Adiponectin and Adiponectin Receptors in Atherosclerosis

Abstract

Adiponectin is an abundantly secreted hormone that communicates information between the adipose tissue, and the immune and cardiovascular systems. In metabolically healthy individuals, adiponectin is usually found at high levels and helps improve insulin responsiveness of peripheral tissues, glucose tolerance, and fatty acid oxidation. Beyond its metabolic functions in insulin-sensitive tissues, adiponectin plays a prominent role in attenuating the development of atherosclerotic plaques, partially through regulating macrophage-mediated responses. In this context, adiponectin binds to its receptors, adiponectin receptor 1 (AdipoR1) and AdipoR2 on the cell surface of macrophages to activate a downstream signaling cascade and induce specific atheroprotective functions. Notably, macrophages modulate the stability of the plaque through their ability to switch between proinflammatory responders, and anti-inflammatory proresolving mediators. Traditionally, the extremes of the macrophage polarization spectrum span from M1 proinflammatory and M2 anti-inflammatory phenotypes. Previous evidence has demonstrated that the adiponectin-AdipoR pathway influences M1-M2 macrophage polarization; adiponectin promotes a shift toward an M2-like state, whereas AdipoR1- and AdipoR2-specific contributions are more nuanced. To explore these concepts in depth, we discuss in this review the effect of adiponectin and AdipoR1/R2 on 1) metabolic and immune responses, and 2) M1-M2 macrophage polarization, including their ability to attenuate atherosclerotic plaque inflammation, and their potential as therapeutic targets for clinical applications.

Graphical Abstract

Graphical Abstract.

ESSENTIAL POINTS.

• Atherosclerosis is a multifactorial disease that occurs in major blood vessels, wherein the physical disruption of unstable atherosclerotic plaques is a predominant cause of dangerous cardiovascular events such as myocardial infarctions and strokes

• Monocyte-derived macrophages are major innate immune cell players that contribute to the stability of the plaque, due to their ability to balance proinflammatory responses with anti-inflammatory proresolving mechanisms, consistent with M1 and M2 macrophage phenotypes, respectively

• Adiponectin exerts its beneficial properties during atherosclerotic plaque development by inhibiting various macrophage-mediated proinflammatory signaling pathways, and by promoting M2 macrophage activation

• Adiponectin acts on its 2 main cell surface receptors, adiponectin receptor 1 (AdipoR1) and AdipoR2, which are also regulated by M1-M2 macrophage polarization

• The adiponectin-AdipoR2 pathway can modulate macrophage polarization by mediating the shift toward an anti-inflammatory M2 phenotype, which could have major implications for plaque stabilization

• Leveraging the beneficial effects of adiponectin-induced AdipoR signaling is more precarious due to the difficulty in producing stable multimeric recombinant adiponectin isoforms

• The discovery of novel ligands that can target AdipoR and be translated into viable drug candidates will be essential to provide new avenues to optimize the medical management of atherosclerosis

Atherosclerosis is a multifactorial disease that occurs in major blood vessels, triggered by an accumulation of lipids and inflammatory-driven response, and is a major contributor to cardiovascular (CV) disease, a global leading cause of death and disability (1). Endothelial dysfunction, an influx of monocytes and T cells, unregulated macrophage-mediated uptake of oxidized low-density lipoprotein (oxLDL) and foam cell formation, vascular smooth muscle cell (vSMC) activity, and extracellular matrix (ECM) deposition are major determinants of plaque instability (2). The physical disruption of unstable atherosclerotic plaques is a predominant cause of dangerous CV events such as myocardial infarctions and strokes (3). In this context, monocyte-derived macrophages are major innate immune cell players that contribute to the stability of the plaque, due to their ability to balance proinflammatory responses with anti-inflammatory, proresolving mechanisms (4-6). Essentially, since macrophages are phenotypic plastic cells, they can switch between M1 proinflammatory to M2 anti-inflammatory phenotypes, consistent with unstable, and stable plaques, respectively (7). To this end, M1 macrophage content has been associated with human ischemic stroke incidence (5), while M2 macrophages have been shown to be important for the repair of infarcted adult murine hearts post myocardial infarction (8). Notably, while the M1-M2 paradigm has proved fundamental in understanding inflammation-driven injury, this nomenclature is not entirely descriptive of in vivo macrophage status (9-11). However, using in vitro models of macrophage polarization that induce an M1 or M2 state with distinct cytokines still represents a common approach to study players involved in producing a proinflammatory vs anti-inflammatory, healing plaque environment (12).

Adipose tissue through its secretion of hormones regulates the production of pro-inflammatory and anti-inflammatory cytokines that mediate the crosstalk between the immune and CV systems (13). In addition, this active endocrine organ produces an array of bioactive molecules, known as adipokines, that are involved in regulating glucose uptake, fatty acid oxidation, and CV inflammation (13). Of interest, adipocyte-derived adiponectin is the most abundantly secreted hormone in metabolically healthy individuals, and exerts insulin-sensitizing, anti-inflammatory, and antiatherogenic properties (13). When the amount of energy to be stored exceeds the storage capacity of the subcutaneous adipose tissue (SAT), where it is supposed to be stored, intra-abdominal adipose tissue volume expands, adiponectin levels are decreased, and adipocyte-secreted factors with proinflammatory properties, such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) levels, are increased (13) (Fig. 1). In this context, the production of adiponectin is markedly reduced in individuals living with metabolically unhealthy obesity (14), where infiltrating macrophages attempting to control adipose tissue expansion, an initially beneficial physiologic response, will induce chronic inflammation in adipose tissue, which in turn can over time become excessive and pathologic (15-19). This can contribute to metabolic disorders, such as insulin resistance, metabolic abnormalities, diabetes, and CV disease (15-19). According to a meta-analysis, higher levels of the insulin sensitizer and anti-inflammatory hormone adiponectin are associated with a decreased risk of type 2 diabetes (T2DM) (20). Furthermore, an inverse relationship exists between circulating adiponectin and markers of systemic inflammation, as well as CV disease (21).

Figure 1.

Multimerization of adiponectin and its roles on various tissues. In healthy adipose tissue, adiponectin transcription is mainly induced by binding of peroxisome proliferator-activated receptor (PPAR)-γ and CCAT/enhancer-binding protein (C/EBP)α to the promoter region. In inflamed adipose tissue, common negative regulators of adiponectin transcription are tumor necrosis (TNF)-α and interleukin (IL)-6. After adiponectin transcription, full-length protein translation will occur and lead to the oligomerization of higher-order isoforms, including low-molecular-weight (LMW), medium-molecular-weight (MMW), and high-molecular-weight (HMW) adiponectin. On secretion into the circulation, adiponectin can exert its beneficial properties on the vasculature, brain, pancreas, kidney, heart, and insulin-sensitive tissues such as liver, muscle, and adipose tissue. Created using BioRender.com.

Adiponectin exerts its beneficial properties during atherosclerotic plaque development by inhibiting various macrophage-mediated proinflammatory signaling pathways, including but not limited to excessive oxLDL uptake and foam cell formation, defective clearance of apoptotic cells, and degradation of the ECM (22). To perform these functions, adiponectin acts on its 2 main cell surface receptors, adiponectin receptor (AdipoR)1 and AdipoR2, which are abundantly expressed in the monocyte-macrophage lineage (22, 23). Previous evidence has shown that adiponectin can enhance an anti-inflammatory M2-like macrophage phenotype in blood-isolated cultured human monocytes from healthy individuals (24). Furthermore, AdipoR expression is also regulated by macrophage polarization, whereby M2 polarization resulted in higher AdipoR1/R2 expression compared to M1 (25). To bridge these concepts, in this review we discuss the role of adiponectin and AdipoR1/R2 on regulating 1) metabolic and immune responses, and 2) M1-M2 macrophage polarization, as well as its therapeutic potential to attenuate atherosclerotic plaque inflammation (Graphical Abstract).

Metabolic Functions of Adiponectin and the Adiponectin Receptors

While adiponectin is mainly produced and secreted by adipocytes, other adiponectin-expressing cells include primary human osteoblasts, kidney cells, cardiomyocytes, epithelial cells, quiescent hepatic stellate cells, and liver parenchymal cells (26-34). Moreover, while some evidence suggests that adiponectin is produced by the placenta (35, 36), other studies reported an absence of adiponectin in placental cell types (37, 38). In its full-length form, adiponectin is a 244-amino acid protein containing 4 main domains: the N-terminal signal sequence (1-18), variable region (18-41), collagenous domain (41-107), and C-terminal globular domain (107-244) (39). The cooperative binding of peroxisome proliferator-activated receptor (PPAR)-γ and CCAT/enhancer-binding protein (C/EBP)α on the promoter region of adiponectin will induce its transcription and subsequent full-length protein translation (40-43). The full-length form of adiponectin is restricted to the cytoplasm, and oligomerizes into higher-order structures assembled by disulfide bonds through conserved cysteine residues (44). Posttranslational modifications include hydroxylation and glycosylation of 4 conserved lysine residues in adiponectin's collagenous domain and are crucial for its insulin-sensitizing activities and required for formation of its high-molecular-weight (HMW) complex (45, 46). In this context, adiponectin mainly exists in three homo-oligomeric complexes in the plasma, notably as its low-molecular-weight (LMW), middle-molecular-weight (MMW) or HMW form (see Fig. 1). Notably, in blood samples from individuals with coronary artery disease (CAD) and severe stenosis, total and HMW adiponectin were significantly increased, while glycated HMW adiponectin concentrations were decreased compared to healthy volunteers (47). Experiments investigating the effect of glycated adiponectin on human vSMCs demonstrated its capacity to inhibit proinflammatory cellular functions, such as oxLDL-induced lipid accumulation, proliferation, and migration; these findings highlighted its critical role in regulating functions against atherosclerosis, compared to nonglycated adiponectin (47).

HMW adiponectin has been reported as the most biologically active form compared to other higher-order complexes (48-50). In this context, low HMW adiponectin levels and the presence of metabolic syndrome (MetS) demonstrated a stronger association in females compared to males (51). Moreover, the HMW to total adiponectin ratio was shown to predict the presence of insulin resistance, and MetS in males and females (52). Another study demonstrated that the HMW to total adiponectin ratio was significantly reduced in individuals with T2DM, while the LMW to total adiponectin ratio was elevated and positively associated with T2DM (53). To this end, decreased HMW adiponectin concentrations have been consistently associated with obesity, insulin resistance, T2DM, and atherosclerosis (14, 54-56), highlighting the importance of adiponectin multimer distribution in determining cardiometabolic outcomes. Along these lines, low levels of HMW and total adiponectin have been predictive of the development of metabolic dysfunction–associated steatotic liver disease (MASLD), independent of obesity and insulin resistance, specifically in females (57). Of note, adiponectin was significantly reduced in individuals with T2DM and MASLD compared to individuals without MASLD, wherein adiponectin was also an independent predictor for the development of MASLD in individuals with T2DM (58). Notably, circulating adiponectin levels (combined with specific serum lipid levels) can differentiate between individuals with MASLD and metabolic dysfunction–associated steatohepatitis (MASH), wherein adiponectin levels were lower in individuals with MASH vs MASLD, highlighting adiponectin's potential as a noninvasive MASLD biomarker (59, 60).

