An adipocyte-derived peptide, adiponectin (also known as GBP28), is decreased in subjects with type 2 diabetes. Recent genome-wide scans have mapped a diabetes susceptibility locus to chromosome 3q27, where the adiponectin gene (APM1) is located. Herein, we present evidence of an association between frequent single nucleotide polymorphisms at positions 45 and 276 in the adiponectin gene and type 2 diabetes (P = 0.003 and P = 0.002, respectively). Subjects with the G/G genotype at position 45 or the G/G genotype at position 276 had a significantly increased risk of type 2 diabetes (odds ratio 1.70 [95% CI 1.09-2.65] and 2.16 [1.22-3.95], respectively) compared with those having the T/T genotype at positions 45 and 276, respectively. In addition, the subjects with the G/G genotype at position 276 had a higher insulin resistance index than those with the T/T genotype (1.61 +/- 0.05 vs. 1.19 +/- 0.12, P = 0.001). The G allele at position 276 was linearly associated with lower plasma adiponectin levels (G/G: 10.4 +/- 0.85 microg/ml, G/T: 13.7 +/- 0.87 microg/ml, T/T: 16.6 +/- 2.24 microg/ml, P = 0.01) in subjects with higher BMIs. Based on these findings together with the observation that adiponectin improves insulin sensitivity in animal models, we conclude that the adiponectin gene may be a susceptibility gene for type 2 diabetes.

By use of its affinity to gelatin-Cellulofine, a novel protein, GBP28 (gelatin-binding protein of 28 kDa), was obtained from human plasma. GBP28 bound to gelatin-Cellulofine could be eluted with 1 M NaCl. By analysis of its amino-terminal amino acid sequences and the peptides obtained by protease digestion, GBP28 was identified as a novel protein. After repeated gel chromatography of the 1 M NaCl eluate from gelatin-Cellulofine, about 50 micrograms of GBP28 was purified from 500 ml of human plasma. On gel chromatography, the protein migrated as a molecule of about 420 kDa. On SDS-PAGE, its molecular mass was 28 kDa under reducing conditions and 68 kDa under nonreducing conditions. Recently, human mRNA specific to adipose tissue, cDNA clone apM1, has been registered [Maeda, K., Okubo, K., Shimomura, I., Funahashi, T., Matsuzawa, Y., and Matsubara, K. (1996) Biochem. Biophys. Res. Commun. 221, 286-289]. The assumed amino acid sequence of cDNA clone apM1 contained all the sequences of GBP28 and its peptides. Therefore, it is evident that the cDNA clone apM1 encodes GBP28 and the protein is specific to adipose tissue. The clone encodes a polypeptide of 244 amino acids with a secretory signal sequence at the amino terminus, a small non-helical region, a stretch of 22 collagen repeats and a globular domain. Thus, GBP28 appears to belong to a family of proteins possessing a collagen-like domain through which they form homo-trimers, which further combine to make oligomeric complexes. Although its biological function is presently unclear, its adipocyte-specific expression suggests that GBP28 may function as an endogenous factor involved in lipid catabolism and storage or whole body metabolism.

Adipocyte-specific secreted molecules, termed adipokines, have dispelled the notion of adipose tissue as an inert storage depot for lipids, and highlighted its role as an active endocrine organ that monitors and alters whole-body metabolism and maintains energy homeostasis. One of these adipokines, adiponectin (also known as Acrp30, AdipoQ, and GBP28), has gained significant attention recently as a mediator of insulin sensitivity. Many clinical reports and genetic studies over the past few years demonstrate decreased circulating levels of this hormone in metabolic dysfunction, such as obesity and insulin resistance, in both humans and animal models. Pharmacologic adiponectin treatments in rodents increase insulin sensitivity, although the primary site and detailed mechanism of action is yet to be determined. The phenotypes of adiponectin-deficient and transgenic adiponectin-overproducing animal models underscore the role of adiponectin in the maintenance of glucose and lipid homeostasis.

