Functional evolution of leptin of Ochotona curzoniae in adaptive thermogenesis driven by cold environmental stress

Background

Environmental stress can accelerate the directional selection and evolutionary rate of specific stress-response proteins to bring about new or altered functions, enhancing an organism''s fitness to challenging environments. Plateau pika (Ochotona curzoniae), an endemic and keystone species on Qinghai-Tibetan Plateau, is a high hypoxia and low temperature tolerant mammal with high resting metabolic rate and non-shivering thermogenesis to cope in this harsh plateau environment. Leptin is a key hormone related to how these animals regulate energy homeostasis. Previous molecular evolutionary analysis helped to generate the hypothesis that adaptive evolution of plateau pika leptin may be driven by cold stress.

Methodology/Principal Findings

To test the hypothesis, recombinant pika leptin was first purified. The thermogenic characteristics of C57BL/6J mice injected with pika leptin under warm (23±1°C) and cold (5±1°C) acclimation is investigated. Expression levels of genes regulating adaptive thermogenesis in brown adipose tissue and the hypothalamus are compared between pika leptin and human leptin treatment, suggesting that pika leptin has adaptively and functionally evolved. Our results show that pika leptin regulates energy homeostasis via reduced food intake and increased energy expenditure under both warm and cold conditions. Compared with human leptin, pika leptin demonstrates a superior induced capacity for adaptive thermogenesis, which is reflected in a more enhanced β-oxidation, mitochondrial biogenesis and heat production. Moreover, leptin treatment combined with cold stimulation has a significant synergistic effect on adaptive thermogenesis, more so than is observed with a single cold exposure or single leptin treatment.

Conclusions/Significance

These findings support the hypothesis that cold stress has driven the functional evolution of plateau pika leptin as an ecological adaptation to the Qinghai-Tibetan Plateau.     

Brown adipose tissue in morbidly obese subjects

Background

Cold-stimulated adaptive thermogenesis in brown adipose tissue (BAT) to increase energy expenditure is suggested as a possible therapeutic target for the treatment of obesity. We have recently shown high prevalence of BAT in adult humans, which was inversely related to body mass index (BMI) and body fat percentage (BF%), suggesting that obesity is associated with lower BAT activity. Here, we examined BAT activity in morbidly obese subjects and its role in cold-induced thermogenesis (CIT) after applying a personalized cooling protocol. We hypothesize that morbidly obese subjects show reduced BAT activity upon cold exposure.

Methods and Findings

After applying a personalized cooling protocol for maximal non-shivering conditions, BAT activity was determined using positron-emission tomography and computed tomography (PET-CT). Cold-induced BAT activity was detected in three out of 15 morbidly obese subjects. Combined with results from lean to morbidly obese subjects (n = 39) from previous study, the collective data show a highly significant correlation between BAT activity and body composition (P<0.001), respectively explaining 64% and 60% of the variance in BMI (r = 0.8; P<0.001) and BF% (r = 0.75; P<0.001). Obese individuals demonstrate a blunted CIT combined with low BAT activity. Only in BAT-positive subjects (n = 26) mean energy expenditure was increased significantly upon cold exposure (51.5±6.7 J/s versus 44.0±5.1 J/s, P = 0.001), and the increase was significantly higher compared to BAT-negative subjects (+15.5±8.9% versus +3.6±8.9%, P = 0.001), indicating a role for BAT in CIT in humans.

Conclusions

This study shows that in an extremely large range of body compositions, BAT activity is highly correlated with BMI and BF%. BAT-positive subjects showed higher CIT, indicating that BAT is also in humans involved in adaptive thermogenesis. Increasing BAT activity could be a therapeutic target in (morbid) obesity.     

Frequent Extreme Cold Exposure and Brown Fat and Cold-Induced Thermogenesis: A Study in a Monozygotic Twin

Introduction

Mild cold acclimation is known to increase brown adipose tissue (BAT) activity and cold-induced thermogenesis (CIT) in humans. We here tested the effect of a lifestyle with frequent exposure to extreme cold on BAT and CIT in a Dutch man known as ‘the Iceman’, who has multiple world records in withstanding extreme cold challenges. Furthermore, his monozygotic twin brother who has a ‘normal’ sedentary lifestyle without extreme cold exposures was measured.

