49 - 60: Targeting Adipose Tissue Inflammation to Treat the Underlying Basis of the Metabolic Complications of Obesity

It was once believed that adipocytes were only involved in the storage of triglycerides, but recent studies have demonstrated that they also act as endocrine organs. Hotamisligil et al. [6] and Karasik's group first showed that proinflammatory cytokine tumor necrosis factor (TNF)-α was produced by adipocytes, induced insulin resistance, and increased with expanding fat volume. The concept of adipose tissue as a site for the production of cytokines and other substances has expanded to include leptin, interleukin (IL)-6, resistin, monocyte chemoattractant protein-1 (MCP-1), plasminogen activator inhibitor-1 (PAI-1), angiotensinogen, visfatin, retinol-binding protein-4, and serum amyloid A. Adiponectin is also produced by adipocytes, but its release decreases with increasing adiposity. Leptin and adiponectin are adipokines that are produced only by adipocytes; while TNF-α, IL-6, MCP-1, visfatin, and PAI-1 are also expressed at high levels in activated macrophages. TNF-α, IL-6, resistin, and other proinflammatory cytokines participate in the induction and maintenance of the acute inflammatory response associated with obesity. Additionally, MCP-1 and other chemokines recruit macrophages to adipose tissue. These cytokines and chemokines activate intracellular pathways that promote the development of insulin resistance, type 2 diabetes and other metabolic complications associated with obesity [5]. Therefore, adipose tissue and macrophages within adipose tissue have been shown to play important roles in the regulation of metabolic pathways through the excretion of adipokines and cytokines.

Despite the observed link between body fat, non-alcoholic fatty liver disease, and type 2 diabetes, some individuals exhibit ‘metabolically benign obesity’ and are protected from the metabolic consequences of excess adiposity, possibly due to differences in adipocyte tissue metabolism and macrophage infiltration. In a study aimed at identifying insulin-resistant individuals, 17% of the overweight and obese participants were found to be insulin sensitive [7]. Additionally, a review by Karelis et al. [4] determined that approximately 20% of the general population can be categorized as obese but metabolically healthy. In contrast to this, 18% of the population were found to have a normal bodyweight but suffered from severe metabolic abnormalities. In other studies among obese adults, the degree of adipose tissue inflammation was closely associated with increased metabolic risk for type 2 diabetes, cardiovascular disease and fatty liver disease, whereas obese adults without adipose tissue inflammation have metabolic risk factors in the healthy range [8, 9]. Despite the fact that most human research has focused on the links between fat distribution and metabolic risk, these findings suggest a role for adipose tissue inflammation. In a previous study among obese young minority adults, we found that approximately 40% of subjects had subcutaneous abdominal adipose tissue with crown-like structures, indicating inflammation, whereas approximately 60% of subjects had no signs of adipose tissue inflammation. Despite the two groups being identical for overall obesity and subcutaneous abdominal adipose tissue volume, those with inflamed adipose tissue had approximately 30% greater visceral adipose tissue and 41% greater liver fat; 53% greater fasting insulin and 23% lower β-cell function, and 22% higher TNF-α. Additionally, adipose tissue from those with inflamed adipose tissue had upregulated nuclear factor-κB (NF-κB) expression activity and downregulation of insulin signaling [9]. Another recent study by Bremer et al. [8] demonstrated that, among overweight and obese adults, those with metabolic syndrome have significantly higher levels of infiltrating macrophages/ crown-like structures in their adipose tissue compared to those without the metabolic syndrome. Given these observations, the disparities in metabolic diseases among obese individuals may be explained by the degree of chronic low-grade inflammation of adipose tissue. Therefore, targeting adipose tissue inflammation has become an important new strategy in treating the metabolic conditions typically associated with obesity.