Adiponectin is a white and brown adipose tissue hormone, also known as gelatin-binding protein-28 (GBP28), AdipoQ, adipocyte complement-related protein (ACRP30), or apM1. Adiponectin circulates in the bloodstream in trimeric, hexameric, and high-molecular-mass species, while different forms of adiponectin have been found to play distinct roles in the balance of energy homoeostasis. Adiponectin is an insulin sensitizing hormone that exerts its action through its receptors AdipoR1, AdipoR2, and T-cadherin. AdipoR1 is expressed abundantly in muscle, whereas AdipoR2 is predominantly expressed in the liver. Adiponectin is inversely proportional to obesity, diabetes, and other insulin-resistant states. In this review we present the current findings regarding the regulation of its production and several new findings pertaining to its biological effects. Adiponectin enhances AMPK and the PPARα pathway in the liver and skeletal muscle. Adiponectin increases fatty acids oxidation, which lowers circulating free fatty acids and prevents insulin resistance. Adiponectin has been reported to exert an antiatherosclerotic effect. It inhibits macrophage activation and foam cell accumulation, while it also augments endothelial nitrous oxide production and protects the vasculature by reducing platelet aggregation and vasodilation. Apart from causing metabolic dysfunction, adiponectin deficiency may also contribute to coronary heart disease, steatohepatitis, insulin resistance, nonalcoholic fatty liver disease, and a wide array of cancers. In this study, we present ample evidence that adiponectin mediates multiple molecular pathways. We therefore support the concept that it shows distinct potential for being of therapeutic value in the treatment of obesity related diseases, ranging from metabolic syndrome to malignancies.

We have screened a subtracted cDNA library in order to identify differentially expressed genes in omental adipose tissue of human patients with Type 2 diabetes. One clone (#1738) showed a marked reduction in omental adipose tissue from patients with Type 2 diabetes. Sequencing and BLAST analysis revealed clone #1738 was the adipocyte-specific secreted protein gene apM1 (synonyms ACRP30, AdipoQ, GBP28). Consistent with the murine orthologue, apM1 mRNA was expressed in cultured human adipocytes and not in preadipocytes. Using RT-PCR we confirmed that apM1 mRNA levels were significantly reduced in omental adipose tissue of obese patients with Type 2 diabetes compared with lean and obese normoglycemic subjects. Although less pronounced, apM1 mRNA levels were reduced in subcutaneous adipose tissue of Type 2 diabetic patients. Whereas the biological function of apM1 is presently unknown, the tissue specific expression, structural similarities to TNFalpha, and the dysregulated expression observed in obese Type 2 diabetic patients suggest that this factor may play a role in the pathogenesis of insulin resistance and Type 2 diabetes.

3T3-L1-adipocytes produce the adipocyte complement related protein of 30 kD (Acrp30), which is also designated as AdipoQ. In order to study the expression and secretion of the human homologue of this protein, apM1 (adipose Most abundant gene transcript 1, also named gelatin-binding protein of 28 kD [GBP28] or adiponectin), a polyclonal antibody was produced. Both expression and secretion can be detected beginning with day 4 after induction of differentiation. The amount of expressed apM1 correlates with the specific activity of the differentiation marker glycerol-3-phosphate dehydrogenase. Secretion of apM1 is increased by the addition of ionomycin. Both the nonhydrolysable dibutyryl-cycloAMP and tumour necrosis factor alpha reduce the expression and secretion of apM1.

In recent years, the simple paradigm of adipose tissue as merely a fat store is rapidly evolving into a complex paradigm of this tissue as multipotential secretory organ, partitioned into a few large depots, including visceral and subcutaneous location, and many small depots, associated with a variety of organs in the human body. The major secretory compartment of adipose tissue consists of adipocytes, fibroblasts, and mast cells. These cells, using endocrine, paracrine and autocrine pathways, secrete multiple bioactive molecules, conceptualized as adipokines or adipocytokines. This review examines current information in adipobiology of various diseases besides obesity and related diseases such as type 2 diabetes, metabolic syndrome, and cardiovascular disease. Finally, we emphasize the possibilities for adipokine-targeted pharmacology in adiponectin (Acrp30, apM1, AdipoQ, GBP28), angiotensin II, estrogens, nerve growth factor, tumor necrosis factor-alpha, and also adipose mast cells.

