Tip60-mediated Rheb acetylation links palmitic acid with mTORC1 activation and insulin resistance

  1. Zengqi Zhao
  2. Qiang Chen
  3. Xiaojun Xiang
  4. Weiwei Dai
  5. Wei Fang
  6. Kun Cui
  7. Baolin Li
  8. Qiangde Liu
  9. Yongtao Liu
  10. Yanan Shen
  11. Yueru Li
  12. Wei Xu
  13. Kangsen Mai
  14. Qinghui Ai

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Abstract

Differences in dietary fatty acid saturation impact glucose homeostasis and insulin sensitivity in vertebrates. Excess dietary intake of saturated fatty acids (SFAs) induces glucose intolerance and metabolic disorders. In contrast, unsaturated fatty acids (UFAs) elicit beneficial effects on insulin sensitivity. However, it remains elusive how SFAs and UFAs signal differentially toward insulin signaling to influence glucose homeostasis. Here, using a croaker model, we report that dietary palmitic acid (PA), but not oleic acid or linoleic acid, leads to dysregulation of mTORC1 signaling which provokes systemic insulin resistance and glucose intolerance. Mechanistically, using croaker primary myocytes, mouse C2C12 myotubes and HEK293T cells, we show that PA-induced mTORC1 activation is dependent on mitochondrial fatty acid β oxidation. Notably, PA profoundly elevates acetyl-CoA derived from mitochondrial fatty acid β oxidation which intensifies Tip60-mediated Rheb acetylation. Subsequently, the induction of Rheb acetylation facilitates hyperactivation of mTORC1 which enhances serine phosphorylation of IRS1 and simultaneously inhibits transcription of IRS1 through impeding TFEB nuclear translocation, leading to impairment of insulin signaling. Furthermore, targeted abrogation of acetyl-CoA produced from fatty acid β oxidation or Tip60-mediated Rheb acetylation by pharmacological inhibition and genetic knockdown rescues PA-induced insulin resistance. Collectively, this study reveals a conserved acetylation-dependent mechanistic insight for understanding the link between fatty acids and insulin resistance, which may provide a potential therapeutic avenue to intervene in the development of T2D.

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    Reply to the reviewers

    Reviewer #1:

    1. The authors claim PA-induced mTORC1 activation is dependent on acetyl-CoA derived from mitochondrial fatty acid oxidation. Using isotope tracing to determine the contribution of PA to acetyl-CoA, would improve.

    Response: We thank the reviewer for this valuable comment. As suggested, we measured the contribution of PA to the total cellular acetyl-CoA pool using metabolic flux assays. The results showed that the incorporation of 13C from [U-13C16]-palmitate into acetyl-CoA was exceeding 60% (Page 40, Figure 4D in the revised version), indicating that exceeding 60% of the acetyl-CoA pool was PA derived. Likewise, we also found that the incorporations of 13C from [U-13C16]-palmitate into 6:0-CoA, 8:0-CoA, 10:0-CoA and 12:0-CoA were all exceeding 50% (Page 40, Figure 4D in the revised version). Thus, these results suggested that a large portion of the PA was used for fatty acid oxidation upon entering the cell. Moreover, we found that fatty acid β oxidation blocked by perhexiline maleate inhibited PA-induced increase of acetyl-CoA, suggesting that the induction of acetyl-CoA content was largely dependent on the fatty acid oxidation of PA. Furthermore, we also demonstrated that inhibition of mitochondrial fatty acid β oxidation by pharmacological inhibitor or genetic knockdown abrogated PA-induced activation of mTORC1. However, using sodium acetate treatment to elevate cellular acetyl-CoA levels rescued impaired mTORC1 activity induced by the inhibition of fatty acid β oxidation under PA condition. Together, these results revealed that PA-induced mTORC1 activation is dependent on acetyl-CoA derived from mitochondrial fatty acid oxidation.

    1. They showed PA increases fatty acid oxidation related gene expression and acetyl-CoA level, while OA and LA could not. why only PA could increases fatty acid oxidation and acetyl-CoA level, considering both of these lipids could be oxidation in mitochondria? Is there any differences in mitochondria among treatment of PA, OA and LA? It is better to monitor fatty acid oxidation in real time using seahorse. And add discussions.

    Response: We thank the reviewer for this constructive question. As suggested, we performed the seahorse real-time cell metabolic analysis and the results showed that PA treatment enhanced mitochondrial OCR and elevated maximal oxygen consumption rates compared with OA or LA treatment in fish myocytes (Page 37, Figure 3B in the revised version). Likewise, we also found that PA-induced increase of fatty acid oxidation-related gene expressions was more robust than OA or LA in vivo and in vitro. Thus, these results indicated that the induction of mitochondrial fatty acid oxidation by pa treatment was stronger than OA or LA treatment.

