Triacylglycerol (TAG) synthesis and storage in tissues such as adipose tissue and liver have important roles in metabolic homeostasis. The molecular identification of genes encoding enzymes that ...catalyze steps in TAG biosynthesis from glycerol 3-phosphate has revealed an unexpected number of protein isoforms of the glycerol phosphate acyltransferase (GPAT), acylglycerolphosphate acyltransferase (AGPAT), and lipin (phosphatidate phosphatase) families that appear to catalyze similar biochemical reactions. However, on the basis of available data for a few members in which genetic deficiencies in mouse and/or human have been studied, we postulate that each GPAT, AGPAT, and lipin family member likely has a specialized role that may be uncovered through careful biochemical and physiological analyses.
Most aquatic invertebrates adapt to environmental osmotic changes primarily by the cellular osmoconforming process, in which osmolytes accumulated in their cells play an essential role. Taurine is ...one of the most widely utilized osmolytes and the most abundant in many molluscs. Here, we report the structure, function and expression of the taurine transporter in the Mediterranean blue mussel (muTAUT), as a key molecule in the cellular osmoconforming process. Deduced amino acid sequence identity among muTAUT and vertebrate taurine transporters is lower (47-51%) than that among vertebrate taurine transporters (>78%). muTAUT has a lower affinity and specificity for taurine and a requirement for higher NaCl concentration than vertebrate taurine transporters. This seems to reflect the internal environment of the mussel; higher NaCl and taurine concentrations. In addition to the hyperosmotic induction that has been reported for cloned taurine transporters, the increase in muTAUT mRNA was unexpectedly observed under hypoosmolality, which was depressed by the addition of taurine to ambient seawater. In view of the decrease in taurine content in mussel tissue under conditions of hypoosmolality reported previously, our results lead to the conclusion that muTAUT does not respond directly to hypoosmolality, but to the consequent decrease in taurine content. By immunohistochemistry, intensive expression of muTAUT was observed in the gill and epithelium of the mantle, which were directly exposed to intensive osmotic changes of ambient seawater.
A cDNA clone encoding a Na
+- and Cl
−-dependent high affinity taurine transporter was isolated from a common carp cell line,
Epithelioma papulosum cyprini (EPC), as a hyperosmotic stress-inducible ...gene by RNA arbitrarily primed PCR. The clone contained a 2.5-kb cDNA fragment including an open reading frame of 1878 bp encoding a protein of 625 amino acids. The deduced amino acid sequence of carp taurine transporter shows 78–80% identity to those of cloned mammalian taurine transporters. The functional characteristics of the cloned transporter were analyzed by expression in COS-7 cells. Transfection with the cDNA induced Na
+- and Cl
−-dependent taurine transport activity with an apparent
K
m of 56 μM. The Na
+/Cl
−/taurine coupling ratio for cloned transporter was estimated as 2:1:1. Uptake of radiolabeled taurine was inhibited by excessive cold taurine, hypotaurine, β-alanine and γ-aminobutyric acid but not by α-alanine, glycine, leucine or glycinebetaine. Taurine transporter mRNA is ubiquitously distributed in carp tissues, and its level decreases in the order: kidney>spleen>heart>fin>eye≈intestine≈gill>skin>brain>muscle>hepatopancreas. Taurine transporter mRNA level increased up to 7.5-fold on raising the ambient osmolality from 300 to 450 mosmol/kgH
2O. These data suggest the significant role of taurine as an osmolyte in carp cells.
Adipose tissue plays a key role in metabolic homeostasis. Disruption of the Lpin1 gene encoding lipin-1 causes impaired adipose tissue development and function in rodents. Lipin-1 functions as a ...phosphatidate phosphatase (PAP) enzyme in the glycerol 3-phosphate pathway for triglyceride storage and as a transcriptional coactivator/corepressor for metabolic nuclear receptors. Previous studies established that lipin-1 is required at an early step in adipocyte differentiation for induction of the adipogenic gene transcription program, including the key regulator peroxisome proliferator-activated receptor γ (PPARγ). Here, we investigate the requirement of lipin-1 PAP versus coactivator function in the establishment of Pparg expression during adipocyte differentiation. We demonstrate that PAP activity supplied by lipin-1, lipin-2, or lipin-3, but not lipin-1 coactivator activity, can rescue Pparg gene expression and lipogenesis during adipogenesis in lipin-1-deficient preadipocytes. In adipose tissue from lipin-1-deficient mice, there is an accumulation of phosphatidate species containing a range of medium chain fatty acids and an activation of the MAPK/extracellular signal-related kinase (ERK) signaling pathway. Phosphatidate inhibits differentiation of cultured adipocytes, and this can be rescued by the expression of lipin-1 PAP activity or by inhibition of ERK signaling. These results emphasize the importance of lipid intermediates as choreographers of gene regulation during adipogenesis, and the results highlight a specific role for lipins as determinants of levels of a phosphatidic acid pool that influences Pparg expression.
Background: Lipin-1-deficient cells cannot differentiate into mature adipocytes.
Results: Lipin-1 phosphatidic acid phosphatase activity, but not its coactivator activity, is required for induction of the transcription factor peroxisome proliferator-activated receptor γ (PPARγ).
