Sachiyo Tanaka

Adiponectin and development of type 2 diabetes in the Pima Indian population

Adiponectin is a collagen-like circulating protein secreted by adipocytes that is proposed to mediate obesity-related resistance to insulin. In a case-control series, we assessed the role of adiponectin in later development of type 2 diabetes in 70 patients who later developed type 2 diabetes and 70 controls, matched for body-mass index, age, and sex. Cases and controls were taken from the longitudinal study of health in the Pima Indian population. At baseline, the concentration of adiponectin was lower in cases than in controls (p=0.01) and individuals with high concentrations of this protein were less likely to develop type 2 diabetes than those with low concentrations (incidence rate ratio 0.63 [95% CI 0.43-0.92]; p=0.02).

Adipose Tissue Hypoxia in Obesity and Its Impact on Adipocytokine Dysregulation

Obesity is linked to a variety of metabolic disorders, such as insulin resistance and atherosclerosis. Dysregulated production of fat-derived secretory factors, adipocytokines, is partly responsible for obesity-linked metabolic disorders. However, the mechanistic role of obesity per se to adipocytokine dysregulation has not been fully elucidated. Here, we show that adipose tissue of obese mice is hypoxic and that local adipose tissue hypoxia dysregulates the production of adipocytokines. Tissue hypoxia was confirmed by an exogenous marker, pimonidazole, and by an elevated concentration of lactate, an endogenous marker. Moreover, local tissue hypoperfusion (measured by colored microspheres) was confirmed in adipose tissue of obese mice. Adiponectin mRNA expression was decreased, and mRNA of C/EBP homologous protein (CHOP), an endoplasmic reticulum (ER) stress–mediated protein, was significantly increased in adipose tissue of obese mice. In 3T3-L1 adipocytes, hypoxia dysregulated the expression of adipocytokines, such as adiponectin and plasminogen activator inhibitor type-1, and increased the mRNAs of ER stress marker genes, CHOP and GRP78 (glucose-regulated protein, 78 kD). Expression of CHOP attenuated adiponectin promoter activity, and RNA interference of CHOP partly reversed hypoxia-induced suppression of adiponectin mRNA expression in adipocytes. Hypoxia also increased instability of adiponectin mRNA. Our results suggest that hypoperfusion and hypoxia in adipose tissues underlie the dysregulated production of adipocytokines and metabolic syndrome in obesity.

Adipocytokines, body composition, and fitness in children

Recent evidence suggests a role for adipose derived cytokines (adipocytokines) such as tumor necrosis factor-α (TNF-α), IL-6, and the recently discovered adiponectin in the mechanism of impaired glucose regulation and atherosclerosis in adults. However, the relationship between adipocytokines and body composition, fasting insulin, and fitness is virtually unknown children. Fasting blood sampling was performed in 30 healthy, predominately Hispanic- and Asian-American children (16 boys, mean age 12.7 ± 0.1 y old) from a lower socioeconomic area in Los Angeles. Adiposity was measured by dual x-ray absorptiometry (DEXA); and peak oxygen uptake using cycle ergometry. Adiponectin (mean 10.8 ± 0.8 μg/mL) was inversely correlated with body mass index (BMI, as percentile by age) (r = −0.48, p = 0.011) and fat mass (r = −0.43, p = 0.03). In contrast, TNF-α and IL-6 were both positively correlated with BMI and fat mass. Adiponectin was inversely correlated with fasting insulin (r = −0.52, p = 0.006), but no correlations were found for insulin and either TNF-α or IL-6. Adiponectin was correlated with HDL (r = 0.448, p = 0.019). Paradoxically, peak oxygen consumption (an indicator of fitness) was negatively correlated with adiponectin levels (r = −0.471, p = 0.013) and positively correlated with TNF-α (r = 0.560, p = 0.002). In children, adipocytokines are correlated with fat mass, insulin sensitivity, and cardiovascular risk factors in a manner that is qualitatively similar to relationships recently observed in adults. In more obese children, the mass of fat tissue may attenuate potentially positive effects of fitness on circulating levels of adiponectin and TNF-α. The novel data on adiponectin suggest that deleterious dysregulation of adipocytokines associated with obesity may occur relatively early in life.

High expression of leptin receptor mRNA in breast cancer tissue predicts poor prognosis for patients with high, but not low, serum leptin levels.

