IRW prevents diet-induced non-alcoholic fatty liver disease by preserving mitochondrial content and enhancing hepatic fatty acid oxidation capacity

doi:10.21203/rs.3.rs-3228961/v1

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IRW prevents diet-induced non-alcoholic fatty liver disease by preserving mitochondrial content and enhancing hepatic fatty acid oxidation capacity

https://doi.org/10.21203/rs.3.rs-3228961/v1

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Non-alcoholic fatty liver disease (NAFLD) the hepatic manifestation of the metabolic syndrome, remains without approved pharmacological treatment, with lifestyle modifications being the first line of therapy. Alternative approaches, including food-derived bioactive peptides can aid in the management of metabolic conditions including hypertension, obesity and insulin resistance. IRW is a tripeptide produced from the egg white protein ovotransferrin with angiotensin converting enzyme-inhibitory properties. Previous studies reveal that IRW supplementation elicits antihypertensive effects, improves skeletal muscle insulin signaling and glucose tolerance, while reducing BW gain. In this study, we hypothesized that IRW45 supplementation would prevent high-fat diet-induced NAFLD by modulating hepatic lipid metabolism and preserving mitochondrial content. We found that IRW45 prevents diet-induced NAFLD, while rosiglitazone (ROSI) treatment worsens it. IRW45 decreases hepatic triglyceride content and lipid droplet size compared to HFD and ROSI. This is accompanied by a trend to increase hepatic Ppargc1a gene expression and increase Cd36 compared to HFD. Moreover, IRW45 increases the hepatic mitochondrial complexes, p-AMPKα and has a trend to increase p-ACC abundance compared to HFD. Therefore, IRW45 prevents diet-induced NAFLD, in part by preserving mitochondrial content and increasing hepatic lipid oxidation capacity.

Biological sciences/Physiology/Metabolism

Biological sciences/Physiology/Metabolism/Metabolic diseases

Health sciences/Diseases/Metabolic disorders

NAFLD is a term used to refer to fatty liver (hepatic steatosis) or to non-alcoholic steatohepatitis (NASH), in which inflammation, cell damage and fibrosis are present ¹. If untreated, NASH leads to non-functional hepatic tissue due to fibrosis development (cirrhosis), which can further progress to liver carcinoma and the need of liver transplantation². NAFLD is highly associated with comorbidities within the metabolic syndrome cluster, such as obesity, dyslipidemia and insulin resistance; in fact, NAFLD is considered the hepatic manifestation of the metabolic syndrome ¹.

Many pathways lead to hepatic lipid accumulation, such as increased non-esterified fatty acid (NEFA) availability in the blood, increased hepatic NEFA uptake, de novo lipogenesis (DNL), decreased fatty acid oxidation or reduced very low-density lipoprotein (VLDL) secretion ³. In addition, inflammation, oxidative stress, fibrosis and mitochondrial dysfunction are involved in the progression of liver steatosis to NASH ^4,5. The most accepted theory for NAFLD pathogenesis is the multi-hit hypothesis. Briefly, it states that increased caloric intake leads to obesity and white adipose tissue (WAT) insulin resistance. Insulin resistance impairs lipolysis inhibition in WAT, increasing NEFA in the circulation, which can travel to and be taken up by the liver. Moreover, insulin resistance is associated with WAT dysfunction leading to inflammation and secretion of inflammatory cytokines. In the liver, insulin resistance induces exaggerated DNL, which combined with the extrahepatic influx of NEFA causes lipid accumulation and lipotoxicity (damage caused by lipid mediators). This in turn initiates mitochondrial dysfunction, reactive oxygen species production and endoplasmic reticulum stress. In addition, according to this theory the gut microbiome, genetics and epigenetic factors also play a role in disease initiation and progression ⁶. With chronic exposure to these detrimental effects, hepatocellular damage and inflammation occur, leading to NASH. NASH can progress to fibrosis causing liver cirrhosis, and eventually hepatocellular carcinoma and the need for liver transplantation ^1,3,6.

Lifestyle modifications (diet and physical activity) leading to weight loss are the first line of treatment for NAFLD ⁷. Despite a global effort to find a pharmacological therapy for NAFLD, many clinical trials failed to advance to the next phase due to null results or adverse side effects. For instance, the peroxisome proliferator-activated receptor (PPAR) agonist, elanafibranor, failed to improve NASH after 72 weeks of treatment ⁸. Null results were also observed with a fibroblast-growth factor analog ⁹ and a caspase inhibitor ¹⁰. Although a recent meta-analysis showed that a thiazolidinedione drug (pioglitazone) improves NAFLD in clinical trials ¹¹, thiazolidinedione usage comes with severe side-effects including weight gain and increased risk for cardiovascular disease ^12,13. There are still clinical trials focusing on NASH and drug repurposing in progress, for example, trials using semaglutide and tirzepatide, which are currently used for obesity and diabetes treatment, respectively ⁴. Nevertheless, currently there is no approved pharmacological treatment for NAFLD, which affects more than 25% of the global adult population ¹⁴ and is a leading cause of liver failure and need for transplant ¹⁵.