Adiponectin is an insulin-sensitizing and anti-inflammatory fat cell hormone that has immense potential as a therapeutic target for a multitude of obesity-associated diseases, including type 2 diabetes, NASH and atherosclerosis (Chandran M, Phillips SA, Ciaraldi T, Henry RR: Adiponectin: more than just another fat cell hormone?Diabetes Care 2003, 26:2442-2450). The adiponectin gene is located in chromosome 3q27, a susceptibility locus for T2DM and metabolic disorders (Saito K, Tobe T, Minoshima S, Asakawa S, Sumiya J, Yoda M, Nakano Y, Shimizu N, Tomita M: Organization of the gene for gelatin-binding protein (GBP28). Gene 1999, 229:67-73). Increased circulating levels of adiponectin are associated with improvement in the metabolic syndrome and reductions are strongly predictive of diabetes risk (Li S, Shin HJ, Ding EL, van Dam RM: Adiponectin levels and risk of type 2 diabetes: a systematic review and meta-analysis. JAMA 2009, 302:179-188. Extensive efforts have been made to understand how adiponectin levels can be elevated. The complex post-translational processing and secretion of adiponectin provides a rich area where pharmacologic manipulation may be developed to increase adiponectin levels in humans. Circulating adiponectin levels are increased by many commonly used drugs, such as statins, angiotensin converting enzyme (ACE) inhibitors, and thiazolidinediones (TZDs) providing an important opportunity to gain insight into the mechanisms underlying their effects. This review describes the cellular processes by which adiponectin is synthesized and secreted, current therapeutics known to affect this pathway and the potential for therapeutic manipulation in human subjects.

GBP28 is a novel human plasma gelatin-binding protein that is encoded by apM1 mRNA, expressed specifically in adipose tissue. Three overlapping clones (two lambda clones and one BAC clone) containing the human plasma gelatin-binding protein (GBP28) gene were isolated and characterized. The GBP28 gene spans 16kb and is composed of three exons from 18bp to 4277bp in size with consensus splice sites. The sizes of the two introns were 0.8 and 12kb, respectively. The gene's regulatory sequences contain putative promoter elements, but no typical TATA box. The third exon of this gene contains a long 3'-untranslated sequence containing three Alu repeats. The exon-intron organization of this gene was very similar to that of obese gene, encoding leptin. We also report the chromosome mapping of this gene by fluorescence in situ hybridization (FISH) using a genomic DNA fragment as a probe. The GBP28 gene was located on human chromosome 3q27. The nucleotide sequence data reported in this paper will appear in the DDBJ/EMBL/GenBank nucleotide sequence databases with the accession numbers ABO12163, ABO12164 or ABO12165.

GBP28 (gelatin-binding protein of 28 kDa)/adiponectin is an adipocyte-producing plasma protein proposed to interact with the extracellular matrix. To examine the production of GBP28 in non-adipose tissues, we herein analyzed its expression and localization in mouse livers before and after CCl(4) treatment. In immunohistochemical analyses, the boundary of hepatocytes provided positive signals for GBP28 after 3-6 h and their cytoplasm was intensely stained after 18 h of CCl(4) treatment. Quantitative RT-PCR and in situ hybridization revealed that GBP28 mRNA expression was markedly elevated in CCl(4)-treated mouse livers. These results suggest that the circulating GBP28 binds the extracellular matrices of hepatocytes during the initial stage of CCl(4)-induced hepatic injury and the damaged hepatocytes themselves started to produce GBP28 thereafter. The induced expression of GBP28 was also observed in human hepatoma HepG2 cells after treatment with IL-6. Thus, GBP28 is also produced by the liver, where it undergoes tissue damage-induced transcriptional regulation.

