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Feeding and Fasting Signals Converge on the LKB1-SIK3 Pathway to Regulate Lipid Metabolism in Drosophila.

Choi S, Lim DS, Chung J - PLoS Genet. (2015)

Bottom Line: Interestingly, we found that the LKB1-SIK3-HDAC4 signaling axis is modulated by dietary conditions.In short-term fasting, the adipokinetic hormone (AKH) pathway, related to the mammalian glucagon pathway, inhibits the kinase activity of LKB1 as shown by decreased SIK3 Thr196 phosphorylation, and consequently induces HDAC4 nuclear localization and brummer gene expression.However, under prolonged fasting conditions, AKH-independent signaling decreases the activity of the LKB1-SIK3 pathway to induce lipolytic responses.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejon, Republic of Korea; National Creative Research Initiatives Center for Energy Homeostasis Regulation, Seoul National University, Seoul, Republic of Korea; Institute of Molecular Biology and Genetics, Seoul National University, Seoul, Republic of Korea.

ABSTRACT
LKB1 plays important roles in governing energy homeostasis by regulating AMP-activated protein kinase (AMPK) and other AMPK-related kinases, including the salt-inducible kinases (SIKs). However, the roles and regulation of LKB1 in lipid metabolism are poorly understood. Here we show that Drosophila LKB1 mutants display decreased lipid storage and increased gene expression of brummer, the Drosophila homolog of adipose triglyceride lipase (ATGL). These phenotypes are consistent with those of SIK3 mutants and are rescued by expression of constitutively active SIK3 in the fat body, suggesting that SIK3 is a key downstream kinase of LKB1. Using genetic and biochemical analyses, we identify HDAC4, a class IIa histone deacetylase, as a lipolytic target of the LKB1-SIK3 pathway. Interestingly, we found that the LKB1-SIK3-HDAC4 signaling axis is modulated by dietary conditions. In short-term fasting, the adipokinetic hormone (AKH) pathway, related to the mammalian glucagon pathway, inhibits the kinase activity of LKB1 as shown by decreased SIK3 Thr196 phosphorylation, and consequently induces HDAC4 nuclear localization and brummer gene expression. However, under prolonged fasting conditions, AKH-independent signaling decreases the activity of the LKB1-SIK3 pathway to induce lipolytic responses. We also identify that the Drosophila insulin-like peptides (DILPs) pathway, related to mammalian insulin pathway, regulates SIK3 activity in feeding conditions independently of increasing LKB1 kinase activity. Overall, these data suggest that fasting stimuli specifically control the kinase activity of LKB1 and establish the LKB1-SIK3 pathway as a converging point between feeding and fasting signals to control lipid homeostasis in Drosophila.

No MeSH data available.


