Motowo Tomita

The fat-derived hormone adiponectin reverses insulin resistance associated with both lipoatrophy and obesity.

Adiponectin is an adipocyte-derived hormone. Recent genome-wide scans have mapped a susceptibility locus for type 2 diabetes and metabolic syndrome to chromosome 3q27, where the gene encoding adiponectin is located. Here we show that decreased expression of adiponectin correlates with insulin resistance in mouse models of altered insulin sensitivity. Adiponectin decreases insulin resistance by decreasing triglyceride content in muscle and liver in obese mice. This effect results from increased expression of molecules involved in both fatty-acid combustion and energy dissipation in muscle. Moreover, insulin resistance in lipoatrophic mice was completely reversed by the combination of physiological doses of adiponectin and leptin, but only partially by either adiponectin or leptin alone. We conclude that decreased adiponectin is implicated in the development of insulin resistance in mouse models of both obesity and lipoatrophy. These data also indicate that the replenishment of adiponectin might provide a novel treatment modality for insulin resistance and type 2 diabetes.

SP-40,40 is a constituent of Alzheimer's amyloid

SummaryCerebrospinal fluid (CSF), serum and seminal plasma contain a small amount of SP-40,40, a modulatory protein of the human complement system. The SP-40,40 in each body fluid was different in molecular size on SDS-PAGE, and glioblastoma cells, hepatoma cells and testicular tumor cells produced SP-40,40, while neuroblastoma cells did not. Therefore, it was estimated that CSF SP-40,40 originated in glia cells, serum SP-40,40 in liver cells and seminal plasma SP-40,40 in testicular cells. SP-40,40 concentrations in CSF of the patients with Alzheimers disease and the patients with cerebral tumor were higher than those of normal donors. β-Amyloid deposits in the brains of the patients with Alzheimers disease were stained with an anti-SP-40,40 monoclonal antibody (mAb) but not with an anti-S-protein mAb, while cellular processes around β-amyloid were stained with an anti-S-protein mAb but not with an anti-SP-40,40 mAb. Therefore, β-amyloid contained SP-40,40 in a form different from that in the soluble membrane attack complex (SMAC, SC5b-9) of the complement, which contains S-protein as well as SP-40,40.

A novel enzyme-linked immunosorbent assay specific for high-molecular-weight adiponectin

Human plasma contains at least three forms of adiponectin: a trimer, a hexamer, and a high-molecular-weight (HMW) multimer. We purified HMW adiponectin from human plasma using its affinity to gelatin and obtained monoclonal antibodies against it. On Western blot analysis, the reactivity of these monoclonal antibodies was shown to be restricted to a non-heat-denatured form of adiponectin molecules. On heating, the collagen-like domain of adiponectin molecules became denatured, and thus the trimer form could not be maintained. From these, monoclonal antibodies against HMW adiponectin were suggested to react with the intact trimer of adiponectin. With these monoclonal antibodies, we developed a sandwich ELISA system for quantifying adiponectin in human serum. Its specificity was verified by analysis of serum fractions separated by gel-filtration chromatography, and our ELISA system was found to be HMW adiponectin-specific. With this novel ELISA, the HMW adiponectin concentrations were 8.4 ± 5.5 μg/ml (mean ± SD) in healthy women and 6.2 ± 3.6 μg/ml in healthy men. Also, serum with a lower HMW adiponectin concentration was shown to have a lower HMW ratio (i.e., HMW adiponectin/total adiponectin).

Distribution and Synthesis of Apolipoprotein J in the Atherosclerotic Aorta

The distribution of apolipoprotein (apo) J during the development of atherosclerosis in the human aorta was evaluated by immununohistochemical observation, together with the other apolipoprotein A-I, A-II, B, C-III, and E. Although apoJ was never observed in the normal aorta (ie, without any intimal lesions or intimal thickening), it was distributed not only in the intima but also in the media of aortas with diffuse, intimal thickening or atherosclerotic lesions. Double immunostaining with antibodies for apoJ and alpha-smooth muscle actin revealed apoJ deposition in smooth muscle cells (SMCs) or the aortic stroma in the vicinity of SMCs. The extent of apoJ distribution in the aortic wall increased with the degree of atherosclerosis development. In addition, the distribution pattern of apoJ was very similar to that of apoA-I and E. In situ hybridization with human apoJ cDNA demonstrated intense signals in cells scattered within the subendothelial space and medial SMCs of the aorta with advanced atherosclerosis but not in those of the normal aorta without intimal thickening. Furthermore, reverse transcriptase-polymerase chain reaction of the cultured human aortic SMCs revealed apoJ mRNA expression in these cells. The results indicate that apoJ in the aortic wall originates from not only apoJ circulated in the plasma but also apoJ produced by SMCs in the aortic wall. Considering the similarities of the distribution between apoJ and apo-A-I or E, we hypothesize that apoJ possibly has a protective role against human atherosclerosis by its involvement with cholesterol transport from the aortic wall to the liver.

