Glucose and insulin modify thrombospondin 1 expression and secretion in primary adipocytes from diet-induced obese rats.

Abstract

Thrombospondin 1 (TSP-1), an antiangiogenic factor and transforming growth factor (TGF)-β activity regulator, has been recently recognized as an adipokine that correlates with obesity, inflammation and insulin resistance processes. In the present study, epididymal adipocytes of rats that were fed a chow or a high-fat diet (HFD) for 50 days were isolated and incubated (24-72 h) in low (5.6 mM) or high (HG; 25 mM) glucose, in the presence or absence of 1.6 nM insulin. Rats fed the HF diet showed an established obesity state. Serum TSP-1 levels and TSP-1 mRNA basal expression of adipocytes from HFD rats were higher than those from controls. Adipocytes from HFD animals presented an insulin resistance state, as suggested by the lower insulin-stimulated glucose uptake as compared to controls. TSP-1 expression in culture was higher in adipocytes from obese animals at 24 h, but when the adipocytes were treated with HG, these expression levels dropped dramatically. Later at 72 h, TSP-1 expression was lower in adipocytes from HFD rats, and no effects of the other treatments were observed. Surprisingly, the secretion levels of this protein at 72 h were increased significantly by the HG treatment in both types of adipocytes, although they were even higher in adipocytes from obese animals. Finally, cell viability was significantly reduced by HG treatment in both types of adipocytes. In summary, TSP-1 expression/secretion was modulated in an in vitro model of insulin-resistant adipocytes. The difference between expression and secretion patterns suggests a posttranscriptional regulation. The present study confirms that TPS-1 is closely associated with obesity-related mechanisms.

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Glucose and insulin modify thrombospondin 1 expression and
secretion in primary adipocytes from diet-induced obese rats

Garcia-Diaz DF, Arellano AV, Milagro FI, Moreno-Aliaga MJ, Portillo MP, Martinez
JA and Campion J

Department of Nutrition and Food Sciences, Physiology and Toxicology
University of Navarra, Pamplona, Spain

Corresponding author: Dr. Javier Campion
Department of Nutrition and Food Sciences,
Physiology and Toxicology
University of Navarra
c/Irunlarrea 1, 31008
Pamplona, Spain
Phone: +34 948425600
Fax: +34 948425649
e-mail address: jcampion@unav.es

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ABSTRACT

Thrombospondin 1 (TSP-1), an anti-angiogenic factor and TGF-β activity regulator, has
been recently recognized as an adipokine that correlates with obesity, inflammation and
insulin-resistance processes. In the present study, epididymal adipocytes of rats that
were fed a chow (C) or a high fat diet (HFD) for 50 days, were isolated and incubated
(24-72 h) in low (LG; 5.6 mM) or high (HG; 25 mM) glucose, in presence or absence of
1.6 nM insulin. Rats fed the HF diet showed an established obesity state. Serum TSP-1
levels and TSP-1 mRNA basal expression of adipocytes from HFD rats were higher
than those from controls. Adipocytes from HFD animals presented an insulin-resistance
state, as suggested by the lower insulin-stimulated glucose uptake as compared to
controls. TSP-1 expression in culture was higher in adipocytes from obese animals at 24
h, but when the adipocytes were treated with HG, these expression levels dropped
dramatically. Later at 72 h, TSP-1 expression was lower in adipocytes from HFD rats,
and no effects of the other treatments were observed. Surprisingly, the secretion levels
of this protein at 72 h were increased significantly by the HG treatment in both types of
adipocytes, although they were even higher in adipocytes from obese animals. Finally,
cell viability was significantly reduced by HG treatment in both types of adipocytes. In
summary, TSP-1 expression/secretion was modulated in an in vitro model of insulin-
resistant adipocytes. The difference between expression and secretion patterns suggests
a post-transcriptional regulation. The present study confirms that TPS-1 is closely
associated with obesity-related mechanisms.