There is an increasing focus on the potential role of food-derived bioactive peptides to complement the management of metabolic diseases as reviewed elsewhere ^16,17; however, the number of studies validating in vitro findings in animal models or in clinical trials is small. Based on previous evidence from our group, IRW (isoleucine-arginine-tryptophan) holds potential to ameliorate metabolic syndrome conditions in vivo. We showed that IRW, an egg white-derived bioactive peptide with angiotensin converting enzyme (ACE) inhibitory activity in vitro ¹⁸, which increased ACE2 activity in vivo ¹⁹, improved glucose tolerance and insulin signaling in skeletal muscle ²⁰ and decreased blood pressure ^19,21 in rodents. Moreover, IRW modulated skeletal muscle glucose homeostasis in an insulin-independent manner, via 5' AMP-activated protein kinase (AMPK)-α activation ²⁰. In cell culture, IRW reversed tumor necrosis factor (TNF)-α-induced insulin resistance in a skeletal muscle cell line ²² and attenuated the inflammatory response in endothelial cells ²³. Because NAFLD patients exhibit insulin resistance and inflammation in addition to fatty liver, an ideal pharmacological treatment would improve insulin resistance and modulate hepatic lipid metabolism, e.g., by increasing fatty acid oxidation via AMPK activation and preserving mitochondria function. Therefore, a role for IRW in NAFLD management is plausible. In this study we hypothesized that IRW would prevent high-fat diet (HFD)-induced hepatic steatosis in mice by modulating lipid metabolism and preventing hepatic mitochondrial dysfunction.

2.1 Body Weight (BW) and composition

HFD animals gained more weight during the first 6 weeks prior to the peptide supplementation than low-fat diet (LFD) fed animals (Fig. 1A). At week 6, at the start of the peptide supplementation, no differences in BW among the HFD groups were observed (Fig. 1B and C). At week 12, the final BW of LFD (p < 0.0001) and IRW45 (p = 0.0003) groups were lower than HFD groups, while the rosiglitazone (ROSI) group had similar final BW as HFD (Fig. 1D). This was due to lower absolute fat mass in the LFD (p < 0.0001) and slight, but not statistically lower mass in the IRW45 group (p = 0.034), compared to HFD. No differences in lean mass were observed (Fig. 1J).

2.2 Glucose tolerance

Glucose tolerance in the HFD group was impaired compared with LFD (p = 0.007) (Fig. 2A and C, left). ROSI (p = 0.05) and IRW45 (p = 0.02) groups presented improved glucose tolerance compared with HFD as shown in Fig. 2B and by the area under the curve (AUC) (Fig. 2C, right). No changes were seen in the incremental AUC (iAUC) among groups (Fig. 2D). Differences in AUC but not iAUC are consistent with fasting blood glucose concentration being lower in the LFD (p = 0.007), ROSI (p = 0.03) and IRW45 (p = 0.002) groups compared to HFD (Fig. 2E).

2.3 Liver lipid content and NAFLD characteristics

The ROSI group exhibited ~ 50% increased liver weight compared to HFD and IRW45 (p < 0.0001) (Fig. 3A). Liver triglyceride (TG) was decreased in LFD (p = 0.0031) and IRW45 (p = 0.015) groups compared to HFD, while ROSI had increased liver TG content versus HFD (p = 0.016) and IRW45 (p < 0.0001) groups (Fig. 3B). In addition, there was a trend for IRW45 to decrease liver cholesterol content compared to HFD and ROSI (p = 0.06, Fig. 3C) but no differences were observed among the other groups. Quantitative analysis of the lipid droplet (LD) number in liver histological samples showed that IRW45 presented a similar LD number as HFD, but ROSI had ~ 50% more LD than HFD (p = 0.006) and IRW45 groups (p = 0.001) (Fig. 3D). IRW45 (p = 0.0012) had a smaller LD area than HFD, while ROSI had a similar LD area to HFD (Fig. 3E). Overall, IRW45 (p = 0.014) had less area of the total image covered by LD compared to HFD (Fig. 3F), while ROSI had more area covered by LD than IRW45 and HFD groups (p < 0.0001) (Fig. 3F). These data are supported by the qualitative assessment of the histological samples (Fig. 3I). No treatment changes were observed in terms of cell size (Fig. 3G) and inflammatory foci (Fig. 3H). The distribution of LD by number and size is shown in Fig. 3J, supporting that HFD exhibited a right shift in LD area that was even more exaggerated in ROSI.

