Liraglutide and not lifestyle intervention reduces soluble CD163 after comparable weight loss in obese participants with prediabetes or type 2 diabetes mellitus

Liraglutide is an analogue of glucagon-like peptide-1 (GLP-1) widely used in the treatment of type 2 diabetes (T2DM) and has been shown to reduce body weight [1]. Further, liraglutide is reported to lower cardiovascular and total mortality in patients with T2DM and high cardiovascular risk [2], but the mechanisms are not fully understood. Macrophages, neutrophils and T-cells express the GLP-1 receptor, and thus GLP-1 and its analogues have the potential to affect a wide spectrum of immune cells [3–5]. Liraglutide affects immune cells in obesity mouse models as well as in human and mouse cells in vitro [6–8]. In clinical trials, liraglutide reduces levels of various inflammatory cytokines such as tumor necrosis factor (TNF) and interleukin (IL)-6 in overweight and obese individuals with T2DM [9–11]. We have previously shown that the inflammatory marker soluble suppression of tumorigenesis-2 (sST2) was decreased after weight loss induced by liraglutide treatment, but not after comparable weight loss due to lifestyle changes [12]. Interestingly, in the same cohort, liraglutide achieved a more pronounced reduction in visceral adipose tissue (VAT) and improvement in beta cell function, independently of weight loss [13]. Monocyte/macrophages are known to be involved in both adipose tissue and islet inflammation, however, the effect of liraglutide treatment on immune cells is still not fully clarified, and it is uncertain whether the anti-inflammatory effects of liraglutide is mainly related to weight reduction.

In the present study, we challenged the idea that immune cells such as monocyte/macrophages, Tcells or neutrophils may be interesting cellular targets of liraglutide, thus contributing to alterations in systemic, adipose tissue and islet cell inflammation. Analyzing plasma from a previous study, comparing liraglutide and lifestyle intervention [13], we explored the regulation of markers reflecting activation of neutrophils: myeloperoxidase (MPO) and neutrophil gelatinase‐associated lipocalin (NGAL), T-cells: soluble (s)CD25 and T-cell immunoglobulin mucin domain-3 (sTIM-3) and monocyte/macrophages: sCD14 and sCD163, before and after similar weight loss induced by liraglutide treatment or lifestyle-changes only.

Liraglutide is an acylated glucagon-like peptide-1 analogue with 97% amino acid homology with endogenous GLP-1 and greatly protracted action. It is widely used for the treatment of T2DM and administered by subcutaneous injection once daily [18]. GLP-1 analogues were initially developed to treat T2DM patients, in whom the effects upon glycemia and, also weight loss, were evident. Recently, the latter effect of these drugs has received much attention [19].

CD163 is a member of the scavenger receptor superfamily, categorized into class B, and its soluble form, sCD163, is regarded as a marker of activated M2 macrophages, and thus potentially a systemic marker reflecting a counteracting mechanism of pro-inflammatory activation of tissue-resident macrophages [20]. sCD163 is cleaved upon activation by a myriad of stimuli, and plasma levels are most likely due to a combination of CD163 expression, altered clearance rate and increased shedding [21]. Increased sCD163 has been shown in obesity [22, 23], metabolic dysregulation and visceral adiposity [24], and is considered a marker of insulin resistance and future T2DM [25, 26]. In keeping with this, we found that baseline sCD163 was associated with several markers of metabolic dysfunction (e.g., a negative association with insulin sensitivity, as measured by Matsuda index) and with markers of inflammation such as CRP. In a recent study, and in agreement with our data, serum levels of sCD163 were higher in patients with obesity and metabolic syndrome as compared to controls [27]. Furthermore, a decrease in serum concentrations of sCD163 and fewer inflammatory macrophages has been previously demonstrated in patients with T2DM treated for 6 months with liraglutide [10], but the present study is, as far as we know, the first to compare levels of immune cell markers after liraglutide treatment and lifestyle intervention with comparable weight loss.

The mechanisms for the sCD163-reducing effect of liraglutide are not known, but we have previously shown improved beta-cell function and reduced VAT after liraglutide treatment, and reduced macrophagic sCD163 might be connected to both these events [13]. Indeed, macrophages are the primary immune cells involved in obesity-associated islet inflammation in both mice and humans [28, 29]. Along these lines, in the same cohort of obese subjects with prediabetes or early diabetes we showed that individuals treated with liraglutide experienced a larger reduction in VAT [10], as compared to individuals who achieved the same extent of weight loss receiving lifestyle intervention. As VAT is regarded as more inflammatory than the subcutaneous adipose tissue (SAT) compartment [30], macrophages from VAT may be a primary source of sCD163 and VAT reduction with liraglutide treatment may mirror reduced macrophage inflammation, as assessed by decreased circulating sCD163. In contrast, Pastel et al. showed that treatment with liraglutide, compared to dietary restriction-based weight loss, increased adipose tissue inflammation, measured as CD14, MCP-1, TNF and IL-6 gene expression and concluded that despite a stronger improvement of glycemic control, liraglutide was not effective in amelioration of obesity-associated adipose tissue dysfunction [31]. However, in that study, the degree of weight reduction between the groups was not equal and gene expression may not necessarily reflect the protein levels, which both could be significant confounding factors. Nonetheless, the reduction in sCD163 in the liraglutide arm and not in the lifestyle intervention arm with comparable weight loss, as well as lack of association between changes in sCD163 and changes in any of metabolic parameters such as BMI, HbA1c, fasting plasma insulin and Matsuda index, support an immune-modulating effect of this GLP-1 analog at least partly independent of the weight loss and related metabolic changes.