Methods

The Iceman (subject A) and his brother (subject B) were studied during mild cold (13°C) and thermoneutral conditions (31°C). Measurements included BAT activity and respiratory muscle activity by [¹⁸F]FDG-PET/CT imaging and energy expenditure through indirect calorimetry. In addition, body temperatures, cardiovascular parameters, skin perfusion, and thermal sensation and comfort were measured. Finally, we determined polymorphisms for uncoupling protein-1 and β3-adrenergic receptor.

Results

Subjects had comparable BAT activity (A: 1144 SUV_total and B: 1325 SUV_total), within the range previously observed in young adult men. They were genotyped with the polymorphism for uncoupling protein-1 (G/G). CIT was relatively high (A: 40.1% and B: 41.9%), but unlike during our previous cold exposure tests in young adult men, here both subjects practiced a g-Tummo like breathing technique, which involves vigorous respiratory muscle activity. This was confirmed by high [¹⁸F]FDG-uptake in respiratory muscle.

Conclusion

No significant differences were found between the two subjects, indicating that a lifestyle with frequent exposures to extreme cold does not seem to affect BAT activity and CIT. In both subjects, BAT was not higher compared to earlier observations, whereas CIT was very high, suggesting that g-Tummo like breathing during cold exposure may cause additional heat production by vigorous isometric respiratory muscle contraction. The results must be interpreted with caution given the low subject number and the fact that both participants practised the g-Tummo like breathing technique.     

High fat diet-induced changes in mouse muscle mitochondrial phospholipids do not impair mitochondrial respiration despite insulin resistance

Background

Type 2 diabetes mellitus and muscle insulin resistance have been associated with reduced capacity of skeletal muscle mitochondria, possibly as a result of increased intake of dietary fat. Here, we examined the hypothesis that a prolonged high-fat diet consumption (HFD) increases the saturation of muscle mitochondrial membrane phospholipids causing impaired mitochondrial oxidative capacity and possibly insulin resistance.

Methodology

C57BL/6J mice were fed an 8-week or 20-week low fat diet (10 kcal%; LFD) or HFD (45 kcal%). Skeletal muscle mitochondria were isolated and fatty acid (FA) composition of skeletal muscle mitochondrial phospholipids was analyzed by thin-layer chromatography followed by GC. High-resolution respirometry was used to assess oxidation of pyruvate and fatty acids by mitochondria. Insulin sensitivity was estimated by HOMA-IR.

Principal Findings

At 8 weeks, mono-unsaturated FA (16∶1n7, 18∶1n7 and 18∶1n9) were decreased (−4.0%, p<0.001), whereas saturated FA (16∶0) were increased (+3.2%, p<0.001) in phospholipids of HFD vs. LFD mitochondria. Interestingly, 20 weeks of HFD descreased mono-unsaturated FA while n-6 poly-unsaturated FA (18∶2n6, 20∶4n6, 22∶5n6) showed a pronounced increase (+4.0%, p<0.001). Despite increased saturation of muscle mitochondrial phospholipids after the 8-week HFD, mitochondrial oxidation of both pyruvate and fatty acids were similar between LFD and HFD mice. After 20 weeks of HFD, the increase in n-6 poly-unsaturated FA was accompanied by enhanced maximal capacity of the electron transport chain (+49%, p = 0.002) and a tendency for increased ADP-stimulated respiration, but only when fuelled by a lipid-derived substrate. Insulin sensitivity in HFD mice was reduced at both 8 and 20 weeks.

Conclusions/Interpretation

Our findings do not support the concept that prolonged HF feeding leads to increased saturation of skeletal muscle mitochondrial phospholipids resulting in a decrease in mitochondrial fat oxidative capacity and (muscle) insulin resistance.     

