w w w. n a t u r e . c o m / n a t u r e | 1

SuPPLementarY InFormatIondoi:10.1038/nature09584

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d controla LiTsc1KO controlb LIRKOcontrolcLiRapKOfed AL 95 ± 10.6 112.3 ± 7.1fasted 66.8 ± 7.0 63.7 ± 6.8fed AL 3.12 ± 1.4 2.33 ± 1.3fasted 0.43 ± 0.27 0.21 ± 0.13

fasted TG (mg/dl) 83 ± 12.3 108 ± 14.8fasted FFA (mM) 0.81 ± 0.07 0.76 ± 0.05

fasted ketones (mM) 0.96 ± 0.19 0.37 ± 0.12*

96.9 ± 8.671.2 ± 4.84.10 ± 1.7

0.52 ± 0.2192 ± 10.41.1 ± 0.18

1.23 ± 0.29

339 ± 22.7*110 ± 15.4*64.5 ± 9.5*3.7 ± 0.86*62 ± 11.2*

0.64 ± 0.11*1.59 ± 0.37

102 ± 8.471.5 ± 12.72.84 ± 1.5

0.24 ± 0.13108 ± 12.60.68 ± 0.080.91 ± 0.17

94.2 ± 10.173.5 ± 12.63.21 ± 1.4

0.33 ± 0.1777 ± 16.9

0.62 ± 0.080.86 ± 0.13

glucose (mg/dl)

insulin (ng/ml)

a = TSC1LoxP/LoxP mice injected with empty adenovirus; b = raptorLoxP/LoxP mice; c = Insulin ReceptorLoxP/LoxP

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Figure S1. Additional characterization of LiTsc1KO, LiRapKO, and rapamycin-treated mice (A) Bar graph shows mean ± S.D. fold change in the intensity of phospho-240/44 S6 immunofluorescent staining of liver sections from mice fasted for 24 hours or fasted and refed for 45 minutes for n = 4 livers and asterisk indicates p < 0.05. (B) For livers analyzed in Figure 1E, changes in liver size correlate with those in hepatocyte size. Bar graph shows mean ± S.D. hepatocyte size in arbitrary units (a.u.) for n ≥ 100 cells. Asterisks indicate p < 0.05 compared to cells in respective control mice fed ad lib. (C) Total protein content from livers from fed and fasted LiTsc1KO and LiRapKO mice and their respective controls. Bar graphs show mean ± S.D. for n ≥ 5. (D) Levels of indicated serum metabolites and insulin in fed or fasted LiTsc1KO, LIRKO, and LiRapKO mice and their respective control mice. Fed mice were given ad libitum access to food and sacrificed at the beginning of the day. Fasted mice were denied food for 24 hours and sacrificed at the same time of day as the fed mice. Values are mean ± S.D. for n ≥ 6 and asterisks indicate p < 0.05 compared to respective control mice. (E) Loss of Tsc1 leads to decreased Akt phosphorylation. (F) Hepatic triglyceride levels from fasted LiTsc1KO, LiRapKO, and control mice. Bar graph show mean ± S.D. for n ≥ 5. (G) Total serum glucagon levels from fasted LiTsc1KO mice and their respective controls. Bar graphs show mean ± S.D. for n ≥ 5. (H) Indicated mice were fasted for 32 hours in individual cages in which locomotor activity was monitored by measuring beam breaks (see methods). Representative tracings are shown for one control and one LiTsc1KO mouse. (I) Mouse rectal temperature was monitored at the initiation of the fast and at the indicated times of the fast.

Figure S2. The ketogenic defect in LiTsc1KO mice is liver autonomous and resistant to PPARα agonist treatment; LiRapKO and rapamycin treated mice can generate ketones under fed conditions (A) Fasted LiTsc1KO mice fail to produce ketones even when given an exogenous ketogenic substrate. Control and LiTsc1KO mice were fasted for 18 hours, administered sodium octanoate, and total ketone levels determined in tail bleeds taken at the indicated times. Values are mean ± S.D. for n = 5. Asterisks indicate p < 0.05 compared to fasted control mice. (B) When given an exogenous ketogenic substrate, LiRapKO mice produce ketones in the fed state. Control and LiRapKO mice were fasted for 18 hours and then refed at the same time they were administered sodium octanoate. At the indicated times, total serum ketones were measured from tail bleeds. Values are mean ± S.D. for n = 4. Asterisks indicate p < 0.05 compared to refed control mice. (C-D) Hepatic raptor loss or injection with rapamycin for one-hour prior to refeeding delays the decrease in total serum ketones (E) A synthetic PPARα agonist does not activate ketone production in LiTsc1KO mice. Control and LiTsc1KO mice were administered WY-14643 via oral gavage for 5 days, fasted for 24 hours, and total serum ketone levels measured. Values are mean ± S.D. values for n = 5. Asterisk indicates p < 0.05 compared to control mice treated with WY-14463. (F) A synthetic PPARα agonist activates PPARα-target gene expression in the small intestine, but not in the liver, of LiTsc1KO mice. Indicated mice were treated as in (E) and mRNA levels measured as in Figure 2D. Values are mean ± S.D. for n = 5. Asterisks indicate p < 0.05 compared to control mice treated with WY-14463. (G) Hepatic raptor loss or injection with rapamycin for one-hour prior to refeeding delays the decrease of mRNA levels of PPARα targets upon refeeding for 2 hours. Asterisk indicate p < 0.05 compared to refed controlb or vehicle-treated mice for n ≥ 4 mice.

