Unprocessed microscopy and immunoblotting images for data presented in Ahsan et al. Molecular Cell Volume 86 Issue 13 (2026), "Reductive death is averted by a conserved de novo lipogenic switch"

Description

Biguanides, including metformin, the world’s most prescribed oral hypoglycemic, extend health span and lifespan in vertebrates and invertebrates. Given the widespread use and apparent safety of metformin, it is assumed that its effects are not associated with toxicity, except when in marked excess. Here we determine that accumulation of damaging reducing equivalents is an unanticipated toxicity associated with biguanides, the defense against which requires post-transcriptional protection of de novo fatty acid biosynthesis. We demonstrate that biguanide treatment during impaired fatty acid biosynthesis drives NADPH toxicity, leading to catastrophic elevation of NADH/GSH reducing equivalents and accelerated death across metazoans. Multiple NADPH-generating interventions require fatty acid biosynthesis to prevent markedly shortened survival, indicating that this defense mechanism is broadly leveraged. We propose that fatty acid biosynthesis is a tunable rheostat which can minimize biguanide-induced reductive stress whilst maximizing its pro-longevity outcomes and serve as an exploitable vulnerability in reductive stress sensitive cancers.

Steps to reproduce

Immunoblotting was performed as previously described. Worm and cell lysates were collected into 1x RIPA buffer (containing 50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 1% (v/v) Triton X-100 detergent, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 1 mM EDTA) supplemented with EDTA-free protease inhibitor tablets (Roche) and a phosphatase inhibitor cocktail containing sodium fluoride, sodium pyrophosphate, sodium orthovanadate, β-glycerophosphate, and okadaic acid. Samples were sonicated at 40% amplitude in a QSonica Q800R3 water bath sonicator at 30 s ON/30 s OFF intervals for a total of 10 minutes at 4°C. Lysates were cleared by centrifugation at 21,000xg at 4°C for 10 minutes and supernatants were retained in clean microcentrifuge tubes for protein analysis. Protein concentration quantification was performed using a Pierce BCA Protein Assay Kit (Thermo Fisher) and samples were diluted to equal concentration using RIPA lysis buffer. Lysates were diluted in 4x Laemmli Sample Buffer (Bio-Rad) supplemented with 10% (v/v) β-mercaptoethanol and boiled for 5 minutes in a 95°C dry bath prior to SDS-PAGE. SDS-PAGE was performed using the Bio-Rad Mini Protean Tetra Cell gel system with 4-15% Mini-PROTEAN TGX precast gels at 150V, using Bio-Rad Precision Plus Dual Color standards. Electrophoretic transfer to nitrocellulose membranes was performed at 100V for 1 hour at 4°C using a 20% (v/v) methanol-based transfer solution. For primary antibody immunodetection, a 1:5000 dilution of Goat anti-Rabbit HRP conjugate or Goat anti-Mouse HRP conjugate (GE Healthcare) in a 5% bovine serum albumin (BSA) 1x TBST solution was used, incubating membranes for 1 hour rocking at room temperature. Membranes were washed 3 times with 1x TBST, and HRP detection was performed using the SuperSignal West Pico PLUS chemiluminescent substrate. Chemiluminescence was detected and imaged on membranes using the iBright imaging system (Thermo Fisher Scientific). Images were obtained using a chemiluminescence and membrane overlay and compiled using Adobe Illustrator and Microsoft Office PowerPoint. For all fluorescence imaging studies, animals of the indicated stage, genotype, or treatment were manually transferred to a droplet of 1 mg/mL levamisole in M9 buffer on top of a 2% (w/v) agarose pad embedded on a microscope slide. Animals were observed for cessation of thrashing and then covered with a coverslip immediately prior to imaging. For whole worm fluorescence imaging studies, slides were imaged at 5x magnification on a Leica DM6 B microscope with Thunder Imaging. For lipid droplet imaging studies, slides were imaged on a Zeiss LSM 800 confocal microscope equipped with an Airyscan detector, centering the posterior intestinal cell nuclei under DIC imaging. Animals were paralyzed and mounted immediately prior to imaging to prevent fluorescence bleaching or excessive levamisole exposure.

Institutions

• Harvard Medical School

  MA, Boston
• Massachusetts General Hospital

  MA, Boston

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