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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

The AKH pathway regulates LKB1-SIK3-HDAC4 signaling to control lipid homeostasis.(A-B) Effects of AKHR gene disruption on TAG amounts (A) and bmm gene expression (B) in LKB1 and SIK3  mutants. Genotypes are as follows: WT (w1118), LKB1X5 (LKB1X5/LKB1X5), SIK3Δ5–31 (SIK3Δ5–31/SIK3Δ5–31), AKHR1 (AKHR1/AKHR1), LKB1X5,AKHR1 (AKHR1/AKHR1;LKB1X5/LKB1X5), and SIK3Δ5–31,AKHR1 (SIK3Δ5–31,AKHR1/SIK3Δ5–31,AKHR1). (C) Immunoblot analyses showing the effect of 4 hr fasting and AKH on Thr196 phosphorylation of SIK3 protein in larvae (top four panels). Densitometric analyses of phospho-SIK3 bands (bottom panel). FB-Gal4 was used to drive transgene expression in the fat body. Anti-phospho-Thr196 SIK3, -Myc (SIK3 protein), -AKH and -β-tubulin (TUB) antibodies were used. (D) Immunohistochemical analyses of HDAC4 (anti-FLAG antibody, green) in AKHR mutant (AKHR1) L3 larvae in feeding or 4 hr fasting condition as denoted in figures. Cell nuclei were stained by Hoechst 33258 (blue). Genotypes are as follows: Control (FB-Gal4,AKHR1/AKHR1) and FLAG-HDAC4,AKHR1 (FB-Gal4,AKHR1/UAS-HDAC4,AKHR1). The graphs showed the staining intensity profile for each antibody along the red lines. Scale bars, 20 μm. (E-F) Effect of the fat body-specific expression of constitutively active HDAC4 (HDAC3A) on TAG amounts (E) and bmm gene expression (F) in AKHR mutant. Genotypes: FB> (FB-Gal4/+), AKHR1,FB> (FB-Gal4,AKHR1/AKHR1), FB>HDAC43A (FB-Gal4/UAS-HDAC4 3A), and AKHR1,FB>HDAC43A (FB-Gal4,AKHR1/UAS-HDAC4 3A,AKHR1). Data are presented as mean ± SEM (*P < 0.05; **P < 0.01; ***P < 0.001; NS, non-significant).
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pgen.1005263.g004: The AKH pathway regulates LKB1-SIK3-HDAC4 signaling to control lipid homeostasis.(A-B) Effects of AKHR gene disruption on TAG amounts (A) and bmm gene expression (B) in LKB1 and SIK3 mutants. Genotypes are as follows: WT (w1118), LKB1X5 (LKB1X5/LKB1X5), SIK3Δ5–31 (SIK3Δ5–31/SIK3Δ5–31), AKHR1 (AKHR1/AKHR1), LKB1X5,AKHR1 (AKHR1/AKHR1;LKB1X5/LKB1X5), and SIK3Δ5–31,AKHR1 (SIK3Δ5–31,AKHR1/SIK3Δ5–31,AKHR1). (C) Immunoblot analyses showing the effect of 4 hr fasting and AKH on Thr196 phosphorylation of SIK3 protein in larvae (top four panels). Densitometric analyses of phospho-SIK3 bands (bottom panel). FB-Gal4 was used to drive transgene expression in the fat body. Anti-phospho-Thr196 SIK3, -Myc (SIK3 protein), -AKH and -β-tubulin (TUB) antibodies were used. (D) Immunohistochemical analyses of HDAC4 (anti-FLAG antibody, green) in AKHR mutant (AKHR1) L3 larvae in feeding or 4 hr fasting condition as denoted in figures. Cell nuclei were stained by Hoechst 33258 (blue). Genotypes are as follows: Control (FB-Gal4,AKHR1/AKHR1) and FLAG-HDAC4,AKHR1 (FB-Gal4,AKHR1/UAS-HDAC4,AKHR1). The graphs showed the staining intensity profile for each antibody along the red lines. Scale bars, 20 μm. (E-F) Effect of the fat body-specific expression of constitutively active HDAC4 (HDAC3A) on TAG amounts (E) and bmm gene expression (F) in AKHR mutant. Genotypes: FB> (FB-Gal4/+), AKHR1,FB> (FB-Gal4,AKHR1/AKHR1), FB>HDAC43A (FB-Gal4/UAS-HDAC4 3A), and AKHR1,FB>HDAC43A (FB-Gal4,AKHR1/UAS-HDAC4 3A,AKHR1). Data are presented as mean ± SEM (*P < 0.05; **P < 0.01; ***P < 0.001; NS, non-significant).

Mentions: Under fasting conditions, AKH activates the mobilization of fat body triglyceride by triggering AKHR and consequent activation of cAMP signaling in the fat body [7]. Consistently, we showed that AKHR mutation highly increased TAG levels (Fig 4A) and decreased bmm gene expression (Fig 4B). To determine the functional interaction between AKHR signaling and the LKB1-SIK3 signaling pathway, we crossed LKB1 or SIK3 mutant flies with AKHR mutant flies. Interestingly, deletion of LKB1 or SIK3 reversed both the lipid accumulation and the reduced bmm expression phenotypes of AKHR mutant flies (Fig 4A and 4B, respectively), suggesting that the LKB1-SIK3 pathway likely acts downstream of AKHR. Furthermore, SIK3 Thr196 phosphorylation was reduced in both fasting and AKH overexpression conditions compared to that in feeding conditions (Fig 4C), supporting that the AKH pathway inhibits the kinase activity of LKB1 as shown by decreased SIK3 Thr196 phosphorylation.