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Figure S1. Additional characterization of LiTsc1KO, LiRapKO, and rapamycin-treated mice (A) Bar graph shows mean ± S.D. fold change in the intensity of phospho-240/44 S6 immunofluorescent staining of liver sections from mice fasted for 24 hours or fasted and refed for 45 minutes for n = 4 livers and asterisk indicates p < 0.05. (B) For livers analyzed in Figure 1E, changes in liver size correlate with those in hepatocyte size. Bar graph shows mean ± S.D. hepatocyte size in arbitrary units (a.u.) for n ≥ 100 cells. Asterisks indicate p < 0.05 compared to cells in respective control mice fed ad lib. (C) Total protein content from livers from fed and fasted LiTsc1KO and LiRapKO mice and their respective controls. Bar graphs show mean ± S.D. for n ≥ 5. (D) Levels of indicated serum metabolites and insulin in fed or fasted LiTsc1KO, LIRKO, and LiRapKO mice and their respective control mice. Fed mice were given ad libitum access to food and sacrificed at the beginning of the day. Fasted mice were denied food for 24 hours and sacrificed at the same time of day as the fed mice. Values are mean ± S.D. for n ≥ 6 and asterisks indicate p < 0.05 compared to respective control mice. (E) Loss of Tsc1 leads to decreased Akt phosphorylation. (F) Hepatic triglyceride levels from fasted LiTsc1KO, LiRapKO, and control mice. Bar graph show mean ± S.D. for n ≥ 5. (G) Total serum glucagon levels from fasted LiTsc1KO mice and their respective controls. Bar graphs show mean ± S.D. for n ≥ 5. (H) Indicated mice were fasted for 32 hours in individual cages in which locomotor activity was monitored by measuring beam breaks (see methods). Representative tracings are shown for one control and one LiTsc1KO mouse. (I) Mouse rectal temperature was monitored at the initiation of the fast and at the indicated times of the fast.

Figure S2. The ketogenic defect in LiTsc1KO mice is liver autonomous and resistant to PPARα agonist treatment; LiRapKO and rapamycin treated mice can generate ketones under fed conditions (A) Fasted LiTsc1KO mice fail to produce ketones even when given an exogenous ketogenic substrate. Control and LiTsc1KO mice were fasted for 18 hours, administered sodium octanoate, and total ketone levels determined in tail bleeds taken at the indicated times. Values are mean ± S.D. for n = 5. Asterisks indicate p < 0.05 compared to fasted control mice. (B) When given an exogenous ketogenic substrate, LiRapKO mice produce ketones in the fed state. Control and LiRapKO mice were fasted for 18 hours and then refed at the same time they were administered sodium octanoate. At the indicated times, total serum ketones were measured from tail bleeds. Values are mean ± S.D. for n = 4. Asterisks indicate p < 0.05 compared to refed control mice. (C-D) Hepatic raptor loss or injection with rapamycin for one-hour prior to refeeding delays the decrease in total serum ketones (E) A synthetic PPARα agonist does not activate ketone production in LiTsc1KO mice. Control and LiTsc1KO mice were administered WY-14643 via oral gavage for 5 days, fasted for 24 hours, and total serum ketone levels measured. Values are mean ± S.D. values for n = 5. Asterisk indicates p < 0.05 compared to control mice treated with WY-14463. (F) A synthetic PPARα agonist activates PPARα-target gene expression in the small intestine, but not in the liver, of LiTsc1KO mice. Indicated mice were treated as in (E) and mRNA levels measured as in Figure 2D. Values are mean ± S.D. for n = 5. Asterisks indicate p < 0.05 compared to control mice treated with WY-14463. (G) Hepatic raptor loss or injection with rapamycin for one-hour prior to refeeding delays the decrease of mRNA levels of PPARα targets upon refeeding for 2 hours. Asterisk indicate p < 0.05 compared to refed controlb or vehicle-treated mice for n ≥ 4 mice.