The association of mRNA expression levels of leptin receptors (long isoform: Lep‐R(L) and short isoform: Lep‐R(S)) in breast cancer tissue with patient prognosis was studied with special reference to the serum leptin level or the leptin mRNA level in tumor tissue. Lep‐R(L), Lep‐R(S) and leptin mRNA levels in breast cancer tissue (n = 91) were determined with a real‐time PCR assay, and serum leptin levels in breast cancer patients (n = 67) with an enzyme‐linked immunosorbent assay. Neither Lep‐R(L) nor Lep‐R(S) mRNA levels in tumor tissue were significantly associated with patient prognosis, but both intratumoral Lep‐R(L) and Lep‐R(S) mRNA high tumors were significantly (p < 0.01) associated with a poor prognosis. Multivariate analysis showed that a high level of both Lep‐R (L) and Lep‐R (S) mRNA in tumor tissue was a significant risk factor, independent of other risk factors. The subset analysis demonstrated that both intratumoral Lep‐R(L) and Lep‐R(S) mRNA high tumors were significantly associated with a poor prognosis for the subset of patients with high serum leptin or high intratumoral leptin mRNA levels but not in the subset of patients with low serum leptin or low intratumoral leptin mRNA levels. The association between both intratumoral Lep‐R(L) and Lep‐R(S) mRNA high tumors and a poor prognosis in the presence of high serum leptin or high intratumoral leptin mRNA levels seems to suggest that the leptin and Lep‐R(L)/Lep‐R(S) pathways are implicated in the growth stimulation of breast tumors. The well‐established finding that obesity serves as a risk factor for relapse in breast cancer patients may thus be partially explained by the high serum leptin level seen in obese women.

Disturbed secretion of mutant adiponectin associated with the metabolic syndrome.

Adiponectin, an adipocyte-derived protein, consists of collagen-like fibrous and complement C1q-like globular domains, and circulates in human plasma in a multimeric form. The protein exhibits anti-diabetic and anti-atherogenic activities. However, adiponectin plasma concentrations are low in obese subjects, and hypoadiponectinemia is associated with the metabolic syndrome, which is a cluster of insulin resistance, type 2 diabetes mellitus, hypertension, and dyslipidemia. We have recently reported a missense mutation in the adiponectin gene, in which isoleucine at position 164 in the globular domain is substituted with threonine (I164T). Subjects with this mutation showed markedly low level of plasma adiponectin and clinical features of the metabolic syndrome. Here, we examined the molecular characteristics of the mutant protein associated with a genetic cause of hypoadiponectinemia. The current study revealed (1) the mutant protein showed an oligomerization state similar to the wild-type as determined by gel filtration chromatography and, (2) the mutant protein exhibited normal insulin-sensitizing activity, but (3) pulse-chase study showed abnormal secretion of the mutant protein from adipose tissues. Our results suggest that I164T mutation is associated with hypoadiponectinemia through disturbed secretion into plasma, which may contribute to the development of the metabolic syndrome.

The Genes Influencing Adiponectin Levels Also Influence Risk Factors for Metabolic Syndrome and Type 2 Diabetes

ABSTRACT Results from previous studies suggest that adiponectin levels are associated with risk factors for cardiovascular disease and type 2 diabetes mellitus; however, the genetic and/or environmental components of this relationship have not been characterized. The aims of this study were (1) to assess the presence of pleiotropy between adiponectin levels and risk factors for cardiovascular disease and (2) to study the association of circulating levels of adiponectin with risk factors for cardiovascular disease in the absence and presence of obesity in Mexican American adults from the San Antonio Family Heart Study. Body composition and circulating levels of adiponectin, leptin, and lipid subfractions and measurements of glucose metabolism were measured in 898 subjects. The mean and standard error of the circulating levels of adiponectin was 8.7 ±3.2 μg/ml. Bivariate quantitative analyses between adiponectin levels and phenotypes related to cardiovascular disease and type 2 diabetes mellitus were conducted using the variance decomposition approach implemented in SOLAR. A second analysis in unrelated subjects compared these risk factors between sex and agematched lean and obese subjects with high and low adiponectin levels. We found significant evidence of pleiotropy (i.e., shared genetic effects) between plasma levels of adiponectin and wellestablished risk factors for cardiovascular disease and type 2 diabetes mellitus. Individuals with low adiponectin levels per body weight had more adverse risk profiles. These findings offer new insights into the genetic connection between increasing adiposity and risk for cardiovascular disease and type 2 diabetes mellitus, and they suggest that adiponectin may be an important risk factor for the development of these conditions.

Collection of homozygous mutant mouse embryonic stem cells arising from autodiploidization during haploid gene trap mutagenesis

Abstract Haploid mouse embryonic stem cells (ESCs), in which a single hit mutation is sufficient to produce loss-of-function phenotypes, have provided a powerful tool for forward genetic screening. This strategy, however, can be hampered by undesired autodiploidization of haploid ESCs. To overcome this obstacle, we designed a new methodology that facilitates enrichment of homozygous mutant ESC clones arising from autodiploidization during haploid gene trap mutagenesis. Haploid mouse ESCs were purified by fluorescence-activated cell sorting to maintain their haploid property and then transfected with the Tol2 transposon-based biallelically polyA-trapping (BPATrap) vector that carries an invertible G418 plus puromycin double selection cassette. G418 plus puromycin double selection enriched biallelic mutant clones that had undergone autodiploidization following a single vector insertion into the haploid genome. Using this method, we successfully generated 222 homozygous mutant ESCs from 2208 clones by excluding heterozygous ESCs and ESCs with multiple vector insertions. This relatively low efficiency of generating homozygous mutant ESCs was partially overcome by cell sorting of haploid ESCs after Tol2 BPATrap transfection. These results demonstrate the feasibility of our approach to provide an efficient platform for mutagenesis of ESCs and functional analysis of the mammalian genome.