Conclusion: Lipin-1 modulation of phosphatidic acid levels is required for early steps in adipogenesis.
Significance: The levels of signaling lipids are important in adipogenesis prior to fat storage.
Post‐mortem muscle tenderization during chilled storage is thought to result from loss of physical strength due to the disintegration of intramuscular connective tissue. Although metalloproteinases ...may be involved in the disintegration, details of the proteolytic processes occurring during chilled storage remain to be elucidated. To determine the involvement of matrix metalloproteinase‐9 (MMP‐9) in the post‐mortem disintegration of intramuscular connective tissue, we produced recombinant Japanese flounder MMP‐9 (jfMMP‐9) in COS‐7 cells. The recombinant jfMMP‐9 showed gelatinolytic activity in the zymographic analysis at chilled temperature, but no apparent proteolytic activity against the triple helical domain of types I and type V collagens. In contrast, recombinant jfMMP‐9 solubilized type I collagen from a crude connective tissue preparation at chilled temperature. This finding, together with the fact that a protein corresponding to MMP‐9 was immunologically detected in the muscle extract, indicates that MMP‐9 may be involved in the disintegration of the intramuscular connective tissue that induces the post‐mortem tenderization of fish muscle during chilled storage.
Trimethylamine- N -oxide (TMAO) is abundant in marine fish. Formaldehyde synthesis by TMAO demethylation during storage markedly deteriorates
fish meat. In the present work, we cloned the extremely ...aspartic acid-rich proteins from skeletal muscle of a commercially
important species, walleye pollack, in the course of molecular identification of trimethylamine- N -oxide demethylase (TMAOase). One of the cDNAs, designated as aspolin1, encodes an extremely aspartic acid-rich protein of
228 amino acids which is converted to the TMAOase after processing between Ala 42 and Asp 43 . Mature aspolin1/TMAOase protein contains 179 Asp in 186 total amino acids. The other cDNA, designated as aspolin2, has a
common nucleotide sequence with aspolin1 in the 5â² part and encodes a protein which has an additional Asp polymer and a C-terminal
cysteine-rich region. The amino acid sequence of the C-terminal cysteine-rich region of aspolin2 is highly homologous to the
mammalian histidine-rich Ca 2+ -binding protein. Aspolin1/TMAOase and aspolin2 mRNA was most abundant in the skeletal muscle. A lower level of the mRNA was
also detected in kidney, heart, spleen, and brain. Synthetic Asp polymer showed marked TMAOase activity in the presence of
Fe 2+ , whereas a monomer and oligomers did not. Purified TMAOase protein bound to Fe 2+ with low affinity, which may be responsible for the catalytic activity. Poly aspartic acid-Fe 2+ complex generated after death would be involved in formaldehyde synthesis by the demethylation of TMAO during the storage
of fish meat.
We have cloned a cDNA encoding the MMP‐9 from a carp epidermal cell (EPC) cDNA
library. The clone contains a 2025‐base pair (bp) open reading frame encoding a protein
of 674 amino acids. The deduced ...amino acid sequence shares 68% and 69%
identity with medaka and Japanese flounder MMP‐9. The hinge domain of the carp MMP‐9,
like those of the other non‐mammalian species, lacks a type V collagen‐like region
that is typical of mammalian MMP‐9. Gelatin zymography and immunoblot analysis of
conditioned media of EPC cells and cDNA‐transfected COS‐7 cells detected a 76‐kDa
gelatinase. The apparent molecular mass of the carp zymogen is much smaller than
those of its mammalian counterparts while almost identical with that of chicken 75‐kDa
gelatinase B‐like enzyme. Although hypo‐osmotic stress induced the elevation of MMP‐9
mRNA level in EPC cells, no significant change in the protein in conditioned medium
was detected during hypo‐osmotic stress. Northern blot analysis detected a large
amount of MMP‐9 mRNA in carp kidney and spleen, suggesting the high expression
of MMP‐9 in blood cells, neutrophils, and macrophages. The smaller amount
of MMP‐9 mRNA was detected in gill, heart, fin, and eye, whereas none of the mRNA was detected in the hepatopancreas, intestine, brain, muscle, and skin.
Toughness is one of the most important elements that define the commercial value of the raw meat of fish. Degradation of the extracellular matrix is thought to be a cause of postmortem tenderization ...of fish meat. A previous study has suggested that this tenderization is caused mainly by metalloproteinases. The present study seeks to identify the proteinase(s) involved in tenderization; hence, cloned cDNA of two gelatinases from Japanese flounder, which showed high homology with mammalian matrix metalloproteinase (MMP)‐2 and MMP‐9, were designated as jfMMP‐2 and
jfMMP‐9, respectively. Northern blot analysis revealed that jfMMP‐2
mRNA was expressed almost ubiquitously in adult tissues including the brain, muscle,
gill, heart, gut, kidney, spleen, testis, and ovary. In contrast, the expression
of jfMMP‐9 mRNA was observed in those tissues which were abundant in blood cells, such as kidney, spleen, heart, and gill. Both recombinant proteins (jfMMP‐2 and jfMMP‐9) produced with the COS‐7 cell system exhibited gelatin‐degrading activity that was sensitive to 1,10‐phenanthroline, a typical metalloproteinase inhibitor.
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