Lipopolysaccharide, high glucose and saturated fatty acids induce endoplasmic reticulum stress in cultured primary human adipocytes: Salicylate alleviates this stress

doi:10.1016/j.bbrc.2010.05.138

Biochemical and Biophysical Research Communications

Volume 397, Issue 3, 2 July 2010, Pages 472-478

https://doi.org/10.1016/j.bbrc.2010.05.138 Get rights and content

Abstract

Recent findings indicate that endoplasmic reticulum (ER) stress is significantly increased in adipose tissue of obese human subjects and is critical to the initiation and integration of pathways of inflammation and insulin action. But the factors inducing ER stress in human adipose tissue are unknown. The common factors increased in obesity and linked to insulin resistance are hyperglycaemia, hyperlipidemia and also endotoxemia. Therefore, our aims were to investigate: (1) the role of lipopolysaccharide (LPS), high glucose (HG) and saturated fatty acids (SFA) as inducers of ER stress in primary human adipocytes and (2) whether salicylate, a known anti-inflammatory compound, can alleviate this effect. Components of the ER stress pathways were studied in human abdominal subcutaneous (AbSc) adipose tissue (AT) from obese and lean. Following the culture and differentiation of primary human preadipocytes, these adipocytes were treated with LPS, HG, tunicamycin (Tun) and SFA either alone or in combination with sodium salicylate (Sal). Markers of ER stress were significantly increased in AbSc AT of obese. Differentiated human adipocytes treated with LPS, Tun, HG and SFA showed significant activation of eukaryotic translation initiation factor 2α (eIF2α) and activating transcription factor 6 (ATF6) and their down-stream targets. Sal alleviated this effect and activated AktSer473 phosphorylation. This study presents important evidence that: (1) there is increased ER stress in adipose tissue of obese individuals, (2) LPS, hyperglycaemia and saturated fatty acids induce significant ER stress in primary human adipocytes and (3) this induction is alleviated by salicylate.

Introduction

Obesity-associated inflammation is a key contributory factor in the pathogenesis of type 2 diabetes mellitus (T2D) and cardiovascular disease (CVD) but the fundamental mechanisms responsible for activating innate immune inflammatory pathways and insulin resistance are currently unclear. Murine studies have revealed that one key link is increased endoplasmic reticulum (ER) stress [1]. The ER has a central role in lipid and protein biosynthesis. During pathological nutrient excess, proteins formed in the ER may fail to attain correct conformation and accumulation of misfolded proteins in the ER causes stress and activates the Unfolded Protein Response (UPR) signal [2].

The UPR signals through three ER transmembrane sensors: PKR-like ER-regulated kinase (PERK), inositol requiring enzyme1α (IRE1α) and activating transcription factor 6 (ATF6) [3]. These activate an adaptive response that results in inhibition of protein translation and increase in transcription of protein-folding chaperones and ER-associated degradation genes [2], [4]. PERK phosphorylates the eukaryotic translation initiation factor 2α (p-eIF2α) [5]. p-eIF2α then attenuates protein synthesis and reduces ER protein overload and also activates activation transcription factor4 (ATF4), which induces expression of many genes, including those involved in apoptosis: C/EBP homologous protein (CHOP) [6]. ATF6 activates transcription of ER chaperones: glucose regulated protein (Grp)78/Bip, protein disulfide isomerase (PDI), Ero1-Lα and calnexin to augment the protein folding capacity [1].

An enhanced level of the UPR has been demonstrated in obese, insulin-resistant human adipose tissues [7], [8], [9]. ER stress and the UPR are linked to major inflammatory and stress-signalling networks, including the activation of JNK and IKK-NFκB pathways which play a central role in obesity-induced inflammation and metabolic abnormalities [10]. High doses of salicylates have been shown to lower blood glucose concentrations [11]. Severely obese rodents when treated with salicylates demonstrated reduced signalling through IKKβ pathway and this was accompanied by improved insulin sensitivity in vivo[12], [13].

Although ER stress and metabolic dysfunction is associated with obesity in rodent models, the importance of ER stress and the potential inducers of ER stress in human adipocytes are not known. Therefore, the objective of the present study was to show the existence of ER stress in obese human adipose tissue, identify the potential originators of this stress and also demonstrate the role of anti-inflammatory agent, sodium salicylate on ER stress in primary human adipocytes. The primary human preadipocytes were cultured and fully differentiated adipocytes were treated with most probable factors: lipopolysaccharides (LPS), high glucose (HG), tunicamycin (Tun) and saturated fatty acids (SFA) with and without salicylate (Sal). ER stress pathways and Akt activation were studied.

Section snippets

Subjects

Human Abdominal Subcutaneous (AbSc) adipose tissue (AT) was collected from patients (age: 40.8 (mean ± SD) ± 5yrs; Lean BMI: 22.04 ± 2.6 kg/m² and obese BMI 30 ± 3.5 kg/m²) undergoing liposuction surgery with informed consent obtained in accordance with LREC guidelines and with ethics committee approval. All tissue samples were flash frozen and/or utilized for isolation of human preadipocytes as detailed [14].

Cell culture

Human AbSc AT (BMI 25.04 ± 0.6 kg/m²; n = 3–6) were digested with collagenase to isolate

ER stress markers are up-regulated in obese human AbSc AT

Protein expression of the ER stress markers was measured in four obese and four lean human AbSc AT. The p-PERK and IRE1α proteins were increased in obese subjects – although this was only significant for IRE1α expression compared with lean (Fig. 1A). ATF6, which regulates the third ER stress pathway, was investigated by examining mRNA expression via real-time PCR, as this proved a more reliable method [18]. ATF6 mRNA expression from AbSc AT of 10 lean and 10 obese subjects showed to be

Discussion

The results of the present study demonstrate that firstly, there is increased ER stress in obese human AbSc AT, secondly, the factors inducing this response could be LPS, hyperglycaemia and SFA and thirdly, this stress response is alleviated by salicylates and could contribute to increased insulin sensitivity in adipocytes. LPS [20], hyperglycaemia [21] and free-fatty acids [22] have all been shown to be elevated in blood during obesity and have been linked to increased inflammation and insulin

Acknowledgments

We would like to thank Research Council UK for supporting G.T. We would also like to thank Government of UAE for funding S.A.A. K.C.M. was funded by Department of Health, UK and A.L.H. is funded by British Heart Foundation UK.

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¹: These authors contributed equally to this work.

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Lipopolysaccharide, high glucose and saturated fatty acids induce endoplasmic reticulum stress in cultured primary human adipocytes: Salicylate alleviates this stress

Abstract

Introduction

Section snippets

Subjects

Cell culture

ER stress markers are up-regulated in obese human AbSc AT

Discussion

Acknowledgments

Cell

Mol. Cell

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

Metabolism

J. Biol. Chem.

J. Biol. Chem.

Cell Signal

J. Surg. Res.

Endoplasmic reticulum stress links obesity, insulin action, and type 2 diabetes

Science

The unfolded protein response: a pathway that links insulin demand with beta-cell failure and diabetes

Endocr. Rev.

Endoplasmic reticulum stress-induced apoptosis in the development of diabetes: is there a role for adipose tissue and liver?

Apoptosis

Protein translation and folding are coupled by an endoplasmic-reticulum-resident kinase

Nature

Increase in endoplasmic reticulum stress-related proteins and genes in adipose tissue of obese, insulin-resistant individuals

Diabetes