High Endogenously Synthesized N-3 Polyunsaturated Fatty Acids in Fat-1 Mice Attenuate High-Fat Diet-Induced Insulin Resistance by Inhibiting NLRP3 Inflammasome Activation via Akt/GSK-3β/TXNIP Pathway

As shown in Figure 1A, compared to the Ctrl diet, the HF diet induced a statistically significant weight gain in fat-1 and WT mice although weight gain was relatively lower in fat-1 mice. Obesity is known to be associated with impaired glucose tolerance. Our results showed that HF diet-fed fat-1 (Fat-1 HF) mice had significantly lower fasting glucose, and were also protected against impaired glucose tolerance observed in HF diet-fed WT (WT HF) mice (Figure 1B,C). In accordance with this remarkable resistance to impaired glucose tolerance, the insulin tolerance test (ITT) showed that compared with WT HF mice, Fat-1 HF mice elicited a significant improvement in insulin sensitivity and remained comparable to Ctrl diet-fed mice (Figure 1D,E).

Adipose tissue H&E staining showed that Fat-1 HF mice displayed adipose hyperplasia compared with WT HF mice, the adipocyte size from the WT HF group was much larger than that from the Fat-1HF group and control-diet group (Figure 2A,B). It is well known that obesity is closely associated with inflammation, especially in promoting the secretion of pro-inflammatory cytokines [14]. Our results demonstrated that WT HF mice displayed increased IL-1β secretion to a greater extent in adipose tissue than Fat-1 HF mice. The culture media concentration of IL-1β was (36.12 ± 1.98) and (6.56 + 0.57) pg/mL in WT HF and Fat-1 HF adipose tissue, respectively (Figure 2C). As shown in Figure 2D, adipose tissue IL-1β mRNA expression was also significantly upregulated in WT HF mice compared with Fat-1 HF mice. As expected, the HF diet significantly induced IL-18 secretion from adipose tissue in WT mice compared with fat-1 mice (Figure 2E). To test whether IL-1β secretion was mediated by NLRP3 inflammasome, adipose tissue was treated with NLRP3 inflammasome inhibitor MCC950. Indeed, MCC950 significantly decreased IL-1β levels in WT HF mice to an extent comparable to those in Fat-1 HF mice, suggesting that NLRP3 inflammasome is involved in controlling IL-1β secretion (Figure 2F).

We next studied which cells in adipose tissue control IL-1β production. Adipose tissue SVF from WT HF mice secreted significantly higher amounts of ATP-induced IL-1β compared with that from Fat-1 HF mice, whereas adipocytes produced negligible levels of IL-1β (Figure 3A). Because IL-1β production depends on caspase-1 activity, caspase-1 activity levels were then determined in adipose tissue SVF. As shown in Figure 3B, ATP treatment significantly activated caspase-1 in SVF from WT HF mice compared with that from Fat-1 HF mice. We further assessed whether IL-1β is also secreted from preadipocytes isolated from SVF. As shown in Figure 3C, preadipocytes from WT HF mice displayed enhanced IL-1β levels in response to ATP as observed in SVF, compared with those from Fat-1 HF mice. Additionally, a significant increase in caspase-1 activity was observed in preadipocytes from WT HF mice compared with those from Fat-1 HF mice (Figure 3D). We further assessed whether NLRP3 inflammasome is responsible for IL-1β secretion in preadipocytes as observed in adipose tissue. As shown in Figure 3E, MCC950, the NLRP3 inflammasome inhibitor, significantly suppressed ATP-induced IL-1β secretion in WT HF-derived preadipocytes to an extent comparable to that in Fat-1 HF-derived preadipocytes.

As shown in Figure 4A,B, the fatty acid composition of the total lipids extracted from adipose tissues showed distinct lipid profiles between fat-1 and WT mice. Significantly higher levels of n-3 PUFAs, including EPA (20:5 n-3), docosapentaenoic acid (DPA) (22:5 n-3), and DHA (22:6 n-3), whereas much lower concentrations of arachidonic acid (AA; 20:4 n-6) were observed in adipose tissues from fat-1 mice than from WT littermates regardless of whether the mice fed a Ctrl or HF diet. Of note, DHA was the most abundant endogenously synthesized n-3 PUFA in fat-1 mice. Additionally, the endogenous n-6/n-3 PUFA ratio in adipose tissue was significantly lower in Fat-1 Ctrl (4.56 ± 0.43) and Fat-1 HF mice (4.78 ± 0.51) than in WT Ctrl (17.22 ± 2.00) and WT HF mice (19.83 ± 1.93) whatever they fed a Ctrl or HF diet (Figure 4C). Similar data were also observed in adipose tissue preadipocytes (Figure 4D–F).