In females, a lower risk of coronary heart disease was associated with high levels of HMW, total adiponectin, and an HMW to total adiponectin ratio (61). Interestingly, these associations were lost after adjusting for diabetes, high-density lipoprotein cholesterol, C-reactive protein (CRP), and glycated hemoglobin A1c, thereby demonstrating that glucose and lipid metabolism, as well as inflammation, are mediators of adiponectin effects (61). Moreover, plasma concentrations of HMW adiponectin in pregnant females was likewise the most prevalent multimer regardless of gestational age or body mass index (BMI) status, while no significant differences were observed for total, MMW, and LMW adiponectin with advancing gestation (62). Among pregnant females, the median HMW adiponectin concentration, and HMW to total adiponectin ratio, were significantly higher compared to nonpregnant females (62). Likewise, it was the HMW to total adiponectin ratio in maternal serum that was independently and inversely associated with infant birthweight (63). Low plasma concentrations of adiponectin were observed in pregnant females with obesity compared to females with normal BMI (64). Moreover, HMW adiponectin, which was positively associated with insulin sensitivity, was reduced in females with gestational diabetes (GDM) compared to females without GDM (65). In line with this, a meta-analysis reported that measuring circulating adiponectin levels at prepregnancy and early pregnancy time points may serve as an early prediction tool for females at high risk of developing GDM (66).

Adiponectin's Presence and Half-life in the Circulation of Males and Females

Adiponectin is structurally similar to TNF-α, and is part of a bigger family of adipocytokines called C1q-TNF-α-related proteins (67). Adiponectin can be cleaved into a globular form through leukocyte elastase in THP-1 and U937 monocyte cell lines (68). It is important to note that the globular form is present in the plasma in only very small quantities (<1% of total adiponectin) due to its very short half-life and fast clearance (69), and for this reason has been predominantly investigated within in vitro (70), and in vivo (71) systems rather than in the circulation. On the other hand, multimers of adiponectin are the primary form in plasma, wherein concentrations range from 5 to 30 μg/mL (0.01% of total plasma) (72), higher than levels of other major hormones such as leptin, cortisone, and inflammatory cytokines TNF-α and IL-6 (73, 74). Interestingly, among females with and without T2DM, high coffee consumption was associated with increased adiponectin concentrations and reduced levels of inflammatory markers compared to females who did not drink coffee regularly (75). Moreover, previous evidence has demonstrated higher circulating adiponectin concentrations in females compared to males (76-79). Notably, higher adiponectin levels in females were present along with higher BMI, waist/height ratio, and body fat percentage compared to males (78). In parallel, despite higher BMI, a greater abundance of SAT in females may contribute toward the variance in adiponectin levels (76). Furthermore, elevated adiponectin levels in females compared to males may possibly be due to testosterone's ability to inhibit HMW adiponectin production, which was shown in male animal models (80-84). In a clinical study including 28 healthy males aged 18 to 35 years, decreased testosterone levels were observed on treatment with a potent gonadotropin-releasing-hormone antagonist, acyline, which led to increased serum adiponectin levels, despite no significant changes in BMI (85). Additionally, another study including 25 men aged 55 to 85 years, treatment with high-dose testosterone resulted in a significant reduction in adiponectin, whereas BMI slightly increased (85). A small study demonstrated that age was positively correlated with adiponectin in males, while no relationship was observed in females (78). However, a larger study showed that serum adiponectin levels were significantly and positively associated with age both in males and females (86).

There are numerous sex-specific mechanisms that could be contributing to diverse associations between adiponectin and age; specifically, adiponectin levels follow a U-shaped trajectory as females transition from a premenopausal, perimenopausal, to early and late postmenopausal state (87, 88). Notably, low adiponectin levels during menopause transition were associated with relative excess androgen (88), while in females who were postmenopausal (1 year), adiponectin levels were negatively correlated with testosterone (87). Furthermore, postmenopausal females with low plasma adiponectin levels showed the highest risk for MetS, which differed considerably from premenopausal females (89). Notably, serum adiponectin concentrations were significantly decreased in females with obesity of childbearing or perimenopausal age compared to females without obesity (90). More specifically, a significant reduction in adiponectin and estrogen levels were observed among females with obesity of perimenopausal compared to childbearing age, while a significant increase in visceral/subcutaneous fat area was noted (90). These findings highlight that a higher distribution of visceral adipose tissue (VAT) compared to SAT, in addition to decreased adiponectin and estrogen levels may be characteristic changes in perimenopausal females with obesity (90). Moreover, in a small, population-based prospective cohort study, circulating adiponectin levels were lower in females who gave birth to a large-for-gestational age (LGA) infant, wherein changes in adiponectin levels was a predictor of birth weight, independent of BMI and insulin resistance (91). On the other hand, in a large population-based cohort, maternal adiponectin levels in early mid-pregnancy demonstrated an inverse relationship with infant birthweight and the likelihood of giving birth to an LGA infant; for each 1-unit increase in adiponectin, the odds of giving birth to an LGA infant decreased by 9% (92). However, this association was lost when adjusted for maternal BMI (92). Of note, a significant interaction was revealed between maternal adiponectin and birth size in female infants, while no association was found in males (92). Therefore, it is possible that the link between maternal adipose tissue accumulation and fetal growth, in addition to subsequent birth size in female infants, is partially mediated by adiponectin (92). In line with this, another study demonstrated that maternal obesity in the absence of GDM was associated with downregulated adiponectin levels, which has a significant effect on placental development (93).

Despite variations in adiponectin levels in the circulation, its half-life was similar both in male and female FVB mice, with a clearance rate of approximately 75 minutes, which suggests differential adiponectin production rates (94). When comparing clearance rates between adiponectin complexes, a longer half-life emerged with larger adiponectin isoforms, wherein the HMW isoform clears the slowest (∼85 minutes), and the trimer the fastest (∼32 minutes) (94). Interestingly, in ob/ob or high-fat diet–fed male mice with decreased adiponectin plasma levels, clearance rates were increased to approximately 120 and 130 minutes, respectively, highlighting a potentially reduced production rate (94). Therefore, these results demonstrated that rapid clearance may be linked to adiponectin's bioavailability and activity, whereas delayed clearance of adiponectin in obese or high-fat diet murine models may be an indicator of metabolic dysfunction (94). Furthermore, this study demonstrated the liver as the main tissue for clearance, and to a lesser degree the kidney, through which the final degradation products are excreted (94). Previous evidence has shown that adiponectin concentrations remain relatively stable in whole blood obtained from male and female volunteers, as storage of blood samples on ice for up to 36 hours was possible without substantial losses due to degradation (95). It has been suggested that indeed a clearance-based mechanism rather than degradation in the circulation is the primary method for elimination of adiponectin (94). In SAT obtained from individuals who are obese compared to those who are lean, through outpatient needle biopsy (96), adiponectin messenger RNA (mRNA) was reduced and was positively correlated with serum adiponectin (97). Together, this may suggest a connection between reduced adiponectin mRNA production and corresponding secretory release with a slower clearance observed in obesity (94, 97). Furthermore, adiponectin internalization was studied in muscle cells, wherein full-length adiponectin and globular adiponectin were endocytosed in clathrin-dependent and clathrin-independent pathways, respectively (98). Several studies have reported that elevated adiponectin levels in patients with cardiometabolic disorders, heart failure, and advanced age are associated with a higher mortality rate, often referred to as the adiponectin paradox (60, 99-103). In this context, circulating adiponectin concentrations are dictated by the balance between its production and clearance, and as such one possible explanation for its disproportionally higher levels in conditions with CV dysfunction could be a liver-mediated delay in adiponectin clearance rather than increased production (60, 99). On the other hand, the presence of high adiponectin levels in individuals with CV disorders may instead reflect a compensatory response that is activated to restore CV function (60, 99). More recently, a study demonstrated that adiponectin levels were strongly related to diabetic vascular complications in participants with T2DM that were not obese, while exhibiting a modest association in individuals with obesity (104). While these results suggest that the paradoxical elevation of adiponectin may be compromised by fat accumulation (104), further research is required to understand the mechanistic links between adiponectin clearance and production in healthy and pathological conditions to fully rule out a possible proinflammatory function of adiponectin in accelerating the progression of CV disorders (99).

Adiponectin's Expression in Specific Adipose Tissue Depots in Healthy and Pathological Conditions

Adiponectin exerts its anti-inflammatory actions on various tissues, such as the liver, skeletal muscle, pancreas, heart, kidney, and brain; on immune and nonimmune cells in the vasculature in an endocrine manner; and on adipose tissue cells in an autocrine and paracrine manner (50, 105) (see Fig. 1). Of note, adiponectin acts locally on neighboring adipocytes and surrounding stromal vascular cells to suppress TNF-α– and IL-6–induced secretion, and in a paracrine manner to reduce macrophage infiltration (105-107). In this context, in human multipotent adipose-derived stem cells differentiated into adipocytes that were treated with conditioned media from polarized M1-like proinflammatory and M2-like anti-inflammatory THP-1 macrophages, adiponectin mRNA levels were significantly increased in the M2 compared to M1 media condition (108). Moreover, adiponectin and C/EBPα mRNA was significantly elevated in freshly isolated subcutaneous compared to omental adipose tissue, obtained via adipose tissue biopsies from healthy individuals without obesity (109). On the other hand, in vitro analyses of differentiated adipose tissue cells demonstrated elevated adiponectin secretion into the culture medium of visceral compared to subcutaneous adipocytes (109). These findings suggest that adipose tissue depot-related differences exist in adiponectin gene expression compared to protein secretion from healthy individuals (109). Importantly, adiponectin protein secretion can be regulated by other factors, such as insulin, which, in addition to PPAR-γ agonist rosiglitazone, was shown to increase adiponectin concentrations in omental compared to subcutaneous adipocytes obtained from patients who underwent elective benign surgery or gastric banding for obesity (110). However, depot-specific secretory characteristics differ in people who are lean compared to obese (111). Specifically, in adipocytes isolated from subcutaneous and omental adipose tissue samples obtained from females, adiponectin release was similar across both subcutaneous and omental cells in those who were lean (111). On the other hand, in females who were obese, adiponectin secretion was instead significantly reduced in omental cells and negatively correlated with VAT area (111). Nonetheless, it must be noted that in vitro studies using isolated adipocytes do not entirely reflect the adipose tissue depot-specific environment, as adipocyte secretions are largely influenced by surrounding stromal and inflammatory cells (112). As such, a study using cultured SAT and VAT samples from individuals who underwent elective abdominal surgery revealed that in individuals with BMI less than 40 no depot-specific adiponectin secretion differences were observed (112). However, in participants with BMI greater than 40 adiponectin secretion from VAT was significantly reduced compared to SAT and was negatively correlated with VAT percentage (112). Moreover, a large study including males and females demonstrated that plasma adiponectin was significantly associated with SAT-to-VAT ratios (113). Taken together, depot-specific biology must be considered, as adipose tissue accumulation in subcutaneous depots does not carry the same metabolic disease risk as visceral depots (112-115). Notably, in a community-based asymptomatic population, the incidence of MetS was most prominent in participants with high visceral adiposity and low serum adiponectin concentrations (116).

A systematic investigation examining the effect of sex on inflammation and MetS in males and females who are overweight revealed that females had lower concentrations of adiponectin, while males had higher concentrations of proinflammatory factors such as leptin and IL-6, as well as elevated hyperresponsive circulating immune cells (117). These findings highlight that in the presence of MetS females have defective anti-inflammatory mechanisms, while males have more profound proinflammatory mechanisms (117). In individuals with elevated blood pressure and in the context of MetS, females showed a relatively greater decrease in adiponectin levels compared to males (118). This could be partially due to the greater accumulation of SAT in females compared to VAT in males, given that adiponectin is more highly expressed in SAT than VAT (119). However, females with obesity in the context of MetS were characterized by higher VAT (120), which may explain their decreased adiponectin production (117). In line with this, metabolically unhealthy obesity with ectopic fat distribution is a crucial risk factor for the development of CV diseases (121), wherein distinct adipose tissue distribution (VAT/SAT area ratio) is an independent predictor of CV disease in individuals with T2DM (122). In individuals living with obesity, a lower risk for higher coronary plaque severity was demonstrated in the group with low VAT/high SAT (123). These findings highlight the importance of characterizing the distribution of adipose tissue depots to better evaluate CV risk. In this context, along with previous evidence demonstrating that the adiponectin/leptin ratio correlates with inflammation, insulin resistance, oxidative stress, and numerous cardiometabolic risk factors (124, 125), it was also reported as a measure of adipose tissue dysfunction among older adults with obesity (126). Notably, the adiponectin/leptin ratio was positively associated with high-density lipoprotein cholesterol, and inversely correlated with CRP and IL-6 in females with obesity (126). Therefore, since a low adiponectin/leptin ratio is reflective of dysfunctional adipose tissue per overall fat mass ratio, it may be a clinically useful biomarker to identify individuals that are susceptible to CV disease; this may provide an opportunity for therapeutic intervention before disease onset and progression (126).

