LncRNA Snhg3 Aggravates Hepatic Steatosis via PPARγ Signaling

Reviewer #1 (Public Review):

Summary:

In this manuscript the authors investigate the contributions of the long noncoding RNA snhg3 in liver metabolism and MAFLD. The authors conclude that liver-specific loss or overexpression of Snhg3 impacts hepatic lipid content and obesity through epigenetic mechanisms. More specifically, the authors invoke that nuclear activity of Snhg3 aggravates hepatic steatosis by altering the balance of activating and repressive chromatin marks at the Pparg gene locus. This regulatory circuit is dependent on a transcriptional regulator SNG1.

Strengths:

The authors developed a tissue specific lncRNA knockout and KI models. This effort is certainly appreciated as few lncRNA knockouts have been generated in the context of metabolism. Furthermore, lncRNA effects can be compensated in a whole organism or show subtle effects in acute versus chronic perturbation, rendering the focus on in vivo function important and highly relevant. In addition, Snhg3 was identified through a screening strategy and as a general rule the authors the authors attempt to follow unbiased approaches to decipher the mechanisms of Snhg3.

Weaknesses:

Despite efforts at generating a liver-specific knockout, the phenotypic characterization is not focused on the key readouts. Notably missing are rigorous lipid flux studies and targeted gene expression/protein measurement that would underpin why loss of Snhg3 protects from lipid accumulation. Along those lines, claims linking the Snhg3 to MAFLD would be better supported with careful interrogation of markers of fibrosis and advanced liver disease. In other areas, significance is limited since the presented data is either not clear or rigorous enough. Finally, there is an important conceptual limitation to the work since PPARG is not established to play a major role in the liver.

Reviewer #2 (Public Review):

Through RNA analysis, Xie et al found LncRNA Snhg3 was one of the most down-regulated Snhgs by high fat diet (HFD) in mouse liver. Consequently, the authors sought to examine the mechanism through which Snhg3 is involved in the progression of metabolic dysfunction-associated fatty liver diseases (MASLD) in HFD-induced obese (DIO) mice. Interestingly, liver-specific Sngh3 knockout reduced, while Sngh3 over-expression potentiated fatty liver in mice on a HFD. Using the RNA pull-down approach, the authors identified SND1 as a potential Sngh3 interacting protein. SND1 is a component of the RNA-induced silencing complex (RISC). The authors found that Sngh3 increased SND1 ubiquitination to enhance SND1 protein stability, which then reduced the level of repressive chromatin H3K27me3 on PPARg promoter. The upregulation of PPARg, a lipogenic transcription factor, thus contributed to hepatic fat accumulation.

The authors propose a signaling cascade that explains how LncRNA sngh3 may promote hepatic steatosis. Multiple molecular approaches have been employed to identify molecular targets of the proposed mechanism, which is a strength of the study. There are, however, several potential issues to consider before jumping to the conclusion.

(1) First of all, it's important to ensure the robustness and rigor of each study. The manuscript was not carefully put together. The image qualities for several figures were poor, making it difficult for the readers to evaluate the results with confidence. The biological replicates and numbers of experimental repeats for cell-based assays were not described. When possible, the entire immunoblot imaging used for quantification should be presented (rather than showing n=1 representative). There were multiple mis-labels in figure panels or figure legends (e.g., Fig. 2I, Fig. 2K and Fig. 3K). The b-actin immunoblot image was reused in Fig. 4J, Fig. 5G and Fig. 7B with different exposure times. These might be from the same cohort of mice. If the immunoblots were run at different times, the loading control should be included on the same blot as well.

(2) The authors can do a better job in explaining the logic for how they came up with the potential function of each component of the signaling cascade. Sngh3 is down-regulated by HFD. However, the evidence presented indicates its involvement in promoting steatosis. In Fig. 1C, one would expect PPARg expression to be up-regulated (when Sngh3 was down-regulated). If so, the physiological observation conflicts with the proposed mechanism. In addition, SND1 is known to regulate RNA/miRNA processing. How do the authors rule out this potential mechanism? How about the hosting snoRNA, Snord17? Does it involve in the progression of NASLD?

(3) The role of PPARg in fatty liver diseases might be a rodent-specific phenomenon. PPARg agonist treatment in humans may actually reduce ectopic fat deposition by increasing fat storage in adipose tissues. The relevance of the finding to human diseases should be discussed.

Author response:

The following is the authors’ response to the original reviews.

Public Reviews:

Reviewer #1 (Public Review):

Summary:

In this manuscript, the authors investigate the contributions of the long noncoding RNA snhg3 in liver metabolism and MAFLD. The authors conclude that liver-specific loss or overexpression of Snhg3 impacts hepatic lipid content and obesity through epigenetic mechanisms. More specifically, the authors invoke that the nuclear activity of Snhg3 aggravates hepatic steatosis by altering the balance of activating and repressive chromatin marks at the Pparg gene locus. This regulatory circuit is dependent on a transcriptional regulator SND1.

Strengths:

The authors developed a tissue-specific lncRNA knockout and KI models. This effort is certainly appreciated as few lncRNA knockouts have been generated in the context of metabolism. Furthermore, lncRNA effects can be compensated in a whole organism or show subtle effects in acute versus chronic perturbation, rendering the focus on in vivo function important and highly relevant. In addition, Snhg3 was identified through a screening strategy and as a general rule the authors the authors attempt to follow unbiased approaches to decipher the mechanisms of Snhg3.

Weaknesses:

Despite efforts at generating a liver-specific knockout, the phenotypic characterization is not focused on the key readouts. Notably missing are rigorous lipid flux studies and targeted gene expression/protein measurement that would underpin why the loss of Snhg3 protects from lipid accumulation. Along those lines, claims linking the Snhg3 to MAFLD would be better supported with careful interrogation of markers of fibrosis and advanced liver disease. In other areas, significance is limited since the presented data is either not clear or rigorous enough. Finally, there is an important conceptual limitation to the work since PPARG is not established to play a major role in the liver.

We thank the reviewer for the detailed comment. In this study, hepatocyte-specific Snhg3 deficiency decreased body and liver weight and alleviated hepatic steatosis in DIO mice, whereas overexpression induced the opposite effect (Figure 2 and 3). Furthermore, we investigated the hepatic differentially expressed genes (DEGs) between the DIO Snhg3-HKI and control WT mice using RNA-Seq and revealed that Snhg3 exerts a global effect on the expression of genes involved in fatty acid metabolism using GSEA (Figure 4B). We validated the expression of some DEGs involved in fatty acid metabolism by RT-qPCR. The results showed that the hepatic expression levels of some genes involved in fatty acid metabolism, including Cd36, Cidea/c and Scd1/2 were upregulated in Snhg3-HKO mice and were downregulated in Snhg3-HKI mice compared to the controls (Figure 4C), respectively. Please check them in the first paragraph in p8.