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* * *Figure S1. Additional characterization of LiTsc1KO, LiRapKO, and rapamycin-treated mice (A) Bar graph shows mean ± S.D. fold change in the intensity of phospho-240/44 S6 immunofluorescent staining of liver sections from mice fasted for 24 hours or fasted and refed for 45 minutes for n = 4 livers and asterisk indicates p < 0.05. (B) For livers analyzed in Figure 1E, changes in liver size correlate with those in hepatocyte size. Bar graph shows mean ± S.D. hepatocyte size in arbitrary units (a.u.) for n ≥ 100 cells. Asterisks indicate p < 0.05 compared to cells in respective control mice fed ad lib. (C) Total protein content from livers from fed and fasted LiTsc1KO and LiRapKO mice and their respective controls. Bar graphs show mean ± S.D. for n ≥ 5. (D) Levels of indicated serum metabolites and insulin in fed or fasted LiTsc1KO, LIRKO, and LiRapKO mice and their respective control mice. Fed mice were given ad libitum access to food and sacrificed at the beginning of the day. Fasted mice were denied food for 24 hours and sacrificed at the same time of day as the fed mice. Values are mean ± S.D. for n ≥ 6 and asterisks indicate p < 0.05 compared to respective control mice. (E) Loss of Tsc1 leads to decreased Akt phosphorylation. (F) Hepatic triglyceride levels from fasted LiTsc1KO, LiRapKO, and control mice. Bar graph show mean ± S.D. for n ≥ 5. (G) Total serum glucagon levels from fasted LiTsc1KO mice and their respective controls. Bar graphs show mean ± S.D. for n ≥ 5. (H) Indicated mice were fasted for 32 hours in individual cages in which locomotor activity was monitored by measuring beam breaks (see methods). Representative tracings are shown for one control and one LiTsc1KO mouse. (I) Mouse rectal temperature was monitored at the initiation of the fast and at the indicated times of the fast.

Figure S2. The ketogenic defect in LiTsc1KO mice is liver autonomous and resistant to PPARα agonist treatment; LiRapKO and rapamycin treated mice can generate ketones under fed conditions (A) Fasted LiTsc1KO mice fail to produce ketones even when given an exogenous ketogenic substrate. Control and LiTsc1KO mice were fasted for 18 hours, administered sodium octanoate, and total ketone levels determined in tail bleeds taken at the indicated times. Values are mean ± S.D. for n = 5. Asterisks indicate p < 0.05 compared to fasted control mice. (B) When given an exogenous ketogenic substrate, LiRapKO mice produce ketones in the fed state. Control and LiRapKO mice were fasted for 18 hours and then refed at the same time they were administered sodium octanoate. At the indicated times, total serum ketones were measured from tail bleeds. Values are mean ± S.D. for n = 4. Asterisks indicate p < 0.05 compared to refed control mice. (C-D) Hepatic raptor loss or injection with rapamycin for one-hour prior to refeeding delays the decrease in total serum ketones (E) A synthetic PPARα agonist does not activate ketone production in LiTsc1KO mice. Control and LiTsc1KO mice were administered WY-14643 via oral gavage for 5 days, fasted for 24 hours, and total serum ketone levels measured. Values are mean ± S.D. values for n = 5. Asterisk indicates p < 0.05 compared to control mice treated with WY-14463. (F) A synthetic PPARα agonist activates PPARα-target gene expression in the small intestine, but not in the liver, of LiTsc1KO mice. Indicated mice were treated as in (E) and mRNA levels measured as in Figure 2D. Values are mean ± S.D. for n = 5. Asterisks indicate p < 0.05 compared to control mice treated with WY-14463. (G) Hepatic raptor loss or injection with rapamycin for one-hour prior to refeeding delays the decrease of mRNA levels of PPARα targets upon refeeding for 2 hours. Asterisk indicate p < 0.05 compared to refed controlb or vehicle-treated mice for n ≥ 4 mice.

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Figure S1. Additional characterization of LiTsc1KO, LiRapKO, and rapamycin-treated mice (A) Bar graph shows mean ± S.D. fold change in the intensity of phospho-240/44 S6 immunofluorescent staining of liver sections from mice fasted for 24 hours or fasted and refed for 45 minutes for n = 4 livers and asterisk indicates p < 0.05. (B) For livers analyzed in Figure 1E, changes in liver size correlate with those in hepatocyte size. Bar graph shows mean ± S.D. hepatocyte size in arbitrary units (a.u.) for n ≥ 100 cells. Asterisks indicate p < 0.05 compared to cells in respective control mice fed ad lib. (C) Total protein content from livers from fed and fasted LiTsc1KO and LiRapKO mice and their respective controls. Bar graphs show mean ± S.D. for n ≥ 5. (D) Levels of indicated serum metabolites and insulin in fed or fasted LiTsc1KO, LIRKO, and LiRapKO mice and their respective control mice. Fed mice were given ad libitum access to food and sacrificed at the beginning of the day. Fasted mice were denied food for 24 hours and sacrificed at the same time of day as the fed mice. Values are mean ± S.D. for n ≥ 6 and asterisks indicate p < 0.05 compared to respective control mice. (E) Loss of Tsc1 leads to decreased Akt phosphorylation. (F) Hepatic triglyceride levels from fasted LiTsc1KO, LiRapKO, and control mice. Bar graph show mean ± S.D. for n ≥ 5. (G) Total serum glucagon levels from fasted LiTsc1KO mice and their respective controls. Bar graphs show mean ± S.D. for n ≥ 5. (H) Indicated mice were fasted for 32 hours in individual cages in which locomotor activity was monitored by measuring beam breaks (see methods). Representative tracings are shown for one control and one LiTsc1KO mouse. (I) Mouse rectal temperature was monitored at the initiation of the fast and at the indicated times of the fast.

