Ketone bodies (KBs) are an alternative energy source under starvation and play multiple roles as signaling molecules regulating energy and metabolic homeostasis. The mechanism by which KBs influence visceral white adipose tissue physiology is only partially known, and our study aimed to shed light on the effects they exert on such tissue. To this aim, we administered 1,3-butanediol (BD) to rats since it rapidly enhances β-hydroxybutyrate serum levels, and we evaluated the effect it induces within 3 h or after 14 days of treatment. After 14 days of treatment, rats showed a decrease in body weight gain, energy intake, gonadal-WAT (gWAT) weight, and adipocyte size compared to the control. BD exerted a pronounced antioxidant effect and directed redox homeostasis toward reductive stress, already evident within 3 h after its administration. BD lowered tissue ROS levels and oxidative damage to lipids and proteins and enhanced tissue soluble and enzymatic antioxidant capacity as well as nuclear erythroid factor-2 protein levels. BD also reduced specific mitochondrial maximal oxidative capacity and induced endoplasmic reticulum stress as well as interrelated processes, leading to changes in the level of adipokines/cytokines involved in inflammation, macrophage infiltration into gWAT, adipocyte differentiation, and lipolysis.

Brown adipose tissue (BAT) participates in thermogenesis and energy homeostasis. Studies on factors capable of influencing BAT function, such as a high-fat diet (HFD) or exposure to environmental pollutants, could be useful for finding metabolic targets for maintaining energy homeostasis. We evaluated the effect of chronic exposure to dichlorodiphenyldichloroethylene (DDE), the major metabolite of dichlorodiphenyltrichloroethane (DDT), and/or a HFD on BAT morphology, mitochondrial mass, dynamics, and oxidative stress in rats. To this end, male Wistar rats were treated for 4 weeks with a standard diet, or a HFD alone, or together with DDE. An increase in paucilocular adipocytes and the lipid droplet size were observed in HFD-treated rats, which was associated with a reduction in mitochondrial mass and in mitochondrial fragmentation, as well as with increased oxidative stress and upregulation of the superoxide dismutase-2. DDE administration mimics most of the effects induced by a HFD on BAT, and it aggravates the increase in the lipid droplet size when administered together with a HFD. Considering the known role of oxidative stress in altering BAT functionality, it could underlie the ability of both DDE and a HFD to induce similar metabolic adaptations in BAT, leading to reduced tissue thermogenesis, which can result in a predisposition to the onset of energy homeostasis disorders.

Combination therapy has shown promise in treating aggressive cancers by using several drugs simultaneously to target different biological pathways, with the added benefit of potentially reducing toxicity. Given the critical role of mitochondrial dysfunction in tumor progression, targeting mitochondrial metabolism represents a promising therapeutic avenue. In this study, we developed a mitochondria-targeted nanofiber based on self-assembling peptides, engineered to co-deliver two complementary therapeutic agents for glioblastoma treatment. The nanofiber carries 1,3-bromopyruvate (BrP), a glycolysis inhibitor, and temozolomide, an alkylating chemotherapeutic, conjugated via a matrix metalloproteinase-9 (MMP-9)-responsive linker for controlled, on-demand release. To enhance selectivity for glioblastoma cells, the nanofiber surface was functionalized with the targeting peptide falGea binding specifically to EGFRvIII, commonly overexpressed in tumor cells, and gH625, a cell-penetrating peptide known to facilitate the blood-brain barrier (BBB) transport. The nanofibers were comprehensively characterized for their aggregation behavior, structural stability, and morphology. Mitochondrial targeting and functional effects were evaluated by using isolated rat brain mitochondria. Therapeutic efficacy was assessed in U-87 MG glioblastoma cells cultured in both 2D and 3D systems. Additionally, BBB permeability was examined by using a dynamic 3D in vitro BBB model, demonstrating the transport-enhancing role of gH625. These findings support the potential of multifunctional, mitochondria-targeted nanofibers as an effective platform for glioblastoma therapy, offering both precision targeting and enhanced drug delivery across the BBB.

