Nutrient-sensitive mitochondrial NAD+ levels dictate cell survival

Abstract

A major cause of cell death caused by genotoxic stress is thought to be due to the depletion of NAD(+) from the nucleus and the cytoplasm. Here we show that NAD(+) levels in mitochondria remain at physiological levels following genotoxic stress and can maintain cell viability even when nuclear and cytoplasmic pools of NAD(+) are depleted. Rodents fasted for 48 hr show increased levels of the NAD(+) biosynthetic enzyme Nampt and a concomitant increase in mitochondrial NAD(+). Increased Nampt provides protection against cell death and requires an intact mitochondrial NAD(+) salvage pathway as well as the mitochondrial NAD(+)-dependent deacetylases SIRT3 and SIRT4. We discuss the relevance of these findings to understanding how nutrition modulates physiology and to the evolution of apoptosis.

Figure 1. Nampt Is a Stress- and Nutrient-Responsive Gene that Protects Cells against the Genotoxic Agent MMS

(A and B) Nampt levels in human fibrosarcoma HT1080 cells in the presence or absence of 10% FBS (A) or of liver tissue extracts from fed or 2 day-fasted Sprague-Dawley rats (B). Actin and tubulin were used as loading controls. (C and D) Nampt protein (C) and mRNA levels of Nampt (D) in livers of fasted rats (n = 4; bars represent the mean of three experiments ± standard deviations [SD] using Student’s t test). (E) Western blot of Nampt in primary rat cardiomyocytes under hypoxia and/or serum starvation. (F and G) Survival of HT1080 cells stably expressing human (F) or transiently expressing mouse Nampt (G) following treatment with 1.2 mM methylmethanesulfonate (MMS). (H) Survival of human kidney HEK293 cells stably expressing human Nampt treated with MMS as in (F). Always, bars represent the mean of three experiments ± SD.

Figure 2. Nampt Protects against Apoptotic Cell Death Induced by Topoisomerase Inhibitors

(A) Sensitivity of HT1080 with siRNA-mediated knockdown of NAMPT after MMS exposure. (B) Stable overexpression of Nampt enhances survival of HEK293 cells following MMS treatment and the effect is blocked by the Nampt-inhibitor FK866. (C) Survival of WT or Nampt knockdown HT1080 cells after serum deprivation for 22 hr and then exposure to MMS for 17 hr. Serum deprivation upregulates Nampt and enhances survival of WT but not Nampt knockdown HT1080 cells. (D and E) Survival of HEK293 stably overexpressing Nampt (D) or HT1080 with siRNA knockdown of Nampt (E) following etoposide treatment. (F) Survival of HT1080 Nampt knockdown cells after camptothecin treatment. Apoptosis was assessed by western blot analysis of cleaved Caspase-3. Bars represent the mean of three experiments ± SD.

Figure 3. Nampt-Mediated Protection against Genotoxicity Requires SIRT3 and SIRT4

(A) Survival of HEK293 cells stably expressing Nampt following exposure to MMS in the presence or absence of the SIRT1-specific inhibitor EX-527. (B) siRNA knockdown of SIRT1 using a pool of four siRNA oligos, compared to nontargeting siRNA controls. Cells were cotransfected with FAM-tagged fluorescent oligos and percentage cell death was determined by FACS as a ratio of PI/FAM-positive cells versus total FAM-positive cells. (C) HEK293 cells were treated with 100 μM sirtinol, a pan sirtuin inhibitor. All experiments were carried out three times in triplicate. (D and E) SIRT3 or SIRT4 was knocked down in HEK293 cells stably overexpressing Nampt using pools of specific siRNA oligos, and cells were then treated with MMS and scored for survival. (F) Cells from (D) were probed by western blotting for cleaved caspase-3, an indicator of apoptosis. (G) Immunoprecipitation (IP) of AceCS2 from cell lysates of control and Nampt-overexpressing HEK293 cells transfected with control vector or AceCS2-HA for 48 hr. The levels of acetylated AceCS2 in IPs were analyzed by western blotting. Bars represent the mean of three experiments ± SD.