Related in: MedlinePlus

LKB1 and its downstream kinase SIK3 are required for lipid homeostasis.(A) qPCR analysis of LKB1 and its cofactors required for the catalytic activity, STRAD and MO25, in Drosophila larvae under feeding condition. (B) TAG amounts of wild-type and LKB1 mutant larvae (n = 10 per genotype). (C) qPCR analysis for lipogenic genes (SREBP, FAS and ACC) and lipolytic genes (bmm and HSL) in wild-type and LKB1 mutant larvae at mid-to-late L2 (60 hr AEL) stage under feeding conditions. (D) qPCR analysis of LKB1, SIKs (SIK2 and SIK3), and AMPK complex (AMPKα, AMPKβ, and AMPKγ) in larvae. (E) TAG amounts in LKB1 mutants following fat body-specific expression of wild-type, kinase-dead (K201I) LKB1, constitutively active (T196E) SIK3 or constitutively active (T184D) AMPK. Genotypes are as follows: FB> (FB-Gal4/+), LKB1X5,FB> (FB-Gal4/+;LKB1X5/LKB1X5), LKB1X5,FB>LKB1WT (FB-Gal4/UAS-LKB1;LKB1X5/LKB1X5), LKB1X5,FB>LKB1KI (FB-Gal4/UAS-LKB1 K201I;LKB1X5/LKB1X5), LKB1X5,FB>SIK3TE (FB-Gal4/UAS-SIK3 T196E;LKB1X5/LKB1X5), and LKB1X5,FB>AMPKTD (FB-Gal4/UAS-AMPK T184D;LKB1X5/LKB1X5) (n = 10 per genotype). (F) qPCR analysis of bmm gene expression in LKB1 mutants following fat body-specific expression of wild-type LKB1 or constitutively active (T196E) SIK3 at mid-to-late L2 stage under feeding condition. Genotypes are as follows: FB> (FB-Gal4/+), LKB1X5,FB> (FB-Gal4/+;LKB1X5/LKB1X5), LKB1X5,FB>LKB1WT (FB-Gal4/UAS-LKB1;LKB1X5/LKB1X5), and LKB1X5,FB>SIK3TE (FB-Gal4/UAS-SIK3 T196E;LKB1X5/LKB1X5). (G) Immunoblot analyses showing the effect of LKB1 on Thr196 phosphorylation of SIK3 protein in larvae. Wild-type and kinase-dead (K70M) SIK3 were highly phosphorylated at Thr196 by LKB1 (second panel). SIK3T196A was used as a control. FB-Gal4 was used to drive transgene expression in the fat body. (H) Immunoblot analyses showing relative amounts of SIK3 Thr196 phosphorylation in wild-type and LKB1X5 mutant larvae. The phosphorylation was absolutely dependent on LKB1 (first panel). FB-Gal4 was used to drive transgene expression. (G-H) Anti-LKB1, -phospho-Thr196 SIK3, -Myc (SIK3 protein), and -β-tubulin (TUB) antibodies were used. Data are presented as mean ± SEM (*P < 0.05).
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pgen.1005263.g001: LKB1 and its downstream kinase SIK3 are required for lipid homeostasis.(A) qPCR analysis of LKB1 and its cofactors required for the catalytic activity, STRAD and MO25, in Drosophila larvae under feeding condition. (B) TAG amounts of wild-type and LKB1 mutant larvae (n = 10 per genotype). (C) qPCR analysis for lipogenic genes (SREBP, FAS and ACC) and lipolytic genes (bmm and HSL) in wild-type and LKB1 mutant larvae at mid-to-late L2 (60 hr AEL) stage under feeding conditions. (D) qPCR analysis of LKB1, SIKs (SIK2 and SIK3), and AMPK complex (AMPKα, AMPKβ, and AMPKγ) in larvae. (E) TAG amounts in LKB1 mutants following fat body-specific expression of wild-type, kinase-dead (K201I) LKB1, constitutively active (T196E) SIK3 or constitutively active (T184D) AMPK. Genotypes are as follows: FB> (FB-Gal4/+), LKB1X5,FB> (FB-Gal4/+;LKB1X5/LKB1X5), LKB1X5,FB>LKB1WT (FB-Gal4/UAS-LKB1;LKB1X5/LKB1X5), LKB1X5,FB>LKB1KI (FB-Gal4/UAS-LKB1 K201I;LKB1X5/LKB1X5), LKB1X5,FB>SIK3TE (FB-Gal4/UAS-SIK3 T196E;LKB1X5/LKB1X5), and LKB1X5,FB>AMPKTD (FB-Gal4/UAS-AMPK T184D;LKB1X5/LKB1X5) (n = 10 per genotype). (F) qPCR analysis of bmm gene expression in LKB1 mutants following fat body-specific expression of wild-type LKB1 or constitutively active (T196E) SIK3 at mid-to-late L2 stage under feeding condition. Genotypes are as follows: FB> (FB-Gal4/+), LKB1X5,FB> (FB-Gal4/+;LKB1X5/LKB1X5), LKB1X5,FB>LKB1WT (FB-Gal4/UAS-LKB1;LKB1X5/LKB1X5), and LKB1X5,FB>SIK3TE (FB-Gal4/UAS-SIK3 T196E;LKB1X5/LKB1X5). (G) Immunoblot analyses showing the effect of LKB1 on Thr196 phosphorylation of SIK3 protein in larvae. Wild-type and kinase-dead (K70M) SIK3 were highly phosphorylated at Thr196 by LKB1 (second panel). SIK3T196A was used as a control. FB-Gal4 was used to drive transgene expression in the fat body. (H) Immunoblot analyses showing relative amounts of SIK3 Thr196 phosphorylation in wild-type and LKB1X5 mutant larvae. The phosphorylation was absolutely dependent on LKB1 (first panel). FB-Gal4 was used to drive transgene expression. (G-H) Anti-LKB1, -phospho-Thr196 SIK3, -Myc (SIK3 protein), and -β-tubulin (TUB) antibodies were used. Data are presented as mean ± SEM (*P < 0.05).

