Non-alcoholic fatty liver disease, vascular inflammation and insulin resistance are exacerbated by TRAIL deletion in mice

Non-alcoholic fatty liver disease (NAFLD) incorporates steatosis, non-alcoholic steato-hepatitis (NASH) and liver cirrhosis, associating with diabetes and cardiovascular disease (CVD). TNF-related apoptosis-inducing ligand (TRAIL) is protective of CVD. We aimed to determine whether TRAIL protects against insulin resistance, NAFLD and vascular injury. Twelve-week high fat diet (HFD)-fed Trail −/− mice had increased plasma cholesterol, insulin and glucose compared to wildtype. Insulin tolerance was impaired with TRAIL-deletion, with reduced p-Akt, GLUT4 expression and glucose uptake in skeletal muscle. Hepatic triglyceride content, inflammation and fibrosis were increased with TRAIL-deletion, with elevated expression of genes regulating lipogenesis and gluconeogenesis. Moreover, Trail −/− mice exhibited reduced aortic vasorelaxation, impaired insulin signaling, and >20-fold increased mRNA expression for IL-1β, IL-6, and TNF-α. In vitro, palmitate treatment of hepatocytes increased lipid accumulation, inflammation and fibrosis, with TRAIL mRNA significantly reduced. TRAIL administration inhibited palmitate-induced hepatocyte lipid uptake. Finally, patients with NASH had significantly reduced plasma TRAIL compared to control, simple steatosis or obese individuals. These findings suggest that TRAIL protects against insulin resistance, NAFLD and vascular inflammation. Increasing TRAIL levels may be an attractive therapeutic strategy, to reduce features of diabetes, as well as liver and vascular injury, so commonly observed in individuals with NAFLD.


NAFLD reduces circulating TRAIL and alters TRAIL receptor expression in vitro and in vivo.
To directly delineate the effect of free fatty acids on hepatocytes and TRAIL expression, we used an in vitro model of NAFLD 11 . As expected, treatment of HepG2 cells with BSA-conjugated palmitate significantly increased oil red O staining, which is indicative of triglyceride content (Fig. 2a). This increase in triglyceride-content was also associated with increased expression of inflammatory and fibrotic markers (Fig. 2b,c). In contrast, lipid loading significantly reduced (~50%) TRAIL mRNA expression (Fig. 2d), while DR4 and DR5 expression was elevated, with only DR4 reaching significance (Fig. 2e,f). Importantly, exogenous TRAIL treatment of hepatocytes significantly reduced palmitate-induced lipid uptake in vitro by ~30% (Fig. 2g).
Trail −/− mice display altered plasma chemistries. To assess whether TRAIL deletion alone affects metabolic changes, 12 w HFD wildtype were compared with 12 w HFD Trail −/− mice. In contrast to our previous findings in Trail −/− Apoe −/− vs. Apoe −/− mice 10 , there was no observed difference in body weight (Supplemental Table 2), food intake or total energy expenditure (Supplemental Table 3) between wildtype and Trail −/− mice. 12 w HFD Trail −/− mice did however, display an increase in rearing activity compared to wildtype (Supplemental Table 3). Importantly, Trail −/− had elevated plasma glucose, insulin and cholesterol levels, with a significant reduction in plasma triglycerides when compared to 12 w HFD wildtype mice (Supplemental Table 3). Collectively these findings indicate that HFD-fed Trail −/− mice have a more profound T2D phenotype.  Trail −/− mice have impaired insulin sensitivity. 12 w HFD Trail −/− mice did not respond to an insulin challenge, such that their plasma glucose levels remained markedly higher over 2 h (Fig. 3a). TRAIL-deletion at baseline (0 w HFD) also resulted in reduced sensitivity to insulin at 60 min following an insulin bolus (Fig. 3b). No change in glucose tolerance was observed (Fig. 3c,d). Skeletal muscle from 12 w HFD Trail −/− mice had impaired insulin-induced p-Akt (Fig. 3e), as well as reduced glucose transporter-4 (GLUT4) expression ( Fig. 3f) and glucose uptake ex vivo at baseline (Fig. 3g). Insulin resistance in skeletal muscle of 12 w HFD Trail −/− mice was associated with significantly increased expression of TNF-α (Fig. 3h). No change in monocyte chemo-attractant protein-1 (MCP-1), interleukin-1β (IL-1β), interleukin-6 (IL-6) and TNF-α expression was observed in muscle tissue at baseline (Fig. 3i). Importantly, adipose tissue did not compensate for the skeletal muscle effects, since insulin-inducible glucose uptake was also impaired in WAT ex vivo (Supplemental Fig. 2). These findings suggest that the presence of TRAIL improves insulin sensitivity, and that TRAIL-gene deletion impairs insulin signaling and promotes insulin resistance.
