Melanocortin 1 receptor regulates cholesterol and bile acid metabolism in the liver
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The significance of this manuscript is that is provides useful information for the field of hepatology and endocrinology on the regulatory mechanisms of cholesterol homeostasis by melanocortin. The authors provide solid evidence utilizing both in vivo and in vitro molecular, cellular, and biochemical approaches to support their claims.
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Abstract
Melanocortin 1 receptor (MC1-R) is widely expressed in melanocytes and leukocytes and is thus strongly implicated in the regulation of skin pigmentation and inflammation. MC1-R has also been found in the rat and human liver, but its functional role has remained elusive. We hypothesized that MC1-R is functionally active in the liver and involved in the regulation of cholesterol and bile acid metabolism. We generated hepatocyte-specific MC1-R knock-out (Mc1r LKO) mice and phenotyped the mouse model for lipid profiles, liver histology, and bile acid levels. Mc1r LKO mice had significantly increased liver weight, which was accompanied by elevated levels of total cholesterol and triglycerides in the liver as well as in the plasma. These mice demonstrated also enhanced liver fibrosis and a disturbance in bile acid metabolism as evidenced by markedly reduced bile acid levels in the plasma and feces. Mechanistically, using HepG2 cells as an in vitro model, we found that selective activation of MC1-R in HepG2 cells reduced cellular cholesterol content and enhanced uptake of low- and high-density lipoprotein particles via a cAMP-independent mechanism. In conclusion, the present results demonstrate that MC1-R signaling in hepatocytes regulates cholesterol and bile acid metabolism and its deficiency leads to hypercholesterolemia and enhanced lipid accumulation and fibrosis in the liver.
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Author Response
Reviewer #1 (Public Review):
The work described herein would have an impact on the field in multiple ways. Firstly, it demonstrates a novel metabolic role for MSH in the regulation of hepatic cholesterol metabolism. This may prove to be a viable therapeutic strategy for the treatment of dyslipidemia. Furthermore, the authors demonstrate an alternative signaling cascade elicited by MSH independent of cAMP, but rather relying on AMPK. This novel interaction between AMPK and MC1R could have more widespread implications beyond the control of hepatic cholesterol metabolism.
For the most part, the conclusions offered by the authors are supported by the data that is presented. There are, however, a number of concerns in the current version of this manuscript detailed below.
We thank the reviewer for the encouraging and …
Author Response
Reviewer #1 (Public Review):
The work described herein would have an impact on the field in multiple ways. Firstly, it demonstrates a novel metabolic role for MSH in the regulation of hepatic cholesterol metabolism. This may prove to be a viable therapeutic strategy for the treatment of dyslipidemia. Furthermore, the authors demonstrate an alternative signaling cascade elicited by MSH independent of cAMP, but rather relying on AMPK. This novel interaction between AMPK and MC1R could have more widespread implications beyond the control of hepatic cholesterol metabolism.
For the most part, the conclusions offered by the authors are supported by the data that is presented. There are, however, a number of concerns in the current version of this manuscript detailed below.
We thank the reviewer for the encouraging and insightful comments, and we are pleased to read that the manuscript has raised considerable interest.
- The authors demonstrate the expression of MC1R in hepatocytes through IHC staining and western blot analysis. Furthermore, the authors show an alteration in systemic bile acid homeostasis in MC1R KO mice. However, no mention of MC1R expression or function in cholangiocytes is discussed. This is important to assess both experimentally and within the discussion given the profound role of the biliary epithelium in modulating bile acid homeostasis. Furthermore, in figure 1 the authors validate the MC1R knockdown only through mRNA expression. Given panels A and C of figure 1 shows there is clearly a functional antibody for MC1R, validation of protein knockdown is needed.
The reviewer raises an important point, which we addressed by performing immunofluorescence staining using an antibody against the cholangiocyte marker cytokeratin 19 (CK-19). These colocalization studies demonstrate the presence of MC1-R in CK19-positive cholangiocytes (Figure 1-figure supplement 1). Furthermore, we have now added a discussion on the possible role of MC1-R in modulating bile acid homestasis in cholangiocytes (page 12, lines 456-462).
