Research Items (84)
- Apr 2019
Suppression of Dicer, an endoribonuclease that regulates microRNAs(miRNA), has evolved as a viral mechanism to enhance host HIV infectivity and may have unintended metabolic consequences. Animal knockout models of adipose-specific dicer(ADicer) acquire lipodystrophy accompanied by severe metabolic abnormalities. Data show adipose is a source of exosomal miRNAs, functioning as adipokines influencing metabolic homeostasis. We hypothesized a unique miRNA profile among individuals well-phenotyped for HIV lipodystrophy and reduced ADicer expression. We evaluated >1000 miRNAs from exosomes derived from sera among the 27 male individuals [9 HIV lipodystrophy(HIV/lipo), 9 HIV without lipodystrophy(HIV/non-lipo), 9 non-HIV] whom we previously showed variations in ADicer: most suppressed among HIV lipo, followed by HIV non-lipo and non-HIV (2.49[0.02,4.88] vs. 11.20[4.83,21.45] vs. 17.69[10.72,47.91],P=.002). To estimate miRNA abundance, data was normalized using the geomean of expressed Ct values. A false discovery rate analysis(FDR) was applied. We investigated whether significant exosomal miRNAs demonstrated a similar pattern in corresponding subcutaneous adipose tissue(SAT) samples. Using target prediction databases (TargetScan, miRDB, Diana), we identified genes related to fat biology (target score ≥85%) with clinical relevance to lipodystrophic phenotypes and further evaluated these in the adipose depot. HIV/lipo individuals(age 56±3years, BMI 30±1kg/m², duration HIV 24±2years, duration ART 20±2years, CD4+ count 482±90cells/μl, undetectable VL 67%) were similar to HIV/non-lipo(age 52±3 years, BMI 30±1 kg/m²) and non-HIV(age 55±3years, BMI 30±1kg/m²) individuals. Reduced ADicer expression was significantly related to reduced CD4⁺ count(r=0.55,P=.02), duration ART use(r=-0.70,P=.001) and tended to be related to duration HIV(r=-0.44,P=.07) and reduced CD8⁺ count(r=0.42,P=.08). Applying FDR, we detected exosomal miRNA-20a-3p(P=.0026), 324-5p(P=.0059), and 186-5p(P=.0977) were expressed differentially in HIV/lipo vs. non-HIV and 324-5p(P=.0348) in HIV/lipo vs. HIV/non-lipo. We further confirmed that miRNA-20a-3p was significantly and differentially expressed in the SAT, expressed highest in HIV lipo followed by HIV non-lipo and non-HIV [9.73±12.38 vs. 2.24±1.84 vs. 1.02±0.19, P<0.05] in a pattern similar to the exosomal profiles. Relevant miRNA and their target genes which tended to be differentially expressed in the SAT of HIV lipo vs. HIV non-lipo include: 20a-3p (TDG, P=0.07) and 186-5p (PDE10A, PID1; P<0.05). Altered ADicer expression and specific exosomal miRNAs may affect gene expression of regulators important to fat biology and metabolic homeostasis in HIV. In particular, miRNA-20a-3p may function as an adipokine demonstrating similar expression profiles in exosomes and adipose tissue of individuals with HIV lipodystrophy.
In endothermic animals, brown adipose tissue (BAT) is activated to produce heat for defending body temperature in response to cold. BAT's ability to expend energy has made it a potential target for novel therapies to ameliorate obesity and associated metabolic disorders in humans. Though this tissue has been well studied in small animals, BAT's thermogenic capacity in humans remains largely unknown due to the difficulties of measuring its volume, activity, and distribution. Identifying and quantifying active human BAT is commonly performed using 18F-Fluorodeoxyglucose (18F-FDG) positron emission tomography and computed tomography (PET/CT) scans following cold-exposure or pharmacological activation. Here we describe a detailed image-analysis approach to quantify total-body human BAT from 18F-FDG PET/CT scans using an open-source software. We demonstrate the drawing of user-specified regions of interest to identify metabolically active adipose tissue while avoiding common non-BAT tissues, to measure BAT volume and activity, and to further characterize its anatomical distribution. Although this rigorous approach is time-consuming, we believe it will ultimately provide a foundation to develop future automated BAT quantification algorithms.
