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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

HDAC4 is the responsible target of LKB1-SIK3 signaling for controlling lipid homeostasis.(A-B) Effects of the fat body-specific knockdown of HDAC4 (HDAC4 RNAi) on TAG amounts (A) and bmm gene expression (B) in LKB1 and SIK3  mutants. Genotypes are as follows: FB> (FB-Gal4/+), LKB1X5,FB> (FB-Gal4/+;LKB1X5/LKB1X5), SIK3Δ5–31,FB> (FB-Gal4,SIK3Δ5–31/SIK3Δ5–31), FB>HDAC4 RNAi (FB-Gal4/+;UAS-HDAC4 RNAi/+), LKB1X5,FB>HDAC4 RNAi (FB-Gal4/+;LKB1X5/LKB1X5,UAS-HDAC4 RNAi), and SIK3Δ5–31,FB>HDAC4 RNAi (FB-Gal4,SIK3Δ5–31/SIK3Δ5–31;UAS-HDAC4 RNAi). (C) Drosophila HDAC4 protein structure showing three SIK3 phosphorylation sites and an HDAC class IIa domain (top panel). Immunoblot analyses showing the effect of wild-type and constitutively active (T196E) SIK3 on HDAC4 Ser239 phosphorylation in larvae (middle four panels). Densitometric analyses of phospho-HDAC4 bands (bottom panel). FB-Gal4 was used to drive transgene expression. Anti-phospho-Ser239 HDAC4, -FLAG (HDAC protein), -Myc (SIK3 protein), and -β-tubulin (TUB) antibodies were used. (D) Immunohistochemical analyses of HDAC4 (anti-FLAG antibody, green) in the fat body cells of wild type (first, second, and third rows), LKB1 mutant (LKB1X5) (fourth row) and SIK3 mutant (SIK3Δ5–31) (fifth row) L3 larvae in feeding or 4 hr fasting condition as denoted in figures. Similar experiments were also conducted for phosphorylation-defective and constitutively active HDAC4 (HDAC3A) in wild type L3 larvae in feeding (sixth row) or 4 hr fasting condition (bottom row). Cell nuclei were stained by Hoechst 33258 (blue). The graphs on the right of each image showed the intensity plot profile for each antibody staining along the red lines. Genotypes are as follows: Control (FB-Gal4/+), FLAG-HDAC4 (FB-Gal4/UAS-HDAC4), FLAG-HDAC43A (FB-Gal4/UAS-HDAC4 3A), FLAG-HDAC4,LKB1X5 (FB-Gal4/UAS-HDAC4;LKB1X5/LKB1X5), and FLAG-HDAC4,SIK3Δ5–31 (FB-Gal4,SIK3Δ5–31/UAS-HDAC4,SIK3Δ5–31). Scale bars, 20 μm. (E) Effect of fat body-specific expression of constitutively active SIK3 (SIK3 T196E) on bmm gene expression in larvae expressing wild-type HDAC4 in the fat body. Genotypes are as follows: FB> (FB-Gal4/+), FB>HDAC4WT (FB-Gal4/UAS-HDAC4), FB>SIK3TE (FB-Gal4/+;UAS-SIK3 T196E/+), and FB>HDAC4 WT,SIK3TE (FB-Gal4/UAS-HDAC4;UAS-SIK3 T196E/+). (F-G) Effects of the fat body-specific knockdown of bmm (bmm RNAi) on TAG amounts (F) and bmm gene expression (G) in LKB1 and SIK3  mutants. Genotypes are as follows: FB> (FB-Gal4/+), LKB1X5,FB> (FB-Gal4/+;LKB1X5/LKB1X5), SIK3Δ5–31,FB> (FB-Gal4,SIK3Δ5–31/SIK3Δ5–31), FB>bmm RNAi (FB-Gal4/;UAS-bmm RNAi/+), LKB1X5,FB>bmm RNAi (FB-Gal4/+;LKB1X5/LKB1X5,UAS-bmm RNAi) and SIK3Δ5–31,FB>bmm RNAi (FB-Gal4,SIK3Δ5–31/SIK3Δ5–31;UAS-bmm RNAi). Data are presented as mean ± SEM (*P < 0.05; **P < 0.01; ***P < 0.001).