    In this study, using LC–MS, we showed that PA treatment increased the contents of short/medium-chain acyl-CoA and acylcarnitine in comparison with OA or LA treatment. Thus, these results suggested that although all three fatty acids can be oxidation in mitochondria, PA may be preferred to enter the mitochondria for fatty acid β oxidation, compared with OA or LA. Previous studies have found that OA is more inclined to synthesize triglycerides to induce the formation of lipid droplets than PA (Chen et al., 2023; Plötz et al., 2016). Likewise, we also found that OA significantly increased the contents of 18:1-CoA in comparison with PA. Thus, we speculate that, after entering the cell, OA is more preferentially synthesized to triglyceride for storage than fatty acid oxidation. Moreover, LA is considered to be a precursor of arachidonic acid, and can be converted to a myriad of bioactive compounds called eicosanoids (Whelan & Fritsche, 2013). Similarly, we found that LA markedly elevated the contents of 18:2-CoA/18:3-CoA. Thus, we conjecture that LA preferentially synthesizes functional lipids compared to entering mitochondria for fatty acid oxidation. Together, differences in the levels of acetyl-CoA produced by these three fatty acids may be related to their metabolic pathway preferences.

    There may be two reasons for why PA prefers to enter mitochondrial for fatty acid oxidation. On one hand, due to differences in the structure of PA, OA and LA, the substrate affinity of CPT1B to these fatty acyl-CoAs may be different, that may contribute to the different rates of fatty acid to enter into mitochondria. On the other hand, in contrast to the β-oxidation of SFAs, the β-oxidation of UFAs requires the involvement of 2,4-dienoyl-CoA reductase (You et al., 1989), and thus the β-oxidation of SFAs may be more efficient.

    At present, the understanding of differences in fatty acid oxidation between SFAs and UFAs is insufficient, so more studies are needed in the future to further explore the underling mechanisms behind these differences. The reviewers have raised a very important direction for research, and so we will continue to address this issue in future.

    We have expanded this section of the Discussion (Page 14, line 388-412 in the revised version).

    1. The authors present lots of western blot images, suggest to provide quantification data of these blots.

    Response: We thank the reviewer for their careful assessment of our study. We apologize for not providing quantification data for western blot images in our initial manuscript. To support our conclusions, we have now added a densitomentric and statistical analysis of all western blots in the revised version (Page 33, Figure 1 in the revised version; Page 35, Figure 2 in the revised version; Page 37, Figure 3 in the revised version; Page 40, Figure 4 in the revised version; Page 43, Figure 5 in the revised version; Page 45, Figure 6 in the revised version; Page 47, Figure 7 in the revised version; Page 51, Figure S1 in the revised version; Page 53, Figure S2 in the revised version; Page 54, Figure S3 in the revised version; Page 56, Figure S4 in the revised version; Page 57, Figure S5 in the revised version).

    1. It is better to discuss the relationship between fatty acid oxidation and mTOR signaling.

    Response: The reviewer’s comments are very valuable. We apologize for not discussing enough for the relationship between fatty acid oxidation and mTOR signaling in our initial manuscript. We have now expanded this section of the Discussion (Page 13, line 366-387 in the revised version, see below).

    Growing lines of evidence suggested a strong link between mitochondrial fatty acid oxidation and mTORC1 signaling (Ricoult & Manning, 2013). As a central regulator of anabolism, mTORC1 is considered to inhibit fatty acid β oxidation pathway for energy storage or ketogenesis (Aguilar et al., 2007). Several studies revealed that restrained mTORC1 by rapamycin induced fatty acid β oxidation in rat hepatocytes through increasing expression of fatty acid β oxidation related enzymes (Brown et al., 2007; Peng et al., 2002). Likewise, mice with whole-body knockout of S6K1 showed enhanced fatty acid β oxidation and increased expression levels of CPT1 in isolated adipocytes (Um et al., 2004), and S6K1/S6K2 double-knockout mice also exhibited elevated fatty acid β oxidation of fatty acids in isolated myoblasts by activating AMPK (Aguilar et al., 2007). Furthermore, a recent study has established that FOXK1 can mediate the inhibition of fatty acid β oxidation by mTORC1 (Fujinuma et al., 2023). Thus, these collective data revealed that fatty acid β oxidation was restrained by mTORC1. However, conversely, the role of mitochondrial fatty acid oxidation in the regulation of mTORC1 is still controversy. A study in prostate cancer cells suggested that inhibited fatty acid β oxidation by etomoxir reduced mTORC1 activity (Schlaepfer et al., 2014), and another study found that deleting CPT1B specifically in skeletal muscle of mice suppressed mTORC1 by provoking AMPK activation (Vandanmagsar et al., 2016). Consistent with these studies, our results showed that acetyl-CoA derived from mitochondrial fatty acid β oxidation induced mTORC1 activation under PA treatment, indicating that acetyl-CoA may be a novel insight linking fatty acid β oxidation and mTORC1 signaling. Paradoxically, unlike other studies, a recent study found that mice with heart-specific CPT2-deficient exhibited induction of mTORC1 pathway. Thus, the effects of fatty acid β oxidation on mTORC1 pathway are complicated and may differ under variable physiological and pathological conditions. Further studies are needed to determine the sophisticated mechanisms underlying the regulation of fatty acid β oxidation on mTORC1 signaling.

    Reviewer #2:

    Major comments:

    Initial experiment: Among several fatty acid-rich diets, fish were fed a palmitic acid (PA) rich (PO) diet for 10 weeks, and the PO diet significantly raised fasting blood glucose levels compared to control diet (fish oil of equal lipid content). The PO diet also impaired the fish's glucose and insulin tolerance. The PO diet also led to decreased phosphorylation levels of AKT, which regulates glucose metabolism. Therefore, the researchers initially concluded that a palmitic acid-rich diet leads to systemic insulin resistance in fish.