Diabetes. 2018 Jul 6. pii: db180462. doi: 10.2337/db18-0462. [Epub ahead of print] Regulation of Human Adipose Tissue Activation, Gallbladder Size, and Bile Acid Metabolism by a β3-Adrenergic Receptor Agonist. Baskin AS1, Linderman JD1, Brychta RJ1, McGehee S1, Anflick-Chames E1, Cero C1, Johnson JW1, O'Mara AE1, Fletcher LA1, Leitner BP1, Duckworth CJ1, Huang S1, Cai H2, Garraffo HM2, Millo CM3, Dieckmann W3, Tolstikov V4, Chen EY4, Gao F4, Narain NR4, Kiebish MA4, Walter PJ2, Herscovitch P3, Chen KY1, Cypess AM5. Author information Abstract β3-adrenergic receptor (AR) agonists are approved to treat only overactive bladder. However, rodent studies suggest that these drugs could have other beneficial effects on human metabolism. We performed tissue receptor profiling and showed that the human β3-AR mRNA is also highly expressed in gallbladder and brown adipose tissue (BAT). We next studied the clinical implications of this distribution in twelve healthy men given one-time randomized doses of placebo; the approved dose of 50 mg; and 200 mg of the β3-AR agonist mirabegron. There was a more-than-dose proportional increase in BAT metabolic activity as measured by 18F-FDG PET/CT (medians 0.0 vs. 18.2 vs. 305.6 mL*SUVmean*g/mL). Only the 200 mg dose elevated both non-esterified fatty acids (+68%) and resting energy expenditure (+5.8%). Previously undescribed increases in gallbladder size (+35%) and reductions in conjugated bile acids were also discovered. Therefore, besides urinary bladder relaxation, the human β3-AR contributes to WAT lipolysis, BAT thermogenesis, gallbladder relaxation, and bile acid metabolism. This physiology should be considered in the development of more selective β3-AR agonists to treat obesity-related complications. PMID: 29980535 DOI: 10.2337/db18-0462 Share on FacebookShare on TwitterShare on Google+
Thermogenic fat expends energy during cold for temperature homeostasis, and its activity regulates nutrient metabolism and insulin sensitivity. We measured cold-activated lipid landscapes in circulation and in adipose tissue by MS/MSALL shotgun lipidomics. We created an interactive online viewer to visualize the changes of specific lipid species in response to cold. In adipose tissue, among the approximately 1,600 lipid species profiled, we identified the biosynthetic pathway of the mitochondrial phospholipid cardiolipin as coordinately activated in brown and beige fat by cold in wild-type and transgenic mice with enhanced browning of white fat. Together, these data provide a comprehensive lipid bio-signature of thermogenic fat activation in circulation and tissue and suggest pathways regulated by cold exposure.
Brown adipose tissue (BAT) has been identified as a potential target in the treatment and prevention of obesity and metabolic disease. The precise kinetics of BAT activation and the duration of stimulus required to recruit metabolically active BAT, and its subsequent deactivation, are not well-understood. In this clinical trial, 19 healthy adults (BMI: 23.7 ± 0.7 kg/m2, Age: 31.2 ± 2.8 year, 12 female) underwent three different cooling procedures to stimulate BAT glucose uptake, and active BAT volume was determined using 18F-Fluorodeoxyglucose (FDG) PET/CT imaging. We found that 20 min of pre-injection cooling produces activation similar to the standard 60 min (39.9 mL vs. 44.2 mL, p = 0.52), indicating that BAT activity approaches its peak function soon after the initiation of cooling. Furthermore, upon removal of cold exposure, active BAT volume declines (13.6 mL vs. 44.2 mL, p = 0.002), but the deactivation process persists even hours following cessation of cooling. Thus, the kinetics of human BAT thermogenesis are characterized by a rapid increase soon after cold stimulation but a more gradual decline after rewarming. These characteristics reinforce the feasibility of developing mild, short-duration cold exposure to activate BAT and treat obesity and metabolic disease.