Adiponectin is one of the most important adipocytokines secreted by adipocytes and is called a "guardian angel adipocytokine" owing to its unique biological functions. Adiponectin inversely correlates with body fat mass and visceral adiposity. Identified independently by four different research groups, adiponectin has multiple names; Acrp30, apM1, GBP28, and AdipoQ. Adiponectin mediates its biological functions via three known receptors, AdipoR1, AdipoR2, and T-cadherin, which are distributed throughout the body. Biological functions of adiponectin are multifold ranging from anti-diabetic, anti-atherogenic, anti-inflammatory to anti-cancer. Lower adiponectin levels have been associated with metabolic syndrome, type 2 diabetes, insulin resistance, cardiovascular diseases, and hypertension. A plethora of experimental evidence supports the role of obesity and increased adiposity in multiple cancers including breast, liver, pancreatic, prostrate, ovarian, and colorectal cancers. Obesity mediates its effect on cancer progression via dysregulation of adipocytokines including increased production of oncogenic adipokine leptin along with decreased production of adiponectin. Multiple studies have shown the protective role of adiponectin in obesity-associated diseases and cancer. Adiponectin modulates multiple signaling pathways to exert its physiological and protective functions. Many studies over the years have shown the beneficial effect of adiponectin in cancer regression and put forth various innovative ways to increase adiponectin levels.

Adiponectin (APN), also known as apM1, Acrp30, GBP28 and adipoQ, is a circulating hormone that is predominantly produced by adipose tissue. Many pharmacological studies have demonstrated that this protein possesses potent anti-diabetic, anti-atherogenic and anti-inflammatory properties. Although several studies have demonstrated the antioxidative activity of this protein, the regulatory mechanisms have not yet been defined in skeletal muscles. The aim of the present study was to examine the cytoprotective effects of APN against damage induced by oxidative stress in mouse-derived C2C12 myoblasts. APN attenuated H2O2-induced growth inhibition and exhibited scavenging activity against intracellular reactive oxygen species that were induced by H2O2. Furthermore, treating C2C12 cells with APN significantly induced heme oxygenase-1 (HO-1) and nuclear factor-erythroid 2 related factor 2 (Nrf2). APN also suppressed H2O2-induced mitophagy and partially inhibited the colocalization of mitochondria with autophagosomes/lysosomes, correlating with the expression of Pink1 and Parkin and mtDNA. Moreover, APN protected C2C12 myoblasts against oxidative stress-induced apoptosis. Furthermore, APN significantly reduced the mRNA and protein expression levels of Bax. These data suggest that APN has a moderate regulatory role in oxidative stress-induced mitophagy and suppresses apoptosis. These findings demonstrate the antioxidant potential of APN in oxidative stress-associated skeletal muscle diseases.

OBJECTIVE

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.

RESULTS

Myocardial tissues obtained from 47 autopsied hearts with infarction were immunostained with antibodies against GBP28, fibronectin, type III and IV collagens, and prolyl 4-hydroxylase. GBP28 was distributed in interstitium of infarcted lesions at an early stage. GBP28 was linearly found both along the border with vital myocardium and at the periphery of surviving cardiomyocyte around the lesion at granulative stage. However, it was not observed in the scar. GBP28 distribution patterns were similar to those of fibronectin in infarcted lesions and those of type IV collagen at the periphery of cardiomyocyte. Type III collagen was not recognized in the early-stage lesion but increased along with scar maturation. Prolyl 4-hydroxylase was found in surviving cardiomyocytes around the lesion during all stages and in interstitial cells appeared in granulation tissue.

CONCLUSIONS

GBP28 plays a role as a scaffold of newly formed collagen in myocardial remodelling after ischaemic injury, and GBP28 immunostaining may assist in a diagnosis of healing stage.

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.

METHODS

Mice were fed a high-fat diet for 4 weeks, fasted for 48 h or exposed at 4 degrees C.

METHODS

C57BL/6J mouse, male, 4--6 weeks old.

METHODS

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.