As a transcription regulator of Cd36 and Cidea/c, it is well known that PPARγ plays major adipogenic and lipogenic roles in adipose tissue. Although the expression of PPARγ in the liver is very low under healthy conditions, induced expression of PPARγ in both hepatocytes and non-parenchymal cells (Kupffer cells, immune cells, and HSCs) in the liver has a crucial role in the pathophysiology of MASLD (Lee et al., 2023b, Chen et al., 2023, Gross et al., 2017). The activation of PPARγ in the liver induces the adipogenic program to store fatty acids in lipid droplets as observed in adipocytes (Lee et al., 2018). Moreover, the inactivation of liver PPARγ abolished rosiglitazone-induced an increase in hepatic TG and improved hepatic steatosis in lipoatrophic AZIP mice (Gavrilova et al., 2003). Furthermore, there is a strong correlation between the onset of hepatic steatosis and hepatocyte-specific PPARγ expression. Clinical trials have also indicated that increased insulin resistance and hepatic PPARγ expressions were associated with NASH scores in some obese patients (Lee et al., 2023a, Mukherjee et al., 2022). Even though PPARγ’s primary function is in adipose tissue, patients with MASLD have much higher hepatic expression levels of PPARγ, reflecting the fact that PPARγ plays different roles in different tissues and cell types (Mukherjee et al., 2022). As these studies mentioned above, our result also hinted at the importance of PPARγ in the pathophysiology of MASLD. Snhg3 deficiency or overexpression respectively induced the decrease or increase in hepatic PPARγ. Moreover, administration of PPARγ antagonist T0070907 mitigated the hepatic Cd36 and Cidea/c increase and improved Snhg3-induced hepatic steatosis. However, conflicting findings suggest that the expression of hepatic PPARγ is not increased as steatosis develops in humans and in clinical studies and that PPARγ agonists administration didn’t aggravate liver steatosis (Gross et al., 2017). Thus, understanding how the hepatic PPARγ expression is regulated may provide a new avenue to prevent and treat the MASLD (Lee et al., 2018). We also discussed it in revised manuscript, please refer the first paragraph in the section of Discussion in p13.

Hepatotoxicity accelerates the development of progressive inflammation, oxidative stress and fibrosis (Roehlen et al., 2020). Chronic liver injury including MASLD can progress to liver fibrosis with the formation of a fibrous scar. Injured hepatocytes can secrete fibrogenic factors or exosomes containing miRNAs that activate HSCs, the major source of the fibrous scar in liver fibrosis (Kisseleva and Brenner, 2021). Apart from promoting lipogenesis, PPARγ has also a crucial function in improving inflammation and fibrosis (Chen et al., 2023). In this study, no hepatic fibrosis phenotype was seen in Snhg3-HKO and Snhg3-HKI mice (figures supplement 1D and 2D). Moreover, deficiency and overexpression of Snhg3 respectively decreased and increased the expression of profibrotic genes, such as collagen type I alpha 1/2 (Col1a1 and Col1a2), but had no effects on the pro-inflammatory factors, including transforming growth factor β1 (Tgfβ1), tumor necrosis factor α (Tnfα), interleukin 6 and 1β (Il6 and Il1β) (figures supplement 3A and B). Inflammation is an absolute requirement for fibrosis because factors from injured hepatocytes alone are not sufficient to directly activate HSCs and lead to fibrosis (Kisseleva and Brenner, 2021). Additionally, previous studies indicated that exposure to HFD for more 24 weeks causes less severe fibrosis (Alshawsh et al., 2022). In future, the effect of Snhg3 on hepatic fibrosis in mice need to be elucidated by prolonged high-fat feeding or by adopting methionine- and choline deficient diet (MCD) feeding. Please check them in the second paragraph in the section of Discussion in p13.

References

ALSHAWSH, M. A., ALSALAHI, A., ALSHEHADE, S. A., SAGHIR, S. A. M., AHMEDA, A. F., AL ZARZOUR, R. H. & MAHMOUD, A. M. 2022. A Comparison of the Gene Expression Profiles of Non-Alcoholic Fatty Liver Disease between Animal Models of a High-Fat Diet and Methionine-Choline-Deficient Diet. Molecules, 27. DIO:10.3390/molecules27030858, PMID:35164140

CHEN, H., TAN, H., WAN, J., ZENG, Y., WANG, J., WANG, H. & LU, X. 2023. PPAR-gamma signaling in nonalcoholic fatty liver disease: Pathogenesis and therapeutic targets. Pharmacol Ther, 245, 108391. DIO:10.1016/j.pharmthera.2023.108391, PMID:36963510

GAVRILOVA, O., HALUZIK, M., MATSUSUE, K., CUTSON, J. J., JOHNSON, L., DIETZ, K. R., NICOL, C. J., VINSON, C., GONZALEZ, F. J. & REITMAN, M. L. 2003. Liver peroxisome proliferator-activated receptor gamma contributes to hepatic steatosis, triglyceride clearance, and regulation of body fat mass. J Biol Chem, 278, 34268-76. DIO:10.1074/jbc.M300043200, PMID:12805374

GROSS, B., PAWLAK, M., LEFEBVRE, P. & STAELS, B. 2017. PPARs in obesity-induced T2DM, dyslipidaemia and NAFLD. Nat Rev Endocrinol, 13, 36-49. DIO:10.1038/nrendo.2016.135, PMID:27636730

KISSELEVA, T. & BRENNER, D. 2021. Molecular and cellular mechanisms of liver fibrosis and its regression. Nat Rev Gastroenterol Hepatol, 18, 151-166. DIO:10.1038/s41575-020-00372-7, PMID:33128017

LEE, S. M., MURATALLA, J., KARIMI, S., DIAZ-RUIZ, A., FRUTOS, M. D., GUZMAN, G., RAMOS-MOLINA, B. & CORDOBA-CHACON, J. 2023a. Hepatocyte PPARgamma contributes to the progression of non-alcoholic steatohepatitis in male and female obese mice. Cell Mol Life Sci, 80, 39. DIO:10.1007/s00018-022-04629-z, PMID:36629912