- Mar 2018
Patients with psychotic disorders are at high risk for type 2 diabetes mellitus, and there is increasing evidence that patients display glucose metabolism abnormalities before significant antipsychotic medication exposure. In the present study, we examined insulin action by quantifying insulin sensitivity in first-episode psychosis (FEP) patients and unaffected siblings, compared to healthy individuals, using a physiological-based model and comprehensive assessment battery. Twenty-two unaffected siblings, 18 FEP patients, and 15 healthy unrelated controls were evaluated using a 2-h oral glucose tolerance test (OGTT), with 7 samples of plasma glucose and serum insulin concentration measurements. Insulin sensitivity was quantified using the oral minimal model method. Lipid, leptin, free fatty acids, and inflammatory marker levels were also measured. Anthropometric, nutrient, and activity assessments were conducted; total body composition and fat distribution were determined using whole-body dual-energy X-ray absorptiometry. Insulin sensitivity significantly differed among groups (F = 6.01 and 0.004), with patients and siblings showing lower insulin sensitivity, compared to controls (P = 0.006 and 0.002, respectively). Body mass index, visceral adipose tissue area (cm2), lipids, leptin, free fatty acids, inflammatory markers, and activity ratings were not significantly different among groups. There was a significant difference in nutrient intake with lower total kilocalories/kilogram body weight in patients, compared to siblings and controls. Overall, the findings suggest that familial abnormal glucose metabolism or a primary insulin signaling pathway abnormality is related to risk for psychosis, independent of disease expression and treatment effects. Future studies should examine underlying biological mechanisms of insulin signaling abnormalities in psychotic disorders.
The identification of brown adipose deposits in adults has led to significant interest in targeting this metabolically active tissue for treatment of obesity and diabetes. Improved methods for the direct measurement of heat production as the signature function of brown adipocytes (BAs), particularly at the single cell level, would be of substantial benefit to these ongoing efforts. Here, we report the first application of a small molecule-type thermosensitive fluorescent dye, ERthermAC, to monitor thermogenesis in BAs derived from murine brown fat precursors and in human brown fat cells differentiated from human neck brown preadipocytes. ERthermAC accumulated in the endoplasmic reticulum of BAs and displayed a marked change in fluorescence intensity in response to adrenergic stimulation of cells, which corresponded to temperature change. ERthermAC fluorescence intensity profiles were congruent with mitochondrial depolarisation events visualised by the JC-1 probe. Moreover, the averaged fluorescence intensity changes across a population of cells correlated well with dynamic changes such as thermal power, oxygen consumption, and extracellular acidification rates. These findings suggest ERthermAC as a promising new tool for studying thermogenic function in brown adipocytes of both murine and human origins.
PurposeBrown adipose tissue (BAT) in adult humans has been recently rediscovered and intensively investigated as a new potential therapeutic target for obesity and type 2 diabetes (T2D). However, reliable assessment of BAT mass in vivo represents a considerable challenge. The purpose of this investigation is to demonstrate for the first time that human BAT depots can be imaged with a translocator protein (TSPO)-specific positron emission tomography (PET) tracer [11C]PBR28 under thermoneutral conditions. ProceduresIn this retrospective analysis, we analyzed the images of three healthy volunteers who underwent PET/magnetic resonance (MR) imaging after injection of 14 m Ci of [11C]PBR28 at room temperature. Thirty-minute static PET images were reconstructed from the data obtained 60–90 min after the injection of the tracer. Results[11C]PBR28 uptake in the neck/supraclavicular regions was identified, which was parallel to the known distribution pattern of human BAT depots. These areas co-localized with the areas of hyperintensity and corresponded to fat on T1-weighted MR images. Standardized uptake value (SUV) was used to quantify [11C]PBR28 signal in BAT depots. The average (± SD) SUV(mean) and SUVmax for BAT depots was 2.13 (± 0.33) and 3.19 (± 0.34), respectively, while the average SUV(mean) for muscle and subcutaneous adipose tissue was 0.79 (± 0.1) and 0.18 (± 0.04), respectively. Conclusions In this brief article, we provide the first evidence suggesting that [11C]PBR28, a widely available TSPO-specific PET tracer, can be used for imaging human BAT mass under thermoneutral conditions.