    1. I have a couple of questions on this initial experiment on which all the subsequent studies are based. In Figure 1A, the body weight was identical in control and PO group. Don't you expect PO feeding lead to obesity in fish, as HFD induces obesity in mice?

    Response: We thank the reviewer for this constructive question. In our study, we found that dietary PO diet for 10 weeks failed to affect the body weight of fish, compared with CON diet. Similar to our results, a study in human also found that there was no significant differences in the body weight and body mass index (BMI) between saturated fat diet and monounsaturated fat diet (Vessby et al., 2001). Unlike high-fat diet, the lipid content level of PO diet was not elevated, but only the fatty acid composition was altered, with palmitic acid composition being significantly increased in comparison with CON diet (Page 60, Table S1). Thus, this may be the reason of why the PO diet did not induce weight gain.

    Although accumulating evidence showed that the onset of insulin resistance was often accompanied by weight gain and obesity (Kahn & Flier, 2000; Shoelson et al., 2007), some studies also found that insulin resistance occurred without obesity. A recent study found that mice with liver knockout of Lpcat3 exhibited improved insulin sensitivity without a change in the body weight (Tian et al., 2023). Moreover, another study in mice showed that dietary phenylalanine-rich diet induced insulin resistance, but had no effects on the body weight (Zhou et al., 2022). Likewise, our study also found that dietary PO diet provoked systemic insulin resistance, while did not affect the body weight in fish. Thus, these studies indicated that the development of insulin resistance may not always be entirely accompanied by obesity.

    1. Figure 1G and 1H show glucose and insulin tolerance after PO feeding for 10 weeks. The area under curve (AUC) should be compared to determine if GTT and ITT were statistically different. The ITT curve is particularly interesting as the control fish did not seem to respond to insulin, while the PO-fed fish responded more robustly. The only difference is the initial glucose level. Are the GTT and ITT done after fasting? How long is the fasting? The curves suggest that even though PO increased (fasting) blood glucose levels, it improved insulin sensitivity - therefore the premise that PO induces insulin resistance is not supported here. The lack of insulin induced response in the control group is worrisome. I suggest that the measures should be retaken, and AUC should be used to support if there are any differences in GTT and ITT.

    Response: We thank the reviewer for this valuable comment. We apologize for making this confusion in the initial manuscript and we thank the reviewer for providing this opportunity to correct our manuscript. As suggested, to further investigate whether dietary PO could cause impairment of insulin sensitivity, we have re-performed the GTT and ITT assays. Considering that fish have a poor capacity to utilize glucose, we extended the assay time to 8 h. To make the results more accurate, we also added the biological replicates. Moreover, before injection of glucose or insulin, fish were fasted for 24 h. Furthermore, we added area under curve (AUC) of GTT and ITT, and performed statistical analyses of the AUC data.

    Our results showed that dietary PO diet reduced glucose tolerance and insulin tolerance in fish (Page 33, Figure 1G and 1H in the revised version). Moreover, compared with CON diet, the AUC of GTT and ITT were significantly increased in PO diet (Page 33, Figure 1G and 1H in the revised version). Similarly, we found that dietary PO diet elevated fasting blood glucose levels and plasma insulin concentrations. Furthermore, we showed that dietary PO diet decreased the phosphorylation levels of AKT in the liver and skeletal muscle. In addition, we also demonstrated that PA treatment could induce cellular insulin resistance in fish myocytes and C2C12 myotubes. Thus, in our opinion, the above results could indicate that dietary PO induced insulin resistance in fish.

    1. Based on the assumption that PO induces IR (which needs to be confirmed based on the previous comments), the researchers attempted to understand how PA triggers IR through a series of experiments, predominantly western blot analysis. All the Western blots should be quantified. The model is that PA activates FAO in mitochondrial that elevates cytosolic acetyl-coA, which acetylates Rheh to activate mTORC1. mTORC1 on one hand alters IRS1 phosphorylation and on the other hand inhibits transcriptional activity of TFEB to reduce Irs1 mRNA level. Together reduces IRS1 leads to Insulin Resistance.

    Response: The reviewer’s comments were very important to verify the validity of our findings. We have now added a densitomentric and statistical analysis of all western blots in the revised version (Page 33, Figure 1 in the revised version; Page 35, Figure 2 in the revised version; Page 37, Figure 3 in the revised version; Page 40, Figure 4 in the revised version; Page 43, Figure 5 in the revised version; Page 45, Figure 6 in the revised version; Page 47, Figure 7 in the revised version; Page 51, Figure S1 in the revised version; Page 53, Figure S2 in the revised version; Page 54, Figure S3 in the revised version; Page 56, Figure S4 in the revised version; Page 57, Figure S5 in the revised version).

    1. Figure 1. PA reduces basal and insulin stimulated AKT phosphorylation in fish liver and muscle, as well as in culture fish and murine myocytes (Fig. 1I-M). The results appear to be solid but need to be quantified.

    Response: We thank the reviewer for this kind suggestion. We have now added a densitomentric and statistical analysis of all western blots in Figure 1 (Page 33, Figure 1 in the revised version).

    1. Figure 2 shows that PA provoked hyperactivation of mTORC1 (indicated by elevated phosphorylated S6K levels. This effect was abolished by Rapamycin treatment (an mTORC1 inhibitor) and also abolished by insulin stimulation (2F). Again, the western blots should be quantified.