2.4 Liver lipid metabolism gene expression and protein content

Measurement of hepatic expression of genes involved in lipid metabolism revealed that Cd36 and Cidea expression was upregulated 2-fold and more than 15-fold in the IRW45 (p = 0.013 and 0.0027, respectively) and ROSI (p = 0.071 and 0.001, respectively) groups compared to HFD, respectively. On the other hand, Cpt1a, Mttp and Acaca gene expression was downregulated in the IRW45 (p = 0.017, 0.0135 and 0.029, respectively) group compared to HFD (Fig. 4A). Despite a 50% reduction in Pparg2 expression, it did not reach statistical significance when IRW45 was compared to HFD but had a strong trend to be lower than the ROSI group (p = 0.06) (Fig. 4A). Western blot was conducted to determine if changes in gene expression were confirmed at the protein level. While fatty acid synthase (FAS) protein abundance showed a trend (p = 0.099) to be higher in the IRW45 group and microsomal triglyceride transfer protein (MTP) showed a slight increase (p = 0.03), western blot of other key proteins involved in lipid metabolism (carnitine palmitoyl transferase 1α (CPT1α), PPARγ, total acetyl-CoA carboxylase (ACC) and fatty acid translocase (CD36)) were not different among the HFD groups (Fig. 4B-F). This suggests that some of differences observed in mRNA expression did not correspond to the protein being translated in the IRW45 group or that post-transcriptional regulation may limit protein abundance in these mice. p-ACC protein abundance was enhanced in the IRW45 group compared to ROSI (p = 0.006) and had a strong trend to be increased when compared to HFD (p = 0.06) (Fig. 4E). AMPK subunits were probed to examine a potential insulin-independent regulation of lipid metabolism. p-AMPKβ and total AMPKβ protein abundance did not change among the HFD treatment groups (Fig. 5A-C). p-AMPKα/AMPKα was similar between IRW45 and HFD groups but was increased in IRW45 compared to ROSI (p < 0.01) (Fig. 5B); similarly, p-AMPKα/β-actin was significantly higher in IRW45 compared to the other groups (p < 0.05) (Fig. 5F).

2.5 Mitochondria content and citrate synthase activity

Mitochondrial-related genes Vdac1 (p = 0.01) and Mfn1 (p = 0.06) were downregulated in IRW45 compared to HFD (Fig. 6A), while Opa1 showed a trend (p = 0.098) to be reduced in IRW45 in comparison to HFD. ROSI had reduced Vdac1 compared to HFD (p = 0.036). Both Bax and Ppargc1a gene expression was upregulated in the ROSI compared to HFD (p = 0.025 and 0.048, respectively), while IRW45 showed a trend (p = 0.087) to increased Ppargc1a gene expression compared to HFD. The protein abundance of mitochondrial complexes V, IV, III and II were increased in the IRW45 group compared to HFD (p < 0.05) (Fig. 6B-E) and Complex I showed a consistent, similar trend (p = 0.07) (Fig. 6F). Citrate synthase activity was higher in the IRW45 group than in HFD (p = 0.0037) and a trend to higher (p = 0.06) compared to ROSI (Fig. 6H).

2.6 VAT adipocyte size and adipogenesis markers

ROSI and IRW45 groups had increased epidydimal WAT (eWAT) mass compared with HFD (p < 0.001) (Fig. 7A) but similar adipocyte size as HFD (Fig. 7B). Protein abundance of perilipin 1, fatty acid binding protein 4 (FAB4), PPARγ and adiponectin was similar among the HFD treatment groups (Fig. 7C-F), but a trend to increased PPARγ protein abundance was seen in ROSI vs HFD (p = 0.09) (Fig. 7E). Representative images of the western blots and adipocyte size in eWAT are shown in Fig. 7G and H, respectively.

In terms of retroperitoneal WAT (rWAT), no changes were seen in rWAT mass among the HFD groups (Fig. 7I). Adipocyte size and adipogenesis markers were not different among HFD groups (Fig. 7J-M). Adiponectin was increased in the IRW45 group compared to HFD (p = 0.032) (Fig. 7N). Representative images of the western blots and adipocyte size in rWAT are shown in Fig. 7O and P, respectively.

The complex pathophysiology of NAFLD and the lack of understanding about the triggers leading to the onset of NASH complicates the development of therapeutic agents to treat this condition. Thus, NAFLD currently remains without approved pharmacological treatment. While diet and exercise are recommended to decrease BW and improve hepatic steatosis ⁷, there is keen interest to find novel approaches to complement NAFLD management. The underlying pathogenesis of NAFLD involves multiple detrimental “hits” in an organ crosstalk manner, including insulin resistance in WAT, increased influx of lipids in the liver and DNL, decreased lipid oxidation and export of VLDL. These ultimately cause inflammation, impairment in mitochondria dynamics, ER stress and apoptosis, which culminates in hepatic steatosis, inflammation and NASH ^1,6. We show in this study that a small peptide derived from the egg ovotransferrin protein, namely IRW (isoleucine-arginine-tryptophan), prevented HFD-induced hepatic steatosis, in part by increasing the capacity for fatty acid oxidation via increased abundance of mitochondrial complexes, enzymes involved in fatty acid oxidation and citrate synthase activity. We previously showed that IRW45 improved skeletal muscle insulin signaling and glucose tolerance in HFD-induced obese mice ²⁰, therefore, IRW could be a novel therapy to manage metabolic conditions and prevent one of the first (insulin resistance) and later hits (mitochondrial dysfunction) involved in NAFLD progression.

