The number of known proteins associated with plant lipid droplets (LDs) is small compared with other organelles. Many aspects of LD biosynthesis and degradation are unknown, and identifying and characterizing candidate LD proteins could help elucidate these processes. Here, we analyzed the proteome of LD-enriched fractions isolated from tobacco (Nicotiana tabacum) pollen tubes. Proteins that were highly enriched in comparison with the total or cytosolic fraction were further tested for LD localization via transient expression in pollen tubes. One of these proteins, PLANT UBX DOMAIN-CONTAINING PROTEIN10 (PUX10), is a member of the plant UBX domain-containing (PUX) protein family. This protein localizes to LDs via a unique hydrophobic polypeptide sequence and can recruit the AAA-type ATPase CELL DIVISION CYCLE48 (CDC48) protein via its UBX domain. PUX10 is conserved in Arabidopsis thaliana and expressed in embryos, pollen tubes, and seedlings. In pux10 knockout mutants in Arabidopsis, LD size is significantly increased. Proteomic analysis of pux10 mutants revealed a delayed degradation of known LD proteins, some of which possessed ubiquitination sites. We propose that PUX10 is involved in a protein degradation pathway at LDs, mediating an interaction between polyubiquitinated proteins targeted for degradation and downstream effectors such as CDC48.
Context Cold exposure (CE) has been shown to enhance energy expenditure by activating brown adipose tissue thermogenesis and metabolism in humans. However, it remains to be elucidated if there are gender-specific differences in cold-induced thermogenesis and metabolism. Objective To study the impact of mild CE on resting energy expenditure (REE) and metabolism in males compared with females. Setting A cross-sectional study. Participants 117 healthy young Caucasians participated in this study (58 males). Mean age was 25.1 ± 3.6 years and mean body mass index 22.3 ± 1.7 kg/m2. Intervention Participants underwent a short-term CE using water perfused mattresses to activate nonshivering thermogenesis. Main Outcome Measures REE was assessed before and 2 hours after CE followed by blood sampling. Selected metabolites and hormones were measured. Skin temperatures were monitored at various sites throughout the experiment. Results Participants showed a significant increase in REE after CE (6.5%, P < .001). This increase did not differ between genders (P = .908). However, there were differences between males and females in changes of plasma glucose (–5.1% versus –7.4%, P = .024), leptin (–14.3% versus –30.1%, P < .001) and adiponectin (5.4% versus 12.8%, P = .018) after CE. We observed a significant decrease of the supraclavicular skin temperature in men (–0.3%, P = .034), but not in women (0.3%, P = .326)(P = .019 between genders). Conclusions We did not observe a difference in the thermogenic response, measured as change of REE, to CE in women compared with men. However, we found that some metabolic and hormonal changes were more pronounced in women than in men suggesting a gender-specific response to cold.
ObjectiveObesity and type-2 diabetes (T2D) are metabolic diseases that represent a critical health problem worldwide. Metabolic disease is differentially associated with fat distribution, while visceral white adipose tissue (VAT) is particularly prone to obesity-associated inflammation. Next to their canonical function of immune suppression, regulatory T cells (Tregs) are key in controlling adipose tissue homeostasis. Towards understanding the molecular underpinnings of metabolic disease, we focus on how environmental-metabolic stimuli impinge on the functional interplay between Tregs and adipose tissue. Here, cold exposure or beta3-adrenergic signaling are a promising tool to increase energy expenditure by activating brown adipose tissue, as well as by reducing local inflammation within fat depots by supporting immunosuppressive Tregs. However, in humans, the underlying mechanisms that enable the environmental-immune crosstalk in the periphery and in the respective tissue remain currently unknown.MethodsWe used combinatorial approaches of next generation humanized mouse models and in vitro and in vivo experiments together with beta3-adrenergic stimulation to dissect the underlying mechanisms of human Treg induction exposed to environmental stimuli such as cold. To test the translational relevance of our findings, we analyzed samples from the FREECE study in which human subjects were exposed to individualized cooling protocols. Samples were analyzed ex vivo and after in vitro Treg induction using qRT-PCR, immunofluorescence, as well as with multicolor flow cytometry and cell sorting.ResultsIn vivo application of the beta3-adrenergic receptor agonist mirabegron in humanized mice induced thermogenesis and improved the Treg induction capacity of naïve T cells isolated from these animals. Using samples from the human FREECE study, we demonstrate that a short-term cold stimulus supports human Treg induction in vitro and in vivo. Mechanistically, we identify BORCS6 encoding the Ragulator-interacting protein C17orf59 to be significantly induced in human CD4+ T cells upon short-term cold exposure. Strong mTOR signaling is known to limit successful Treg induction and thus likely by interfering with mTOR activation at lysosomal surfaces, C17orf59 improves the Treg induction capacity of human naïve T cells upon cold exposure.ConclusionsThese novel insights into the molecular underpinnings of human Treg induction suggest an important role of Tregs in linking environmental stimuli with adipose tissue function and metabolic diseases. Moreover, these discoveries shed new light on potential approaches towards tailored anti-inflammatory concepts that support human adipose tissue homeostasis by enabling Tregs.
Activation of brown adipose tissue may increase energy expenditure by non-shivering thermogenesis. Cold exposure is one of the options to activate brown adipocytes. To link changes in energy metabolism with microRNA expression (miRNAs), we analyzed 158 miRNAs in serum of 169 healthy individuals before and after cold exposure. Validating the results of a miRNA array, a significant down-regulation of miR-375 after cold exposure (P < 0.0001) was detected. These changes went along with a significant negative correlation between miR-375 and visceral adipose tissue (VAT) mass (P < 0.0001), implicating a specific function of miR-375 in this depot. Significantly higher expression levels of miR-375 were found in VAT in comparison to subcutaneous fat (SAT). Using in silico prediction, we identified putative miR-375 target genes involved in the thermogenesis pathway. Cold-stimulation of subcutaneous and visceral pre-adipocytes (PACs) led to significantly higher expression levels of FABP4, FGF21, PPARGC1A and PRDM16 in VC-PACs. Analyzing miR-375 knock down and cold stimulated VC-PACs revealed a significant up-regulation of thermogenesis associated genes PPARGC1A, ELOVL3 and PRDM16. In summary, our findings identified miR-375 as a potential adipogenic and thermogenesis-associated miRNA exclusively acting in visceral adipose tissue.
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