Once, these pro-inflammatory mediators are released, they induce tissue-specific inflammation due to which IR in peripheral tissues and impaired insulin secretion in pancreatic islets occur that ultimately lead to overt T2DM. Adapted from Akash et al. Development of IR is one of the major hallmark for pathogenesis of T2DM.

To control the propagation of IR is one of the most important targeted treatment. For the development of IR, several factors are involved Fig.

Several treatment strategies have been used to overcome the development of IR. The most important ones have been described here in the following sub-sections. Interleukin-1 receptor antagonist IL-1Ra is naturally occurring anti-inflammatory cytokine of interleukin-1 family.

It competitively binds with IL-1RI and prevent the binding of IL-1β and antagonizes its effects. It has been evidenced from several experimental studies that imbalance between IL-1Ra and IL-1β generates inflammation in various parts of the body where IL-1RI is present [ 4 , 12 ].

Moreover, it has also been found that expression of IL-1Ra is strongly correlated with the development of IR, impaired insulin secretion and T2DM [ 4 , ].

Treatment of human recombinant IL-1Ra improves normoglycemia, insulin sensitivity in adipose and peripheral tissues, and insulin secretion from β-cells of pancreatic islets impairs [ 31 , , ].

This is one of the most important treatment strategy that anti-inflammatory agent might indeed prevent the development of IR and improves glycemia.

One of the main shortcoming of IL-1Ra is its short biological half-life and to overcome this problem, high doses with frequent dosing intervals are required to achieve desired therapeutic effects.

To overcome this problem, several treatment strategies have been applied to prolong the biological half-life and therapeutic effects of IL-1Ra [ 29 ]. Salicylates are an important class of anti-inflammatory agents.

They are used in variety of inflammatory diseases and syndromes. Inflammation plays a crucial role for the development of IR and T2DM, therefore, by using salicylates as an alternate treatment strategy, it has been found that salicylates can imporve insulin sensitivity via inhibition of NF-κB and IKKβ [ 82 ] and glucose tolerance [ , ].

In the above sections, it has been briefly described that TNF-α is one of the most important pro-inflammatory mediator that is responsible to induce IR in adipocytes and peripheral tissues.

Inhibition of TNF-α production might be one of the choice to prevent the development of IR and pathogenesis of T2DM [ 4 ]. Recently, infliximab has been demonstrated to improve insulin signaling and inflammation especially in the liver in rodent model of diet-induced IR [ ].

Similarly, using anti-TNF-α antibodies also improve the insulin sensitivity in peripheral tissues [ ]. Lo et al. demonstrated that etanercept therapy can also improve total concentration of adiponectin which is anti-inflammatory adipokine and improved insulin sensitivity [ ].

Keeping in view the decisive role of TNF-α in pathogenesis of IR, several anti-TNF-α treatment strategies have been utilized to prevent the pathogeneis of IR and development of T2DM.

Similarly, anti-TNF-α treatment has also shown to prevent the IR in Sprague—Dawley rats [ ] while neutralization of TNF-α also prevented IR in hepatocytes [ ]. Few controversial studies have also demonstrated that using TNF-α blockade has no effect on IR [ ] which indicates that TNF-α blockade is not a treatment of choice as its production is dependent on the generation of IL-1β and activation of various transcriptional mediated pathways.

It has been thought that chemokines activately participate in the development of IR by potentiating the inflammation in adipocytes. Moreover, genetic inactivation of these chemokine signaling [ 52 , 53 , ] or inhibition of their axis [ , ] by pharmacological approaches have been shown to improve the insulin sensitivity in adipocytes and peripheral tissues.

ER stress, as mentioned in the above sections, is a key link between IR and T2DM [ ]. Blockade of ER stress is one of the treatment option to prevent the development of IR and pathogenesis of T2DM.

In the recent years, various pharmaceutical chaperones, notably endogenous bile acids and the derivatives of these bile acids such as ursodeoxycholic acid UDCA , 4-phenyl butyric acid PBA have been investigated that have proven to have the ability to modulate the normal functioning of ER and its folding capacity [ 28 ].

Ozcan et al. The results of this study indicated that UDCA significantly improved insulin sensitivity and normoglycemia. Thiazolidinediones also known as glitazones, are one of the most important insulin sensitisers.

They are the agonists of peroxisome proliferator-activated receptors-gamma PPARγ. It has been found that thaizolidinediones have the ability to improve insulin action and decrease IR [ , ].

Inflammatory responses are induced through the activation of various pro-inflammatory and oxidative stress mediators via involment of various transcriptional mediated pathways.

To stop the inflammatory responses in IR development is one of the key treatment strategy. In this areticle, we have comprehensively highlighted the up-to-date scientific knowlesge of role of inflammatory responses in IR development and its treatment strategies. IR plays a crucial role for the pathogenesis and development of T2DM and its associated complicaitons.

Based on the findings mentioned in above sections, anti-inflammatory treatment strategies are one of the best choice to prevent the the pathogenesis of IR, but the studies conducted to investigate the role of anti-inflammatory strategies for the prevention of IR are still in their beginning stages and need to be focused further in future studies for more better and improved clinical outcomes.

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These findings suggest that insulin-resistant visceral WAT, via MCP1 expression, recruits monocytes, which then differentiate into M1 macrophages. A Adipokine array of eWAT from HFD-fed AdRiKO and control mice.

Immunoblots show the reduction of RICTOR expression and mTORC2 signaling. B MCP1 protein levels in eWAT from HFD-fed AdRiKO and control mice. C MCP1 protein levels in plasma from HFD-fed AdRiKO and control mice. D Ccr2 mRNA levels in SVCs isolated from eWAT of HFD-fed AdRiKO and control mice.

Mice were fed a HFD for 8 weeks and treated with a control or MCP1-neutralizing antibody for 2 weeks with ongoing HFD feeding. F Percentage of inflammatory monocytes in peripheral blood mononuclear cells PBMCs. Mice were treated as in E. We next tested whether the increase in MCP1 is responsible for M1 macrophage accumulation in AdRiKO eWAT.

Mice were fed a HFD for 8 weeks and then treated with an MCP1-neutralizing or control antibody for 2 weeks along with ongoing HFD feeding.

The antibody treatments had no impact on body weight Supplemental Figure 7B. Thus, MCP1 appears to mediate the increase in M1 macrophages in AdRiKO eWAT. Altogether, our data suggest that mTORC2 inhibition in WAT results in Mcp1 expression, followed by infiltration of monocytes in an MCP1-CCR2—dependent manner.

Expression of the Mcp1 gene was increased in the eWAT of HFD-fed AdRiKO and i-AdRiKO mice compared with expression levels in control eWAT Figure 5, A and B , suggesting that MCP1 is upregulated in AdRiKO WAT at the transcriptional level.

Furthermore, we note that the increase in Mcp1 expression Figure 5A coincided with an increase in the number of M1 macrophages in AdRiKO eWAT Figure 2F.

The number of macrophages and expression levels of Mcp1 were unchanged in AdRiKO and control eWAT in ND-fed mice Supplemental Figure 3 and Supplemental Figure 8A. To identify the cells in which Mcp1 expression was induced, we measured Mcp1 mRNA levels in adipocytes and SVCs isolated from eWAT of HFD-fed AdRiKO and control mice Supplemental Figure 2B.

AdRiKO adipocytes, but not SVCs, showed increased Mcp1 expression Figure 5, C and D. To determine whether the regulation of Mcp1 transcription by mTORC2 is cell autonomous, we first treated 3T3-L1 adipocytes with the mTOR inhibitor torin 1 Next, we generated 2 Rictor -knockout 3T3-L1 adipocyte cell lines Figure 5F and Supplemental Figure 8B using the genome-editing CRISPR-Cas9 system Rictor- knockout 3T3-L1 adipocytes were able to differentiate, albeit at a slower rate compared with control cells Supplemental Figure 8C.

Consistent with our in vivo data, Mcp1 expression was increased in the Rictor -knockout 3T3-L1 adipocytes Figure 5G and Supplemental Figure 8B. Serum and insulin treatment suppressed Mcp1 expression in control but not Rictor- knockout 3T3-L1 adipocytes Figure 5H and Supplemental Figure 8D.

In WT mice, Mcp1 expression increased by 10 weeks, but not 4 weeks, of HFD feeding Supplemental Figure 8E.

These data support the notion that insulin resistance precedes and promotes Mcp1 transcription in adipocytes. We note that Rictor knockout in liver LiRiKO did not result in hepatic Mcp1 expression Supplemental Figure 8F , consistent with our above finding that LiRiKO failed to stimulate inflammation in liver.

A Mcp1 mRNA levels in eWAT from AdRiKO and control mice during the HFD time course. B Mcp1 mRNA levels in eWAT from HFD-fed i-AdRiKO and control mice. C and D Mcp1 mRNA levels in adipocytes C and SVCs D isolated from eWAT of HFD-fed AdRiKO and control mice.

