Hiroyuki Kawachi

Cellular regulation of bovine intramuscular adipose tissue development and composition.

It is well documented that grain feeding stimulates adipogenesis in beef cattle, whereas pasture feeding depresses the development of adipose tissues, including intramuscular (i.m.) adipose tissue. Additionally, production practices that depress adipocyte differentiation also limit the synthesis of MUFA. Marbling scores and MUFA increase in parallel suggesting that stearoyl-coenzyme A desaturase (SCD) gene expression is closely associated with and necessary for marbling adipocyte differentiation. Similarly, marbling scores and fatty acid indices of SCD activity are depressed in response to dietary vitamin A restriction. In bovine preadipocytes, vitamins A and D both decrease glycerol-3-phosphate dehydrogenase (GPDH) activity, an index of adipocyte differentiation, whereas incubation of bovine preadipocytes with l-ascorbic acid-2-phosphate increases GPDH activity. Exposing bovine preadipocytes to zinc also stimulates adipogenesis, putatively by inhibiting nitric oxide (NO) production. However, incubation of bovine preadipocytes with arginine, a biological precursor of NO, strongly promotes differentiation in concert with increased SCD expression. This suggests that the effect of either arginine or zinc on adipogenesis is independent of NO synthesis in bovine preadipocytes. Enhanced expression of SCD is associated with a greater accumulation of MUFA both in bovine preadipocyte cultures and during development in growing steers. In bovine preadipocytes, trans-10, cis-12 CLA strongly depresses adipocyte differentiation and SCD gene expression, thereby reducing MUFA concentrations. The bovine preadipocyte culture studies suggest that any production practice that elevates vitamins A or D or trans-10, cis-12 CLA in bovine adipose tissue will reduce i.m. adipose tissue development. Conversely, supplementation with vitamin C or zinc may promote the development of i.m. adipose tissue.

Activin A inhibits differentiation of 3T3-L1 preadipocyte

We investigated the effect of activin A on differentiation of 3T3-L1 preadipocyte. Activin A suppressed the induction of terminal differentiation markers such as glycerol-3-phosphate dehydrogenase (GPDH) activity, lipid accumulation, and the expression of adipocyte fatty acid-binding protein (aP2) mRNA when the cells were treated with activin A throughout the differentiation period. Activin A treatment during the early phase decreased GPDH activity and aP2 mRNA level, and also reduced the expression of peroxisome proliferator-activated receptor (PPAR) gamma and CCAAT/enhancer binding protein (C/EBP) alpha mRNAs without affecting the expressions of the active isoforms of C/EBPbeta and its mRNA. On the other hand, activin A treatment had no effect on the mitotic clonal expansion. These results indicate that activin A inhibits adipogenesis via affecting the transcriptional factor cascade upstream of PPARgamma expression.

Receptor expression modulates the specificity of transforming growth factor-β signaling pathways

In current models of transforming growth factor‐β (TGF‐β) family signaling, type II receptors activate specific activin receptor‐like kinase (ALK) type I receptors. These serine/threonine kinases activate ligand‐dependent receptor regulated (R)‐Smad by phosphorylating carboxy‐terminal serines. We found that the receptor expression levels affected the phosphorylation and activation of the two R‐Smad subclasses, activin/TGF‐β‐specific (AR‐Smad) and bone morphogenetic protein (BMP)‐specific (BR‐Smad). Co‐expressing constitutively active type I and type II receptors in COS7 cells resulted in the phosphorylation of both R‐Smad subclasses in a ligand‐independent manner. This was verified using in vitro kinase assays. In untransfected B16 melanoma cells, TGF‐β1 and BMP‐2 induced phosphorylation of both R‐Smad subclasses, and TGF‐β1 up‐regulated the inhibitor of differentiation (Id) gene, which is usually regulated by BMP. By contrast, BMP‐2 up‐regulated plasminogen activator inhibitor‐1 (PAI‐1), which is an AR‐Smad‐regulated gene. Except for ALK4 and ALK6, levels of type I and type II receptor mRNAs were higher in B16 cells than in HeLa and HepG2 cells, in which TGF‐β1 and BMP‐2 induced phosphorylation of only the expected R‐Smad. These results help to explain the diverse effects of this ligand family.

Subcutaneous and intramuscular adipose tissue stearoyl-coenzyme A desaturase gene expression and fatty acid composition in calf- and yearling-fed Angus steers.

