FLSs Are Sensitive to Insulin

A previous study suggested that FLSs are vital for osteoarthritis pathogenesis and that synoviocyte hyperplasia and abnormal synoviocyte proinflammatory cytokine production were both important pathological hallmarks of OA [2,3,4,5]. CCK-8 assays were applied to assess the effects of insulin (0, 100, 200, or 500 nM) on the viability of FLS. In response to insulin (200 or 500 nM), cell viability was significantly enhanced at 24 h (Fig. 1a) and 48 h (Fig. 1b). Next, the regulatory effects of insulin on proinflammatory cytokines and MMP mechanisms in FLSs were investigated. To determine whether insulin can directly regulate proinflammatory cytokine expression, FLSs were treated with incremental concentrations of insulin (0, 100, 200, or 500 nM). The qRT-PCR results (Fig. 1c–g) showed that IL-1β, IL-6, TNF-α, MMP-9, and MMP-13 expression levels were increased in a concentration-dependent manner compared with those of the control group (P < 0.05) at 6 h, 12 h, and 24 h after treatment with insulin (0, 100, 200, or 500 nM), and these levels were increased most significantly at 24 h. A similar increase in FLS secretion in response to insulin stimulation was observed by ELISA (Fig. 1h–l).

Fig. 1 Cell viability and proinflammatory cytokines were both increased in response to high insulin in FLSs. a, b CCK-8 technology was used to assess cell viability of FLSs following insulin treatment (0, 100, 200, 500 nM) for 24 or 48 h; a representative of three independent experiments is shown. The figure of optical density (OD) detected by CCK-8 was used to reflect the degree of cell viability. FLSs were treated with insulin (0, 100, 200, 500 nM) for 6, 12, or 24 h. c–g The relative expression of IL-1β, IL-6, TNF-α, MMP-9, and MMP-13 were measured by RT-qPCR. h–l The secretion of IL-1β, IL-6, TNF-α, MMP-9, and MMP-13 were measured by ELISA. All the results are expressed as the mean ± SEM of three experiments conducted in triplicate separately. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. control group. Full size image

Insulin Enhanced FLS-Mediated Chemotaxis in Macrophages

Because macrophage infiltration is a significant pathological feature of OA, macrophages can also contribute to OA [4, 5]. Chemokines are the major drivers of leukocyte adhesion and cell migration in inflammatory disease development [32, 33]. Among the chemokines, CXCL12, CCL2/MCP-1, and CCL5/RANTES can induce macrophage chemotaxis and are closely involved in OA development [34,35,36]. It is unclear whether insulin can regulate FLS-mediated macrophage infiltration and chemokine production. Transwell assays were employed to analyze the role of insulin in macrophage infiltration. The results suggest that the number of transmigrated macrophages was significantly increased at 24 h in the presence of FLSs treated with high insulin (500 nM). In addition, ELISA was used to detect CXCL12, CCL2/MCP-1, and CCL5/RANTES secretion by FLSs after 24 h. It was observed that insulin could independently attract macrophages in the absence of FLSs (Fig. 2a). Moreover, CXCL12, CCL2/MCP-1, and CCL5/RANTES secretion increased following insulin stimulation (500 nM, Fig. 2b–d).

Fig. 2 Effect of insulin on chemotaxis of FLSs to macrophages. FLSs received the treatment of high insulin (500 nM) for 24 h. a The chemotactic ability of FLSs was performed by Transwell assay and the average number of macrophage cells that invaded through the filter was quantified. Migration capacity of macrophage was measured by Transwell assay. b–d ELISA were performed to detect the secretion of CXCL12, CCL2/MCP-1, and CCL5/RANTES by high insulin (500 nM) after 24 h. All the results are expressed as the mean ± SEM of three experiments performed in triplicate separately. ***P < 0.001 vs. control group. Full size image

Insulin Activates the PI3K/mTOR/Akt and NF-ĸB Pathways and Mediates Autophagy Inhibition

