The in vitro effects of resistin on the innate immune signaling pathway in isolated human subcutaneous adipocytes

Context: Obesity-associated inflammation is a contributory factor in the pathogenesis of type 2 diabetes mellitus (T2DM); the mechanisms underlying the progression to T2DM are unclear. The adipokine resistin has demonstrated pro-inflammatory properties in relation to obesity and T2DM. Objective: To characterize resistin expression in human obesity and address the role of resistin in the innate immune pathway. Furthermore, examine the influence of lipopolysaccharide, recombinant human resistin (rhResistin), insulin and rosiglitazone in human adipocytes. Finally, analyze the effect of rhResistin on the expression of components of the NF-κB pathway and insulin signaling cascade. Methods: Abdominal subcutaneous adipose tissue was obtained from patients undergoing elective liposuction surgery (n = 35, aged: 36-49 yr; BMI: 26.5 ± 5.9 kg/m2). Isolated adipocytes were cultured with rhResistin (10-50 ng/ml). The level of cytokine secretion from isolated adipocytes was examined by ELISA. The effect of rhResistin on protein expression of components of the innate immune pathway was examined by Western blot. Results: In-vitro studies demonstrated that antigenic stimuli increase resistin secretion (P < 0.001) from isolated adipocytes. Pro-inflammatory cytokine levels were increased in response to rhResistin (P < 0.001); this was attenuated by rosiglitazone (P < 0.01). When examining components of the innate immune pathway, rhResistin stimulated Toll-like receptor-2 protein expression. Similarly, mediators of the insulin signaling pathway, phosphospecific JNK1 and JNK2, were upregulated in response to rhResistin. Conclusion: Resistin may participate in more than one mechanism to influence pro-inflammatory cytokine release from human adipocytes; potentially via the integration of NF-κB and JNK signaling pathways

Determining the mechanism contributing to sub-clinical inflammation in metabolic disease

This thesis aims to investigate the role of innate immunity and the bacterial endotoxin lipopolysaccharide (LPS) in inflammation in adipose tissue. It attempts to do this by: 1) Describing the presence of Toll-like Receptors (TLR) in adipose tissue and adipocytes and their response to stimulation by LPS. It was demonstrated that both Toll-like receptor 2 and 4 were present in adipose tissue and that the receptors were present in greater amount in subjects with diabetes at a protein level and in subjects with obesity at both a RNA and protein level. It was also shown that these receptors responded to stimulation by LPS and that TLR-2 could be seen to be upregulated. 2) It was further shown that LPS induced production of inflammatory cytokines in cultured whole adipose tissue and adipocytes and caused downregulation of adiponectin in whole adiposes tissue. Blockade of NFκB in adipocytes was shown to regulate the levels of inflammatory cytokines produced and to differentially effect this levels dependent on whether whole adipose tissue or isolated adipocytes were cultured. 3) It was shown that LPS levels are significantly higher in the serum of T2DM subjects than controls and that these levels appeared to correlate with insulin. It was also seen that the LPS could be seen to correlate with inflammatory cytokines. It was also demonstrated that treatment with rosiglitazone significantly reduced both insulin and LPS levels in tandem. 4) It was also shown that LPS is significantly higher in NAFLD subjects, correlated with inflammatory cytokines and can be reduced by the use of Orlistat, an intestinal lipase inhibitor

A–E. Photographs of Immunological Staining of Visfatin Protein.

<p>This figure shows the immunological staining of visfatin in (A) human kidney negative (primary antibody omitted); (B) human kidney positive control; (C) human AT PBEF negative (primary antibody omitted); (D) human Om AT; (E) human Abd Sc AT. Red arrows highlight areas of red staining to show the presence of the visfatin protein.</p

Clinical and Biochemical Characteristics of T2DM Subjects Compared with Control Subjects.

<p>Data are presented as mean (±SD), unless otherwise stated. Non-parametric data have been log transformed in which case they are presented as mean (interquartile range).</p

Visfatin Protein Expression in Relation to Adiposity and Depot Specificity.

<p>This figure shows the mean relative fold protein expression of visfatin in omental (Om) versus abdominal (Abd) adipose tissue depots in matched pairs, from a cohort of lean and obese subjects, where the Abd Sc depot has been standardized. The representative Western blots are shown above. Lean samples: n = 7 & Obese samples: n = 6; p-values: p<0.001.</p

The Effects of JNK and NF-κB Inhibition on Visfatin Protein Expression in Human Adipocytes.

<p>This figure shows the effect of JNK and NF-κB blockade, alone and in combination, on the mean relative fold protein expression (±SEM) of visfatin in isolated Abd Sc adipocytes compared with control (untreated adipocytes), given an arbitrary value of one. The representative Western blots are shown above (n = 4, p-values: p<0.05*, p<0.01**).</p

Clinical and Biochemical Characteristics of T2DM Subjects Pre and Post 10 week Rosiglitazone Treatment.

<p>Data are presented as mean (±SD). Non-parametric data have been log transformed in which case they are presented as mean (interquartile range).</p

A&B. The Effect of Insulin Alone and in Combination with RSG on Visfatin Protein Expression in Isolated Adipocytes.

<p>These figures show the mean relative protein expression (±SEM) of visfatin in isolated human adipocytes treated with insulin (1 nM and 100 nM, respectively, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020287#pone-0020287-g003" target="_blank">Figure 3A</a>) and insulin in combination with RSG (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020287#pone-0020287-g003" target="_blank">Figure 3B</a>), compared with control (untreated adipocytes) as an arbitrary value of 1. The representative Western blots are shown above (n = 4, p-values: p<0.05*, p<0.01**).</p

A&B. The Effects of Insulin and RSG on IKKβ and JNK1/2 Protein Expression.

<p>These figures shows the effect of insulin, alone (100 nM), and insulin in combination with RSG (Ins 100 nM/RSG 10 nM) on the mean relative fold protein expression (± SEM) of IKKβ (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020287#pone-0020287-g004" target="_blank">Figure 4A</a>) and JNK 1/2 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020287#pone-0020287-g004" target="_blank">Figure 4B</a>). The representative Western blots are shown above (n = 4, p-values: p<0.05*, p<0.001***).</p