Liver and muscle in morbid obesity: the interplay of fatty liver and insulin resistance

Introduction

Nonalcoholic fatty liver disease (NAFLD) can be seen as a manifestation of overnutrition. The muscle is a central player in the adaptation to energy overload, and there is an association between fatty-muscle and -liver. We aimed to correlate muscle morphology, mitochondrial function and insulin signaling with NAFLD severity in morbid obese patients.

Methods

Liver and deltoid muscle biopsies were collected during bariatric surgery in NAFLD patients. NAFLD Activity Score and Younossi''s classification for nonalcoholic steatohepatitis (NASH) were applied to liver histology. Muscle evaluation included morphology studies, respiratory chain complex I to IV enzyme assays, and analysis of the insulin signaling cascade. A healthy lean control group was included for muscle morphology and mitochondrial function analyses.

Results

Fifty one NAFLD patients were included of whom 43% had NASH. Intramyocellular lipids (IMCL) were associated with the presence of NASH (OR 12.5, p<0.001), progressive hepatic inflammation (p = 0.029) and fibrosis severity (p = 0.010). There was a trend to an association between IMCL and decreased Akt phosphorylation (p = 0.059), despite no association with insulin resistance. In turn, hepatic steatosis (p = 0.015) and inflammation (p = 0.013) were associated with decreased Akt phosphoryation. Citrate synthase activity was lower in obese patients (p = 0.047) whereas complex I (p = 0.040) and III (p = 0.036) activities were higher, compared with controls. Finally, in obese patients, complex I activity increased with progressive steatosis (p = 0.049) and with a trend with fibrosis severity (p = 0.056).

Conclusions

In morbid obese patients, presence of IMCL associates with NASH and advanced fibrosis. Muscle mitochondrial dysfunction does not appear to be a major driving force contributing to muscle fat accumulation, insulin resistance or liver disease. Importantly, insulin resistance in muscle might occur at a late point in the insulin signaling cascade and be associated with IMCL and NAFLD severity.     

Implications of nonshivering thermogenesis for energy balance regulation in humans

The incidence of the metabolic syndrome has reached epidemic levels in the Western world. With respect to the energy balance, most attention has been given to reducing energy (food) intake. Increasing energy expenditure is an important alternative strategy. Facultative thermogenesis, which is the increase in energy expenditure in response to cold or diet, may be an effective way to affect the energy balance. The recent identification of functional brown adipose tissue (BAT) in adult humans promoted a renewed interest in nonshivering thermogenesis (NST). The purpose of this review is to highlight the recent insight in NST, general aspects of its regulation, the major tissues involved, and its metabolic consequences. Sustainable NST in adult humans amounts to 15% of the average daily energy expenditure. Calculations based on the limited available literature show that BAT thermogenesis can amount to 5% of the basal metabolic rate. It is likely that at least a substantial part of NST can be attributed to BAT, but it is possible that other tissues contribute to NST. Several studies on mitochondrial uncoupling indicate that skeletal muscle is another potential contributor to facultative thermogenesis in humans. The general and synergistic role of the sympathetic nervous system and the thyroid axis in relation to NST is discussed. Finally, perspectives on BAT and skeletal muscle NST are given.     

Adaptive thermogenesis with weight loss in humans

Adaptive thermogenesis (AT) with weight loss refers to underfeeding‐associated fall in resting and non‐resting energy expenditure (REE, non‐REE); this is independent of body weight and body composition. In humans, the existence of AT was inconsistently shown and its clinical significance has been questioned.

Objectives:

Discrepant findings are mainly due to different definitions of AT, the use of various and nonstandardized study protocols, and the limits of accuracy of methods to assess energy expenditure. With controlled underfeeding, AT takes more than 2 wk to develop. AT accounts to an average of 0.5 MJ (or 120 kcal) with a considerable between subject variance.

