Long-term oltipraz administration prevents HFD feeding-induced whole-body insulin resistance
Insulin resistance in C57BL/6J mice has often been induced in the short term by feeding a diet containing poly-saturated fatty acid (45%) or with a mixed fatty acid diet with 60% energy from fat. Here we employed soybean oil as the fat source in the HFD. In addition, the fat content of the HFD was relatively low (45%). In this HFD mouse model, the development of obesity and insulin resistance was slow (see below). The mice were fed with LFD, HFD or HFD plus oltipraz for 28 weeks. We measured levels of plasma glucose, insulin, leptin, adiponectin, triacylglycerol and HDL. As shown in Table 1, the fasting blood glucose level, total cholesterol and NEFA showed no significant differences among the three groups of animals, while neither HFD nor oltipraz administration had any significant effect on plasma HDL or adiponectin levels. However, oltipraz significantly attenuated HFD-induced elevation of plasma insulin and leptin levels. The triacylglycerol level was significantly higher in HFD mice than in LFD mice, while oltipraz administration showed a trend towards reducing plasma triacylglycerol content. Together, although the deleterious effects of HFD were modest and not all variables examined showed significant alterations, the protective effect of oltipraz was appreciable.
To evaluate the functional impact of long-term oltipraz administration on whole-body glucose metabolism, we conducted an intraperitoneal glucose tolerance test (IPGTT). A representative IPGTT performed at 20 weeks is presented in Fig. 1a. Blood glucose levels were higher in HFD mice than in LFD mice after glucose challenge, while oltipraz treatment significantly improved glucose tolerance in the former. To determine insulin sensitivity more directly, an intraperitoneal insulin tolerance test (IPITT) was performed. Insulin was less effective in lowering glucose level in the HFD mice, while oltipraz administration significantly enhanced insulin-mediated glucose-lowering (Fig. 1b). We then assessed whether the liver was responsible for the improved glucose disposal using an intraperitoneal pyruvate tolerance test (IPPTT). As shown in Fig. 1c, glucose production following pyruvate administration was significantly enhanced in the HFD animals, while oltipraz administration partially inhibited this effect, indicating that increased liver gluconeogenesis induced by HFD can be inhibited by oltipraz.
Oltipraz administration prevents HFD-induced obesity and lipid accumulation in muscle
In this animal model, the effect of HFD feeding on body weight gain was gradual. A significant difference in body weight appeared at 16 weeks after HFD vs LFD feeding (Fig. 2a). Importantly, oltipraz administration completely inhibited the effect of HFD on body weight gain during the entire experimental period. To specifically examine fat mass, we used MRI. In the LFD-fed animals, the average total fat volume was approximately 3.7 ml. HFD caused an increase to 9.8 ml, while oltipraz treatment of HFD mice reduced this to 4.1 ml (Fig. 2b). Consistent with total fat mass, the weight of epididymal fat pads, representative of visceral fat, was significantly increased by HFD feeding, while oltipraz blocked this stimulation (Fig. 2c). Indeed, the size of adipocytes in HFD-fed mice was greater than in the LFD-fed mice, while oltipraz administration reduced adipocyte size, as shown by haematoxylin and eosin staining (Fig. 2d). Figure 2e shows representative RARE images of cross-sections in the mice with fat displayed as hyperintense tissue.
There was no significant difference in liver weight between LFD and HFD mice, while the oltipraz group showed reduced liver weight compared with the HFD group (ESM Fig. 1a). Liver triacylglycerol content was slightly increased by HFD, while the mice treated with HFD and oltipraz showed a triacylglycerol level similar to that of LFD mice, although there were no significantly statistical differences between them (ESM Fig. 1b). MRI assessment revealed that total liver lipid content was significantly increased by HFD, while oltipraz tended to block the effect of HFD (ESM Fig. 1c, d). Together, the above observations suggest that this long-term HFD feeding generated a model of moderate insulin resistance, characterised by a significant increase in fat mass and a modest increase of hepatic lipid content. We also observed a significant increase in liver TNFα induced by HFD feeding and a repressive effect of oltipraz administration on this increase (data not shown). However, this increase did not result in increased plasma TNFα in the HFD group (Table 1).
HFD feeding was also shown to increase muscle triacylglycerol content, which was blocked by oltipraz administration (ESM Fig. 2a). We then assessed intra-muscle lipid content by MRI. As presented in ESM Fig. 2b, c, HFD-fed mice had elevated total lipid content in the gastrocnemius muscle, although the increase did not reach statistical significance. Oltipraz showed a trend to reduce lipid content in mouse muscle. There was no apparent pathological change in liver sections of the three groups by haematoxylin and eosin staining, in contrast to sections of corresponding C57BL/6J mouse groups subjected to HFD with 60% energy from fat (ESM Fig. 3). In our experimental animals, oltipraz administration prevented not only HFD-induced obesity, but also lipid accumulation in insulin-sensitive tissues other than the adipose tissue.
