Helen Budge

Therapeutic activation of thermogenic brown adipose tissue (BAT) may be feasible to prevent, or treat, cardiometabolic disease. However, rodents are commonly housed below thermoneutrality (~20 °C) which can modulate their metabolism and physiology including the hyperactivation of brown (BAT) and beige white adipose tissue. We housed animals at thermoneutrality from weaning to chronically supress BAT, mimic human physiology and explore the efficacy of chronic, mild cold exposure (20 °C) and β3-adrenoreceptor agonism (YM-178) under these conditions. Using metabolic phenotyping and exploratory proteomics we show that transfer from 28 °C to 20 °C drives weight gain and a 125% increase in subcutaneous fat mass, an effect not seen with YM-178 administration, thus suggesting a direct effect of a cool ambient temperature in promoting weight gain and further adiposity in obese rats. Following chronic suppression of BAT, uncoupling protein 1 mRNA was undetectable in the subcutaneous inguinal white adipose tissue (IWAT) in all groups. Using exploratory adipose tissue proteomics, we reveal novel gene ontology terms associated with cold-induced weight gain in BAT and IWAT whilst Reactome pathway analysis highlights the regulation of mitotic (i.e., G2/M transition) and metabolism of amino acids and derivatives pathways. Conversely, YM-178 had minimal metabolic-related effects but modified pathways involved in proteolysis (i.e., eukaryotic translation initiation) and RNA surveillance across both tissues. Taken together these findings are indicative of a novel mechanism whereby animals increase body weight and fat mass following chronic suppression of adaptive thermogenesis from weaning. In addition, treatment with a B3-adrenoreceptor agonist did not improve metabolic health in obese animals raised at thermoneutrality.

Objective: The interaction between thyroid status and brown adipose tissue (BAT) activation is complex. We assessed the effect of autoimmune hypothyroidism (ATD) in female children on BAT activation, measured using infrared thermography.Design: Twenty-six female participants (14 with ATD and 12 healthy controls) between 5 and 17 years of age attended a single study session. Thermal images were taken of the supraclavicular region before, and after, the introduction of a cool stimulus.Results: Participants with ATD had lower resting (hypothyroid, 34.9 ± 0.7°C; control, 35.4 ± 0.5°C; P = 0.03) and stimulated (hypothyroid, 35.0 ± 0.6°C; control, 35.5 ± 0.5°C; P = 0.04) supraclavicular temperatures compared with controls, but there was no difference between groups in the temperature increase with stimulation. BAT activation, calculated as the relative temperature change comparing the supraclavicular temperature to a sternal reference region, was reduced in participants with ATD (hypothyroid, 0.1 ± 0.1°C; control, 0.2 ± 0.2°C; P = 0.04). Children with ATD were frequently biochemically euthyroid due to replacement therapy, but, despite this, increased relative supraclavicular temperature was closely associated with increased TSH (r = 0.7, P = 0.01) concentrations.Conclusions: Girls with ATD had an attenuated thermogenic response to cold stimulation compared with healthy controls, but, contrary to expectation, those with suboptimal biochemical control (with higher TSH) showed increased BAT activation. This suggests that the underlying disease process may have a negative effect on BAT response, but high levels of TSH can mitigate, and even stimulate, BAT activity. In summary, thyroid status is a complex determinant of BAT activity in girls with ATD.

Therapeutic activation of thermogenic brown adipose tissue (BAT) may be feasible to prevent, or treat, cardiometabolic disease. However, rodents are commonly housed below thermoneutrality (∼20°C) which can modulate their metabolism and physiology including the hyperactivation of brown (BAT) and beige white adipose tissue. We housed animals at thermoneutrality from weaning to chronically supress BAT, mimic human physiology and explore the efficacy of chronic, mild cold-exposure and β3-adrenoreceptor agonism under these conditions. Using metabolic phenotyping and exploratory proteomics we show that transfer from 28°C to 20°C drives weight gain and a 125% increase in subcutaneous fat mass, an effect not seen with YM-178 administration thus suggesting a direct effect of a cool ambient temperature in promoting weight gain and further adiposity in obese rats. Following chronic suppression of BAT, uncoupling protein 1 mRNA was undetectable in IWAT in all groups. Using exploratory adipose tissue proteomics, we reveal novel gene ontology terms associated with cold-induced weight gain in BAT and IWAT whilst Reactome pathway analysis highlights the regulation of mitotic (i.e. G2/M transition) and metabolism of amino acids and derivatives pathways. Conversely, YM-178 had minimal metabolic-related effects but modified pathways involved in proteolysis (i.e. eukaryotic translation initiation) and RNA surveillance across both tissues. Taken together these findings are indicative of a novel mechanism whereby animals increase body weight and fat mass following chronic suppression of adaptive thermogenesis from weaning. In addition, treatment with a B3-adrenoreceptor agonist did not improve metabolic health in obese animals raised at thermoneutrality.