Mentions: LKB1 functions in a complex with two scaffolding proteins, STE20-related adaptor (STRAD) and mouse protein 25 (MO25) [20,21]. As the first step toward elucidation of the role of LKB1 in lipid metabolism, we demonstrated the gene expression of each component of the LKB1 complex in the fat body (Fig 1A), suggesting that Drosophila LKB1 forms the heterotrimeric complex when activated in tissues. Additionally, we characterized an LKB1- mutant line, LKB1X5 [22], and found that these flies showed markedly decreased lipid storage compared to wild-type flies, despite having similar food intake and retaining expression of the lipogenic genes (SREBP, FAS, and ACC) (Figs 1B, 1C and S1A). However, expression of bmm and lipolysis activity were elevated in LKB1X5 mutants (Figs 1C and S1B, respectively). Moreover, transgenic expression of wild-type LKB1 with two different fat body drivers (FB-Gal4 and cg-Gal4) rescued the decreased lipid levels and increased bmm expression phenotypes of LKB1X5 mutants, whereas expression of the kinase-dead form of LKB1 (LKB1 K201I) did not (Figs 1E, 1F, S2A and S2B). Additionally, overexpression of LKB1 induced significant increases in the lipid levels and decreases in bmm expression in a dose-dependent manner (S3A and S3B Fig). The implication behind these observations is that LKB1 plays a critical role in lipid storage in Drosophila by regulating the lipolysis pathway in a kinase activity-dependent manner.


Feeding and Fasting Signals Converge on the LKB1-SIK3 Pathway to Regulate Lipid Metabolism in Drosophila.

Choi S, Lim DS, Chung J - PLoS Genet. (2015)