    Response: We thank the reviewer for this excellent suggestion. We have now added a densitomentric and statistical analysis of all western blots in Figure 2 (Page 35, Figure 2 in the revised version).

    1. Figure 6: the researchers measured the effect of PA treatment on IRS1 phosphorylation in order to understand the mechanism of insulin resistance induced by mTORC1 activation under PA treatment. A PO diet intensified S636/S639 phosphorylation in fish muscle. In fish myocytes and C2C12 myotubes, PA treatment elevated S636/S639 phosphorylation but decreased the Y612 phosphorylation of IRS1 in a dose-dependent manner. Treatment of fish myocytes and C2C12 myotubes with an mTOR inhibitor blocked increased IRS1 S636/S639 phosphorylation levels under PA treatment. Also, PA specifically reduced mRNA levels of Irs1. This indicates that PA-induced, mTOR-dependent alteration of IRS1 phosphorylation and transcription may have contributed to insulin resistance. It is unclear how mTORC induces either increase or decrease in IRS1 phosphorylation depending on the residuals.

    Response: We appreciate the reviewers for this important question. In fact, previous studies have clearly explored how mTORC1 pathway affects S636/S639 phosphorylation of IRS1. On one hand, as a kinase complex, mTORC1 could directly induce S636/S639 phosphorylation of IRS1 in vitro (Ozes et al., 2001). On the other hand, mTORC1 could activate S6K to promote S636/S639 phosphorylation of IRS1 (Shah & Hunter, 2006; Um et al., 2004). In addition, considering that the serine/threonine phosphorylation status of IRS has been shown to affect its tyrosine phosphorylation and protein degradation (Copps & White, 2012), we speculate that the decrease of Y612 phosphorylation of IRS1 is dependent on the induction of IRS1 S636/S639 phosphorylation.

    In this study, we found that PA could induce S636/S639 phosphorylation of IRS1 in a mTORC1-dependent manner. Considering that previous studies have explored the mechanism by which mTORC1 induced IRS1 S636/S639 phosphorylation, we did not conduct further studies on this issue. Notably, we found that mTORC1 could also regulate the transcription of IRS1, so we subsequently investigated the mechanism by which mTORC1 inhibited IRS1 transcription.

    1. Figure 7 shows that PA inhibits nuclear translocation of TFEB to suppress IRS1 transcription. The EMSA in 7D is not convincing.

    Response: We thank the reviewer for this valuable comment and we apologize for providing unclear blots in the initial manuscript. To support our conclusions, we have now re-performed the EMSA assays. The results suggested that TFEB can directly bind to the IRS1 promoter at these two sites (Page 47, Figure 7D in the revised version).

    Minor comments:

    1. Some data appears to weaken the results and/or contradictory. For example, the paper initially showed reduced AKT phosphorylation to support PA induced IR, but shouldn't a lower level of pAKT reduces mTORC activation? But then the rest of the manuscript explores how PA activates mTOR. Part of the IR is manifested by impaired mTORC1 activation, yet the PA activates mTORC1. The authors should present the rationale and flow of the ideas in a better way.

    Response: We thank the reviewer for this excellent suggestion. We appreciate the points that in some insulin resistance conditions, as a downstream of the insulin pathway, mTORC1 activity is manifested to be inhibited. However, mTORC1 activity showed different under other insulin resistance conditions.

    In fact, multiple negative feedback signals exist in cells to maintain cellular homeostasis under diverse environmental challenges and stimulations (Kearney et al., 2021). However, aberrant of negative feedback can lead to impaired intracellular signaling pathway and induce a variety of diseases (Nguyen & Kholodenko, 2016). Similarly, numerous negative feedback mechanisms also exist in insulin signaling to prevent the development of cancers that may be induced by hyperactivation of insulin pathway. The negative feedback of insulin pathway is mainly mediated by mTORC1, which has been found to inhibit insulin signaling transduction by directly or indirectly affecting IRS1 phosphorylation (Copps & White, 2012; Shah & Hunter, 2006; Um et al., 2004). However, under some pathological or stress conditions, mTORC1 is over-activated, resulting in the amplification of the negative feedback of insulin pathway and the development of insulin resistance. A recent study found that imidazole propionate, a metabolite produced by the gut microbiota, provoked insulin resistance through inducing mTORC1 activation and phosphorylation of IRS1 (Koh et al., 2018). Other studies also showed that elevated abundance of branched-chain amino acids (BCAAs) or branched-chain α-keto acid (BCKA) could cause insulin resistance by boosting mTORC1 pathway (Zhou et al., 2019). Thus, mTORC1 activation induced-negative feedback inhibition of insulin pathway may be a critical factor in the development of insulin resistance.

    Consistently, our study found that PA could activate mTORC1 in an acetylation modification-dependent manner. Moreover, activation of mTORC1 inhibited the phosphorylation of AKT and caused insulin resistance by affecting the phosphorylation and transcription of IRS1.Indeed, AKT is considered to activate mTORC1 in multiple manners, and inhibition of AKT results in the reduction of mTORC1 activity. However, mTORC1 activity is not only affected by AKT, but is also regulated by a diverse set of upstream signals (Saxton & Sabatini, 2017). Thus, we considered that the activating effect of PA on mTORC1 activity is higher than the negative effect of mTORC1 activity produced by AKT inhibition. This also led to the fact that mTORC1 remained in an activated state despite the inhibition of AKT in PA condition.

