While immune responses in the adipose tissue of mice fed on a high-fat diet over a relatively short period of time are studied extensively, little is known about advanced stages of adipose tissue inflammation in long-standing obesity. Here, we examined the effects of long-term high-fat feeding on adipocytokine gene expression in the epididymal fat of mice with long-standing obesity and correlated the findings with the extent of apoptosis and inflammatory cell infiltration. We showed that extensive loss of adipocytes in this fat depot was associated with reduced fat mass and diminished leptin, adiponectin, IL-6, and MCP-1 gene expression levels. We also showed that cell death and inflammatory cell infiltration occurred predominantly in the rostral zone of the epididymal fat of obese mice. In addition, we found an increase in the expression levels of TNF-α and IL-10 genes in the epididymal fat of obese mice. However, TNF-α and IL-10 expression levels relative to those of F4/80 and mMCP-6 was significantly lower in the heavily infiltrated rostral than in caudal epididymal fat.
There are structural and functional differences between the adipose tissue found in different anatomical locations. For example, subcutaneous and visceral fat differ in immune cell composition, insulin sensitivity, lipolysis, expression of glucose transporters, and obesity-associated adipocyte death and attendant inflammation [3, 15–17]. Biological and immunological differences also exist among various visceral fat depots. For example, we have shown that the prevalence of both mast cells and macrophage CLS are higher in epididymal and perinephric fat than in mesenteric fat of diet-induced obese mice . Emerging evidence indicates that there is also substantial heterogeneity in the structure and function of superficial and deep abdominal subcutaneous fat . Here, we show for the first time that there are distinct zones (regions) within an abdominal fat depot that exhibit different level of susceptibility to metabolic challenges. Although the mechanisms underlying adipocyte injury and death in obesity are under investigation, it has been proposed that exaggerated metabolic demands trigger stress responses in adipocytes that could lead to cell injury and death . In addition, the disequilibrium between oxygen demand and supply in expanding adipose tissues in obesity can lead to local hypoxia, which might have a role in the pathogenesis of adipocyte dysfunction, altered adipokine expression, and adipose tissue inflammation . Based on these observations, it could be argued that, within an abdominal fat depot, there are functionally diverse populations of adipocytes that show different levels of tolerance to metabolic challenges. Alternatively, adipose tissue expansion in obesity might not be uniform throughout the same depot leading to lower oxygen tension in areas of fat depots undergoing a more rapid expansion. Considering the severity of pathological changes in the present study, we elected not to determine the degree of hypoxia in the caudal and rostral epididymal fat of obese mice.
Consistent with a previous report, the loss of the epididymal fat mass in obese mice was inversely related to the weight of the liver . Therefore, it could be argued that lipids released from dead adipocytes, if not locally metabolized by phagocytes, would possibly find their way to the liver and/or other fat depots. Alternatively, the diminished ability of a smaller epididymal fat depot containing many dysfunctional or dead adipocytes would result in energy storage in the liver and/or other fat depots.
Inflammatory cytokines play a significant role in the pathogenesis of insulin resistance . IL-6 is reported to induce insulin resistance in the liver, the skeletal muscle, and 3T3-L1 adipocytes [21–23]. While IL-6 secretion from explanted adipose tissue from obese human subjects is well documented , studies comparing IL-6 protein and/or gene expression between lean and obese subjects are rather sparse. Nevertheless, a recent study showed that IL-6 gene expression was increased in the epididymal fat of mice fed on a high-fat diet (42% calories from fat) for 6 weeks . Similarly, the perigonadal adipose tissue of 8-week old obese KK-A
mice, a model of genetic obesity, demonstrated elevated IL-6 gene expression levels . Loss-of function and gain-of-function mutations showed that MCP-1 play a role in macrophage accumulation and insulin sensitivity in the adipose tissue . MCP-1 was overexpressed in white adipose tissue of mice fed on a high-fat diet (56% calories from fat) for 12 weeks . In addition, compared to mice on control diet, MCP-1 gene expression was greater in the epididymal fat of 20-week old mice after 16 weeks of high fat feeding (45% calories from fat) . In contrast to these studies, here we used mice fed on a diet with higher fat content (60% calories from fat) for a longer period of time (20 weeks) and showed a reduction in IL-6 and MCP-1 gene expression in the epididymal fat. Strissel et al. randomized 5-week-old C57BL/6 mice to either a low-fat diet (10% calories from fat) or a high-fat diet (60% calories from fat) for up to 20 weeks and compared adipocytokine gene expression in the epididymal fat of obese mice of different age . They observed a trend for decreased MCP-1 and IL-6 gene expression between weeks 16 and 20 of high-fat feeding . Since IL-6 and MCP-1 are secreted mainly by the stromal vascular fraction of the adipose tissue , we argue that reduced IL-6 and MCP-1 gene expression in the epididymal fat of mice with long-standing obesity is a manifestation of reduced pro-inflammatory activity of immune cells in the adipose tissue.