Brown fat dissipates energy as heat and protects against obesity. Here, we identified nuclear factor I-A (NFIA) as a transcriptional regulator of brown fat by a genome-wide open chromatin analysis of murine brown and white fat followed by motif analysis of brown-fat-specific open chromatin regions. NFIA and the master transcriptional regulator of adipogenesis, PPARγ, co-localize at the brown-fat-specific enhancers. Moreover, the binding of NFIA precedes and facilitates the binding of PPARγ, leading to increased chromatin accessibility and active transcription. Introduction of NFIA into myoblasts results in brown adipocyte differentiation. Conversely, the brown fat of NFIA-knockout mice displays impaired expression of the brown-fat-specific genes and reciprocal elevation of muscle genes. Finally, expression of NFIA and the brown-fat-specific genes is positively correlated in human brown fat. These results indicate that NFIA activates the cell-type-specific enhancers and facilitates the binding of PPARγ to control the brown fat gene program.
Human brown adipose tissue (BAT) can be activated to increase glucose uptake and energy expenditure, making it a potential target for treating obesity and metabolic disease. Data on the functional and anatomic characteristics of BAT are limited, however. In 20 healthy young men [12 lean, mean body mass index (BMI) 23.2 ± 1.9 kg/m(2); 8 obese, BMI 34.8 ± 3.3 kg/m(2)] after 5 h of tolerable cold exposure, we measured BAT volume and activity by (18)F-labeled fluorodeoxyglucose positron emission tomography/computerized tomography (PET/CT). Obese men had less activated BAT than lean men (mean, 130 vs. 334 mL) but more fat in BAT-containing depots (mean, 1,646 vs. 855 mL) with a wide range (0.1-71%) in the ratio of activated BAT to inactive fat between individuals. Six anatomic regions had activated BAT-cervical, supraclavicular, axillary, mediastinal, paraspinal, and abdominal-with 67 ± 20% of all activated BAT concentrated in a continuous fascial layer comprising the first three depots in the upper torso. These nonsubcutaneous fat depots amounted to 1.5% of total body mass (4.3% of total fat mass), and up to 90% of each depot could be activated BAT. The amount and activity of BAT was significantly influenced by region of interest selection methods, PET threshold criteria, and PET resolutions. The present study suggests that active BAT can be found in specific adipose depots in adult humans, but less than one-half of the fat in these depots is stimulated by acute cold exposure, demonstrating a previously underappreciated thermogenic potential.
- Mar 2017
Brown adipose tissue (BAT) and beige adipose tissue combust fuels for heat production in adult humans, and so constitute an appealing target for the treatment of metabolic disorders such as obesity, diabetes and hyperlipidemia. Cold exposure can enhance energy expenditure by activating BAT, and it has been shown to improve nutrient metabolism. These therapies, however, are time consuming and uncomfortable, demonstrating the need for pharmacological interventions. Recently, lipids have been identified that are released from tissues and act locally or systemically to promote insulin sensitivity and glucose tolerance; as a class, these lipids are referred to as 'lipokines'. Because BAT is a specialized metabolic tissue that takes up and burns lipids and is linked to systemic metabolic homeostasis, we hypothesized that there might be thermogenic lipokines that activate BAT in response to cold. Here we show that the lipid 12,13-dihydroxy-9Z-octadecenoic acid (12,13-diHOME) is a stimulator of BAT activity, and that its levels are negatively correlated with body-mass index and insulin sensitivity. Using a global lipidomic analysis, we found that 12,13-diHOME was increased in the circulation of humans and mice exposed to cold. Furthermore, we found that the enzymes that produce 12,13-diHOME were uniquely induced in BAT by cold stimulation. The injection of 12,13-diHOME acutely activated BAT fuel uptake and enhanced cold tolerance, which resulted in decreased levels of serum triglycerides. Mechanistically, 12,13-diHOME increased fatty acid (FA) uptake into brown adipocytes by promoting the translocation of the FA transporters FATP1 and CD36 to the cell membrane. These data suggest that 12,13-diHOME, or a functional analog, could be developed as a treatment for metabolic disorders.