E Mcp1 mRNA levels in 3T3-L1 adipocytes treated with DMSO or nM torin 1 for 6 hours. F 2DGP accumulation in insulin-stimulated Rictor -knockout or control 3T3-L1 adipocytes treated with DMSO or the JNK inhibitor SP 20 μM.

G Mcp1 mRNA levels in Rictor -knockout and control 3T3-L1 adipocytes. H Mcp1 mRNA levels in Rictor -knockout and control 3T3-L1 adipocytes treated with or without serum and insulin. I Mcp1 mRNA levels in Rictor -knockout and control 3T3-L1 cells treated with DMSO or the JNK inhibitor SP 20 μM for 6 hours.

J Immunoblots of Rictor -knockout and control 3T3-L1 adipocytes treated with DMSO or the JNK inhibitor SP 20 μM for 6 hours. K In vitro JNK kinase assay. Active JNK was immunoprecipitated from Rictor -knockout or control 3T3-L1 adipocytes, and JNK activity was assessed toward its substrate cJUN.

SP treatment served as a negative control. How does mTORC2 loss lead to Mcp1 transcription? It has been suggested that JNK is required for MCP1 expression and secretion in cultured 3T3-L1 adipocytes Consistent with that report, treatment with the JNK inhibitor SP reduced Mcp1 expression in Rictor -knockout 3T3-L1 cells Figure 5I.

Inhibition of JNK was confirmed by loss of cJUN Ser73 phosphorylation Figure 5J. SP did not restore AKT Ser phosphorylation Figure 5J or insulin-stimulated glucose uptake Figure 5F in the Rictor- knockout 3T3-L1 cells, indicating that the effect of the drug was independent of mTORC2 and insulin resistance.

Furthermore, JNK activity was unaffected by Rictor knockout Figure 5, J and K. Thus, mTORC2 and JNK control Mcp1 expression independently. MCP1 mRNA levels in omental WAT oWAT correlate with BMI in obese human subjects 9 , However, how MCP1 transcription is regulated in human adipose tissue is unknown.

To this end, oWAT samples were collected from 20 lean and 30 obese human patients who were under general anesthesia Figure 6A and Supplemental Table 2. The obese patients were insulin resistant as determined by high homeostatic model assessment of insulin resistance HOMA-IR Figure 6B and Supplemental Figure 9A.

Nevertheless, in oWAT, AKT2 Ser phosphorylation, a readout for mTORC2 signaling, was lower in obese patients than in lean patients Figure 6, C and D. AKT2 Ser phosphorylation negatively correlated with BMI Figure 6E. MCP1 expression was higher in obese subjects and positively correlated with BMI Figure 6, F and G , and Supplemental Figure 9B.

CD68 expression was also higher in the obese subjects and positively correlated with BMI Figure 6, H and I , and Supplemental Figure 9C. MCP1 and CD68 expression levels also correlated Figure 6J.

However, approximately one-third of the obese patients 9 of 30 had low AKT Ser phosphorylation and high HOMA-IR, MCP1 , and CD68 Figure 6K , suggesting that AdRiKO mice may phenocopy this subgroup of patients. A and B BMI and HOMA-IR of lean and obese patients.

See also Supplemental Table 2. C Representative immunoblots for p-AKT2 Ser and AKT2 in human oWAT. D Quantification of p-AKT2 Ser normalized to total AKT2. F MCP1 mRNA levels in human oWAT. G MCP1 positively correlated with BMI.

H CD68 mRNA levels in human oWAT. I and J CD68 positively correlated with BMI I and MCP1 levels J. Differentiated human primary adipocytes were treated with DMSO or nM torin 1 for 6 hours.

We provide 2 lines of evidence that insulin resistance promotes the accumulation of M1 macrophages and thereby fosters inflammation Figure 7. First, we show that knockout of mTORC2, i. As a consequence, monocytes were recruited to visceral WAT, where they differentiated into M1 macrophages and caused inflammation.

Second, HFD-induced insulin resistance in WT mice preceded the accumulation of proinflammatory M1 macrophages. We also show that oWAT from obese, insulin-resistant patients had low mTORC2 signaling, high MCP1 expression, and high macrophage content, suggesting that our findings in mice have human relevance Figure 6.

Insulin resistance causes inflammation in adipose tissue. MCP1 in turn recruits monocytes and activates proinflammatory M1 macrophages. Our findings are consistent with observations made in mice genetically modified in other components of the insulin signaling pathway. Two studies demonstrated that deletion of PTEN or PIK3R1, negative regulators of insulin signaling, causes enhanced insulin sensitivity and a reduced number of macrophages in adipose tissue 51 , More recently, Shearin et al.

Obesity induces insulin resistance, via a yet-to-be defined mechanism, which in turn promotes inflammation. As suggested previously 16 , this inflammation may contribute to adipose tissue remodeling and expansion to maintain glucose hemostasis. It has been suggested that activated M1 macrophages undergo metabolic reprogramming from oxidative phosphorylation to glycolysis 54 , Thus, a physiological role of M1 macrophages could be to clear excess local glucose.

Our finding that insulin resistance precedes inflammation may account for the observation that inhibition of TNF-α is ineffective in the treatment of obesity-induced insulin resistance 18 — HFD-fed AdRiKO mice showed reduced AKT Ser phosphorylation Figure 1B , decreased glucose uptake Figure 1A , and extensive inflammation Figure 2 in eWAT.

HFD-fed WT mice also displayed decreased glucose uptake Figure 3, A—C , which was followed by mild inflammation Figure 3D. Unexpectedly, AKT Ser phosphorylation was not reduced in WT mice fed a HFD for 4 or 10 weeks Supplemental Figure 5, E and F 56 , although we still observed mild inflammation in eWAT by week 10 of HFD feeding.

We note that the number of M1 macrophages in AdRiKO eWAT was much higher than that in control eWAT by week 10 of the HFD Figure 2F. These findings suggest that obesity-induced insulin resistance promotes mild inflammation downstream or independently of mTORC2, whereas chronic insulin resistance leads to mTORC2 inhibition and therefore extensive inflammation, as observed in AdRiKO mice and obese patients.

Our experiments reveal that loss of mTORC2 leads to increased Mcp1 expression in adipocytes Figure 5. What is the downstream effector through which mTORC2 controls Mcp1 transcription? One candidate is the phosphatidic acid phosphatase LIPIN1, whose knockdown results in Mcp1 expression in 3T3-L1 adipocytes LIPIN1, independently of its phosphatase activity, also functions as a transcriptional repressor We found that Lipin1 expression was reduced in Rictor -knockout eWAT and adipocytes Supplemental Figure 10, A and B , suggesting that mTORC2 may negatively control Mcp1 transcription via LIPIN1.

An adipose-specific transcription factor is another candidate through which mTORC2 may control Mcp1 expression. Rictor knockout increased Mcp1 expression in adipocytes, but not in liver or fibroblasts Supplemental Figure 8F and Supplemental Figure 10C , indicating that the regulation of Mcp1 transcription by mTORC2 is specific to adipocytes.

Why does AdRiKO eWAT accumulate a disproportionately high number of M1 macrophages only in response to obesity? The increase in Mcp1 transcription in mTORC2-knockout adipocytes required JNK activity Figure 5I , which is high only in WAT from obese mice 59 , Thus, obesity might be a prerequisite for JNK activation, which in turn stimulates Mcp1 expression in conjunction with loss of mTORC2.

We note that mTORC2 did not control JNK Figure 5, J and K. In summary, we propose that obesity-mediated insulin resistance is a cause of inflammation in visceral WAT.

Although our findings do not rule out the possibility that inflammation promotes insulin resistance in other tissues or conditions, they bring into question whether antiinflammation therapy in adipose tissue is an effective strategy in the prevention of type 2 diabetes.

Adipose tissue—specific Rictor -knockout AdRiKO and liver-specific Rictor -knockout LiRiKO mice were described previously 32 , 35 , For experiments with AdRiKO mice, age-matched Cre -negative males were used as controls.

For experiments with LiRiKO and i-AdRiKO mice, Cre -negative littermate male mice were used as controls. For i-AdRiKO mice, Rictor knockout was induced by i. The HFD-feeding experiment was conducted for 10 weeks unless otherwise specified. Cre -negative animals were used as a control. For i-AdRiKO mouse experiments, control mice were also treated with tamoxifen.

Mice were randomly assigned for each experiment. Only male, 6- to week-old mice were used for experiments. Cell culture.

For differentiation, cells were maintained in M1 medium for 2 days after reaching confluence. The medium was replaced with M2 medium M1 medium supplemented with 1.

After 2 days, the medium was replaced with M3 medium M1 with 1. The medium was replaced with M2 on day 4 of differentiation. From day 6, cells were maintained in M3 with a medium change every 2 days.