IRW45 improved histological and biochemical parameters in the liver of obese, glucose intolerant mice. IRW45-treated mice had parallel decreases in hepatic TG content, hepatic LD size and LD-covered area compared to HFD, demonstrating an improvement in hepatic steatosis. On the other hand, rosiglitazone-treated animals had worsened hepatic TG accumulation and LD-covered area. Rosiglitazone, a PPARγ agonist, was included in our study as a positive control for insulin sensitization. Therefore, even though both IRW45 and rosiglitazone improved glucose homeostasis and insulin resistance in our previous experiments ^20,24, only IRW45 prevented HFD-induced liver steatosis. Differently from its effects in humans, rosiglitazone treatment in mice induces hepatic lipid accumulation via PPARγ activation in the liver ^25,26, which is consistent with our findings. Because we previously saw an increase in skeletal muscle PPARγ protein abundance in IRW45-treated animals ²⁰, we wanted to further investigate the effects of IRW45 in the liver and if it would prevent hepatic steatosis differentiating it from the effects promoted by rosiglitazone.

Initially we thought that IRW45 might reduce lipid uptake by the liver, thereby preventing hepatic lipid accumulation, while increasing lipid accumulation in the WAT, as a safer storage depot to prevent metabolic complications ^27,28. However, despite an increase in eWAT in the IRW45 and ROSI groups compared to HFD, absolute fat mass was not different between the IRW45 group compared to HFD at the end of the study. In fact, BW was marginally reduced in the IRW45 group. In addition, the ROSI group had no differences compared to HFD in terms of BW and fat mass. Moreover, individual adipocyte size did not change among the HFD-treated groups, nor did the adipogenesis markers. This suggests that lipid accumulation in WAT is similar among the groups and that IRW45 is probably not inducing adipogenesis to prevent hepatic lipid accumulation. It is worth noting that in the rWAT, adiponectin protein abundance was higher in the IRW45 group compared to HFD, which may contribute to IRW45’s beneficial effects, since adiponectin agonists are shown to improve NASH in rodents ²⁹, and plasma adiponectin is inversely correlated hepatic lipid accumulation in humans ³⁰.

We hypothesized that IRW would modulate hepatic lipid metabolism and mitochondrial content to prevent NAFLD. Although IRW elicited changes in lipid metabolism it seems that, contrary to our initial hypothesis, it was not due to decreased fatty acid uptake because both IRW45 and ROSI groups had upregulated hepatic expression of genes involved in lipid uptake (Cd36) and downregulated expression of genes involved in VLDL assembly (Mttp) and in the transport of fatty acids into the mitochondria (Cpt1a). In addition, gene expression of targets involved in DNL were downregulated or unchanged in the IRW45 group compared to HFD (Acaca and Fasn, respectively). However, total protein content of CPT1α, CD36 and ACC (encoded by Acaca) was similar among the groups, while FAS and MTP protein abundance showed a trend to be increased in the IRW45 group. Moreover, ACC phosphorylation (Ser79) was increased by IRW45. ACC is a rate-limiting enzyme in DNL and its inhibition by AMPKα-mediated phosphorylation of Ser79 is essential to decrease lipogenesis by reducing malonyl-CoA synthesis, thus decreasing hepatic lipid accumulation ³¹. Therefore, enhanced p-ACC in the IRW45 group suggests decreased lipogenesis in this group, which could contribute, in part, to the lower hepatic TG content and smaller LD. Despite a suggested increase in hepatic lipid uptake by the qPCR results, image analysis and protein results support the hypothesis of less lipid accumulation in the IRW45 group.

We then investigated other pathways that could be working to decrease hepatic lipid accumulation, despite increased uptake, in particular fatty acid oxidation. AMPKα is a known lipid metabolism regulator and its activation, besides leading to inhibition of lipogenesis, also induces fatty acid uptake. Via AMPKα-catalyzed phosphorylation of ACC, malonyl-CoA availability is decreased, which relieves inhibition of the rate-limiting enzyme shuttling fatty acid into the mitochondria, CPT1α. Therefore, more fatty acids can enter cells and be directed for β-oxidation as reviewed elsewhere ³². Here we showed that liver Cd36 expression was increased in IRW45 group, while protein amount although increased did not reach statistical significance, suggesting increased capacity for fatty acid uptake. Moreover, we previously found that in skeletal muscle of HFD-fed animals, IRW45 supplementation increased p-AMPKα, an effect not seen in WAT ²⁰. In this study, hepatic p-AMPKα/AMPKα ratio did not change between IRW45 and HFD group, but it was increased compared to ROSI group. Interestingly, p-AMPKα/β-actin and AMPKα/β-actin were more than 50% higher in the IRW45 group compared to HFD, suggesting a greater initial capacity for AMPKα activation ( i.e., V_max), which in turn increases the capacity for activating AMPKα-related targets. In fact, in another study, compared to HFD alone, IRW45 increased hepatic silent mating-type information regulation 2 homolog 1 (SIRT1) protein abundance, a protein known to interact with AMPKα ³³. This effect was accompanied by increased nicotinamide phosphoribosyltransferase (NAMPT) and forkhead box O3 (FOXO3) protein content in the liver and increased nicotinamide adenine dinucleotide (NAD⁺) concentration in the liver and in plasma ³³, consistent with improved metabolic regulation and possibly enhanced mitochondrial oxidative phosphorylation in the IRW45-supplemented mice. This is supported by our finding that citrate synthase activity was increased in the IRW45 group, which is consistent with enhanced hepatic oxidative metabolism. The role of NAMPT and SIRT1 in NAFLD is already shown, for example, SIRT1 is decreased in models of HFD-induced NAFLD ³⁴, while overexpression of SIRT1 prevents hepatic TG accumulation and inflammation ^35,36. On the other hand, pharmacological inhibition of NAMPT worsens hepatic TG accumulation via SIRT1/AMPKα in an HFD-induced hepatic steatosis model ³⁷. In addition, NAD⁺ deficiency induces hepatic steatosis, inflammation, fibrosis and apoptosis in HFD-fed mice, therefore contributing to the progression to NASH ³⁸. Combined, this provides evidence that IRW45 may prevent HFD-induced hepatic steatosis by improving the function of the NAMPT/SIRT1/AMPKα pathway.