We proposed that stearoyl-CoA desaturase (SCD) activity dictates fatty acid composition of adipose tissue and muscle in beef cattle, regardless of ruminal or hepatic fatty acid hydrogenation or desaturation. Twelve Angus steers were assigned to a calf-fed (CF) group and slaughtered at weaning (8 mo of age; n=4), 12 mo of age (n=4), or 16 mo of age (n=4). Twelve steers were assigned to a yearling-fed (YF) group and slaughtered at 12 mo of age (n=4), 16 mo of age (n=4), and 17.5 mo of age (n=4; 525 kg, market weight). Data were analyzed based on time on the corn-based finishing diet, with terminal age as a covariate, and orthogonal polynomial contrasts were tested on the main effects of treatment group and time on the finishing diet. Fatty acids from duodenal digesta, plasma, liver, LM, and subcutaneous and intramuscular adipose tissue were measured, and SCD gene expression was measured in intramuscular and subcutaneous adipose tissues. In duodenal digesta, palmitic and linoleic acids increased by 100% over the sampling period, α-linolenic acid decreased over the sampling period, and trans-vaccenic acid was greater in YF than in CF steers (all P < 0.01). The proportion of α-linolenic acid decreased over time in all tissues, including liver. The SCD index (ratio of SCD fatty acid products to SCD fatty acid substrates) increased over time in LM and in intramuscular and subcutaneous adipose tissues. The SCD:glyceraldehyde 3-phosphate dehydrogenase mRNA ratio was virtually undetectable at the initial sampling periods in subcutaneous adipose tissue of YF and CF steers, and it increased over time (P < 0.01). The SCD index and SCD:glyceraldehyde 3-phosphate dehydrogenase ratio were greater in intramuscular adipose tissue of CF steers than in that of YF steers. The SCD index did not change over time in liver and decreased over time in duodenal digesta. We conclude that, unlike essential fatty acids, the SFA and MUFA composition of adipose tissue is regulated by adipose tissue fatty acid desaturation, with little contribution from hepatic or duodenal fatty acids.

PCR detection of bovine mitochondrial DNA derived from meat and bone meal in feed

Because bovine meat and bone meal (MBM) is thought to be a major source of bovine spongiform encephalopathy, we developed a PCR-based method for detection of bovine MBM in animal feed. We isolated bone particles from feed containing bovine MBM using a separation technique based on specific gravity and then washed bone particles with sodium hypochlorite solution and an EDTA-proteinase K solution. The mitochondrial DNA was extracted from bone particles and amplified using PCR with cattle-specific primers. Bovine DNA was not detected in a milk replacer containing dried skim milk and dried whey, but bovine DNA was detected in the milk replacer that was mixed with bovine MBM. Other cattle-derived materials in feeds did not interfere with the selective detection of bovine MBM. This method allowed detection of bovine mitochondrial DNA in feed with 0.1% added bovine MBM. When the treatment with sodium hypochlorite was excluded, bovine DNA derived from MBM could not be distinguished from bovine DNA derived from other bovine materials. However, the exclusion of this treatment improved the detection limit of bovine MBM in feed. This method appears suitable for the selective detection of bovine MBM in feed.

Bmp4 expressed in preadipocytes is required for the onset of adipocyte differentiation

We previously revealed that endogenous bone morphogenetic protein (Bmp) activity is required for lipid accumulation in 3T3-L1 adipocytes. The present study characterized the role of endogenous Bmp activity in preadipocytes. Endogenous Bmp activity was monitored by analyzing the level of phosphorylation of Smad1/5/8, downstream molecules in the Bmp pathway. Higher levels of phosphorylated Smad1/5/8 were detected in adipogenic cells but not in non-adipogenic cells prior to differentiation induction. The inhibition of the Bmp pathway during this period decreased the expression of Pparγ2 and C/ebpα, which are transcription factors responsible for adipocyte differentiation. The expression of these transcription factors were also down-regulated by Bmp4 knockdown. In addition, endogenous Bmp4 was required for the repression of Intrleukin-11 expression. Endogenous Bmp4 in preadipocytes is indispensable for the onset of the adipogenic program, and may help to maintain the preadipocytic state during adipocyte differentiation.

Nitric oxide suppresses preadipocyte differentiation in 3T3-L1 culture

Nitric oxide (NO) is an important chemical messenger controlling many physiological functions, involving cell proliferation, and differentiation. The purpose of this study was to investigate the effect of NO on adipocyte differentiation using a murine preadipocyte cell line, 3T3-L1. The treatment with a NO donor, 1-hydroxy-2-oxo-3,3-bis(2-aminoethyl)-1-triazene (NOC18), reduced some markers of adipocyte differentiation such as glycerol-3-phosphate dehydrogenase activity, and intracellular lipid accumulation. To examine whether these effects of NOC18 on adipocyte differentiation markers are due to its cytotoxity, lactate dehydrogenase (LDH) release from the cells were measured. NOC18 did not affect LDH release into the culture medium. Thus, the suppressive actions of NO donor were unlikely to result from its cytotoxicity. Peroxisome proliferator-activated receptor (PPAR) γ is a critical transcription factor for adipocyte differentiation and adipocyte fatty acid binding protein (aP2) gene is one of its targets. Protein expression of PPARγ was not diminished by NOC18 treatment, although mRNA expression of aP2 was reduced. Electrophoretic mobility shift assay showed that NOC18 interfered with the DNA binding activity of PPARγ. Therefore, the present experiment suggest that NO suppresses adipocyte differentiation through suppressing the transcriptional activity of PPARγ, without suppressing its expression level.