For all experiments, cells differentiated for 10 to 14 days were used. Torin 1 was purchased from Tocris Bioscience and dissolved in DMSO. Human oWAT biopsies.

Patients were fasted overnight and then underwent general anesthesia. All oWAT samples were obtained between and , snap-frozen in liquid nitrogen, and stored at —80°C. Human primary adipocytes. Human primary visceral preadipocytes from a year-oldwoman with a BMI of 23 were obtained from Lonza.

After 14 days of differentiation, cells were treated with DMSO or nM torin1 for 6 hours. For this purpose, several studies have been conducted on human and transgenic animal models demonstrating a correlative and causative association between dietary excess and activation of the innate and adaptive immune system in organs that control systemic energy homeostasis Lumeng et al.

The initial mechanistic evidence supporting the inflammatory origin of obesity and diabetes comes from human and animal studies carried out in the early s.

In these studies, AT from obese rodents and humans show inflammatory modifications and enhanced secretion of pro-inflammatory cytokine TNF-α able to induce IR by inactivating the IRS-1 Hotamisligil et al.

The pivotal role of TNF-α is significantly supported by evidence establishing that TNF-α neutralization in obese mice improves insulin sensitivity and glucose metabolism Hotamisligil et al.

Low-grade chronic AT inflammation also noted as meta-inflammation is strongly and consistently associated with excess body fat mass and is characterized by infiltration and activation of pro-inflammatory macrophages and other immune cells that produce and secrete pro-inflammatory cytokines and chemokines Chawla et al.

Macrophages change not only their number during obesity i. While in normal weight subjects the macrophages show anti-inflammatory properties, the polarization of AT macrophages ATMs in obese AT shifts to a pro-inflammatory phenotype Lumeng et al.

In obesity, macrophages surround dead adipocytes i. The inflammatory triggers are still almost unknown; however, obesity-induced AT remodeling provides a plethora of intrinsic signals e.

The role of inflammation in T2D pathogenesis and associated metabolic complications has led to a growing interest in targeting inflammatory mediators or pathways to prevent and treat T2D Shoelson et al.

In this review, we address the primary role played by the loss of immune regulation in the AT inflammation and the development of obesity-associated disorders, providing details on molecular aspects. We highlight the cellular and molecular triggers for obesity-induced inflammation and finally give some insights into the new anti-inflammatory therapeutic strategies.

Insulin is an anabolic hormone secreted by β-cells that plays a crucial role not only in carbohydrate metabolism but also in protein and lipid anabolic regulation, cell growth, and proliferation Fu et al. Blood glucose concentrations stimulate insulin synthesis and release; its effects on whole-body metabolism result from its binding to the cell membrane receptor, which is activated by autophosphorylation of specific tyrosine residues.

The activated insulin receptor phosphorylates and recruits intracellular proteins, also known as IRSs. Downstream of IRS proteins, PI3K mediates insulin functions mainly by activating PKB and protein kinase C cascades i. Obesity association with T2D has long been recognized, and the primary reason is the ability of obesity to promote IR, the main pathophysiological aspect of T2D Kahn and Flier, IR is a metabolic complication in which the three major insulin-sensitive tissues skeletal muscle, liver, and AT become less responsive to insulin action.

IR is characterized by serious failures in glucose uptake, glycogen synthesis, and, to a lesser extent, glucose oxidation Ormazabal et al.

In this scenario, the β-cells compensate for IR by increasing insulin secretion and restoring blood glucose concentration within the normal range. A further decline in insulin sensitivity makes the β-cells exhausted, and this results in persistent hyperglycemia and T2D Shulman, A number of studies have been performed to identify the causal factors responsible for obesity-induced IR.

One of the most accepted theories considers chronic systemic inflammation induced by obesity as a preponderant mechanism Weisberg et al.

This theory is strongly supported by many findings and clinical evidence; for instance, inflammatory markers such as CRP, TNF-α, and interleukin 6 IL-6 are elevated in obese and insulin-resistant subjects Dandona et al. The first evidence of an association between IR and inflammation has been hypothesized when, following the administration of anti-inflammatory agents, an improvement in glucose homeostasis has been observed in T2D patients Williamson, ; Reid et al.

Further studies in the mids have shown that the white AT WAT of obese rodents and humans exhibited changes in the levels of pro-inflammatory molecules e.

Such inflammatory mediators modulate IR either directly by affecting insulin signaling or indirectly by stimulating inflammatory pathways Tilg and Moschen, Other studies have shown that hypoxia, which occurs in AT during obesity, is directly responsible for IR induction in both human and murine models Regazzetti et al.

Animal and human studies have identified WAT as the primary site where obesity-related chronic inflammation is initiated and exacerbated Weisberg et al.

AT remodeling during obesity provides a plethora of intrinsic and extrinsic signals capable of triggering an inflammatory response Chawla et al. These triggers, discussed later in the review, converge on the activation of the JNK and NF-κB signaling pathways Nakatani et al.

The activation of these signaling pathways increases the production of pro-inflammatory cytokines, endothelial adhesion molecules, and chemotactic mediators that promote the infiltration of monocytes in AT and the differentiation into pro-inflammatory M1 macrophages Shoelson et al.

Infiltrating macrophages produce and secrete many inflammatory mediators that promote local and systemic pro-inflammatory status and impair insulin signaling Haase et al. The effects of these cytokines are mediated by stimulation of IκB kinase β IKK β and JNK1 , expressed in myeloid and insulin-targeted cells McLaughlin et al.

JNK is one of the most investigated signal transducers in obesity models of IR. It is activated after exposure to many inflammatory stimuli including cytokines, free fatty acids, and activation of cellular pathways, such as UPR Aguirre et al.

The role of Jnk1 in adipocytes has been investigated using tissue-specific Jnk1 -deficient mice. These mice are protected against the development of IR when fed a HFD.

This effect is tissue specific because Jnk1 deficiency in adipocytes does not affect muscle insulin sensitivity Hirosumi et al. Obesity is also associated with the activation of NF-κB inflammatory pathway.

In physiological conditions, NF-κB proteins are retained in the cytoplasm of myeloid and insulin-targeted cells by a family of inhibitors called inhibitors of κB IκBs McLaughlin et al. Activation of IKK kinase complex that contains IKKα and IKKβ subunits induces proteasomal degradation of IκBα, leading to NF-κB nuclear translocation.

This culminates in the increased expression of several NF-κB target genes [e. IKKβ deficiency in adipocytes totally prevents the expression of IL-6 and TNF- α induced by free fatty acid, while its activation inhibits the expression of anti-inflammatory cytokines such as adiponectin and leptin Jiao et al.

Therapeutic approaches capable of targeting these pathways and improving insulin sensitivity in obese subjects will be further discussed below in this review. Macrophages represent another important cell type in mediating the obesity-induced inflammation in the AT.

During obesity, macrophages infiltrate the AT and secrete many pro-inflammatory cytokines Weisberg et al. These mediators have local effects on adipocytes and resident immune cells e. Myeloid cells activate another molecular pathway, called inflammasome, in obesity Lee and Lee, Macrophages and other innate immune cells may trigger inflammatory responses by detecting pathogen- or danger-associated molecular patterns PAMPs or DAMPs using a broad variety of pattern-recognition receptors such as TLRs and NLRs Pedra et al.

Compelling evidence shows that NLRP3 the most studied member of the NLR family activation by DAMPs generated by nutrient excess in obesity plays a key role behind the chronic inflammation characteristic of obesity and IR Stienstra et al. NLRP3 is present in several tissues and cell types Vandanmagsar et al.

Once activated, NLRP3 interacts with procaspase-1 through an adaptive protein forming the NLRP3 inflammasome Schroder et al. This results in the processing and activation of caspase-1, which mediates the maturation and secretion of IL-1β and IL by macrophages Shoelson et al.

Caloric and exercise-mediated weight loss in obese people with T2D reduces NLPR3 and IL-1 β gene expression in abdominal subcutaneous AT and improves systemic insulin sensitivity Vandanmagsar et al. Inflammasome-activated IL-1β is a major cytokine produced by macrophages Sims and Smith, Its enhanced production in pancreatic islets and insulin-sensitive tissues is associated to T2D Hotamisligil et al.

In obesity, chronic rise in circulating nutrients such as glucose and free fatty acids FFAs resulted in over-expression of IL-1β in pancreatic β-cells Maedler et al. It is now clear that IL-1β is a key cytokine in the etiology of T2D since it has been implicated in IR, β-cell dysfunction, and death Eizirik and Mandrup-Poulsen, ; Donath et al.

IL-1β alters the insulin sensitivity of AT by suppressing insulin signaling; exposure to IL-1β of murine and human adipocytes decreases insulin-stimulated glucose uptake and lipogenesis Lagathu et al. The pro-apoptotic effects of IL-1β on β-cells derive from a complex network of signaling events triggered by IL-1β binding to its cognate receptor, whose expression is higher in β-cells than in other tissues Böni-Schnetzler et al.