Background and aim: Exercise training elicits diverse effects on brown (BAT) and white adipose tissue (WAT) physiology in rodents. However, these animals are typically housed below their thermoneutral zone (i.e. 28-32°C). In these conditions, BAT is chronically hyperactive and, unlike human residence, closer to thermoneutrality. Therefore, we set out to determine the effects of exercise training in obese animals at 28°C (i.e. thermoneutrality) on BAT and WAT in its basal (i.e. inactive) state. Methods: Sprague-Dawley rats (n=12) were housed at thermoneutrality from 3 weeks of age and fed a high-fat diet (HFD). At 12 weeks of age half these animals were randomised to 4-weeks of exercise exercise training, i.e. swim-training (1 hour/day, 5 days per week). Metabolic assessment was undertaken during the final 48h and was followed by interscapular and perivascular BAT and inguinal (I)WAT sampling for the analysis of thermogenic genes and the proteome. Results: Exercise attenuated weight gain but did not affect fat mass or general metabolic parameters (i.e. fasting insulin and glucose). Interestingly, although BAT mass was increased, there was no change in thermogenic gene expression. Differentially regulated proteins in BAT enriched gene ontology (GO) terms including 2-oxoglutarate metabolic process, cytochrome-c activity and mitochondrial respiratory chain complex IV. This was accompanied by an upregulation of multiple proteins and GO terms involved in skeletal muscle physiology suggesting an adipocyte to myocyte switch in BAT. UCP1 mRNA was undetectable in IWAT despite an increase in classical ‘browning’ markers (i.e. PGC1a and ADRB3) with exercise. Enriched GO terms in IWAT included DNA binding and positive regulation of apoptosis. Impact analysis highlighted carbon metabolism and OXPHOS pathways were regulated by exercise in BAT whilst HIF-1 signalling and cytokine-cytokine receptor interaction were among those modified in IWAT. Conclusion: Exercise training reduces weight gain in obese animals at thermoneutrality and is accompanied by an oxidative, myogenic signature in BAT, rather than induction of thermogenic genes. This may represent a new, UCP1-independent pathway through which BAT regulates body weight at thermoneutrality.

Brown adipose tissue (BAT) function may depend on its anatomical location and developmental origin. Interscapular BAT (iBAT) regulates acute macronutrient metabolism, whilst perivascular BAT (PVAT) regulates vascular function. Although phenotypically similar, whether these depots respond differently to acute nutrient excess is unclear. Given their distinct anatomical locations and developmental origins and we hypothesised that iBAT and PVAT would respond differently to brief period of nutrient excess. Sprague-Dawley rats aged 12 weeks (n=12) were fed either a standard (10% fat, n=6) or high fat diet (HFD: 45% fat, n=6) for 72h and housed at thermoneutrality. Following an assessment of whole body physiology, fat was collected from both depots for analysis of gene expression and the proteome. HFD consumption for 72h induced rapid weight gain (c. 2.6%) and reduced serum non-esterified fatty acids (NEFA) with no change in either total adipose or depot mass. In iBAT, an upregulation of genes involved in insulin signalling and lipid metabolism was accompanied by enrichment of lipid-related processes and functions, plus glucagon and peroxisome proliferator-activated receptor (PPAR) signalling pathways. In PVAT, HFD induced a pronounced down-regulation of multiple metabolic pathways which was accompanied with increased abundance of proteins involved in apoptosis (e.g., Hdgf and Ywaq) and toll-like receptor signalling (Ube2n). There was also an enrichment of DNA-related processes and functions (e.g., nucleosome assembly and histone exchange) and RNA degradation and cell adhesion pathways. In conclusion, we show that iBAT and PVAT elicit divergent responses to short-term nutrient excess highlighting early adaptations in these depots before changes in fat mass.