Design and Methods:

Low‐sympathetic nervous system activity, 3,5,3′‐tri‐iodothyronine (T3) and leptin are likely to add to AT; however, the kinetic changes of their plasma levels with underfeeding differ from the time course of AT and controlled intervention studies substituting and titrating these hormones are rare in humans. AT in response to underfeeding is independent of thermogenesis in response to either diet or cold. Although fat‐free mass (FFM) and, thus, liver, and skeletal muscle are considered as major sites of AT, cold‐induced nonshivering thermogenesis relates to the metabolism of brown adipose tissue (BAT). In humans, diet‐induced thermogenesis is related to postprandial substrate metabolism of FFM with a questionable role of BAT. Obviously, the REE component of AT differs from and its non‐REE component with respect to organ contribution as well as mechanisms. Thus, AT cannot be considered as unique.

Conclusions:

AT should be characterized based on individual components of daily energy expenditure, detailed body composition analyses, and mathematical modeling. The biological basis of AT as well as the influences of age, sex, obesity, stress, and inflammation remain to be established in humans.     

The Relationships between Body Composition and the Systemic Inflammatory Response in Patients with Primary Operable Colorectal Cancer

Background

Weight loss is recognised as a marker of poor prognosis in patients with cancer but the aetiology of cancer cachexia remains unclear. The aim of the present study was to examine the relationships between CT measured parameters of body composition and the systemic inflammatory response in patients with primary operable colorectal cancer.

Patient and Methods

174 patients with primary operable colorectal cancer who underwent resection with curative intent (2003–2010). Image analysis of CT scans was used to measure total fat index (cm²/m²), subcutaneous fat index (cm²/m²), visceral fat index (cm²/m²) and skeletal muscle index (cm²/m²). Systemic inflammatory response was measured by serum white cell count (WCC), neutrophil:lymphocyte ratio (NLR) and the Glasgow Prognostic Score (mGPS).

Results

There were no relationships between any parameter of body composition and serum WCC or NLR. There was a significant relationship between low skeletal muscle index and an elevated systemic inflammatory response, as measured by the mGPS (p = 0.001). This was confirmed by linear relationships between skeletal muscle index and both C-reactive protein (r = −0.21, p = 0.005) and albumin (r = 0.31, p<0.001). There was no association between skeletal muscle index and tumour stage.

Conclusions

The present study highlights a direct relationship between low levels of skeletal muscle and the presence of a systemic inflammatory response in patients with primary operable colorectal cancer.     

Seasonal effects of the UCP3 and the RPTOR gene polymorphisms on obesity traits in Japanese adults

Background

Non-shivering thermogenesis (NST) involves a substantial amount of energy expenditure in humans and, thus, contributes to reducing the risk for obesity. Molecular evolutionary studies have reported that SNPs in/near the uncoupling protein 3 gene (UCP3) and the regulatory associated protein of mTOR complex 1 gene (RPTOR) might influence NST and confer adaptive advantages for modern human dispersal into cold environments. In the present study, the impact of these SNPs on obesity-related traits was investigated.

Methods

Study subjects consisted of 2,834 Japanese adults (percentage of female: 46%, mean age: 51.5). Associations of the UCP3-55C/T and the RPTOR-26934C/T - the 2 potential genetic variations involved in cold adaptation and thermogenic mechanisms in mammals, with quantitative obesity-related traits including body mass index (BMI), waist circumference, visceral fat area (VFA), VFA adjusted for BMI, and selected blood parameters - were tested using multiple linear regression models. Sliding windowsampling analysis was applied to depict seasonal effects of the SNPs on the obesity-related phenotypes.

Results

UCP3-55C/T and the RPTOR-26934C/T did not show any association with obesity traits and blood chemical parameters in multiple linear regression models consisting of the whole subjects. Moreover, sliding window sampling-based association analyses involving seasonality also failed to find associations between these two SNPs and obesity-related traits.

Conclusions

UCP3-55C/T and the RPTOR-26934C/T may only have subtle effects on the development of obesity-related traits in the present humans. These two SNPs might be irrelevant to inter-individual variations in energy metabolism and efficiency of NST.     