Adiponectin is also highly expressed in other forms of ectopic fat deposition such as epicardial adipose tissue (EAT) surrounding major coronary vessels, as well as in perivascular adipose tissue (PVAT) that serves as an adipose tissue depot around the vascular wall (127). A direct correlation exists between dysfunctional EAT and CAD (128), conditions wherein levels of adiponectin were found to be extremely low (129). Likewise, Western blot analyses revealed that adiponectin protein expression was significantly lower in individuals with CAD compared to individuals without CAD (130). Another study demonstrated lower and higher expression of adiponectin and IL-6 mRNA, respectively, in EAT of participants with CAD compared to those without CAD (131). Moreover, in pathological conditions, a decreased adiponectin/leptin ratio in EAT was demonstrated as a risk factor for CAD (132, 133). Due to its anatomical location, PVAT-derived adiponectin can enter atherosclerotic plaques and act on various vascular cells, such as endothelial cells, SMCs, and inflammatory cells, notably macrophages (127, 134). In this context, using apolipoprotein E (apoE)-knockout (KO) mice with PVAT derived from adiponectin-KO mice, accelerated plaque volume and attenuated macrophage autophagy were observed compared to apoE-KO transplanted with wild-type littermate tissue (135). These findings highlighted the key function of PVAT-secreted adiponectin to help suppress plaque formation (135).

Pivotal Role of Constitutively Expressed Adiponectin Receptor 1 and Adiponectin Receptor 2 for Human Health

Adiponectin binds to integral membrane proteins AdipoR1 and AdipoR2 that are expressed ubiquitously (136-138), but predominantly in skeletal muscle and the liver, respectively (136). Adiponectin can also bind to a third receptor, T-cadherin (T-cad), which exerts cardioprotective effects (139) and is present in vascular cells, such as endothelial cells, SMCs, cardiomyocytes, and pericytes, but not in major immune cells, such as monocytes/macrophages, T, B, and natural killer (NK)-cells derived from the human aortic wall (139, 140). Of note, AdipoR1 and AdipoR2 are cell surface proteins spanning 7 transmembrane domains with an intracellular N-terminus and extracellular C-terminus in contrast to classic G protein–coupled receptors (GPCRs) (136). Notably, the globular isoform of adiponectin preferentially binds to AdipoR1, while its HMW isoform binds both to AdipoR1 and AdipoR2 with similar affinities (136) (Fig. 2). Furthermore, HMW isoforms of adiponectin bind to T-cad, rather than the globular form (141).

Figure 2.

Binding interaction of adiponectin to its receptors induces its signaling cascade. Globular adiponectin has a higher affinity for adiponectin receptor 1 (AdipoR1), while low-molecular-weight (LMW), medium-molecular-weight (MMW), and high-molecular-weight (HMW) isoforms have equal affinities for both AdipoR1 and AdipoR2. On ligand-receptor binding, adaptor protein containing pleckstrin homology domain, phosphotyrosine binding domain, and leucine zipper motif 1 (APPL1) will mediate downstream signaling pathways of AdipoR1 and AdipoR2, which mainly activate 5′ adenosine monophosphate–activated protein kinase (AMPK) and peroxisome proliferator-activated receptor (PPAR)-α, respectively. Other AdipoR-dependent pathways include activation of insulin receptor susbstrate-1/2 (IRS1/2) that binds to the insulin receptor (IR), phosphoinositide-3-kinase/protein kinase B (PI3K/Akt), p38 mitogen-activated protein kinase (MAPK), and Rab5, which promotes the translocation of glucose transporter (GLUT)4 and glucose uptake. Furthermore, the AdipoRs possess intrinsic ceramidase functions and regulate membrane fluidity. Created using BioRender.com.

In steady-state, experiments conducted in HEK293 cells demonstrated that AdipoR1 is a resident cell-surface protein, while the majority of AdipoR2 was not present at the plasma membrane but rather at the endoplasmic reticulum (ER) (142). When comparing nonpermeabilized and permeabilized conditions, no changes were observed for AdipoR1, while AdipoR2 was undetectable in nonpermeabilized cells and exclusively present during permeabilization (142). Furthermore, AdipoR1 and AdipoR2 share overall 68% homology at the amino acid level; their N-terminal regions consisting of residues of AdipoR1 (1-70) and AdipoR2 (1-81) show only 17% homology, while their remaining sequences AdipoR1 (71-136) and AdipoR2 (82-147) demonstrate 95% homology (142). Interestingly, truncated constructs demonstrated that these nonconserved, N-terminal cytoplasmic domains explain the differences in cell-surface levels of AdipoR1 and AdipoR2 (142, 143). Of note, cell-surface expression of AdipoR2 was restricted by its N-terminal residues 1 to 81 (142). Through AdipoR1 and AdipoR2 cotransfection experiments in CHO cells, coimmunoprecipitation demonstrated that AdipoR1 and AdipoR2 can indeed form homodimers and heterodimers, and that AdipoR1 particularly promotes cell-surface expression of AdipoR2 (142).

Through fluorescence resonance energy transfer experiments in HEK293 cells, it was demonstrated that AdipoR1 and AdipoR2 form stable homodimers in unstimulated conditions and quickly dissociate on adiponectin binding (144), similarly to GPCRs (145). AdipoR1 and AdipoR2 can rapidly reassociate into homodimers shortly after adiponectin-induced receptor dissociation (144). Of note, heterodimeric interactions between AdipoR1 and AdipoR2 similarly undergo conformational changes on ligand binding, but at a faster rate than the homodimers, which could imply that heterodimeric interactions are less stable (144). Ligand-induced internalization of AdipoR1/R2 primarily leads to lysosomal degradation, therefore decreasing the response to adiponectin due to reduced recycling of AdipoR to the membrane (144). Specifically, immunostaining of AdipoR1- and AdipoR2-transfected cells demonstrated colocalization of AdipoRs with lysosomal-associated membrane protein 1 after 30 to 60 minutes’ exposure with globular and full-length adiponectin (144). Therefore, it seems that ligand-mediated internalization of the AdipoRs may be targeting them for degradation to the lysosomal pathway to avoid overstimulation and maintain homeostasis, or rather could suggest a form of adiponectin resistance in pathological conditions (144). In line with this, another study in HEK293 cells demonstrated that adiponectin reduced AdipoR1 homodimerization in a concentration-dependent manner, mainly mediated through amino acid residues 60 to 89 in its collagen domain (146). Furthermore, similarly to full-length adiponectin, AdipoR1 is endocytosed in a clathrin- and Rab5-dependent pathway in HEK293 cells (98). However, inhibition of AdipoR1 endocytosis in muscle cells demonstrated enhanced and sustained adiponectin-induced 5′ adenosine monophosphate-activated protein kinase (AMPK) phosphorylation; this highlighted that AdipoR1 endocytosis may be necessary to modulate the quantity of cell surface receptors and duration of downstream signaling (98).

Fluorescence recovery after photobleaching experiments in HEK293 cells demonstrated the key role of constitutively expressed cell surface AdipoR1 and AdipoR2 to maintain membrane fluidity (147). When challenged with high concentrations of exogenous saturated fatty acid palmitic acid (PA) to induce lipotoxicity, only inhibiting single AdipoR2 and double AdipoR1/R2 resulted in loss of membrane fluidity (147). Therefore, maintaining intact AdipoR2 seems to be important in preventing cell membrane rigidification under stress-induced conditions. Of note, the addition of unsaturated fatty acids, namely oleic acid and eicosapentaenoic acid, suppressed the loss of membrane fluidity during PA-induced AdipoR knockdown conditions by restoring the balance of fatty acids (147). Furthermore, the capacity of the AdipoRs to maintain membrane fluidity was independent of their putative ligand, adiponectin, in HEK293, HepG2, 1231N1, human umbilical vein endothelial cells (HUVECs), and human adipose tissue (147). These findings were consistent with another study conducted in PA-treated HEK293 cells, wherein small-interfering RNA (siRNA)-mediated knockdown of AdipoR2, but not AdpoR1, resulted in a dramatic decrease in membrane fluidity and increase in saturated fatty acid content compared to nontargeting siRNA control (148). Of note, PA is a precursor for ceramide synthesis, which was decreased in PA-treated HEK293 cells during AdipoR2 knockdown compared to control (148). This contrasted with in vivo studies that highlighted that AdipoR-mediated ceramidase activity was in fact dependent on adiponectin to promote ceramide degradation to sphingosine and sphingosine-1-phosphate (S1P) (149, 150). Therefore, it is possible that AdipoR1 compensates with its ceramidase function during siRNA-mediated AdipoR2 knockdown to reduce the abundance of ceramides (148). In this context, an in-depth biochemical and computational analysis of AdipoR1 and AdipoR2 crystal structures confirmed that the receptors possess adiponectin-sensitive low ceramidase activity (151). Thus, adiponectin's in vivo effect on ceramide metabolism may be partly attributed to the intrinsic AdipoR ceramidase function (151) (Fig. 2). Consistent with this, it was identified that PA-induced membrane rigidification promotes AdipoR intrinsic ceramidase activity, primarily AdipoR2, and a conserved mechanistic pathway that leads to increased expression of stearoyl-CoA desaturase that helps restore membrane fluidity (152). This process relies on AdipoR2's downstream signaling cascade, which activates sterol regulatory-element binding protein 1 and PPAR-γ that in turn promote the transcription of desaturases (149, 152, 153).

Notably, 2 recently published CRISPR/Cas9 screens, identifying genes that prevent PA-associated or desaturase inhibition toxicity, exhibited that AdipoR2 ranked 25th out of approximately 3000 metabolism genes (154) and fourth out of approximately 20 000 genes (155), respectively. The important role of AdipoR in maintaining membrane homeostasis was further confirmed in another study using HEK293T AdipoR2-KO cells (156). Intriguingly, using a chemical screen, tyloxapol, a nonionic detergent that is a US Food and Drug Administration (FDA)-approved inhaled surfactant used for removing and liquefying bronchopulmonary secretions with mucus and pus, was the primary identified hit among membrane-fluidizing compounds investigated to rescue the C elegans PAQR-2 (AdipoR2 mammalian homologue) mutant with rigid membranes (157). Using CRISPR/Cas9, the efficacy of tyloxapol was further confirmed in AdipoR2-KO HAP1 cells (157). However, while tyloxapol is not a favorable option to treat conditions associated with membrane rigidification as it is also used to induce hyperlipidemia in animal models (158, 159), accompanied by genomic instability (160), this study showcased an avenue to investigate FDA-approved compounds (membrane fluidizers) using C elegans (157). Collectively, these results are not to discount AdipoR1 as a fluidity regulator but rather suggest that AdipoR2 acts as the main receptor responsible to protect against membrane rigidification (157, 161, 162) (see Fig. 2).

