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The Causative Gene in Chanarian Dorfman Syndrome Regulates Lipid Droplet Homeostasis in C. elegans.

Xie M, Roy R - PLoS Genet. (2015)

Bottom Line: We found that the compromise of one of the three C. elegans orthologues of human cgi-58 significantly improves the survival of AMPK-deficient dauers.We also provide evidence that C. elegans CGI-58 acts as a co-activator of ATGL-1, while it also functions cooperatively to maintain regular lipid droplet structure.Surprisingly, we show that it also acts independently of ATGL-1 to restrict lipid droplet coalescence by altering the surface abundance and composition of long chain (C20) polyunsaturated fatty acids (PUFAs).

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, McGill University, Penfield, Montreal, Quebec, Canada.

ABSTRACT
AMP-activated kinase (AMPK) is a key regulator of many cellular mechanisms required for adjustment to various stresses induced by the changing environment. In C. elegans dauer larvae AMPK- mutants expire prematurely due to hyperactive Adipose Triglyceride Lipase (ATGL-1) followed by rapid depletion of triglyceride stores. We found that the compromise of one of the three C. elegans orthologues of human cgi-58 significantly improves the survival of AMPK-deficient dauers. We also provide evidence that C. elegans CGI-58 acts as a co-activator of ATGL-1, while it also functions cooperatively to maintain regular lipid droplet structure. Surprisingly, we show that it also acts independently of ATGL-1 to restrict lipid droplet coalescence by altering the surface abundance and composition of long chain (C20) polyunsaturated fatty acids (PUFAs). Our data reveal a novel structural role of CGI-58 in maintaining lipid droplet homeostasis through its effects on droplet composition, morphology and lipid hydrolysis; a conserved function that may account for some of the ATGL-1-independent features unique to Chanarin-Dorfman Syndrome.

No MeSH data available.