Obesity promotes local replication of islet-resident macrophages and recruits circulating monocytes [29]. Islet macrophages in obese mice have been shown to dampen beta cell insulin secretion and promote beta cell proliferation [28]. Thus, targeting islet macrophages is a potential therapeutic approach to modulate beta cell function and prevent development of T2DM. In this regard we previously reported, in patients with T2DM, increased circulating levels of the inflammatory mediator LIGHT/TNFSF14 largely derived from activated monocytes and platelets [17]. We further showed that receptors for LIGHT on islet cells are upregulated and can induce beta cell death and impair insulin secretion from human pancreatic islets in vitro [17], thus contributing to occurrence of overt diabetes and its progression. Interestingly, in the present cohort LIGHT and sCD163 were both increased in obese subjects vs. controls, directly related with each other at baseline, and both were significantly reduced after liraglutide treatment but not during life-style intervention. Thus, we can speculate that GLP-1RAs may act on circulating monocyte/islet macrophages, decrease the release of LIGHT, thus reducing the extent of systemic and local inflammation, leading to improved beta cell function, as assessed by beta-index [13].

MPO has also been shown to be causally linked to development of obesity and insulin resistance [32]. In humans, MPO is upregulated in obesity, independently of T2DM status [33]. In contrast to these findings, baseline MPO was herein not significantly increased in individuals with obesity, as compared to controls, and was negatively correlated with WHR. Nevertheless, the liraglutide group experienced a decrease in MPO levels during weight reduction which was not seen in the lifestyle intervention group. Thus, our data may suggest specific anti-inflammatory effects of liraglutide in T2DM, independent of weight reduction, potentially also involving attenuated neutrophil responses. In cardiovascular safety trials in T2DM patients, with most individuals presenting with cardiovascular disease and excess weight, GLP-1RAs decreased cardiovascular risk [34]. In the case of the LEADER study, major adverse cardiovascular events (MACE) decreased by 13 percent with liraglutide [2]. The mechanism behind the cardiovascular protection observed with human GLP-1RAs in T2DM is not fully known. Carotid plaque inflammation is mitigated in patients treated with GLP-1RAs [35], pointing to reduced inflammation as a possible mechanism underlying the reduction in cardiovascular risk. Gaining insight into the complex mechanisms through which GLP-1RAs produce their anti-inflammatory effects will improve our understanding of their therapeutic potential and facilitate the creation of new anti-inflammatory approaches [36]. Both MPO and LIGHT are associated with increased cardiovascular disease risk and in particular sCD163 is associated with both incidence and death from cardiovascular disease [37–41], and thus reduced levels of these could be one of the mechanisms contributing to the cardioprotective effects of liraglutide.

The present study has some limitations such as a relatively low number of participants. Despite the small sample size, we adjusted for VAT and triglycerides, values that were significantly different between arms at baseline, but we intentionally refrained from conducting further adjustments which may not yield reliable results and could potentially introduce spurious associations. Nonetheless, the lack of correction for several potentially confounding factors is a limitation of the present study. Univariate correlation analyses cannot be used to make assumptions about a causal relationship, and analyzing a large number of variables increases the likelihood of false positive correlations. Another limitation is that there were no measures of diet nor exercise compliance during the intervention.

Finally, our understanding of the inherent functions of the soluble immune cell markers is lacking. Strengths of our study includes the randomized design of the study and the comparable weight reduction between the intervention groups, excluding the confounding effect of different weight loss on the markers in study, as well as the detailed characterization of the metabolic features of participants.

Nevertheless, these data provide novel insight to the regulation of immune cell markers after liraglutide treatment, and a potential mechanism for the observed metabolic benefits seen with liraglutide as compared with lifestyle intervention for the treatment of T2DM and obesity. These data are also interesting in relation to the more widely use of GLP-1 analogs in obese patients to obtain weight reduction independent on the presence of diabetes. Moreover, reduced levels of sCD163, LIGHT and MPO could reflect the observed decrease in VAT, preservation of beta cell function as well as cardiovascular protection by liraglutide treatment, but this needs to be confirmed in larger studies and validation cohorts.