Allelic variants of melanocortin 3 receptor gene (MC3R) and weight loss in obesity: a randomised trial of hypo-energetic high- versus low-fat diets

Introduction

The melanocortin system plays an important role in energy homeostasis. Mice genetically deficient in the melanocortin-3 receptor gene have a normal body weight with increased body fat, mild hypophagia compared to wild-type mice. In humans, Thr6Lys and Val81Ile variants of the melanocortin-3 receptor gene (MC3R) have been associated with childhood obesity, higher BMI Z-score and elevated body fat percentage compared to non-carriers. The aim of this study is to assess the association in adults between allelic variants of MC3R with weight loss induced by energy-restricted diets.

Subjects and Methods

This research is based on the NUGENOB study, a trial conducted to assess weight loss during a 10-week dietary intervention involving two different hypo-energetic (high-fat and low-fat) diets. A total of 760 obese patients were genotyped for 10 single nucleotide polymorphisms covering the single exon of MC3R gene and its flanking regions, including the missense variants Thr6Lys and Val81Ile. Linear mixed models and haplotype-based analysis were carried out to assess the potential association between genetic polymorphisms and differential weight loss, fat mass loss, waist change and resting energy expenditure changes.

Results

No differences in drop-out rate were found by MC3R genotypes. The rs6014646 polymorphism was significantly associated with weight loss using co-dominant (p = 0.04) and dominant models (p = 0.03). These p-values were not statistically significant after strict control for multiple testing. Haplotype-based multivariate analysis using permutations showed that rs3827103–rs1543873 (p = 0.06), rs6014646–rs6024730 (p = 0.05) and rs3746619–rs3827103 (p = 0.10) displayed near-statistical significant results in relation to weight loss. No other significant associations or gene*diet interactions were detected for weight loss, fat mass loss, waist change and resting energy expenditure changes.

Conclusion

The study provided overall sufficient evidence to support that there is no major effect of genetic variants of MC3R and differential weight loss after a 10-week dietary intervention with hypo-energetic diets in obese Europeans.     

Cold exposure differently influences mitochondrial energy efficiency in rat liver and skeletal muscle

This study deals with mitochondrial energy efficiency in liver and skeletal muscle mitochondria in 15 days cold exposed rats. Cold exposure strongly increases the sensitivity to uncoupling by added palmitate of skeletal muscle but not liver mitochondria, while mitochondrial energy coupling in the absence of fatty acids is only slightly affected by cold in liver and skeletal muscle. In addition, uncoupling protein 3 content does not follow changes in skeletal muscle mitochondrial coupling. It is therefore concluded that skeletal muscle could play a direct thermogenic role based on fatty acid-induced mild uncoupling of mitochondrial oxidative phosphorylation.     

Skeletal muscle phosphodiester content relates to body mass and glycemic control

Background

Aging and insulin resistance have been related to reduced mitochondrial function and oxidative stress. Muscular phosphodiesters (PDE) are comprised of metabolites of phospholipid breakdown and may reflect membrane damage. We aimed to test the hypothesis that myocellular PDE are increased in patients with type 2 diabetes (T2D) and correlate inversely with mitochondrial ATP turnover.

Methods

A Cross-sectional study in the Clinical Research Facility of an University hospital was performed. 10 nonobese middle-aged patients with T2D, 10 healthy humans matched for sex, age and physical activity index (CONm) and 18 young healthy humans (CONy) were included. Myocellular PDE and unidirectional flux through ATP synthase (fATP) were measured with ³¹P magnetic resonance spectroscopy (MRS). Intramyocellular (IMCL) and hepatocellular lipid deposition (HCL) were quantified with ¹H MRS. Insulin sensitivity (Rd) was assessed from hyperinsulinemic-euglycemic clamp tests in 10 T2D, 10 CONm and 11 CONy.

Results

During fasting, T2D and CONm had 1.5 fold greater PDE than CONy (2.8±0.2, 2.5±0.2, 1.7±0.1 mmol/l, P = 0.004). Stimulation by insulin did not affect PDE in any group. PDE correlated negatively with Rd (r = −0.552, p<0.005) and fATP (r = −0.396, p<0.05) and positively with age (r = 0.656, p<0.001) and body mass (r = 0.597, p<0.001). PDE also related positively to HbA1c (r = 0.674, p<0.001) and fasting plasma glucose (r = 0.629, p<0.001) within T2D and across all participants.