LKB1 and its downstream kinase SIK3 are required for lipid homeostasis.(A) qPCR analysis of LKB1 and its cofactors required for the catalytic activity, STRAD and MO25, in Drosophila larvae under feeding condition. (B) TAG amounts of wild-type and LKB1 mutant larvae (n = 10 per genotype). (C) qPCR analysis for lipogenic genes (SREBP, FAS and ACC) and lipolytic genes (bmm and HSL) in wild-type and LKB1 mutant larvae at mid-to-late L2 (60 hr AEL) stage under feeding conditions. (D) qPCR analysis of LKB1, SIKs (SIK2 and SIK3), and AMPK complex (AMPKα, AMPKβ, and AMPKγ) in larvae. (E) TAG amounts in LKB1 mutants following fat body-specific expression of wild-type, kinase-dead (K201I) LKB1, constitutively active (T196E) SIK3 or constitutively active (T184D) AMPK. Genotypes are as follows: FB> (FB-Gal4/+), LKB1X5,FB> (FB-Gal4/+;LKB1X5/LKB1X5), LKB1X5,FB>LKB1WT (FB-Gal4/UAS-LKB1;LKB1X5/LKB1X5), LKB1X5,FB>LKB1KI (FB-Gal4/UAS-LKB1 K201I;LKB1X5/LKB1X5), LKB1X5,FB>SIK3TE (FB-Gal4/UAS-SIK3 T196E;LKB1X5/LKB1X5), and LKB1X5,FB>AMPKTD (FB-Gal4/UAS-AMPK T184D;LKB1X5/LKB1X5) (n = 10 per genotype). (F) qPCR analysis of bmm gene expression in LKB1 mutants following fat body-specific expression of wild-type LKB1 or constitutively active (T196E) SIK3 at mid-to-late L2 stage under feeding condition. Genotypes are as follows: FB> (FB-Gal4/+), LKB1X5,FB> (FB-Gal4/+;LKB1X5/LKB1X5), LKB1X5,FB>LKB1WT (FB-Gal4/UAS-LKB1;LKB1X5/LKB1X5), and LKB1X5,FB>SIK3TE (FB-Gal4/UAS-SIK3 T196E;LKB1X5/LKB1X5). (G) Immunoblot analyses showing the effect of LKB1 on Thr196 phosphorylation of SIK3 protein in larvae. Wild-type and kinase-dead (K70M) SIK3 were highly phosphorylated at Thr196 by LKB1 (second panel). SIK3T196A was used as a control. FB-Gal4 was used to drive transgene expression in the fat body. (H) Immunoblot analyses showing relative amounts of SIK3 Thr196 phosphorylation in wild-type and LKB1X5 mutant larvae. The phosphorylation was absolutely dependent on LKB1 (first panel). FB-Gal4 was used to drive transgene expression. (G-H) Anti-LKB1, -phospho-Thr196 SIK3, -Myc (SIK3 protein), and -β-tubulin (TUB) antibodies were used. Data are presented as mean ± SEM (*P < 0.05).
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pgen.1005263.g001: LKB1 and its downstream kinase SIK3 are required for lipid homeostasis.(A) qPCR analysis of LKB1 and its cofactors required for the catalytic activity, STRAD and MO25, in Drosophila larvae under feeding condition. (B) TAG amounts of wild-type and LKB1 mutant larvae (n = 10 per genotype). (C) qPCR analysis for lipogenic genes (SREBP, FAS and ACC) and lipolytic genes (bmm and HSL) in wild-type and LKB1 mutant larvae at mid-to-late L2 (60 hr AEL) stage under feeding conditions. (D) qPCR analysis of LKB1, SIKs (SIK2 and SIK3), and AMPK complex (AMPKα, AMPKβ, and AMPKγ) in larvae. (E) TAG amounts in LKB1 mutants following fat body-specific expression of wild-type, kinase-dead (K201I) LKB1, constitutively active (T196E) SIK3 or constitutively active (T184D) AMPK. Genotypes are as follows: FB> (FB-Gal4/+), LKB1X5,FB> (FB-Gal4/+;LKB1X5/LKB1X5), LKB1X5,FB>LKB1WT (FB-Gal4/UAS-LKB1;LKB1X5/LKB1X5), LKB1X5,FB>LKB1KI (FB-Gal4/UAS-LKB1 K201I;LKB1X5/LKB1X5), LKB1X5,FB>SIK3TE (FB-Gal4/UAS-SIK3 T196E;LKB1X5/LKB1X5), and LKB1X5,FB>AMPKTD (FB-Gal4/UAS-AMPK T184D;LKB1X5/LKB1X5) (n = 10 per genotype). (F) qPCR analysis of bmm gene expression in LKB1 mutants following fat body-specific expression of wild-type LKB1 or constitutively active (T196E) SIK3 at mid-to-late L2 stage under feeding condition. Genotypes are as follows: FB> (FB-Gal4/+), LKB1X5,FB> (FB-Gal4/+;LKB1X5/LKB1X5), LKB1X5,FB>LKB1WT (FB-Gal4/UAS-LKB1;LKB1X5/LKB1X5), and LKB1X5,FB>SIK3TE (FB-Gal4/UAS-SIK3 T196E;LKB1X5/LKB1X5). (G) Immunoblot analyses showing the effect of LKB1 on Thr196 phosphorylation of SIK3 protein in larvae. Wild-type and kinase-dead (K70M) SIK3 were highly phosphorylated at Thr196 by LKB1 (second panel). SIK3T196A was used as a control. FB-Gal4 was used to drive transgene expression in the fat body. (H) Immunoblot analyses showing relative amounts of SIK3 Thr196 phosphorylation in wild-type and LKB1X5 mutant larvae. The phosphorylation was absolutely dependent on LKB1 (first panel). FB-Gal4 was used to drive transgene expression. (G-H) Anti-LKB1, -phospho-Thr196 SIK3, -Myc (SIK3 protein), and -β-tubulin (TUB) antibodies were used. Data are presented as mean ± SEM (*P < 0.05).
Mentions: LKB1 functions in a complex with two scaffolding proteins, STE20-related adaptor (STRAD) and mouse protein 25 (MO25) [20,21]. As the first step toward elucidation of the role of LKB1 in lipid metabolism, we demonstrated the gene expression of each component of the LKB1 complex in the fat body (Fig 1A), suggesting that Drosophila LKB1 forms the heterotrimeric complex when activated in tissues. Additionally, we characterized an LKB1- mutant line, LKB1X5 [22], and found that these flies showed markedly decreased lipid storage compared to wild-type flies, despite having similar food intake and retaining expression of the lipogenic genes (SREBP, FAS, and ACC) (Figs 1B, 1C and S1A). However, expression of bmm and lipolysis activity were elevated in LKB1X5 mutants (Figs 1C and S1B, respectively). Moreover, transgenic expression of wild-type LKB1 with two different fat body drivers (FB-Gal4 and cg-Gal4) rescued the decreased lipid levels and increased bmm expression phenotypes of LKB1X5 mutants, whereas expression of the kinase-dead form of LKB1 (LKB1 K201I) did not (Figs 1E, 1F, S2A and S2B). Additionally, overexpression of LKB1 induced significant increases in the lipid levels and decreases in bmm expression in a dose-dependent manner (S3A and S3B Fig). The implication behind these observations is that LKB1 plays a critical role in lipid storage in Drosophila by regulating the lipolysis pathway in a kinase activity-dependent manner.