A potent anti-inflammatory cytokine, IL-10 is secreted by macrophages, dendritic cells, mast cells, and subsets of B- and T-lymphocytes . Recent evidence suggests that IL-10 has favorable effects on insulin sensitivity and adipocyte function. Hyperinsulinemic-euglycemic clamp studies showed that IL-10 prevents IL-6-induced insulin resistance in the liver and the skeletal muscle in mice . In addition, IL-10 decreased MCP-1 production and ameliorated TNF-α-induced inhibition of insulin-stimulated glucose uptake by 3T3-L1 cells . Consistent with previous reports, we found increased IL-10 and TNF-α gene expression in the epididymal fat of obese mice [30, 31]. However, IL-10 and TNF-α gene expression relative to those of F4/80 and mMCP6 was reduced in the rostral epididymal fat where macrophages and mast cells were abundant. Since IL-10 and TNF-α are expressed and released mainly from the stromal vascular fraction of the adipose tissue , these findings provide further evidence for altered inflammatory activity of immune cells in the epididymal fat depot of mice with long-standing obesity.
Adiponectin and leptin are cytokines produced mainly by adipocytes. Adiponectin promotes insulin sensitivity and, by modulating innate and adaptive immune responses, exerts potent anti-inflammatory effects . Consistent with previous reports, here we found reduced adiponectin gene expression in the epididymal fat of obese mice [33, 34]. However, our finding of reduced leptin gene expression in the epididymal fat of mice with long-standing obesity is not consistent with previous reports [20, 35]. We argue that high numbers of dysfunctional or dead adipocytes in a fat depot could lead to the reduced expression of proteins that are primarily synthesized by adipocytes. In fact, the loss of adipocytes, at least in part by apoptosis, in the epididymal fat of obese mice in the present study was so severe that it led to a decrease in fat mass, a finding that has also been observed by others . Consistent with these findings, we recently reported reduced adiponectin and leptin gene expression in the visceral and inguinal subcutaneous fat of aP2-nSREBP-1c transgenic mice, a model of lipodystrophy . Since mice have several distinct fat depots in various anatomical locations, the contribution of diminished leptin and adiponectin gene expression in the epididymal fat to their circulating concentrations in obese mice remains to be determined. However, considering paracrine actions of leptin and adiponectin, it could be argued that decreased leptin and adiponectin expression would affect the behavior of adipocytes and cells of the stromal vascular fraction, including immune cells, present in the adipose tissue. This is of particular interest considering the differences in relative TNF-α and IL-10 expression between the caudal and the rostral epididymal fat of obese mice in the present study.
The underlying mechanisms that render adipocytes susceptible to metabolic challenges and provoke immune responses in the adipose tissue remain largely elusive. Zonal (regional) distribution of metabolically challenged adipocytes and associated immune responses in a single fat depot could serve as a useful model for studying the underlying mechanisms. Furthermore, an understanding of the mechanisms involved in the downregulation of immune cell activity in inflamed adipose tissue in obesity might help identify molecular targets of therapeutic relevance.