To further support the role of functional AdipoR2 to promote these processes, it was demonstrated that membrane homeostasis is achieved through nonautonomous regulation, wherein AdipoR2-expressing HEK293 cells normalize membrane fluidity in distant AdipoR2-deficient cells (163). Likewise, another study reported that AdipoR2 siRNA-treated HEK293 cells lost their ability to elongate, desaturate, and incorporate polyunsaturated fatty acids, such as linoleic acid, into phospholipids compared to nontargeting siRNA control (153). Using a mass spectrometry proteomics approach, immunoprecipitation experiments in HEK293 cells or whole C elegans of tagged AdipoR2 and PAQR-2, respectively, identified that key coimmunoprecipitated proteins were indeed involved in fatty acid elongation and incorporation into phospholipids that aim to restore membrane fluidity (153). Of interest, 3 key ER-resident enzymes were found to interact with AdipoR2/PAQR-2, which are required to form a fatty acid elongation complex: namely HACD3, HSD17B12, and ELOVL3/6 (mammalian homologues to C elegans HPO-8, LET-767-, and ELO-2, respectively) (153). These findings suggest that AdipoR2/PAQR-2 are either ER-bound proteins, or actively recruiting ER-resident enzymes to the plasma membrane (153).

Comprehensive mRNA expression analysis through reverse-transcription quantitative polymerase chain reaction of murine tissue samples revealed that while both AdipoR1 and AdipoR2 were expressed across somatic tissues, AdipoR2 alone was expressed in the testis (164). In fact, spermatogenesis defects were revealed in AdipoR2-KO male mice that led to the complete loss of male fertility (164). Of note, adiponectin-KO mice have been previously reported to be fertile (153), suggesting that AdipoR2's function in the testes occurs without adiponectin binding, similar to previous reports that highlighted adiponectin-independent roles of the AdipoRs (147). It was particularly AdipoR2's transcriptional and posttranscriptional upregulation of ELOVL2 that contributed toward ensuring membrane fluidization for faithful meiosis in male germlines (164). On the other hand, in male mice fed a high-fat diet, AdipoR1 mRNA and protein in the testis were significantly decreased compared to those fed a normal chow diet, with no significant difference in AdipoR2 expression (165). In this context, AdipoR1-KO mice testes weight was significantly decreased and seminiferous tubules were atrophied, while sperm count, sperm motility, and phosphorylation of AMPK were reduced compared to those in wild-type mice (165). These findings suggest that functional AdipoR1 is crucial to help prevent particularly obesity-induced male infertility mediated through its downstream signaling cascade, while AdipoR2 seems to be required for male reproduction through its capacity to maintain membrane fluidity (164, 165).

In the context of regulating cellular homeostasis, failure of sustaining plasma membrane integrity is associated with many acute and chronic pathologies, including diabetes, insulin resistance, and atherosclerosis (166, 167). Efficient phagocytic activity of macrophages is highly dependent on membrane fatty acid composition, wherein a higher ratio of unsaturated/saturated fatty acids showed an increased phagocytosis rate (168). To this end, it would be interesting to investigate the effect of AdipoRs on macrophage membrane fluidity during polarization toward M1 or M2 phenotypes to identify which receptor is more pivotal in the regulation of immune-mediated mechanisms. Nonetheless, these findings suggest a critical role for AdipoR1 and AdipoR2 at the cell surface to maintain plasma membrane homeostasis, beyond the functional benefits of their receptor-specific intracellular signaling cascades (147). In this line, it has been demonstrated that AdipoR1/AdipoR2 double-KO in mice leads to embryonic lethality (169). More recently, in a murine model wherein Vav1-Cre; AdipoR1^fl/fl;AdipoR2^fl/fl bone marrow cells were transplanted into AdipoR1^fl/fl;AdipoR2^fl/fl control donor cells, the absence of the AdipoRs led to elevated levels of multiple proinflammatory cytokines, resulting in the reduction of hematopoietic stem cell (HSC) self-renewal and increased HSC depletion during aging (170). Notably, the source of inflammation in the bone marrow was derived from lymphoid (CD4+ and CD8+ T cells) and myeloid (monocytes) cells, and further investigation demonstrated that the AdipoRs acted nonautonomously in immune cells to suppress the proinflammatory phenotype (170). Therefore, not only are AdipoRs vital for development (169) and cell membrane integrity (147), but they are also important throughout adulthood to protect HSCs from inflammation by sustaining their quiescence and preventing their premature depletion (170). In general, organelle-specific roles in AdipoR1/R2-deficient conditions have not been extensively investigated, and merit further research to assess the intracellular trafficking and recycling capacities of the AdipoRs and how these properties contribute toward various pathologies.

Key Signaling Pathways of the Adiponectin Receptors

Early evidence has demonstrated that AdipoR1 predominantly activates AMPK and promotes glucose uptake, as well as reduces gluconeogenesis and lipid synthesis, while AdipoR2 activates PPAR-α signaling to stimulate fatty acid oxidation (136, 171). Other AdipoR-dependent signaling pathways include activation of p38 mitogen-activated protein kinase (MAPK), which regulates cell proliferation, and immune responses (25), insulin receptor substrate (IRS)1/2 to improve insulin sensitivity (172), phosphoinositide-3-kinase/protein kinase B (PI3K/Akt) to induce endothelial nitric oxide synthase (eNOS) activation and insulin signaling, in addition to Rab5 to promote translocation of the glucose transporter (GLUT) (173) (see Fig. 2). Of note, using yeast 2-hybrid technology, it was identified that adiponectin's downstream signaling events through AdipoR1/R2 are mediated by a key intracellular binding partner, adaptor protein containing pleckstrin homology domain, phosphotyrosine binding domain (PTB), and leucine zipper motif 1 (APPL1) (174) (see Fig. 2). Specifically, through its C-terminal PTB and coiled-coil domains, APPL1 directly binds to the intracellular domains of AdipoR1 and AdipoR2 at their N-terminus (174). The overexpression of APPL1 in muscle cells led to a significant increase in the activation of phosphorylated p38 MAPK and AMPK, which was lost in APPL1-deficient cells (174). Furthermore, adiponectin-stimulated GLUT4 membrane translocation was mediated via the interaction between APPL1 and the GTP-bound form of Rab5 (175), which is likewise blocked during overexpression of dominant-negative Rab5 (174). In line with a previous report that showed APPL1 interacting with insulin-activated PI3K/Akt (176), siRNA-mediated knockdown of APPL1 in muscle cells similarly resulted in a significant reduction in the synergistic effect of adiponectin on insulin-stimulated Akt phosphorylation (174). Therefore, the insulin-sensitizing effect of adiponectin in muscle cells is at least partly due to the crosstalk between adiponectin- and insulin-stimulated signaling pathways (174). Consistent with this, another study showed that APPL1 indeed forms a complex with IRS1/2 and facilitates its recruitment to the insulin receptor (IR) on adiponectin-, or insulin-stimulated conditions, which is abolished in APPL1-KO mice (177). In HUVECs, APPL1 was likewise shown to act as an immediate downstream effector of the AdipoRs to mediate adiponectin-stimulated phosphorylation of AMPK and eNOS while promoting the association of eNOS with HSP90 to enhance NO production (178). Additionally, the protective effect of adiponectin-induced NO signaling relied on AdipoR1 in arterioles collected from subjects with CAD, as confirmed through siRNA-mediated AdipoR1 silencing during exogenous adiponectin treatment (179). Beyond observing APPL1 as a key downstream signal for the AdipoRs in muscle (174) and endothelial cells (178), another study reported its role in THP-1 monocyte-derived macrophages (22), a commonly used cell line to study atherosclerosis (180). APPL1 short hairpin RNA knockdown significantly reduced adiponectin's ability to attenuate intracellular lipid accumulation in THP-1 macrophages that were pretreated with adiponectin, followed by oxLDL uptake (22). Additionally, in adult rat cardiomyocytes, coimmunoprecipitation experiments demonstrated that adiponectin likewise enhanced the association of AdipoR1 with APPL1 and subsequent binding of APPL1 with AMPKα2, which led to increased fatty acid uptake and oxidation (181). These metabolic functions were lost during APPL1 siRNA-mediated knockdown in neonatal rat cardiomyocytes (181). These data affirm the importance of APPL1 in mediating adiponectin's downstream signaling pathways in key vascular and immune cells (22, 174, 175, 177, 178, 181, 182).

APPL2, an isoform of APPL1, is also able to bind AdipoR1 and AdipoR2, and is expressed in several insulin-sensitive tissues such as the brain, liver, skeletal muscle, pancreas, and adipose tissue (182). Interestingly, overexpression of APPL2 negatively regulated adiponectin-stimulated AMPK and p38 MAPK phosphorylation by heterodimerizing to APPL1 and preventing its binding interaction to AdipoR1 (182). To this end, the activated forms of AMPK and p38 MAPK were significantly increased during APPL2-RNAi adenovirus conditions in adiponectin-stimulated muscle cells (182). On the other hand, suppressing APPL1 led to increased APPL2-AdipoR1 interactions, blocking the activation of downstream signaling events (182). It was reported that the subcellular localization differs between APPL1 and APPL2, wherein the majority of APPL1 is cytosolic under basal conditions, while APPL2 is plasma-membrane associated (182). The cellular localization was altered on stimulation with adiponectin, promoting cytosolic translocation of APPL2, and membrane translocation for APPL1; a similar effect was observed on treatment with the AMPK activator metformin (182). As such, adiponectin treatment, as well as metformin, induced the dissociation between APPL1-APPL2, which further supports that the regulation of adiponectin signaling heavily relies on the relative expression of APPL isoforms since they exert opposite actions (182). Likewise, APPL1 knockdown in THP-1 cells demonstrated an increase in APPL2 protein expression, further corroborating that APPL2 may be competing with APPL1 for binding with AdipoR1 and AdipoR2 (22). While most AdipoR-dependent pathways are mediated through the interaction with APPL1, the capacity of AdipoR1 and AdipoR2 to reduce cellular ceramide levels, and thereby susceptibility to palmitate-induced cell death (149), instead stems from their intrinsic ceramidase activity (151) (see Fig. 2).

In ob/ob mice, AdipoR1/R2 expression was significantly decreased in skeletal muscle and adipose tissue (183). Downregulated AdipoR1/R2 in skeletal muscle was also coupled with reduced adiponectin binding to the membrane fractions, and stimulation of AMPK (183). Expression levels of AdipoR1/R2 seem to regulate the degree of adiponectin binding and activation of downstream molecules, thereby regulating adiponectin sensitivity (183). Furthermore, overexpression of AdipoR1 or AdipoR2 in skeletal muscle of lean mice affirmed their capacity to increase phosphorylation of downstream effectors, as well as increase GLUT4 expression, while only AdipoR2 overexpression resulted in enhanced PPAR-α expression (184). On the other hand, in high-fat diet–induced obese mice, activation of downstream effectors was attenuated, despite overexpression of either receptor and maintenance or increase of circulating adiponectin levels (184). Nonetheless, AdipoR2 overexpression in obese mice resulted in a significant decrease in weight gain, adipose tissue mass, and inflammation; however, circulating adiponectin levels were increased (184). Therefore, while adiponectin sensitivity seems to be enhanced during overexpression, obesity-induced adiponectin resistance persists, as confirmed by decreased activation of downstream signaling (184). Adiponectin resistance observed in skeletal muscle has also been reported in rodent models, wherein the loss of adiponectin's stimulatory effect led to impaired insulin-induced glucose transport during high-fat feeding (185, 186). Additionally, in human skeletal muscle cells isolated from people with obesity compared to those that are lean, activation of AMPK, glucose uptake, and fatty acid oxidation were blunted despite the presence of globular adiponectin (187). A similar phenotype was observed in globular adiponectin-stimulated cultured muscle cells from people with obesity and T2DM, exhibiting that the inhibition of AMPK signaling was not derived from reduced AdipoR expression (188). Taken together, these findings suggest that downstream adiponectin-induced signaling is blunted in obesity, and often despite moderate levels of cell-surface AdipoR1 and AdipoR2, further strengthening the notion of adiponectin resistance (184).