- Oct 2016
Background: In addition to its role in adaptive thermogenesis, brown adipose tissue (BAT) may protect from weight gain, insulin resistance/diabetes and metabolic syndrome. Prior studies have shown contradictory results regarding the influence of thyroid hormone (TH) levels on BAT volume and activity. The aim of this pilot study was to gain further insight regarding the effect of TH treatment on BAT function in adult humans by evaluating the BAT mass and activity prospectively in six patients, first in the hypothyroid and then in the thyrotoxic phase. Methods: The study subjects underwent 18F-FDG PET-CT scanning after cold exposure to measure BAT mass and activity while undergoing treatment for differentiated thyroid cancer, first while hypothyroid following thyroid hormone withdrawal at the time of the radioactive iodine treatment and then 3-6 months after starting TH suppressive treatment when they were iatrogenically thyrotoxic. We measured thermogenic and metabolic parameters in both phases. Results: All study subjects had detectable BAT under cold stimulation in both the hypothyroid and thyrotoxic state. The majority but not all (4 out of 6) subjects showed an increase in detectable BAT volume and activity under cold stimulation between the hypothyroid and thyrotoxic phase (total BAT volume: 72.0 ± 21.0 vs. 87.7 ± 16.5 mL, P = 0.25; total BAT activity 158.1 ± 72.8 vs. 189.0 ± 55.5 SUV*g/mL, P = 0.34). Importantly, circulating T3 was a stronger predictor of energy expenditure changes compared to cold-induced BAT activity. Conclusions: Iatrogenic hypothyroidism lasting 2-4 weeks does not prevent cold-induced BAT activation, while the use of TH to induce thyrotoxicosis does not consistently increase cold-induced BAT activity. It remains to be determined which physiological factors besides TH play a role in regulating BAT function.
- Sep 2016
“Beige” adipocytes reside in white adipose tissue (WAT) and dissipate energy as heat. Several studies have shown that cold temperature can activate pro-opiomelanocortin-expressing (POMC) neurons and increase sympathetic neuronal tone to regulate WAT beiging. WAT, however, is traditionally known to be sparsely innervated. Details regarding the neuronal innervation and, more importantly, the propagation of the signal within the population of “beige” adipocytes are sparse. Here, we demonstrate that beige adipocytes display an increased cell-to-cell coupling via connexin 43 (Cx43) gap junction channels. Blocking of Cx43 channels by 18α-glycyrrhetinic acid decreases POMC-activation-induced adipose tissue beiging. Adipocyte-specific deletion of Cx43 reduces WAT beiging to a level similar to that observed in denervated fat pads. In contrast, overexpression of Cx43 is sufficient to promote beiging even with mild cold stimuli. These data reveal the importance of cell-to-cell communication, effective in cold-induced WAT beiging, for the propagation of limited neuronal inputs in adipose tissue.
Human brown adipose tissue (BAT) presence, metabolic activity, and estimated mass are typically measured by imaging [18F]fluorodeoxyglucose (FDG) uptake in response to cold exposure in regions of the body expected to contain BAT, using positron emission tomography combined with X-ray computed tomography (FDG-PET/CT). Efforts to describe the epidemiology and biology of human BAT are hampered by diverse experimental practices, making it difficult to directly compare results among laboratories. An expert panel was assembled by the National Institute of Diabetes and Digestive and Kidney Diseases on November 4, 2014 to discuss minimal requirements for conducting FDG-PET/CT experiments of human BAT, data analysis, and publication of results. This resulted in Brown Adipose Reporting Criteria in Imaging STudies (BARCIST 1.0). Since there are no fully validated best practices at this time, panel recommendations are meant to enhance comparability across experiments, but not to constrain experimental design or the questions that can be asked.
- Jul 2016
The rates of diabetes, obesity, and metabolic disease have reached epidemic proportions worldwide. In recent years there has been renewed interest in combating these diseases not only by modifying energy intake and lifestyle factors, but also by inducing endogenous energy expenditure. This approach has largely been stimulated by the recent recognition that brown adipose tissue (BAT)-long known to promote heat production and energy expenditure in infants and hibernating mammals-also exists in adult humans. This landmark finding relied on the use of clinical fluorine 18 fluorodeoxyglucose positron emission tomography/computed tomography, and imaging techniques continue to play a crucial and increasingly central role in understanding BAT physiology and function. Herein, the authors review the origins of BAT imaging, discuss current preclinical and clinical strategies for imaging BAT, and discuss imaging methods that will provide crucial insight into metabolic disease and how it may be treated by modulating BAT activity. (©) RSNA, 2016.