To bring insight into the benefits of endogenous n-3 PUFA on impairing IL-1β production via controlling NLRP3 inflammasome activation, we assessed the NLRP3 inflammasome pathway proteins by immunoblotting. As shown in Figure 5A,B, the HF diet significantly induced the protein expression of NLRP3, pro-caspase-1 and cleaved caspase-1 in adipose tissue of WT mice, whereas these proteins were comparable among that of WT Ctrl, Fat-1 Ctrl and Fat-1 HF mice. Interestingly, although the HF diet induced IL-1β mRNA expression compared with the Ctrl diet (Figure 2D), no difference in pro-IL-1β protein expression was observed whether the mice were fed a Ctrl or HF diet (Figure 5A). In addition, previous adipose tissue culture showed that endogenous n-3 PUFAs in fat-1 mice markedly inhibited HF diet-induced IL-1β secretion (Figure 2C). These results suggested that endogenous n-3 PUFAs impaired IL-1β production by inhibiting NLRP3 inflammasome activation.

Because reactive oxygen species (ROS) are proximal signals for inflammasome activation [23], we determined the ROS levels in adipose tissue. ROS were significantly elevated in adipose tissue from WT HF mice (2.25 ± 0.11) compared with from Fat-1 HF mice (1.37 ± 0.07) (Figure 5C). Due to the key role of ROS in modulating NLRP3 inflammasome activation, we detected whether endogenous n-3 PUFAs suppressed ROS production and NLRP3 inflammasome activation via the Nrf2/Trx1 antioxidant axis. As shown in Figure 5D,E, Nrf2 and Trx1 protein levels were significantly down-regulated in adipose tissue from WT HF mice, whereas these two proteins were restored in adipose tissue from Fat-1 HF mice. Under oxidative stress conditions, increased ROS facilitate Trx1-TXNIP dissociation, then promoting TXNIP-NLRP3 interaction and inducing IL-1β production [24]. As shown in Figure 5D,E, the HF diet significantly up-regulated cytoplasmic TXNIP protein levels accompanied by decreased nuclear TXNIP levels in adipose tissue from WT HF mice, but not in adipose tissue from Fat-1 HF mice. Previous studies demonstrated that the Nrf2 activity is inhibited by GSK-3β, whereas the inhibitory activity of GSK-3β was blocked by being phosphorylated by Akt directly or AMPK indirectly [25,26]. As shown in Figure 5F,G, the HF diet markedly decreased the phosphorylation of Akt, AMPK and GSK-3β in adipose tissue of WT HF mice, whereas AMPK phosphorylation was partially and GSK-3β phosphorylation was completely restored in adipose tissue of Fat-1 HF mice. Meanwhile, Akt phosphorylation was significantly upregulated in the adipose tissue of fat-1 mice compared with that of WT mice.

Because IL-1β was secreted from nonadipocytes of adipose tissue such as preadipocytes (Figure 3C), we next explored the underlying mechanisms by which endogenous n-3 PUFAs antagonize HF diet-mediated NLRP3 inflammasome activation using 3T3-L1 murine preadipocytes. PA is the main saturated fatty acid in plasma and the HF diet, and DHA is the most abundant n-3 PUFA in adipose tissue of fat-1 mice. Therefore, these two fatty acids were used in the following mechanistic studies. To confirm whether reduced GSK-3β phosphorylation in vivo by HF diet can be restored by endogenous n-3 PUFA-mediated Akt phosphorylation in adipose tissue from Fat-1 HF mice, 3T3-L1 preadipocytes cultured in vitro were treated with PA or DHA alone or in combination with PI3K inhibitor alpelisib. As shown in Figure 6A,B, the phosphorylation levels of AMPK, Akt and GSK-3β were significantly reduced with PA treatment, whereas AMPK, Akt and GSK-3β were unchanged. In addition, DHA-treated 3T3-L1 cells exhibited higher Akt phosphorylation levels compared with Ctrl or PA-treated cells, consistent with those findings in adipose tissue. As expected, alpelisib treatment markedly abrogated DHA-mediated Akt phosphorylation. Correspondingly, GSK-3β phosphorylation was significantly suppressed in alpelisib-treated cells and remained comparable to that in PA-treated cells, suggesting GSK-3β phosphorylation is tightly regulated by Akt activity. However, AMPK phosphorylation was hardly affected by alpelisib treatment. Simultaneously, alpelisib treatment can rescue IL-1β secretion from 3T3-L1 preadipocytes in the presence of DHA (Figure 6C). We further assessed whether reduced Akt phosphorylation observed in adipose tissue of WT HF mice or in PA-treated cells was mediated by AMPK, 3T3-L1 preadipocytes were treated with PA or a widely used pharmacological AMPK activator, 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) alone or in combination. As shown in Figure 6D,E, AICAR reversed PA-mediated decreased phosphorylation levels of AMPK and Akt, suggesting that PA-mediated decreased AMPK activity, at least partly, contributes to reduced Akt phosphorylation.