Adiponectin's third receptor T-cad lacks an intracellular signaling domain, thus it is unclear how adiponectin transduces its signal, and has rather been suggested as an adiponectin-binding protein (141, 189, 190). The prominent association of adiponectin and T-cad was also detected in cell lysates of cultured HUVECs and human aortic SMCs, wherein the loss of adiponectin binding was revealed on siRNA-mediated knockdown of T-cad (191). Likewise, the adiponectin-T-cad association was paramount to protect against neointimal and atherosclerotic plaque formation in vivo (191, 192). Transient overexpression of the 3 adiponectin receptors in HEK293 cells confirmed that human serum adiponectin binds only to T-cad–expressing cells, depicted via native page analysis (193). In addition to this, adiponectin-stimulated exosome production from endothelial cells was shown to uniquely depend on T-cad, rather than AdipoR1 or AdipoR2, and further decreased cellular ceramide levels through ceramide efflux in exosomes (194). Adiponectin similarly decreased ceramide levels in mouse aorta (the most T-cad abundant tissue) in a T-cad–dependent manner (194). Thus, in response to HMW adiponectin, T-cad may regulate cellular ceramide content in a tissue-dependent manner, in addition to the adiponectin-AdipoR axis (194). Furthermore, a murine model of T-cad deficiency showed a causal relationship with endothelial dysfunction, reflected by impaired acetylcholine-induced vasodilation, and reduced NO accumulation compared to wild-type mice (195). Another study demonstrated that, in a model of hind limb ischemia, T-cad–deficient mice phenocopy adiponectin-deficient mice, wherein both mice showed impaired blood flow recovery compared to wild-type controls (196). Additionally, delivery of exogenous adiponectin in these models rescued the impaired revascularization phenotype only in adiponectin-deficient, rather than T-cad–deficient, mice (196). These findings were further supported by an in vitro model, wherein adiponectin's functions were prevented in siRNA-mediated T-cad knockdown conditions in cultured endothelial cells (196). In T-cad–deficient mice, adiponectin was no longer able to bind cardiac tissue, and instead dramatically increased in the circulation, further supporting the importance of the ligand/receptor relationship to activate adiponectin's downstream protective signaling (139). Together, these findings confirm that functional T-cad is essential to mediate the vascular actions of adiponectin (139, 196, 197). Interestingly, in human osteosarcoma cells, transcriptional and posttranscriptional regulation of T-cad was modulated by serum levels of key hormones such as estradiol, and progesterone (198). Therefore, during the postmenopausal period wherein estrogen and progesterone levels decline, reduced T-cad expression may partly explain the presence of accumulated plasma adiponectin levels, since ligand-receptor binding decreases (87, 88, 198).

Immunomodulatory Functions of the Adiponectin–Adiponectin Receptor Pathway

The development and progression of atherosclerotic plaques can be attributed to various risk factors, including numerous modifiable biological risk factors (obesity, diabetes, hypertension, dyslipidemia), lifestyle risk factors (diet, smoking, exercise, pollution, stress, sleep deprivation), as well as nonmodifiable risk factors (age, sex, family history) (199, 200). In early stages of atherosclerosis, physical insults to the vascular wall damage the endothelium (201), wherein circulating LDL can become trapped in the arterial subendothelial space and get oxidized by reactive oxygen species; this will stimulate a chemotactic gradient that will attract immune cells to the site of vascular injury (202). Cellular adhesion molecules will be expressed along the dysfunctional endothelium, which mediate the binding to integrins on the monocyte surface, facilitating their rolling, adhesion, and transmigration properties (201). Specifically, infiltrating monocytes will differentiate into macrophages, mediated by macrophage-colony stimulating factor, followed by their uncontrolled uptake of oxLDL and transformation into foam cells, which they will attempt to efflux through cholesterol exporter ATP-binding cassette transporter A1 (ABCA1) (203). Furthermore, coincubation of apoptotic Jurkat cells with J774 macrophages (in vitro) or with primary resident peritoneal macrophages (ex vivo) leads to an upregulation of macrophage ABCA1 mRNA and protein levels and efflux, highlighting the importance of ABCA1 in mediating apoptotic cellular signaling and efflux (204, 205). Defective efflux of foamy macrophages and impaired efferocytosis, that is, clearance of apoptotic cells, leads to the accumulation of necrotic cells and perpetuates a highly proinflammatory plaque environment (204, 206, 207). Previous reports in human and murine models have demonstrated that foam cells are largely derived from vSMCs that engulf excess oxLDL and populate the plaque core but exhibit reduced expression of ABCA1 compared to macrophages (208, 209). On the other hand, in response to various proinflammatory cues in the atherosclerotic milieu, vSMCs can lose their classic contractile lineage markers, adopt a synthetic phenotype, and thereby gain proliferative and migrating properties while secreting proinflammatory molecules and matrix metalloproteinases (MMPs), further compromising the stability of the plaque (210). Likewise, inflammatory macrophages are a major source of MMP secretion, which can degrade the ECM and further reduce collagen content as well as fibrous cap thickness (211, 212). Collectively, the imbalance between functional macrophage-mediated efflux, efferocytosis, and secretion of proinflammatory signals can destabilize the plaque, lead to its rupture, and underlie subsequent myocardial infarctions and strokes (11). Identifying pathways that can stabilize atherosclerotic plaques to prevent their rupture is crucial to advance the development of therapeutics that can help reduce the risk of dangerous clinical events (11). In this context, adipokines are biologically active factors that exert their functions in an autocrine, paracrine, and endocrine manner, and are highly involved in atherosclerosis (213). Interestingly, a recent review classified the diverse adipokines into 5 categories: protective, deteriorating, dual-acting, indeterminate, and adipose tissue–derived bioactive materials (213). Among protective adipokines is adiponectin, which has been shown to attenuate plaque progression, through its binding to AdipoR1 and AdipoR2 on major vascular and immune cells such as endothelial cells, macrophages, and vSMCs (213).

Anti-Inflammatory Actions of Adiponectin in Atherosclerosis

Experimental studies have shown that adiponectin can protect against many stages of atherosclerotic plaque development, including endothelial dysfunction, macrophage-mediated inflammation, vSMC dedifferentiation, and ECM degradation (214-216). More specifically, while numerous mechanisms can collectively destabilize the plaque and lead to its rupture, the key events include increased expression of adhesion molecules along the endothelium, macrophage-to-foam cell transformation through excessive engulfment of oxLDL, defective efflux and efferocytosis, phenotypic switching between contractile to synthetic vSMC phenotypes, and MMP-mediated degradation of the ECM (6, 216) (Fig. 3). An apoE-KO murine model demonstrated that adenovirus-adiponectin treatment reduces the lesion size through decreased gene expression of vascular cell adhesion molecule-1 (VCAM-1), TNF-α, and class A1 scavenger receptor to prevent further oxLDL uptake (217). In a similar in vivo model, in addition to these markers, a reduction of IL-6 and serum levels of MMP-9 was observed, along with an increase of anti-inflammatory eNOS and IL-10 (218). Furthermore, through the use of an adhesion assay, human aortic endothelial cells that were pretreated with adiponectin inhibited TNF-α–induced THP-1 adhesion, as well as VCAM-1, endothelial-leukocyte adhesion molecule 1, and intercellular adhesion molecule-1 (ICAM-1) expression in a dose-dependent manner (219). Likewise, in HUVECs overexpressing AdipoR1 and AdipoR2 that were pretreated with globular adiponectin, TNF-α–induced VCAM-1 and ICAM-1 mRNA activation was significantly reduced compared to mock-transfected cells (220). Furthermore, ICAM-1 mRNA reduction was lost during siRNA-mediated knockdown of PPAR-α in pretreated HUVECs compared to siRNA control, highlighting the importance of PPAR-α signaling in mediating adiponectin's anti-inflammatory effects on endothelial cells (220). Experiments conducted in bovine aortic endothelial cells affirmed adiponectin's capacity to increase NO production using PI3K-dependent pathways, involving eNOS phosphorylation by AMPK (221). In line with this, APPL1 siRNA significantly attenuated adiponectin-stimulated AMPK phosphorylation and complex formation of eNOS and HSP90 in diabetic mice, thereby reducing NO production and endothelium-dependent vasodilation (178). In male subjects with stable angina, serum CCL2 was identified as an adiponectin-binding protein, wherein plaque burden was positively correlated with CCL2 and negatively with adiponectin (222). The binding interaction between CCL2 and adiponectin was further confirmed in vitro, whereby pretreatment with adiponectin significantly reduced CCL2-induced THP-1 monocyte migratory capacity (222). These findings highlight adiponectin's ability to attenuate endothelial dysfunction, an early hallmark of atherosclerotic plaque development (39).

Figure 3.

Adiponectin receptors are abundantly expressed on monocytes and exert anti-inflammatory macrophage-mediated functions in atherosclerosis. In peripheral blood mononuclear cells isolated from healthy blood donors, adiponectin receptor 1 (AdipoR1) and AdipoR2 were most highly expressed on monocytes, followed by B cells, natural killer (NK)-cells, and T cells. Monocyte-to-macrophage differentiation will occur on entry into the plaque from the circulation, wherein adiponectin can bind to AdipoR1/R2 and perform various anti-inflammatory and atheroprotective functions. Activities between local macrophages include attenuation of uncontrollable oxidized low-density lipoprotein (oxLDL) uptake and necrosis and promoting phagocytosis of apoptotic macrophages. Furthermore, macrophages will also affect other (a)cellular components of the plaque, such as preventing phenotypic switching of contractile to synthetic vascular smooth muscle cells and promoting secretion of tissue inhibitor of matrix metalloproteinase-1 that helps maintain the integrity of the extracellular matrix. Together, these macrophage-specific activities mediated by the adiponectin-AdipoR pathway help prevent the destabilization of atherosclerotic plaques. Created using BioRender.com.

In line with reducing macrophage lipid burden, our previous work revealed that adiponectin significantly increases ABCA1 protein expression and cholesterol efflux from THP-1 macrophages that were treated with cholesterol (223, 224). In fact, using J774 macrophage-like cells, we have demonstrated that circulating adiponectin levels were strongly associated with increased cholesterol efflux capacity in individuals with severe carotid atherosclerosis (225). Furthermore, a robust and independent correlation of adiponectin with cholesterol efflux capacity was likewise observed in an adult cohort, irrespective of BMI and adipose tissue distribution (226). While these studies cannot infer causality, they do nonetheless suggest that adiponectin is an important determinant of cholesterol efflux capacity (225-227). Additionally, in adiponectin-treated murine bone marrow-derived macrophages (BMDMs) that were incubated with apoptotic Jurkat cells, efferocytosis was improved (228). Therefore, studying how adiponectin mediates the interplay between efflux and efferocytosis would further illuminate its atheroprotective role in the ABCA1-efferocytosis axis (229). In the plaque, as lipid influx outweighs efflux, efferocytosis is impaired and cells undergo necrosis; this process fuels the formation of the plaque core, which contains lipids, cytokines, and proteases that can degrade the fibrous cap, making it more prone to rupture (230, 231). To overcome fibrous cap thinning, it was demonstrated in human aortic SMCs that globular adiponectin treatment indeed suppresses vSMC proliferation through AdipoR1- and AdipoR2-dependent pathways (232). RNA sequencing of adiponectin-deficient vSMCs isolated from aortas of male mice likewise revealed a synthetic phenotype and an altered ECM composition in comparison to adiponectin-positive vSMCs (233). The phenotypic switch from a contractile to synthetic phenotype was also accompanied by impaired oxidative phosphorylation, and increased expression of glycolytic enzymes, suggesting that adiponectin-deficient vSMCs preferentially use glycolysis to fuel their proliferation and migration (233). Furthermore, in human monocyte-derived macrophages (hMDMs), adiponectin treatment upregulated IL-10 mRNA and protein, while mediating increased gene and protein levels of tissue inhibitor of MMP-1 (TIMP-1) (215), which reduces MMP activity and plaque instability (234). Coculture experiments that examine whether the crosstalk between macrophages and vSMCs is partly mediated by adiponectin would help connect its role across plaque cells (235).