Once cytokine binds its receptor, the co-receptor is recruited, and this results in the formation of the heterodimer receptor transmembrane complex. This leads to activation of MAPK and NF-κB signaling pathways. The activation of these two signaling pathways causes variations in gene expression, therefore triggering the apoptotic cell death program in β-cells Donath et al.

The pro-apoptotic effects mediated by NF-kB depend on the cell type, nature, and duration of the stimulus.

Indeed, NF-kB activation in β-cells is more marked, rapid, and sustained than in other cell types Ortis et al. MAPKs also take part in β-cell apoptosis through transcription-independent mechanisms, such as regulating B-cell lymphoma 2 protein activity Donath et al. The combined use of IFN-γ and IL-1β induces the activation of an additional mechanism, the so-called non-canonical NF-kB pathway, also implicated in the pro-apoptotic effects of IL-1β on β-cells Meyerovich et al.

IL-1β is also implicated in cardiovascular and microvascular long-term complications nephropathy, retinopathy, and polyneuropathy of diabetes Herder et al. Endothelial cell damage is a crucial and an early manifestation of diabetic-associated vascular complications van den Oever et al.

Among the multiple and potential mechanisms that contribute to this phenomenon, a crucial role is played by chronic low-grade inflammation. IL-1β has been reported to cause endothelial cell damage in isolated mesenteric rat micro-vessels Vila and Salaices, ; Shashkin et al.

Vallejo et al. Over-activation of NADPH oxidase has also been associated with excess ROS production and the development of atherosclerosis in diabetic vasculopathy Olukman et al. IL is another pro-inflammatory mediator activated by inflammasome and produced and released by human AT Wood et al.

IL plasma levels are increased in obese people and in individuals with T2D Moriwaki et al. It is a powerful pro-inflammatory cytokine that increases the maturation of T and NKs, as well as the production of other pro-inflammatory cytokines, exacerbating the obesity-induced systemic inflammation Weisberg et al.

Likewise, IL-6 has been suggested to be involved in the development of obesity-related and T2D-related IR Fève and Bastard, IL-6 leads to impaired insulin signaling, and this occurs primarily by inhibition of insulin-stimulated tyrosine phosphorylation of IRSs both in the liver and in AT Senn et al.

Nonetheless, conflicting results have been reported for IL-6 action on skeletal muscle Fève and Bastard, Carey et al. Nevertheless, it has also been shown that in murine skeletal muscle cells, IL-6 is capable of reducing insulin-stimulated glucose uptake through JNK activation Nieto-Vazquez et al.

In pancreas, IL-6 impairs insulin secretion and has pro-apoptotic effects on β-cells Ellingsgaard et al. An opposite effect is carried out on α-cells; IL-6 prevents α-cells apoptosis and induces the secretion of glucagon-like peptide This could be considered an adaptive mechanism to compensate for β-cell failure Ellingsgaard et al.

Such findings support the tissue-specific effect of IL-6 on glucose homeostasis, which depends on several factors, such as concentrations, targets, and signaling pathways activated. The IL-6 signaling cascade involves activation of the Janus kinase JAK -signal transducer and activator of transcription STAT pathway Fève and Bastard, ; Dodington et al.

It serves as a crucial downstream mediator for a variety of hormones, cytokines Gadina et al. There are four identified members in the JAK kinase family JAKs and Tyk2 , which associate with cytokine and growth factor receptors.

JAK-mediated signaling leads to the activation of seven STAT family members STATs , 5A, 5B, and 6. STAT proteins have cell- and tissue-specific distribution that influences their specificity and function Schindler and Darnell, ; Richard and Stephens, , This signaling pathway mediates the action of several hormones that have profound effects on adipocyte development and function.

Adipocytes also produce hormones that utilize this pathway Richard and Stephens, The expression of several STATs is modulated during adipogenesis Richard and Stephens, JAK2, STAT3, and STAT5 are essential for signaling through both the growth hormone and leptin receptors and have been characterized in WAT Dodington et al.

As the major upstream kinases required for STAT activity, it is not surprising that JAK proteins also play important roles in the control of AT function Gurzov et al.

Adipose-specific Jak2 KO mice have demonstrated defective lipolysis, increased body weight and adiposity compared to controls, leading to IR Nordstrom et al. Similarly, loss of either Stat3 or Stat5 in AT contributes to increased weight gain, adiposity, and impaired lipolysis Dodington et al.

Some studies have shown IR Shi et al. This inconsistency might be due to a variety of factors including tissue specificity and cell stage-dependent expression of the cre transgene, mouse genetic background, physiologic status, and other environmental factors in which the experiments were performed Dodington et al.

Although the direct role of STAT1 in the anti-adipogenic action of IFN-γ was not investigated, experiments using pharmacological inhibitors show that the JAK-STAT1 pathway plays a key role in the ability of IFN-γ to induce IR, decline triglyceride stores, and down-regulate expression of lipogenic genes in mature human adipocytes Richard and Stephens, The increased IFN-γ levels and JAK-STAT1 signaling in obesity contribute to AT dysfunction and IR Gurzov et al.

Obesity increases levels of IL-6 in WAT that, in turn, chronically activate intracellular JAK-STAT3 signaling. Chronic JAK-STAT3 signaling induced by IL-6 leads to the increased expression of suppressor of cytokine signaling-3 that not only negatively regulates IL-6 signaling but also hinders insulin action, eventually resulting in obesity and IR Wunderlich et al.

This complexity highlights the need for validation of the relative contribution of STAT proteins in human samples. Further studies will also be required to reveal the complex roles of the JAK-STAT pathway in adipocytes, obesity, and IR. Manipulation of this pathway within AT is a novel therapeutic approach for the treatment of obesity and diabetes.

Systemic inflammation is characterized by high circulating levels of inflammatory mediators and immune cells that infiltrate insulin-dependent tissues Weisberg et al. As has already been discussed in the review, WAT is the main site where low-grade systemic inflammation begins Weisberg et al.

Accumulation of lipids that occurs in AT during obesity triggers an inflammatory response that results in an increased secretion of several inflammatory cytokines Haase et al. Such molecules can also activate JNK and NF-κB signaling pathways in the liver and skeletal muscle, thus inhibiting systemic insulin signaling Hotamisligil et al.

Obesity-induced inflammation initiates in WAT and then spreads to other tissues, resulting in low-grade systemic inflammation. In obesity, both liver and skeletal muscle exhibit signs of local inflammation Figure 1. Figure 1. Pathways linking local obesity-induced inflammation to systemic insulin resistance.

Obesity results in the activation of the inflammatory signaling pathways mediated by JNK and nuclear factor-kappa B NF-κB. Once activated, these pathways induce the production of several pro-inflammatory cytokines in adipocytes, which contribute to insulin resistance and pro-inflammatory macrophages infiltration.

Instead, NF-κB signaling pathway activation culminates in the increased expression of several NF-κB target genes such as tumor necrosis factor-α TNF-α , interleukin-6 IL-6 , and monocyte chemotactic protein-1 MCP-1 , which leads to serine phosphorylation of IRS-1, therefore preventing insulin signaling.

The inflammatory mediators including free fatty acids FFA , IL-6, TNF-α, and MCP-1 also spread through systemic circulation and activate JNK and NF-κB signaling pathways in the liver and skeletal muscle, inhibiting systemic insulin signaling.

GLUT4, glucose transporter type 4. Skeletal muscle is the principal organ for insulin-stimulated glucose uptake i.

Obesity contributes to the development of chronic muscle inflammation, characterized by increased pro-inflammatory M1 macrophage infiltration Fink et al. These macrophages secrete many cytokines, which have been shown to trigger inflammatory pathways within myocyte, culminating in decreased insulin signaling Varma et al.

In the liver, obesity leads to increased infiltration and pro-inflammatory activation of two major macrophage groups: Kupffer cells i. Further work indicates that, during obesity, the number of Kupffer cells remains unaffected, whereas the accumulation of monocyte-derived recruited macrophages increases several times Morinaga et al.

It has been demonstrated that ATM-released inflammatory mediators lead to hepatic IR by reducing insulin signaling Morinaga et al.

These inflammatory mediators also contribute to liver steatosis by promoting lipogenesis and toxic ceramide biosynthesis Schubert et al. Recently, as specified above, particular attention has been paid to the role played by macrophages in AT inflammation.

Nevertheless, many other immune cells both innate and adaptive immune systems are involved in the development of local and systemic inflammation and IR.

During obesity, different types of both innate and adaptive immune cells accumulate in AT Lackey and Olefsky, They are suggested to be the major source of pro-inflammatory cytokines Samuel and Shulman, , which can cause IR Xu et al.