The inhibitory effect of GSK3β on the Nrf2/Trx1 antioxidant pathway is reduced by its low expression or being phosphorylated by other kinases [27]. To assess whether GSK-3β plays a pivotal role in which DHA antagonizes PA-mediated NLRP3 inflammasome activity, we selectively knocked down GSK-3β expression using GSK-3β specific shRNAs. GSK-3β protein expression was significantly suppressed following transfection with the GSK-3β shRNAs, but not with the control shRNA (Figure 7A). PA-mediated caspase-1 activity was inhibited by DHA in 3T3-L1 preadipocytes transfected with control shRNA or by knockdown of GSK-3β in cells transfected with GSK-3β shRNA (Figure 7B). In addition, PA suppressed the protein expression of Nrf2 and Trx1, which was reversed by DHA or by knockdown of GSK-3β with GSK-3β shRNA, whereas the expression of NLRP3 was comparable across groups (Figure 7C,D). Simultaneously, PA-induced ROS and cytoplasmic translocation of TXNIP accompanied by increased IL-1β secretion were also abrogated by DHA or by knockdown of GSK-3β (Figure 7C,E–G). These results demonstrated that DHA suppresses PA-induced NLRP3 inflammasome activity by blocking the inhibitory activity of GSK-3β.

All animal studies were performed in accordance with the use and care of laboratory animals and approved by the Ethics Committee of Ningbo University (Ningbo, China). The fat-1 transgenic mice were kindly provided by Dr. Jing X. Kang at Massachusetts General Hospital and Harvard Medical School (Boston, Massachusetts, the United States of America). Six-week-old male heterozygous fat-1 (n = 24) and non-transgenic littermate controls (WT, n = 24) were fed a control (Ctrl) diet or a HF diet for 4 months until sacrifice. Mice were maintained under specific pathogen-free conditions in standard cages in temperature- and humidity-controlled conditions with a 12 h light/dark cycle. The Ctrl diet contained (g/100 g diet) 4.5 g sucrose, 18.6 g casein, 8.6 g cellulose, 50 g wheat starch, 0.3 g DL-methionine, 7 g mineral mix, 1 g vitamin mix, and 10 g safflower oil. The HF diet contained (g/100 g diet) 4.5 g sucrose, 18.6 g casein, 8.6 g cellulose, 15 g wheat starch, 35 g lard, 0.3 g DL-methionine, 7 g mineral mix, 1 g vitamin mix, and 10 g safflower oil.

Oral glucose tolerance test (OGTT) and insulin tolerance test (ITT) were performed as described previously [40]. Briefly, for the OGTT, mice were fasted for 16 h, followed by a gavage of glucose (1.5 g kg⁻¹). For the ITT, mice were fasted for 4 h and injected intraperitoneally with insulin at a final concentration of 0.75 U/kg body weight. Blood glucose concentrations were determined immediately at the indicated time points (i.e., at 0, 30, 60, 90 and 120 min) before and after a glucose/insulin challenge with a commercial glucose meter.

Epididymal adipose tissues were isolated and washed in cold PBS. These tissue fragments were then cultured in 12-well plates in opti-MEM medium (100 mg/mL) supplemented with 1% penicillin/streptomycin. After 24 h, culture media was collected and analyzed for IL-1β and IL-18 according to the manufacturer’s instructions.

Primary stromal vascular fraction (SVF) and adipocytes were fractionated from white adipose tissue from 22-week-old fat-1 or WT mice as described previously [43]. Epididymal adipose tissues from fat-1 or WT mice were isolated, minced, and digested by type II collagenase (2 mg/mL) in Krebs–Ringer bicarbonate buffer containing 10 mM HEPES (pH7.4) and 5% FBS for 30 min at 37 °C with shaking. The collagenase digests were filtered through a 200-mesh strainer and centrifuged at 300× g for 5 min to separate suspended adipocytes and SVF. The SVF cells (1 × 10⁶ cells/mL), and adipocytes (200 μL packed volume/mL) were seeded, cultured in DMEM containing 10% FBS, and 1% penicillin/streptomycin and treated with or without ATP for 24 h. Protein lysates and culture media were prepared for further analysis. Separately, in order to separate preadipocytes from adipose tissue, adipose tissue was digested by type II collagenase (2 mg/mL) in Krebs–Ringer bicarbonate buffer containing 10 mM HEPES (pH7.4) and 5% FBS at 37 °C for least 1 h with shaking. The collagenase digests were centrifuged at 350× g for 5 min to obtain sedimented stromal cells. These stromal cells were then suspended in an erythrocyte-lysing buffer for 5 min, washed and seeded in DMEM containing 10% FBS. Nonadherent cells were removed after 16–20 h for cell attachment.