Characterization of the intron-containing citrate synthase gene from the alkanotrophic yeast Candida tropicalis: cloning and expression in Saccharomyces cerevisiae.

Abstract Citrate synthase, an essential enzyme of the tricarboxylic acid cycle in mitochondria, was purified from acetate-grown Candida tropicalis. Results from SDS-PAGE and gel filtration showed that this enzyme was a dimer composed of 45-kDa subunits. A citrate synthase cDNA fragment was amplified by the 5′-RACE method. Nucleotide sequence analysis of this cDNA fragment revealed that the deduced amino acid sequence contained an extended leader sequence which is suggested to be a mitochondrial targeting signal, as judged from helical wheel analysis. Using this cDNA probe, one genomic citrate synthase clone was isolated from a yeast λEMBL3 library. The nucleotide sequence of the gene encoding C. tropicalis citrate synthase, CtCIT, revealed the presence of a 79-bp intron in the N-terminal region. Sequences essential as yeast splicing motifs were present in this intron. When the CtCIT gene including its intron was introduced into Saccharomyces cerevisiae using the promoter UPR-ICL, citrate synthase activity was highly induced, which strongly indicated that this intron was correctly spliced in S. cerevisiae.

Immunochemically distinct NADP-linked isocitrate dehydrogenase isozymes in mitochondria and peroxisomes of Candida tropicalis

Abstract Although peroxisomal localization of NADP-linked isocitrate dehydrogenase (Idp) was first demonstrated in Candida tropicalis, the mitochondrial isozyme has not been found in this yeast. Here we report that the presence of mitochondrial Idp in the yeast was demonstrated by screening for its gene with a DNA probe containing conserved sequences of Idps from various organisms. The nucleotide sequence of the gene (CtIDP1) revealed a 1,290-bp open reading frame corresponding to a 430-amino-acid protein with a high similarity to previously reported Idps. Overexpression of CtIDP1 in Saccharomyces cerevisiae gave a high intracellular Idp activity, and the purified recombinant Idp was shown to be a homodimer with a subunit molecular mass of approximately 44 kDa, different from that of peroxisomal Idp (45 kDa) previously purified from C. tropicalis. Western blot analysis of the subcellular fractions from acetate-grown C. tropicalis with polyclonal antibodies raised against the recombinant CtIdp1 showed that the CtIdp1 in C. tropicalis was localized in mitochondria but not in peroxisomes. Similar levels of CtIDP1 mRNA and its protein product were detected in cells grown on glucose, acetate, and n-alkane, although a slight decrease was observed in n-alkane-grown cells. From these results, CtIdp1 was demonstrated to be mitochondrial Idp. The properties of mitochondrial Idp and peroxisomal Idp isozymes were proven to be similar, but they were immunochemically distinct, suggesting the presence of another gene responsible for peroxisomal Idp in C. tropicalis.

Establishment of a quantitative in vivo method for estimating adipose tissue volumes and the effects of dietary soy sauce oil on adipogenesis in medaka, Oryzias latipes

Adipose tissue, which is conserved in higher eukaryotes, plays central roles in controlling the body’s energy balance, including excess energy storage and energy expenditure during starvation. In adipogenesis, intranuclear receptor, peroxisome proliferator–activated receptor gamma (PPARγ) is a key molecule, and PPARγ agonists can promote adipogenesis. Many studies on the in vitro screening of PPARγ agonists with compounds derived from various materials have been reported; however, in vivo assays for quick examination of these feeding effects have not been established. In this study, we developed a technique using a lipophilic fluorescent reagent, Nile red to quantitatively estimate the adipose tissue volumes by using Japanese rice fish, medaka (Oryzias latipes) and studied effects of dietary soy sauce oil (SSO), which is a discarded by-product from Japanese traditional food and is known to have PPARγ-agonistic activity, on adipogenesis. We found that SSO feeding increased the adipose tissue volumes, and the expression levels of adipogenesis-related genes increased in these medaka larvae. These results suggest that SSO feeding increases the adipose tissue volumes through adipogenesis promotion by PPARγ-agonistic activity in medaka, and medaka is a powerful model for studying adipogenesis. Furthermore, our study also demonstrates the availability of SSO as a dietary additive for farmed fish.