The above-mentioned data suggest that insulin could increase the inflammatory effect of FLSs. The PI3K/Akt/mTOR signaling pathway is involved in the pathogenesis of inflammation, including OA [28,29,30,31], and NF-ĸB is a known signaling pathway of OA inflammation [11, 12]. Thus, whether insulin can regulate PI3K/Akt/mTOR and NF-ĸB signaling pathway activation in FLSs was verified. Western blotting was used to evaluate the phosphorylation of PI3K/Akt/mTOR and p50/p65 (NF-ĸB subunits). According to the Western blotting results, the phosphorylation of PI3K/Akt/mTOR (Fig. 3a) and p50/p65 (Fig. 3b) was increased remarkably in FLSs treated with high insulin concentrations (100, 200, or 500 nM) for 24 h. These results verified PI3K/Akt/mTOR and NF-ĸB signaling pathway activation by high insulin conditions in FLSs. Immunofluorescence confirmed that a high insulin level (500 nM) could improve p50 and p65 (Fig. 3c, d) translocation into the nucleus in FLSs.

Fig. 3 Activation of PI3K/Akt/Mtor/NF-ĸB pathway and autophagy deficiency by insulin. a, b Western blot analysis was used to examine the phosphorylation level of PI3K/Akt/mTOR and p50/p65 in FLSs treated with insulin (INS: 0, 100, 200, 500 nM) for 24 h. c, d Effects of insulin (INS: 500 nM) on p50 and p65 nuclear translocation in FLSs were observed using confocal fluorescence microscopy. a Western blot analysis was used to examine ratio of LC3 I/II in FLSs treated with insulin (Ins; 0, 100, 200, 500 nM) for 24 h. Results are expressed as the mean ± SEM of three experiments conducted in triplicate separately. All the results are expressed as the mean ± SEM of three experiments conducted in triplicate separately. Full size image

Autophagy is a critical cellular process that maintains homeostasis, and autophagy inhibition is closely associated with OA [21,22,23,24,25]. To evaluate how insulin regulates autophagy activation in FLSs, Western blotting was employed to detect the expression of LC3II, a major autophagy effector. LC3II expression and the LC3II/LC3I ratio were decreased with concomitantly increased PI3K/Akt/mTOR phosphorylation levels (Fig. 3a).

Insulin Exacerbates Inflammatory Responses and Inhibits Autophagy in a Manner Dependent on the PI3K/mTOR/Akt and NF-ĸB Pathways

According to the above-mentioned data, insulin could activate the PI3K/Akt/mTOR and NF-ĸB signaling pathways in FLSs (Fig. 3a–c). In addition, the PI3K/Akt/mTOR pathway is a fundamental intracellular signaling pathway that is widely involved in autophagy regulation [28, 29]. To further verify the regulation of both signaling pathways by insulin, the effects of specific inhibitors on insulin-induced inflammatory response exacerbation and autophagy inhibition were also studied. The data revealed that inhibiting the PI3K/Akt/mTOR signaling pathways could eliminate the increased FLS viability following insulin treatment (0, 100, 200, or 500 nM) for 24 or 48 h (Fig. 4a, b). Furthermore, the decreases in LC3II expression and the LC3II/LC3I ratio caused by insulin (500 nM) could be restored by inhibitor treatment. The results demonstrated that the PI3K/Akt/mTOR pathway plays a role in autophagy regulation in FLSs (Fig. 4c).

Fig. 4 High insulin-mediated inflammatory responses are dependent on PI3K/Akt/mTOR and NF-ĸB pathway. a, b FLSs were treated with insulin (INS; 0, 100, 200, 500 nM) in the presence of inhibitor targeting PI3K/Akt (LY294002, 10 μM) and mTOR (Rapamycin, 10 μM) signaling. CCK-8 technology was used to assess cell viability of FLSs treated with a duration of 24 h or 48 h. c Various inhibitors targeting PI3K/Akt (LY294002, 10 μM) and mTOR (Rapamycin, 10 μM) were pre-treated for 6 h respectively to antagonize the effects of high insulin (500 nM) stimulation with a duration of 24 h. Ratio of LC3 I/II was determined by WB. d–f ELISA were performed to detect the secretion of CXCL12, CCL2/MCP-1, and CCL5/RANTES by high insulin (500 nM) in the presence of NF-ĸB inhibitor (PDTC, 10 μM) after 24 h. Various inhibitors targeting NF-ĸB (PDTC, 10 μM), PI3K/Akt (LY294002, 10 μM), and mTOR (Rapamycin, 10 μM) were pre-treated for 6 h to antagonize the effects of insulin stimulation with a duration of 24 h. g–k RT-qPCR was performed to ascertain the relative expression of IL-1β, IL-6, TNF-α, MMP-9, and MMP-13 in FLSs. l–p ELISA was performed to ascertain the secretion of IL-1β, IL-6, TNF-α, MMP-9, and MMP-13 in FLSs. Results are expressed as the mean ± SEM of three experiments conducted in triplicate separately. All the results are expressed as the mean ± SEM of three experiments conducted in triplicate separately. *P < 0.05. **P < 0.01. ***P < 0.001 vs. control group. Full size image

