Supplementary Material for: Impact of Sodium Butyrate Supplementation on Insulin Resistance and Adipose Tissue Modulation in Murine Models of Polycystic Ovary Syndrome

Objectives: Polycystic ovary syndrome (PCOS) is an endocrine-metabolic disorder affecting up to 10% of women of reproductive age, commonly associated with visceral obesity, insulin resistance, and chronic low-grade inflammation. Sodium butyrate, a microbial-derived short-chain fatty acid, has been proposed as a therapeutic agent due to its anti-inflammatory and metabolic regulatory properties. However, its efficacy under hyperandrogenic and proinflammatory conditions remains uncertain. Design: Experimental study using female BALB/c mice. Forty animals were allocated into five groups: control (CT), high-fat diet (HFD), dehydroepiandrosterone (DHEA), butyrate without PCOS (BUT), and butyrate with PCOS (DHEA+BUT). Participants/Materials, Setting, Methods: Animals received a high-fat diet (except CT), and hyperandrogenism was induced by DHEA. Sodium butyrate was administered via oral gavage during the last 40 days of the protocol. Body weight, glucose tolerance, adipose tissue mass and morphology, serum adipokines, inflammatory gene expression, and gut microbiota profiles were assessed. Results: Butyrate-treated groups, particularly DHEA+BUT, exhibited greater body weight gain and significant expansion of subcutaneous, visceral, and brown adipose depots (p < 0.01). Adipose morphometry showed reduced adipocyte number with preserved diameter, indicative of hypertrophy, accompanied by elevated IL-6 and TNF-α expression in visceral fat (p < 0.01). Leptin levels tended to increase, while adiponectin was significantly reduced in the DHEA+BUT group. Glucose tolerance was impaired in all high-fat diet groups, with the BUT group showing the highest glycemic peaks. Although butyrate increased gut microbial diversity, it failed to prevent metabolic or inflammatory dysfunction. Limitations: The modest number of animals per group reduced statistical power, reflecting constraints in breeding and availability of age-matched BALB/c mice. Their relatively small adipose depots limited tissue yield for molecular and histological assays. Daily oral gavage, required for precise dosing, was technically challenging and led to the loss of one DHEA+BUT animal. Microbiota sequencing was performed in only one animal per group due to high costs, limiting the strength of conclusions regarding microbial changes. Finally, lean mass composition was not directly assessed, although depot weights strongly support adipose expansion. Conclusions: In this model of pre-established obesity and hyperandrogenism, butyrate supplementation was not metabolically protective. Instead, it was associated with worsened adipose inflammation, altered adipokine profiles, and impaired glucose regulation. These findings highlight the context-dependent nature of butyrate’s effects and caution against its generalization as a universally beneficial metabolic modulator.