Giovanna de Brito Rodrigues Rosa^1,2, Mariana Feitosa Custódio¹, Bruna Silva de Lacerda¹, Israelle Netto Freitas², Kênia Moreno de Oliveira², Ana Valéria Colnaghi Simionato¹, Everardo Magalhães Carneiro²

INTRODUCTION:

Malnutrition is a serious public health problem that commonly affects children in developing countries. There is evidence that protein restriction during the early stages of development predisposes to peripheral insulin resistance, type 2 diabetes, and increased periepididimal, retroperitoneal, inguinal, and visceral fat stores despite low weight. In this context, experimental evidence indicates that bile acids (BAs) exert neuroendocrine effects that regulate intermediary metabolism and energy expenditure. Among the various BAs with such actions, tauroursodeoxycholic acid (TUDCA) stands out. Therefore, this work aims to investigate the untargeted metabolomic profile of serum from male C57Bl/6 mice subjected to a low-protein diet intervention and subsequently treated intraperitoneally with 300 mg/kg of TUDCA during the last 15 days of life, to identify biomarkers related to these pathogenic processes and therapeutic response.

METHODS:

In accordance with ethical guidelines for animal use (Ethics Committee approval nº 6190/2023), two experimental groups of male C57BL/6 mice were formed (n = 6 per group): one group was fed with a normoproteic diet (14% protein, Control) and the other group was fed with a low-protein diet (6% protein, Restricted) for 105 days of life. After this period, blood samples were collected by cardiac puncture at the time of euthanasia and centrifuged to obtain serum, which was then prepared for metabolite extraction and derivatization, and analyzed by gas chromatography–mass spectrometry (GC-MS) following Fiehn library protocols for untargeted metabolomic analysis. It is noteworthy that, at this stage, data from the TUDCA-treated groups will not be presented; although serum from these animals was collected at the same time point, experiments involving these groups have not yet been initiated.

RESULTS:

Chromatograms were processed in MS-DIAL 4.93 to generate a feature table, and the data were first corrected for signal drift using non-parametric smoothing (LOWESS v1.1). In Excel, features detected in less than 70% of QC samples and those with robust coefficients of variation above 30% in QCs were discarded. Subsequently, the data were analyzed in MetaboAnalyst 6.0. Remaining missing values were imputed using the KNN (sample-wise) method. Feature filtering was performed using a 5% threshold based on median absolute deviation (MAD). Sample normalization was carried out by sum of intensities, followed by auto-scaling. Multivariate analysis was performed using PCA, which showed appropriate QC clustering and analytical repeatability after removing one QC injection replicate. PLS-DA was able to separate the Control and Restricted groups, with R² = 0.99, Q² = 0.37, and Accuracy = 0.75 using five latent variables, after removing features that were not contributing to group discrimination. Talose, mannose, and glucose showed the highest VIP scores (close to 2) and were more abundant in the Control group, suggesting their potential role in group separation. The study is currently at the data processing stage, and biological interpretations are still pending.

 CONCLUSION:

The biochemical interpretation of the results is the final step toward understanding the overall metabolic alterations associated with the studied condition. Preliminary analyses suggest improved metabolic outcomes and indicate that the restricted diet may reduce certain blood carbohydrate levels.

Agradecimentos: The authors acknowledge funding from the São Paulo Research Foundation (FAPESP), the National Council for Scientific and Technological Development (CNPq), and the Coordination for the Improvement of Higher Education Personnel (CAPES), as well as the University of Campinas (Unicamp) for providing the infrastructure necessary to support this research.