Figure S2. The ketogenic defect in LiTsc1KO mice is liver autonomous and resistant to PPARα agonist treatment; LiRapKO and rapamycin treated mice can generate ketones under fed conditions (A) Fasted LiTsc1KO mice fail to produce ketones even when given an exogenous ketogenic substrate. Control and LiTsc1KO mice were fasted for 18 hours, administered sodium octanoate, and total ketone levels determined in tail bleeds taken at the indicated times. Values are mean ± S.D. for n = 5. Asterisks indicate p < 0.05 compared to fasted control mice. (B) When given an exogenous ketogenic substrate, LiRapKO mice produce ketones in the fed state. Control and LiRapKO mice were fasted for 18 hours and then refed at the same time they were administered sodium octanoate. At the indicated times, total serum ketones were measured from tail bleeds. Values are mean ± S.D. for n = 4. Asterisks indicate p < 0.05 compared to refed control mice. (C-D) Hepatic raptor loss or injection with rapamycin for one-hour prior to refeeding delays the decrease in total serum ketones (E) A synthetic PPARα agonist does not activate ketone production in LiTsc1KO mice. Control and LiTsc1KO mice were administered WY-14643 via oral gavage for 5 days, fasted for 24 hours, and total serum ketone levels measured. Values are mean ± S.D. values for n = 5. Asterisk indicates p < 0.05 compared to control mice treated with WY-14463. (F) A synthetic PPARα agonist activates PPARα-target gene expression in the small intestine, but not in the liver, of LiTsc1KO mice. Indicated mice were treated as in (E) and mRNA levels measured as in Figure 2D. Values are mean ± S.D. for n = 5. Asterisks indicate p < 0.05 compared to control mice treated with WY-14463. (G) Hepatic raptor loss or injection with rapamycin for one-hour prior to refeeding delays the decrease of mRNA levels of PPARα targets upon refeeding for 2 hours. Asterisk indicate p < 0.05 compared to refed controlb or vehicle-treated mice for n ≥ 4 mice.

w w w. n a t u r e . c o m / n a t u r e | 5

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Figure S3. Validation of the shRNAs used in the study and that aged LiTsc1KO and LiRapKO mice maintain hepatic loss of TSC1 and raptor, respectively. (A, B) qRT-PCR validation of indicated mRNAs in AML12 cells expressing indicated shRNAs. For sequences of each shRNA, see methods. (D, E) Immunoblot analyses of liver extracts from indicated mice strains expressing or not expressing Cre in their livers.

Figure S4. Overexpression of PPARα does not rescue inhibition of ketogenesis and PPARα signaling caused by mTORC1 activation. (A-C) Control and LiTsc1KO mice (4-6 months of age) were injected with high-titer adenovirus expressing either GFP (Ad-GFP) or PPARα (Ad- PPARα). Four days later, total serum ketones (A) were determined in fed mice or mice fasted for 24 hours. Asterisks indicate p < 0.05 compared to fasted livers infected with Ad-GFP. qRT-PCR analyses confirmed that PPARα was overexpressed in livers from both control and LiTsc1KO mice (B). Relative mRNA levels of indicated genes (C) from fasted livers of control and LiTsc1KO mice with hepatic overexpression of GFP or PPARα were determined as in Figure 2J. Asterisks indicate p < 0.05 compared to livers expressing Ad-GFP in control mice. (D-F) Confluent murine AML12 hepatocyte cells stably expressing validated lentiviral shRNAs targeting RFP or TSC2 were infected with adenovirus expressing either GFP or PPARα (1010 pfu/ml) at a MOI of 100. RT-PCR analyses confirmed that PPARα was overexpressed in cells (E). After bright GFP fluorescence was observed, cells were placed in control or ketogenic media for 3 days and total media ketones (D) and indicated mRNAs levels (F) were determined as in Figure 2J. Asterisks indicate p < 0.05 compared to shRFP-expressing cells cultured in ketogenic media (D) or from shRFP-expressing cells also infected with Ad-GFP (F).

Figure S5. mTORC1 modulates the association of NCoR1 with PPRE-containing promoters in vivo and in cells in culture. (A-B) At PPRE-containing promoters, mTORC1 inhibition prevents refeeding-induced deacetylation of Histone H4 (A) and recruitment of NCoR1 (B). Asterisks indicate p < 0.05 compared to controlb mice refed for 2 hours. (C) PPARα occupancy at PPRE-containing promoters is unchanged in livers from fed and fasted controla and LiTsc1KO mice. ChIP assays were performed on indicated liver extracts as described in methods. Values are mean ± S.D. for n ≥ 5. (D) In AML12 cells, mTORC1 activation inhibits the disassociation of NCoR1 from PPRE-containing promoters in response to ketogenic media containing WY-14,643. ChIP assays were performed on indicated cell extracts as described in methods. Values are mean ± S.D. for n ≥ 5.