3T3-L1 pre-adipocytes were purchased from the American Type Culture Collection (Manassas, VA, the United States of America) and cultured in Dulbecco’s modified Eagle’s Medium (DMEM) supplemented with 10% foetal bovine serum (FBS), 1% penicillin/streptomycin.

Adipose tissue from WT or fat-1 mice was isolated, minced, and digested by type II collagenase (2 mg/mL) in PBS for 30 min at 37 °C with shaking. A total of 5% FBS was then added into the collagenase digests to terminate the digestion process. The collagenase digests were filtered through a 200-mesh strainer to remove tissue blocks. The cell suspension or cultured 3T3-L1 preadipocytes were then incubated with 50 μM of DCFH-DA (Beyotime, Jiangsu, China) at 37 °C in a dark place for 30 min. DCF fluorescence intensities were detected by a fluorescence microplate reader (Thermo Fisher Scientific, Massachusetts, MA, USA).

For real-time PCR analysis, total RNA was extracted from adipose tissue using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) in accordance with the manufacturer’s instructions. A total of 1 μg RNA was transcribed to complementary DNA using a reverse transcriptase system (Bio-Rad, Hercules, CA, USA). RT-PCR was performed on an Agilent Mx3000P QPCR System (Agilent, CA, USA) with SYBR Green Supermix (Bio-Rad, Hercules, CA, USA). The following primer was used in this study: IL-1β (forward 5′-CTCACAAGCAGAGCACAAGC-3′, reverse 5′-CTCAGTGCAGGCTATGACCA-3′).

Control shRNA and GSK-3β shRNA lentiviruses were obtained from Xizhao Biotech (Shanghai, China). 3T3-L1 preadipocytes were transfected with control shRNA and GSK-3β shRNA lentiviruses using 5 μg/mL polybrene transfection reagent according to the protocol of the shRNA transfection kit (Xizhao Biotech, Shanghai, China). After 72 h, cells were harvested, and western blotting analysis was used to determine GSK-3β expression.

Adipose tissues isolated from mice were homogenized in ice-cold RIPA buffer without Triton X-100. After low-speed centrifugation (6000× g at 4 °C) for 15 min, the fat cake was removed, and Triton X-100 was added to a final concentration of 1% (v/v). 3T3-L1 preadipocytes were directly homogenized and lysed in Triton X-100 containing buffer. The extracts were sonicated and centrifuged at 12,000× g at 4 °C for 15 min. Protein concentrations were determined by a bicinchoninic acid (BCA) protein assay kit (Biyuntian Biotech, Haimen, China). Protein extracts were separated by 10% SDS-PAGE and transferred onto nitrocellulose membranes (Millipore, Massachusetts, MA, USA). Membranes were blocked in 5% fat-free milk at room temperature for 1 h and incubated with primary antibodies overnight at 4 °C, followed by incubation with the HRP-conjugated secondary antibodies for 1 h at room temperature. Proteins were visualized by chemiluminescence reagent ECL. The protein band densities were quantified using Image-Pro Plus 6.0 (IPP 6.0) software (Media Cybernetics, Massachusetts, MA, USA). The primary antibodies used in this study: rabbit anti-pro-IL-1β, rabbit anti-IL-1β, rabbit anti-pro-caspase-1, rabbit anti-cleaved caspase-1, rabbit anti-TXNIP, rabbit anti-Akt, rabbit anti-phosphor-Akt (Thr308), rabbit anti-NLRP3, rabbit anti-AMPK, rabbit anti-phosphor-AMPK (Thr172), rabbit anti-GSK3β, rabbit anti-phosphor-GSK3β (Ser9), rabbit anti-Nrf2, rabbit anti-Trx1, mouse anti-β-actin, and the HRP-conjugated secondary antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA).

The caspase-1 activity in the lysates of SVF, 3T3-L1 preadipocytes and adipose tissue preadipocytes was determined using a caspase-1 fluorometric kit (Biovision, San Francisco, CA, USA) as previously described [44]. Cells were lysed in hypotonic cell lysis buffer on ice for 10 min and centrifuged to remove the insoluble fraction (12,000× g, 10 min). A total of 50 μL SVF lysate was incubated with 50 μM peptide YVAD-AFC and reaction buffer (50 μL) for 2 h. The fluorescence of the cleaved substrate was measured using a fluorometer at set time intervals.

The fatty acid composition in adipose tissues and adipose tissue preadipocytes was determined by gas chromatography, as described previously [34].

For histological investigations, adipose tissues were fixed in 4% paraformaldehyde in phosphate buffer overnight and then embedded in paraffin for hematoxylin and eosin (H&E) staining.

Data were expressed as mean ± SEM and statistical differences were evaluated by one-way ANOVA followed by Newman–Keuls test. Statistical significance was presented as p < 0.05.

Raw data available upon reasonable request.