Obesity is associated with the recruitment of M1-polarized macrophages, which secrete pro-inflammatory cytokines such as TNF-α and IL-1β Han and Levings, Inflamed AT is characterized by the combination of an increase in total macrophages and an increased ratio of M1 to M2 anti-inflammatory macrophages, which comes along obesity and is linked with the development of IR Lumeng et al.

However, we should consider that macrophage inflammation in response to obesity is not identical to the classic M1 activation state observed in inflammation associated with acute infection.

As ATMs express a different set of surface markers, the pro-inflammatory activation in the setting of obesity has been referred to as metabolic activation, or Me, rather than M1 Reilly and Saltiel, The pro-inflammatory macrophages in obese AT also upregulate the expression of genes that encode proteins involved in lipid metabolism.

Hence, they can be also distinguished from classically activated macrophages Xu et al. Neutrophils are the leukocyte subpopulation Chmelar et al.

However, neutrophils are among the first responders recruited to AT in mice as early as 3 days after the initiation of HFD. Talukdar et al. We should take into account that another study already showed that this migration is transient Elgazar-Carmon et al.

Neutrophils stimulate AT inflammation by producing TNF-α and MCP-1 Dam et al. Neutrophils also produce elastase, which impairs glucose uptake in AT Wang et al. The activity of elastase is also increased in the AT of HFD mice, corresponding to the number of infiltrated neutrophils Talukdar et al.

Genetic deletion of elastase attenuates macrophage influx into the AT of obese mice and results in improved insulin sensitivity Talukdar et al. Dendritic cells are specialized antigen-presenting cells that link the innate and adaptive immunity Bertola et al.

Dendritic cells accumulate in AT of mice fed an HFD and in the subcutaneous AT of obese humans Cho et al. They likely induce the pro-inflammatory microenvironment through macrophage recruitment and IL-6 production Stefanovic-Racic et al. Blocking their accumulation improves insulin sensitivity in obese mice Cho et al.

DCs inhibit healthy expansion of AT, and depletion of these cells improves glucose homeostasis in mice Hotamisligil, Adipose-recruited DCs have been shown to be associated with the deregulation of chemerin, a particular adipokine Ghosh et al.

Altogether, these studies suggest a pathogenic role for DCs in the development of obesity in mice and humans. Mice with deletion of Fms-like tyrosine kinase 3 ligand Flt3L , that lack DCs, revealed reduced macrophage number in the AT and liver as well as improved insulin sensitivity in diet-induced obesity.

Administration of recombinant Flt3L to these mice reversed this phenotype Chung et al. Mast cells are innate immune cells Liu et al. AT is a reservoir of mast cells Zelechowska et al. There is a significant increase in the number of mast cells in the WAT of mice and humans with obesity Liu et al.

Mast cells promote AT low-grade inflammation in obesity Liu et al. They mediate the macrophage infiltration Liu et al.

Interestingly, mast cells are regulated by IL-6 and IFN-γ but not via TNF-α Liu et al. IL-6 and IFN-γ play a crucial role in the ability of mast cells to regulate metabolism, and they may mediate diet-induced obesity and diabetes Zelechowska et al.

Immature mast cells that infiltrate into AT the non-obese stage progressively mature and promote obesity and diabetes progression Hirai et al. Mast cells tend to degranulate Zelechowska et al. Hence, they have a key role in allergic responses and AT homeostasis Sun et al.

Mast cell deficiency is associated with improved insulin sensitivity McLaughlin et al. B cells are an important component of the adaptive immunity that release immunoglobulins or antibodies to recognize the cognate antigen.

This feature differs from the cell-mediated immunity where T cells recognize processed antigenic peptides presented by antigen-presenting cells Sun et al. B cells in AT are phenotypically different from B cells found in other tissues, as B cells in AT have unique genetic markers Dam et al.

They are present across all known AT depots but are less well characterized than T cells Kane and Lynch, B cells have also been shown to be pathogenic in obesity Kane and Lynch, B cells accumulate in the AT of obese mice relative to lean mice and become more inflammatory, producing chemokines that promote the recruitment of neutrophils, T cells, and monocytes Kane and Lynch, B cells promote pro-inflammatory activation of ATMs and T cells Winer et al.

B cells modulate IR by accumulating in the AT of obese mice Winer et al. It has been reported that B cell accumulation precedes T cell accumulation during the development of obesity Lau et al.

B cells might contribute to AT inflammation by producing immunoglobulin G antibodies or pro-inflammatory cytokines. The B cells from obese mice release a more inflammatory repertoire of cytokines Michelsen et al.

Obese mice with B cell deficiency reduce IR DeFuria et al. Transfer of B cells from obese donor mice causes impaired insulin sensitivity and glucose homeostasis in the recipients Winer et al. By contrast, there are also tolerance-promoting B regulatory cells in AT, and their number is decreased in models of obesity Nishimura et al.

T cells can be divided into two subtypes depending on the markers on their surface, CD4 and CD8 T cells Pennock et al. Treg population in lean AT is characterized by high peroxisome proliferator-activated receptor γ PPAR γ expression.

These Tregs play a critical role in maintaining AT inflammatory tone and insulin sensitivity Lee et al. The decline in the numbers of AT Treg cells during obesity contributes to increased AT inflammation Zhou et al.

Invariant natural killer T cells iNKTs are lipid-antigen-reactive T cells restricted by the major histocompatibility complex-like molecule CD1d Lee et al.

iNKT cells form a subset of lymphocytes in normal AT. The number of iNKTs is reduced in obesity Lee et al. Furthermore, mice lacking iNKTs shows increased weight gain, larger adipocytes, and IR on HFD.

This is associated with increased infiltration of macrophages into AT Lynch et al. Collectively, the network of T and B cells has crucial effects to influence macrophage infiltration. Thus, pro-inflammatory macrophages are the final effector cells that induce IR Lee et al.

There is a limited understanding of how obesity-induced inflammation in AT is triggered. However, potential mechanisms identified include dysregulation of fatty acids homeostasis, increased adipose cell size and death, local hypoxia, mitochondrial dysfunction, ER stress, and mechanical stress Figure 2 Heilbronn and Liu, ; Reilly and Saltiel, These mechanisms are recognized as the link between chronic caloric excess and AT inflammation or as factors that may perpetuate chronic tissue inflammation Burhans et al.

The list of potential mechanical links mentioned here is not complete, and it is likely that the triggers leading to AT inflammation have not yet been identified.

Figure 2. Obesity triggers inflammation. Obesity provides a plethora of intrinsic and extrinsic signals capable of triggering an inflammatory response in AT. These mechanisms are commonly considered the link between chronic caloric excess and adipose tissue inflammation.

Some of these mechanisms include dysregulation of fatty acid homeostasis, increased adipose cell size and death, local hypoxia, mitochondrial dysfunction, endoplasmic reticulum ER , and mechanical stress. These triggers converge on the activation of the c-Jun N-terminal kinase JNK and nuclear factor-kappa B NF-κB pathways, commonly considered signaling hubs.

The activation of these pathways increases the production of pro-inflammatory cytokines and promotes the infiltration of pro-inflammatory M1 macrophages. TLR2, Toll like receptor 2; TLR4, Toll like receptor 4; FFA, free fatty acids; UPR, unfolded protein response; HIF-1α, hypoxia-inducible factor-1α; RhoA, ras homolog gene family, member A; TNF-α, tumor necrosis factor-α; IL-6, interleukin-6; MCP-1, monocyte chemotactic protein-1; ECM, extracellular matrix.

Saturated fatty acids promote inflammatory activation of macrophages, partially mediated by indirect binding to TLR4 and TLR2 Konner and Bruning, , resulting in the activation of NF-κB and JNK pathways Shi et al. Once these pathways have been stimulated, many chemokines e.

In obesity, in addition to an increased intake of saturated fatty acids, TLR4 and TLR2 expression are increased in the AT, further supporting the role of these receptors in obesity-associated inflammatory signaling Husam et al.

In regard to this, acute lipid infusion is enough to stimulate AT inflammation and systemic IR in wild-type mice, and these effects are prevented in TLR4 -deficient mice Shi et al. Based on these findings, TLR4 appears to be an interesting candidate for linking dietary fatty acids with AT inflammation and IR Poggi et al.

Despite saturated fatty acids, unsaturated omega-3 and -9 fatty acids have beneficial effects and alleviate AT inflammation Oliveira et al. WAT plays a major role in regulating systemic energy homeostasis, which acts as a safe reservoir for fat storage.

In response to changes in nutritional status, AT expands by increasing the number hyperplasia and size of the adipocytes hypertrophy Sun et al.

Thus, the evidence indicating that adipocyte hypertrophy certainly contributes to AT inflammation is quite convincing at the present. Increased adipocyte size is characterized by a higher rate of adipocyte death and macrophage recruitment. Larger adipocytes exhibit an altered chemoattractant and immune-related proteins secretion that may promote pro-inflammatory macrophage infiltration Jernas et al.