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pgen.1005263.g003: HDAC4 is the responsible target of LKB1-SIK3 signaling for controlling lipid homeostasis.(A-B) Effects of the fat body-specific knockdown of HDAC4 (HDAC4 RNAi) on TAG amounts (A) and bmm gene expression (B) in LKB1 and SIK3 mutants. Genotypes are as follows: FB> (FB-Gal4/+), LKB1X5,FB> (FB-Gal4/+;LKB1X5/LKB1X5), SIK3Δ5–31,FB> (FB-Gal4,SIK3Δ5–31/SIK3Δ5–31), FB>HDAC4 RNAi (FB-Gal4/+;UAS-HDAC4 RNAi/+), LKB1X5,FB>HDAC4 RNAi (FB-Gal4/+;LKB1X5/LKB1X5,UAS-HDAC4 RNAi), and SIK3Δ5–31,FB>HDAC4 RNAi (FB-Gal4,SIK3Δ5–31/SIK3Δ5–31;UAS-HDAC4 RNAi). (C) Drosophila HDAC4 protein structure showing three SIK3 phosphorylation sites and an HDAC class IIa domain (top panel). Immunoblot analyses showing the effect of wild-type and constitutively active (T196E) SIK3 on HDAC4 Ser239 phosphorylation in larvae (middle four panels). Densitometric analyses of phospho-HDAC4 bands (bottom panel). FB-Gal4 was used to drive transgene expression. Anti-phospho-Ser239 HDAC4, -FLAG (HDAC protein), -Myc (SIK3 protein), and -β-tubulin (TUB) antibodies were used. (D) Immunohistochemical analyses of HDAC4 (anti-FLAG antibody, green) in the fat body cells of wild type (first, second, and third rows), LKB1 mutant (LKB1X5) (fourth row) and SIK3 mutant (SIK3Δ5–31) (fifth row) L3 larvae in feeding or 4 hr fasting condition as denoted in figures. Similar experiments were also conducted for phosphorylation-defective and constitutively active HDAC4 (HDAC3A) in wild type L3 larvae in feeding (sixth row) or 4 hr fasting condition (bottom row). Cell nuclei were stained by Hoechst 33258 (blue). The graphs on the right of each image showed the intensity plot profile for each antibody staining along the red lines. Genotypes are as follows: Control (FB-Gal4/+), FLAG-HDAC4 (FB-Gal4/UAS-HDAC4), FLAG-HDAC43A (FB-Gal4/UAS-HDAC4 3A), FLAG-HDAC4,LKB1X5 (FB-Gal4/UAS-HDAC4;LKB1X5/LKB1X5), and FLAG-HDAC4,SIK3Δ5–31 (FB-Gal4,SIK3Δ5–31/UAS-HDAC4,SIK3Δ5–31). Scale bars, 20 μm. (E) Effect of fat body-specific expression of constitutively active SIK3 (SIK3 T196E) on bmm gene expression in larvae expressing wild-type HDAC4 in the fat body. Genotypes are as follows: FB> (FB-Gal4/+), FB>HDAC4WT (FB-Gal4/UAS-HDAC4), FB>SIK3TE (FB-Gal4/+;UAS-SIK3 T196E/+), and FB>HDAC4 WT,SIK3TE (FB-Gal4/UAS-HDAC4;UAS-SIK3 T196E/+). (F-G) Effects of the fat body-specific knockdown of bmm (bmm RNAi) on TAG amounts (F) and bmm gene expression (G) in LKB1 and SIK3 mutants. Genotypes are as follows: FB> (FB-Gal4/+), LKB1X5,FB> (FB-Gal4/+;LKB1X5/LKB1X5), SIK3Δ5–31,FB> (FB-Gal4,SIK3Δ5–31/SIK3Δ5–31), FB>bmm RNAi (FB-Gal4/;UAS-bmm RNAi/+), LKB1X5,FB>bmm RNAi (FB-Gal4/+;LKB1X5/LKB1X5,UAS-bmm RNAi) and SIK3Δ5–31,FB>bmm RNAi (FB-Gal4,SIK3Δ5–31/SIK3Δ5–31;UAS-bmm RNAi). Data are presented as mean ± SEM (*P < 0.05; **P < 0.01; ***P < 0.001).

Mentions: To evaluate whether HDAC4 is crucial for the regulation of lipid storage by LKB1 and SIK3, we expressed HDAC4 RNAi in the fat body of LKB1 and SIK3 mutants. Surprisingly, knockdown of HDAC4 in the fat body fully rescued the TAG levels and bmm gene expression of LKB1 and SIK3 mutants (Fig 3A and 3B, respectively), indicating that HDAC4 is indeed a critical downstream target of LKB1 and SIK3 in lipid metabolism of Drosophila.