Role of the Adiponectin–Adiponectin Receptor Pathway in Monocytes and Macrophages

Clinical evidence suggests that high levels of adiponectin are associated with a reduced risk of CV disease (236). However, according to meta-analyses by our group and others, this association is controversial; in individuals with established CV disease, elevated adiponectin levels were positively and significantly associated with a higher risk of all-cause and CV mortality (237, 238). In contrast to studies in healthy individuals, a large prospective multicenter cohort study including patients with first-ever acute ischemic stroke demonstrated that elevated serum levels of adiponectin were similarly associated with an increased risk of major adverse CV and cerebrovascular events, including death (239). Intriguingly, treatment with globular adiponectin in human macrophages from individuals with CAD exhibited a more proinflammatory cytokine profile compared to macrophages from individuals without CAD (240). Similarly, in globular adiponectin-stimulated RAW264.7 macrophages, activation of nuclear factor-κB secretion of TNF-α and IL-6 were observed at earlier time points (70). The expression of AdipoR1/R2 was not reported in this study, making it difficult to draw conclusions as to whether this effect was mediated by modulation of receptor levels and/or their downstream signaling pathways (70). Nonetheless, it has been reported that neutrophil elastase is abundantly colocalized with macrophages in vulnerable plaques (241), which may explain the presence of the globular fragment in inflammatory regions. Moreover, it has been speculated that adiponectin's paradoxical effects may be attributed to the impaired biological response to adiponectin (adiponectin resistance) due to downregulation of the AdipoRs (242). This may be caused by inefficient recycling of the AdipoRs to the membrane and overtargeting to the lysosomal pathway (144), decreasing their functional benefits and leading to pathological changes. However, further research is needed to understand how distinct metabolic and inflammatory phenotypes are linked between circulating adiponectin levels, AdipoR expression, and downstream signaling.

In healthy individuals, AdipoR1 and AdipoR2 are most highly expressed in the monocyte/macrophage lineage (93%) compared to other peripheral blood mononuclear cells, such as B cells, NK cells, and T cells (23) (see Fig. 3). Thus, adiponectin exerts its anti-inflammatory actions during atherosclerotic plaque development largely through its binding to AdipoRs on monocytes/macrophages (25). In lipid-rich atherosclerotic plaques isolated from 4 patients who underwent a carotid endarterectomy, AdipoR1 and AdipoR2 gene expression was more prominent in the lesion areas compared to the healthy carotid zone (243). AdipoR1/AdipoR2 gene expression on monocyte-derived macrophages originated from healthy normolipidemic donors was similar in primary human aortic SMCs and human microvascular endothelial cells, key vascular cells involved in atherosclerosis (243). Notably, AdipoR1 was more abundant than AdipoR2 in primary macrophages, and further decreased on the monocyte-to-macrophage differentiation process, while AdipoR2 was unaffected (243). In contrast, AdipoR1 remained unchanged across THP-1 monocytes, macrophages, and foam cells, while AdipoR2 was gradually reduced (22), which may be attributed to intrinsic differences between THP-1 cells and hMDMs (244). However, similar to primary cells, AdipoR2 is much less expressed at baseline compared to AdipoR1 across THP-1 monocytes, macrophages, and foam cells, whereas T-cad is very minimally expressed in general (22). For this reason, it is of greater interest to study the functions of AdipoR1/R2, rather than T-cad, specifically in the myeloid lineage, due to macrophages’ capacity to reduce lipid accumulation and attenuate inflammatory responses (22). Specifically, it was reported that activation of the AdipoR1-R2-APPL1 axis was essential to transduce adiponectin's signal and reduce macrophage lipid accumulation and foam cell formation in THP-1 cells (22).

While many studies have reported the circulating levels of adiponectin in various CV diseases, the assessment of AdipoR expression in monocytes/macrophages is largely lacking. Of the few studies, plasma adiponectin levels in addition to AdipoR1 and AdipoR2 protein levels were decreased in peripheral monocytes isolated from individuals with CAD who were overweight/obese compared to those without CAD, while no differences were observed for AdipoR1/R2 mRNA levels (245). In line with this, AdipoR1 and AdipoR2 protein levels were decreased in monocytes from individuals with T2DM compared to normal-weight controls, while AdipoR1 and AdipoR2 mRNA levels were elevated in monocytes isolated from participants with obesity and those with T2DM compared to normal-weight controls (246). In contrast, we have demonstrated decreased AdipoR gene expression in circulating monocytes from individuals with severe carotid atherosclerosis treated with statins compared to statin-naive individuals (247). While this finding seems paradoxical given the antiatherosclerotic and anti-inflammatory function of statins, it should be stressed that among individuals treated with statins, significant AdipoR downregulation particularly occurred at higher dosages of atorvastatin (40-80 mg) and rosuvastatin (20-40 mg), while remaining unchanged at low dosages compared to statin-naive participants (247). Therefore, it seems that higher statin dosages compromise AdipoR expression, which was further supported by in vitro analyses in THP-1 macrophages; higher dosages of each statin resulted in elevated proinflammatory cytokine levels, notably TNF-α, IL-6, and IL-1β (247). Previous evidence has shown that statins promote a dose-dependent secretory release of IL-1β from macrophages, largely mediated via activation of the NLRP3 inflammasome (248-250). In addition, in murine and human adipose cells, it was demonstrated that IL-1β is a potent inhibitor of adiponectin production (251). Therefore, it is possible that a statin-induced increase of the NLRP3 inflammasome-IL-1β pathway negatively modulates AdipoR expression. On the other hand, increased monocyte AdipoR1 and AdipoR2 mRNA expression was noted in individuals with multivessel vs single-vessel CAD, despite the use of statins (252). Elevated AdipoR gene expression was also observed in human monocytes from individuals with insulin resistance, while having low plasma adiponectin levels (25). Therefore, according to these studies, AdipoR expression in monocytes/macrophages is differentially regulated in various pathological conditions and depending on statin therapy, thus combining its assessment with other clinical characteristics would provide a more comprehensive understanding of the adiponectin-AdipoR pathway in CV disease. Moreover, studies would also benefit from sex-specific analyses to better understand the biological behavior of adiponectin and the AdipoRs between men and women.

Adiponectin and Adiponectin Receptor 1/Receptor 2 Mediate M1- and M2-like Macrophage Phenotypes

The effect of adiponectin on macrophage polarization was assessed for the first time in 2010 by Ohashi et al (253), who identified the presence of increased proinflammatory M1 and decreased anti-inflammatory M2 peritoneal macrophages isolated from adiponectin-KO mice (Table 1). To corroborate their result, overexpression of adiponectin in wild-type and adiponectin-KO mice further confirmed increased levels of M2 markers in both models (253). While this study showed an increased M2 phenotype in adiponectin-treated hMDMs compared to the vehicle control through elevated IL-10, mannose receptor (MR), and CD163 mRNA levels (253), classic cell surface M1 and M2 markers were not measured through flow cytometry, which would have strengthened their phenotypical characterization. In addition, the contribution of each AdipoR was not considered. Nevertheless, this study provided important biological insights of adiponectin's role in regulating macrophage polarization and prompted further research. Thereafter, a study using hMDMs from healthy volunteers showed that adiponectin treatment in IL-4–primed macrophages further amplified MR levels compared to IL-4 treatment alone through qRT-PCR and flow cytometry (see Table 1) (24). Furthermore, AMPK gene and protein levels were unchanged, while PPAR-α was significantly increased in adiponectin-treated IL-4–primed macrophages compared to IL-4 treatment alone (24). Although this study did not assess AdipoR1 or AdipoR2 mRNA or protein expression (24), these results may suggest a more predominant role for AdipoR2-specific PPAR-α signaling (136) in regulating M2 macrophage polarization. Moreover, although adiponectin could not phenotypically switch from M1 to M2 macrophages, coculture of adiponectin-induced M2-derived media with M1 macrophages reversed the M1 phenotype (24). Through these findings, it seems that ongoing M2 macrophage stimuli are necessary to sustain or revert to an M2 state, highlighting macrophage plasticity (254). Together, these studies provided a first glance at adiponectin's ability in regulating macrophage phenotypic properties, and its potential to promote an M2 anti-inflammatory environment.

Table 1.

Effect of the adiponectin–adiponectin receptor pathway on macrophage polarization in various murine and human cellular models

	Cellular model	Treatment	Marker levels	Methodological assessment
Ohashi et al (253)	Peritoneal macrophages from adiponectin-KO mice compared to wild-type mice	No additional treatment	Increase in M1 (TNF-α, MCP-1, IL-6)	qRT-PCR Western blot*
			Decrease in M2 (Arginase-1*, Mgl-1, IL-10)
	Peritoneal macrophages from wild-type mice	Recombinant adiponectin treatment compared to untreated	Decrease in M1 (TNF-α, MCP-1)
			Increase in M2 (Arginase-1*, Mgl-1, IL-10)
	Human monocyte-derived macrophages	Recombinant adiponectin treatment compared to untreated	Decrease in M1 (TNF-α, MCP-1)
			Increase in M2 (MR, IL-10, CD163)
Lovren et al (24)	Human monocyte-derived macrophages	Adiponectin treatment in IL-4 primed macrophages compared to IL-4 treatment alone	Increase in M2 (MR)	qRT-PCR Flow cytometry
Mandal et al (255)	RAW264.7 mouse macrophages	Scramble siRNA + full-length or globular adiponectin compared to untreated	Increase in M2 (arginase-1, CD36, Mgl-1, Mgl-2, secreted lectins, IL-4Rα, CD206*)	qRT-PCR *Flow cytometry
		AdipoR1-siRNA + full-length adiponectin compared to scramble siRNA + full-length adiponectin	No effect on M2 (arginase-1, Mgl-2, IL-4Rα)
		AdipoR2-siRNA + full-length adiponectin compared to scramble siRNA + full-length adiponectin	Decrease in M2 (Arginase-1, Mgl-2, IL-4Rα)
van Stijn et al (25)	Murine bone marrow-derived macrophages	M1 stimulation compared to control	Decrease in AdipoR1/R2	qRT-PCR
		M2 stimulation compared to control
		M2 stimulation compared to M1	Increase in AdipoR1/R2
		M1 stimulation + recombinant adiponectin treatment compared to untreated	Increase in M1 (TNF-α, IL-6, IL-12p40) and AdipoR1/R2
		M2 stimulation + recombinant adiponectin treatment compared to untreated	Increase in M2 (MR, IL-10) and no changes in AdipoR1/R2
	Mouse peritoneal macrophages	M1 stimulation compared to control	Decrease in AdipoR1/R2	qRT-PCR Western blot
		M2 stimulation compared to control	No change in AdipoR1, increase in AdipoR2
		M2 stimulation compared to M1	Increase in AdipoR1/R2
Galvan et al (228)	Murine bone marrow-derived macrophages coincubated with Jurkat cells	Recombinant adiponectin treatment compared to control	Increase in M2 (MerTK)	Western blot
	Bone marrow-derived macrophages from AdipoR1-KO and AdipoR2-KO mice

Abbreviations: AdipoR1, adiponectin receptor 1; AdipoR2, adiponectin receptor 2; IL, interleukin; KO, knockout; Mgl-1, macrophage galactose N-acetyl-galactosamine-specific lectin-1; MR, mannose receptor; qRT-PCR, quantitative reverse-transcription polymerase chain reaction; siRNA, small-interfering RNA; TNF, tumor necrosis factor.