Bottom Line: Interestingly, we found that the LKB1-SIK3-HDAC4 signaling axis is modulated by dietary conditions.In short-term fasting, the adipokinetic hormone (AKH) pathway, related to the mammalian glucagon pathway, inhibits the kinase activity of LKB1 as shown by decreased SIK3 Thr196 phosphorylation, and consequently induces HDAC4 nuclear localization and brummer gene expression.However, under prolonged fasting conditions, AKH-independent signaling decreases the activity of the LKB1-SIK3 pathway to induce lipolytic responses.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejon, Republic of Korea; National Creative Research Initiatives Center for Energy Homeostasis Regulation, Seoul National University, Seoul, Republic of Korea; Institute of Molecular Biology and Genetics, Seoul National University, Seoul, Republic of Korea.

ABSTRACT
LKB1 plays important roles in governing energy homeostasis by regulating AMP-activated protein kinase (AMPK) and other AMPK-related kinases, including the salt-inducible kinases (SIKs). However, the roles and regulation of LKB1 in lipid metabolism are poorly understood. Here we show that Drosophila LKB1 mutants display decreased lipid storage and increased gene expression of brummer, the Drosophila homolog of adipose triglyceride lipase (ATGL). These phenotypes are consistent with those of SIK3 mutants and are rescued by expression of constitutively active SIK3 in the fat body, suggesting that SIK3 is a key downstream kinase of LKB1. Using genetic and biochemical analyses, we identify HDAC4, a class IIa histone deacetylase, as a lipolytic target of the LKB1-SIK3 pathway. Interestingly, we found that the LKB1-SIK3-HDAC4 signaling axis is modulated by dietary conditions. In short-term fasting, the adipokinetic hormone (AKH) pathway, related to the mammalian glucagon pathway, inhibits the kinase activity of LKB1 as shown by decreased SIK3 Thr196 phosphorylation, and consequently induces HDAC4 nuclear localization and brummer gene expression. However, under prolonged fasting conditions, AKH-independent signaling decreases the activity of the LKB1-SIK3 pathway to induce lipolytic responses. We also identify that the Drosophila insulin-like peptides (DILPs) pathway, related to mammalian insulin pathway, regulates SIK3 activity in feeding conditions independently of increasing LKB1 kinase activity. Overall, these data suggest that fasting stimuli specifically control the kinase activity of LKB1 and establish the LKB1-SIK3 pathway as a converging point between feeding and fasting signals to control lipid homeostasis in Drosophila.

No MeSH data available.


Related in: MedlinePlus