We also quantified MC1-R protein expression by Western blotting in the liver of LMc1r-/- mice. MC1-R protein level was significantly reduced in L-Mc1r-/- mice compared to L-Mc1+/- mice (Figure 2-figure supplement 2).- Figure 2 demonstrates a steatotic effect of MC1R knockdown in hepatocytes. The authors attempt to provide mechanistic insight into this phenomenon through assessing the mRNA expression of genes involved in cholesterol and fatty acid synthesis. The data provided is modest at the gene level and no protein validation was provided to demonstrate functional alterations of these proteins in MC1R KO mice. Key proteins proposed such as SREBP2 and HMGCR need to be validated via a western blot of IHC analysis.
As requested by the reviewer, we quantified the expression of key proteins in the liver of L-Mc1r-/- mice by Western blotting. We observed that the protein levels of HMGCR and DHCR7 as well as the ratio between the mature and precursor forms of SREBP2 were reduced in L-Mc1r-/- mice (Figure 2F-H, page 6/lines 182-191 & page 10-11/lines 390-401). This is likely a result of the feedback regulation, whereby cholesterol accumulation suppresses the cleavage of SREBP2 and leads to a consequent downregulation of the key cholesterol synthesis enzymes such as HMGCR and DHCR7 (Brown S & Goldstein JL, Cell. 1997 May 2;89(3):331-40).
We discussed in the original submission (page 11) as follows: ‘In the presence of excess cellular cholesterol, transcriptional induction and posttranslational activation of SREBP-2 should be attenuated, which in turn downregulates Hmgcr and Dhcr7 and reduces cholesterol synthesis as a counterregulatory mechanism. Therefore, given the increase in hepatic cholesterol content, it was unexpected that Srebp2 expression was upregulated in the liver of L-Mc1r-/- mice’. The finding of reduced SREBP2/HMGCR protein expression is thus more logical, but admittedly, it is discordant with increased Srebp2/Hmgcr mRNA expression (as reported in the original submission), which might be a compensatory response to suppressed SREBP2 cleavage. Taking into account that activation of MC1-R did not affect the protein expression of HMGCR or DHCR7 in HepG2 cells, it is plausible that hepatic cholesterol accumulation in L-Mc1r-/- mice is driven by a defect in bile acid metabolism, rather than by a direct effect of MC1-R signaling on cholesterol synthesis. To avoid unnecessary confusion, we decided to omit the qPCR data and related text parts from the manuscript and report the protein expression data instead.
- The authors suggest the involvement of AMPK in mediating the cholesterol-lowering effects of MSH. However, MSH is still able to lower free cholesterol levels even in the presence of an AMPK inhibitor. This suggests that MSH does not in fact rely on the activation of AMPK to elicit these cholesterol-lowering effects. The authors' conclusions are stronger than the actual data support. Furthermore, the authors claim LD211 phenocopies the effects of MSH in the presence of an AMPK inhibitor. However, the authors only measured the phosphorylation of Akt as their outcome. This begs the question, does LD211 still lower total cholesterol in the presence of AMPK inhibitors? This experiment is essential to conclude whether or not LD211 phenocopies the effects of MSH.
The reviewer may have missed that we postulate in the manuscript that ‘MC1-R activation engages multiple signaling mechanisms to regulate cholesterol metabolism in HepG2 cells’ (manuscript page 8, lines 310-311 & page 13, lines 498508), since low concentration of a-MSH was still able to lower free cholesterol level in the presence of the AMPK inhibitor dorsomorphin. We have been careful not to claim that the effects of a-MSH are solely dependent on AMPK phosphorylation. Likewise, we have not claimed in the original submission that LD211 phenocopies the effects of MSH in the presence of an AMPK inhibitor. However, as suggested by the reviewer, we performed new experiments to investigate the effects of LD211 on cellular cholesterol levels in the absence and presence of dorsomorphin. We found that AMPK inhibition with dorsomorphin completely abolished the cholesterollowering effect of LD211 (Figure 7-figure supplement 2), which might indicate that this synthetic agonist has a stronger signaling bias toward the AMPK pathway compared to α-MSH.