Conclusions

Muscular PDE concentrations associate with age, lower resting mitochondrial activity and insulin resistance, which is determined mainly by body mass and glycemia.     

Zidovudine Exposure in HIV-1 Infected Tanzanian Women Increases Mitochondrial DNA Levels in Placenta and Umbilical Cords

Background

Zidovudine (AZT) constitutes part of the recommended regimens for prevention and treatment of HIV-1 infection. At the same time, AZT as well as HIV-1 infection itself may induce mitochondrial damage. In this study, we analyzed the impact of prenatal AZT-exposure on mitochondrial alterations in HIV-infected women and their infants.

Methods

Mitochondrial DNA (mtDNA) levels in placentas of HIV-1 infected Tanzanian women with and without prenatal AZT exposure, and in the umbilical cords of their AZT-exposed/unexposed infants were quantified using real-time PCR. Furthermore, we checked for the most common mitochondrial deletion in humans, the 4977 base pair deletion (dmtDNA4977) as a marker for mitochondrial stress.

Results

83 women fulfilled the inclusion criteria. 30 women had been treated with AZT (median duration 56 days; IQR 43–70 days) while 53 women had not taken AZT during pregnancy. Baseline maternal characteristics in the two groups were similar. The median mtDNA levels in placentas and umbilical cords of women (311 copies/cell) and infants (190 copies/cell) exposed to AZT were significantly higher than in AZT-unexposed women (187 copies/cell; p = 0.021) and infants (127 copies/cell; p = 0.037). The dmtDNA4977 was found in placentas of one woman of each group and in 3 umbilical cords of AZT-unexposed infants but not in umbilical cords of AZT-exposed infants.

Conclusions

Antenatal AZT intake did not increase the risk for the common mitochondrial deletion dmtDNA4977. Our data suggests that AZT exposure elevates mtDNA levels in placentas and umbilical cords possibly by positively influencing the course of maternal HIV-1 infection.     

Muscle Mitochondrial Uncoupling Dismantles Neuromuscular Junction and Triggers Distal Degeneration of Motor Neurons

Background

Amyotrophic lateral sclerosis (ALS), the most frequent adult onset motor neuron disease, is associated with hypermetabolism linked to defects in muscle mitochondrial energy metabolism such as ATP depletion and increased oxygen consumption. It remains unknown whether muscle abnormalities in energy metabolism are causally involved in the destruction of neuromuscular junction (NMJ) and subsequent motor neuron degeneration during ALS.

Methodology/Principal Findings

We studied transgenic mice with muscular overexpression of uncoupling protein 1 (UCP1), a potent mitochondrial uncoupler, as a model of muscle restricted hypermetabolism. These animals displayed age-dependent deterioration of the NMJ that correlated with progressive signs of denervation and a mild late-onset motor neuron pathology. NMJ regeneration and functional recovery were profoundly delayed following injury of the sciatic nerve and muscle mitochondrial uncoupling exacerbated the pathology of an ALS animal model.

Conclusions/Significance

These findings provide the proof of principle that a muscle restricted mitochondrial defect is sufficient to generate motor neuron degeneration and suggest that therapeutic strategies targeted at muscle metabolism might prove useful for motor neuron diseases.     

Bile acid sequestration reduces plasma glucose levels in db/db mice by increasing its metabolic clearance rate

Aims/Hypothesis

Bile acid sequestrants (BAS) reduce plasma glucose levels in type II diabetics and in murine models of diabetes but the mechanism herein is unknown. We hypothesized that sequestrant-induced changes in hepatic glucose metabolism would underlie reduced plasma glucose levels. Therefore, in vivo glucose metabolism was assessed in db/db mice on and off BAS using tracer methodology.