Keywords: diet-induced obesity, adipokine, cell culture, adipose tissue

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INTRODUCTION

An excessive body fat accumulation could drive to several associated clinical
complications such as type 2 diabetes, metabolic syndrome features, cardiovascular
diseases, inflammatory disturbances, etc [8]. These manifestations have been linked to
an impaired production and secretion of endogenous products by the enlarged
adipocytes or the accompanying macrophages from the white adipose tissue (WAT)
stroma-vascular fraction [8]. Actually, several inflammatory products derived from this
tissue (TNF-α, IL-6, MCP-1, iNOS) have shown positive associations with body
adiposity [16]. Besides the pro-inflammatory molecules secreted by WAT, this tissue
also produces numerous adipokines that may have also important local and systemic
effects [15].
Thrombospondin 1 (TSP-1) is a molecule that was first identified as a thrombin-
sensitive protein released upon activation of platelets by thrombin [6]. It was initially
isolated from platelets and megakaryocytes [28], but was later detected in several cell
types such as macrophages and adipocytes [21, 22, 30, 32]. Nowadays, it is known that
TSP-1 is a multifunctional protein composed by multiple structural domains [5, 10], and
it has been especially related with several anti-angiogenic pathways [20, 23, 24].
Recently, this protein has been included among the group of adipokines, since its
expression is increased in WAT of obese and insulin-resistant subjects, presenting
positive correlations with the level of adiposity [35, 41]. Furthermore, it has been
reported as an important factor in the adipocyte- and macrophage-driven inflammation
in the adipose tissue, and that could mediate the elevation of PAI-1, promoting a
prothrombotic state [41]. Also, TSP-1 has been described as a major regulator of the
transforming growth factor (TGF)-β activity [11, 31], which, together with the increased

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PAI-1 levels, are correlated with different features of obesity, insulin-resistance, and
metabolic syndrome [1, 29, 37]. Finally, it has been described higher TSP-1 gene
expression in visceral respect to subcutaneous adipose tissue in obese subjects [35].
Moreover, it has been described that the expression of TSP-1 in blood vessels is
increased in diabetes, perhaps by a direct effect of the elevated glucose levels in blood
[39]. Nevertheless, no effects of glucose incubation have been studied so far in
adipocytes from obese specimens. Thus, the purpose of this study was to characterize
TSP-1 expression and secretion patterns in adipocytes isolated from either lean or diet-
induced obese rats, in order to demonstrate that this important protein is involved in the
modulation of the inflammatory processes occurring in WAT.


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MATERIALS AND METHODS

Materials
Dulbecco’s modified eagle’s medium (DMEM), 100X minimal essential medium
(MEM) non-essential amino acids, penicillin/streptomycin, heat-inactivated fetal bovine
serum (FBS), 10X MEM, nystatin, Trizol Reagent, and M-MLV reverse transcriptase
were obtained from Invitrogen (Paisley, UK). Bovine serum albumin (BSA), HEPES,
insulin and 2′,7′-dichlorofluorescein were all obtained from Sigma-Aldrich Company
(St. Louis, USA). Collagen (Purecol) was purchased from Nutacon (Leimuiden, The
Netherlands). Type I collagenase was supplied by Worthington Biochemical
Corporation (Lakewood, USA). Glycerol-3-phosphate dehydrogenase (GPDH), glycerol
kinase (GK), adenosine triphosphate (ATP), and nicotinamide adenine dinucleotide
(NAD) were obtained from Roche Diagnostics (Mannheim, Germany).

Animals and dietary treatment
Eight-week-old male Wistar rats (n=20) supplied by the Center for Applied
Pharmacobiology Research (CIFA, Pamplona, Spain), were housed in a temperature-
controlled room at 21-23 ºC with a 12 h light cycle (lights goes off at 8 pm). The
animals were assigned into two different dietary groups: a control group (n=10) that was
fed a standard chow diet (2014 Tekland Global 14% Protein Rodent Maintenance Diet;
Harlan Iberica, Barcelona, Spain) containing 16.6% of energy as protein, 73.1% of
energy as carbohydrate (7% as simple sugars) and 10.3% of energy as lipid by dry
weight, and a high fat diet-fed group (HFD, n=10) that was fed a high fat diet (D12330
diet, 58 kcal% fat w/cornstarch Surwit Diet; Research Diets Inc., New Brunswick,
USA) containing 16.4% of energy as protein, 25.5% of energy as carbohydrate (4.5% as

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