- The authors initiate the project by showing high-fat diet disrupts the expression of MC1R. However, all of the subsequent experiments in hepatic MC1R KO mice are performed under normal chow. This begs the question of what is the phenotype of the hepatic MC1R KO mice fed a high-fat diet. Does KO of MC1R in the liver exacerbate HFD-induced obesity, glucose intolerance, and dyslipidemia? Inversely, can WT mice challenged with an HFD be rescued metabolically by treatment with either MSH or LD211? Providing data along these lines of investigation will provide physiological/clinical relevance to their findings.
As suggested by the reviewer, we phenotyped the hepatic MC1R KO (LMc1r-/-) mice after feeding them a cholesterol- and fat-rich Western diet for 12 weeks (RD Western Diet, D12079B, Research Diets Inc, NJ, USA). This was exactly the same dietary regimen (product and duration) that was used to study the changes in hepatic MC1-R expression in wild-type C57Bl mice (Figure 1B&C). We observed that 12-week Western diet feeding induced a significant gain in body weight and total fat mass as well as an increase in plasma and hepatic cholesterol and TG levels (Figure 2-figure supplement 2). L-Mc1r-/- mice did not show a difference in body weight gain, but the weight gain was attributable to enhanced gain in fat mass and a blunted increase in lean mass compared to control Mc1rfl/fl mice (Figure 2-figure supplement 2A, D & E). Furthermore, liver weight and plasma cholesterol and TG concentrations were unchanged in HFD-fed L-Mc1r-/- mice (Figure 2-figure supplement 2B, C, F & G). Importantly, recapitulating the phenotype observed in chow-fed mice, hepatic cholesterol and TG content was significantly increased in LMc1r-/- mice after a HFD challenge (Figure 2-figure supplement 2H & I). Taken together, it appears that the phenotype of HFD-fed L-Mc1r-/- mice was slightly diluted compared to the phenotype observed in chow-fed L-Mc1r-/- mice. This phenotypic difference might relate to the finding that Western diet feeding reduced the hepatic expression of MC1-R, thus limiting the incremental effect of genetically induced MC1-R deficiency on hypercholesterolemia and hepatic lipid accumulation.
We have previously studied the effects of pharmacological MC1-R activation in Western diet-fed mice and observed that chronic treatment with a selective MC1-R agonist reduced plasma cholesterol level and upregulated hepatic Ldlr expression without affecting body weight gain (Rinne P et al, Circulation. 2017 Jul 4;136(1):8397.). These findings are also discussed on manuscript page 12, lines 475-478. Although the selective MC1-R agonist was different in that particular study, it is expected that LD211 would also elicit a similar cholesterol-lowering effect in Western diet-fed mice. Chronic treatment with a-MSH, on the other hand, would likely produce wide-ranging metabolic effects. In addition to MC1-R activation in hepatocytes and its consequent effect on liver cholesterol metabolism, a-MSH would affect feeding, energy expenditure and cholesterol metabolism via MC4-R activation in the central nervous system as well as fatty acid and glucose metabolism via MC5-R activation in the skeletal muscle. Therefore, the phenotype associated with a-MSH treatment would be complex and mediated by multiple mechanisms and MC-R subtypes, thus making it difficult to interpret the exact contribution of hepatic MC1-R signaling to the observed phenotype.
Reviewer #2 (Public Review):
Keshav Thapa et al. investigated the role of melanocortin 1 receptor (MC1-R) in cholesterol and bile acid metabolism in the liver. First, they observed that MC1-R is present in the mouse liver and that its expression is reduced in response to a cholesterolrich diet. To determine the role of MC1-R in the liver, they generated hepatocyte-specific MC1-R KO mice (L-Mc1r-/-). These animals exhibited a significant increase in liver weight, lipid accumulation, triglycerides and cholesterol levels, and fibrosis in comparison with control mice. By performing liquid chromatography-mass spectrometry, the authors also found that L-Mc1r-/- mice also have fewer bile acids in the plasma and faeces, but not in the liver. In accordance with these findings, mRNA/protein expression of different genes involved in these processes were altered in L-Mc1r-/- animals.