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)

The AKH pathway regulates LKB1-SIK3-HDAC4 signaling to control lipid homeostasis.(A-B) Effects of AKHR gene disruption on TAG amounts (A) and bmm gene expression (B) in LKB1 and SIK3  mutants. Genotypes are as follows: WT (w1118), LKB1X5 (LKB1X5/LKB1X5), SIK3Δ5–31 (SIK3Δ5–31/SIK3Δ5–31), AKHR1 (AKHR1/AKHR1), LKB1X5,AKHR1 (AKHR1/AKHR1;LKB1X5/LKB1X5), and SIK3Δ5–31,AKHR1 (SIK3Δ5–31,AKHR1/SIK3Δ5–31,AKHR1). (C) Immunoblot analyses showing the effect of 4 hr fasting and AKH on Thr196 phosphorylation of SIK3 protein in larvae (top four panels). Densitometric analyses of phospho-SIK3 bands (bottom panel). FB-Gal4 was used to drive transgene expression in the fat body. Anti-phospho-Thr196 SIK3, -Myc (SIK3 protein), -AKH and -β-tubulin (TUB) antibodies were used. (D) Immunohistochemical analyses of HDAC4 (anti-FLAG antibody, green) in AKHR mutant (AKHR1) L3 larvae in feeding or 4 hr fasting condition as denoted in figures. Cell nuclei were stained by Hoechst 33258 (blue). Genotypes are as follows: Control (FB-Gal4,AKHR1/AKHR1) and FLAG-HDAC4,AKHR1 (FB-Gal4,AKHR1/UAS-HDAC4,AKHR1). The graphs showed the staining intensity profile for each antibody along the red lines. Scale bars, 20 μm. (E-F) Effect of the fat body-specific expression of constitutively active HDAC4 (HDAC3A) on TAG amounts (E) and bmm gene expression (F) in AKHR mutant. Genotypes: FB> (FB-Gal4/+), AKHR1,FB> (FB-Gal4,AKHR1/AKHR1), FB>HDAC43A (FB-Gal4/UAS-HDAC4 3A), and AKHR1,FB>HDAC43A (FB-Gal4,AKHR1/UAS-HDAC4 3A,AKHR1). Data are presented as mean ± SEM (*P < 0.05; **P < 0.01; ***P < 0.001; NS, non-significant).
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pgen.1005263.g004: The AKH pathway regulates LKB1-SIK3-HDAC4 signaling to control lipid homeostasis.(A-B) Effects of AKHR gene disruption on TAG amounts (A) and bmm gene expression (B) in LKB1 and SIK3 mutants. Genotypes are as follows: WT (w1118), LKB1X5 (LKB1X5/LKB1X5), SIK3Δ5–31 (SIK3Δ5–31/SIK3Δ5–31), AKHR1 (AKHR1/AKHR1), LKB1X5,AKHR1 (AKHR1/AKHR1;LKB1X5/LKB1X5), and SIK3Δ5–31,AKHR1 (SIK3Δ5–31,AKHR1/SIK3Δ5–31,AKHR1). (C) Immunoblot analyses showing the effect of 4 hr fasting and AKH on Thr196 phosphorylation of SIK3 protein in larvae (top four panels). Densitometric analyses of phospho-SIK3 bands (bottom panel). FB-Gal4 was used to drive transgene expression in the fat body. Anti-phospho-Thr196 SIK3, -Myc (SIK3 protein), -AKH and -β-tubulin (TUB) antibodies were used. (D) Immunohistochemical analyses of HDAC4 (anti-FLAG antibody, green) in AKHR mutant (AKHR1) L3 larvae in feeding or 4 hr fasting condition as denoted in figures. Cell nuclei were stained by Hoechst 33258 (blue). Genotypes are as follows: Control (FB-Gal4,AKHR1/AKHR1) and FLAG-HDAC4,AKHR1 (FB-Gal4,AKHR1/UAS-HDAC4,AKHR1). The graphs showed the staining intensity profile for each antibody along the red lines. Scale bars, 20 μm. (E-F) Effect of the fat body-specific expression of constitutively active HDAC4 (HDAC3A) on TAG amounts (E) and bmm gene expression (F) in AKHR mutant. Genotypes: FB> (FB-Gal4/+), AKHR1,FB> (FB-Gal4,AKHR1/AKHR1), FB>HDAC43A (FB-Gal4/UAS-HDAC4 3A), and AKHR1,FB>HDAC43A (FB-Gal4,AKHR1/UAS-HDAC4 3A,AKHR1). Data are presented as mean ± SEM (*P < 0.05; **P < 0.01; ***P < 0.001; NS, non-significant).
Mentions: Under fasting conditions, AKH activates the mobilization of fat body triglyceride by triggering AKHR and consequent activation of cAMP signaling in the fat body [7]. Consistently, we showed that AKHR mutation highly increased TAG levels (Fig 4A) and decreased bmm gene expression (Fig 4B). To determine the functional interaction between AKHR signaling and the LKB1-SIK3 signaling pathway, we crossed LKB1 or SIK3 mutant flies with AKHR mutant flies. Interestingly, deletion of LKB1 or SIK3 reversed both the lipid accumulation and the reduced bmm expression phenotypes of AKHR mutant flies (Fig 4A and 4B, respectively), suggesting that the LKB1-SIK3 pathway likely acts downstream of AKHR. Furthermore, SIK3 Thr196 phosphorylation was reduced in both fasting and AKH overexpression conditions compared to that in feeding conditions (Fig 4C), supporting that the AKH pathway inhibits the kinase activity of LKB1 as shown by decreased SIK3 Thr196 phosphorylation.

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