Related in: MedlinePlus

Elimination of cgi-58 rescued both triglyceride levels and excretory defects in daf-2; aak(0) mutant Dauer larvae.(A) Protein alignment of the two isoforms of C. elegans CGI-58 (C37H5.a and C37H5.b) with human CGI-58. The red underlined amino acids indicate the deleted region in the ok3245 allele. (B) Elimination of cgi-58 by RNAi feeding or by using the ok3245 allele significantly enhanced the survival of daf-2; aak-1; aak-2 mutant dauer larvae. aak-1; aak-2 is represented as aak(0) in all Figs. A Log-rank (Mantel-Cox) test was used to statistically compare the survival curves in all Figs. (C) Mutation of cgi-58 in control daf-2 animals significantly enhanced their survival during dauer stage (P = 0.0003). (D)cgi-58 protects the triglyceride stores from depletion in daf-2; aak(0) mutant dauer larvae. Colorimetric analysis of triglyceride content in day 4 dauer larvae. ** indicates statistical significance (P<0.01) compared to all four of the other genotypes using one-way ANOVA followed by a Tukey HSD test. Error bars indicate SD of three independent experiments. The same statistical analysis was applied for all subsequent experiments performed hereafter. (E) Elimination of cgi-58 protects the triglyceride stockpile in daf-2; aak(0) mutant dauer larvae. Oil Red O staining of day 4 dauer larvae. Arrowhead indicates the junction between the pharynx (left) and the intestine (right). Oil Red O staining intensity was evaluated by measuring optical density. * and ** indicate statistical significance (P<0.05 and P<0.01, respectively) compared to daf-2; aak(0) mutant dauer larvae. Error bars indicate SD of 20 animals. Scale bar = 10 μm. (F) Osmoregulatory defects typical of AMPK dauer larvae were corrected by reducing cgi-58 function. cgi-58 compromise restores osmoresistance of day 4 daf-2; aak(0) mutant dauer larvae following culture in varying NaCl concentrations for 24 hours at 25°C. The data represent a pool of three independent experiments. Survival of daf-2, daf-2; aak(0) cgi-58(RNAi) and daf-2; aak(0); cgi-58 dauers is significantly higher than daf-2; aak(0) dauers at concentrations of both 1M and 1.5M NaCl, analyzed using one-way ANOVA followed by a Tukey HSD test.
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pgen.1005284.g001: Elimination of cgi-58 rescued both triglyceride levels and excretory defects in daf-2; aak(0) mutant Dauer larvae.(A) Protein alignment of the two isoforms of C. elegans CGI-58 (C37H5.a and C37H5.b) with human CGI-58. The red underlined amino acids indicate the deleted region in the ok3245 allele. (B) Elimination of cgi-58 by RNAi feeding or by using the ok3245 allele significantly enhanced the survival of daf-2; aak-1; aak-2 mutant dauer larvae. aak-1; aak-2 is represented as aak(0) in all Figs. A Log-rank (Mantel-Cox) test was used to statistically compare the survival curves in all Figs. (C) Mutation of cgi-58 in control daf-2 animals significantly enhanced their survival during dauer stage (P = 0.0003). (D)cgi-58 protects the triglyceride stores from depletion in daf-2; aak(0) mutant dauer larvae. Colorimetric analysis of triglyceride content in day 4 dauer larvae. ** indicates statistical significance (P<0.01) compared to all four of the other genotypes using one-way ANOVA followed by a Tukey HSD test. Error bars indicate SD of three independent experiments. The same statistical analysis was applied for all subsequent experiments performed hereafter. (E) Elimination of cgi-58 protects the triglyceride stockpile in daf-2; aak(0) mutant dauer larvae. Oil Red O staining of day 4 dauer larvae. Arrowhead indicates the junction between the pharynx (left) and the intestine (right). Oil Red O staining intensity was evaluated by measuring optical density. * and ** indicate statistical significance (P<0.05 and P<0.01, respectively) compared to daf-2; aak(0) mutant dauer larvae. Error bars indicate SD of 20 animals. Scale bar = 10 μm. (F) Osmoregulatory defects typical of AMPK dauer larvae were corrected by reducing cgi-58 function. cgi-58 compromise restores osmoresistance of day 4 daf-2; aak(0) mutant dauer larvae following culture in varying NaCl concentrations for 24 hours at 25°C. The data represent a pool of three independent experiments. Survival of daf-2, daf-2; aak(0) cgi-58(RNAi) and daf-2; aak(0); cgi-58 dauers is significantly higher than daf-2; aak(0) dauers at concentrations of both 1M and 1.5M NaCl, analyzed using one-way ANOVA followed by a Tukey HSD test.

Mentions: Although AMPK negatively regulates ATGL-1 during the dauer stage, we and others have identified factors that are required for, or enhance ATGL-1 activity [15], [17], [18]. In mammalian cells ATGL is regulated by a complex relay mechanism between lipid droplet-associated proteins such as the Perilipins, G0S2 and CGI-58. At present, no clear orthologue of G0S2, or the Perilipin protein family has been identified in the annotation of the C. elegans genome sequence. To explore how ATGL-1 may be regulated during the dauer stage, we characterized the function of the C. elegans orthologue of mammalian CGI-58, which we identified in a genome-wide survey for genes that phenocopied atgl-1(RNAi) by enhancing dauer survival of animals with mutations in both isoforms of their AMPK α subunit (aak-1 and aak-2, which will be presented as aak(0) throughout the study) [17]. The predicted C. elegans gene C37H5.3 is expressed as two alternative transcripts, both of which encode a protein that is 46.1% and 58.3% similar to the human CGI-58 protein, respectively (Fig 1A). Importantly, one of the N-terminal tryptophan residues previously shown to be critical for the correct localization and ATGL-activating function of mammalian CGI-58 is conserved in C. elegans [15]. There are two other genes in the C. elegans genome that share sequence homology with the mammalian CGI-58, lid-1 and C37H5.2, where the former has recently been shown to regulate ATGL-1-dependant lipolysis during fasting in C. elegans [19]. However, neither of these two genes were recovered from our genome-wide RNAi survey [17]. Furthermore, in our directed RNAi assays, elimination of either lid-1 or C37H5.2 did not enhance dauer survival in insulin-signalling compromised (daf-2) dauer larvae that lacked AMPK function (S1 Fig). For this reason we chose to further investigate C37H5.3 function during dauer development and will be referred to hereafter as cgi-58 throughout our study.