Background: The effect of chronic exercise activity on brown adipose tissue (BAT) is not clear, with some studies showing positive and others showing negative associations. Chronic exercise is associated with increased resting energy expenditure (REE) secondary to increased lean mass and a probable increase in BAT. Many athletes are in a state of relative energy deficit suggested by lower fat mass and hypothalamic amenorrhea. States of severe energy deficit such as anorexia nervosa are associated with reduced BAT. There are no data regarding the impact of chronic exercise activity on BAT volume or activity in young women and it is unclear whether relative energy deficiency modifies the effects of exercise on BAT. Purpose: We assessed cold induced BAT volume and activity in young female athletes compared with non-athletes, and further evaluated associations of BAT with measures of REE, body composition and menstrual status. Methods: The protocol was approved by our Institutional Review Board. Written informed consent was obtained from all participants prior to study initiation. This was a cross-sectional study of 24 women (16 athletes and8 non-athletes) between 18-25 years of age. Athletes were either oligo-amenorrheic (n = 8) or eumenorrheic (n = 8).We used PET/CT scans to determine cold induced BAT activity, VMAX Encore 29 metabolic cart to obtain measures of REE, and DXA for body composition. Results: Athletes and non-athletes did not differ for age or BMI. Compared with non-athletes, athletes had lower percent body fat (p = 0.002), higher percent lean mass (p = 0.01) and trended higher in REE (p = 0.09). BAT volume and activity in athletes trended lower than in non-athletes (p = 0.06; p = 0.07, respectively). We found negative associations of BAT activity with duration of amenorrhea (r = -0.46, p = 0.02).BAT volume correlated inversely with lean mass (r = -0.46, p = 0.02), and positively with percent body fat, irisin and thyroid hormones. Conclusions: Our study shows a trend for lower BAT in young female athletes compared with non-athletes, and shows associations of brown fat with menstrual status and body composition. Brown fat may undergo adaptive reductions with increasing energy deficit.
Evidence suggests abnormal bioenergetic status throughout the body in psychotic disorders. The present study examined predictors of elevated body mass index (BMI) across diagnostic categories of schizophrenia, schizoaffective and bipolar disorders. In a cross-sectional study, we studied demographic and clinical risk factors for overweight and obesity in a well-characterized sample of 262 inpatients and outpatients with schizophrenia (n=59), schizoaffective disorder (n=81), and bipolar I disorder (n=122). Across the three diagnostic categories, the prevalence of overweight (29.4%) and obesity (33.2%) combined was 62.6% (164/262). Logistic regression analyses, adjusted for age, sex and ethnicity, showed that schizoaffective disorder, lifetime major depressive episode, presence of prior suicide attempt, and more than 5 lifetime hospitalizations were significantly associated with BMI≥25. Patients with schizophrenia had significantly lower risk for overweight and obesity. Overall, we found that affective components of illness were associated with elevated BMI in our cross-diagnostic sample. Our results show that patients with schizoaffective disorder have a greater risk for obesity. Identifying predictors of elevated BMI in patients with psychotic and mood disorders will help prevent obesity and related cardiovascular and cerebral complications. Future studies are needed to elucidate the mechanistic nature of the relationship between obesity and psychiatric illness.