SIRT1 also interacts with AMPKα and induces PGC1α activation, which is a key factor in mitochondrial biogenesis ³⁹. Mitochondrial dysfunction is implicated in NAFLD development and progression ⁴⁰. Moreover, HFD decreases mitochondrial complexes (oxphos) and therefore, reduces capacity for fatty acid oxidation and increases lipid accumulation, processes involved in the progression of NAFLD ^41,42. In addition, Ppargc1a overexpression in vitro and in vivo increases fatty acid oxidation, decreases hepatic TG accumulation and circulating TG concentration ⁴³. Our observation of decreased Mttp gene expression, together with a trend for increased hepatic Ppargc1a mRNA and increased content of mitochondrial complexes in the IRW45 compared to HFD, is consistent with the literature. This indicates increased mitochondrial content in the IRW45 group and a higher capacity for fatty acid oxidation leading to reduced hepatic lipid accumulation, as observed.

Mitochondrial dynamics is another factor under debate in relation to NAFLD ⁴⁴. Mitochondrial fission and fusion are essential mechanisms to prevent mitochondrial dysfunction and a balance between these processes maintains healthy mitochondrial function. The process of mitochondrial fusion is regulated mainly by mitofusin-1 and OPA1 and occurs when mitochondrial function is impaired, acting as a protective mechanism to prevent further damage and maintain mitochondrial homeostasis ⁴⁵. Increased fusion leads to enlarged mitochondria, a characteristic seen in the liver of individuals with NAFLD ^46,47 and liver-specific deletion of OPA1 prevents methionine-choline-deficient diet-induced hepatic damage and mitochondrial enlargement ⁴⁸. Conversely, others found that HFD decreases mitofusin-1 ⁴⁹. Moreover, mitofusin-2 plays a key role in transferring lipid between the endoplasmic reticulum and mitochondria, and a role for decreased mitofusin-2 in NAFLD progression is suggested ⁵⁰. We found decreased Mfn1 and a trend for decreased Opa1 gene expression in the liver of IRW45-treated animals compared to HFD, and no changes in terms of Mfn2 expression. These results are consistent with a compensatory effect occurring in the HFD group, whereby mitochondrial fusion is used as a mechanism to stem the increased lipid influx into the liver and prevent further HFD-mediated mitochondrial damage. Conversely, IRW45 treatment enhanced mitochondria content and function, as indicated by higher oxphos enzymes and citrate synthase activity, which accommodated the extra lipid influx and relieved the stress imposed by the HFD, feeding back to lower expression of Mfn1 and Opa1. This is also consistent with the increased lipid accumulation in the ROSI group, which could reflect more lipid storage via PPARγ activation as a protective mechanism against fat deposition in skeletal muscle. Fat accumulation without induction of mitochondria activity would not result in mitochondrial damage but would increase hepatic lipid deposition, as observed. Moreover, Vdac1 mRNA was upregulated in the HFD group. VDAC1 regulates many cellular processes including calcium transport, energy metabolism, apoptosis and inflammation (inducing cytokine release) ⁵¹. Therefore, enhanced expression of Vdac1 in the HFD group may indicate impaired mitochondrial homeostasis, including impaired mitophagy and inflammatory processes. However, future functional assays must be done to investigate mitochondrial membrane potential, permeability, biogenesis, morphological aspects and stability to fully elucidate the role of IRW in this context.

