Hideki Kizawa

Free fatty acids regulate insulin secretion from pancreatic beta cells through GPR40.

Diabetes, a disease in which carbohydrate and lipid metabolism are regulated improperly by insulin, is a serious worldwide health issue. Insulin is secreted from pancreatic β cells in response to elevated plasma glucose, with various factors modifying its secretion. Free fatty acids (FFAs) provide an important energy source as nutrients, and they also act as signalling molecules in various cellular processes, including insulin secretion. Although FFAs are thought to promote insulin secretion in an acute phase, this mechanism is not clearly understood. Here we show that a G-protein-coupled receptor, GPR40, which is abundantly expressed in the pancreas, functions as a receptor for long-chain FFAs. Furthermore, we show that long-chain FFAs amplify glucose-stimulated insulin secretion from pancreatic β cells by activating GPR40. Our results indicate that GPR40 agonists and/or antagonists show potential for the development of new anti-diabetic drugs.

An aspartic acid repeat polymorphism in asporin inhibits chondrogenesis and increases susceptibility to osteoarthritis.

Osteoarthritis is the most common form of human arthritis. We investigated the potential role of asporin, an extracellular matrix component expressed abundantly in the articular cartilage of individuals with osteoarthritis, in the pathogenesis of osteoarthritis. Here we report a significant association between a polymorphism in the aspartic acid (D) repeat of the gene encoding asporin (ASPN) and osteoarthritis. In two independent populations of individuals with knee osteoarthritis, the D14 allele of ASPN is over-represented relative to the common D13 allele, and its frequency increases with disease severity. The D14 allele is also over-represented in individuals with hip osteoarthritis. Asporin suppresses TGF-β–mediated expression of the genes aggrecan (AGC1) and type II collagen (COL2A1) and reduced proteoglycan accumulation in an in vitro model of chondrogenesis. The effect on TGF-β activity is allele-specific, with the D14 allele resulting in greater inhibition than other alleles. In vitro binding assays showed a direct interaction between asporin and TGF-β. Taken together, these findings provide another functional link between extracellular matrix proteins, TGF-β activity and disease, suggesting new therapeutic strategies for osteoarthritis.

A functional SNP in CILP , encoding cartilage intermediate layer protein, is associated with susceptibility to lumbar disc disease

Lumbar disc disease (LDD) is caused by degeneration of intervertebral discs of the lumbar spine. One of the most common musculoskeletal disorders, LDD has strong genetic determinants. Using a case-control association study, we identified a functional SNP (1184T → C, resulting in the amino acid substitution I395T) in CILP, which encodes the cartilage intermediate layer protein, that acts as a modulator of LDD susceptibility. CILP was expressed abundantly in intervertebral discs, and its expression increased as disc degeneration progressed. CILP colocalized with TGF-β1 in clustering chondrocytes and their territorial matrices in intervertebral discs. CILP inhibited TGF-β1–mediated induction of cartilage matrix genes through direct interaction with TGF-β1 and inhibition of TGF-β1 signaling. The susceptibility-associated 1184C allele showed increased binding and inhibition of TGF-β1. Therefore, we conclude that the extracellular matrix protein CILP regulates TGF-β signaling and that this regulation has a crucial role in the etiology and pathogenesis of LDD. Our study also adds to the list of connective tissue diseases that are associated with TGF-β.

Mechanisms for Asporin Function and Regulation in Articular Cartilage

Osteoarthritis (OA), the most prevalent form of skeletal disease, represents a leading cause of disability following middle age. OA is characterized by the loss of articular cartilage; however, the details of its etiology and pathogenesis remain unclear. Recently, we demonstrated a genetic association between the cartilage extracellular matrix protein asporin and OA (Kizawa, H., Kou, I., Iida, A., Sudo, A., Miyamoto, Y., Fukuda, A., Mabuchi, A., Kotani, A., Kawakami, A., Yamamoto, S., Uchida, A., Nakamura, K., Notoya, K., Nakamura, Y., and Ikegawa, S. (2005) Nat. Genet. 37, 138-144). Furthermore, we showed that asporin binds to transforming growth factor-β (TGF-β), a key cytokine in OA pathogenesis, and inhibits TGF-β-induced chondrogenesis. To date, functional data for asporin have come primarily from mouse cell culture models of developing cartilage rather than from human articular cartilage cells, in which OA occurs. Here, we describe mechanisms for asporin function and regulation in human articular cartilage. Asporin blocks chondrogenesis and inhibits TGF-β1-induced expression of matrix genes and the resulting chondrocyte phenotypes. Small interfering RNA-mediated knockdown of asporin increases the expression of cartilage marker genes and TGF-β1; in turn, TGF-β1 stimulates asporin expression in articular cartilage cells, suggesting that asporin and TGF-β1 form a regulatory feedback loop. Asporin inhibits TGF-β/Smad signaling upstream of TGF-β type I receptor activation in vivo by co-localizing with TGF-β1 on the cell surface and blocking its interaction with the TGF-β type II receptor. Our results provide a basis for elucidating the role of asporin in the molecular pathogenesis of OA.