LEE, S. M., MURATALLA, J., SIERRA-CRUZ, M. & CORDOBA-CHACON, J. 2023b. Role of hepatic peroxisome proliferator-activated receptor gamma in non-alcoholic fatty liver disease. J Endocrinol, 257. DIO:10.1530/JOE-22-0155, PMID:36688873

LEE, Y. K., PARK, J. E., LEE, M. & HARDWICK, J. P. 2018. Hepatic lipid homeostasis by peroxisome proliferator-activated receptor gamma 2. Liver Res, 2, 209-215. DIO:10.1016/j.livres.2018.12.001, PMID:31245168

MUKHERJEE, A. G., WANJARI, U. R., GOPALAKRISHNAN, A. V., KATTURAJAN, R., KANNAMPUZHA, S., MURALI, R., NAMACHIVAYAM, A., GANESAN, R., RENU, K., DEY, A., VELLINGIRI, B. & PRINCE, S. E. 2022. Exploring the Regulatory Role of ncRNA in NAFLD: A Particular Focus on PPARs. Cells, 11. DIO:10.3390/cells11243959, PMID:36552725

ROEHLEN, N., CROUCHET, E. & BAUMERT, T. F. 2020. Liver Fibrosis: Mechanistic Concepts and Therapeutic Perspectives. Cells, 9. DIO:10.3390/cells9040875, PMID:32260126

Reviewer #2 (Public Review):

Through RNA analysis, Xie et al found LncRNA Snhg3 was one of the most down-regulated Snhgs by a high-fat diet (HFD) in mouse liver. Consequently, the authors sought to examine the mechanism through which Snhg3 is involved in the progression of metabolic dysfunction-associated fatty liver diseases (MASLD) in HFD-induced obese (DIO) mice. Interestingly, liver-specific Snhg3 knockout was reduced, while Snhg3 over-expression potentiated fatty liver in mice on an HFD. Using the RNA pull-down approach, the authors identified SND1 as a potential Sngh3 interacting protein. SND1 is a component of the RNA-induced silencing complex (RISC). The authors found that Sngh3 increased SND1 ubiquitination to enhance SND1 protein stability, which then reduced the level of repressive chromatin H3K27me3 on PPARg promoter. The upregulation of PPARg, a lipogenic transcription factor, thus contributed to hepatic fat accumulation.

The authors propose a signaling cascade that explains how LncRNA sngh3 may promote hepatic steatosis. Multiple molecular approaches have been employed to identify molecular targets of the proposed mechanism, which is a strength of the study. There are, however, several potential issues to consider before jumping to a conclusion.

(1) First of all, it's important to ensure the robustness and rigor of each study. The manuscript was not carefully put together. The image qualities for several figures were poor, making it difficult for the readers to evaluate the results with confidence. The biological replicates and numbers of experimental repeats for cell-based assays were not described. When possible, the entire immunoblot imaging used for quantification should be presented (rather than showing n=1 representative). There were multiple mislabels in figure panels or figure legends (e.g., Figure 2I, Figure 2K, and Figure 3K). The b-actin immunoblot image was reused in Figure 4J, Figure 5G, and Figure 7B with different exposure times. These might be from the same cohort of mice. If the immunoblots were run at different times, the loading control should be included on the same blot as well.

We thank the reviewer for the detailed comment. We have provided the clear figures in revised manuscript, please check them.

The biological replicates and numbers of experimental repeats for cell-based assays had been updated and please check them in the manuscript.

The entire immunoblot imaging used for quantification had been provided in the primary data. Please check them.

The original Figure 2I, Figure 2K, Figure 3K have been revised and replaced with new Figure 2F, Figure 2H, Figure 3H, and their corresponding figure legends has also been corrected in revised manuscript.

The protein levels of CD36, PPARγ and β-ACTIN were examined at the same time and we had revised the manuscript, please check them in revised Figure 7B and 7C.

(2) The authors can do a better job in explaining the logic for how they came up with the potential function of each component of the signaling cascade. Snhg3 is down-regulated by HFD. However, the evidence presented indicates its involvement in promoting steatosis. In Figure 1C, one would expect PPARg expression to be up-regulated (when Sngh3 was down-regulated). If so, the physiological observation conflicts with the proposed mechanism. In addition, SND1 is known to regulate RNA/miRNA processing. How do the authors rule out this potential mechanism? How about the hosting snoRNA, Snord17? Does it involve the progression of NASLD?

We thank the reviewer for the detailed comment. Our results showed that the expression of Snhg3 was decreased in DIO mice which led us to speculate that the downregulation of Snhg3 in DIO mice might be a stress protective reaction to high nutritional state, but the specific details need to be clarified. This is probably similar to fibroblast growth factor 21 (FGF21) and growth differentiation factor 15 (GDF15), whose endogenous expression and circulating levels are elevated in obese humans and mice despite their beneficial effects on obesity and related metabolic complications (Keipert and Ost, 2021). Although FGF21 can be induced by oxidative stress and be activated in obese mice and in NASH patients, elevated FGF21 paradoxically protects against oxidative stress and reduces hepatic steatosis (Tillman and Rolph, 2020). We had added the content the section of Discussion, please check it in the second paragraph in p12.

SND1 has multiple roles through associating with different types of RNA molecules, including mRNA, miRNA, circRNA, dsRNA and lncRNA. SND1 could bind negative-sense SARS-CoV-2 RNA and promoted viral RNA synthesis, and to promote viral RNA synthesis (Schmidt et al., 2023). SND1 is also involved in hypoxia by negatively regulating hypoxia‐related miRNAs (Saarikettu et al., 2023). Furthermore, a recent study revealed that lncRNA SNAI3-AS1 can competitively bind to SND1 and perturb the m6A-dependent recognition of Nrf2 mRNA 3'UTR by SND1, thereby reducing the mRNA stability of Nrf2 (Zheng et al., 2023). Huang et al. also reported that circMETTL9 can directly bind to and increase the expression of SND1 in astrocytes, leading to enhanced neuroinflammation (Huang et al., 2023). However, whether there is an independent-histone methylation role of SND1/lncRNA-Snhg3 involved in lipid metabolism in the liver needs to be further investigated. We also discussed the limitation in the manuscript and please refer the section of Discussion in the third paragraph in p17.

