Adipose tissue can rapidly expand in response to excess energy intake through both adipocyte hypertrophy (cell size increase) and hyperplasia (cell number increase). Excessive adipose tissue expansion in obesity is associated with adipokine dysregulation, inflammation, ECM remodeling and fibrosis particulary in visceral adipose depots. However, the time-course of these pathophysiological events and the underlying transcriptional changes in adipose tissue are not fully understood. We show adipose tissue expansion occurs in multiple visceral WAT depots early during the development of diet-induced obesity. Pro-inflammatory adipokine levels also increase early after 2–4 weeks, while anti-inflammatory adipokine levels are reduced after 20 weeks in HFD fed mice. Time-course microarray profiling revealed early and prolonged up-regulation of inflammation and immune system associated genes in the epididymal WAT depot and the smaller mesenteric WAT depot. In the later stages of diet-induced obesity, fibrosis was evident in the epididymal WAT depot. Hence the long-term diet-induced obesity model used in the present study provides a unique insight into the timing of events in visceral adipose tissue during the development of obesity.
Adipocytokine dysregulation is reported to be closely associated with obesity and plasma proinflammatory adipokines are widely reported to be elevated in obese animals. Adipokines are secreted from visceral adipose depots, hence expansion of visceral adipose tissue depots is correlated with plasma adipokine levels, but it is less well established whether adipokine are transcriptionally regulated in obesity. In the present study, plasma leptin and resistin levels were increased consistently from 4 to 24 weeks. Time-course transcriptional profiling revealed leptin mRNA levels were increased in both the epididymal and mesenteric WAT depots over 24 weeks, but changes in resistin mRNA levels were less clear. Notably, leptin mRNA levels were higher in the mesenteric WAT depot than in the epididymal WAT depot. From a structural perspective, leptin is a member of the cytokine family, and leptin receptors belong to the class-I cytokine receptor family [22, 23]. Leptin can control the production and activation of pro-inflammatory cytokines such as IL-6 and TNF-α by macrophages [24, 25], and is a key regulatory factor expressed in both zebrafish and mammalian obesity in lipid metabolism . Resistin is also known as an adipose tissue-specific secretory factor, participating in the pathogenesis of insulin resistance, adipogenesis and inflammation in mice [27, 28]. However, the time-course of plasma resistin level changes, was largely dissociated from resistin mRNA levels in the epididymal and mesenteric WAT depots. Jackson et al.  reported similar observations, largely that resistin mRNA levels were very low or undetectable in adipose tissue, which suggests that visceral adipocytes are not a major cell type producing resistin. Plasma resistin levels may by due to resistin production by circulating monocytes and macrophages. Like many other pro-inflammatory cytokines, resistin stimulates intracellular signalling through NF-κB activation, which in turn promotes the synthesis of other pro-inflammatory cytokines, including MCP-1, vascular cell adhesion molecule-1 (VCAM-1), as well as intercellular adhesion molecule-1 (ICAM-1), which are involved in chemotactic pathways directing leukocyte recruitment to sites of infection [30, 31]. Therefore, while resistin appears not to be produced by visceral adipose tissue depots, plasma resistin may still be a player in early visceral adipose tissue inflammation via activation of pro-inflammatory signalling and leukocyte recruitment.
Circulating plasma adiponectin and adiponectin mRNA levels are reportedly decreased by HFD and inversely correlated with fat mass, and directly correlated with insulin sensitivity [32, 33]. In the present study, a markedly different time-course in plasma adiponectin levels was evident with late decreases in plasma adiponectin observed, along with concomitant down-regulation of adiponectin mRNA levels in both the epididymal and mesenteric WAT depots. There have been reports indicating adiponectin is probably not a useful early biomarker of obesity. Sumiyoshi and colleagues reported that high fat feeding (45% fat, wt/wt) for 55 weeks did not alter circulating adiponectin level in C57BL/6 J mice compared with a low fat diet (3% fat, wt/wt) . In accordance with our results, Park and colleague’s reported that circulating adiponectin level was significantly reduced after 20 weeks of high-fat feeding (55 kcal% from fat) compared with a normal diet . The lack of any early changes in plasma adiponectin and visceral adipose tissue adiponectin mRNA levels in HFD fed mice may be part of an adaptive response to protect against early inflammatory changes. There were prominent changes in adiponectin levels in both HFD and ND fed mice around 6 weeks, which we speculate may be related to sexual maturation or ageing.