Context: HIV patients are at increased risk for cardiometabolic disease secondary to depot-specific alterations in adipose function, but mechanisms remain poorly understood. Objective: The endoribonuclease Dicer has been linked to modulation of brown and white adipocyte differentiation. We previously demonstrated that Dicer-knockout mice undergo transformation of brown adipose tissue (BAT) to white adipose tissue (WAT) and develop a lipodystrophic phenotype. We hypothesized reduced Dicer and BAT gene expression from non-lipomatous subcutaneous (SC) fat among HIV patients with a lipodystrophic phenotype. Design: 18 HIV (9 with and without lipodystrophic changes in fat distribution, characterized by excess dorsocervical adipose tissue [DCAT]) and 9 non-HIV subjects underwent punch biopsy of abdominal SC fat to determine expression of Dicer and other adipose-related genes. Results: HIV subjects with long duration antiretroviral use demonstrated excess DCAT vs. non-HIV subjects (9.8±1.0 vs. 6.6±0.8cm(2), P=0.02) with similar BMI. Dicer expression was decreased in abdominal SC fat of HIV vs. non-HIV (4.88 [1.91, 11.93] vs.17.69 [10.72, 47.91], P=0.01), as were PGC1α, ZIC1, PRDM16, DIO2, and HSP60 (all P≤0.03). Moreover, expression of Dicer (2.49 [0.02, 4.88] vs. 11.20 [4.83, 21.45], P=0.006), brown fat [PGC1α (P=0.002), ZIC1 (P=0.004), LHX6 (P=0.03), PRDM16 (P=0.0008), PAT2 (P=0.008), P2RX5 (P=0.02)], beige fat [TMEM26 (P=0.004), CD137 (P=0.008)] and other genes [DIO2 (P=0.002), leptin (P=0.003), HSP60 (P=0.0004)] was further decreased in abdominal SC fat comparing HIV subjects with vs. without excess DCAT. Downregulation of Dicer in the abdominal SC fat correlated with downregulation of all brown and beige fat genes (all P≤0.01). Conclusion: Our results demonstrate dysfunctional SC adipose tissue marked by reduced Dicer in relationship to downregulation of brown and beige fat related genes in lipodystrophic HIV patients and may provide a novel mechanism for metabolic dysregulation. A strategy to increase "browning" of WAT may improve cardiometabolic health in HIV.
Brown adipose tissue (BAT) dissipates chemical energy as heat and can counteract obesity. MicroRNAs are emerging as key regulators in development and disease. Combining microRNA and mRNA microarray profiling followed by bioinformatic analyses, we identified miR-455 as a new regulator of brown adipogenesis. miR-455 exhibits a BAT-specific expression pattern and is induced by cold and the browning inducer BMP7. In vitro gain- and loss-of-function studies show that miR-455 regulates brown adipocyte differentiation and thermogenesis. Adipose-specific miR-455 transgenic mice display marked browning of subcutaneous white fat upon cold exposure. miR-455 activates AMPKα1 by targeting HIF1an, and AMPK promotes the brown adipogenic program and mitochondrial biogenesis. Concomitantly, miR-455 also targets the adipogenic suppressors Runx1t1 and Necdin, initiating adipogenic differentiation. Taken together, the data reveal a novel microRNA-regulated signaling network that controls brown adipogenesis and may be a potential therapeutic target for human metabolic disorders.
Targeting brown adipose tissue (BAT) content or activity has therapeutic potential for treating obesity and the metabolic syndrome by increasing energy expenditure. However, both inter- and intra-individual differences contribute to heterogeneity in human BAT and potentially to differential thermogenic capacity in human populations. Here we generated clones of brown and white preadipocytes from human neck fat and characterized their adipogenic and thermogenic differentiation. We combined an uncoupling protein 1 (UCP1) reporter system and expression profiling to define novel sets of gene signatures in human preadipocytes that could predict the thermogenic potential of the cells once they were maturated. Knocking out the positive UCP1 regulators, PREX1 and EDNRB, in brown preadipocytes using CRISPR-Cas9 markedly abolished the high level of UCP1 in brown adipocytes differentiated from the preadipocytes. Finally, we were able to prospectively isolate adipose progenitors with great thermogenic potential using the cell surface marker CD29. These data provide new insights into the cellular heterogeneity in human fat and offer potential biomarkers for identifying thermogenically competent preadipocytes.
Brown adipose tissue (BAT) acts in mammals as a natural defense system against hypothermia, and its activation to a state of increased energy expenditure is believed to protect against the development of obesity. Even though the existence of BAT in adult humans has been widely appreciated, its cellular origin and molecular identity remain elusive largely because of high cellular heterogeneity within various adipose tissue depots. To understand the nature of adult human brown adipocytes at single cell resolution, we isolated clonally derived adipocytes from stromal vascular fractions of adult human BAT from two individuals and globally analyzed their molecular signatures. We used RNA sequencing followed by unbiased genome-wide expression analyses and found that a population of uncoupling protein 1 (UCP1)-positive human adipocytes possessed molecular signatures resembling those of a recruitable form of thermogenic adipocytes (that is, beige adipocytes). In addition, we identified molecular markers that were highly enriched in UCP1-positive human adipocytes, a set that included potassium channel K3 (KCNK3) and mitochondrial tumor suppressor 1 (MTUS1). Further, we functionally characterized these two markers using a loss-of-function approach and found that KCNK3 and MTUS1 were required for beige adipocyte differentiation and thermogenic function. The results of this study present new opportunities for human BAT research, such as facilitating cell-based disease modeling and unbiased screens for thermogenic regulators.