Snhg3 serves as host gene for producing intronic U17 snoRNAs, the H/ACA snoRNA. A previous study found that cholesterol trafficking phenotype was not due to reduced Snhg3 expression, but rather to haploinsufficiency of U17 snoRNA. Upregulation of hypoxia-upregulated mitochondrial movement regulator (HUMMR) in U17 snoRNA-deficient cells promoted the formation of ER-mitochondrial contacts, resulting in decreasing cholesterol esterification and facilitating cholesterol trafficking to mitochondria (Jinn et al., 2015). Additionally, disruption of U17 snoRNA caused resistance to lipid-induced cell death and general oxidative stress in cultured cells. Furthermore, knockdown of U17 snoRNA in vivo protected against hepatic steatosis and lipid-induced oxidative stress and inflammation (Sletten et al., 2021). We determined the expression of hepatic U17 snoRNA and its effect on SND1 and PPARγ. The results showed that the expression of U17 snoRNA decreased in the liver of DIO Snhg3-HKO mice and unchanged in the liver of DIO Snhg3-HKI mice, but overexpression of U17 snoRNA had no effect on the expression of SND1 and PPARγ (figure supplement 5A-C), indicating that Sngh3 induced hepatic steatosis was independent on U17 snoRNA. We also discussed it in revised manuscript, please refer the section of Discussion in p15.

References

HUANG, C., SUN, L., XIAO, C., YOU, W., SUN, L., WANG, S., ZHANG, Z. & LIU, S. 2023. Circular RNA METTL9 contributes to neuroinflammation following traumatic brain injury by complexing with astrocytic SND1. J Neuroinflammation, 20, 39. DIO:10.1186/s12974-023-02716-x, PMID:36803376

JINN, S., BRANDIS, K. A., REN, A., CHACKO, A., DUDLEY-RUCKER, N., GALE, S. E., SIDHU, R., FUJIWARA, H., JIANG, H., OLSEN, B. N., SCHAFFER, J. E. & ORY, D. S. 2015. snoRNA U17 regulates cellular cholesterol trafficking. Cell Metab, 21, 855-67. DIO:10.1016/j.cmet.2015.04.010, PMID:25980348

KEIPERT, S. & OST, M. 2021. Stress-induced FGF21 and GDF15 in obesity and obesity resistance. Trends Endocrinol Metab, 32, 904-915. DIO:10.1016/j.tem.2021.08.008, PMID:34526227

SAARIKETTU, J., LEHMUSVAARA, S., PESU, M., JUNTTILA, I., PARTANEN, J., SIPILA, P., POUTANEN, M., YANG, J., HAIKARAINEN, T. & SILVENNOINEN, O. 2023. The RNA-binding protein Snd1/Tudor-SN regulates hypoxia-responsive gene expression. FASEB Bioadv, 5, 183-198. DIO:10.1096/fba.2022-00115, PMID:37151849

SCHMIDT, N., GANSKIH, S., WEI, Y., GABEL, A., ZIELINSKI, S., KESHISHIAN, H., LAREAU, C. A., ZIMMERMANN, L., MAKROCZYOVA, J., PEARCE, C., KREY, K., HENNIG, T., STEGMAIER, S., MOYON, L., HORLACHER, M., WERNER, S., AYDIN, J., OLGUIN-NAVA, M., POTABATTULA, R., KIBE, A., DOLKEN, L., SMYTH, R. P., CALISKAN, N., MARSICO, A., KREMPL, C., BODEM, J., PICHLMAIR, A., CARR, S. A., CHLANDA, P., ERHARD, F. & MUNSCHAUER, M. 2023. SND1 binds SARS-CoV-2 negative-sense RNA and promotes viral RNA synthesis through NSP9. Cell, 186, 4834-4850 e23. DIO:10.1016/j.cell.2023.09.002, PMID:37794589

SLETTEN, A. C., DAVIDSON, J. W., YAGABASAN, B., MOORES, S., SCHWAIGER-HABER, M., FUJIWARA, H., GALE, S., JIANG, X., SIDHU, R., GELMAN, S. J., ZHAO, S., PATTI, G. J., ORY, D. S. & SCHAFFER, J. E. 2021. Loss of SNORA73 reprograms cellular metabolism and protects against steatohepatitis. Nat Commun, 12, 5214. DIO:10.1038/s41467-021-25457-y, PMID:34471131

TILLMAN, E. J. & ROLPH, T. 2020. FGF21: An Emerging Therapeutic Target for Non-Alcoholic Steatohepatitis and Related Metabolic Diseases. Front Endocrinol (Lausanne), 11, 601290. DIO:10.3389/fendo.2020.601290, PMID:33381084

ZHENG, J., ZHANG, Q., ZHAO, Z., QIU, Y., ZHOU, Y., WU, Z., JIANG, C., WANG, X. & JIANG, X. 2023. Epigenetically silenced lncRNA SNAI3-AS1 promotes ferroptosis in glioma via perturbing the m(6)A-dependent recognition of Nrf2 mRNA mediated by SND1. J Exp Clin Cancer Res, 42, 127. DIO:10.1186/s13046-023-02684-3, PMID:37202791

(3) The role of PPARg in fatty liver diseases might be a rodent-specific phenomenon. PPARg agonist treatment in humans may actually reduce ectopic fat deposition by increasing fat storage in adipose tissues. The relevance of the findings to human diseases should be discussed.

We thank the reviewer for the detailed comment. As a transcription regulator of Cd36 and Cidea/c, it is well known that PPARγ plays major adipogenic and lipogenic roles in adipose tissue. Although the expression of PPARγ in the liver is very low under healthy conditions, induced expression of PPARγ in both hepatocytes and non-parenchymal cells (Kupffer cells, immune cells, and hepatic stellate cells (HSCs)) in the liver has a crucial role in the pathophysiology of MASLD (Lee et al., 2023b, Chen et al., 2023, Gross et al., 2017). The activation of PPARγ in the liver induces the adipogenic program to store fatty acids in lipid droplets as observed in adipocytes (Lee et al., 2018). Moreover, the inactivation of liver PPARγ abolished rosiglitazone-induced an increase in hepatic TG and improved hepatic steatosis in lipoatrophic AZIP mice (Gavrilova et al., 2003). Apart from promoting lipogenesis, PPARγ has also a crucial function in improving inflammation and fibrosis (Chen et al., 2023). Furthermore, there is a strong correlation between the onset of hepatic steatosis and hepatocyte-specific PPARγ expression. Clinical trials have also indicated that increased insulin resistance and hepatic PPARγ expressions were associated with NASH scores in some obese patients (Lee et al., 2023a, Mukherjee et al., 2022). Even though PPARγ’s primary function is in adipose tissue, patients with MASLD have much higher hepatic expression levels of PPARγ, reflecting the fact that PPARγ plays different roles in different tissues and cell types (Mukherjee et al., 2022). As these studies mentioned above, our result also hinted at the importance of PPARγ in the pathophysiology of MASLD. Snhg3 deficiency or overexpression respectively induced the decrease or increase in hepatic PPARγ. Moreover, administration of PPARγ antagonist T0070907 mitigated the hepatic Cd36 and Cidea/c increase and improved Snhg3-induced hepatic steatosis. However, conflicting findings suggest that the expression of hepatic PPARγ is not increased as steatosis develops in humans and in clinical studies and that PPARγ agonists administration didn’t aggravate liver steatosis (Gross et al., 2017). Thus, understanding how the hepatic PPARγ expression is regulated may provide a new avenue to prevent and treat the MASLD (Lee et al., 2018). We also discussed it in revised manuscript, please refer the first paragraph in the section of Discussion in p13.