Trail −/− mice have altered glucose and lipid metabolism. Given that TRAIL-deletion promoted a T2D phenotype, we next wanted to examine whether hepatic glucose and lipid metabolism was altered in these mice. While glucose uptake in liver was significantly impaired with TRAIL-deletion at baseline (Fig. 4a), no change in glucose-6-phosphatase or GLUT2 expression was observed (Fig. 4b). Baseline Trail −/− mice challenged with insulin showed significantly increased hepatic phosphoenolpyruvate carboxykinase (PEPCK) mRNA (Fig. 4c), and increased glycogen content (Fig. 4d). Furthermore, there was a significant increase in hepatic expression of 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGCoAR; Fig. 4f), with a trend for increased sterol regulatory element-binding protein-1 (SREBP1; Fig. 4e) expression. These data imply that the presence of TRAIL improves hepatic glucose and lipid metabolism.
Liver from Trail −/− exhibit altered liver pathology in response to a HFD. We next examined liver of 12 w HFD Trail −/− vs. wildtype mice for evidence of NAFLD pathology. Fat engorged hepatocytes were clearly seen in both genotypes (Fig. 5a). Importantly, oil red O staining was significantly elevated with TRAIL deletion (Fig. 5b). 12 w HFD Trail −/− liver also displayed increased fibrosis and apoptosis (Fig. 5c,d), with no change in mDR5 expression (Fig. 5e). Hepatic stellate cell activation plays a key role in the development of fibrosis 13 ; these

Dietary cholesterol is essential for TRAIL-dependent NAFLD/NASH. It has been proposed that
increased dietary cholesterol is critical in the development of NAFLD/NASH in humans 16 and in experimental models 17 . As the HFD employed in this study contains 0.15% cholesterol, we next assessed whether added cholesterol in the diet is critical for TRAIL-dependent NAFLD/NASH. For this we placed wildtype and Trail −/− mice on a lard diet for 12 w; containing a similar fat content to the HFD, without cholesterol. While 12 w lard Trail −/− mice had significantly increased plasma glucose levels compared to wildtype, no changes in body weight, plasma cholesterol, triglycerides, insulin or NEFA were observed (Supplemental Table 4). No change in GTT or ITT was also evident (Supplemental Fig. 3a,b). Importantly, there was no change in hepatic triglyceride content in these mice (Supplemental Fig. 3c). Collectively, these suggest that dietary cholesterol is essential for the development and severity of NAFLD with TRAIL-deletion.

Trail −/− vascular tissue is insulin resistant and displays inflammation.
NAFLD is strongly associated with T2D and vascular injury, with CVD an independent risk factor 3 . Insulin-induced aortic vasodilation was impaired with TRAIL deletion at baseline (Fig. 6a), and more profoundly at 12 w HFD (Fig. 6b). In contrast, acetylcholine (Ach) or sodium nitroprusside (SNP)-induced vasodilation was unaltered (Fig. 6c). Importantly, insulin signaling was impaired, with aortic p-Akt expression in response to insulin, markedly reduced in 12 w HFD Trail −/− mice (Fig. 6d). 12 w HFD Trail −/− mice also had ~50-70% reduced insulin receptor and GLUT4 mRNA (Fig. 6e). The effect of a HFD on TRAIL expression in vascular tissues is not known and low-grade chronic inflammation is important in the development of vascular disease. In response to a HFD, wildtype mice had 2 to 5-fold increases in aortic expression of MCP-1, TNF-α, IL-1β, IL-6 and TRAIL (Fig. 7a). Compared to 12 w HFD wildtype mice however, TRAIL-deletion resulted in marked elevation of aortic TNF-α (22-fold), IL-1β (20-fold) and IL-6 (31-fold) (Fig. 7b). Of note, only vascular MCP-1, IL-1β (Fig. 7c) and VCAM-1 (Fig. 7d) expression were significantly elevated with TRAIL-deletion at baseline. These findings suggest that Trail −/− vessels are insulin resistant and have impaired downstream insulin signals. Moreover, Trail −/− vessels are more susceptible to inflammation, which is accelerated in response to a HFD.