Most of these infiltrated macrophages surround necrotic adipocytes and form crown-like structures. In obese rodents as well as humans, necrosis-related factors further attract monocytes in AT where they uptake the lipids released by dead adipocytes Cinti et al. As described above, the recruited monocytes have a pro-inflammatory phenotype and secrete cytokines and reactive oxygen species in neighboring adipocytes that interfere with insulin signaling Shapiro et al.

An increase in the number of dead adipocytes has been recognized to prevent normal AT functions and cause inflammation Choe et al. During adipocyte hypertrophy, angiogenesis is initiated to supply oxygen to the expanding tissue.

If the AT expansion is very rapid, the vasculature cannot fulfill the oxygen requirement and hypoxia occurs Gealekman et al. Hypoxia is a strong metabolic stressor. Current evidence reveals that hypoxia develops as AT expands because of a relative under perfusion of the enlarged AT or increased oxygen utilization Gealekman et al.

Cellular hypoxia can initiate inflammation by activating hypoxia-inducible factor-1 HIF-1 gene program. Activated HIF-1α translocates to the nucleus where it recognizes and binds the HREs on DNA. The binding to HREs promotes not only the expression of many genes involved in the angiogenesis but also inflammation Trayhurn, ; Fiory et al.

These include vascular endothelial growth factor, insulin-like growth factor 2, transforming growth factor α, as well as nuclear factor of kappa light polypeptide gene enhancer in B-cells 1 and inflammatory cytokines such as interleukin and 18 Shi and Fang, It has been shown that adipocyte-specific HIF-1 α deletion prevents obesity-induced inflammation and IR Lee et al.

Mitochondria are present in almost all eukaryotic cells and are responsible for cellular energy production, calcium signaling, and apoptosis Osellame et al. Alterations in mitochondrial functions are capable of causing inflammation, oxidative stress, cell death, and metabolic dysfunction Hock and Kralli, ; Kim et al.

A number of studies in obese mice and human subjects have shown that mitochondrial dysfunction is strongly associated with pathological conditions such as inflammation, IR, and T2D Silva et al.

A comparable decrease in mitochondrial activity has also been observed in human AT from obese individuals Yin et al. The mitochondrial dysfunction leads to inflammation through modulating redox-sensitive inflammatory mechanisms such as NF-κB or direct inflammasome activation Vaamonde-García et al.

The activation of both pathways induces an upregulation of inflammatory cytokines and adhesion molecules secretion, resulting in a substantial amplification of the inflammatory response Escames et al.

Petersen et al. Woo et al. ER is a cellular organelle that exhibits high sensitivity to cellular nutrients and energy status Hummasti and Hotamisligil, Many genetic and environmental hits can alter the functions of ER and therefore contribute to ER stress Hummasti and Hotamisligil, Several studies have shown that the incorrect functioning of the UPR i.

It has been shown in mice that obesity results in increased ER stress, particularly in the liver and AT. Indeed, the expression of most ER stress markers and chaperones is strongly BMI-related and associated with AT insulin sensitivity Sharma et al.

Additionally, a weight-loss gastric bypass surgery has been shown to enhance insulin sensitivity and decrease ER stress in obese Gregor et al. Inflammation is the predominant mechanism by which ER stress negatively affects metabolic homeostasis.

The primary mechanisms by which ER stress establishes inflammatory mechanisms in AT involve the activation of NF-κB, JNK, and apoptosis signaling pathways. In response to ER stress, the three UPR branches are activated. The activation of two branches is mediated by protein kinase RNA PKR -like ER kinase PERK and activating transcription factor 6 ATF6.

This activation stimulates NF-κB signaling pathway, resulting in the subsequent inhibition of insulin action via IRS-1 phosphorylation. In addition, the branch mediated by inositol-requiring enzyme 1 results in the activation of the JNK signaling pathway Hotamisligil, ; Hummasti and Hotamisligil, There is also crosstalk between the three branches.

For example, spliced X-box binding protein 1, as well as activating transcription factor 4, induces the production of the inflammatory cytokines IL-6, interleukin-8, and MCP-1 by human endothelial cells Hotamisligil, A further important function of UPR is to activate pro-apoptotic signaling pathways in order to prevent the release and accumulation of misfolded proteins, which may have adverse effects on cellular functions Hotamisligil, However, ER stress-induced apoptosis may also contribute to increased inflammatory signaling and other aspects of metabolic diseases.

For instance, adipocyte death in obesity has been suggested as a potential trigger for the recruitment of macrophages and other inflammatory cells Cinti et al.

Evidence also indicates that ER stress is essential for β-cell development and survival Harding et al. In , we have reported that UPR hyper-activation by glucose insult leads to a pro-inflammatory phenotype in preadipocytes.

Cells exposed to hyperglycemia release an increased amount of pro-inflammatory cytokines, chemokines and IL lymphokine, which can trigger inflammation by affecting inflammatory cells. However, such effects are prevented by a chemical chaperone such as 4-phenyl butyric acid Longo et al.

ER stress pharmacological inhibition can reverse metabolic dysfunction also in other tissues, including liver and brain Ozcan et al. Meta-inflammation and ER dysfunction are emerging as critical mechanisms. If these mechanisms are targeted therapeutically, they can enhance multiple metabolic parameters, as shown in preclinical and clinical studies Hummasti and Hotamisligil, The protein composition and dynamics of the ECM are crucial for the adipocyte function.

ECM remodeling is essential for the expansion and contraction of adipocytes to accommodate changes in energy stores Rutkowski et al.

During a positive energy balance, ECM accumulation occurs in AT, which contributes to fibrosis and impairs its role as a nutrient storage organ Lee et al.

Abnormal accumulation of ECM components in AT has been shown to cause obesity-associated IR Lin et al. Excessive ECM deposition in AT is suggested for triggering adipocyte necrosis, which attracts pro-inflammatory macrophages and causes AT inflammation and metabolic dysfunction.

In addition, excess ECM deposition causes adipocyte death and AT inflammation by activation of integrins and CD44 signaling pathways Lin et al. Lipid accumulation occurring in obesity may also cause ECM instability and induce various mechanical stresses on these cells.

The mechanisms governed by these mechanical stresses in adipocytes have not yet been fully explained, but certain pathways such as RhoA, and NF-κB have been evaluated.

RhoA signaling, for instance, inhibits adipogenesis through PPAR γ suppression and stimulates the secretion of pro-inflammatory cytokines McBeath et al. Meanwhile, Li et al. As mentioned above, some of the potential mechanisms involved in AT inflammation have been identified; however, it is likely that there are still unknown triggers.

The temporal sequence of events leading to AT inflammation, as well as the contribution of each mechanism described above, has not yet been fully established.

In our opinion, adipocyte hypertrophy may be the primary and initial event causing AT inflammation. In obesity, adipocytes respond to excess energy by storing lipids inside and undergoing dramatic changes in size hypertrophy.

Hypertrophy is associated with hypoxia, cellular and tissue stress, and cell death due to the activation of both necrotic and apoptotic mechanisms. Hypertrophic adipocytes are also characterized by excessive lipolysis, resulting in increased release of FFAs acting on TLR4, as previously indicated.

All the above mechanisms promote adipocyte dysfunction, characterized by an altered cytokine secretion pattern. These mechanisms play a dual role; they are able both to trigger individually inflammatory responses and to induce downstream processes, amplifying and eliciting chronic systemic inflammation and thus promoting systemic IR.

The temporal sequence of events suggested here and the relevance that we attribute to adipocyte hypertrophy in the initiation of AT inflammation needs to be further verified. The role of chronic inflammation, particularly in the AT, in the pathogenesis of T2D and associated complications, is now well established.

The association between obesity, AT inflammation, and metabolic disease makes inflammatory pathways an appealing target to treat metabolic disorders.

Inflammation is recognized as the pathologic mediator of these frequently common comorbidities. Several anti-inflammatory approaches have been tested in clinical studies of obese individuals with IR, but clinical trials to confirm the therapeutic potential are still ongoing Goldfine and Shoelson, The number of available drugs that can target different components of the immune system and improve different metabolic aspects is increasing rapidly Donath, Based on the mechanism of action, therapeutic approaches to target inflammation in IR and T2D can be divided into i pharmacologic approaches that directly target inflammation and ii diabetes drugs with anti-inflammatory properties.

Salsalate is an analog of salicylate that belongs to the non-steroidal anti-inflammatory drug classes. Independent studies have shown that salsalate can improve glycemic control in T2D patients.

The mechanism of action of salsalate in reverse hyperglycemia in obese mice is through the inhibition of NF-κB pathway and has been identified in by Shoelson Yuan et al. Goldfine then translated this initial finding to the clinical study and showed that salsalate decreases fasting glucose and triglyceride levels, increases adiponectin levels and glucose utilization in diabetic patients during hyperinsulinemic—euglycemic clamp, and improves insulin clearance Goldfine et al.