References

CHEN, H., TAN, H., WAN, J., ZENG, Y., WANG, J., WANG, H. & LU, X. 2023. PPAR-gamma signaling in nonalcoholic fatty liver disease: Pathogenesis and therapeutic targets. Pharmacol Ther, 245, 108391. DIO:10.1016/j.pharmthera.2023.108391, PMID:36963510

GAVRILOVA, O., HALUZIK, M., MATSUSUE, K., CUTSON, J. J., JOHNSON, L., DIETZ, K. R., NICOL, C. J., VINSON, C., GONZALEZ, F. J. & REITMAN, M. L. 2003. Liver peroxisome proliferator-activated receptor gamma contributes to hepatic steatosis, triglyceride clearance, and regulation of body fat mass. J Biol Chem, 278, 34268-76. DIO:10.1074/jbc.M300043200, PMID:12805374

GROSS, B., PAWLAK, M., LEFEBVRE, P. & STAELS, B. 2017. PPARs in obesity-induced T2DM, dyslipidaemia and NAFLD. Nat Rev Endocrinol, 13, 36-49. DIO:10.1038/nrendo.2016.135, PMID:27636730

LEE, S. M., MURATALLA, J., KARIMI, S., DIAZ-RUIZ, A., FRUTOS, M. D., GUZMAN, G., RAMOS-MOLINA, B. & CORDOBA-CHACON, J. 2023a. Hepatocyte PPARgamma contributes to the progression of non-alcoholic steatohepatitis in male and female obese mice. Cell Mol Life Sci, 80, 39. DIO:10.1007/s00018-022-04629-z, PMID:36629912

LEE, S. M., MURATALLA, J., SIERRA-CRUZ, M. & CORDOBA-CHACON, J. 2023b. Role of hepatic peroxisome proliferator-activated receptor gamma in non-alcoholic fatty liver disease. J Endocrinol, 257. DIO:10.1530/JOE-22-0155, PMID:36688873

LEE, Y. K., PARK, J. E., LEE, M. & HARDWICK, J. P. 2018. Hepatic lipid homeostasis by peroxisome proliferator-activated receptor gamma 2. Liver Res, 2, 209-215. DIO:10.1016/j.livres.2018.12.001, PMID:31245168

MUKHERJEE, A. G., WANJARI, U. R., GOPALAKRISHNAN, A. V., KATTURAJAN, R., KANNAMPUZHA, S., MURALI, R., NAMACHIVAYAM, A., GANESAN, R., RENU, K., DEY, A., VELLINGIRI, B. & PRINCE, S. E. 2022. Exploring the Regulatory Role of ncRNA in NAFLD: A Particular Focus on PPARs. Cells, 11. DIO:10.3390/cells11243959, PMID:36552725

Recommendations for the authors:

Reviewer #1 (Recommendations For The Authors):

As a general strategy for the revision, I would advise the authors to focus on strengthening the analysis of the liver with the two most important figures being Figure 2 and Figure 3. The mechanism as it stands is problematic which reduces the impact of the animal studies despite substantial efforts from the authors. Consider removing or toning down some of the studies focused on mechanisms in the nucleus, including changing the title.

We thank the reviewer for the detailed comment. In this study, hepatocyte-specific Snhg3 deficiency decreased body and liver weight, alleviated hepatic steatosis and promoted hepatic fatty acid metabolism in DIO mice, whereas overexpression induced the opposite effect. The hepatic differentially expressed genes (DEGs) between the DIO Snhg3-HKI and control WT mice using RNA-Seq and revealed that Snhg3 exerts a global effect on the expression of genes involved in fatty acid metabolism using GSEA (Figure 4B). RT-qPCR analysis confirmed that the hepatic expression levels of some genes involved in fatty acid metabolism, including Cd36, Cidea/c and Scd1/2, were upregulated in Snhg3-HKO mice and were downregulated in Snhg3-HKI mice compared to the controls (Figure 4C). Moreover, deficiency and overexpression of Snhg3 respectively decreased and increased the expression of profibrotic genes, such as Col1a1 and Col1a2, but had no effects on the pro-inflammatory factors, including Tgfβ1, Tnfα, Il6 and Il1β (figure supplement 3A and B). The results indicated that Snhg3 involved in hepatic steatosis through regulating fatty acid metabolism. Furthermore, PPARγ was selected to study its role in Snhg3-induced hepatic steatosis by integrated analyzing the data from CUT&Tag-Seq, ATAC-Seq and RNA-Seq. Finally, inhibition of PPARγ with T0070907 alleviated Snhg3 induced Cd36 and Cidea/c increases and improved Snhg3-aggravated hepatic steatosis. In summary, we confirmed that SND1/H3K27me3/PPARγ is partially responsible for Sngh3-inuced hepatic steatosis. As the reviewer suggested, we replaced the title with “LncRNA-Snhg3 Aggravates Hepatic Steatosis via PPARγ Signaling”.

(1) How is steatosis changing in the liver? Is this due to a change in fatty acid uptake, lipogenesis/synthesis, beta-oxidation, trig secretion, etc..? The analysis in Figures 2 and 3 is mostly focused on metabolic chamber studies which seem distracting, particularly in the absence of a mechanism and given a liver-specific perturbation. The authors should use a combination of targeted gene expression, protein blots, and lipid flux measurements to provide better insights here. The histology in Figure 2H suggests a very dramatic effect but does match with lipid measurements in 2I.