    1. There are also many run-on sentences and grammar issues, making it very hard to read. The writing can be improved.

    Response: We thank the reviewer for this valuable comment and we apologize for these grammar mistakes in the initial manuscript. Following the reviewer’s suggestion, we have invited native speaker to guide the English writing and carefully corrected these run-on sentences and grammar issues. We thank the reviewer for this careful evaluation of our manuscript.

    Reviewer #3:

    Major issues affecting the conclusions:

    1. The conclusions are supported by the data. However, I suggest to perform a densitomentric and statistical analysis of western blots, especially when the authors report a representative blot, showing samples loaded in single.

    Response: We thank the reviewer for this excellent suggestion. We agree that it would be important to verify the validity of our findings and we apologize for not providing quantification data for western blot images in our initial manuscript. We have now added a densitomentric and statistical analysis of all western blots in the revised version (Page 33, Figure 1 in the revised version; Page 35, Figure 2 in the revised version; Page 37, Figure 3 in the revised version; Page 40, Figure 4 in the revised version; Page 43, Figure 5 in the revised version; Page 45, Figure 6 in the revised version; Page 47, Figure 7 in the revised version; Page 51, Figure S1 in the revised version; Page 53, Figure S2 in the revised version; Page 54, Figure S3 in the revised version; Page 56, Figure S4 in the revised version; Page 57, Figure S5 in the revised version).

    1. The methods are clear and reproducible. The authors should better explain how they have dissolved all the powders (i.e. fatty acids) to obtain the stock solutions next diluted (from what concentration?) in the media

    Response: We thank the reviewer for this valuable comment. We apologize for not explaining how to dissolved all the powders in the media. As suggested, we have now provided a detailed explanation of how to dissolved all the powders to obtain the stock solutions in the Methods (Page 19, line 537-586 in the revised version, see below).

    For PA, OA or LA in vitro treatment, fatty acid free BSA (Equitech-Bio, USA) was dissolved in FBS-free DMEM at room temperature according the ratio 1:100 (1 g fatty-acid free BSA: 100 ml FBS-free DMEM). 500 mg PA (Merck, Cat#P0500), OA (Merck, Cat#O1008) or LA (Merck, Cat#L1376) was dissolved in 10 ml ethanol to obtain PA, OA or LA stock solution respectively. Then PA, OA or LA stock solution was blow-drying with nitrogen gas and was dissolved in 0.1 M NaOH and warming at 75°C until clear to obtain 100 mM PA, OA or LA solution. Subsequently, 100 mM PA, OA or LA solution was added to 1% BSA solution according the ratio 1:100 (100 mM PA:1% BSA, v/v) at 50°C. Finally, the mixture was filtered using a 0.45 μM filter and stored at -20°C. For insulin in vitro treatment, insulin powder (Merck, USA) was dissolved in hydrochloric acid (pH=2) to obtain 1 mg/ml stock solution. For rapamycin or Torin1 in vitro treatment, rapamycin (Med Chem Express, #HY-10219, USA) or Torin1 (Med Chem Express, #HY-13003, USA) was dissolved in dimethyl sulfoxide (DMSO, Solarbio, China) to obtain 1 mM stock solution respectively. For MHY1485 in vitro treatment, MHY1485 (Med Chem Express, #HY-B0795, USA) was dissolved in DMSO (Solarbio, China) to obtain 10 mM stock solutions.

    For etomoxir or perhexiline maleate in vitro treatments, etomoxir (Med Chem Express, #HY-50202, USA) or perhexiline maleate (Med Chem Express, #HY-B1334A, USA) was dissolved in DMSO (Solarbio, China) to obtain 50 mM stock solution respectively. For BMS-303141 treatment, BMS-303141 (Med Chem Express, #HY-16107, USA) was dissolved in DMSO (Solarbio, China) to obtain 25 mM stock solutions. For sodium acetate treatment, sodium acetate (Merck, #S2889, USA) was dissolved in ultrapure water from a Milli-Q water system to obtain 5M stock solution. For C646, spermidine or MB-3 treatment, C646 (Med Chem Express, #HY-13823, USA), spermidine (Med Chem Express, #HY-B1776, USA) or MB-3 (Merck, #M2449, USA) was dissolved in DMSO (Solarbio, China) to obtain 50 mM stock solution respectively. For MG149 treatment, MG149 (Med Chem Express, #HY-15887, USA) was dissolved in DMSO (Solarbio, China) to obtain 150 mM stock solution. For TFEB activator 1 treatment, TFEB activator 1 (Med Chem Express, #HY-135825) was dissolved in DMSO (Solarbio, China) to obtain 10 mM stock solution.

    1. Anova analysis should be performed to analyze western blot densitometries.

    Response: The reviewer raises an important point and we appreciate this comment. As suggested, we have now added statistical analyses of all western blot densitometries in the revised version. The data are presented as the means ± SEM and were analyzed using independent t-tests for two groups and one-way ANOVA with Tukey’s test for multiple groups (Page 33, Figure 1 in the revised version; Page 35, Figure 2 in the revised version; Page 37, Figure 3 in the revised version; Page 40, Figure 4 in the revised version; Page 43, Figure 5 in the revised version; Page 45, Figure 6 in the revised version; Page 47, Figure 7 in the revised version; Page 51, Figure S1 in the revised version; Page 53, Figure S2 in the revised version; Page 54, Figure S3 in the revised version; Page 56, Figure S4 in the revised version; Page 57, Figure S5 in the revised version).