Discussion
The key novel findings of this study are: first, that plasma TRAIL levels are significantly reduced in patients with the severe form of NAFLD (i.e. NASH), negatively associating with plasma ALT, even after adjustment for diabetes, BMI, age and sex. Second, in response to a HFD with cholesterol, mice with TRAIL-deletion develop a marked increase in hepatic steatosis, display altered hepatic cholesterol and glucose homeostasis, and many features of NASH. Mechanistically, free-fatty acid overload in hepatocytes was associated with reduced TRAIL mRNA, with TRAIL administration inhibiting lipid accumulation in vitro. Third, we show that Trail −/− mice are more susceptible to insulin resistance, inflammation, develop T2D and vascular injury in response to a HFD. This is the first report showing that TRAIL protects against hepatic steatosis, inflammation and fibrosis; features of NASH. These findings are significant because increased consumption of energy-dense food has resulted in a pandemic of NAFLD, diabetes and CVD affecting approximately 30% of the world's population.
The clinical importance of TRAIL in people with NAFLD is uncertain, but does, on balance, favor a beneficial effect. One small study examined soluble TRAIL levels in NAFLD patients from China 18 . This study indicated that the C/G allele polymorphisms in the TRAIL gene at position 1525/1595 were higher in NAFLD than healthy individuals, associating with increased serum TRAIL 18 . On the other hand, the same group showed that the AATT genotype frequencies at 1525/1595 were lower with NAFLD, associating with reduced soluble TRAIL levels 18 . Notably, this report was unable to determine the association between TRAIL levels, NASH and simple steatosis. Here, we show that circulating TRAIL levels are significantly reduced in people with NASH but not with obese individuals, and strongly correlate with levels of ALT.
Hepatic steatosis occurs when there is an imbalance in hepatic fatty acid and triglyceride acquisition and removal. The main sources of free fatty acids in the liver are (i) NEFA released from adipose stores; (ii) de novo lipogenesis (e.g. from glucose) involving transcriptional regulation by factors including SREBP-1; and (iii) free fatty acids from dietary intake. Surprisingly, and in contrast to our previous findings in 12 w HFD Trail −/− Apoe −/− mice 10 , no changes in food intake, body weight and adipose tissue weight (not shown) were observed between Trail −/− and wildtype mice. The surplus lipids observed in Trail −/− Apoe −/− (e.g. plasma cholesterol, triglycerides, LDL and VLDL 10 ), may further promote an increase in adipocyte hypertrophy and body weight, not evident with TRAIL-deletion alone. Indeed, excessive lipid can stimulate adipocyte hypertrophy 19 , and promote adiposity in people 20 .
We found no change in plasma NEFA with TRAIL-deletion alone, nor between 12 w HFD Trail −/− Apoe −/− vs. Apoe −/− mice (not shown), suggesting that NEFA released from fat stores does not affect hepatic triglyceride levels in these mice. Increases in hepatic HMGCoAR (and to some extent SREBP-1) with TRAIL deletion, imply that cholesterol and triglyceride synthesis is increased in the liver. Moreover, TRAIL-deletion alone impaired the ability of the liver to remove triglyceride, since more was evident by oil red O staining in HFD Trail −/− mice. Importantly, these changes in Trail −/− mice were only evident in response to a HFD with cholesterol. Of note, dietary fat and cholesterol are known to synergistically interact, enhancing NASH and metabolic changes in mice by an ~2-fold greater extent, than achieved with a fat or cholesterol diet alone 21 ; a finding also supported in humans with NASH 16 . Therefore, added dietary cholesterol in a HFD is essential for the NAFLD/NASH we observe with TRAIL-deletion in mice. In humans, TRAIL signaling occurs upon binding and trimerization of its death receptors, DR4 and DR5. In mice, there is only one TRAIL death receptor, mDR5 with ~60% homology to both DR4 and DR5. Both TRAIL and DR5 are expressed in human hepatocytes in vitro 22 . TRAIL itself can induce apoptosis of normal human hepatocytes at high, non-physiological, concentrations 23 ; with free-fatty acids sensitizing human hepatocytes to TRAIL-mediated apoptosis via DR5 24 . Mice fed a fibrogenic methionine and choline-deficient diet develop NAFLD, with an increase in hepatic mDR5 expression and apoptosis 25 , suggesting that mDR5 signals promote a NAFLD phenotype. Furthermore, Dr5 −/− mice fed a high fat, high sucrose and high cholesterol diet for 3 months, not dissimilar to our HFD, had reduced hepatic steatosis, inflammation and markers of fibrosis 26 . Fas has also been implicated in NAFLD in people 27 , and in experimental models 28,29 . Reduced hepatic steatosis was also observed in mice with Fas deletion specifically from adipocytes 30 . These data imply that TNF ligands may promote NAFLD pathogenesis via their cognate receptors. However, the direct effect of TRAIL in experimental models of NAFLD is unknown. We found that global TRAIL-gene deletion in mice promoted a more severe NAFLD phenotype with increased lipid accumulation, inflammation and fibrosis. This apparently contradictory finding may be explained by ligand-independent receptor activation. While no changes in mDR5 mRNA between genotype in the liver was observed, Trail −/− hepatic tissues had significantly increased apoptosis. Intriguingly, an overload of lipid in hepatocytes promotes DR5 localization into lipid rafts, stimulating cell death independent of TRAIL binding 31 and further, ligand independent hepatocyte apoptosis has also been observed for Fas 32,33 . Whether TRAIL or ligand-independent mechanisms promote liver injury in this setting is unclear and requires further elucidation.