Oltipraz prevents HFD-induced impairments in insulin signalling and GLUT4 depletion in adipose tissue
To explore mechanisms underlying the protective effects of oltipraz during HFD feeding, we first investigated insulin signalling by determining PKB Ser473 phosphorylation in response to insulin in adipose tissue, and soleus and gastrocnemius muscles. In adipose tissue, insulin-stimulated PKB phosphorylation was clearly impaired by HFD, while oltipraz administration restored the response (Fig. 3a). However, this impairment was not observed in soleus (ESM Fig. 4a) or gastrocnemius muscles (ESM Fig. 4b). These results support the notion that adipose tissue is the initial site affected by HFD feeding. Thus, the following investigations were focused on the adipose tissue.
Mammalian target of rapamycin (mTOR), along with its downstream targets, p70S6K and 4E-BP1, may function as a negative feedback loop of insulin signalling, while AMPK improves insulin sensitivity by inhibiting the mTOR/p70S6 kinase pathway [25, 26]. In hepatocytes, oltipraz was shown to enhance insulin signalling via AMPK activation-mediated p70S6K inhibition . Here we tested whether the stimulatory effect of oltipraz on AMPK can be detected in our experimental setting in adipose tissue. As shown in Fig. 3b, HFD inhibited AMPK T172 phosphorylation, while oltipraz administration at least partially attenuated the effect of HFD. Correspondingly, the level of phosphorylated acetyl-CoA carboxylase, a known target of AMPK , was barely detectable in HFD-fed mice, while oltipraz administration restored its appearance (Fig. 3b). In addition, insulin-stimulated p-4E-BP1 and p-70S6K levels were higher in HFD-fed mice than in LFD-fed mice, while oltipraz partially attenuated the effect of HFD (Fig. 3c, d).
To further investigate the mechanisms by which oltipraz increases the capacity of mice to lower blood glucose, we examined GLUT4 content in adipose tissue in the three groups of mice. The level of GLUT4, which is responsible for insulin-stimulated glucose uptake , was reduced in HFD mice, while oltipraz administration restored it (Fig. 3e).
Oltipraz reverses HFD-induced NRF2 repression and oxidative stress
To investigate the involvement of oxidative stress and the endogenous NRF2 system in the development of insulin resistance induced by HFD, we first measured plasma GSH:GSSG ratio, an index of redox status. As shown in Fig. 4a, HFD feeding reduced the GSH:GSSG ratio by approximately 27%, while oltipraz administration completely prevented the reduction. In adipose tissue, HFD feeding also significantly reduced the GSH:GSSG ratio, while oltipraz administration showed a partial, although not statistically significant reversal (Fig. 4b). To detect protein carboxylation (an indicator of protein oxidation) in adipose tissue, we used dinitrophenol antibody. As shown in Fig. 4c, the total protein oxidation level was elevated in HFD mice, while oltipraz administration clearly reduced protein oxidation.
We then tested whether endogenous NRF2 machinery in adipose tissue was impaired after HFD feeding. Both nuclear NRF2 levels and the levels of two known targets of NRF2, HO-1 and SOD, were chosen as indicators of the status of the NRF2 system. As shown in Fig. 4d, nuclear NRF2 levels in HFD mice were lower than in LFD mice, while oltipraz partially restored nuclear NRF2 content. Consistently, the levels of HO-1 and SOD were significantly reduced by HFD feeding, while oltipraz restored them (Fig. 4e). These changes were accompanied by increased KEAP1 production following HFD feeding and its reduction by oltipraz administration (Fig. 4f).
Oltipraz prevents HFD-induced inflammation and ER stress in adipose tissue
Oxidative stress affects other redox-sensitive signalling, such as inflammation and ER function. Increased inflammatory signalling and ER stress have been associated with the development of insulin resistance [11, 12, 29]. We did not see significant alterations in plasma TNFα content with HFD feeding or oltipraz administration (Table 1). In fat tissue, however, the content of IκB, a negative modulator of the inflammatory mediator nuclear factor kappa-B, was significantly reduced by HFD feeding and significantly increased by oltipraz administration (Fig. 5a). Furthermore, iNOS, the nitric oxide synthase isoform that is elevated in response to inflammation, was increased by HFD feeding, and this increase was prevented by oltipraz administration (Fig. 5b). This was associated with inhibition of macrophage infiltration by oltipraz administration, as detected by immunostaining using the F4/80 antibody (Fig. 5c). Finally, in adipose tissue, eIF2α Ser51, an indicator of ER stress, was activated by HFD feeding, while oltipraz partially attenuated this effect (Fig. 5d). Neither apparent changes in IκB content, nor alterations of the ER stress indicator eIF2α Ser51 were observed in the liver (data not shown).
Our observations collectively suggest that oltipraz regulates insulin signalling via stimulation of the endogenous NRF2 machinery. For the exploration of underlying mechanisms, extensive in vitro investigations will be needed. Up to this stage, we have conducted Nrf2 siRNA knockdown in a hepatocyte cell line, showing that reduced Nrf2 production led to impaired insulin signalling and attenuated response to oltipraz protection (ESM Fig. 5).