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Figure S3. Validation of the shRNAs used in the study and that aged LiTsc1KO and LiRapKO mice maintain hepatic loss of TSC1 and raptor, respectively. (A, B) qRT-PCR validation of indicated mRNAs in AML12 cells expressing indicated shRNAs. For sequences of each shRNA, see methods. (D, E) Immunoblot analyses of liver extracts from indicated mice strains expressing or not expressing Cre in their livers.

Figure S4. Overexpression of PPARα does not rescue inhibition of ketogenesis and PPARα signaling caused by mTORC1 activation. (A-C) Control and LiTsc1KO mice (4-6 months of age) were injected with high-titer adenovirus expressing either GFP (Ad-GFP) or PPARα (Ad- PPARα). Four days later, total serum ketones (A) were determined in fed mice or mice fasted for 24 hours. Asterisks indicate p < 0.05 compared to fasted livers infected with Ad-GFP. qRT-PCR analyses confirmed that PPARα was overexpressed in livers from both control and LiTsc1KO mice (B). Relative mRNA levels of indicated genes (C) from fasted livers of control and LiTsc1KO mice with hepatic overexpression of GFP or PPARα were determined as in Figure 2J. Asterisks indicate p < 0.05 compared to livers expressing Ad-GFP in control mice. (D-F) Confluent murine AML12 hepatocyte cells stably expressing validated lentiviral shRNAs targeting RFP or TSC2 were infected with adenovirus expressing either GFP or PPARα (1010 pfu/ml) at a MOI of 100. RT-PCR analyses confirmed that PPARα was overexpressed in cells (E). After bright GFP fluorescence was observed, cells were placed in control or ketogenic media for 3 days and total media ketones (D) and indicated mRNAs levels (F) were determined as in Figure 2J. Asterisks indicate p < 0.05 compared to shRFP-expressing cells cultured in ketogenic media (D) or from shRFP-expressing cells also infected with Ad-GFP (F).

Figure S5. mTORC1 modulates the association of NCoR1 with PPRE-containing promoters in vivo and in cells in culture. (A-B) At PPRE-containing promoters, mTORC1 inhibition prevents refeeding-induced deacetylation of Histone H4 (A) and recruitment of NCoR1 (B). Asterisks indicate p < 0.05 compared to controlb mice refed for 2 hours. (C) PPARα occupancy at PPRE-containing promoters is unchanged in livers from fed and fasted controla and LiTsc1KO mice. ChIP assays were performed on indicated liver extracts as described in methods. Values are mean ± S.D. for n ≥ 5. (D) In AML12 cells, mTORC1 activation inhibits the disassociation of NCoR1 from PPRE-containing promoters in response to ketogenic media containing WY-14,643. ChIP assays were performed on indicated cell extracts as described in methods. Values are mean ± S.D. for n ≥ 5.

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* *

Figure S3. Validation of the shRNAs used in the study and that aged LiTsc1KO and LiRapKO mice maintain hepatic loss of TSC1 and raptor, respectively. (A, B) qRT-PCR validation of indicated mRNAs in AML12 cells expressing indicated shRNAs. For sequences of each shRNA, see methods. (D, E) Immunoblot analyses of liver extracts from indicated mice strains expressing or not expressing Cre in their livers.

Figure S4. Overexpression of PPARα does not rescue inhibition of ketogenesis and PPARα signaling caused by mTORC1 activation. (A-C) Control and LiTsc1KO mice (4-6 months of age) were injected with high-titer adenovirus expressing either GFP (Ad-GFP) or PPARα (Ad- PPARα). Four days later, total serum ketones (A) were determined in fed mice or mice fasted for 24 hours. Asterisks indicate p < 0.05 compared to fasted livers infected with Ad-GFP. qRT-PCR analyses confirmed that PPARα was overexpressed in livers from both control and LiTsc1KO mice (B). Relative mRNA levels of indicated genes (C) from fasted livers of control and LiTsc1KO mice with hepatic overexpression of GFP or PPARα were determined as in Figure 2J. Asterisks indicate p < 0.05 compared to livers expressing Ad-GFP in control mice. (D-F) Confluent murine AML12 hepatocyte cells stably expressing validated lentiviral shRNAs targeting RFP or TSC2 were infected with adenovirus expressing either GFP or PPARα (1010 pfu/ml) at a MOI of 100. RT-PCR analyses confirmed that PPARα was overexpressed in cells (E). After bright GFP fluorescence was observed, cells were placed in control or ketogenic media for 3 days and total media ketones (D) and indicated mRNAs levels (F) were determined as in Figure 2J. Asterisks indicate p < 0.05 compared to shRFP-expressing cells cultured in ketogenic media (D) or from shRFP-expressing cells also infected with Ad-GFP (F).