    Minor comments:

    Prior studies are referenced appropriately, text and figures are clear. I suggest to add in the abstract all the model systems used. HEK293 also should be inserted in the description of the results. Please add the reference to figure 8 in the text. Please, describe cell origin.

    Response: We thank the reviewer for this careful assessment of our study. We apologize for not making this clearer in our initial manuscript. We have now added all the model systems used in the Abstract (Page 2, line 24-28 in the revised version, see below).

    Here, using a croaker model, we report that dietary palmitic acid (PA), but not oleic acid or linoleic acid, leads to dysregulation of mTORC1 signaling which provokes systemic insulin resistance and glucose intolerance. Mechanistically, using croaker primary myocytes, mouse C2C12 myotubes and HEK293T cells, we show that PA-induced mTORC1 activation is dependent on mitochondrial fatty acid β oxidation.

    Moreover, we have now added the description of HEK293T cells in the Results (Page 10, line 261-265 in the revised version; Page 11-12, line 309-315 in the revised version, see below).

    To further investigate whether the regulation of mTORC1 by Tip60 is dependent on the acetylation of Rheb, the interaction between Tip60 and Rheb was analyzed via co-immunoprecipitation assays, and the results showed that Tip60 can interact with Rheb in HEK293T cells (Figure 5E). Moreover, overexpressed Tip60 reinforced the acetylation of Rheb and phosphorylation levels of S6K in HEK293T cells (Figure 5F).

    Dual luciferase experiments in HEK293T cells showed that TFEB had the strongest ability to elevate the luciferase activity of the IRS1 promoter among the crucial downstream transcription factors of mTORC1 (Figure 7A). Moreover, TFEB enhanced the promoter activity of IRS1 in a dose-dependent manner (Figure 7B) and mutations of the predicted TFEB binding site 4 and site 6 in the IRS1 promoter significantly reduced the promoter activity of IRS1 in HEK293T cells (Figure 7C). Furthermore, ChIP and EMSA experiments in HEK293T cells verified that TFEB can directly bind to the IRS1 promoter at site 4 and site 6 (Figures 7D and 7E).

    As suggested, we have added the reference to figure 8 in the Discussion (Page 17, line 483-487 in the revised version, see below).

    In summary, our work unveils an evolutionarily conserved mechanism by which mitochondrial fatty acid β oxidation flux of acetyl-CoA induces mTORC1 activation through enhancing Tip60-mediated Rheb acetylation under PA condition. Subsequently, hyperactivation of mTORC1 boosted serine phosphorylation of IRS1 and inhibited TFEB-mediated transcription of IRS1, leading to insulin resistance (Figure 8).

    As suggested, we have added the description of cell origin in the Methods (Page 19, line 526-527 in the revised version; Page 19, line 533-534 in the revised version, see below).

    Mouse C2C12 myoblast cells were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China).

    HEK293T cells were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China).

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    Referee #3

    Evidence, reproducibility and clarity

    The manuscript by Zhao et al aimed to elucidate the mechanisms by which palmitic acids drives insulin resistance. The authors performed experiments in croaker, fish myocytes and mouse differentiated C2C12. They manage in vivo metabolic assays, enzymatic assays, immunoblot procedures, double luciferase assays, RNA analysis, pharmacological inhibitions and genetic knockdown. By using these model systems and procedures, the authors demonstrate that palmitic acid, but not oleic and linoleic acids, induces systemic and cellular insulin resistance through the hyper activation of mTORC1. They show that palmitic acid stimulates the mitochondrial fatty acid β oxidation, increasing the acetyl-CoA levels which enhances the acetylation of Rheb, a well known activator of mTORC1, by Tip60. Moreover, the authors show that mTORC1, beside reinforcing IRS1 phosphorylation, inhibits nuclear translocation of TFEB, thus preventing IRS1 transcription.

    Major issues affecting the conclusions:

    The conclusions are supported by the data. However, I suggest to perform a densitomentric and statistical analysis of western blots, especially when the authors report a representative blot, showing samples loaded in single.
    The methods are clear and reproducible. The authors should better explain how they have dissolved all the powders (i.e. fatty acids) to obtain the stock solutions next diluted (from what concentration?) in the media
    Anova analysis should be performed to analyze western blot densitometries.
    Minor comments:
    Prior studies are referenced appropriately, text and figures are clear. I suggest to add in the abstract all the model systems used. HEK293 also should be inserted in the description of the results. Please add the reference to figure 8 in the text. Please, describe cell origin.

    Referee Cross-commenting

    All reviewers have requested densitomentric (and statistical) analysis of western blot to prove the strength of the results. This is the major point to be addressed. Other points should also be only discussed.

    Significance

    The study extends the knowledge in the field of fatty acids-induced insulin resistance, that is a field studied by many researchers from many years, but with a lot of unclear mechanisms yet. Thus, the nature of the advance is conceptual and mechanistic. The only limitation is the lack of evidence in human samples/cells.

