Discovering Metabolic Dynamics Regulated by Anti-Aging Metformin Through Subcellular Multimodal Imaging

Introduction: Aging is the gradual deterioration of physiological functions which alters lipid metabolism. Metformin, a dimethylbiguanide antihyperglycemic drug commonly prescribed for the treatment of type 2 diabetes, activates a similar signaling pathway to dietary restriction (DR), rendering it a putative DR mimetic drug. However, despite the drug’s prevalence within the medical realm, its prospects at the subcellular level are significantly discounted for. On account of this, we utilized a multimodal optical imaging system, which integrates deuterium oxide (D2O)-probed stimulated Raman scattering (DO-SRS) and two photon excitation fluorescence (2PEF) microscopies, to study metformin’s contributions to age retardation. Conveniently, Drosophila, or fruit flies, not only possess a short lifespan but also share over 60% of their genes with humans, characterizing them as ideal model organisms for gerontological studies. Furthermore, by implementing the non-invasive optical imaging methods, we effectively observed subcellular metabolic changes in the lipid droplets (LDs) inside the fat body tissues of metformin-treated flies.

Materials and

Methods: Wild type w1118 flies were maintained on standard cornmeal diet. Newly-eclosed flies were collected over a 24-hour period. They were allowed to mature and mate in the cornmeal diet for 48 hours before they were shortly anesthetized with carbon dioxide (CO2) and sorted into single sexes. Only female flies were selected for experiments. The selected female flies were maintained on either a control sugar/yeast/agar (SYA) medium or an SYA medium consisting of metformin. The flies were transferred to culture vials with fresh diet every 3 days. For each of the two diet conditions, 40 female flies were arbitrarily allocated in vials at a density of 10 flies per vial. The changes of lipid turnover rate in w1118 flies at different ages were labeled by simultaneously transferring the 2- and 30-day old female flies to their respective diets containing 20% (v/v) deuterium oxide (D2O) for 5 days without flipping. This probing allows for the newly synthesized lipids to be identified by their carbon-deuterium (C-D) bonds as opposed to original lipids which contain only carbon-hydrogen (C-H) bonds. After the D2O labeling period, 10 randomly selected 7-day old flies, and 10 randomly selected 35-day old flies from each diet type (40 flies in total) were sacrificed. Their fat bodies were dissected and fixed in a 4% methanol-free paraformaldehyde solution diluted by 1× phosphate-buffered saline (PBS) for 30 minutes. These samples were subjected to Raman and 2PEF imaging within 24 hours of dissection and stored at 4˚C when not in use.

Results, Conclusions, and Discussions: Our study showed that metformin treatment extended the median lifespan of Drosophila flies by approximately 3 days. The results indicated that metformin reduced fatty acid synthesis through the inhibition of enzymes of the mitochondrial respiratory complex 1 (MT-Complex 1). This inhibition also led to an upregulation of lysosomal autophagy, which encouraged lipolytic activity. Moreover, metformin promoted NADH reduction, resulting in an increased optical redox ratio in the metformin-treated group. Additionally, the results indicated that the concentration of saturated fatty acids (SFA) was reduced through metformin’s restoration of autophagic flux, thereby mitigating inflammatory responses as a corollary. The increase in lipid saturation ratio was supported by a lowered amount of reactive oxygen species, which consequently increased unsaturated lipid content in the fat body. Thus, with 2PEF, we discovered that the drug rescued the fat body tissues from oxidative stress. Unprecedentedly, through the implementation of non-invasive multimodal imaging techniques of DO-SRS and 2PEF, we have concluded that metformin exhibits anti-aging properties by restoring vital metabolic functions. These conclusions exemplify the prospects of implementing DO-SRS and 2PEF in future pharmacology-related endeavors towards visualizing in situ metabolic activity with subcellular resolution and spatiotemporal information.

Acknowledgements (Optional): We thank Dr. Lingyan Shi, Dr. Hongje Jang, Dr. Anthony A. Fung, Zhi Li, and Shi Lab members for helpful discussions. We acknowledge support from UC San Diego Startup Funds, Hellman Fellowship, Sloan Research Fellowship, NIH U54CA132378, NIH 5R01NS111039, Summer URS Eureka! 2023 Research Scholarship, 2024 Triton Research and Experiential Learning Scholarships, UC San Diego Undergraduate Research Hub (URH), Shu Chien-Gene Lay Department of Bioengineering Undergraduate Travel Award, UC San Diego Undergraduate Research Hub Development Grant, Jacobs School of Engineering Student Travel Fund, and Revelle College Travel Fund.