Figure S5. mTORC1 modulates the association of NCoR1 with PPRE-containing promoters in vivo and in cells in culture. (A-B) At PPRE-containing promoters, mTORC1 inhibition prevents refeeding-induced deacetylation of Histone H4 (A) and recruitment of NCoR1 (B). Asterisks indicate p < 0.05 compared to controlb mice refed for 2 hours. (C) PPARα occupancy at PPRE-containing promoters is unchanged in livers from fed and fasted controla and LiTsc1KO mice. ChIP assays were performed on indicated liver extracts as described in methods. Values are mean ± S.D. for n ≥ 5. (D) In AML12 cells, mTORC1 activation inhibits the disassociation of NCoR1 from PPRE-containing promoters in response to ketogenic media containing WY-14,643. ChIP assays were performed on indicated cell extracts as described in methods. Values are mean ± S.D. for n ≥ 5.

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8 | w w w. n a t u r e . c o m / n a t u r e

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120

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Figure S6. Reduction in NCoR1 levels or use of the HDAC-inhibitor trichostatin A suppresses mTORC1-mediated inhibition of ketogenesis in mice and AML12 cells.(A-C) A 60% reduction in NCoR1 levels increases levels of serum ketones and suppresses the defect on PPARα signaing induced by mTORC1 activation. qRT-PCR validation of NCoR1 mRNA in livers from mice administered adenoviruses expressing indicated shRNAs (A). Controla and LiTsc1KO mice were administered adenoviruses expressing shRNAs targeting either lacZ or NCoR1; mice were fasted 6 days later and total serum ketones (B) and indicated mRNA levels (C) were determined as in Figure 2J. Asterisk indicates p < 0.05 compared to LiTsc1KO mice administered adenovirus expressing shlacZ. (D) AML12 cells stably expressing lentiviral shRNAs targeting GFP, NCoR1, or TSC1 were incubated in ketogenic media for 3 days plus ethanol vehicle or 100 nM of trichostatin A (TSA), and total media ketones were measured as described in the methods. Asterisk indicates p < 0.05 compared to shGFP-expressing cells treated with ethanol vehicle.

Figure S7. Immunofluorescence signal obtained with NCoR1 antibody decreases with an RNAi-mediated NCoR1 knockdown, and mTORC1 regulates the localization of recombinant epitope-tagged NCoR1. (A) Images of AML12 cells stably expressing lentiviral shRNAs targeting GFP or NCoR1 and co-stained for NCoR1 (red) and DNA (blue). (B) Images of AML12 cells transfected with an expression cDNA for FLAG-NCoR1 and incubated for 24 hours in control media, control media with 20 nM rapamycin, or ketogenic media and co-stained for the FLAG epitope (red) and DNA (blue).

Figure S8. mTORC1 controls the fasting-induced exit of NCoR1 from the nucleus.(A) Images of liver sections from control and LiTsc1KO mice given ad libitum access to food (fed AL) or fasted for 24 hours (fasted) and co-stained for NCoR1 (red) and DNA (blue). Insets show high magnification images of individual hepatocytes. (B) Images of liver sections from control and LiRapKO mice fasted for 24 hours or fasted and refed for 2 hours and co-stained for NCoR1 (red) and DNA (blue). (C) Images of AML12 cells expressing indicated shRNAs and incubated for 24 hours in control media, ketogenic media, or control media with 20 nM rapamycin sections and co-stained for NCoR1 (red) and DNA (blue). Insets show high magnification images of cells. (A-C) To the right of all images, graphs show mean ± S.D. of NCoR1 staining intensity in the nuclear and peri-nuclear areas, as determined as described in the methods. Dark and light blue shaded areas depict the mean and one standard deviation of the mean of the hepatocyte nuclear diameter, respectively.

Figure S9. Loss of raptor in the liver prevents the aging-induced decline in fasting serum ketones. (A) Aged control, but not LiRapKO, mice have a defect in the fasting-induced exit of NCoR1 from the nucleus. Images of liver sections from indicated mice and co-stained for NCoR1 (red) and DNA (blue). Insets show high magnification images of individual hepatocytes. Mice were fed and fasted as described in Figure 2A and images were quantified as described in methods. (B) Total serum ketones were measured in fasted controlb and LiRapKO mice of the indicated ages. Values are mean ±S.D. for n ≥ 5. Asterisks indicate p < 0.05 compared to fasted 2 month-old controlb mice.

w w w. n a t u r e . c o m / n a t u r e | 9

SUPPLEMENTARY INFORMATION RESEARCH

Complete Media (CM) Ketogenic MediaCM + Rapamycin

a

Figure S7

shGFP shNCoR1_1

b

NCoR1

FLAG-NCoR1

Figure S6. Reduction in NCoR1 levels or use of the HDAC-inhibitor trichostatin A suppresses mTORC1-mediated inhibition of ketogenesis in mice and AML12 cells.(A-C) A 60% reduction in NCoR1 levels increases levels of serum ketones and suppresses the defect on PPARα signaing induced by mTORC1 activation. qRT-PCR validation of NCoR1 mRNA in livers from mice administered adenoviruses expressing indicated shRNAs (A). Controla and LiTsc1KO mice were administered adenoviruses expressing shRNAs targeting either lacZ or NCoR1; mice were fasted 6 days later and total serum ketones (B) and indicated mRNA levels (C) were determined as in Figure 2J. Asterisk indicates p < 0.05 compared to LiTsc1KO mice administered adenovirus expressing shlacZ. (D) AML12 cells stably expressing lentiviral shRNAs targeting GFP, NCoR1, or TSC1 were incubated in ketogenic media for 3 days plus ethanol vehicle or 100 nM of trichostatin A (TSA), and total media ketones were measured as described in the methods. Asterisk indicates p < 0.05 compared to shGFP-expressing cells treated with ethanol vehicle.