Figure 4. Nampt Regulates Total NAD⁺

(A) Synthesis of isotope-labeled ¹⁸O-NAD⁺, a reference compound used in NAD⁺ measurement. ¹⁸O-NAM was synthesized by hydrolyzing 3-cyanopyridine in ¹⁸O-H₂O and was then used as a substrate in the enzymatic reaction catalyzed by CD38, a NAD⁺ glycohydrolase, to generate ¹⁸O-NAD⁺. (B and C) Total endogenous ¹⁶O-NAD⁺ and spiked-in NAD reference ¹⁸O-NAD⁺ were isolated by HPLC then subjected to MALDI-MS. The ion intensity of the reference peaks of ¹⁸O-NAD⁺ were normalized to 100 in all cases. The ratio of ¹⁶O-NAD⁺ peaks reflects the relative amount of NAD⁺ in the two samples. Experiments were performed at least three times. Total NAD⁺ spectra from HEK293 are shown for vector controls and cells stably overexpressing Nampt (B) as well as total NAD⁺ spectra from HT1080 vector controls and siRNA-Nampt stable cells (C). (D) Overexpression of Nampt cannot prevent total cellular NAD⁺ depletion by MMS as determined by MALDI-MS spectra of endogenous ¹⁶O-NAD and reference ¹⁸O-NAD after 2 hr MMS treatment of HEK293 WT and Nampt-overexpressing cells. (E) Time course of cell death induced by 1.2 mM MMS treatment. Percent cell death was determined by FACS analysis. (F) Total cellular NAD⁺ as measured by MALDI-MS during the time course in (E). Bars represent the mean of three experiments ± SD.

Figure 5. Mammalian Mitochondria Maintain Mitochondrial NAD⁺ Levels during Genotoxic Stress

(A and B) Nampt regulates mitochondrial NAD⁺ levels. Mitochondrial NAD⁺ was isolated and analyzed as described in Figures 4B and 4C. Spectra from HEK293 are shown for vector controls and cells stably overexpressing Nampt (A), as well as spectra from HT1080 vector controls and siRNA-Nampt stable cells (B). (C) Additional Nampt greatly attenuates mitochondrial NAD⁺ depletion by MMS treatment, as determined by MALDI-MS after 2 hr MMS treatment of HEK293 WT and Nampt-overexpressing cells. (D and E) Western blotting analysis of Nampt in highly purified cytosolic and mitochondrial fractions. Mitochondiral fractions were isolated from HEK293 cells or from rat livers using two different protocols, and their purity was assessed by probing for Hsp90, calreticulin, and/or lactate dehydrogenase (exclusively cytoplasmic proteins), and CoxIV or cytochrome C (mitochondrial matrix markers). The same blot was probed for lamin A/C to test for contamination of the mitochondrial fractions with nuclei. The experiment was performed three times on HEK293 cells and on liver tissue. The same pattern was observed each time and representative blots are shown. (F) Mitochondria from rat livers were prepared and exposed to methylmethane sulfonate (MMS), a genotoxic DNA alkylating agent, or the Nampt inhibitor FK866, or both. NAD⁺ levels in isolated mitochondria were determined using MALDI-MS, as above. (G) NAD⁺ levels in isolated mitochondria are reduced by exposure to MMS and FK866. Similar data were obtained using a different mitochondrial isolation protocol (see Figure S5). (H) Knocking down expression of Nmnat-3 reduces the ability of Nampt to provide resistance to MMS. (I) Knocking down expression of a putative human mitochondrial NAD⁺ transporter, hMFT, does not affect survival of Nampt-overexpressing cells treated with MMS. Bars represent the mean of three experiments ± SD.

Figure 6. Fasting Increases Hepatic Mitochondrial NAD⁺ and Nampt Levels

(A) Overexpression of Nampt in HEK293 cells inhibits the localization of AIF to the nucleus after MMS treatment for the times indicated, as assessed by western blotting. (B) Western blotting analysis of Nampt in mitochondria from rats fed AL or fasted for 48 hr. (C) Relative mitochondrial NAD⁺ levels in liver tissues from rats fed AL or fasted for 48 hr. Mitochondrial NAD⁺ levels were measured by MALDI-MS. Bars represent the mean of three experiments ± SD.