Interestingly, Cidea gene expression was drastically increased in the IRW45 and ROSI groups compared to HFD. Cell death-inducing DNA fragmentation factor-α-like effectors A (Cidea) is a lipid droplet-associated protein. Its expression is regulated by PGC1α ⁵² and its hepatic expression is elevated during NAFLD ⁵³. PPARα agonists increase Cidea expression in the liver ^54,55 and in WAT ⁵⁶. Moreover, PPARγ-dependent hepatic steatosis increases Cidea expression independently of PPARα, whereas choline deficiency-induced steatosis (which is independent of PPARγ activation) does not ⁵⁴. This suggests that both PPARα and PPARγ can directly enhance Cidea transcription and is consistent with the Cidea overexpression in the ROSI group because rosiglitazone is a potent PPARγ agonist. In the liver, Pparg2 gene expression had a strong trend (p = 0.06) to be lower in the IRW group compared to ROSI group, and our histological analysis is consistent with IRW45 not activating hepatic PPARγ. However, Cidea expression was more than 15-fold higher in the IRW45 group as well. Because PPARα upregulates Cidea transcription, it is possible that IRW45 is enhancing fatty acid oxidation via PPARα activation. This would explain, in part, the differences between IRW45 and ROSI groups. However, Ppara expression did not change among the groups and PPAR transcriptional activation was not investigated in this study. Although Cidea and Cidec/FSP27 are induced during liver steatosis, it was recently reported that Cidea expression is reduced during the progression from hepatic steatosis to NASH ⁵³. Thus, it raises the possibility of HFD animals being in a more advanced stage of progression to NASH compared to IRW45 and ROSI.

We cannot ignore the effect of IRW45 in decreasing BW gain, which may indirectly improve NAFLD. As mentioned before, lifestyle changes improve NAFLD mainly through decreased BW ⁷. In this study, IRW45 decreased BW and fat mass (both by approximately 9%) of HFD-induced obese, glucose intolerant mice independently of changes in food intake, consistent with previous results using IRW45 ²⁰. Moreover, we consistently observed improvements in OGTT and fasting blood glucose concentration (²⁰ and this study), demonstrating that IRW45-supplemented animals exhibit a healthier metabolic phenotype compared to HFD animals. In contrast, although rosiglitazone improved OGTT and fasting glucose, it promoted no changes in BW in this study, which is partially consistent with the well-known side-effect of thiazolidinedione to induce weight gain in humans ^57–59, and consistent with the controversial effect of rosiglitazone on BW in studies using rodent models ^26,60,61.

Our findings are, in part, consistent with a recent study published by Liu et al. ⁶². In their study, IRW supplementation for 3 weeks decreased blood glucose concentration, improved glucose tolerance and insulin resistance, without changes in plasma TG concentration ⁶². Moreover, IRW decreased the expression of inflammatory genes in the liver, together with downregulation of Dgat1 and Dgat2 gene expression, supporting the reduced liver TG seen in our study. However, the decrease in liver TG in their study was not statistically significant. Despite no changes in BW, relative eWAT and iWAT mass was reduced by IRW. The authors attributed many of the effects of IRW to changes in the gut microbiota ⁶². The differences seen between our studies may be due to the supplementation period, 8 weeks versus 3 weeks. Moreover, we used a dosage of approximately 45mg/Kg BW based on weekly food intake and BW, while they used a fixed dosage of 0.03 g/L of water. In addition, the authors did not specify the dosage based on BW and water intake nor provide the diet macronutrient content, making it difficult to compare the trials. Nevertheless, in both trials IRW reduces the metabolic complications of HFD, and together suggest that a longer period of supplementation may be required for IRW to promote significant changes in BW and liver TG accumulation.

This is the first study showing that IRW, a small peptide that improves skeletal muscle insulin signaling, is also protective against HFD-induced NAFLD. Although research is still needed to fully elucidate the complete mechanism of action of IRW in this respect, we present evidence that IRW supplementation improves NAFLD via both a enhancement in insulin signaling in skeletal muscle as previously reported ²⁰, and a possible effect in the liver leading to increased capacity for fatty acid uptake and oxidation by preserving mitochondrial content and function. Together, these mechanisms would reduce the substrates for DNL and increase fatty acid oxidation to ameliorate HFD-induced NAFLD. It is worth mentioning that the effects observed after IRW supplementation are, in part, similar to the effects observed with thyroid receptor β-agonists, which have been used in clinical trials to treat NAFLD and promote increased hepatic fatty acid uptake and oxidation, enhanced mitochondrial biogenesis and energy expenditure, and modulation of cholesterol and bile acid metabolism, as reviewed ⁶³. In addition, a recent study using another tripeptide named DT-109 (Gly-Gly-Leu) shows that daily gavage of DT-109 for 12 weeks (mice) and 5 months (nonhuman primates) improves NAFLD by inducing fatty acid oxidation, increasing antioxidant capacity through glutathione biosynthesis and modulating bile acid metabolism ⁶⁴. The most effective dosage of DT-109 was 450 mg/Kg/day in mice ⁶⁴, which is 10-fold higher than the IRW dosage used in our study.

Our study has some limitations, for example the transcriptional activation of PPARs was not investigated and we did not directly measure fatty acid flux or oxidation. In addition, our dietary model, although efficient in promoting hepatic steatosis, is not an established model of NASH and the interpretation of the results should be taken with care if translated in the context of NASH.