We also found Trail −/− mice were insulin resistant, with TRAIL deletion impairing insulin signaling and glucose homeostasis. PEPCK is an enzyme that controls the rate of glucose synthesis with the ability to modulate plasma glucose levels 34 . Overexpression of PEPCK promotes T2D 34 . The increase in plasma glucose levels and glycogen content in Trail −/− mice may reflect the role PEPCK plays in regulating hepatic glucose (and cholesterol) homeostasis. Collectively, our study implies that TRAIL improves insulin sensitivity by modulating expression of genes involved in glucose metabolism and insulin signaling. Consistent with this, plasma TRAIL levels in T2D patients after gastric banding surgery were increased and correlated with improved β-cell function 35 . TNF ligand regulation of insulin signaling and glucose homeostasis may in fact be a common theme in diabetics, since TNF-α and FasL have also been implicated in insulin resistance 36,37 .
Insulin resistance can also develop in cardiovascular tissues where insulin can contribute to the development of CVD, hypertension and metabolic diseases 38 . In wildtype mice, we observed a significant reduction in circulating TRAIL levels in response to a HFD, correlating with an elevation of vascular expression of IL-1β, MCP-1, IL-6 and TNF-α, pro-inflammatory cytokines known to play key roles in insulin resistance and diabetes 39 . In contrast to circulating TRAIL levels, vascular TRAIL expression (including in liver and fat) was significantly increased in these mice. Discrepancies between circulating vs. tissue TRAIL expression have been observed in other inflammatory diseases. For example, plasma TRAIL levels are reduced in patients with chronic kidney disease 40 , and increased TRAIL in kidneys is associated with disease severity in diabetic nephropathy 41 . While this suggests that TRAIL may play an inflammatory role and promote disease, importantly, our global TRAIL knockout studies in nephropathy 42 and here, demonstrate the opposite. In fact, compared to HFD-fed wildtype mice, TRAIL-deletion resulted in >20-fold elevation of inflammatory marker mRNA in the vessel wall. Increased expression of inflammatory markers was also observed in the liver and in fat (not shown). Thus, Trail −/− mice are more susceptible to inflammation, suggesting that early inflammatory events occur with TRAIL deletion alone. Our findings are of major importance. Rather than promoting inflammation and disease, the presence of TRAIL may in fact exert an overall anti-inflammatory action in damaged or injured tissues, resulting in protection against injury.
This study has revealed a critical role of TRAIL in blunting metabolic diseases. Our findings show for the first time that deletion of TRAIL causes systemic insulin resistance, increases hepatic cholesterol and glucose production and renders the liver more susceptible to triglyceride accumulation and injury, promoting a more severe form of NAFLD in response to a HFD. Importantly, these metabolic changes are associated with increased dysfunction and inflammation in blood vessels. Taken together, we speculate that these findings may translate into a therapeutic use for TRAIL as a novel anti-inflammatory agent with vascular protective actions. Indeed, the therapeutic potential of TRAIL in reducing HFD-induced inflammation, adiposity and improving glucose sensitivity in mice has previously been reported 43 . It is therefore possible that in the future, TRAIL and related agents, may offer new therapeutic options for patients with NAFLD, diabetes and CVD.