Secondly, in an attempt to evaluate the underlying mechanisms, they measured the expression of MC1-R in HepG2 cells under different treatments (i.e., palmitic acid, LDL, and atorvastatin). Moreover, they stimulated these cells with the endogenous MC1-R agonist - MSH, where they show that this molecule decreases the free cholesterol content, whereas increasing LDL and HDL uptake, as well as recapitulates some previously observed phenotypes in the proportions of bile acids. These effects were also encountered when using a selective agonist for MC1-R (i.e., LD211), further supporting the specific role of MC1-R. Finally, some experiments indicated that -MSH evokes not one single, but multiple intracellular signalling cascades for which MC1-R activation effects might take place.
Overall, this work provides novel and interesting findings on the role of MC1-R in cholesterol and bile acid metabolism in the liver, which undoubtedly will have some crucial implications for future research. Nevertheless, some experimental details should be better explained for the correct interpretation of the data. Besides, discrepant results exist regarding the molecular mechanisms behind MC1-R action that requires additional experimentation to support the conclusions drawn.
We thank the reviewer for the encouraging and insightful comments, and we are pleased to read that the manuscript has raised considerable interest.
-
eLife assessment
The significance of this manuscript is that is provides useful information for the field of hepatology and endocrinology on the regulatory mechanisms of cholesterol homeostasis by melanocortin. The authors provide solid evidence utilizing both in vivo and in vitro molecular, cellular, and biochemical approaches to support their claims.
-
Reviewer #1 (Public Review):
"Melanocortin 1 receptor regulates cholesterol and bile acid metabolism in the liver" by Thapa et al. extends previous findings that MC1R global knockout mice have dysregulated lipid metabolism in APOE KO mice. The authors generated a hepatocyte-specific MC1R KO mouse to assess the hepatic effects of MC1R on the regulation of lipid metabolism. Thapa et al. go on to show that hepatic MC1R deletion leads to dyslipidemia and hepatic steatosis. The authors subsequently show that altered cholesterol homeostasis disrupts bile acid metabolism in hepatic MC1R KO mice. Finally, the authors provide data to suggest a role for AMPK in mediating the effects of MSH on hepatic cholesterol metabolism. The authors designed rigorous experiments using multiple different models (in vivo and in vitro) as well as different …
Reviewer #1 (Public Review):
"Melanocortin 1 receptor regulates cholesterol and bile acid metabolism in the liver" by Thapa et al. extends previous findings that MC1R global knockout mice have dysregulated lipid metabolism in APOE KO mice. The authors generated a hepatocyte-specific MC1R KO mouse to assess the hepatic effects of MC1R on the regulation of lipid metabolism. Thapa et al. go on to show that hepatic MC1R deletion leads to dyslipidemia and hepatic steatosis. The authors subsequently show that altered cholesterol homeostasis disrupts bile acid metabolism in hepatic MC1R KO mice. Finally, the authors provide data to suggest a role for AMPK in mediating the effects of MSH on hepatic cholesterol metabolism. The authors designed rigorous experiments using multiple different models (in vivo and in vitro) as well as different approaches (genetic and pharmacological).
The work described herein would have an impact on the field in multiple ways. Firstly, it demonstrates a novel metabolic role for MSH in the regulation of hepatic cholesterol metabolism. This may prove to be a viable therapeutic strategy for the treatment of dyslipidemia. Furthermore, the authors demonstrate an alternative signaling cascade elicited by MSH independent of cAMP, but rather relying on AMPK. This novel interaction between AMPK and MC1R could have more widespread implications beyond the control of hepatic cholesterol metabolism.
For the most part, the conclusions offered by the authors are supported by the data that is presented. There are, however, a number of concerns in the current version of this manuscript detailed below:
The authors demonstrate the expression of MC1R in hepatocytes through IHC staining and western blot analysis. Furthermore, the authors show an alteration in systemic bile acid homeostasis in MC1R KO mice. However, no mention of MC1R expression or function in cholangiocytes is discussed. This is important to assess both experimentally and within the discussion given the profound role of the biliary epithelium in modulating bile acid homeostasis. Furthermore, in figure 1 the authors validate the MC1R knockdown only through mRNA expression. Given panels A and C of figure 1 shows there is clearly a functional antibody for MC1R, validation of protein knockdown is needed.