In conclusion, while both IRW45 and rosiglitazone improved glucose tolerance and insulin resistance in previous studies ^20,24, our data indicate that IRW45 uniquely acts in the liver to protect against NAFLD by preserving mitochondrial content. This in turn enhances mitochondria oxidative capacity potentially leading to increased fatty acid oxidation, while decreasing lipogenesis, all of which prevent HFD-induced NAFLD (Fig. 8). On the other hand, the evidence for rosiglitazone is consistent with activation of hepatic PPARγ, which induces lipogenic pathways and lipid accumulation as a mechanism to protect against lipid-induced insulin resistance in skeletal muscle and improve systemic insulin sensitivity. Therefore, while both IRW45 and ROSI improve whole body insulin sensitivity, IRW45 has a protective effect against hepatic steatosis while rosiglitazone worsens the HFD-induced lipid accumulation in the liver. The proposed mechanisms of the direct action of IRW in the liver to promote the observed effects are shown in Fig. 8.

4.1 Animals and diet

This study was approved by the Animal Care and Use Committee of the University of Alberta (Protocol #1472) in accordance with guidelines issued by the Canadian Council on Animal Care. Male C57BL/6 mice age 5 weeks (Charles River Canada, St. Constant, QC, Canada) were fed LFD (10% kcal fat) or HFD (45% kcal fat) for 6 weeks to induce obesity and glucose intolerance ²⁴. After that, animals receiving HFD were randomly assigned to continue receiving only HFD or HFD supplemented with IRW at 45 mg/Kg BW or 95 µmol/Kg BW (IRW45) in their diet or HFD + rosiglitazone at 2.5 µmol/Kg BW in their drinking water (ROSI) for 8 weeks. Rosiglitazone was added as a positive control for PPARγ agonism. LFD group received the same diet throughout the entire period. Mice had ad libitum access to food and water and were housed 4 per cage with a 12:12 light cycle, 60% humidity and 23°C temperature. After a total of 14 weeks, after overnight fasting half the animals were injected with (2 IU/kg BW) insulin or saline, then after 10 minutes animals were euthanized using CO₂. IRW was synthesized by Genscript (Piscataway, NJ, USA) and the dosage selected was based on a previous study by our group ²⁰. Rosiglitazone was purchased from Sigma-Aldrich (St. Louis, MA, USA). Diet composition is shown in Table S1.

4.2 BW, body composition and biological samples

Animals were weighed weekly throughout the study. At week 14, body composition was assessed in overnight-fasted mice using ECHO magnetic resonance imaging (ECHO MRI) (Echo Medical Systems LLC, Houston, TX, USA). Blood was collected via cardiac puncture after euthanasia. Liver, eWAT and rWAT adipose tissues were collected, snap frozen in liquid nitrogen and stored at − 80°C. In addition, a sample of each tissue was fixed with buffered formalin and preserved in paraffin blocks for histological analysis.

4.3 Oral glucose tolerance test (OGTT) and fasting glucose

At week 13, all the animals underwent an OGTT as previously described ²⁴. Briefly, a bolus of glucose (1g/kg BW) was orally gavaged to overnight-fasted mice and blood glucose measured from the tail vein using a glucometer (Contour^®Next, Mississauga, ON, CA) at 0-, 15-, 30-, 60-, 90-, and 120-min. Fasting blood glucose was measured after overnight fasting from the tail vein.

4.4 Histological analysis

Paraffin blocks were cut in 5µm sections and the prepared slides stained for hematoxylin & eosin as previously reported ²⁴. Liver characterization: 3 random photomicroscopic images per mice were taken by one researcher (20×, Axio Vision 4.8 software) and a second researcher blinded to group allocation used the ImageJ software “freehand selections” tool to quantify LD area. Cell number and inflammatory foci (clusters with > 5 immune cells) were counted. Each image was divided into 4 equal quadrants and the top left section analyzed. Adipose tissue characterization: 10 random photomicroscopic images per animal were taken (20× objective lens and Axio Vision 4.8 software). Adipocyte area (mm²) was measured using the ImageJ software “freehand selections” tool. A total of 300 cells or 10 images per sample was measured, whichever was reached first.

4.5 Hepatic lipid content

Liver lipid content was extracted as previously described ^24,65. Briefly, 100 mg of tissue was homogenized in 1 mL of NaCl solution. Half of the homogenate (500 mL) was mixed with 2 mL of Folch solution (chloroform:methanol (2:1)), centrifuged at 3000 rpm for 10 min and the lower phase collected. Dried samples were resuspended in 1 mL of 2% TritonX-100 solution in chloroform and dried under nitrogen one more time. The final sample was resuspended in ddH₂O and kept at − 20°C until further analysis. Liver TG and cholesterol content were measured using commercial kits (Infinity^™, Thermo Scientific, Waltham, MA, USA).

4.6 Citrate synthase activity

Hepatic citrate activity was measured in liver lysate using a commercial kit specific for rodents (MAK193, Sigma-Aldrich, St. Louis, MA, USA). 30mg of tissue was homogenized in 300 µL of assay buffer and 25 µL of sample was used in each well following the manufacturer’s protocol.

