The work sits at the intersection of several fields: the study of biosignatures and metabolic plasticity, visualization of cellular processes, and the analysis of aging. Nearby in the archive are studies that examine metabolism at the level of tissues or individual cells, for example, how enzymes work in a dense gel or how an electron microscope emerges from vacuum. Our work takes a step further: it combines isotope labeling with ultrastructural imaging to see metabolic fluxes simultaneously at several scales in an intact animal. This allows us to ask how dietary restriction redistributes resources between tissues and organelles, which is important for understanding the mechanisms of longevity.
The researchers developed a protocol for labeling C. elegans nematodes with the stable isotopes ¹³C and ¹⁵N via bacterial food. E. coli bacteria were grown on a medium where the sole carbon source was labeled glucose and the sole nitrogen source was labeled ammonium chloride, ensuring uniform incorporation of the isotopes into all macromolecules. The animals were grown on labeled food from egg to the first day of adulthood, then transferred to unlabeled food under conditions of either unlimited feeding or caloric restriction. After 0, 8, 24, 48, and 72 hours, the animals were frozen under high pressure to preserve ultrastructure, and thin sections were prepared. The sections were first scanned in an electron microscope to obtain morphology, then in a secondary ion mass spectrometer (MIMS) to map the isotope ratios ¹³C/¹²C and ¹⁵N/¹⁴N with a resolution of about 60 nanometers. The resulting images were aligned and manually segmented into muscle, hypodermis, and intestine, as well as individual mitochondria. Turnover kinetics were described by a one-phase exponential model, and the decay constants were compared between diets.
Under normal feeding, carbon in the intestine turned over faster (half-life 6.65 h) than in muscle (12.7 h) and hypodermis (12.3 h). Dietary restriction accelerated carbon turnover in muscle (5.47 h) and hypodermis (5.08 h), but did not change it in the intestine (5.08 h versus 6.65 h, p=0.363). For nitrogen, the picture was different: in muscle, dietary restriction accelerated turnover (5.62 h versus 13.8 h, p=0.0447), in the intestine it did too (5.22 h versus 11.1 h, p=0.0112), while in the hypodermis the trend did not reach significance. Mitochondria turned over faster than the tissue as a whole, especially for carbon, and dietary restriction accelerated their turnover similarly to the tissue level. Analysis of mitochondrial heterogeneity showed that the coefficient of variation of isotope composition remained stable over time (0.22–0.27) and did not depend on diet. Spatial analysis revealed that neighboring mitochondria are more similar in isotope composition than distant ones, and the characteristic correlation length grew over time from less than 2 µm to about 9 µm, indicating local mixing of mitochondrial contents.
The work differs from neighboring studies in that it applies MIMS-EM to a whole multicellular organism for the first time, allowing simultaneous measurement of biomass turnover in different tissues and organelles with nanometer resolution. This makes it possible to test hypotheses about resource redistribution during dietary interventions that were previously based on averaged biochemical data. The results show that dietary restriction does not simply accelerate overall material turnover, but selectively modulates carbon and nitrogen turnover depending on the tissue, which is consistent with a model of selective remodeling rather than global recycling.
The MIMS-EM platform for C. elegans opens the way to systematic study of how genetic, dietary, and age-related changes rearrange metabolic fluxes from organelles to the whole organism. In the future, this method can be combined with biochemical analysis of purified macromolecular pools to link elemental signals to specific proteins and lipids. It is also possible to use specific labeled precursors for individual classes of molecules.
The method and the data obtained will be useful for gerontology, which studies the mechanisms of life extension by dietary restriction, for cell biology, which investigates mitochondrial dynamics, and for systems biology, which builds models of metabolic networks in multicellular organisms.
The immediate next steps include direct manipulation of mitochondrial division and fusion to test the hypothesis of content mixing, as well as conducting similar experiments on aging animals to identify age-related heterogeneity.
The work is directly related to the unresolved question of how dietary restriction redistributes resources between tissues and organelles to extend life. Existing contradictions between data on proteome turnover (slowing of turnover for many proteins) and activation of autophagy require spatial resolution, which MIMS-EM provides. Our results show that elemental turnover can accelerate even when the turnover of individual proteins slows down, which reconciles competing models.
The worm's cuticle, composed mainly of collagen, after labeling showed a ¹⁵N/¹⁴N ratio of about 15,000, which is thousands of times higher than the natural level and demonstrates almost complete saturation with isotopes.
Not published on arXiv. Here are its page, the PDF and the formalization: you can check it yourself, not only read our account.