Because NF-ĸB signaling could significcytokines (e.g., IL-1β, IL-6, and TNF-α) directly and MMPs indirectly [11,12,13,14], we speculated that the overproduction of proinflammatory factors and chemokines following insulin treatment might result from NF-ĸB signaling pathway activation. To verify the mechanism of high insulin-induced proinflammatory factor and chemokine production, an inhibitor targeting the NF-ĸB (PDTC, 10 μM) signaling pathway was used. To evaluate the role of different pathways involved in OA progression, inhibitors targeting the PI3K/Akt/mTOR signaling pathway (LY294002, rapamycin) were employed. Subsequently, the inhibition of NF-ĸB signaling pathway could inhibit the CXCL12, CCL2/MCP-1, and CCL5/RANTES oversecretion induced by a high insulin concentration (500 nM) at 24 h (Fig. 4d–f). Similar results showing that inhibiting the PI3K/AKT/mTOR and NF-ĸB signaling pathways could reduce the overexpression (Fig. 4g–k) and secretion (Fig. 4l–p) of IL-1β, IL-6, TNF-α, MMP-9, and MMP-13 induced by a high insulin concentration (500 nM) at 24 h were also obtained. These experiments provide evidence that the proinflammatory effect of insulin on FLSs could be exerted through PI3K/mTOR/Akt and NF-ĸB pathway activation.

Insulin Sensitizes FLSs to Inflammatory Factors by Upregulating Their Surface Receptors

NF-ĸB contributes to the production of proinflammatory cytokines (e.g., IL-1β, IL-6, and TNF-α), and in turn, the proinflammatory effects of IL-1β, IL-6, and TNF-α are mediated through the activation of several signaling pathways, including NF-ĸB signaling [2, 11, 12]. Thus, a feedback mechanism exists among inflammatory cytokines and signaling pathways, which can activate each other and have a close relationship. Based on the fact that insulin is a positive regulator of proinflammatory cytokine production and was found to upregulate the downstream targets of PI3K/mTOR/Akt/NF-ĸB signaling in FLSs, we hypothesized that insulin might be involved in the interaction between IL-1β/IL-6/TNF-α and PI3K/mTOR/Akt/NF-ĸB signaling. Cytokines act as important mediators of intercellular communication through specific cytokine receptors on the cell surface and initiate a series of specific biochemical reactions. The receptors responsible for the proinflammatory effects of IL-1β/IL-6/TNF-α-activated intracellular signaling pathways include interleukin-1 receptor 1 (IL-1R1), interleukin-1 receptor 3 (IL-1R3, interleukin-1 receptor accessory chain), interleukin-6 receptor (IL-6R), glycoprotein 130 (GP130, interleukin-6 coreceptor), tumor necrosis factor receptor 1 (TNFR1 or p55), and tumor necrosis factor receptor 2 (TNFR2 or p75) [37,38,39]. Thus, we measured the expression of these proinflammatory cytokine receptors in insulin-stimulated FLSs by qRT-PCR. The results showed that IL-1R1, IL-1R3, IL-6R, GP130, TNFR1, and TNFR2 expression levels were increased in a concentration-dependent manner compared with those of the control group (P < 0.05) at 24 h after treatment with insulin (0, 100, 200, or 500 nM); these receptors were upregulated most significantly at the 500 nM concentration (Fig. 5a–f). To explore the association of cytokine receptor upregulation with PI3K/mTOR/Akt/NF-ĸB signaling in FLSs, the effect of inhibitors targeting the PI3K/Akt (LY294002, 10 μM), mTOR (rapamycin, 10 μM), and NF-ĸB (PDTC, 10 μM) signaling pathways on insulin-induced cytokine receptor expression was studied. The data revealed that inhibiting the PI3K/Akt/mTOR and NF-ĸB signaling pathways could reduce the IL-1R1, IL-1R3, IL-6R, GP130, TNFR1, and TNFR2 overexpression induced by high insulin (500 nM) at 24 h (Fig. 5g–l). These experiments provide evidence that when FLSs are stimulated with insulin, the expression of proinflammatory cytokine (e.g., IL­1-β, IL­6, and TNF-α) receptors depends on PI3K/Akt/mTOR and NF-ĸB signaling pathway activation.

