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.

Mitochondrial turnover: a phenotype distinguishing brown adipocytes from interscapular brown adipose tissue and white adipose tissue.

To determine the differences between brown adipocytes from interscapular brown tissue (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 °C to 4 °C and the quantitative relationship between UCP1 and selected subunits of mitochondrial respiratory complex 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 inguinal WAT (ingWAT); the former showed minimal changes in protein content, whereas the latter showed 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 °C 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 ∼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 respiratory complex 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.

Binding studies of truncated variants of the Aß peptide to the V-domain of the RAGE receptor reveal Aß residues responsible for binding.

Alzheimer's disease (AD) symptoms correlate with the concentration of soluble, although not necessarily monomeric forms of Aß peptide in the brain parenchyma. The RAGE receptor has been implicated as the protein responsible for active transport of Aß from blood circulation to the brain. In murine models of AD, inhibition of the Aß:RAGE interaction decreases the levels of Aß in the brain. Inhibition of the Aß:RAGE interaction would be a promising alternative for the therapy of AD. Rational design of an Aß:RAGE interaction blocker requires detailed knowledge of the structure of the complex. However, the binding domain of RAGE is natively unfolded in physiological conditions, which severely hampers the application of classic methods of protein structure analysis to the design of an antagonist. Here, alternative methods are used to characterize the structural properties of the RAGE-ligand binding domain and to monitor the binding of a series of truncated variants of Aß. Using intrinsic RAGE tryptophan fluorescence and mass spectrometry of non-covalent protein-ligand complexes we have identified shorter versions of Aß that bind to the RAGE V-domain. We have also shown in cell culture experiments that a selected shortened version of Aß effectively inhibits full-length Aß, RAGE-mediated, cell uptake. Thus, a truncated version of Aß capable of blocking its receptor-mediated internalization was established, revealing the binding code and providing the lead compound in the process of drug design.

Intertwined structured and unstructured regions of exRAGE identified by monitoring hydrogen-deuterium exchange.

Receptor for advanced glycation end products (RAGE) is a multiligand receptor that is engaged in many pathological processes. Potentially beneficial modification of its activity requires sound knowledge of its structural properties. However, up to now, only the structures of its separated domains have been published or deposited in databases. In this work, we used hydrogen-deuterium exchange and mass spectrometry to gain insight into the structural properties of exRAGE (extracellular region of RAGE)--the full extracellular part of the protein. The present work indicates the common and disparate features of full exRAGE as compared to the structural models of its separate domains. The highlight of the present study is the contrasting behavior of the different regions of the protein, with the protected regions neighboring fully exposed parts especially in the N-terminal V domain.