Adipsin, a serine protease is synthesized and secreted by adipose cells and is found in the bloodstream . In the current study, plasma adipsin levels were significantly higher in the HFD group compared to ND group from the week 2 to 24. However, it is not clear whether the higher concentration of adipsin in blood reflects increased adiposity in the HFD fed mice, because fat mass was increased over time but plasma adipsin decreased after 6 weeks regardless of diet. Previous studies have reported that blood adipsin levels are substantially increased in obese humans [37, 38], but decreased in murine models of obesity . Taken together, the present in-vivo observations of the time-course of various plasma adipokine levels and visceral WAT depot adipokine mRNA levels suggests leptin gene expression is up-regulated early, while adiponectin gene expression is down-regulated later in the epididymal and mesenteric WAT depots contributing to time-dependent changes in plasma adipokine levels.
We conducted in-depth time-course microarray profiling. The early and prolonged changes in the global transcriptome of both epididymal and mesenteric WAT revealed up-regulation of genes predominantly associated with immune and inflammatory system function enriched across all time-points in HFD fed mice. On the other hand, down-regulated genes were predominantly associated with monosaccharide, hexose and glucose metabolism across all time-points. Interestingly, down-regulated genes were also associated with lipid and cholesterol metabolism across all time-points although predominantly in the mesenteric WAT depot, rather than the epididymal WAT depot. In agreement with other diet-induced obesity models, functional ontology terms including sterol metabolism and acetyl-CoA biosynthesis were reportedly highly enriched among regulated genes in the mesenteric WAT depot but not in the epididymal WAT depot , and transcripts involved in lipid biosynthesis were up-regulated only by low fat meals . Genes associated with the inflammatory response that were significantly up-regulated in the epididymal WAT depot during diet-induced obesity included pro-inflammatory cytokine and chemokine genes (Tnf
Ccl3, Ccl4, Ccl6, Ccl7 and Ccl9) and their upstream signalling pathway genes such as TLRs, Irf5 and Cd14. In accordance with our study, many inflammatory genes including Ccl3, Ccl2, Saa3, Tlr13, which were highly expressed in adipose tissue of fat mouse, have been linked with obesity and/or insulin resistance , furthermore Emr1 and Ccl7 mRNA expression are associated with body weight gain as well as obesity-related inflammation. Adipose tissue consists of multiple cell types including adipocytes, fibroblasts, macrophages and endothelial cells. Therefore, the genes identified in the present study as differentially expressed during the development of diet-induced induced obesity will reflect overall changes in visceral adipose tissue depots rather than specific cell-types.
Recent studies have suggested some interplay between TLRs and adipokines. Lin et al.  suggested adipocyte Tlr2 synthesis increases upon stimulation with Tnf. Also, the expression of Tlr1-9 and Tlr11-13 in murine adipose tissue appears to be strongly regulated by leptin . In the present study, the expression of Tlr1, Tlr2, Tlr6, Tlr7, Tlr8 and Tlr13 were significantly up-regulated by HFD in the epididymal WAT depot, along with Tnf and Lep expression, while expression of Adipoq was down-regulated. HFD also significantly up-regulated Irf5 in the later stages of diet-induced obesity. Irf5 is generally involved downstream of the TLR–MyD88 signalling pathway for induction of pro-inflammatory cytokines, and Irf5 is a potential target of therapeutic interventions to control harmful immune responses .
Of the thirteen TLRs, TLR4 was characterized first, working in conjunction with the LPS receptor CD14, TLR4 is the primary signalling receptor for gram negative bacterial lipopolysaccharide (LPS) . CD14 is crucial for LPS recognition by TLR4 and cooperates with other TLRs, including TLR2 and TLR3 [46, 47]. Cd180 also belongs to the family of pathogen receptors. Cd180 takes part in B-cell recognition and LPS signalling . In this study, we show diet-induced obesity leads to significant induction of Cd14
Cd68 and other Cd antigen genes active in the immune and inflammation system of animals. Therefore, expression of Cd antigen genes may be used as markers of visceral adipose tissue inflammation during the development of diet-induced obesity. Taken together these findings suggest that, adipose tissue expansion, and concomitant activation of TLR-mediated inflammatory signalling cascades and induction of CD antigens, causes increased expression of pro-inflammatory cytokines and chemokines, which results in chronic low-grade inflammation.