Fig. 5 Insulin upregulated surface receptors of inflammatory factors through PI3K/Akt/mTOR and NF-ĸB pathway in FLSs. a–f FLSs were treated with insulin (0, 100, 200, 500 nM) for 6, 12, or 24 h. The expression and secretion of IL1R1, ILR3, IL6R, GP130, TNFR1, and TNFR2 were measured by qRT-PCR. g–l Various inhibitors targeting NF-ĸB (PDTC, 10 μM), PI3K/Akt (LY294002, 10 μM), and mTOR (Rapamycin, 10 μM) were pre-treated for 6 h to antagonize the effects of insulin stimulation with a duration of 24 h. To ascertain the expression and secretion of IL1R1, ILR3, IL6R, GP130, TNFR1, and TNFR2 in FLSs, qRT-PCR was performed. All the results are expressed as the mean ± SEM of three experiments conducted in triplicate separately. *P < 0.05. **P < 0.01. ***P < 0.001 vs. control group. Full size image

Insulin and Inflammatory Factors Synergistically Exacerbated the Inflammatory Phenotype of OA FLSs

In synovial joint pathology, a major hallmark in response to increased levels of IL-1β, IL-6 and TNF-α is the overexpression of MMPs, including MMP-9 and MMP-13, which contributes to cartilage degradation [13, 14]. To determine whether insulin can directly regulate cytokine-mediated inflammatory reactions, we chose MMP-9 and MMP-13 expression as a functional readout for the effect of insulin. FLSs were treated with incremental concentrations of IL-1β, IL-6 and TNF-α (0, 0.01, 0.1, 1, 10, or 50 ng/ml) or were cotreated with high insulin (500 nM) and all three cytokines separately for comparison. The qRT-PCR results showed that the expression of MMP-9 and MMP-13 increased (P < 0.05) after treatment with IL-1β, IL-6 and TNF-α (0, 0.01, 0.1, 1, 10, or 50 ng/ml) at 24 h; the MMPs were increased most significantly (P < 0.01) at the 50 ng/ml concentration, and the response was further increased by the presence of insulin (Fig. 6a–f).

Fig. 6 Effects of insulin on expression of MMPs and regulation of signaling pathways by inflammatory cytokines. a–f FLSs were treated for 24 h with IL-1β, IL-6, and TNF-α (0, 0.01, 0.1, 1, 10, 50 ng/ml) separately, or insulin (500 nM) was pre-treated for 12 h to affect the effects of inflammatory cytokines stimulation with a duration of 24 h. The expression of MMP-9 and MMP-13 were measured by qRT-PCR. g FLSs were treated for 24 h with IL-1β, IL-6, and TNF-α (50 ng/ml) separately, or insulin (INS:500 nM) was pre-treated for 12 h to affect the effects of inflammatory cytokines stimulation with a duration of 24 h. The phosphorylation level of PI3K/Akt/mTOR and p50/p65 were measured by Western blotting. All the results are expressed as the mean ± SEM of three experiments conducted in triplicate separately. **P < 0.01. ***P < 0.001 vs. control group. Full size image

Finally, we sought to investigate whether insulin could affect IL-1β, IL-6 and TNF-α-mediated signaling activation in FLSs, as suggested by the increase in cytokine-induced MMP expression. FLSs were treated with inflammatory cytokines (IL-1β, IL-6 and TNF-α, 50 ng/ml) and insulin (500 nM) separately or cotreated with the two stimuli simultaneously. Western blotting was used to test the phosphorylation of PI3K/Akt/mTOR and p50/p65 in different groups. After treatment, the PI3K/Akt/mTOR and p50/p65 phosphorylation levels in the three groups increased significantly and were even higher in the cotreatment group than in the other two groups (Fig. 6g). These experiments showed that insulin sensitized cellular signaling transduction activation by inflammatory cytokines. Based on the above-mentioned research, we suggest that insulin is involved in a positive feedback loop between IL-1β/IL-6/TNF-α and PI3K/mTOR/Akt/NF-ĸB signaling in FLSs.