obesity-related hypertension via afferent vagal signals from the liver.

Characterization of mouse metastasis-associated gene 2: genomic structure, nuclear localization signal, and alternative potentials as transcriptional activator and repressor.

We characterized the mouse metastasis-associated gene 2 product (mmta2), which is a homolog of the metastasis-associated gene 1 product (MTA1). We revealed that the mmta2 gene spanned approximately 10 kb and was separated into 18 exons. The transcription start site of mmta2 was located 377 bp upstream from the putative initiation codon. The subcellular location of the mmta2 protein was the nucleus, and nuclear localization signals were identified in the region between amino acids 456 and 497. To obtain data on the transcription-regulating potential of mmta2, various constructs containing different portions were fused to the GAL4 DNA-binding domain. The entire mmta2 protein repressed the transcription of the reporter genes, whereas treatment with a histone deacetylase inhibitor, trichostatin A (TSA), led to recovery from the repression and to transcriptional activation. However, the N terminus of mmta2 activated transcriptional activity in the absence of TSA. These results suggest that mmta2 has the potential to both repress and activate gene transcription and that its transcription repression activity might be related to histone deacetylation.

Involvement of CXCL14 in osteolytic bone metastasis from lung cancer.

To investigate the molecular mechanisms of lung cancer-induced bone metastasis, we established a bone-seeking subclone (HARA-B4) from a human squamous lung cancer cell line (HARA) using an in vivo selection method. We compared comprehensive gene expression profiles between HARA and HARA-B4, and identified the critical factors for the formation of bone metastasis using in vitro and in vivo assays. The number of bone metastatic colonies in the hind legs was significantly higher in HARA-B4-inoculated mice than in HARA-inoculated mice at 4 weeks after inoculation. In addition, visceral (adrenal) metastases were not found in HARA-B4-inoculated mice at autopsy, suggesting an increase in cancer cell tropism to bone in HARA-B4. Based on a comprehensive gene expression analysis, the expression level of CXC chemokine ligand 14 (CXCL14) was 5-fold greater in HARA-B4 than in HARA. Results of a soft agar colony formation assay showed that anchorage-independent growth ability was 4.5-fold higher with HARA-B4 than with HARA. The murine pre-osteoblast cell line MC3T3-E1 and the pre-osteoclast/macrophage cell line RAW264.7 migrated faster toward cultured HARA-B4 cells than toward HARA cells in a transwell cell migration assay. Interestingly, CXCL14 was shown to be involved in all events (enhancement of cancer cell tropism to the bone, anchorage-independent growth and/or recruitment of bone marrow cells) based on siRNA experiments in HARA-B4 cells. Furthermore, in clinical specimens of lung cancer-induced bone metastasis, expression of CXCL14 was observed in the tumor cells infiltrated in bone marrow in all specimens examined. CXCL14 was able to promote bone metastasis through enhancement of cancer cell tropism to the bone and/or recruitment of bone marrow cells around metastatic cancer cells.