These observations have been confirmed in two multicenter, randomized, placebo-controlled trials in subjects with T2D Goldfine et al. In the first study, treatment with this drug improves insulin sensitivity and decreases HbA1c levels by 0.

This treatment also decreases levels of glycation end products Barzilay et al. Other studies also suggest that metabolic improvement, induced by salsalate treatment, is mediated through AMPK activation Hawley et al. Although the effects on glycemic control are modest, the salsalate is not expensive and has a very safety profile.

In , a preclinical study clearly showed the role of TNF-α in the pathophysiology of IR in the AT Hotamisligil et al. However, the results of clinical studies have so far been disappointing. For instance, TNF-α neutralizing antibodies have been shown to be effective for the treatment of many other inflammatory diseases, and some patients have shown slight improvements in glycemic control Ofei et al.

However, prospective studies in T2D patients have been confusing. In spite of valuable effects in mice, a human clinical trial showed that anti-TNF-α therapy leads to no improvements in insulin sensitivity in patients with T2D Ofei et al.

In contrast, a study performed in obese subjects without T2D showed that an inhibition of TNF-α for 6 months is able to reduce fasting glucose and increase adiponectin levels Stanley et al.

IL-1β is a strong mediator of the obesity-induced inflammation and participates in the pathogenesis of T2D, mediating the adverse consequences of hyperglycemia on pancreatic β-cells Maedler et al. Antagonism of IL-1R for 13 weeks, in a proof-of-concept study of patients with T2D, shows an improved glycemic control and secretory function of the pancreatic β-cells and the reduced markers of systemic inflammation Larsen et al.

The follow-up study on the same population proves that 39 weeks after the last IL-1R antagonist administration, β-cell insulin secretion is still increased and CRP decreased Larsen et al. The long-term effects are probably due to the block of IL-1β auto induction mechanism Böni-Schnetzler et al.

Further studies have also noted that the use of antibodies directed against IL-1β has potential benefits in the treatment of T2D, as it significantly reduces HbA1c levels Cavelti-Weder et al.

Recently, a multicenter randomized controlled trial, specifically designed to evaluate the glycemic outcome, enrolled participants, with RA and T2D followed up for 6 months.

Thirty-nine participants were randomized to IL-1R antagonist anakinra or TNF inhibitors TNFi to assess the efficacy of these drugs in controlling glucose alterations of T2D Ruscitti et al.

Regarding RA, there has been a gradual reduction in disease activity in both groups. In conclusion, results of this research indicate a specific effect of IL-1 inhibition in subjects with RA and T2D, reaching the therapeutic targets of both disorders and improving the main outcome of enrolled participants.

A clearer reduction of HbA1c, comparing this to the previous study on T2D Larsen et al. On this basis, IL-1 pathway can be considered a shared pathogenic mechanism, and a single treatment that manages both diseases appears to be a promising option for improving the care of RA and T2D patients Giacomelli et al.

Thiazolidinediones TZDs are antidiabetic drugs that improve insulin sensitivity and glycemia, as they function as agonists for PPARγ nuclear receptor Yki-Järvinen, TZDs have also anti-inflammatory effects; they repress NF-κB action and reduce the expression of its target genes Pascual et al.

The inhibition of NF-κB pathway reduces ATM content Esterson et al. Furthermore, the ability of TZDs to reduce circulating inflammatory mediators such as CRP and MCP-1 seems to be independent of glycemic control Pfützner et al.

Therefore, TZDs act through different mechanisms and the anti-inflammatory properties of these drugs are not definitely established. The mechanism of metformin action is not completely explained, but it decreases glycemia by reducing hepatic glucose production and raising glucose uptake in peripheral tissues Inzucchi et al.

In addition to its clear metabolic effects, metformin has also anti-inflammatory properties; for instance, it directly inhibits the production of reactive oxygen species in the mitochondria and can reduce the production of many cytokines Wheaton et al.

Emerging evidence supports the novel hypothesis that metformin can exhibit immune-modulatory features. Decreased ATP concentration causes AMPK activation, and among several targets, AMPK inhibits the mammalian target of rapamycin mTOR Zhou et al. mTOR is crucial for cellular metabolism, cytokine responses, antigen presentation, macrophage polarization, and cell migration Weichhart et al.

Metformin can also regulate other pathways relevant to immune cells, including NF-kB Hattori et al. Indeed, other studies have proved that metformin is able to inhibit TNF-α-induced activation of the NF-κB axis and IL-6 production Huang et al.

Metformin, in a dose-dependent manner, reduces IL-1β production in lipopolysaccharide-activated macrophages, and the effect is independent of AMPK activation Kelly et al. Moreover, metformin concurrently decreases circulating inflammatory proteins, including CRP, in impaired glucose tolerance and T2D patients De Jager et al.

The anti-inflammatory effects of metformin, like TZDs, appear to be independent of glycemic control Caballero et al.

In murine models, the attenuation of the inflammatory state has been shown to be effective in improving the obesity-induced IR; however, there are ongoing clinical trials in humans to confirm the therapeutic potential of metformin. This issue represents an essential step in proving the translational relevance of these observations.

T2D is a heterogeneous disorder, and the absence of clinical biomarkers, showing whether the treatments have anti-inflammatory effects in the AT, is a potential issue complicating the analysis Donath, The identification and profiling of these biomarkers in T2D patients would allow us to predict those that should respond to an anti-inflammatory therapy.

G Mcp1 mRNA levels in Rictor -knockout and control 3T3-L1 adipocytes. H Mcp1 mRNA levels in Rictor -knockout and control 3T3-L1 adipocytes treated with or without serum and insulin. I Mcp1 mRNA levels in Rictor -knockout and control 3T3-L1 cells treated with DMSO or the JNK inhibitor SP 20 μM for 6 hours.

J Immunoblots of Rictor -knockout and control 3T3-L1 adipocytes treated with DMSO or the JNK inhibitor SP 20 μM for 6 hours. K In vitro JNK kinase assay. Active JNK was immunoprecipitated from Rictor -knockout or control 3T3-L1 adipocytes, and JNK activity was assessed toward its substrate cJUN.

SP treatment served as a negative control. How does mTORC2 loss lead to Mcp1 transcription? It has been suggested that JNK is required for MCP1 expression and secretion in cultured 3T3-L1 adipocytes Consistent with that report, treatment with the JNK inhibitor SP reduced Mcp1 expression in Rictor -knockout 3T3-L1 cells Figure 5I.

Inhibition of JNK was confirmed by loss of cJUN Ser73 phosphorylation Figure 5J. SP did not restore AKT Ser phosphorylation Figure 5J or insulin-stimulated glucose uptake Figure 5F in the Rictor- knockout 3T3-L1 cells, indicating that the effect of the drug was independent of mTORC2 and insulin resistance.

Furthermore, JNK activity was unaffected by Rictor knockout Figure 5, J and K. Thus, mTORC2 and JNK control Mcp1 expression independently. MCP1 mRNA levels in omental WAT oWAT correlate with BMI in obese human subjects 9 , However, how MCP1 transcription is regulated in human adipose tissue is unknown.

To this end, oWAT samples were collected from 20 lean and 30 obese human patients who were under general anesthesia Figure 6A and Supplemental Table 2. The obese patients were insulin resistant as determined by high homeostatic model assessment of insulin resistance HOMA-IR Figure 6B and Supplemental Figure 9A.

Nevertheless, in oWAT, AKT2 Ser phosphorylation, a readout for mTORC2 signaling, was lower in obese patients than in lean patients Figure 6, C and D. AKT2 Ser phosphorylation negatively correlated with BMI Figure 6E. MCP1 expression was higher in obese subjects and positively correlated with BMI Figure 6, F and G , and Supplemental Figure 9B.

CD68 expression was also higher in the obese subjects and positively correlated with BMI Figure 6, H and I , and Supplemental Figure 9C. MCP1 and CD68 expression levels also correlated Figure 6J.

However, approximately one-third of the obese patients 9 of 30 had low AKT Ser phosphorylation and high HOMA-IR, MCP1 , and CD68 Figure 6K , suggesting that AdRiKO mice may phenocopy this subgroup of patients. A and B BMI and HOMA-IR of lean and obese patients. See also Supplemental Table 2.

C Representative immunoblots for p-AKT2 Ser and AKT2 in human oWAT. D Quantification of p-AKT2 Ser normalized to total AKT2. F MCP1 mRNA levels in human oWAT. G MCP1 positively correlated with BMI. H CD68 mRNA levels in human oWAT. I and J CD68 positively correlated with BMI I and MCP1 levels J.

Differentiated human primary adipocytes were treated with DMSO or nM torin 1 for 6 hours. We provide 2 lines of evidence that insulin resistance promotes the accumulation of M1 macrophages and thereby fosters inflammation Figure 7.