The Causative Gene in Chanarian Dorfman Syndrome Regulates Lipid Droplet Homeostasis in C. elegans.

Xie M, Roy R - PLoS Genet. (2015)

Elimination of cgi-58 rescued both triglyceride levels and excretory defects in daf-2; aak(0) mutant Dauer larvae.(A) Protein alignment of the two isoforms of C. elegans CGI-58 (C37H5.a and C37H5.b) with human CGI-58. The red underlined amino acids indicate the deleted region in the ok3245 allele. (B) Elimination of cgi-58 by RNAi feeding or by using the ok3245 allele significantly enhanced the survival of daf-2; aak-1; aak-2 mutant dauer larvae. aak-1; aak-2 is represented as aak(0) in all Figs. A Log-rank (Mantel-Cox) test was used to statistically compare the survival curves in all Figs. (C) Mutation of cgi-58 in control daf-2 animals significantly enhanced their survival during dauer stage (P = 0.0003). (D)cgi-58 protects the triglyceride stores from depletion in daf-2; aak(0) mutant dauer larvae. Colorimetric analysis of triglyceride content in day 4 dauer larvae. ** indicates statistical significance (P<0.01) compared to all four of the other genotypes using one-way ANOVA followed by a Tukey HSD test. Error bars indicate SD of three independent experiments. The same statistical analysis was applied for all subsequent experiments performed hereafter. (E) Elimination of cgi-58 protects the triglyceride stockpile in daf-2; aak(0) mutant dauer larvae. Oil Red O staining of day 4 dauer larvae. Arrowhead indicates the junction between the pharynx (left) and the intestine (right). Oil Red O staining intensity was evaluated by measuring optical density. * and ** indicate statistical significance (P<0.05 and P<0.01, respectively) compared to daf-2; aak(0) mutant dauer larvae. Error bars indicate SD of 20 animals. Scale bar = 10 μm. (F) Osmoregulatory defects typical of AMPK dauer larvae were corrected by reducing cgi-58 function. cgi-58 compromise restores osmoresistance of day 4 daf-2; aak(0) mutant dauer larvae following culture in varying NaCl concentrations for 24 hours at 25°C. The data represent a pool of three independent experiments. Survival of daf-2, daf-2; aak(0) cgi-58(RNAi) and daf-2; aak(0); cgi-58 dauers is significantly higher than daf-2; aak(0) dauers at concentrations of both 1M and 1.5M NaCl, analyzed using one-way ANOVA followed by a Tukey HSD test.
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pgen.1005284.g001: Elimination of cgi-58 rescued both triglyceride levels and excretory defects in daf-2; aak(0) mutant Dauer larvae.(A) Protein alignment of the two isoforms of C. elegans CGI-58 (C37H5.a and C37H5.b) with human CGI-58. The red underlined amino acids indicate the deleted region in the ok3245 allele. (B) Elimination of cgi-58 by RNAi feeding or by using the ok3245 allele significantly enhanced the survival of daf-2; aak-1; aak-2 mutant dauer larvae. aak-1; aak-2 is represented as aak(0) in all Figs. A Log-rank (Mantel-Cox) test was used to statistically compare the survival curves in all Figs. (C) Mutation of cgi-58 in control daf-2 animals significantly enhanced their survival during dauer stage (P = 0.0003). (D)cgi-58 protects the triglyceride stores from depletion in daf-2; aak(0) mutant dauer larvae. Colorimetric analysis of triglyceride content in day 4 dauer larvae. ** indicates statistical significance (P<0.01) compared to all four of the other genotypes using one-way ANOVA followed by a Tukey HSD test. Error bars indicate SD of three independent experiments. The same statistical analysis was applied for all subsequent experiments performed hereafter. (E) Elimination of cgi-58 protects the triglyceride stockpile in daf-2; aak(0) mutant dauer larvae. Oil Red O staining of day 4 dauer larvae. Arrowhead indicates the junction between the pharynx (left) and the intestine (right). Oil Red O staining intensity was evaluated by measuring optical density. * and ** indicate statistical significance (P<0.05 and P<0.01, respectively) compared to daf-2; aak(0) mutant dauer larvae. Error bars indicate SD of 20 animals. Scale bar = 10 μm. (F) Osmoregulatory defects typical of AMPK dauer larvae were corrected by reducing cgi-58 function. cgi-58 compromise restores osmoresistance of day 4 daf-2; aak(0) mutant dauer larvae following culture in varying NaCl concentrations for 24 hours at 25°C. The data represent a pool of three independent experiments. Survival of daf-2, daf-2; aak(0) cgi-58(RNAi) and daf-2; aak(0); cgi-58 dauers is significantly higher than daf-2; aak(0) dauers at concentrations of both 1M and 1.5M NaCl, analyzed using one-way ANOVA followed by a Tukey HSD test.
Mentions: Although AMPK negatively regulates ATGL-1 during the dauer stage, we and others have identified factors that are required for, or enhance ATGL-1 activity [15], [17], [18]. In mammalian cells ATGL is regulated by a complex relay mechanism between lipid droplet-associated proteins such as the Perilipins, G0S2 and CGI-58. At present, no clear orthologue of G0S2, or the Perilipin protein family has been identified in the annotation of the C. elegans genome sequence. To explore how ATGL-1 may be regulated during the dauer stage, we characterized the function of the C. elegans orthologue of mammalian CGI-58, which we identified in a genome-wide survey for genes that phenocopied atgl-1(RNAi) by enhancing dauer survival of animals with mutations in both isoforms of their AMPK α subunit (aak-1 and aak-2, which will be presented as aak(0) throughout the study) [17]. The predicted C. elegans gene C37H5.3 is expressed as two alternative transcripts, both of which encode a protein that is 46.1% and 58.3% similar to the human CGI-58 protein, respectively (Fig 1A). Importantly, one of the N-terminal tryptophan residues previously shown to be critical for the correct localization and ATGL-activating function of mammalian CGI-58 is conserved in C. elegans [15]. There are two other genes in the C. elegans genome that share sequence homology with the mammalian CGI-58, lid-1 and C37H5.2, where the former has recently been shown to regulate ATGL-1-dependant lipolysis during fasting in C. elegans [19]. However, neither of these two genes were recovered from our genome-wide RNAi survey [17]. Furthermore, in our directed RNAi assays, elimination of either lid-1 or C37H5.2 did not enhance dauer survival in insulin-signalling compromised (daf-2) dauer larvae that lacked AMPK function (S1 Fig). For this reason we chose to further investigate C37H5.3 function during dauer development and will be referred to hereafter as cgi-58 throughout our study.