Methods

Lean and diabetic db/db mice were treated with 2% (wt/wt in diet) Colesevelam HCl (BAS) for 2 weeks. Parameters of in vivo glucose metabolism were assessed by infusing [U-¹³C]-glucose, [2-¹³C]-glycerol, [1-²H]-galactose and paracetamol for 6 hours, followed by mass isotopologue distribution analysis, and related to metabolic parameters as well as gene expression patterns.

Results

Compared to lean mice, db/db mice displayed an almost 3-fold lower metabolic clearance rate of glucose (p = 0.0001), a ∼300% increased glucokinase flux (p = 0.001) and a ∼200% increased total hepatic glucose production rate (p = 0.0002). BAS treatment increased glucose metabolic clearance rate by ∼37% but had no effects on glucokinase flux nor total hepatic or endogenous glucose production. Strikingly, BAS-treated db/db mice displayed reduced long-chain acylcarnitine content in skeletal muscle (p = 0.0317) but not in liver (p = 0.189). Unexpectedly, BAS treatment increased hepatic FGF21 mRNA expression 2-fold in lean mice (p = 0.030) and 3-fold in db/db mice (p = 0.002).

Conclusions/Interpretation

BAS induced plasma glucose lowering in db/db mice by increasing metabolic clearance rate of glucose in peripheral tissues, which coincided with decreased skeletal muscle long-chain acylcarnitine content.     

In vivo determination of mouse olfactory mucus cation concentrations in normal and inflammatory States

Objective

Olfaction is impaired in chronic rhinosinusitis (CRS). The study has two aims: (1) to determine whether changes in cation concentration occur in the olfactory mucus of mice with CRS, which may affect chemo-electrical transduction, (2) and to examine whether these alterations are physiologically significant in humans.

Study Design

Animal study in mice and translational study in humans.

Methods

Inflammation was induced by sensitization and chronic exposure of 16 C57BL/6 mice to Aspergillus fumigatus. The control group included 16 untreated mice. Ion-selective microelectrodes were used to measure free cation concentrations in the olfactory mucus of 8 mice from each treatment group, while the remaining mice were sacrificed for histology. To validate the findings in the animal model, olfactory threshold was measured in 11 healthy human participants using Sniffin’ Sticks before and after nasal irrigation with solutions that were composed of either of the cation concentrations.

Results

In 8 mice, olfactory mucus of chronically inflamed mice had lower [Na⁺] (84.8±4.45 mM versus 93.73±3.06 mM, p = 0.02), and higher [K⁺] (7.2±0.65 mM versus 5.7±0.20 mM, p = 0.04) than controls. No difference existed in [Ca²⁺] (0.50±0.12 mM versus 0.54±0.06 mM, p = 0.39). In humans, rinsing with solutions replicating ion concentrations of the mouse mucosa with chronic inflammation caused a significant elevation in the median olfactory threshold (9.0 to 4.8, p = 0.003) but not with the control solution (8.3 to 7.8, p = 0.75).

Conclusion

Chronic inflammation elevates potassium and lowers sodium ion concentration in mice olfactory mucus. Nasal irrigation with a corresponding solution induced olfactory threshold shift in humans.     

The effect of ACACB cis-variants on gene expression and metabolic traits

Background

Acetyl Coenzyme A carboxylase β (ACACB) is the rate-limiting enzyme in fatty acid oxidation, and continuous fatty acid oxidation in Acacb knock-out mice increases insulin sensitivity. Systematic human studies have not been performed to evaluate whether ACACB variants regulate gene expression and insulin sensitivity in skeletal muscle and adipose tissues. We sought to determine whether ACACB transcribed variants were associated with ACACB gene expression and insulin sensitivity in non-diabetic African American (AA) and European American (EA) adults.

Methods

ACACB transcribed single nucleotide polymorphisms (SNPs) were genotyped in 105 EAs and 46 AAs whose body mass index (BMI), lipid profiles and ACACB gene expression in subcutaneous adipose and skeletal muscle had been measured. Allelic expression imbalance (AEI) was assessed in lymphoblast cell lines from heterozygous subjects in an additional EA sample (n = 95). Selected SNPs were further examined for association with insulin sensitivity in a cohort of 417 EAs and 153 AAs.