Ketogenic diet has a wide range of beneficial effects but presents practical limitations due to its low compliance, hence dietary supplements have been developed to induce ketosis without nutrient deprivation. The alcohol 1,3-butanediol (BD) is a promising molecule for its ability to induce ketosis, but its effects on brain have been investigated so far only in disease models, but never in physiological conditions. To support BD use to preserve brain health, the analysis of its activity is mandatory. Therefore, we investigated, in healthy rats, the effect of a fourteen-days BD-administration on the hippocampus, an area particularly vulnerable to oxidative and inflammatory damage. Since BD treatment has been reported to reduce energy intake, results were compared with those obtained from rats undergoing a restricted dietary regimen, isoenergetic with BD group (pair fed, PF). Reduced pro-inflammatory signaling pathways and glial activation were revealed in hippocampus of BD treated rats in comparison to control (C) and PF groups. ROS content and the extent of protein oxidative damage were lower in BD and PF groups than in C. Interestingly, higher amounts of nuclear factor erythroid 2-related factor 2 (Nrf2), decreased level of lipid hydroperoxides, lower susceptibility to oxidative insult, higher amounts of superoxide dismutase-2, glutathione reductase and glutathione peroxidase (GPx), and increased GPx activity were observed in BD animals. BD administration, but not dietary restriction, attenuated endoplasmic reticulum stress, reduced autophagic response activation, and was associated with an increase of both the neurotrophin BDNF and pre-synaptic proteins synaptophysin and synaptotagmin. Our results highlight that BD plays a neuroprotective role in healthy conditions, thus emerging as an effective strategy to support brain function without the need of implementing ketogenic nutritional interventions.

Some studies indicate that brown adipose tissue (BAT) represents a promising target in the fight against dysmetabolic diseases, with indications suggesting it as a potential target for the effects of ketone bodies. We investigate whether the elevation of plasma levels of the ketone body β‐hydroxybutyrate, achieved through the in vivo administration of its precursor 1,3‐butanediol (BD) to rats, could impact interscapular BAT (iBAT) mitochondrial biochemistry and functionality. We examined the effects induced by BD within 3 h and after 2 weeks of treatment. A large‐scale quantitative proteomics approach, coupling liquid chromatography with tandem mass spectrometry (LC–MS/MS) analysis, and Western blot associated with functional studies by respirometry allowed us to evaluate the changes in iBAT mitochondrial protein expression and bioenergetics induced by BD. BD administration increased β‐hydroxybutyrate plasma levels, which correlated with an enhancement in iBAT mitochondrial respiration rate, likely due to the activation of the respiratory chain and uncoupling protein‐1. The proteomic analysis demonstrated that BD influenced the mitochondrial levels of specific subunits belonging to the five respiratory complexes, uncoupling protein‐1, and proteins involved in propanoate metabolism. BD administration also induced lysine β‐hydroxybutyrylation of mitochondrial proteins, including specific subunits of the respiratory chain complexes and uncoupling protein‐1. Most of the BD‐induced effects were observed within 3 h of its administration and persisted/increased after 2 weeks of treatment. In conclusion, by using BD to increase β‐hydroxybutyrate levels, we provide evidence supporting the role of β‐hydroxybutyrate as a signaling molecule capable of rapidly modulating BAT physiology by acting at the mitochondrial level.

Calorie restriction (CR) without malnutrition improves metabolic health, slows the aging process, and prolongs longevity. Brown adipose tissue (BAT), which specializes in burning energy to produce heat, also contributes to metabolic health; however, the effects of CR on BAT function are only partially understood, and even less is known about those exerted by mild CR for a short period of time. We studied the multifaceted relationship between mild short‐term CR (15% applied for 2 weeks) and intrascapular BAT (iBAT) in rats and provided insights into the adaptations occurring in the tissue through multiple complementary approaches: histology, mitochondrial respirometry, comprehensive quantitative proteomics, and targeted metabolomics. Our data indicate that mild short‐term CR induces structural and metabolic adaptations in iBAT aimed at the deposition of triglycerides within the adipocyte, which appear paucilocular. CR increases the levels of enzymes involved in glycolysis while simultaneously reducing glycolytic intermediate metabolites. It also improves the levels of enzymes involved in de novo lipogenesis, in providing reducing power for lipid synthesis, and in lipid droplet dynamics. Although CR reduced mitochondrial iBAT content, tissue thermogenic potential was maintained, as it increased mitochondrial activity, compensating for their lower abundance. Overall, our data indicate that mild CR, within 2 weeks, affects BAT metabolism, inducing physiological adaptation that preserves its thermogenic capacity under conditions of energy deficit. These adaptations may represent a key mechanism underlying the systemic benefits of CR, offering new insights into the role of even mild and short‐term CR in maintaining overall health.