Bottom Line: We found that the compromise of one of the three C. elegans orthologues of human cgi-58 significantly improves the survival of AMPK-deficient dauers.We also provide evidence that C. elegans CGI-58 acts as a co-activator of ATGL-1, while it also functions cooperatively to maintain regular lipid droplet structure.Surprisingly, we show that it also acts independently of ATGL-1 to restrict lipid droplet coalescence by altering the surface abundance and composition of long chain (C20) polyunsaturated fatty acids (PUFAs).

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, McGill University, Penfield, Montreal, Quebec, Canada.

ABSTRACT
AMP-activated kinase (AMPK) is a key regulator of many cellular mechanisms required for adjustment to various stresses induced by the changing environment. In C. elegans dauer larvae AMPK- mutants expire prematurely due to hyperactive Adipose Triglyceride Lipase (ATGL-1) followed by rapid depletion of triglyceride stores. We found that the compromise of one of the three C. elegans orthologues of human cgi-58 significantly improves the survival of AMPK-deficient dauers. We also provide evidence that C. elegans CGI-58 acts as a co-activator of ATGL-1, while it also functions cooperatively to maintain regular lipid droplet structure. Surprisingly, we show that it also acts independently of ATGL-1 to restrict lipid droplet coalescence by altering the surface abundance and composition of long chain (C20) polyunsaturated fatty acids (PUFAs). Our data reveal a novel structural role of CGI-58 in maintaining lipid droplet homeostasis through its effects on droplet composition, morphology and lipid hydrolysis; a conserved function that may account for some of the ATGL-1-independent features unique to Chanarin-Dorfman Syndrome.

No MeSH data available.


Related in: MedlinePlus