Obesity is in most, but not all humans, associated with marked changes in the secretory function of adipocytes and macrophages, together with chronic low-grade inflammation and an increased risk of developing fibrosis. A complex network of inflammatory cytokines, adipokines, transcription factors, and receptor molecules is involved in the development of adipose tissue fibrosis. In adipose tissue, fibrosis appears to be initiated in response to adipocyte hypertrophy. Adipose tissue ECM remodelling, which plays a pivotal role in adipogenesis and tissue architecture , is crucial to accommodate obesity-induced cellular alterations . However, the persistence of an inflammatory stimulus in adipose tissue may be responsible for the excessive synthesis of ECM components and subsequent interstitial deposition of fibrotic material. Fibrosis, attributed to excessive deposition of ECM proteins, is a ubiquitous tissue response to chronic inflammation . In this study, ECM gene expression was altered by HFD in a time-dependent manner. Furthermore, there was evidence of accumulation of fibrillar collagens, interspersed in between adipocytes in the visceral WAT of HFD-fed mice. Lumican (Lum) has a stimulatory effect on the epithelial-mesenchymal transition state of fibrosis, while decorin (Dcn) has an inhibitory role in transforming growth factor β (TGF-β)-induced fibrosis [52, 53]. Lum mRNA levels were up-regulated, while Dcn mRNA levels were down-regulated at an early stage during the development of diet-induced obesity. Tgfb1 mRNA levels were also up-regulated in the visceral WAT of HFD-fed mice. The up-regulation of Lum and Tgfb1 mRNA levels and the down-regulation of Dcn mRNA levels were consistent with the observed development of fibrosis in visceral WAT of HFD-fed mice. Many metalloproteases (MMPs) were also altered at the mRNA level by HFD. Mmp2, 3, Mmp12 and Mmp13 are reportedly positively correlated with the degree of obesity and were up-regulated in the visceral WAT of HFD fed mice . Interestingly, our detailed time-course microarray analysis over 24 weeks of HFD feeding showed that collagen mRNA levels were up-regulated shortly after the initiation of HFD feeding and then down-regulated by an unknown mechanism after 2 weeks. We suggest that up-regulation of collagen mRNA levels by HFD during the early stages of diet-induced obesity may trigger the accumulation of ECM components that ultimately leads to fibrosis which was evident in the later stages of diet-induced obesity.
The proteolytic and destructive properties of the lysosomal cathepsins play a role in degenerative, as well as chronic inflammatory diseases. Furthermore, proteases of the cathepsin family are involved in the remodelling of extracellular matrix (ECM) proteins . This study indicates that members of the cathepsin family, including Ctsb
Ctss and Ctsz as well as Ctsa may contribute to the pro-inflammatory and extracellular matrix remodelling in visceral WAT depots during the development of diet-induced obesity. Destruction of elastin-rich tissues during the inflammatory response is associated with local accumulation of macrophages that contain high levels of elastinolytic enzymes, such as Ctsb and Ctsl . Ctss, known to degrade several components of the ECM, is also now well established as a novel biomarker of adiposity . The function of cathepsin A in obesity is relatively unknown, however increases in expression of cathepsin B, D, K, S, and Z have previously been observed in obesity .
It is possible that some of differences in this study compared to others could be due to the lower fat content and different diet composition. Especially, while other studies used 60 kcal% fat diet consisting of soybean oil and lard, we used 39.2 kcal% from fat diet consisting of corn oil and lard. One example, several previous studies have found that hypoxia has been observed in obese adipose tissue and HIF1α (hypoxia-inducible factor 1α), a master signal mediator of hypoxia response, is significantly elevated in obese adipose tissue [4, 59, 60]. We also found that HIF1α gene expression tended to be higher (fold change 1.36 at HFD vs ND) at 24 week of high-fat feeding (about 39.2 kcal% from fat), but there was no statistically significant difference between the groups. However, in another experiment of ours that used commercial high fat diet with higher fat content and different fat composition (Research diets, Product No. D12451, 45 kcal% fat, 2.9% soybean oil and 20.7% lard), high fat feeding for 16 weeks significantly up-regulated HIF1α expression in adipose tissue of C57BL/6 J mice (Y. J. Kim, M. S. Choi, U. J. Jung, unpublished paper).