    Basic researchers and experts in translational medicine will be interested by this research.
    This is the point of view of a basic researcher, mainly interested in the molecular mechanisms underlining type 2 diabetes/obesity and cancer.

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    Referee #2

    Evidence, reproducibility and clarity

    Summary:

    The goal of this study was to investigate how saturated fat induce insulin resistance through activating mTOR. More specifically, the researchers show that palmitate (saturated fat) activates mTORC1 to induce insulin via transcriptional and posttranslational suppression of IRS1. Specifically, the researchers show that PA stimulates FAO to raise cytosolic acetyl-coA levels, promoting Tip60-mediated acetylation of Rheb to activate mTORC1 activity. To study the relationship between mTORC1 activity, fatty acid stimulation and insulin resistance, the researchers decided to conduct their study in fish, as fish are known to be glucose intolerant by nature, making them an appropriate model organism for studying insulin resistance. Mouse C2C12 cell line and fish primary myocytes were also used to validate key results.

    Major comments:

    Initial experiment: Among several fatty acid-rich diets, fish were fed a palmitic acid (PA) rich (PO) diet for 10 weeks, and the PO diet significantly raised fasting blood glucose levels compared to control diet (fish oil of equal lipid content). The PO diet also impaired the fish's glucose and insulin tolerance. The PO diet also led to decreased phosphorylation levels of AKT, which regulates glucose metabolism. Therefore, the researchers initially concluded that a palmitic acid-rich diet leads to systemic insulin resistance in fish.

    I have a couple of questions on this initial experiment on which all the subsequent studies are based. In Figure 1A, the body weight was identical in control and PO group. Don't you expect PO feeding lead to obesity in fish, as HFD induces obesity in mice?
    Figure 1G and 1H show glucose and insulin tolerance after PO feeding for 10 weeks. The area under curve (AUC) should be compared to determine if GTT and ITT were statistically different. The ITT curve is particularly interesting as the control fish did not seem to respond to insulin, while the PO-fed fish responded more robustly. The only difference is the initial glucose level. Are the GTT and ITT done after fasting? How long is the fasting? The curves suggest that even though PO increased (fasting) blood glucose levels, it improved insulin sensitivity - therefore the premise that PO induces insulin resistance is not supported here. The lack of insulin induced response in the control group is worrisome. I suggest that the measures should be retaken, and AUC should be used to support if there are any differences in GTT and ITT.

    Based on the assumption that PO induces IR (which needs to be confirmed based on the previous comments), the researchers attempted to understand how PA triggers IR through a series of experiments, predominantly western blot analysis. All the Western blots should be quantified. The model is that PA activates FAO in mitochondrial that elevates cytosolic acetyl-coA, which acetylates Rheh to activate mTORC1. mTORC1 on one hand alters IRS1 phosphorylation and on the other hand inhibits transcriptional activity of TFEB to reduce Irs1 mRNA level. Together reduces IRS1 leads to Insulin Resistance.

    Figure 1. PA reduces basal and insulin stimulated AKT phosphorylation in fish liver and muscle, as well as in culture fish and murine myocytes (Fig. 1I-M). The results appear to be solid but need to be quantified.

    Figure 2 shows that PA provoked hyperactivation of mTORC1 (indicated by elevated phosphorylated S6K levels. This effect was abolished by Rapamycin treatment (an mTORC1 inhibitor) and also abolished by insulin stimulation (2F). Again, the western blots should be quantified.

    Figure 3: PA treatment increases mRNA expression levels of fatty acid beta oxidation genes in fish myocytes and C2C12 myotubes, and subsequent suppression of CPT1B and CPT2 (rate-limiting enzymes of FAO) inhibited mTORC1 activity and signaling in muscle, C2C12 myotubes and fish myocytes under PA treatment. This suggests PA-induced mTORC1 activation is dependent on mitochondrial FAO. Inhibition of CPT1 improved suppression of insulin stimulated phosphorylation of AKT under PA treatment in fish myocytes and C2C12 myotubes, indicating that mitochondrial FAO is heavily involved in PA-induced mTORC1 activation that contributes to insulin resistance.