We thank the reviewer for the detailed comment. The pathogenesis of MASLD has not been entirely elucidated. Multifarious factors such as genetic and epigenetic factors, nutritional factors, insulin resistance, lipotoxicity, microbiome, fibrogenesis and hormones secreted from the adipose tissue, are recognized to be involved in the development and progression of MASLD (Buzzetti et al., 2016, Lee et al., 2017, Rada et al., 2020, Sakurai et al., 2021, Friedman et al., 2018). In this study, we investigated the hepatic differentially expressed genes (DEGs) between the DIO Snhg3-HKI and control WT mice using RNA-Seq and revealed that Snhg3 exerts a global effect on the expression of genes involved in fatty acid metabolism using GSEA (Figure 4B). We validated the expression of some DEGs involved in fatty acid metabolism by RT-qPCR. The results showed that the hepatic expression levels of some genes involved in fatty acid metabolism, including Cd36, Cidea/c and Scd1/2 were upregulated in Snhg3-HKO mice and were downregulated in Snhg3-HKI mice compared to the controls (Figure 4C), respectively. Additionally, we re-analyzed the metabolic chamber data using CalR and the results showed that there were no obvious differences in heat production, total oxygen consumption, carbon dioxide production or RER between DIO Snhg3-HKO or DIO Snhg3-HKI and the corresponding control mice (figure supplement 1C and 2C). Unfortunately, we did not detect lipid flux due to limited experimental conditions. However, in summary, our results indicated that Snhg3 is involved in hepatic steatosis by regulating fatty acid metabolism. Please check them in the first paragraph in p8.

Additionally, we determined the hepatic TC levels in other batch of DIO Snhg3-HKO and control mice and found there was no difference in hepatic TC (as below) between DIO Snhg3-HKO and control mice fed HFD 18 weeks. Perhaps the apparent difference in TC requires a prolonged high-fat diet feeding time.

Author response image 1.

Hepatic TC contents of in DIO Snhg3-Flox and Snhg3-HKO mice.

References

BUZZETTI, E., PINZANI, M. & TSOCHATZIS, E. A. 2016. The multiple-hit pathogenesis of non-alcoholic fatty liver disease (NAFLD). Metabolism, 65, 1038-48. DIO:10.1016/j.metabol.2015.12.012, PMID:26823198

FRIEDMAN, S. L., NEUSCHWANDER-TETRI, B. A., RINELLA, M. & SANYAL, A. J. 2018. Mechanisms of NAFLD development and therapeutic strategies. Nat Med, 24, 908-922. DIO:10.1038/s41591-018-0104-9, PMID:29967350

LEE, J., KIM, Y., FRISO, S. & CHOI, S. W. 2017. Epigenetics in non-alcoholic fatty liver disease. Mol Aspects Med, 54, 78-88. DIO:10.1016/j.mam.2016.11.008, PMID:27889327

RADA, P., GONZALEZ-RODRIGUEZ, A., GARCIA-MONZON, C. & VALVERDE, A. M. 2020. Understanding lipotoxicity in NAFLD pathogenesis: is CD36 a key driver? Cell Death Dis, 11, 802. DIO:10.1038/s41419-020-03003-w, PMID:32978374

SAKURAI, Y., KUBOTA, N., YAMAUCHI, T. & KADOWAKI, T. 2021. Role of Insulin Resistance in MAFLD. Int J Mol Sci, 22. DIO:10.3390/ijms22084156, PMID:33923817

(2) Throughout the manuscript the authors make claims about liver disease models, but this is not well supported since markers of advanced liver disease are not examined. The authors should stain and show expression for fibrosis and inflammation.

We thank the reviewer for the detailed comment. Metabolic dysfunction-associated fatty liver disease (MASLD) is characterized by excess liver fat in the absence of significant alcohol consumption. It can progress from simple steatosis to metabolic dysfunction-associated steatohepatitis (MASH) and fibrosis and eventually to chronic progressive diseases such as cirrhosis, end-stage liver failure, and hepatocellular carcinoma (Loomba et al., 2021). As the reviewer suggested, we detected the effect of Snhg3 on liver fibrosis and inflammation. The results showed no hepatic fibrosis phenotype was seen in Snhg3-HKO and Snhg3-HKI mice (figures supplement 1D and 2D). Moreover, deficiency and overexpression of Snhg3 respectively decreased and increased the expression of profibrotic genes, such as collagen type I alpha 1/2 (Col1a1 and Col1a2), but had no effects on the pro-inflammatory factors including Tgf-β, Tnf-α, Il-6 and Il-1β (figure supplement 3A and 3B). Inflammation is an absolute requirement for fibrosis because factors from injured hepatocytes alone are not sufficient to directly activate HSCs and lead to fibrosis (Kisseleva and Brenner, 2021). Additionally, previous studies indicated that exposure to HFD for more 24 weeks causes less severe fibrosis (Alshawsh et al., 2022). In future, the effect of Snhg3 on hepatic fibrosis in mice need to be elucidated by prolonged high-fat feeding or by adopting methionine- and choline deficient diet (MCD) feeding. Please check them in the second paragraph in the section of Discussion in p13.

References

ALSHAWSH, M. A., ALSALAHI, A., ALSHEHADE, S. A., SAGHIR, S. A. M., AHMEDA, A. F., AL ZARZOUR, R. H. & MAHMOUD, A. M. 2022. A Comparison of the Gene Expression Profiles of Non-Alcoholic Fatty Liver Disease between Animal Models of a High-Fat Diet and Methionine-Choline-Deficient Diet. Molecules, 27. DIO:10.3390/molecules27030858, PMID:35164140

KISSELEVA, T. & BRENNER, D. 2021. Molecular and cellular mechanisms of liver fibrosis and its regression. Nat Rev Gastroenterol Hepatol, 18, 151-166. DIO:10.1038/s41575-020-00372-7, PMID:33128017

LOOMBA, R., FRIEDMAN, S. L. & SHULMAN, G. I. 2021. Mechanisms and disease consequences of nonalcoholic fatty liver disease. Cell, 184, 2537-2564. DIO:10.1016/j.cell.2021.04.015, PMID:33989548

(3) Publicly available datasets show that PPARG protein is not expressed in the liver (Science 2015 347(6220):1260419, PMID: 25613900). Are the authors sure this is not an effect on another PPAR isoform like alpha? ChIP and RNA-seq pathway readouts do not distinguish between different isoforms.