Results

ACACB transcribed SNP rs2075260 (A/G) was associated with adipose ACACB messenger RNA expression in EAs and AAs (p = 3.8×10⁻⁵, dominant model in meta-analysis, Stouffer method), with the (A) allele representing lower gene expression in adipose and higher insulin sensitivity in EAs (p = 0.04). In EAs, adipose ACACB expression was negatively associated with age and sex-adjusted BMI (r = −0.35, p = 0.0002).

Conclusions

Common variants within the ACACB locus appear to regulate adipose gene expression in humans. Body fat (represented by BMI) may further regulate adipose ACACB gene expression in the EA population.     

Metabolic Flux Analysis of Mitochondrial Uncoupling in 3T3-L1 Adipocytes

Background

Increasing energy expenditure at the cellular level offers an attractive option to limit adiposity and improve whole body energy balance. In vivo and in vitro observations have correlated mitochondrial uncoupling protein-1 (UCP1) expression with reduced white adipose tissue triglyceride (TG) content. The metabolic basis for this correlation remains unclear.

Methodology/Principal Findings

This study tested the hypothesis that mitochondrial uncoupling requires the cell to compensate for the decreased oxidation phosphorylation efficiency by up-regulating lactate production, thus redirecting carbon flux away from TG synthesis. Metabolic flux analysis was used to characterize the effects of non-lethal, long-term mitochondrial uncoupling (up to 18 days) on the pathways of intermediary metabolism in differentiating 3T3-L1 adipocytes. Uncoupling was induced by forced expression of UCP1 and chemical (FCCP) treatment. Chemical uncoupling significantly decreased TG content by ca. 35%. A reduction in the ATP level suggested diminished oxidative phosphorylation efficiency in the uncoupled adipocytes. Flux analysis estimated significant up-regulation of glycolysis and down-regulation of fatty acid synthesis, with chemical uncoupling exerting quantitatively larger effects.

Conclusions/Significance

The results of this study support our hypothesis regarding uncoupling-induced redirection of carbon flux into glycolysis and lactate production, and suggest mitochondrial proton translocation as a potential target for controlling adipocyte lipid metabolism.     

The 24-h energy intake of obese adolescents is spontaneously reduced after intensive exercise: a randomized controlled trial in calorimetric chambers

Background

Physical exercise can modify subsequent energy intake and appetite and may thus be of particular interest in terms of obesity treatment. However, it is still unclear whether an intensive bout of exercise can affect the energy consumption of obese children and adolescents.

Objective

To compare the impact of high vs. moderate intensity exercises on subsequent 24-h energy intake, macronutrient preferences, appetite sensations, energy expenditure and balance in obese adolescent.

Design

This randomized cross-over trial involves 15 obese adolescent boys who were asked to randomly complete three 24-h sessions in a metabolic chamber, each separated by at least 7 days: (1) sedentary (SED); (2) Low-Intensity Exercise (LIE) (40% maximal oxygen uptake, VO₂max); (3) High-Intensity Exercise (HIE) (75%VO₂max).

Results

Despite unchanged appetite sensations, 24-h total energy intake following HIE was 6–11% lower compared to LIE and SED (p<0.05), whereas no differences appeared between SED and LIE. Energy intake at lunch was 9.4% and 8.4% lower after HIE compared to SED and LIE, respectively (p<0.05). At dinner time, it was 20.5% and 19.7% lower after HIE compared to SED and LIE, respectively (p<0.01). 24-h energy expenditure was not significantly altered. Thus, the 24-h energy balance was significantly reduced during HIE compared to SED and LIE (p<0.01), whereas those of SED and LIE did not differ.

Conclusions

In obese adolescent boys, HIE has a beneficial impact on 24-h energy balance, mainly due to the spontaneous decrease in energy intake during lunch and dinner following the exercise bout. Prescribing high-intensity exercises to promote weight loss may therefore provide effective results without affecting appetite sensations and, as a result, food frustrations.

Trial Registration

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