One of the few effective anti-inflammatory treatments for these metabolic diseases is weight loss [10]. Studies in diet-induced obese mice have shown that reductions in adiposity result in decreases in macrophage infiltration of adipose tissue as well as gene expression of pro-inflammatory markers [11, 12]. Specifically, Kosteli et al. [11] found that the murine immune response to weight loss was dynamic. Caloric restriction of high-fat diet-fed mice resulted in an initial increase in adipose tissue macrophages; however, the number of adipose tissue macrophages decreased following an extended period of weight loss. Vieira et al. [12] examined the effects of diet and exercise on inflammation among high-fat diet-induced obese mice. This study examined the effects of weight loss via a low-fat diet, exercise training, or a combination of low-fat diet and exercise on inflammation. All methods of weight loss resulted in a significant attenuation of high-fat diet-induced increases in systemic and adipose tissue inflammation. Additionally, all three interventions improved insulin sensitivity, reduced adiposity, MCP-1, and TNF-α gene expression. Among obese humans, caloric restriction to achieve weight loss also decreases markers of inflammation. For example, a 28-day severe calorie-restrictive diet (800 kcal/ day) among obese females was found to reduce bodyweight by an average of 13 pounds as well as significantly alter markers of inflammation. Specifically, weight loss resulted in a decreased expression of proinflammatory markers (e.g. IL-12a, matrix metalloproteinase-9) in white adipose tissue and increased the expression of anti-inflammatory molecules (e.g. IL-10 and IL-1 receptor antagonist) [13]. Studies using surgical methods to achieve weight loss also improve inflammation among obese patients. A study by Aron-Wisnewsky et al. [10] demonstrated that weight loss following gastric bypass surgery resulted in a mean weight loss of 44 pounds at 3 months. Among the 16 obese females in this study, the activation state of adipose tissue macrophages switched from mostly proinflammatory to anti-inflammatory after weight loss. However, since weight loss is difficult to achieve and maintain, alternative strategies aimed at adipocyte inflammation present a unique target in which to modify metabolic health.

Diets high in sugar and fat have been shown to increase systemic markers of inflammation [14, 15]. Given the link between diet and inflammation, it is not surprising that dietary alterations have been used to examine obesity and obesity-associated inflammation among obese humans [12]. Specifically, healthy adult males given fructose (40 or 80 g/day) or glucose (40 or 80 g/day) sugar-sweetened beverages for 2 weeks demonstrated significant increases in high-sensitivity CRP [14]. In addition to sugar intake, trans fat consumption is related to markers of inflammation. A cross-sectional analysis of 730 women from the Nurse's Health Study found that CRP levels were 73% higher and IL-6 levels were 17% higher among those in the highest quintile of trans fat intake compared to those in the lowest [15]. Among humans, a diet high in fiber (30 g/day), either through diet or fiber supplementation, has been shown to significantly reduce CRP levels [16]. Baseline examination of data from 406 participants from the Finnish Diabetes Prevention Study found that increases in fiber predicted decreases in CRP and IL-6. They also found that changes in fat and carbohydrate intake were either weakly or not related to reductions in CRP and IL-6 [17].

Studies using obese mice have found differing effects of diet composition on inflammation. For example, Wang et al. [18] used mice with high-fat dietinduced obesity to examine the effects of weight loss achieved by switching from high-fat diet to: (1) an ad libitum low-fat normal diet or (2) restricting the high-fat diet intake to match bodyweight of mice with low-fat normal-diet-induced weight loss. Weight loss by either of the two methods resulted in decreased fat mass and liver steatosis; however, effects were greatest among the low-fat normal-diet-induced weight loss than the high-fat diet restriction-induced weight loss. Interestingly, weight loss with the low-fat normal diet, but not the restricted high-fat diet, normalized blood CDC11c+ monocytes and attenuated hepatic inflammation. In contrast, the calorie-restricted high-fat diet significantly reduced chemokine levels and CDC11c+ cells in adipose tissue when compared to low-fat diet-induced weight loss and obese controls. Although these studies demonstrate that changes in diet can affect systemic inflammation associated with obesity, the direct mechanism and critical dietary components are not known. Additionally, the dietary changes necessary to elicit weight loss and decreases in inflammation are likely to be too drastic to sustain over time.

In addition to dietary interventions, studies have examined the effects of anti-inflammatory treatments on systemic markers of inflammation. For example, human studies examining omega-3 polyunsaturated fatty acid (n-3 PUFA) supplementation have shown mixed results in its ability to target systemic inflammation [19], while high-dose aspirin treatments have decreased plasma markers of inflammation [20]. Among adult men and women, a placebo-controlled, double-blind study used 3.5 g/day of fish oil (1.5 g/day n-3 PUFA) for 12 weeks to examine change in plasma markers of inflammation. Compared to placebo, the treatment group did not differ in the median CRP change over the course of the study. In fact, results suggested an increase, not a decrease, in CRP with n-3 PUFA supplementation compared to placebo [19]. Given the disparate findings among animal models and humans, it is not surprising that the potential therapeutic role of n-3 PUFA supplementation to target inflammation has been, for the most part, abandoned. Despite these lackluster findings, researches have begun examining other potential anti-inflammatory treatments that directly target adipose tissue inflammation.