We thank the reviewer for the detailed comment. As a transcription regulator of Cd36 and Cidea/c, it is well known that PPARγ plays major adipogenic and lipogenic roles in adipose tissue. Although the expression of PPARγ in the liver is very low under healthy conditions, induced expression of PPARγ in both hepatocytes and non-parenchymal cells (Kupffer cells, immune cells, and hepatic stellate cells (HSCs)) in the liver has a crucial role in the pathophysiology of MASLD (Lee et al., 2023b, Chen et al., 2023, Gross et al., 2017). The activation of PPARγ in the liver induces the adipogenic program to store fatty acids in lipid droplets as observed in adipocytes (Lee et al., 2018). Moreover, the inactivation of liver PPARγ abolished rosiglitazone-induced an increase in hepatic TG and improved hepatic steatosis in lipoatrophic AZIP mice (Gavrilova et al., 2003). Apart from promoting lipogenesis, PPARγ has also a crucial function in improving inflammation and fibrosis (Chen et al., 2023). Furthermore, there is a strong correlation between the onset of hepatic steatosis and hepatocyte-specific PPARγ expression. Clinical trials have also indicated that increased insulin resistance and hepatic PPARγ expressions were associated with NASH scores in some obese patients (Lee et al., 2023a, Mukherjee et al., 2022). Even though PPARγ’s primary function is in adipose tissue, patients with MASLD have much higher hepatic expression levels of PPARγ, reflecting the fact that PPARγ plays different roles in different tissues and cell types (Mukherjee et al., 2022). As these studies mentioned above, our result also hinted at the importance of PPARγ in the pathophysiology of MASLD. Snhg3 deficiency or overexpression respectively induced the decrease or increase in hepatic PPARγ. Moreover, administration of PPARγ antagonist T0070907 mitigated the hepatic Cd36 and Cidea/c increase and improved Snhg3-induced hepatic steatosis. However, conflicting findings suggest that the expression of hepatic PPARγ is not increased as steatosis develops in humans and in clinical studies and that PPARγ agonists administration didn’t aggravate liver steatosis (Gross et al., 2017). Thus, understanding how the hepatic PPARγ expression is regulated may provide a new avenue to prevent and treat the MASLD (Lee et al., 2018). We also discussed it in revised manuscript, please refer the first paragraph in the section of Discussion in p13 in revised manuscript.

PPARα, most highly expressed in the liver, transcriptionally regulates lipid catabolism by regulating the expression of genes mediating triglyceride hydrolysis, fatty acid transport, and β-oxidation. Activators of PPARα decrease plasma triglycerides by inhibiting its synthesis and accelerating its hydrolysis (Chen et al., 2023). Mice with deletion of the Pparα gene exhibited more hepatic steatosis under HFD induction. As the reviewer suggested, we investigated the effect of Snhg3 on Pparα expression. The result showed that both deficiency of Snhg3 or overexpression of Snhg3 doesn’t affect the mRNA level of Pparα as showing below, indicating that Snhg3-induced lipid accumulation independent on PPARα. Additionally, the exon, upstream 2k, 5’-UTR and intron regions of Pparγ, not Pparα, were enriched with the H3K27me3 mark (fold_enrichment = 4.15697) in the liver of DIO Snhg3-HKO mice using the CUT&Tag assay (table supplement 8), which was further confirmed by ChIP (Figure 6F and G). Therefore, we choose PPARγ to study its role in Sngh3-induced hepatic steatosis by integrated analyzing the data from CUT&Tag-Seq, ATAC-Seq and RNA-Seq.

Author response image 2.

The mRNA levels of hepatic Pparα expression in DIO Snhg3-HKO mice and Snhg3-HKI mice compared to the controls.

References

CHEN, H., TAN, H., WAN, J., ZENG, Y., WANG, J., WANG, H. & LU, X. 2023. PPAR-gamma signaling in nonalcoholic fatty liver disease: Pathogenesis and therapeutic targets. Pharmacol Ther, 245, 108391. DIO:10.1016/j.pharmthera.2023.108391, PMID:36963510

GAVRILOVA, O., HALUZIK, M., MATSUSUE, K., CUTSON, J. J., JOHNSON, L., DIETZ, K. R., NICOL, C. J., VINSON, C., GONZALEZ, F. J. & REITMAN, M. L. 2003. Liver peroxisome proliferator-activated receptor gamma contributes to hepatic steatosis, triglyceride clearance, and regulation of body fat mass. J Biol Chem, 278, 34268-76. DIO:10.1074/jbc.M300043200, PMID:12805374

GROSS, B., PAWLAK, M., LEFEBVRE, P. & STAELS, B. 2017. PPARs in obesity-induced T2DM, dyslipidaemia and NAFLD. Nat Rev Endocrinol, 13, 36-49. DIO:10.1038/nrendo.2016.135, PMID:27636730

LEE, S. M., MURATALLA, J., KARIMI, S., DIAZ-RUIZ, A., FRUTOS, M. D., GUZMAN, G., RAMOS-MOLINA, B. & CORDOBA-CHACON, J. 2023a. Hepatocyte PPARgamma contributes to the progression of non-alcoholic steatohepatitis in male and female obese mice. Cell Mol Life Sci, 80, 39. DIO:10.1007/s00018-022-04629-z, PMID:36629912

LEE, S. M., MURATALLA, J., SIERRA-CRUZ, M. & CORDOBA-CHACON, J. 2023b. Role of hepatic peroxisome proliferator-activated receptor gamma in non-alcoholic fatty liver disease. J Endocrinol, 257. DIO:10.1530/JOE-22-0155, PMID:36688873

LEE, Y. K., PARK, J. E., LEE, M. & HARDWICK, J. P. 2018. Hepatic lipid homeostasis by peroxisome proliferator-activated receptor gamma 2. Liver Res, 2, 209-215. DIO:10.1016/j.livres.2018.12.001, PMID:31245168

MUKHERJEE, A. G., WANJARI, U. R., GOPALAKRISHNAN, A. V., KATTURAJAN, R., KANNAMPUZHA, S., MURALI, R., NAMACHIVAYAM, A., GANESAN, R., RENU, K., DEY, A., VELLINGIRI, B. & PRINCE, S. E. 2022. Exploring the Regulatory Role of ncRNA in NAFLD: A Particular Focus on PPARs. Cells, 11. DIO:10.3390/cells11243959, PMID:36552725

(4) Previous work suggests that SNHG3 regulates its neighboring gene MED18 which is an important regulator of global transcription. Could some of the observed effects be due to changes in MED18 or other neighboring genes?

