Intervention Approaches in Studying the Response to Vitamin D3 Supplementation.

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.

Increasing energy expenditure through exercise and low ambient temperature offers oxidative protection to the hypothalamus after high-fat feeding to mice.

The effects of weight loss produced by increased energy expenditure on measures of oxidative stress and mitochondrial damage have not been investigated in the hypothalamus of diet-induced obese mice. The present study aimed to characterize the effects of either a low housing temperature of 17°C or daily exercise on a treadmill on high-fat diet (HFD)-induced abnormalities in the hypothalamic tissue of mice. Exercise and low ambient temperature protocols were designed to produce energy deficit through increased energy expenditure. Forty mice aged 8 weeks were assigned to one of four conditions: chow diet (n = 10), HFD (n = 10), HFD and 5 weeks of either exercise training (ET; n = 10) or an ambient temperature of 17°C (n = 10). Mice were killed at the age of 31 weeks. In comparison with HFD treatment alone, both interventions reduced body adiposity (14.6% and 27.6% reduction for the ET and 17°C groups, respectively). Moreover, exposing obese mice to ET and 17°C restored mitochondrial DNA content (41.3% and 32.6% increase for the ET and 17°C groups, respectively), decreased level of lipid peroxidation as assessed by the detection of 4-hydroxy-nonenal protein adducts (12.8% and 29.4% reduction for the ET and 17°C groups, respectively) and normalized the expression levels of proinflammatory cytokines (Tnfα: 73.9% and 62%; Il1ß: 54.5% and 39.6%; Il6: 33.1% and 35.6% reduction for the ET and 17°C groups, respectively), as well as several proteins associated with mitochondrial respiratory chain (OxPhos Complex I: 75.7% and 53.9%; Complex III: 33% and 36%; Complex V: 42% and 36.9% reduction for the ET and 17°C groups, respectively) in hypothalamic cells. Negative energy balance induced through either lower ambient temperature or exercise resulted in substantial and similar improvements in markers of inflammation and mitochondrial damage in the hypothalamus of mice with diet-induced obesity, potentially by reducing oxidative stress.

Isolation and identification of endogenous RFamide-related peptides 1 and 3 in the mouse hypothalamus.

Although the RFamide-related peptide (RFRP) preproprotein sequence is known in mice, until now, the molecular structure of the mature, functional peptides processed from the target precursor molecule has not been determined. In the present study, we purified endogenous RFRP1 and RFRP3 peptides from mouse hypothalamic tissue extracts using an immunoaffinity column conjugated with specific antibodies against the mouse C-terminus of RFRP-1 and RFRP-3. Employing liquid chromatography coupled with mass spectrometry, we demonstrated that RFRP1 consists of 15 amino acid residues and RFRP3 consists of 10 amino acid residues (ANKVPHSAANLPLRF-NH2 and SHFPSLPQRF-NH2, respectively). To investigate the distribution of RFRPs in the mouse central nervous system, we performed immunohistochemical staining of the brain sections collected from wild-type and Rfrp knockout animals. These data, together with gene expression in multiple tissues, provide strong confidence that RFRP-immunoreactive neuronal cells are localised in the dorsomedial hypothalamic nucleus (DMH) and between the DMH and the ventromedial hypothalamic nuclei. The identification of RFRP1 and RFRP3 peptides and immunohistochemical visualisation of targeting RFRPs neurones in the mice brain provide the basis for further investigations of the functional biology of RFRPs.