Figure S7. Immunofluorescence signal obtained with NCoR1 antibody decreases with an RNAi-mediated NCoR1 knockdown, and mTORC1 regulates the localization of recombinant epitope-tagged NCoR1. (A) Images of AML12 cells stably expressing lentiviral shRNAs targeting GFP or NCoR1 and co-stained for NCoR1 (red) and DNA (blue). (B) Images of AML12 cells transfected with an expression cDNA for FLAG-NCoR1 and incubated for 24 hours in control media, control media with 20 nM rapamycin, or ketogenic media and co-stained for the FLAG epitope (red) and DNA (blue).

Figure S8. mTORC1 controls the fasting-induced exit of NCoR1 from the nucleus.(A) Images of liver sections from control and LiTsc1KO mice given ad libitum access to food (fed AL) or fasted for 24 hours (fasted) and co-stained for NCoR1 (red) and DNA (blue). Insets show high magnification images of individual hepatocytes. (B) Images of liver sections from control and LiRapKO mice fasted for 24 hours or fasted and refed for 2 hours and co-stained for NCoR1 (red) and DNA (blue). (C) Images of AML12 cells expressing indicated shRNAs and incubated for 24 hours in control media, ketogenic media, or control media with 20 nM rapamycin sections and co-stained for NCoR1 (red) and DNA (blue). Insets show high magnification images of cells. (A-C) To the right of all images, graphs show mean ± S.D. of NCoR1 staining intensity in the nuclear and peri-nuclear areas, as determined as described in the methods. Dark and light blue shaded areas depict the mean and one standard deviation of the mean of the hepatocyte nuclear diameter, respectively.

Figure S9. Loss of raptor in the liver prevents the aging-induced decline in fasting serum ketones. (A) Aged control, but not LiRapKO, mice have a defect in the fasting-induced exit of NCoR1 from the nucleus. Images of liver sections from indicated mice and co-stained for NCoR1 (red) and DNA (blue). Insets show high magnification images of individual hepatocytes. Mice were fed and fasted as described in Figure 2A and images were quantified as described in methods. (B) Total serum ketones were measured in fasted controlb and LiRapKO mice of the indicated ages. Values are mean ±S.D. for n ≥ 5. Asterisks indicate p < 0.05 compared to fasted 2 month-old controlb mice.

SUPPLEMENTARY INFORMATION

1 0 | w w w. n a t u r e . c o m / n a t u r e

RESEARCH

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Figure S6. Reduction in NCoR1 levels or use of the HDAC-inhibitor trichostatin A suppresses mTORC1-mediated inhibition of ketogenesis in mice and AML12 cells.(A-C) A 60% reduction in NCoR1 levels increases levels of serum ketones and suppresses the defect on PPARα signaing induced by mTORC1 activation. qRT-PCR validation of NCoR1 mRNA in livers from mice administered adenoviruses expressing indicated shRNAs (A). Controla and LiTsc1KO mice were administered adenoviruses expressing shRNAs targeting either lacZ or NCoR1; mice were fasted 6 days later and total serum ketones (B) and indicated mRNA levels (C) were determined as in Figure 2J. Asterisk indicates p < 0.05 compared to LiTsc1KO mice administered adenovirus expressing shlacZ. (D) AML12 cells stably expressing lentiviral shRNAs targeting GFP, NCoR1, or TSC1 were incubated in ketogenic media for 3 days plus ethanol vehicle or 100 nM of trichostatin A (TSA), and total media ketones were measured as described in the methods. Asterisk indicates p < 0.05 compared to shGFP-expressing cells treated with ethanol vehicle.

Figure S7. Immunofluorescence signal obtained with NCoR1 antibody decreases with an RNAi-mediated NCoR1 knockdown, and mTORC1 regulates the localization of recombinant epitope-tagged NCoR1. (A) Images of AML12 cells stably expressing lentiviral shRNAs targeting GFP or NCoR1 and co-stained for NCoR1 (red) and DNA (blue). (B) Images of AML12 cells transfected with an expression cDNA for FLAG-NCoR1 and incubated for 24 hours in control media, control media with 20 nM rapamycin, or ketogenic media and co-stained for the FLAG epitope (red) and DNA (blue).