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)

HDAC4 is the responsible target of LKB1-SIK3 signaling for controlling lipid homeostasis.(A-B) Effects of the fat body-specific knockdown of HDAC4 (HDAC4 RNAi) on TAG amounts (A) and bmm gene expression (B) in LKB1 and SIK3  mutants. Genotypes are as follows: FB> (FB-Gal4/+), LKB1X5,FB> (FB-Gal4/+;LKB1X5/LKB1X5), SIK3Δ5–31,FB> (FB-Gal4,SIK3Δ5–31/SIK3Δ5–31), FB>HDAC4 RNAi (FB-Gal4/+;UAS-HDAC4 RNAi/+), LKB1X5,FB>HDAC4 RNAi (FB-Gal4/+;LKB1X5/LKB1X5,UAS-HDAC4 RNAi), and SIK3Δ5–31,FB>HDAC4 RNAi (FB-Gal4,SIK3Δ5–31/SIK3Δ5–31;UAS-HDAC4 RNAi). (C) Drosophila HDAC4 protein structure showing three SIK3 phosphorylation sites and an HDAC class IIa domain (top panel). Immunoblot analyses showing the effect of wild-type and constitutively active (T196E) SIK3 on HDAC4 Ser239 phosphorylation in larvae (middle four panels). Densitometric analyses of phospho-HDAC4 bands (bottom panel). FB-Gal4 was used to drive transgene expression. Anti-phospho-Ser239 HDAC4, -FLAG (HDAC protein), -Myc (SIK3 protein), and -β-tubulin (TUB) antibodies were used. (D) Immunohistochemical analyses of HDAC4 (anti-FLAG antibody, green) in the fat body cells of wild type (first, second, and third rows), LKB1 mutant (LKB1X5) (fourth row) and SIK3 mutant (SIK3Δ5–31) (fifth row) L3 larvae in feeding or 4 hr fasting condition as denoted in figures. Similar experiments were also conducted for phosphorylation-defective and constitutively active HDAC4 (HDAC3A) in wild type L3 larvae in feeding (sixth row) or 4 hr fasting condition (bottom row). Cell nuclei were stained by Hoechst 33258 (blue). The graphs on the right of each image showed the intensity plot profile for each antibody staining along the red lines. Genotypes are as follows: Control (FB-Gal4/+), FLAG-HDAC4 (FB-Gal4/UAS-HDAC4), FLAG-HDAC43A (FB-Gal4/UAS-HDAC4 3A), FLAG-HDAC4,LKB1X5 (FB-Gal4/UAS-HDAC4;LKB1X5/LKB1X5), and FLAG-HDAC4,SIK3Δ5–31 (FB-Gal4,SIK3Δ5–31/UAS-HDAC4,SIK3Δ5–31). Scale bars, 20 μm. (E) Effect of fat body-specific expression of constitutively active SIK3 (SIK3 T196E) on bmm gene expression in larvae expressing wild-type HDAC4 in the fat body. Genotypes are as follows: FB> (FB-Gal4/+), FB>HDAC4WT (FB-Gal4/UAS-HDAC4), FB>SIK3TE (FB-Gal4/+;UAS-SIK3 T196E/+), and FB>HDAC4 WT,SIK3TE (FB-Gal4/UAS-HDAC4;UAS-SIK3 T196E/+). (F-G) Effects of the fat body-specific knockdown of bmm (bmm RNAi) on TAG amounts (F) and bmm gene expression (G) in LKB1 and SIK3  mutants. Genotypes are as follows: FB> (FB-Gal4/+), LKB1X5,FB> (FB-Gal4/+;LKB1X5/LKB1X5), SIK3Δ5–31,FB> (FB-Gal4,SIK3Δ5–31/SIK3Δ5–31), FB>bmm RNAi (FB-Gal4/;UAS-bmm RNAi/+), LKB1X5,FB>bmm RNAi (FB-Gal4/+;LKB1X5/LKB1X5,UAS-bmm RNAi) and SIK3Δ5–31,FB>bmm RNAi (FB-Gal4,SIK3Δ5–31/SIK3Δ5–31;UAS-bmm RNAi). Data are presented as mean ± SEM (*P < 0.05; **P < 0.01; ***P < 0.001).