Figure 2 demonstrates a steatotic effect of MC1R knockdown in hepatocytes. The authors attempt to provide mechanistic insight into this phenomenon through assessing the mRNA expression of genes involved in cholesterol and fatty acid synthesis. The data provided is modest at the gene level and no protein validation was provided to demonstrate functional alterations of these proteins in MC1R KO mice. Key proteins proposed such as SREBP2 and HMGCR need to be validated via a western blot of IHC analysis.
The authors suggest the involvement of AMPK in mediating the cholesterol-lowering effects of MSH. However, MSH is still able to lower free cholesterol levels even in the presence of an AMPK inhibitor. This suggests that MSH does not in fact rely on the activation of AMPK to elicit these cholesterol-lowering effects. The authors' conclusions are stronger than the actual data support. Furthermore, the authors claim LD211 phenocopies the effects of MSH in the presence of an AMPK inhibitor. However, the authors only measured the phosphorylation of Akt as their outcome. This begs the question, does LD211 still lower total cholesterol in the presence of AMPK inhibitors? This experiment is essential to conclude whether or not LD211 phenocopies the effects of MSH.
The authors initiate the project by showing high-fat diet disrupts the expression of MC1R. However, all of the subsequent experiments in hepatic MC1R KO mice are performed under normal chow. This begs the question of what is the phenotype of the hepatic MC1R KO mice fed a high-fat diet. Does KO of MC1R in the liver exacerbate HFD-induced obesity, glucose intolerance, and dyslipidemia? Inversely, can WT mice challenged with an HFD be rescued metabolically by treatment with either MSH or LD211? Providing data along these lines of investigation will provide physiological/clinical relevance to their findings.
-
Reviewer #2 (Public Review):
Keshav Thapa et al. investigated the role of melanocortin 1 receptor (MC1-R) in cholesterol and bile acid metabolism in the liver. First, they observed that MC1-R is present in the mouse liver and that its expression is reduced in response to a cholesterol-rich diet. To determine the role of MC1-R in the liver, they generated hepatocyte-specific MC1-R KO mice (L-Mc1r-/-). These animals exhibited a significant increase in liver weight, lipid accumulation, triglycerides and cholesterol levels, and fibrosis in comparison with control mice. By performing liquid chromatography-mass spectrometry, the authors also found that L-Mc1r-/- mice also have fewer bile acids in the plasma and faeces, but not in the liver. In accordance with these findings, mRNA/protein expression of different genes involved in these …
Reviewer #2 (Public Review):
Keshav Thapa et al. investigated the role of melanocortin 1 receptor (MC1-R) in cholesterol and bile acid metabolism in the liver. First, they observed that MC1-R is present in the mouse liver and that its expression is reduced in response to a cholesterol-rich diet. To determine the role of MC1-R in the liver, they generated hepatocyte-specific MC1-R KO mice (L-Mc1r-/-). These animals exhibited a significant increase in liver weight, lipid accumulation, triglycerides and cholesterol levels, and fibrosis in comparison with control mice. By performing liquid chromatography-mass spectrometry, the authors also found that L-Mc1r-/- mice also have fewer bile acids in the plasma and faeces, but not in the liver. In accordance with these findings, mRNA/protein expression of different genes involved in these processes were altered in L-Mc1r-/- animals.
Secondly, in an attempt to evaluate the underlying mechanisms, they measured the expression of MC1-R in HepG2 cells under different treatments (i.e., palmitic acid, LDL, and atorvastatin). Moreover, they stimulated these cells with the endogenous MC1-R agonist - MSH, where they show that this molecule decreases the free cholesterol content, whereas increasing LDL and HDL uptake, as well as recapitulates some previously observed phenotypes in the proportions of bile acids. These effects were also encountered when using a selective agonist for MC1-R (i.e., LD211), further supporting the specific role of MC1-R. Finally, some experiments indicated that -MSH evokes not one single, but multiple intracellular signalling cascades for which MC1-R activation effects might take place.
Overall, this work provides novel and interesting findings on the role of MC1-R in cholesterol and bile acid metabolism in the liver, which undoubtedly will have some crucial implications for future research. Nevertheless, some experimental details should be better explained for the correct interpretation of the data. Besides, discrepant results exist regarding the molecular mechanisms behind MC1-R action that requires additional experimentation to support the conclusions drawn.
-