Materials and Methods
Human studies. Adults with biopsy-proven NAFLD, obese individuals with normal liver biopsies or healthy controls were recruited. Informed consent was obtained from all subjects. All methods were carried out in accordance with guidelines and regulations from the National Health and Medical Research Council of Australia;  . Healthy controls were defined by the presence of normal liver enzymes and absence of medical conditions requiring medications. Plasma samples were taken following an overnight fast on the day of liver biopsy for patients with NAFLD, or on the day of clinical assessment for healthy controls and frozen at −80 °C. Plasma chemistries were assessed as previously described 44 . Biopsy specimens in NAFLD patients were staged by a liver histopathologist as NASH or non-NASH 45 . Animal studies. Male Trail −/−10 and wildtype mice at 6 w of age, weighing 18-20 g were euthanized for baseline studies, and or randomly grouped and placed on a 'Western' high fat diet (HFD; SF00-219; 22% Fat, 0.15% Cholesterol Semi-Pure Rodent Diet) or the Lard diet (SF04-001; 23.5% Fat Semi-Pure Rodent Diet) for 12 w; both from Specialty Feeds, Glen Forest, Western Australia. After an overnight fast, mice were anaesthetized by i.p. injection of ketamine (100 mg/kg) and xylazine (10 mg/kg), or isoflurane (2%) via nose cone prior to euthanasia by cardiac exsanguination. Blood was collected and plasma stored at −80 °C. Gastrocnemius muscle, epididymal WAT, liver, and aorta were collected, fixed in formaldehyde for immunohistochemistry (IHC) or snap-frozen for gene and protein expression. All methods involving animals were carried out in accordance with guidelines and regulations from the National Health and Medical Research Council of Australia; experimental protocols were approved under the Animal Care and Ethics Committees at the University of New South Wales (11/71B) or the Sydney Local Health District (2013/049), Sydney Australia.
Metabolic cages. The Comprehensive Laboratory Animal Monitoring System (Columbus Instruments, Columbus, OH) was used at 12 w HFD. Oxygen consumption (VO 2 ) and CO 2 production (VCO 2 ) were normalized to body weight. The respiratory exchange ratio was calculated (VCO 2 /VO 2 ). Ambulatory activity was examined where horizontal (XAMB) and vertical (ZTOT) movement were determined. Food intake was measured. All measurements were taken over 24 h. Plasma analysis. Plasma  Glucose and insulin tolerance tests. Glucose and insulin tolerance tests (GTT, ITT) were performed as previously described 42 ; ITTs were performed in non-fasted mice.
Quantification of lipids from frozen liver tissue. Triglyceride accumulation from frozen liver was quantified as described 49 . Tissue Culture. HepG2 cells obtained from ATCC were maintained in Dulbecco's modified Eagle's medium supplemented with 10% (v/v) fetal bovine serum, penicillin (5 U/mL), streptomycin (5 μg/mL) (Lonza) and 200 mM L-glutamine (Lonza) in a humidified atmosphere of 5% CO 2 at 37 °C. Cells were treated with bovine serum albumin-conjugated palmitate (Seahorse Bioscience, Massachusetts USA) to mimic NAFLD in vitro 11 . RNA extraction, cDNA synthesis and qPCR. RNA was extracted from homogenized tissue using the All-Prep DNA/RNA/protein mini kit (Qiagen, Valencia, CA), or TRI reagent (Sigma) for cells. cDNA synthesis and real-time qPCR were performed as described 10,50,51 . Relative mRNA expression was normalized to housekeeping genes HPRT, β-actin,18S or GAPDH 52 . Primer sequences are shown in Supplemental Table 5.
Scientific RepoRts | 7: 1898 | DOI:10.1038/s41598-017-01721-4 Statistics. Results are expressed as mean ± SEM, unless stated otherwise, and analyzed using GraphPad Prism Version 6.0 (GraphPad Software, San Diego, CA, USA) or SSPS (IBM, version 21.0). Statistical comparisons were assessed with Student t-test, Mann Whitney U-test, or ANOVA (one-or two-way) with Bonferroni adjustment for multiple comparisons. Chi-squared test was also used where indicated. Bivariate correlation was assessed between human serum TRAIL and clinical and biochemical factors using Pearson or Spearman correlation coefficients, according to the distribution of data, with adjustment of p values using Bonferroni correction for multiple testing. The relationship between plasma TRAIL and ALT levels was further evaluated using multivariate linear regression analysis with adjustment for possible confounders. A value of p < 0.05 was considered significant.