    Figure 4: PO diet increases acetyl-CoA levels in muscle and PA treatment increases intracellular acetyl-CoA in a dose-dependent manner in fish myocytes. Inhibition of FAO by perhexiline maleate diminished induction of acetyl-CoA under PA treatment. In vivo dsRNA knockdown of ATP citrate lyase (ACLY, catalyze acetyl-CoA synthesis from mitochondrial citrate) decreased mTORC1 activity in muscle, and inhibition of ACLY in fish myocytes and C2C12 myotubes decreased induction of mTORC1 activity under PA treatment. This indicates that palmitic acid promotes mTORC1 activation through acetyl-CoA that is derived from mitochondrial FAO. PA treatment elevates acetylation of Rheb in a dose-dependent manner. Inhibition of FAO by perhexiline maleate attenuated PA-stimulated Rheb acetylation, while fish myocytes and C2C12 myotubes treated with sodium acetate (which can enhance acetyl-CoA) exhibited enhanced Rheb acetylation. The data indicate that acetyl-CoA produced by FAO activates mTORC1 signaling through increased Rheb acetylation. Phosphorylation of AKT were enhanced in muscle with dsACLY knockdown injection, and sodium acetate addition blocked recovery of insulin-stimulated glucose uptake and phosphorylation levels of AKT by perhexiline maleate under PA treatment. ACLY inhibition promoted insulin stimulated phosphorylation of AKT under PA treatment. So, in terms of acetyl-CoA's role in PA-induced insulin resistance, the data suggest that acetyl-CoA derived from FAO mediates PA-induced mTORC1 activation and insulin resistance.
    Figure 5: Acetyl-CoA can activate lysine acetyltransferases, and the researchers found that mRNA expression of tip60 was elevated in fish myocytes and C2C12 myotubes under PA treatment. Cultured fish myocytes and C2C12 myotubes treated with a Tip60 inhibitor prevented the induction of mTORC1 activity under PA treatment. Tip60 knockdown also blocked PA-induced mTORC1 activation in C2C12 myotubes. The researchers then determined that Tip60 regulation of mTORC1 is dependent on the acetylation of Rheb by studying the interaction between the two via a CoIP assay, which indeed indicated that Tip60 and Rheb interact. Additionally, Tip60 knockdown impaired PA-induced acetylation of Rheb, supporting the notion that Tip60 mediates the acetylation of Rheb under PA treatment. Inhibition of Tip60 attenuated PA-induced suppression of insulin-stimulated glucose uptake in C2C12 myotubes, and inhibition of Tip60 also restored insulin-stimulated phosphorylation of AKT under PA treatment. These data suggest that Tip60 mediates the regulation of Rheb acetylation under PA treatment and may be a novel therapeutic target for insulin resistance.

    Figure 6: the researchers measured the effect of PA treatment on IRS1 phosphorylation in order to understand the mechanism of insulin resistance induced by mTORC1 activation under PA treatment. A PO diet intensified S636/S639 phosphorylation in fish muscle. In fish myocytes and C2C12 myotubes, PA treatment elevated S636/S639 phosphorylation but decreased the Y612 phosphorylation of IRS1 in a dose-dependent manner. Treatment of fish myocytes and C2C12 myotubes with an mTOR inhibitor blocked increased IRS1 S636/S639 phosphorylation levels under PA treatment. Also, PA specifically reduced mRNA levels of Irs1. This indicates that PA-induced, mTOR-dependent alteration of IRS1 phosphorylation and transcription may have contributed to insulin resistance. It is unclear how mTORC induces either increase or decrease in IRS1 phosphorylation depending on the residuals.

    Figure 7 shows that PA inhibits nuclear translocation of TFEB to suppress IRS1 transcription. The EMSA in 7D is not convincing.

    Minor comments:

    Some data appears to weaken the results and/or contradictory. For example, the paper initially showed reduced AKT phosphorylation to support PA induced IR, but shouldn't a lower level of pAKT reduces mTORC activation? But then the rest of the manuscript explores how PA activates mTOR. Part of the IR is manifested by impaired mTORC1 activation, yet the PA activates mTORC1. The authors should present the rationale and flow of the ideas in a better way.

    There are also many run-on sentences and grammar issues, making it very hard to read. The writing can be improved.

    Referee cross-commenting

    Other than lacking quantification of western blots, my major concern is the ITT curve in Figure 1G, which does not support the conclusion that PO induces insulin resistance and therefore the rest of the study is based on a faulty premise. The curve shows that insulin reduced blood glucose much more robustly in the PO group than in the control group, suggesting PO increased insulin sensitivity. Area above curve should be calculated to quantify the difference.

    Significance

    Overall, this research was trying to show that PA-induced IR is dependent on hyper activation of mTORC1. More specifically, acetyl-CoA induces mTORC1 activation under a palmitic acid diet, and this is achieved through Tip60-mediated Rheb acetylation, which ultimately leads to insulin resistance through IRS1 suppression. The study is very mechanistic and important for understanding IR that is associated with diets high in saturated fatty acids and could potentially leads to therapeutic targets for combating insulin resistance and glucose intolerance.

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    Referee #1

    Evidence, reproducibility and clarity

    In this work, the authors showed PA induces hyperactivation of mTORC1 and insulin resistance. They found acetyl-CoA derived from mitochondrial fatty acid oxidation is required for PA-induced mTORC1 activation and insulin resistance by increasing TIP60-mediated Rheb acetylation. They also showed PA induced mTORC1 activation enhances IRS1 phosphorylation and inhibits transcription of IRS1 by impeding TFEB nuclear translocation. Overall, the authors did a lot of experiments to prove that PA causes mTORC1 activation and insulin resistance. The results are generally convincing, and the finding is novel and instructive.

    1. The authors claim PA-induced mTORC1 activation is dependent on acetyl-CoA derived from mitochondrial fatty acid oxidation. Using isotope tracing to determine the contribution of PA to acetyl-CoA, would improve.
    2. They showed PA increases fatty acid oxidation related gene expression and acetyl-CoA level, while OA and LA could not. why only PA could increases fatty acid oxidation and acetyl-CoA level, considering both of these lipids could be oxidation in mitochondria? Is there any differences in mitochondria among treatment of PA, OA and LA? It is better to monitor fatty acid oxidation in real time using seahorse. And add discussions.
    3. The authors present lots of western blot images, suggest to provide quantification data of these blots.
    4. It is better to discuss the relationship between fatty acid oxidation and mTOR signaling.

    Significance

    The finding is interesting and significant.

  5. Version published to 10.1101/2023.08.18.553816v1 on bioRxiv