Dysregulation of Subcutaneous White Adipose Tissue Inflammatory Environment Modelling in Non-Insulin Resistant Obesity and Responses to Omega-3 Fatty Acids - A Double Blind, Randomised Clinical Trial

Abstract

Background: Obesity is associated with enhanced lipid accumulation and the expansion of adipose tissue accompanied by hypoxia and inflammatory signalling. Investigation in human subcutaneous white adipose tissue (scWAT) in people living with obesity in which metabolic complications such as insulin resistance are yet to manifest is limited, and the mechanisms by which these processes are dysregulated are not well elucidated. Long chain omega-3 polyunsaturated fatty acids (LC n-3 PUFAs) have been shown to modulate the expression of genes associated with lipid accumulation and collagen deposition and reduce the number of inflammatory macrophages in adipose tissue from individuals with insulin resistance. Therefore, these lipids may have positive actions on obesity associated scWAT hypertrophy and inflammation.

Methods: To evaluate obesity-associated tissue remodelling and responses to LC n-3 PUFAs, abdominal scWAT biopsies were collected from normal weight individuals and those living with obesity prior to and following 12-week intervention with marine LC n-3 PUFAs (1.1 g EPA + 0.8 g DHA daily). RNA sequencing, qRT-PCR, and histochemical staining were used to assess remodelling- and inflammatory-associated gene expression, tissue morphology and macrophage infiltration.

Results: Obesity was associated with scWAT hypertrophy (P < 0.001), hypoxia, remodelling, and inflammatory macrophage infiltration (P = 0.023). Furthermore, we highlight the novel dysregulation of Wnt signalling in scWAT in non-insulin resistant obesity. LC n-3 PUFAs beneficially modulated the scWAT environment through downregulating the expression of genes associated with inflammatory and remodelling pathways (P <0.001), but there were altered outcomes in individuals living with obesity in comparison to normal weight individuals.

Conclusion: Our data identify dysregulation of Wnt signalling, hypoxia, and hypertrophy, and enhanced macrophage infiltration in scWAT in non-insulin resistant obesity. LC n-3 PUFAs modulate some of these processes, especially in normal weight individuals which may be preventative and limit the development of restrictive and inflammatory scWAT in the development of obesity. We conclude that a higher dose or longer duration of LC n-3 PUFA intervention may be needed to reduce obesity-associated scWAT inflammation and promote tissue homeostasis.

Clinical trial registration: www.isrctn.com, identifier ISRCTN96712688.

Keywords: LC n-3 PUFA; Wnt signalling; adipose tissue; inflammation; obesity; tissue remodelling.

Figure 1

CONSORT diagram of participant inclusion and flow through the study.

Figure 2

(A) Histogram of 25 significantly enriched canonical pathways involved in inflammation and immune response in scWAT in individuals living with obesity. (B) Histogram of significantly enriched canonical pathways involved in tissue remodelling and expansion in scWAT in individuals living with obesity. (A, B) The left y-axis represents the z-score (predicted score of activation/inhibition) and the number of genes counted for each of the representative canonical pathways, and the right y-axis represents -log (P-value), significance is deemed -log P> 1.3. Where z-score is not present, there was not enough evidence from current literature to determine if the altered gene expression is likely to result in activation or inhibition of a canonical pathway.

Figure 3

Gene set enrichment analysis (GSEA) of differentially expressed genes in individuals living with obesity at week-0: Enrichment plot for Wnt/β-Catenin signalling.

Figure 4

Regulation of scWATWnt/β-Catenin signalling and differential expression of pathway genes in individuals living with obesity at week-0.

Figure 5

(A) Distribution of adipocyte size in scWAT in normal weight and individuals living with obesity at week-0; (B) Average adipocyte diameter in normal weight and individuals living with obesity at week-0; (C) H&E stained section of scWAT at 10 x magnification from a normal weight individual at week-0; (D) H&E stained section of scWAT at 10 x magnification from an individual living with obesity at week-0. **P < 0.001, *P < 0.050.

Figure 6

(A) Percentage of total fibrosis in scWAT in normal weight and individuals living with obesity at week-0; (B) Percentage of pericellular fibrosis in scWAT in normal weight and individuals living with obesity at week-0; (C) Picro Sirius red stained collagen in a section of scWAT at 10 x magnification from a normal weight individual at week-0; (D) Picro Sirius red stained collagen in a section of scWAT at 10 x magnification from an individual living with obesity at week-0.

Figure 7

(A) Number of macrophages per 100cm² of scWAT from normal weight and individuals living with obesity at week-0; (B) Number of CLS per 100 cm² of scWAT from normal weight and individuals living with obesity at week-0; (C) CD68 stained macrophages in a section of scWAT at 20 x magnification from an individual living with obesity at week-0. *P = 0.023

Figure 8

The regulation of scWAT in obesity in which metabolic complications are yet to manifest. Indication of upregulation or downregulation of mRNA expression is the expression in individuals living with obesity in comparison to that of normal weight individuals at week-0.

Figure 9

Summary of the effects of 12-week LC n-3 PUFA intervention on scWAT in normal weight individuals and individuals living with obesity. ¹ Obesity in which metabolic complications are yet to manifest. Indication of upregulation or downregulation of mRNA expression is the expression at week-12 in comparison to week-0 in each group of individuals.