While the aforementioned research mainly focused on adiponectin's effects during macrophage polarization, the Mandal et al (255) group investigated the adiponectin isoforms and the specific AdipoR that mainly stimulates M2 macrophage activation (see Table 1). Dose-dependent polarization of RAW264.7 mouse macrophages were more efficiently polarized toward M2 through full-length compared to globular adiponectin, in a STAT6- and IL-4–dependent manner (255). Notably, treatment with full-length adiponectin also increased oxidative phosphorylation and lipid oxidation gene expression (255), consistent with the metabolic shift observed in M2 macrophages (256, 257). Furthermore, siRNA-mediated AdipoR1 or AdipoR2 knockdown in RAW264.7 mouse macrophages demonstrated that primarily AdipoR2 was required to shift toward an M2 phenotype (255). More specifically, in response to full-length adiponectin, AdipoR1 siRNA had no effect on M2 markers, while AdipoR2 siRNA reduced M2 marker expression compared to adiponectin-treated scramble siRNA (255). These findings suggest that the adiponectin-AdipoR2 rather than the -AdipoR1 pathway may be regulating M2 macrophage polarization and acting as the main driver in promoting an anti-inflammatory phenotype. Of note, in unstable carotid plaques, which are known to have fewer M2 macrophages (258), we have noted decreased AdipoR2 gene expression and PPAR-α protein expression despite increased adiponectin protein compared to stable carotid plaques, while no association was observed with AdipoR1 (259). Moreover, in AdipoR2/apoE double-KO mice, plaques were more enriched with macrophages and contained a reduced collagen content compared to control apoE-KO mice with functional AdipoR2 (169). Further studies are needed to create mechanistic links between AdipoR2-mediated M2 macrophage polarization, and potential shift toward plaque stabilization.

The van Stijn et al (25) group examined how AdipoR expression is affected in M1- or M2-stimulated murine BMDMs and mouse peritoneal macrophages (see Table 1). Interestingly, promoting an M1 phenotype suppressed AdipoR1/R2 expression compared to control macrophages (25). These findings suggest that a preestablished proinflammatory environment may be inhibiting AdipoR expression. On the other hand, increased or decreased AdipoR1/R2 expression in M2-activated macrophages was noted when compared to M1 macrophages or control macrophages, respectively (25). In this context, increased AdipoR1/R2 mRNA was observed only when comparing an anti-inflammatory to a proinflammatory state, rather than an unpolarized state (25). Furthermore, similar results for AdipoR expression were observed in M1-polarized mouse peritoneal macrophages compared to controls, while in M2-polarized cells, AdipoR1 expression remained unchanged, and AdipoR2 was significantly increased compared to control macrophages (25). However, the control, unpolarized condition is not generally present in vivo since macrophages will rapidly adapt to their immune environment and polarize toward certain phenotypes (260). Further, while there are inherent differences in AdipoR1 and AdipoR2 in M2-polarized mouse macrophages depending on their origin, AdipoR1/R2 consistently decrease on M1 polarization, compared both to control and M2 macrophages (25). Paradoxically, adiponectin-treated M1-polarized BMDMs restored AdipoR levels compared to untreated M1 macrophages, while inducing a proinflammatory response (25). On the other hand, adiponectin-treated M2 macrophages demonstrated unaltered AdipoR1/R2 expression compared to untreated M2, while preserving anti-inflammatory markers MR, and IL-4 (25). Therefore, in this model, adiponectin did not have the ability to reprogram towards M2 from an M1 phenotype, but instead sustained an anti-inflammatory phenotype in M2 macrophages (25). Furthermore, adiponectin-treated murine BMDMs coincubated with apoptotic Jurkat cells exhibited increased mer tyrosine kinase (MerTK) expression and improved phagocytosis (228), important metrics of efficient apoptotic cell clearance. AdipoR1- and/or AdipoR2-KO mice elicited minimal MerTK expression, which was in turn upregulated during adiponectin treatment (228). These results suggest that adiponectin-dependent MerTK expression did not require the presence of AdipoRs; however, studies in hMDMs would provide greater physiological relevance between adiponectin, the AdipoRs, and efferocytosis. There is some literature suggesting a possibility of lipopolysaccharide contamination from preparations of adiponectin (147, 261); hence, this should be considered when interpreting the biological effects that result from recombinant adiponectin treatment in cellular or animal models. The contribution of the adiponectin-AdipoR pathway in promoting specific macrophage phenotypes is still a budding area of research, and further examination will help illuminate their effect on M1-M2 polarization in the plaque environment. However, to date, studies assessing AdipoR1- and AdipoR2-specific functions in macrophage polarization have included only macrophage marker gene and protein characterization using qRT-PCR, Western blotting, and flow cytometry (see Table 1), thereby restricting M1-M2 populations to preselected markers (11). However, with the introduction of single-cell RNA sequencing, transcriptional profiling of atherosclerotic plaques has revealed distinct macrophage phenotypes, often exhibiting overlapping M1 and M2 “signature” markers, highlighting the limitation of employing the M1-M2 paradigm to capture macrophage phenotypic diversity (11, 262). Furthermore, it is also paramount to measure secreted proteins, as macrophages are dynamic cells whose phenotypes are often dictated by paracrine signaling (11). In line with this, using unbiased approaches such single-cell RNA sequencing and single-cell proteomics to examine adiponectin-stimulated AdipoR1 and AdipoR2 siRNA-mediated knockdown conditions would provide a wider scope and deeper characterization of the macrophage phenotypes that are activated or deactivated during AdipoR downregulation.

Harnessing the Adiponectin–Adiponectin Receptor Pathway as a Therapeutic Intervention

While enhancing adiponectin levels in the circulation may be a promising therapeutic strategy to promote plaque stabilization, it is particularly difficult to produce biologically active adiponectin (HMW isoforms) into a viable drug candidate due to 1) its complex multimerization, 2) posttranslational modifications, 3) functional differences between higher-order isoforms, and 4) the very high levels of circulating adiponectin that would necessitate very high levels of compound to be administered via injection given that the molecule would not be absorbable through the gastrointestinal tract (60, 263). As such, designing alternative agonists to activate AdipoR may be more feasible (60, 263). While a meta-analysis reported that statin therapy significantly increased plasma adiponectin levels (264), a residual inflammatory risk remains in patients with CV disease after receiving statins (265). In this context, we have previously demonstrated that individuals with severe carotid atherosclerosis on rosuvastatin had significantly lower plasma total and HMW adiponectin levels, as well as reduced AdipoR1/R2 mRNA expression in monocytes, compared to statin-naive individuals (247). Besides statins, thiazolidinediones (TZDs) such as PPAR-γ agonists pioglitazone and rosiglitazone activate adiponectin promoter activity in adipocytes, which parallels elevated adiponectin levels in the circulation (40, 41, 103, 266-270). Notably, 3-week pioglitazone treatment in wild-type mice reduced neointimal formation, while increasing plasma adiponectin levels and AdipoR2 expression, and suppressing vSMC proliferation (232). Furthermore, 8-week pioglitazone treatment reduced neointimal formation in adiponectin-KO mice at comparable levels to wild-type mice, suggesting that pioglitazone mediates its anti-inflammatory effects via both adiponectin-dependent and adiponectin-independent pathways (232). Similar to pioglitazone, rosiglitazone treatment in hMDMs isolated from healthy donors preferentially activated AdipoR2, but not AdipoR1 expression, in a dose-dependent manner (243). A recent meta-analysis of randomized controlled trials demonstrated that TZDs promote a profound shift in fat redistribution from VAT to SAT in adults, consistent with elevated adiponectin levels, which further explains their insulin-sensitizing mode of action (271). Furthermore, previous evidence demonstrated an improved inflammatory and lipid profile with a combined regimen of TZDs and statins, rather than monotherapies in individuals with various cardiometabolic diseases (272-274). Specifically, combined therapy of pioglitazone and simvastatin significantly reduced CRP levels in individuals with CV disease (272); meanwhile, in individuals with MetS, combined simvastatin/rosiglitazone therapy likewise demonstrated a greater reduction of high-sensitivity CRP compared to the simvastatin/placebo group, while adiponectin levels increased in the combination compared to placebo group (274). However, it must be noted that PPAR-mediated side effects, such as weight gain, fluid retention, edema, hepatotoxicity, and heart failure prevent their wide clinical use (275, 276). Interestingly, a reporter-based high-throughput screening (HTS) assay conducted in insulin-resistant 3T3-L1 preadipocytes identified 6 (out of 100 000) small-molecule compounds that promote adiponectin production without PPAR-γ activity (275). Due to the orally bioactive nature of small molecules (275), this may be a favorable therapeutic approach for the treatment of obesity-linked CV diseases. Furthermore, selective PPAR-γ modulators may demonstrate an improved safety profile by avoiding the adverse reactions of TZDs (277). To this end, INT131 is a novel non-TZD compound that increases adiponectin levels, in addition to improving glycemic control and insulin sensitivity in individuals with T2DM without typical TZD side effects (278-280). In this context, it would be interesting to investigate the role of INT131 in mouse models of atherosclerosis to understand its activity on plaque cells and its potential to reduce proinflammatory mechanisms (277).

Beyond the activation by adiponectin alone, the first orally active small-molecule AdipoR agonist, AdipoRon, was identified to bind both AdipoR1 and AdipoR2 and activate their downstream signaling pathways, AMPK and PPAR-α, respectively (281). AdipoRon treatment in obese mice improved insulin sensitivity and glucose homeostasis, which was abolished in AdipoR1/R2 double-KO mice (281). In primary neurons isolated from pregnant C57BL6J mice subjected to oxygen-glucose deprivation, administration of either adiponectin or AdipoRon was protective against cerebral injury mediated through AdipoR1/AMPK signaling (282). Furthermore, in line with adiponectin-AdipoR–induced internalization (144), administration of AdipoRon in vSMCs from mice similarly increased the expression of lysosomal markers (283). Likewise, oral AdipoRon administration in male mice fed a high-fat diet improved sperm motility and activated AMPK in the testis, highlighting AdipoRon's activation of the AdipoR1 signaling axis (165). In AdipoR-humanized mice on a high-fat diet, generated by cross-breeding muscle-specific human AdipoR1 transgenic mice and AdipoR1-R2-double KO mice, AdipoRon indeed activated AMPK and augmented peroxisome proliferator-activated receptor-gamma coactivator (PGC)-1α, mitochondrial-related gene expression and oxidative stress-detoxification, leading to overall improved glucose tolerance and insulin sensitivity (284). Similarly, male diabetic mice fed a diet containing AdipoRon compared to regular chow diet ameliorated M1-like macrophage inflammation, insulin resistance, fibrosis, and apoptosis in the heart (285). The ability of AdipoRon to regulate oxidative stress, inflammation, and apoptosis was mediated through increased AdipoR expression and ceramidase activity, thereby reducing ceramide levels and increasing S1P levels, as well as activating AMPK-PPARα/PGC-1α pathways (285). To date, there are no studies investigating AdipoRon administration in atherosclerotic (eg, apoE- and/or LDL-receptor-KO) murine models (286), which would further deduce its potential in attenuating plaque progression and instability. However, despite AdipoRon's efficacy in improving insulin sensitivity and glucose tolerance in high-fat conditions, its low aqueous solubility hampers its therapeutic potential (287). In this context, one method to enhance the solubility of lipophilic drugs is the covalent modification with PEG, which was used by one group to generate AdipoRonPEG₅ (287). Notably, compared to AdipoRon, AdipoRonPEG₅ treatment activated the AdipoR1-AMPK pathway, and more profoundly reduced ceramide levels, accompanied by antiapoptotic metabolite S1P formation in the liver of high-fat diet–induced obese mice (287). These results were consistent with previous reports that showed adiponectin's cytoprotective effects are mediated via activation of AdipoR ceramidase (149, 150). This study highlighted that enhanced pharmacokinetics can be achieved with AdipoRon derivatization and may further improve tissue-specific targeting (287).

