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. Author manuscript; available in PMC: 2014 Jun 30.
Published in final edited form as: Cell. 2013 Dec 19;155(7):1624–1638. doi: 10.1016/j.cell.2013.11.037

Figure 3. Loss of SIRT1 Induces a Pseudohypoxic State that Disrupts Mitochondrial-Encoded Genes and Mitochondrial Homeostasis.

Figure 3

(A and B) HK2, PFKM, PKM, and LDHA mRNA (A) and lactate levels (B) of WT and SIRT1 iKO mice (n = 5, *p < 0.05 versus WT).

(C) Immunoblot for HIF-1α and tubulin in WT and SIRT1 iKO mice and in SIRT1 flox/flox Cre-ERT2 primary myoblasts treated with vehicle or OHT to induce SIRT1 excision for 24 hr (SIRT1 iKO).

(D) Immunoblot for HIF-1α and tubulin in primary myoblasts transduced with NMNAT1 or nontargeting shRNA.

(E) Immunoblot of HIF-1α and tubulin in primary myoblasts treated with pyruvate, lactate, or vehicle for 24 hr.

(F) Immunoblot for HIF-1α and tubulin in WT and EglN1 KO mice.

(G) Expression of nuclear- and mitochondrially encoded genes of WT and EglN1 KO mice (n = 5, *p < 0.05 versus WT).

(H) Mitochondrial DNA content of WT and EglN1 KO mice (n = 5, *p < 0.05 versus WT).

(I) Expression of mitochondrially encoded genes in PGC-1α/β KO myotubes treated with adenovirus overexpressing SIRT1 treated with DMSO or the HIF-stabilizing compound DMOG (n = 4, *p < 0.05 versus empty DMSO; #p < 0.05 versus SIRT1 OE DMSO).

(J) Immunoblot for HA tag and tubulin in control and C2C12 cells overexpressing either HIF-1α or HIF-2α with the proline residues mutated (HIF-1α DPA and HIF-2α DPA).

(K) Expression of nuclear- versus mitochondrially encoded genes in HIF-1α DPA or HIF-2α DPA C2C12 cells (n = 6, *p < 0.05 versus empty vector).

(L) Expression of mitochondrially encoded genes in HIF-1α DPA or HIF-2α DPA C2C12 cells with adenovirus overexpressing SIRT1 (n = 4, *p < 0.05 versus empty vector; #p < 0.05 versus SIRT1 OE).

(M) Immunoblot for HIF-1α and tubulin in SIRT1 flox/flox Cre-ERT2 primary myoblasts transduced with HIF-1α or nontargeting shRNA and treated with DMOG.

(N) Mitochondrial DNA in SIRT1 flox/flox Cre-ERT2 primary myoblasts transduced with HIF-1α or nontargeting shRNA, treated with vehicle or OHT to induce SIRT1 excision (SIRT1 iKO) (n = 4, *p < 0.05 versus shNT vehicle; #p < 0.05 versus shNT SIRT1 iKO).

(O) Expression of mitochondrially encoded genes in SIRT1 flox/flox Cre-ERT2 primary myoblasts transduced with HIF-1α or nontargeting shRNA and treated with OHT to induce SIRT1 excision (SIRT1 iKO) (n = 4, *p < 0.05 versus shNT vehicle; #p < 0.05 versus shNT SIRT1 iKO).

(P) ATP content in SIRT1 flox/flox Cre-ERT2 primary myoblasts transduced with HIF-1α or nontargeting shRNA and treated with vehicle or OHT to induce SIRT1 excision (n = 5, *p < 0.05 versus shNT vehicle; #p < 0.05 versus shNT SIRT1 iKO).

Nuclear- and mitochondrially encoded genes were ND1, Cytb, COX1, ATP6 and NDUFS8, SDHb, Uqcrc1, COX5b, ATP5a1, respectively. Tissue samples are gastrocnemius unless otherwise stated. Values are expressed as mean ± SEM. See also Figure S3.