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Comparative Study
, 102 (30), 10610-5

Visceral Adipose Tissue-Derived Serine Protease Inhibitor: A Unique Insulin-Sensitizing Adipocytokine in Obesity

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Comparative Study

Visceral Adipose Tissue-Derived Serine Protease Inhibitor: A Unique Insulin-Sensitizing Adipocytokine in Obesity

Kazuyuki Hida et al. Proc Natl Acad Sci U S A.

Abstract

There is a rapid global rise in obesity, and the link between obesity and diabetes remains somewhat obscure. We identified an adipocytokine, designated as visceral adipose tissue-derived serpin (vaspin), which is a member of serine protease inhibitor family. Vaspin cDNA was isolated by from visceral white adipose tissues (WATs) of Otsuka Long-Evans Tokushima fatty (OLETF) rat, an animal model of abdominal obesity with type 2 diabetes. Rat, mouse, and human vaspins are made up of 392, 394, and 395 amino acids, respectively; exhibit approximately 40% homology with alpha1-antitrypsin; and are related to serine protease inhibitor family. Vaspin was barely detectable in rats at 6 wk and was highly expressed in adipocytes of visceral WATs at 30 wk, the age when obesity, body weight, and insulin levels peak in OLETF rats. The tissue expression of vaspin and its serum levels decrease with worsening of diabetes and body weight loss at 50 wk. The expression and serum levels were normalized with the treatment of insulin or insulin-sensitizing agent, pioglitazone, in OLETF rats. Administration of vaspin to obese CRL:CD-1 (ICR) (ICR) mice fed with high-fat high-sucrose chow improved glucose tolerance and insulin sensitivity reflected by normalized serum glucose levels. It also led to the reversal of altered expression of genes relevant to insulin resistance, e.g., leptin, resistin, TNFalpha, glucose transporter-4, and adiponectin. In DNA chip analyses, vaspin treatment resulted in the reversal of expression in approximately 50% of the high-fat high-sucrose-induced genes in WATs. These findings indicate that vaspin exerts an insulin-sensitizing effect targeted toward WATs in states of obesity.

Figures

Fig. 1.
Fig. 1.
Amino acid sequence, structural analyses, and gene expresion of vaspin in various tissues. (A) Amino acid sequence of rat, mouse, and human vaspins. Signal peptides are underlined, and reactive site loop is boxed. (B) Automated protein structure homology modeling by swiss-model predicted the presence of three β-sheets (blue), nine α-helices (red), and one reactive site loop (yellow). (C) Northern blot analyses of vaspin in various organs of obese 30-wk-old OLETF and visceral adipose tissue of lean 6-wk-old LETO rats. A single transcript is observed in visceral (VIS) fat of OLETF rats. BAT, brown adipose tissue.
Fig. 2.
Fig. 2.
Effect of pioglitazone and insulin on various physiological parameters in OLETF and related strains of rats and expression of vaspins. (A) Body weight of OLETF, LETO, and OLETF rats with EXE, and OLETF rats administered with TZD and insulin (INS). A decline in the body weight of OLETF rats is observed at 50 wk of age after peaking at 30 wk. (B) Hemoglobin A1c levels progressively rise up to 50 wk in OLTEF rats. (C) Fasting immunoreactive insulin (IRI) levels peak at 30 wk and then decline at 50 wk in OLTEF rats. (D) Fat-pad weights peaked at 30 wk in OLETF rats, whereas with TZD and insulin treatment, they progressively rose up to 50 wk. Data included in A-D are mean ± SEM (n = 5). (E) Northern blot analyses of vaspin mRNA in WATs. At 30 wk, the expression is high in visceral retroperitoneal (RET) and MES WATS in OLTEF rats. The expression is high at 30 and 50 wk in SUB WATs from OLTEF rats treated with TZD, whereas the expression of visceral WATs of vaspin was decreased. Insulin had a partial effect on the mRNA expression of vaspin. EPI, epidydimal WATs.
Fig. 3.
Fig. 3.
Expression of vaspin in 293T cells, adipocytes, and stromal vascular cells. (A) Western blot analysis of mouse vaspin by using supernatants of 293T cultured cells transfected with AxCAmOL64 and polyclonal antivaspin Ab. A prominent band of ≈45 kDa is observed. (B) Western blots of purified recombinant human and mouse vaspins, derived from E. coli by using pET expression system. (C) Western blot analyses of vaspin by using sera of OLETF and LETO rats, and OLETF rats with EXE, OLETF rats administered TZD and insulin (INS). A major ≈45-kDa band and a minor ≈50-kDa band are seen at 30 wk. Band intensity is notably less at 50 wk in OLTEF rats, but it seems to be normalized with insulin and TZD treatments. (D) Expression of vaspin in mature adipocytes and stromal vascular cells isolated from epidydimal (EPI), retroperitoneal (RET), MES, and SUB WATs as analyzed by Northern and Western blot analyses. Expression is confined to adipocytes of visceral WATs and not to stromal endothelial or vascular cells. (E and F) Expression in the adipocytes was confirmed by immunofluorescence microscopy in 30-wk-old OLETF rats. (Scale bars: E, 100 μm; F, 25 μm.)
Fig. 4.
Fig. 4.
Effect of vaspin on tryptic activity. (A) Reduced hen-egg lysozyme (HEL), and not native HEL, is sensitive to trypsin treatment. (B) The proteolytic activity of trypsin is normally blocked by its known inhibitor, α1-antitrypsin; however, rhVaspin with or without (His)6-tag failed to inhibit the tryptic activity.
Fig. 5.
Fig. 5.
Profiles of glucose tolerance and insulin sensitization tests after administration of vaspin and insulin. (A) Glucose tolerance test. rhVaspin or PBS was i.p. injected into ICR mice fed STD chow or HFHS chow before glucose administration. Glucose levels were significantly reduced with the administration of vaspin (*, P < 0.01). (B) Insulin levels during the glucose tolerance test were unaltered, and HFHS mice remained hyperinsulinemic. (C) Insulin tolerance test. rhVaspin or PBS was i.p. injected into mice before insulin administration. Blood glucose levels were lowered in the HFHS group receiving insulin plus vaspin (*, P < 0.05; **, P < 0.01). All data are mean ± SEM (n = 10).
Fig. 6.
Fig. 6.
Effect of vaspin on leptin, resistin, TNFα, GLUT4, and adiponectin in obese ICR mice fed with HFHS chow. Vaspin administration reverses gene expression profile of WATs. (A and B) Injection of rhVaspin suppressed gene expression of leptin, resistin, and TNFα and increased the expression of glucose transporter-4 and adiponectin in obese ICR mice with HFHS chow. Data are mean ± SEM (n = 5). *, P < 0.05; **, P < 0.01. (C) Principal component analysis showing that the HFHS-chow-induced gene expression profile in MES and SUB fats is distinct from that of ICR mice with STD chow and after injection of rhVaspin (VAS). (D) Hierarchical clustering analysis seems to divide in two distinct groups, HFHS and STD chow groups. Both the MES and the SUB gene expression profile in rhVaspin-treated ICR mice fed with HFHS chow is on the same branch as mice fed STD chow.

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