Dietary methyl donors, including folate, may modify the placenta and size at birth but the influence of maternal body weight has not been widely investigated. We therefore examined whether maternal or fetal folate status, together with indices of placental folate transport, were modulated by either maternal pre-pregnancy body mass index (BMI i.e., overweight: 25 ≤ BMI < 30 or obesity: BMI ≥ 30 kg/m²) and/or gestational diabetes mellitus (GD). We utilised a sub-sample of 135 pregnant women participating in the Spanish PREOBE survey for our analysis (i.e., 59 healthy normal weight, 29 overweight, 22 obese and 25 GD). They were blood sampled at 34 weeks gestation, and, at delivery, when a placental sample was taken together with maternal and cord blood. Placental gene expression of folate transporters and DNA methyltransferases (DNMT) were all measured. Folate plasma concentrations were determined with an electro-chemiluminescence immunoassay. Food diaries indicated that folate intake was unaffected by BMI or GD and, although all women maintained normal folate concentrations (i.e., 5–16 ng/mL), higher BMIs were associated with reduced maternal folate concentrations at delivery. Umbilical cord folate was not different, reflecting an increased concentration gradient between the mother and her fetus. Placental mRNA abundance for the folate receptor alpha (FOLR1) was reduced with obesity, whilst DNMT1 was increased with raised BMI, responses that were unaffected by GD. Multi-regression analysis to determine the best predictors for placental FOLR1 indicated that pre-gestational BMI had the greatest influence. In conclusion, the placenta’s capacity to maintain fetal folate supply was not compromised by either obesity or GD.

Background: Therapeutic activation of thermogenic brown adipose tissue (BAT) is a potential strategy to prevent obesity and metabolic disease in humans. However, it is now recognised that rodent studies examining BAT physiology are carried out at sub-thermoneutral temperatures (e.g. ~20°C), and are not translationally relevant to humans as BAT is ‘hyperactive’. Therefore, the aim of this study was to determine the effect of common regulators of BAT metabolism when animals were raised at thermoneutrality (28°C). Methods: Thirty weanling Sprague-Dawley rats were housed at thermoneutrality (28°C) and randomised to either chow (C, n=6) or a high-fat diet (HFD, n=24) from 3-weeks of age. At 12 weeks, subgroups (n=6) of HFD were randomised to either mild-cold exposure (20°C), Mirabegron, a selective β3-agonist (0.75 mk/kg per day) or exercise training (1 h/d, 5 d/week). Metabolic assessment was undertaken in CLAMS during the last 48 h to assess energy intake (EI), expenditure (EE) and physical activity (PA) in addition to the acute response to administration of Mirabegron. Key thermogenic and metabolic genes were analysed in interscapular BAT by qPCR in addition to targeted insulin resistance PCR Arrays (86 key genes, n=3). Results: No interventions reduced body weight or fat mass. There was no difference in 24 h EE, EI or PA between groups. Key thermogenic genes in BAT were unchanged by the interventions. CITED1 expression was upregulated by HFD and reduced by all interventions whilst PRDM16 expression was reduced by HFD and increased by exercise. Similarly, expression of PPARA, mTOR and the ‘beige’ marker TBX1 were upregulated by exercise only. Targeted PCR arrays demonstrated an upregulation of inflammatory markers e.g. TLR4, EMR1, CASP1 and IL18R1 and a downregulation of metabolic genes e.g. SCD1, FASN, ACACB, HK2 with HFD. Only FASN, SCD1 and ACACA were upregulated in Cold whilst IL18R1, TLR4 and EMR1 were downregulated. Similarly, β3 increased FASN whilst downregulating IL18R1, IL6 and STAT3. Finally, Exercise upregulated FASN, SCD1, HK, ACACA, ACACB and PDK2 whilst downregulating NLRP3, IL1β and PYCARD. Conclusion: Whilst there is an intervention specific effect on immune genes in BAT we show there is no consistent upregulation of thermogenic genes in response to common stimuli when animals are raised at thermoneutrality. Effects of interventions to activate BAT carried out at sub-thermoneutrality are most likely to be a consequence of chronic mild-cold stress and are unlikely to be translated to humans.