Verapamil Attenuated Prediabetic Neuropathy in High-Fat Diet-Fed Mice through Inhibiting TXNIP-Mediated Apoptosis and Inflammation

Abstract

Diabetic neuropathy (DN) is a common and severe complication of diabetes mellitus. There is still a lack of an effective treatment to DN because of its complex pathogenesis. Thioredoxin-interacting protein (TXNIP), an endogenous inhibitor of thioredoxin, has been shown to be associated with diabetic retinopathy and nephropathy. Herein, we aim to investigate the role of TXNIP in prediabetic neuropathy and therapeutic potential of verapamil which has been shown to inhibit TXNIP expression. The effects of mediating TXNIP on prediabetic neuropathy and its exact mechanism were performed using high-fat diet- (HFD-) induced diabetic mice and palmitate-treated neurons. Our results showed that TXNIP upregulation is associated with prediabetic neuropathy in HFD-fed mice. TXNIP knockdown improved DN in HFD-induced prediabetic mice. Mechanistically, increased TXNIP in dorsal root ganglion is transferred into the cytoplasm and shuttled to the mitochondria. In cytoplasm, TXNIP binding to TRX1 results in the increased oxidative stress and inflammation. In mitochondria, TXNIP binding to TRX2 induced mitochondria dysfunction and apoptosis. TXNIP isolated from TRX2 then shuttles to the cytoplasm and binds to NLRP3, resulting in further increased TXNIP-NLRP3 complex, which induced the release of IL-1β and the development of inflammation. Thus, apoptosis and inflammation of dorsal root ganglion neuron eventually cause neural dysfunction. In addition, we also showed that verapamil, a known inhibitor of calcium channels, improved prediabetic neuropathy in the HFD-fed mice by inhibiting the upregulation of TXNIP. Our finding suggests that TXNIP might be a potential target for the treatment of neuropathy in prediabetic patients with dyslipidemia.

Figure 1

Effects of verapamil treatment on weight, heart rate, and blood pressure of high-fat diet-fed mice. C57/BL6 mice (n = 10) were fed with normal diet (control) or high-fat diet (HF). HF mice and control mice were treated with verapamil (indicated as HF + VP and VP). The body weight (a), heart ratio (b), diastolic pressure (c), systolic pressure (d), and mean blood pressure (e) of mice were measured at weeks 8, 16, and 34, respectively. Each value represents mean ± SEM (n = 10). ^∗∗∗P < 0.001.

Figure 2

Effects of verapamil on glucose intolerance and serum lipid profiles of HF mice. (a) The curve of ipGTT was shown based on plasma glucose concentrations in the indicated time. At weeks 8, 16, and 34, mice were fasted and injected with glucose. The blood was collected at 0 min, 30 min, 60 min, and 120 min from the tail vein, and the blood glucose levels were measured. (b) The AUC of the plasma glucose response was then calculated according to ipGTT results. (c) The serum levels of TG, TC, HDL-C, and LDL-C were determined. In the end of a 34-week study, four groups of mice were fasted for 12 h, and the blood serums were collected and TG, TC, HDL-C, and LDL-C were measured. Each value represents mean ± SEM (n = 10). ^∗∗P < 0.01; ^∗∗∗P < 0.001.

Figure 3

Effects of verapamil on nerve conduction velocity and protein expressions related to metabolism and inflammation in high-fat diet-fed mice. (a) The MNCV and SNCV were detected. After HF mice exhibited IGT for the first time, four groups of mice were anesthetized, and MNCV and SNCV were measured for 3 times with an interval of 15 min. Each value represents mean ± SEM (n = 10). ^∗∗P < 0.01; ^∗∗∗P < 0.001. (b) The expressions of TXNIP, NLRP3, caspase-1, and IL-1β were detected. The sciatic nerve was isolated, and the protein expressions in sciatic nerve including TXNIP, NLRP3, caspase-1, and IL-1β were determined by western blotting analysis, and the target protein expressions relative to GAPDH expression were displayed on the right. The experiments were performed in triplicate, and each value represents mean ± SD. ^∗P < 0.05; ^∗∗P < 0.01; ^∗∗∗P < 0.001.

Figure 4

The effect of TXNIP inhibition on NLRP3, caspase-1, and IL-1β expressions in palmitate-treated neurons. Dorsal root ganglions were isolated from two seven-week-old normal diet-fed mice, and the neurons were cultivated and divided into seven groups: control, HF, HF + VP (50 μM), HF + VP (100 μM), HF + VP (150 μM), HF + NC, and HF + TXNIP-siRNA. (a) The protein expressions of TXNIP, NLRP3, and caspase-1 were detected through western blotting assay. The target protein expressions relative to GAPDH expression were displayed below. The experiments were performed in triplicate, and each value represents mean ± SD. ^∗P < 0.05; ^∗∗P < 0.01; ^∗∗∗P < 0.001. (b) The IL-1β level in supernatant was determined by ELISA assay. The experiments were performed in triplicate, and each value represents mean ± SD. ^∗∗∗P < 0.001.

Figure 5

The effect of TXNIP inhibition on cell apoptosis and promoted cell viability in palmitate-treated neurons. (a) The changes in the Δψm based on JC-1 staining were measured by a flow cytometer. The intensity of green fluorescence delegates JC-1 monomer which is quantified in FL-1 (X-axis), while the intensity of red fluorescence delegates J-aggregates which is quantified in FL-2 (Y-axis). The proportions of green fluorescence and red fluorescence were displayed on the right. The experiments were performed in triplicate. (b) The activity of caspase-3 was measured by pNA, was released from the caspase substrate Ac-DEVD-pNA, and was reported as OD 405 nM/mg protein. (c) The cell viability was measured by MTT assay. The experiments were performed in triplicate, and each value represents mean ± SD. ^∗P < 0.05; ^∗∗P < 0.01; ^∗∗∗P < 0.001.

Figure 6

The distribution and location of TXNIP in palmitate-treated neurons. (a) The distribution of TXNIP in the nucleus, cytosol, and mitochondria after treating neurons with palmitate-BSA for 24 h, 48 h, and 72 h. The expressions were analyzed by western blotting. The target protein expressions relative to GAPDH expression were displayed on the right. The experiments were performed in triplicate, and each value represents mean ± SD. ^∗P < 0.05; ^∗∗∗P < 0.001. (b) The distribution of TXNIP in neurons after treating with palmitate-BSA for 24 h, 48 h, and 72 h, which was detected under the fluorescence microscope. (c) The interaction of TXNIP with TRX1-Cyto, TRX2-Mito, and NLRP3-Cyto, the interaction of TRX1 with ASK1-Cyto, and the interaction of TRX2 with ASK1-Mito were determined by co-IP analysis after treating with palmitate-BSA for 24 h, 48 h, and 72 h. The target protein expressions relative to input were displayed below. The experiments were performed in triplicate, and each value represents mean ± SD. ^∗∗P < 0.01; ^∗∗∗P < 0.001.

Figure 7

The postulated mechanism of verapamil attenuates the inflammation and apoptosis of neurons induced by palmitate through TXNIP inhibition. (a) Under normal conditions: TXNIP rests in the nucleus. TRX1-ASK1 and TRX2-ASK1 binding in the cytosol and mitochondria maintains low ROS. (b) Under high-fat conditions: verapamil inhibits the expression of TXNIP, which shuttles to the cytosol and mitochondria, binds to TRX1 and TRX2, and then transfers to cytosol to combine NLPR3, eventually inhibiting the oxidative stress and apoptosis induced by palmitate treatment.