Figure S8. mTORC1 controls the fasting-induced exit of NCoR1 from the nucleus.(A) Images of liver sections from control and LiTsc1KO mice given ad libitum access to food (fed AL) or fasted for 24 hours (fasted) and co-stained for NCoR1 (red) and DNA (blue). Insets show high magnification images of individual hepatocytes. (B) Images of liver sections from control and LiRapKO mice fasted for 24 hours or fasted and refed for 2 hours and co-stained for NCoR1 (red) and DNA (blue). (C) Images of AML12 cells expressing indicated shRNAs and incubated for 24 hours in control media, ketogenic media, or control media with 20 nM rapamycin sections and co-stained for NCoR1 (red) and DNA (blue). Insets show high magnification images of cells. (A-C) To the right of all images, graphs show mean ± S.D. of NCoR1 staining intensity in the nuclear and peri-nuclear areas, as determined as described in the methods. Dark and light blue shaded areas depict the mean and one standard deviation of the mean of the hepatocyte nuclear diameter, respectively.

Figure S9. Loss of raptor in the liver prevents the aging-induced decline in fasting serum ketones. (A) Aged control, but not LiRapKO, mice have a defect in the fasting-induced exit of NCoR1 from the nucleus. Images of liver sections from indicated mice and co-stained for NCoR1 (red) and DNA (blue). Insets show high magnification images of individual hepatocytes. Mice were fed and fasted as described in Figure 2A and images were quantified as described in methods. (B) Total serum ketones were measured in fasted controlb and LiRapKO mice of the indicated ages. Values are mean ±S.D. for n ≥ 5. Asterisks indicate p < 0.05 compared to fasted 2 month-old controlb mice.

w w w. n a t u r e . c o m / n a t u r e | 1 1

SUPPLEMENTARY INFORMATION RESEARCH

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Figure S6. Reduction in NCoR1 levels or use of the HDAC-inhibitor trichostatin A suppresses mTORC1-mediated inhibition of ketogenesis in mice and AML12 cells.(A-C) A 60% reduction in NCoR1 levels increases levels of serum ketones and suppresses the defect on PPARα signaing induced by mTORC1 activation. qRT-PCR validation of NCoR1 mRNA in livers from mice administered adenoviruses expressing indicated shRNAs (A). Controla and LiTsc1KO mice were administered adenoviruses expressing shRNAs targeting either lacZ or NCoR1; mice were fasted 6 days later and total serum ketones (B) and indicated mRNA levels (C) were determined as in Figure 2J. Asterisk indicates p < 0.05 compared to LiTsc1KO mice administered adenovirus expressing shlacZ. (D) AML12 cells stably expressing lentiviral shRNAs targeting GFP, NCoR1, or TSC1 were incubated in ketogenic media for 3 days plus ethanol vehicle or 100 nM of trichostatin A (TSA), and total media ketones were measured as described in the methods. Asterisk indicates p < 0.05 compared to shGFP-expressing cells treated with ethanol vehicle.

Figure S7. Immunofluorescence signal obtained with NCoR1 antibody decreases with an RNAi-mediated NCoR1 knockdown, and mTORC1 regulates the localization of recombinant epitope-tagged NCoR1. (A) Images of AML12 cells stably expressing lentiviral shRNAs targeting GFP or NCoR1 and co-stained for NCoR1 (red) and DNA (blue). (B) Images of AML12 cells transfected with an expression cDNA for FLAG-NCoR1 and incubated for 24 hours in control media, control media with 20 nM rapamycin, or ketogenic media and co-stained for the FLAG epitope (red) and DNA (blue).

Figure S8. mTORC1 controls the fasting-induced exit of NCoR1 from the nucleus.(A) Images of liver sections from control and LiTsc1KO mice given ad libitum access to food (fed AL) or fasted for 24 hours (fasted) and co-stained for NCoR1 (red) and DNA (blue). Insets show high magnification images of individual hepatocytes. (B) Images of liver sections from control and LiRapKO mice fasted for 24 hours or fasted and refed for 2 hours and co-stained for NCoR1 (red) and DNA (blue). (C) Images of AML12 cells expressing indicated shRNAs and incubated for 24 hours in control media, ketogenic media, or control media with 20 nM rapamycin sections and co-stained for NCoR1 (red) and DNA (blue). Insets show high magnification images of cells. (A-C) To the right of all images, graphs show mean ± S.D. of NCoR1 staining intensity in the nuclear and peri-nuclear areas, as determined as described in the methods. Dark and light blue shaded areas depict the mean and one standard deviation of the mean of the hepatocyte nuclear diameter, respectively.

Figure S9. Loss of raptor in the liver prevents the aging-induced decline in fasting serum ketones. (A) Aged control, but not LiRapKO, mice have a defect in the fasting-induced exit of NCoR1 from the nucleus. Images of liver sections from indicated mice and co-stained for NCoR1 (red) and DNA (blue). Insets show high magnification images of individual hepatocytes. Mice were fed and fasted as described in Figure 2A and images were quantified as described in methods. (B) Total serum ketones were measured in fasted controlb and LiRapKO mice of the indicated ages. Values are mean ±S.D. for n ≥ 5. Asterisks indicate p < 0.05 compared to fasted 2 month-old controlb mice.