Another orally active adiponectin mimetic was discovered and characterized, 6-C-β-D-glucopyranosyl-(2S,3S)-(+)-57,3′,4′-tetrahydroxydihydroflavonol (GTDF), which, similarly to AdipoRon, has a higher affinity for AdipoR1 compared to AdipoR2 (281, 288). In line with this, administration of GTDF into AdipoR-expressing diabetic mice improved their metabolic health, but they ultimately lost these beneficial effects in AdipoR1-deficient conditions (288). Another protein that was found to be structurally and functionally comparable to adiponectin is osmotin, a novel phytohormone that can exert therapeutic effects during obesity, diabetes, fatty liver disease, vascular inflammation, and atherosclerosis (60, 289-293). Similar to AdipoRon and GTDF, osmotin preferentially functions as an agonist for AdipoR1 (290, 294) and suppresses the M1 proinflammatory phenotype in THP-1 macrophages through decreased expression of TNF-α and IL-6, reduced oxLDL foam cell formation, and attenuated development of atherosclerotic lesions in apoE-KO mice (293). Likewise, using an in vitro oxygen and glucose deprivation/reperfusion injury model of rat cardiac myoblast H9c2 cells, osmotin significantly increased the secretion of anti-inflammatory and reduced proinflammatory factors; this effect was preferentially mediated by the AdipoR1/PI3K/Akt signaling pathway (292). Additionally, arterioles from individuals with CAD treated with osmotin resulted in a partial restoration of NO, the mediator of flow-induced dilation, which was inhibited during AdipoR1 silencing (179). Furthermore, a mouse model with Alzheimer disease delivered with osmotin-loaded magnetic nanoparticles demonstrated reduced neurological deficits (295). To this end, it would be interesting to measure the efficacy of this magnetic drug delivery approach to evaluate osmotin's potential as a therapeutic drug candidate in the treatment of atherosclerosis (263).

Emodin is a natural anthraquinone derivative present in many Chinese medicinal herbs that can exert numerous beneficial biological activities, including cardioprotective, lipid-modulating, and anti-inflammatory effects (296, 297). Similar to adiponectin's properties, emodin can attenuate several proatherosclerotic processes by regulating the functions of crucial plaque cells, such as endothelial cells, vSMCs, and macrophages (296-298). Of note, in rat aortic SMCs, emodin inhibited TNF-α–induced migration, proliferation, and suppressed mRNA and protein expression of MMP-2 and MMP-9 (299), which are collectively major plaque-destabilizing features (3, 11). Likewise, in apoE-KO mice fat-fed for 13 weeks, treatment with emodin modulated plaque composition; it reduced the lipid core, lipid-to-collagen content, and MMP-9 expression, and increased PPAR-γ protein (300). However, emodin's fast elimination and low bioavailability in vivo, in addition to the presentation of severe side effects, make it unsuitable for clinical application (297, 301, 302). To this end, a study explored the efficacy of a novel anthraquinone compound, emodin succinate monoethyl ester (ESME), to antagonize AdipoR2 and reduce lipogenesis, as well as promote fatty acid oxidation in a hamster and murine model of metabolic fatty liver disease (303). Interestingly, oral administration of ESME outperformed atorvastatin and emodin in the treatment of liver steatosis in hamsters fed a high-fat diet (303). Furthermore, ESME likewise attenuated hepatic steatosis in an AdipoR2-dependent manner in apoE-KO compared to wild-type mice fed a high-fat and normal diet, respectively (303). While this study confirmed that ESME activated AdipoR2 in hepatocytes (303), it would be interesting to assess whether the ligand-receptor activity is present in macrophages, and thereafter evaluate its plaque-stabilizing effects. While the structure of ESME improves its therapeutic potential (303), whether this oral AdipoR2 agonist can improve CV disease outcomes remains to be investigated.

To further discover novel compounds that agonize AdipoR1 and AdipoR2, a fluorescence polarization-based HTS assay was developed using a 10 000-natural compound library to identify adiponectin ligands (304). The lead compounds were tested in vitro across cancer cell lines to confirm their agonistic activity where 4 natural products upregulated each AdipoR; matairesinol, arctiin, (−)-arctigenin, and gramine for AdipoR1, and parthenolide, taxifoliol, deoxyshizandrin, asyringin for AdipoR2 (304). Evaluating their effect in atherosclerotic immune cells, particularly macrophages, would showcase their potential in attenuating perpetual plaque inflammation. In line with previous evidence that suggests the role of AdipoR2 in M2 macrophage polarization (255) and in-plaque instability (259), it may be useful to identify agonists with higher affinity for AdipoR2 rather than AdipoR1, to ultimately test whether selectively upregulating AdipoR2 can reduce plaque inflammation and promote plaque stability. In line with this, current progress in the field has showcased the development of an HTS protocol that was used to evaluate compounds that can shift M1-polarized macrophages derived from healthy donors toward M2 and vice versa, including bioactive, FDA-approved drugs and natural products (305). While selected compounds from their HTS were used to assess the effect in tumor-activated macrophages (305), testing the identified M2-activating compounds in hMDMs from individuals with CV disease would provide important insights for potential treatment. Bridging the 2 concepts together, it would be of interest to examine whether M2-activating compounds function through an AdipoR1- and/or AdipoR2-specific mechanism in hMDMs. This would offer another validation tool to show whether activation of AdipoR1 and/or AdipoR2 can shift the balance from a proinflammatory to anti-inflammatory, proresolving immune environment. Overall, HTS assays against various compound libraries are a rich source to identify anti-inflammatory agents suitable for the prevention and treatment of CV diseases and provide an important avenue to discover which FDA-approved drugs can be repurposed for CV indications (306). Drug repurposing compared to traditional drug discovery offers several key advantages, including decreased research and development costs and time to market, as well as increased access to existing clinical efficacy and safety profiles (307). Furthermore, along with the importance of studying plaque cells in tandem, cost- and time-effective coculture models were created to reflect early and intermediate stages of atherosclerosis by combining endothelial cells, vSMCs, and macrophages (235). Leveraging coculture models that mimic plaques in vivo further presents an opportunity to provide a more holistic overview of how AdipoR-targeted drug therapies would mediate the crosstalk between vascular and immune components (235, 308).

More recently, an AdipoR-activating monoclonal antibody (AdipoRaMab) was produced by immunizing AdipoR-KO mice with human AdipoR-expressing cells (309). In high-fat diet–fed mice, AdipoRaMab indeed activated the AdipoR1-AMPK and AdipoR2-PPAR-α pathways in skeletal muscle and liver, respectively, and thereby ameliorated glucose tolerance and insulin sensitivity (309). Along with low medication adherence for oral therapies (310), the design of agonistic antibodies against AdipoR1 and AdipoR2 with a long enough half-life provides a potential therapeutic avenue for a once-monthly dosing regimen through their humanization (309). Moreover, with the availability of AdipoR crystal structures (311), computational techniques can be leveraged to identify membrane receptor protein-peptide interactions using surface plasmon resonance and virtual screening (312). Considerations of homodimer and heterodimer AdipoR activity at the cell surface must be considered to better understand ligand-receptor internalization, and recycling back to the surface for continued function (144). Therefore, it is important to also assess the cellular dynamics and compartmentalization of drug-AdipoR interactions to determine whether the desired phenotype is sustained over time and does not plateau due to lack of AdipoR recycling. Furthermore, it is equally essential to analyze how agonistic small molecules and/or antibodies that target AdipoR1/R2 affect age- and/or sex-specific groups, since previous research has demonstrated that adiponectin levels vary among males and females, and across the lifespan (313).

Conclusions

Adiponectin has demonstrated its promise as a potent anti-inflammatory, antiatherogenic factor across various CV disorders (39, 314), while cellular AdipoR1/R2 expression has proven essential for maintaining many vital functions (23, 147, 170, 171). Furthermore, adiponectin-AdipoR–induced signaling promotes specific macrophage responses, which influence various features of the atherosclerotic plaque (ie, inflammatory cell counts and activity [macrophages, SMCs], ECM remodeling), ultimately affecting its stability (25, 169, 259). Previous evidence demonstrated that the adiponectin-AdipoR2 pathway can modulate macrophage polarization by mediating the shift toward an anti-inflammatory M2-like phenotype (24, 25, 255), which could have major implications for plaque stabilization. However, leveraging the beneficial effects of adiponectin-induced AdipoR signaling is more precarious due to the difficulty in producing stable multimeric recombinant adiponectin isoforms (263). For this reason, the discovery of novel ligands that can target AdipoR and be translated into viable drug candidates need to first be evaluated in preclinical models to rule out any unfavorable side effects and rule in a favorable, anti-inflammatory profile; this will be essential to provide new avenues to optimize the medical management of atherosclerosis.

Abbreviations

ABCA1

ATP-binding cassette transporter A1

AdipoR1

adiponectin receptor 1

AdipoR2

adiponectin receptor 2

AdipoRaMab

AdipoR-activating monoclonal antibody

AMPK

5′ adenosine monophosphate-activated protein kinase

apoE

apolipoprotein E

APPL1

PTB domain and leucine zipper motif 1

BMDMs

bone marrow-derived macrophages

BMI

body mass index

CAD

coronary artery disease

C/EBP

CCAT/enhancer-binding protein

CRP

C-reactive protein

CV

cardiovascular

EAT

epicardial adipose tissue

ECM

extracellular matrix

eNOS

endothelial nitric oxide synthase

ER

endoplasmic reticulum

ESME

emodin succinate monoethyl ester

FDA

US Food and Drug Administration

GDM

gestational diabetes

GLUT

glucose transporter

GPCRs

G protein–coupled receptors

GTDF

6-C-β-D-glucopyranosyl-(2S,3S)-(+)-57,3′,4′-tetrahydroxydihydroflavonol

hMDMs

human monocyte-derived macrophages

HMW

high-molecular-weight

HSC

hematopoietic stem cell

HTS

high-throughput screening

HUVECS

human umbilical vein endothelial cells

ICAM-1

intercellular adhesion molecule-1

IL

interleukin

IR

insulin receptor

KO

knockout

LGA

large-for-gestational age

LMW

low-molecular-weight

MAPK

mitogen-activated protein kinase

MASH

metabolic dysfunction–associated steatohepatitis

MASLD

metabolic dysfunction–associated steatotic liver disease

MerTK

mer tyrosine kinase

MetS

metabolic syndrome

Mgl-1

macrophage galactose N-acetyl-galactosamine-specific lectin-1

MMPs

matrix metalloproteinases

MMW

middle-molecular-weight

MR

mannose receptor

mRNA

messenger RNA

oxLDL

oxidized low-density lipoprotein

PA

palmitic acid

PI3K/Akt

phosphoinositide-3-kinase/protein kinase B

PPAR

peroxisome proliferator-activated receptor

PVAT

perivascular adipose tissue

qRT-PCR

quantitative reverse-transcription polymerase chain reaction

S1P

sphingosine-1-phosphate

SAT

subcutaneous adipose tissue

siRNA

small-interfering RNA

T2DM

type 2 diabetes

T-cad

T-cadherin

TNF

tumor necrosis factor

TZDs

thiazolidinediones

VAT

visceral adipose tissue

VCAM-1

vascular cell adhesion molecule-1

vSMC

vascular smooth muscle cell

Funding

This work was supported by the Canadian Institutes of Health Research (PJT-148966, SVB-145589) and the Heart and Stroke Foundation of Canada (G-17-0018755). S.S.D. is a Senior Clinician-Scientist supported by a Fonds de recherche du Québec-Santé Senior salary award. C.S.M. reports grants through his institution from Merck, Massachusetts Life Sciences Center, and Boehringer Ingelheim.

Disclosures

C.S.M. reports personal consulting fees and support through his institution from Ansh Inc; collaborative research support from LabCorp Inc; personal consulting fees from Nestle, Olympus, Genfit, Lumos, Novo Nordisk, Amgen, Corcept, Intercept, 89 Bio, Madrigal, Aligos, Esperion, Biodexa, and Regeneron; educational activity meals through his institution or national conferences from Esperion, Merck, and Boehringer Ingelheim; and travel support and fees from UptoDate, TMIOA, Elsevier, and the Cardio Metabolic Health Conference. None is related to this manuscript. All other authors declare that they have no conflicts of interest.