First, we show that knockout of mTORC2, i. As a consequence, monocytes were recruited to visceral WAT, where they differentiated into M1 macrophages and caused inflammation.

Second, HFD-induced insulin resistance in WT mice preceded the accumulation of proinflammatory M1 macrophages.

We also show that oWAT from obese, insulin-resistant patients had low mTORC2 signaling, high MCP1 expression, and high macrophage content, suggesting that our findings in mice have human relevance Figure 6. Insulin resistance causes inflammation in adipose tissue.

MCP1 in turn recruits monocytes and activates proinflammatory M1 macrophages. Our findings are consistent with observations made in mice genetically modified in other components of the insulin signaling pathway.

Two studies demonstrated that deletion of PTEN or PIK3R1, negative regulators of insulin signaling, causes enhanced insulin sensitivity and a reduced number of macrophages in adipose tissue 51 , More recently, Shearin et al. Obesity induces insulin resistance, via a yet-to-be defined mechanism, which in turn promotes inflammation.

As suggested previously 16 , this inflammation may contribute to adipose tissue remodeling and expansion to maintain glucose hemostasis. It has been suggested that activated M1 macrophages undergo metabolic reprogramming from oxidative phosphorylation to glycolysis 54 , Thus, a physiological role of M1 macrophages could be to clear excess local glucose.

Our finding that insulin resistance precedes inflammation may account for the observation that inhibition of TNF-α is ineffective in the treatment of obesity-induced insulin resistance 18 — HFD-fed AdRiKO mice showed reduced AKT Ser phosphorylation Figure 1B , decreased glucose uptake Figure 1A , and extensive inflammation Figure 2 in eWAT.

HFD-fed WT mice also displayed decreased glucose uptake Figure 3, A—C , which was followed by mild inflammation Figure 3D. Unexpectedly, AKT Ser phosphorylation was not reduced in WT mice fed a HFD for 4 or 10 weeks Supplemental Figure 5, E and F 56 , although we still observed mild inflammation in eWAT by week 10 of HFD feeding.

We note that the number of M1 macrophages in AdRiKO eWAT was much higher than that in control eWAT by week 10 of the HFD Figure 2F. These findings suggest that obesity-induced insulin resistance promotes mild inflammation downstream or independently of mTORC2, whereas chronic insulin resistance leads to mTORC2 inhibition and therefore extensive inflammation, as observed in AdRiKO mice and obese patients.

Our experiments reveal that loss of mTORC2 leads to increased Mcp1 expression in adipocytes Figure 5. What is the downstream effector through which mTORC2 controls Mcp1 transcription? One candidate is the phosphatidic acid phosphatase LIPIN1, whose knockdown results in Mcp1 expression in 3T3-L1 adipocytes LIPIN1, independently of its phosphatase activity, also functions as a transcriptional repressor We found that Lipin1 expression was reduced in Rictor -knockout eWAT and adipocytes Supplemental Figure 10, A and B , suggesting that mTORC2 may negatively control Mcp1 transcription via LIPIN1.

An adipose-specific transcription factor is another candidate through which mTORC2 may control Mcp1 expression. Rictor knockout increased Mcp1 expression in adipocytes, but not in liver or fibroblasts Supplemental Figure 8F and Supplemental Figure 10C , indicating that the regulation of Mcp1 transcription by mTORC2 is specific to adipocytes.

Why does AdRiKO eWAT accumulate a disproportionately high number of M1 macrophages only in response to obesity? The increase in Mcp1 transcription in mTORC2-knockout adipocytes required JNK activity Figure 5I , which is high only in WAT from obese mice 59 , Thus, obesity might be a prerequisite for JNK activation, which in turn stimulates Mcp1 expression in conjunction with loss of mTORC2.

We note that mTORC2 did not control JNK Figure 5, J and K. In summary, we propose that obesity-mediated insulin resistance is a cause of inflammation in visceral WAT. Although our findings do not rule out the possibility that inflammation promotes insulin resistance in other tissues or conditions, they bring into question whether antiinflammation therapy in adipose tissue is an effective strategy in the prevention of type 2 diabetes.

Adipose tissue—specific Rictor -knockout AdRiKO and liver-specific Rictor -knockout LiRiKO mice were described previously 32 , 35 , For experiments with AdRiKO mice, age-matched Cre -negative males were used as controls.

For experiments with LiRiKO and i-AdRiKO mice, Cre -negative littermate male mice were used as controls. For i-AdRiKO mice, Rictor knockout was induced by i.

The HFD-feeding experiment was conducted for 10 weeks unless otherwise specified. Cre -negative animals were used as a control. For i-AdRiKO mouse experiments, control mice were also treated with tamoxifen.

Mice were randomly assigned for each experiment. Only male, 6- to week-old mice were used for experiments. Cell culture. For differentiation, cells were maintained in M1 medium for 2 days after reaching confluence.

The medium was replaced with M2 medium M1 medium supplemented with 1. After 2 days, the medium was replaced with M3 medium M1 with 1. The medium was replaced with M2 on day 4 of differentiation. From day 6, cells were maintained in M3 with a medium change every 2 days. For all experiments, cells differentiated for 10 to 14 days were used.

Torin 1 was purchased from Tocris Bioscience and dissolved in DMSO. Human oWAT biopsies. Patients were fasted overnight and then underwent general anesthesia.

All oWAT samples were obtained between and , snap-frozen in liquid nitrogen, and stored at —80°C. Human primary adipocytes. Human primary visceral preadipocytes from a year-oldwoman with a BMI of 23 were obtained from Lonza.

After 14 days of differentiation, cells were treated with DMSO or nM torin1 for 6 hours. Oligonucleotides containing single-guide RNAs sgRNAs Rictor. Plasmids were amplified by bacterial transformation and isolated by Miniprep Zymo Research.

LentiCRISPRv2 plasmids were cotransfected with psPAX2 Addgene plasmid ; a gift of Didier Trono, EPFL, Lausanne, Switzerland and pCMV-VSV-G 63 Addgene plasmid ; a gift of Robert Weinberg, MIT, Cambridge, Massachusetts, USA into HEKT cells Hall laboratory stock.

Supernatants containing lentiviruses were collected 1 day after transfection and used to transduce undifferentiated 3T3-L1 cells. Transduced cells were selected by puromycin. Mice were fed a ND or HFD for 4 weeks, fasted for 5 hours, and given Humalog insulin i.

Lilly; 0. After 10 minutes, 2-deoxyglucose 2DG Sigma-Aldrich was given i. For in vitro 2DG uptake, differentiated adipocytes were cultured in serum-free M1 medium, washed 3 times with HKRP buffer 1.

Cells were stimulated with nM insulin for 20 minutes and subsequently cultured with 1 mM 2DG for 20 minutes. Tissues or cells were lysed in 10 mM Tris-HCL, pH 8.

MCP1 ELISA and adipokine array. MCP1-neutralizing antibody treatment. Mice were fed a HFD for 8 weeks. Mice were given i. Mice were fasted for 5 hours, Humalog insulin Lilly was given i.

In vivo insulin stimulation. Mice were fasted for 5 hours, Humalog insulin Lilly was administered i. Isolation of adipocytes and SVCs and flow cytometric analysis.

SVCs were isolated and stained with antibody as previously described After digestion, final 10 mM EDTA was added and incubated for 10 minutes to dissociate SVCs.

The resulting suspension was filtered through a μm cell strainer Corning and centrifuged at g for 10 minutes. After centrifugation, floating adipocytes were collected, and SVC-containing pellets were subjected to red blood cell lysis in 1× Red Blood Cell Lysis Buffer eBioscience.

Stained SVCs were analyzed using the FACSCanto II BD Biosciences or sorted with a FACSAria IIIu BD Biosciences. Images were obtained using DMB Leica and analyzed with Fiji software ImageJ; NIH RNA isolation and quantitative real-time PCR.

Total RNA was isolated with TRIzol Reagent Sigma-Aldrich and an RNeasy Kit QIAGEN. For RNA isolation from sorted macrophages, an RNeasy Micro Kit QIAGEN was used. RNA was reverse transcribed to cDNA using an iScript cDNA Synthesis Kit Bio-Rad. Semiquantitative real-time PCR analysis was performed using Fast SYBR Green Applied Biosystems.

Relative expression levels were determined by normalizing each Ct value to either Polr2a , Tbp , or Rpl7 expression for mice and RNA18S5 for human samples using the ΔΔCt method.

The primer sequences used in this study are shown in Table 1. Protein isolation and immunoblots. Linking gut microbiota and inflammation to obesity and insulin resistance. Physiology Bethesda. doi: Bastard JP, Maachi M, Lagathu C, et al. Recent advances in the relationship between obesity, inflammation, and insulin resistance.

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