4.7 Protein extraction and western blot

WAT protein extraction was performed using a commercial kit (AT-022, Invent Biotechnologies, Plymouth, MN, USA), using 100 mg of tissue. Liver protein extracts were obtained using lysis buffer (50 mmol/L Tris HCL pH:8.0, 150 mmol/L NaCl, 0.1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS) supplemented with protease and phosphatase inhibitors (2 µg/mL aprotinin (Calbiochem), 5 mmol/L sodium fluoride, 5 mmol/L sodium orthovanadate, and protease inhibitor cocktail (FastPrep^®-24, MP Biomedicals)). Protein extracts were stored at -80°C. Thawed samples were separated using a SDS-PAGE 8% or 12% polyacrylamide gels, transferred to nitrocellulose membranes as previously described ²⁴ and probed overnight at 4°C for PPARγ (CS2435), perilipin 1 (CS9349), FAS (CS3180), adiponectin (CS2789), oxphos (ab110413), FAB4 (CS2120), ACC (CS3676), p-ACC (Ser79) (CS11818), CPT1α (CS97361), AMPK-β (CS12063), p-AMPKβ (Ser108) (CS23021), p-AMPKα (Thr172) (C2531S), AMPKα (CS2603S) and β-actin (Sigma-Aldrich A5441). The next day membranes were incubated with fluorescent secondary antibodies (Li-cor Biosciences) for 1 h at room temperature. Images were captured using Li-cor scanners and analyzed using Image Studio Lite software (Li-cor Biosciences, Lincoln, NE, USA). Bands that were below of the quantification threshold were given a value of 0 (zero). Total proteins were normalized to β-actin and phosphorylated proteins were normalized to their respective total protein.

4.8 RNA extraction and qPCR

RNA extraction was performed using the QIAGEN RNeasy Mini Plus kit following the manufacturer’s instructions, with the following modifications as previously described ²⁴: 5-100 mg of frozen tissue was homogenized using 1 mL of TRIzol and after 5 min at RT, 0.2 mL of chloroform per mL of TRIzol was added. Samples were incubated at room temperature for 3 min, followed by centrifugation at 12,000 × g for 10 min at 2–8°C. The supernatant was collected, and the manufacturer’s instructions were followed for the remaining steps. RNA concentration was measured using a Nanodrop (Thermo Fisher, Waltham, MA, USA) and cDNA synthesis was performed using the high-capacity cDNA RT kit (Applied Biosystems, Waltham, MA, USA) using 2 µg RNA per reaction. qPCR was performed using PerfeCTa SYBR Green SuperMix ROX (Quantabio, Beverly, MA, USA) in a QuantStudio3 machine (Applied Biosystems, Waltham, MA, USA) using cyclophilin A as the reference gene. Primers sequences are provided in Table S2.

4.9 Statistical analysis and sample size

A sample size of n = 4 (non- or insulin-injected) to n = 8 (all mice)/group was used in this study as specified in the figure captions. Data are presented as means ± SEM. Statistical analysis was performed using GraphPad Prism software 7.0 (GraphPad Software Inc., San Diego, CA, USA). LFD was compared to HFD group using a two tailed t-test to identify glucose intolerance-related differences. HFD, HFD + IRW and HFD + ROSI groups were compared using one-way ANOVA followed by Bonferroni’s post-hoc test to study treatment effects (IRW and rosiglitazone). OGTT was analyzed using 2-way ANOVA followed by Bonferroni’s post-hoc test. Data were checked for normal distribution by the Shapiro–Wilk test and any identified outliers were removed. Data that did not follow a normal distribution was log transformed prior to the statistical analysis. A p-value ≤ 0.05 was considered statistically significant and a p-value < 0.1 was considered a trend.

Acknowledgments: We thank Dr. Robin Clugston for providing the expertise and equipment necessary to conduct this study.

Authors contributions: S.C.C.Z. wrote the manuscript, designed the study, performed in vivo and ex vivo experiments, created the figures, analyzed data and supervised students; E.B. performed experiments; X.J. performed experiments; A.K. performed experiments; E.A. performed experiments; A.G. performed experiments; J.W. designed the study, supervised students, revised and edited the manuscript and acquired funding; C.B.C, designed the study, supervised students, revised and edited the final version of the manuscript and acquired funding.

Data availability: All data generated or analyzed during this study are included in this published article and its supplementary information files.

Conflicts of interest: The authors declare no conflict of interest.

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IRW prevents diet-induced non-alcoholic fatty liver disease by preserving mitochondrial content and enhancing hepatic fatty acid oxidation capacity

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Abstract

Figures

1. Introduction

2. Results

2.1 Body Weight (BW) and composition

2.2 Glucose tolerance

2.3 Liver lipid content and NAFLD characteristics

2.4 Liver lipid metabolism gene expression and protein content

2.5 Mitochondria content and citrate synthase activity

2.6 VAT adipocyte size and adipogenesis markers

3. Discussion

4. Methods

4.1 Animals and diet

4.2 BW, body composition and biological samples

4.3 Oral glucose tolerance test (OGTT) and fasting glucose

4.4 Histological analysis

4.5 Hepatic lipid content

4.6 Citrate synthase activity

4.7 Protein extraction and western blot

4.8 RNA extraction and qPCR

4.9 Statistical analysis and sample size

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