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pgen.1005263.g003: HDAC4 is the responsible target of LKB1-SIK3 signaling for controlling lipid homeostasis.(A-B) Effects of the fat body-specific knockdown of HDAC4 (HDAC4 RNAi) on TAG amounts (A) and bmm gene expression (B) in LKB1 and SIK3 mutants. Genotypes are as follows: FB> (FB-Gal4/+), LKB1X5,FB> (FB-Gal4/+;LKB1X5/LKB1X5), SIK3Δ5–31,FB> (FB-Gal4,SIK3Δ5–31/SIK3Δ5–31), FB>HDAC4 RNAi (FB-Gal4/+;UAS-HDAC4 RNAi/+), LKB1X5,FB>HDAC4 RNAi (FB-Gal4/+;LKB1X5/LKB1X5,UAS-HDAC4 RNAi), and SIK3Δ5–31,FB>HDAC4 RNAi (FB-Gal4,SIK3Δ5–31/SIK3Δ5–31;UAS-HDAC4 RNAi). (C) Drosophila HDAC4 protein structure showing three SIK3 phosphorylation sites and an HDAC class IIa domain (top panel). Immunoblot analyses showing the effect of wild-type and constitutively active (T196E) SIK3 on HDAC4 Ser239 phosphorylation in larvae (middle four panels). Densitometric analyses of phospho-HDAC4 bands (bottom panel). FB-Gal4 was used to drive transgene expression. Anti-phospho-Ser239 HDAC4, -FLAG (HDAC protein), -Myc (SIK3 protein), and -β-tubulin (TUB) antibodies were used. (D) Immunohistochemical analyses of HDAC4 (anti-FLAG antibody, green) in the fat body cells of wild type (first, second, and third rows), LKB1 mutant (LKB1X5) (fourth row) and SIK3 mutant (SIK3Δ5–31) (fifth row) L3 larvae in feeding or 4 hr fasting condition as denoted in figures. Similar experiments were also conducted for phosphorylation-defective and constitutively active HDAC4 (HDAC3A) in wild type L3 larvae in feeding (sixth row) or 4 hr fasting condition (bottom row). Cell nuclei were stained by Hoechst 33258 (blue). The graphs on the right of each image showed the intensity plot profile for each antibody staining along the red lines. Genotypes are as follows: Control (FB-Gal4/+), FLAG-HDAC4 (FB-Gal4/UAS-HDAC4), FLAG-HDAC43A (FB-Gal4/UAS-HDAC4 3A), FLAG-HDAC4,LKB1X5 (FB-Gal4/UAS-HDAC4;LKB1X5/LKB1X5), and FLAG-HDAC4,SIK3Δ5–31 (FB-Gal4,SIK3Δ5–31/UAS-HDAC4,SIK3Δ5–31). Scale bars, 20 μm. (E) Effect of fat body-specific expression of constitutively active SIK3 (SIK3 T196E) on bmm gene expression in larvae expressing wild-type HDAC4 in the fat body. Genotypes are as follows: FB> (FB-Gal4/+), FB>HDAC4WT (FB-Gal4/UAS-HDAC4), FB>SIK3TE (FB-Gal4/+;UAS-SIK3 T196E/+), and FB>HDAC4 WT,SIK3TE (FB-Gal4/UAS-HDAC4;UAS-SIK3 T196E/+). (F-G) Effects of the fat body-specific knockdown of bmm (bmm RNAi) on TAG amounts (F) and bmm gene expression (G) in LKB1 and SIK3 mutants. Genotypes are as follows: FB> (FB-Gal4/+), LKB1X5,FB> (FB-Gal4/+;LKB1X5/LKB1X5), SIK3Δ5–31,FB> (FB-Gal4,SIK3Δ5–31/SIK3Δ5–31), FB>bmm RNAi (FB-Gal4/;UAS-bmm RNAi/+), LKB1X5,FB>bmm RNAi (FB-Gal4/+;LKB1X5/LKB1X5,UAS-bmm RNAi) and SIK3Δ5–31,FB>bmm RNAi (FB-Gal4,SIK3Δ5–31/SIK3Δ5–31;UAS-bmm RNAi). Data are presented as mean ± SEM (*P < 0.05; **P < 0.01; ***P < 0.001).
Mentions: To evaluate whether HDAC4 is crucial for the regulation of lipid storage by LKB1 and SIK3, we expressed HDAC4 RNAi in the fat body of LKB1 and SIK3 mutants. Surprisingly, knockdown of HDAC4 in the fat body fully rescued the TAG levels and bmm gene expression of LKB1 and SIK3 mutants (Fig 3A and 3B, respectively), indicating that HDAC4 is indeed a critical downstream target of LKB1 and SIK3 in lipid metabolism of Drosophila.

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