We thank the reviewer for the detailed comment. Previous work suggested that human SNHG3 promotes progression of gastric cancer by regulating neighboring MED18 gene methylation (Xuan and Wang, 2019). Here, we studied the effect of mouse Snhg3 on Med18 and the result showed that Snhg3 had no effect on the mRNA levels of Med18 (as below). Additionally, we also tested the effect of mouse Snhg3 on its neighboring gene, regulator of chromosome condensation 1 (Rcc1). Although deficiency of Snhg3 inhibited the mRNA level of Rcc1, overexpression of Snhg3 doesn’t affect the mRNA level of Rcc1 as showing below. RCC1, the only known guanine nucleotide exchange factor in the nucleus for Ran, a nuclear Ras-like G protein, directly participates in cellular processes such as nuclear envelope formation, nucleocytoplasmic transport, and spindle formation (Ren et al., 2020). RCC1 also regulates chromatin condensation in the late S and early M phases of the cell cycle. Many studies have found that RCC1 plays an important role in tumors. Furthermore, whether Rcc1 mediates the alleviated effect on MASLD of Snhg3 needs to be further investigated.

Author response image 3.

The mRNA levels of hepatic Rcc1 and Med18 expression in DIO Snhg3-HKO mice and Snhg3-HKI mice compared to the controls.

References

REN, X., JIANG, K. & ZHANG, F. 2020. The Multifaceted Roles of RCC1 in Tumorigenesis. Front Mol Biosci, 7, 225. DIO:10.3389/fmolb.2020.00225, PMID:33102517

XUAN, Y. & WANG, Y. 2019. Long non-coding RNA SNHG3 promotes progression of gastric cancer by regulating neighboring MED18 gene methylation. Cell Death Dis, 10, 694. DIO:10.1038/s41419-019-1940-3, PMID:31534128

(5) The claim that Snhg3 regulates SND1 protein stability seems subtle. There is data inconsistency between different panels regarding this regulation including Figure 5I, Figure 6A, and Figure 7E. In addition, is ubiquitination happening in the nucleus where Snhg3 is expressed?

We thank the reviewer for the detailed comment. The effect of Snhg3-induced SND1 expression had been confirmed by western blotting, please check them in Figure 5I, Figure 6A, Figure 7E and corresponding primary data. Additionally, Snhg3-induced SND1 protein stability seemed subtle, indicating there may be other mechanism by which Snhg3 promotes SND1, such as riboregulation. We had added it in the section of Discussion, please check it in the second paragraph in p16.

Additionally, we did not detect the sites where SND1 is modified by ubiquitination. Our results showed that Snhg3 was more localized in the nucleus (Figure 1D) and Snhg3 also promoted the nuclear localization of SND1 (Figure 5O). We had revised the diagram of Snhg3 action in Figure 8G. Please check them in revised manuscript.

(6) The authors show that the loss of Snhg3 changes the global H3K27me3 level. Few enzymes modify H3K27me3 levels. Did the authors check for an interaction between EZH2, Jmjd3, UTX, and Snhg3/SND1?

We thank the reviewer for the detailed comment. It is crucial to ascertain whether SND1 itself functions as a new demethylase or if it influences other demethylases, such as Jmjd3, enhancer of zeste homolog 2 (EZH2), and ubiquitously transcribed tetratricopeptide repeat on chromosome X (UTX). The precise mechanism by which SND1 regulates H3K27me3 is still unclear and hence requires further investigation. We had added the limitations in the section of Discussion and please check it in the third paragraph in p17.

(7) Can the authors speculate if the findings related to Snhg3/SND1 extend to humans?

We thank the reviewer for the detailed comment. Since the sequence of Snhg3 is not conserved between mice and humans, the findings in this manuscript may not be applicable to humans, but the detail need to be further exploited.

(8) As a general rule the figures are too small or difficult to read with limited details in the figure legends which limits evaluation. For example, Figure 1B and almost all of 4 cannot read labels. Figure 2, cannot see the snapshots show of mice or livers. What figure is supporting the claim that snhg3KI are more 'hyper-accessible'? Can the authors clarify what Figure 4H is referring to?

We thank the reviewer for the detailed comment. We have provided high quality figures in our revised manuscript.

The ‘hyper-accessible’ state in the liver of Snhg3-HKI mice was inferred by the differentially accessible regions (DARs), that is, we discovered 4305 DARs were more accessible in Snhg3-HKI mice and only 2505 DARs were more accessible in control mice and please refer table supplement 3).

The result of Figure 4H about heatmap for Cd36 was from hepatic RNA-seq of DIO Snhg3-HKI and control WT mice. For avoiding ambiguity, we have removed it.

(9) Authors stated that upon Snhg3 knock out, more genes are upregulated(1028) than downregulated(365). This description does not match Figure 4A. It seems in Figure 4A there are equal numbers of up and downregulated genes.

We thank the reviewer for the detailed question. We apologized for this mistake and have corrected it.

(10) Provide a schematic of the knockout and KI strategy in the supplement.

We thank the reviewer for the detailed comment. We had included the knockout and KI strategy in figure supplement 1A and B, and 2A.

Reviewer #2 (Recommendations For The Authors):

(1) Metabolic cage data need to be reanalyzed with CalR (particularly when the body weights are significantly different).

We thank the reviewer for the detailed comment. We reanalyzed the metabolic cage data using CalR (Mina et al., 2018). The results showed that there were no obvious differences in heat production, total oxygen consumption, carbon dioxide production and the respiratory exchange ratio between DIO Snhg3-HKO and control mice. Similar to DIO Snhg3-HKO mice, there was also no differences in heat production, total oxygen consumption, carbon dioxide production, and RER between DIO Snhg3-HKI mice and WT mice. Please check them in figure supplement 1C and 2C, and Mouse Calorimetry in Materials and Methods.

Reference

MINA, A. I., LECLAIR, R. A., LECLAIR, K. B., COHEN, D. E., LANTIER, L. & BANKS, A. S. 2018. CalR: A Web-Based Analysis Tool for Indirect Calorimetry Experiments. Cell Metab, 28, 656-666 e1. DIO:10.1016/j.cmet.2018.06.019, PMID:30017358

(2) ITT in Figure 2F should also be presented as % of the initial glucose level, which would reveal that there is no difference between WT and KO.

We thank the reviewer for the detailed comment. We repeated ITT experiment and include the new data in revised manuscript, please check it in Figure 2C.

(3) The fasting glucose results are inconsistent between ITT and GTT. Is there any difference in fasting glucose?

We thank the reviewer for the questions. The difference between GTT and ITT was caused owing to different fasting time, that is, mice were fasted for 6 h in ITT and were fasted for 16 h in GTT. It seems that Snhg3 doesn’t affect short- and longer-time fasting glucose levels and please refer Figures 2C and 3C.