CONCLUSIONS

GBP28 may play a role in homeostasis, regulating body temperature and basal metabolic rate in response to changing environmental conditions. International Journal of Obesity (2001) 25, 75-83

Adiponectin is an adipocyte-derived abundant plasma protein, also called Acrp30 (adipocyte complement-related protein), adipoQ, ApM1 (AdiPose Most abundant Gene transcript 1), or GBP28 (gelatin-binding protein-28). Insulin resistance is a primary contributing factor in the pathogenesis of type 2 diabetes. Adiponectin binds to adiponectin receptors AdipoR1 and AdipoR2, and exerts antidiabetic effects via activation of AMPK and PPAR-α pathways, respectively. In the same sense chronic exercise has been showed to induce numerous metabolic factors that can improve insulin resistance. It has been reported that physical exercise training increases adiponectin receptors, which may mediate the improvement of insulin resistance in response to exercise, which is the focus of the present review.

We have previously reported the isolation of human gelatin-binding protein 28 (GBP28) gene which is specifically expressed in adipose tissue. The transcriptional activity of the flanking region of the GBP28 gene was examined by the transient transfection of promoter-luciferase reporter constructs into 3T3 adipocytes and electrophoretic mobility shift assay. This revealed the existence of a protein which binds to the 5'-flanking region of the GBP28 gene in nuclear extracts from human adipose tissue, but not in nuclear extracts from mouse liver. The C/EBP sites contained in this region are thought to take part in the regulation of GBP28 gene expression.

Adipocytokines, such as adiponectin, TNF-alpha, and leptin, are cytokines secreted by visceral adipocytes, and they are associated with metabolic syndrome. Adiponectin is one of the adipocytokines, and is a protein comprised of 244 amino acids. It is known as ACRP30, GBP28, and AdipoQ. Adiponectin is secreted by adipocytes, has three different isoforms, including trimers (low-molecular weight: LMW), hexamers (middle-molecular-weight: MMW), and higher-order oligomeric (high-molecular-weight: HMW) structures, and affects the biological activity. Adiponectin is a clinically relevant parameter measured routinely in subjects at risk of type 2 diabetes and metabolic syndrome. We investigated the adiponectin levels using a number of ELISA assay kits.

Human plasma contains three forms of adiponectin, a trimer, a hexamer, and a high-molecular-weight (HMW) multimer. We previously reported HMW adiponectin was a gelatin-binding protein of 28 kDa (GBP28), it having been purified due to its affinity to gelatin-Cellulofine (Nakano, Y., et al. Isolation and characterization of GBP28, a novel gelatin-binding protein purified from human plasma. J. Biochem. 1996. 120: 803-12). Although HMW adiponectin binds to gelatin-Cellulofine, it cannot bind to gelatin-Sepharose. Gelatin-Cellulofine was made of formyl-Cellulofine and gelatin, and we found that HMW adiponectin binds to reduced formyl-Cellulofine with similar affinity as to gelatin-Cellulofine. Through only two steps using reduced formyl-Cellulofine and DEAE-Sepharose, HMW adiponectin can be effectively purified from human plasma.

Adipose tissue is not simply a store of excess energy, but also secretes a variety of proteins into circulating blood that influence systemic metabolism. These include tumor necrosis factor (TNF-alpha), plasminogen activator inhibitor type 1 (PAI-1), leptin, resistine and adiponectin. These are collectively known as adipocytokines. Adiponectin (also referred to as AdipoQ, Acrp 30, apM1 or GBP28) is a novel adipose-specific protein. A recent genome study mapped a susceptibility locus for type 2 diabetes and the metabolic syndrome on chromosome 3q27, where the adiponectin gene is located. Adiponectin is a peculiar adipocytokine because in contrast to the markedly increased levels of many others, as leptin or TNF-alpha, its level is reduced in obesity and type 2 diabetes. The administration of thiazolidinediones, which are synthetic PPARs-gamma ligands, significantly increases the plasma adiponectin concentrations, an effect that could improve insulin sensitivity. Thus, the administration of adiponectin may provide a novel treatment modality for insulin resistance and type 2 diabetes.