Studies using mice models have successfully used anti-inflammatory treatments, such as n-3 PUFA and resolvin D1 to target adipose tissue inflammation [21, 22]. Todoric et al. [22] treated diabetic mice with a low-fat or a high-fat diet [rich in either saturated/monounsaturated fatty acids (HF/S) or n-3 PUFA] and found that adipose tissue macrophage infiltration was increased and genes involved in inflammation were upregulated in mice fed HF/S compared with the low fat diet. The high-fat diet containing n-3 PUFA completely prevented macrophage infiltration induced by the high-fat diet as well as changes in inflammatory gene expression. Most notably, a recent study has demonstrated the therapeutic potential of resolvins. Resolvins are a newly discovered family of lipid mediators that are generated from n-3 PUFA eicosapentaenoic acid and docosahexaenoic acid. Resolvins have an anti-inflammatory affect by blocking leukocyte infiltration into tissues and decreasing the expression of proinflammatory cytokines. A recent study found that resolvin D1 significantly decreased adipose tissue macrophage accumulation and improved insulin sensitivity in male leptin-deficient mice [21]. Despite these promising results, to our knowledge, this is the only study examining the effects of resolvin D1 on adipose tissue inflammation. Further studies aimed at using resolvin D1 in humans are needed to fully understand the therapeutic potential of this agent.

Among animal models and humans, aspirin has shown promise for decreasing inflammation and improving type 2 diabetes. Aspirin is a non-steroidal antiinflammatory drug that inhibits COX enzymes through the modification of the enzyme's active sites and inhibits prostaglandin synthesis in a similar manner [23]. Specifically, high-dose aspirin studies in Caucasian adults have shown that aspirin inhibits JNK, IκB kinase, and the key enzyme of inflammatory transcription, NF-κB. Additionally, high-doses of aspirin (7 g/day) have been shown to improve peripheral insulin sensitivity and CRP [20]. In obese humans and mice, low-dose aspirin therapy was shown to inhibit systemic IL-6 and reduce IL-6 release from subcutaneous white adipose tissue [24]. In addition to aspirin, statin therapy attenuated increased mRNA expression of pro-inflammatory genes, MCP-1 and IL-6, in adipose tissue of obese mice [25]. Of these treatments, aspirin shows the most promise with regard to reducing adipose tissue inflammation associated with obesity; however, the therapeutic potential of high-dose aspirin is limited by bleeding risk.

Several recent studies, mostly in Caucasian adults, used the anti-inflammatory drug salsalate to address its efficacy and tolerability as a new treatment for insulin resistance and glucose control. Salsalate, unlike aspirin, lacks an acetyl group and does not effectively inhibit COX enzymes [23]; however, salsalate has been shown to inhibit NF-κB [26]. Four trials have examined the effects of salsalate treatment among obese adults. Studies by Fleischman et al. [27], Koska et al. [28], and Goldfine et al. [29–30], have demonstrated the viability of salsalate treatment as a means to decrease markers of inflammation and improve glucose control. Specifically, Fleischman et al. [27] used a doublemasked, placebo-controlled trial of salsalate (4 g/day) for 4 weeks to examine inflammation and metabolic indices among 20 obese adults who were at risk for type 2 diabetes. After treatment, they found that salsalate reduced fasting glucose by 13%, glycemic response after oral glucose challenge by 20%, glycated albumin by 17%, and CRP by 34% [27]. Koska et al. [28] examined 54 obese adults using a randomized, double-blind, placebo controlled trial of 3 g/day of salsalate for 7 days. This shorter study found a reduction in fasting plasma glucose concentration and glucose area under the curve during an oral glucose tolerance test. Lastly, Goldfine et al. [29–30] completed two fundamental studies demonstrating the effects of a moderate and high-dose salsalate on insulin sensitivity as well as the effects of a longer-term clinical trial of salsalate [29, 30]. The first trial consisted of three arms: 4.5 g/day for 2 weeks, 3 g/day for 2 weeks, and 4.5 g/day for 4 weeks. The 4.5 and 3 g/day treatment lasting 2 weeks reduced fasting and post-challenge glucose, while the 4.5 g/day for 4 weeks improved fasting and post-challenge glucose levels and decreased NF-κB activity in peripheral blood mononuclear cells by approximately 65%. The second study by Goldfine et al. [30] included 108 obese adults in a randomized, double-masked placebo controlled trial using 3, 3.5, and 4 g/day of salsalate treatment for 14 weeks [29]. Hemoglobin A1c levels decreased and glycemic control improved in all treatment groups. It is interesting to note that mild hypoglycemic events occurred in 22% of the 3 g/day, 30% of the 3.5 g/day, and 22% in the 4 g/day groups [29].