Production of recombinant human relaxin 3 in AtT20 cells

Relaxin 3 has been reported recently as a member of the insulin/IGF/relaxin family. To clarify the function of relaxin 3, we prepared recombinant human relaxin 3 using a mouse adrenocorticotrophic hormone (ACTH)-secreting cell line, AtT20. To detect a mature form of recombinant human relaxin 3, a competitive enzyme immunoassay (EIA) was developed using a monoclonal antibody (mAb; HK4-144-10), which was raised for the N-terminal peptide of human relaxin 3 A-chain. We detected immunoreactive (ir-) relaxin 3 in the culture supernatant of AtT20 cells stably transfected with human relaxin 3 cDNA. After treatment with 5 microM forskolin for 3 days, the concentration of the ir-relaxin 3 in the culture supernatant reached 12 nM. Ir-relaxin 3 was purified from the culture supernatant by a combination of various chromatographies. By analyses of N-terminal amino acid sequence and electrospray ionization mass spectrometry (ESI-MS), we confirmed that the purified material was a mature form of human relaxin 3. The recombinant human relaxin 3 thereby obtained increased intracellular cAMP production in THP-1 cells. Our results demonstrate that the expression of relaxin 3 cDNA in AtT20 cells is a useful tool to produce a bioactive and mature form of relaxin 3.

Scaffold-free 3D bio-printed human liver tissue stably maintains metabolic functions useful for drug discovery

The liver plays a central role in metabolism. Although many studies have described in vitro liver models for drug discovery, to date, no model has been described that can stably maintain liver function. Here, we used a unique, scaffold-free 3D bio-printing technology to construct a small portion of liver tissue that could stably maintain drug, glucose, and lipid metabolism, in addition to bile acid secretion. This bio-printed normal human liver tissue maintained expression of several kinds of hepatic drug transporters and metabolic enzymes that functioned for several weeks. The bio-printed liver tissue displayed glucose production via cAMP/protein kinase A signaling, which could be suppressed with insulin. Bile acid secretion was also observed from the printed liver tissue, and it accumulated in the culture medium over time. We observed both bile duct and sinusoid-like structures in the bio-printed liver tissue, which suggested that bile acid secretion occurred via a sinusoid-hepatocyte-bile duct route. These results demonstrated that our bio-printed liver tissue was unique, because it exerted diverse liver metabolic functions for several weeks. In future, we expect our bio-printed liver tissue to be applied to developing new models that can be used to improve preclinical predictions of long-term toxicity in humans, generate novel targets for metabolic liver disease, and evaluate biliary excretion in drug development.

High-resolution SNP map of ASPN, a susceptibility gene for osteoarthritis.

AbstractOsteoarthritis (OA) is a very common bone and joint disease characterized by breakdown of cartilage in the joint. We recently found that an aspartic-acid repeat polymorphism of the asporin gene (ASPN) on chromosome 9 is associated with susceptibility to OA in Japanese. We provide here a high-resolution single nucleotide polymorphism (SNP) map within a 33.4-kb genomic region containing ASPN. A total of 19 SNPs were isolated from the region by systematic screening using 48 Japanese patients with OA: 7 SNPs in the 5′ flanking region, 8 in introns, and 4 in the 3′ untranslated region. Nine SNPs were novel. This high-resolution SNP map will be a useful resource for analyzing genes associated with OA and other bone and joint diseases.

Association of CYP17 with HLA-B27-negative seronegative spondyloarthropathy in Japanese males.

Susceptibility genes for seronegative spondyloarthropathy (SNSA) other than HLA‐B27 remain unclarified. Sex hormones are implicated in the pathogenesis of SNSA. Cytochrome P450c17a (CYP17) is a key regulator of androgen biosynthesis, and a single nucleotide polymorphism (SNP) in the 5′‐untranslated region of the CYP17 gene (CYP17), −34C > T, is associated with variety of diseases. We have investigated the association between the CYP17 SNP and SNSA in Japanese males. Genomic DNA was extracted from 149 Japanese male SNSA patients and 380 controls. The CYP17 SNP was genotyped using polymerase chain reaction‐restriction fragment length polymorphism analysis. Allelic and genotypic frequencies of the SNP were compared between SNSA patients and controls, and within SNSA patients. We also computed haplotype frequencies using an expectation‐maximization algorithm, analyzed the difference between SNSA and control groups, and examined the potential association of other known SNPs in the CYP17 gene. The frequency of the −34T allele was significantly increased in HLA‐B27‐negative SNSA, but not in total or HLA‐B27‐positive SNSA when compared to controls. The T allele was more prevalent in HLA‐B27‐negative SNSA than in HLA‐B27‐positive SNSA, and the T/T genotype was over‐represented in HLA‐B27‐negative SNSA. Haplotype analysis did not demonstrate more significant association. The CYP17 SNP is associated with SNSA in HLA‐B27‐negative Japanese males.