Cloning and Characterization of the cDNA Encoding a Novel Brain‐Specific 14‐kDa Protein

Abstract: A new acidic protein with a molecular weight of 14,000 was purified from rat brain, in which it was specifically expressed, and partially sequenced by protein sequencg. On the basis of results obtained from the amino acid sequences, mixed oligonucleotides were synthesized and used as probes to clone a cDNA from a rat brain cDNA library. The cloned cDNA provided the full‐length Sequence of the 14‐kDa protein. Northern blot hybridization using total RNA from several tissues of the rat provided evidence that the 14‐kDa protein was expressed specifically in rat brain. Transfection of this cDNA into mammalian cells resulted in expression of the 14‐kDa protein. The amino acid sequence predicted from the cDNA of the rat brain 14‐kDa protein contained 137 amino acid residues. A hydropathy profile revealed a hydrophobic domain (amino acids 60–80) flanked by highly hydrophilic stretches on both sides. Whereas the N‐terminal region of the 14‐kDa protein contained four repeating motifs, EKTKEGV, the C‐terminal domain was rich in glutamic acid and proline. A computer search of the amino acid sequence of the 14‐kDa protein indicated no homology to any other protein reported so far.

p57KIP2 Modulates Stress-activated Signaling by Inhibiting c-Jun NH2-terminal Kinase/Stress-activated Protein Kinase

p57KIP2, a member of the Cip/Kip family of enzymes that inhibit several cyclin-dependent kinases, plays a role in many biological events including cell proliferation, differentiation, apoptosis, tumorigenesis and developmental changes. The human p57KIP2 gene is located in chromosome 11p15.5, a region implicated in sporadic cancers and Beckwith-Wiedemann syndrome. We here report that p57KIP2 physically interacts with and inhibits c-Jun NH2-terminal kinase/stress-activated protein kinase (JNK/SAPK). The carboxyl-terminal QT domain of p57KIP2 is crucial for the inhibition of JNK/SAPK. Overexpressed p57KIP2 also suppressed UV- and MEKK1-induced apoptotic cell death. p57KIP2 expression during C2C12 myoblast differentiation resulted in repression of the JNK activity stimulated by UV light. Furthermore, UV-stimulated JNK1 activity was higher in mouse embryonic fibroblasts derived from p57–/– mice than in the cells from wild-type mice. Taken together, these findings suggest that p57KIP2 modulates stress-activated signaling by functioning as an endogenous inhibitor of JNK/SAPK.

Changes in the distribution pattern of gelatin-binding protein of 28 kDa (adiponectin) in myocardial remodelling after ischaemic injury.

Aims:  Gelatin‐binding protein of 28 kDa (GBP28) is a collagen‐like plasma protein having a binding capacity with collagens. We investigated GBP28 role on myocardial remodelling as well as the diagnostic significance of GBP28 immunostaining in myocardial infarction.

Sandwich ELISA assay for quantitative measurement of SP-40, 40 in seminal plasma and serum

SP-40,40 was purified from human plasma by PEG fractionation, DEAE-Sephacel, Phenyl-Toyopearl 650M, Bio-Gel A-0.5m and hydroxylapatite chromatographies. Three monoclonal antibodies (IF12, IID9 and IVF4) to this protein were prepared: IF12 and IID9 were specific for the beta subunit and IVF4 for the alpha subunit. The concentrations of SP-40,40 in seminal plasmas and sera were determined using a sandwich ELISA method. The results showed that the average concentrations of SP-40,40 were 438 +/- 285 micrograms/ml in seminal plasmas and 111 +/- 50 micrograms/ml in sera of normal donors. SP-40,40 concentrations in seminal plasmas of Klinefelter and excretory azoospermia patients were similar to those of normal donors. However, those of oligozoospermia and idiopathic azoospermia patients were about half the normal value.

Association of a thyroglobulin gene polymorphism with hashimoto's thyroiditis in the Japanese population

objective  The aetiology of the autoimmune thyroid diseases (AITDs), Graves’ disease (GD) and Hashimotos thyroiditis is largely unknown. However, genetic susceptibility is believed to play a major role. Two whole genome scans from Japan and from the USA identified a locus on chromosome 8q24 which showed evidence for linkage with AITD and HT. Recent studies have demonstrated an association between a Tg polymorphisms and AITD, suggesting that Tg is the susceptibility gene on 8q24.

Characterization of mouse GBP28 and its induction by exposure to cold

OBJECTIVE: To investigate whether the expression of the novel adipose tissue-specific protein GBP28 in adipose tissue and serum are altered in mice under a variety of conditions.DESIGN: Mice were fed a high-fat diet for 4 weeks, fasted for 48 h or exposed at 4°C.SUBJECTS: C57BL/6J mouse, male, 4–6 weeks old.MEASUREMENTS: GBP28 mRNA, GBP28 protein, blood glucose, insulin and fad pad weight of the mice.RESULTS: We first confirmed that the mouse has GBP28 and its characteristics are the same as human GBP28. Serum concentration and mRNA levels of GBP28 significantly increased in the mice exposed to cold.CONCLUSION: GBP28 may play a role in homeostasis, regulating body temperature and basal metabolic rate in response to changing environmental conditions.