To determine the differences between brown adipocytes from iBAT and those induced in white adipose tissue (WAT) with respect to their thermogenic capacity, we examined two essential characteristics: the dynamics of mitochondrial turnover during reversible transitions from 29° to 4°C, and the quantitative relationship between UCP1 and selected subunits of mitochondrial respiratory complex (RC) in the fully recruited state. To follow the kinetics of induction and involution of mitochondria, we determined the expression pattern of UCP1 and other mitochondrial proteins, as well as analyzed mtDNA content, after cold stimulation and reacclimation to thermoneutrality. We showed that UCP1 turnover is very different in iBAT and ingWAT; the former showed minimal changes in protein content, while the latter major changes. Similarly, in iBAT both mtDNA content and the expression of mitochondrial proteins were stable and expressed at similar levels during reversible transitions from 29° to 4°C, whereas ingWAT revealed dynamic changes. Further analysis showed that in iBAT, the expression patterns for UCP1 and other mitochondrial proteins resembled each other, whereas in ingWAT, UCP1 varied about 100-fold during the transition from cold to warmth, and no other mitochondrial proteins matched UCP1. In turn, quantitative analysis of thermogenic capacity determined by estimating the proportion of UCP1 to RC components, showed no significant differences between brown and brite adipocytes, suggesting similar thermogenic potentiality. Our results indicate that dynamics of brown adipocytes turnover during reversible transition from warm to cold may determine the thermogenic capacity of an individual in a changing temperature environment.

Vitamin D intervention studies are designed to evaluate the impact of the micronutrient vitamin D₃ on health and disease. The appropriate design of studies is essential for their quality, successful execution, and interpretation. Randomized controlled trials (RCTs) are considered the “gold standard” for intervention studies. However, the most recent large-scale (up to 25,000 participants), long-term RCTs involving vitamin D₃ did not provide any statistically significant primary results. This may be because they are designed similarly to RCTs of a therapeutic drug but not of a nutritional compound and that only a limited set of parameters per individual were determined. We propose an alternative concept using the segregation of study participants into different groups of responsiveness to vitamin D₃ supplementation and in parallel measuring a larger set of genome-wide parameters over multiple time points. This is in accordance with recently developed mechanistic modeling approaches that do not require a large number of study participants, as in the case of statistical modeling of the results of a RCT. Our experience is based on the vitamin D intervention trials VitDmet, VitDbol, and VitDHiD, which allowed us to distinguish the study participants into high, mid, and low vitamin D responders. In particular, investigating the vulnerable group of low vitamin D responders will provide future studies with more conclusive results both on the clinical and molecular benefits of vitamin D₃ supplementation. In conclusion, our approach suggests a paradigm shift towards detailed investigations of transcriptome and epigenome-wide parameters of a limited set of individuals, who, due to a longitudinal design, can act as their own controls.