Overall, these trials noted improvements in insulin sensitivity, fasting glucose, CRP, and NF-κB activity with a 2- and 4-week high-dose salsalate treatment of 4.0 and 4.5 g/day [27, 28, 30]. These data support the hypothesis that utilizing a non-steroidal anti-inflammatory drug, such as salsalate, to target adipose tissue inflammation may provide a therapeutic route for treating obesity-related diseases. Coupled with the above-mentioned findings, and due to the fact that non-acetylated salicylates do not prolong bleeding times, salsalate may offer a relatively safe and effective treatment for the low-grade inflammation associated with obesity. However, it is important to note the limitations of salsalate treatment. Specifically, the moderately high dose needed to elicit improvements in inflammation has potential side effects that warrant concern. Participants in these trials experienced ringing in the ears, alterations in liver function tests, as well as hypoglycemia. These adverse effects highlight the need to understand the mechanism in which salsalate targets inflammation and improves metabolic indices.

Previous studies in obese individuals show that salsalate decreases plasma markers of inflammation and improves glucose control under conditions of weight stability. However, the mechanism of these effects is not known, and no prior clinical study has examined whether the improvement of metabolic complications is due to reduction in adipose tissue inflammation. Currently, our group is initiating a double-blind, randomized control trial, among obese Hispanic young adults, aimed at determining the effects of 4 weeks of salsalate treatment (4 g/day) on the number of macrophages in adipose tissue. This study will be the first to examine the effects of salsalate therapy, without weight loss, on subcutaneous adipose tissue inflammation. In particular, we will examine the notion that the improvement of metabolic risk after salsalate intervention occurs due to its effect on suppressing adipose tissue inflammation, and that without an improvement in adipose tissue inflammation there will be limited improvement in other metabolic risk factors. Findings from this study have the potential to elucidate the mechanism in which salsalate improves glucose control and decreases inflammation. Once the mechanism(s) is identified, safer and more effective therapies could be designed to target inflammation, and thereby treat the metabolic complications, associated with obesity.

Given the known link between chronic low-grade inflammation and metabolic health, it is becoming increasingly important to understand the biological processes that contribute to inflammation in adipose tissue. We have reviewed the evidence linking dietary composition, physical activity, bodyweight, and possibly race/ethnicity to the inflammatory profile that is observed in most overweight and obese individuals. The effects of dietary interventions, dietary supplements, and weight loss have demonstrated mixed results in their ability to effectively treat the inflammation associated with obesity. Nonsteroidal anti-inflammatory drugs have been found to successfully target markers of inflammation and improve glucose control among obese participants. Studies in animal models, as well as humans, have highlighted the potential contribution of adipose tissue inflammation to metabolic disease risk. These studies underscore the need to examine treatments that have shown success in alleviating the metabolic complications associated with obesity, such as nonsteroidal anti-inflammatory drugs, in order to determine their effects on adipose tissue inflammation. Studies such as these would shed light on the mechanism in which these therapies improve systemic inflammation and metabolic health. By understanding the development of obesity-induced inflammation, as well as potential therapeutic targets, drugs can be engineered to alter the inflammatory process that occurs during obesity. These kinds of treatments may offer important clinical methods that can be used to prevent/treat insulin resistance, type 2 diabetes, non-alcoholic fatty liver disease, and other metabolic conditions associated with obesity and inflammation.

The authors declare that no financial or other conflict of interest exists in relation to the content of the chapter.