- Jan 2015
Increasing energy expenditure through activation of endogenous brown adipose tissue (BAT) is a potential approach to treat obesity and diabetes. The class of β3-adrenergic receptor (AR) agonists stimulates rodent BAT, but this activity has never been demonstrated in humans. Here we determined the ability of 200 mg oral mirabegron (Myrbetriq, Astellas Pharma, Inc.), a β3-AR agonist currently approved to treat overactive bladder, to stimulate BAT as compared to placebo. Mirabegron led to higher BAT metabolic activity as measured via (18)F-fluorodeoxyglucose ((18)F-FDG) using positron emission tomography (PET) combined with computed tomography (CT) in all twelve healthy male subjects (p = 0.001), and it increased resting metabolic rate (RMR) by 203 ± 40 kcal/day (+13%; p = 0.001). BAT metabolic activity was also a significant predictor of the changes in RMR (p = 0.006). Therefore, a β3-AR agonist can stimulate human BAT thermogenesis and may be a promising treatment for metabolic disease. Copyright © 2015 Elsevier Inc. All rights reserved.
- Jul 2014
White, beige, and brown adipocytes are developmentally and functionally distinct but often occur mixed together within individual depots. To target white, beige, and brown adipocytes for diagnostic or therapeutic purposes, a better understanding of the cell surface properties of these cell types is essential. Using a combination of in silico, in vitro, and in vivo methods, we have identified three new cell surface markers of adipose cell types. The amino acid transporter ASC-1 is a white adipocyte-specific cell surface protein, with little or no expression in brown adipocytes, whereas the amino acid transporter PAT2 and the purinergic receptor P2RX5 are cell surface markers expressed in classical brown and beige adipocytes in mice. These markers also selectively mark brown/beige and white adipocytes in human tissue. Thus, ASC-1, PAT2, and P2RX5 are membrane surface proteins that may serve as tools to identify and target white and brown/beige adipocytes for therapeutic purposes.
Brown fat can reduce obesity through the dissipation of calories as heat. Control of thermogenic gene expression occurs via the induction of various coactivators, most notably PGC-1α. In contrast, the transcription factor partner(s) of these cofactors are poorly described. Here, we identify interferon regulatory factor 4 (IRF4) as a dominant transcriptional effector of thermogenesis. IRF4 is induced by cold and cAMP in adipocytes and is sufficient to promote increased thermogenic gene expression, energy expenditure, and cold tolerance. Conversely, knockout of IRF4 in UCP1+ cells causes reduced thermogenic gene expression and energy expenditure, obesity, and cold intolerance. IRF4 also induces the expression of PGC-1α and PRDM16 and interacts with PGC-1α, driving Ucp1 expression. Finally, cold, β-agonists, or forced expression of PGC-1α are unable to cause thermogenic gene expression in the absence of IRF4. These studies establish IRF4 as a transcriptional driver of a program of thermogenic gene expression and energy expenditure.
miRNAs are important regulators of biological processes in many tissues, including the differentiation and function of brown and white adipocytes. The endoribonuclease dicer is a major component of the miRNA-processing pathway, and in adipose tissue, levels of dicer have been shown to decrease with age, increase with caloric restriction, and influence stress resistance. Here, we demonstrated that mice with a fat-specific KO of dicer develop a form of lipodystrophy that is characterized by loss of intra-abdominal and subcutaneous white fat, severe insulin resistance, and enlargement and "whitening" of interscapular brown fat. Additionally, KO of dicer in cultured brown preadipocytes promoted a white adipocyte-like phenotype and reduced expression of several miRNAs. Brown preadipocyte whitening was partially reversed by expression of miR-365, a miRNA known to promote brown fat differentiation; however, introduction of other miRNAs, including miR-346 and miR-362, also contributed to reversal of the loss of the dicer phenotype. Interestingly, fat samples from patients with HIV-related lipodystrophy exhibited a substantial downregulation of dicer mRNA expression. Together, these findings indicate the importance of miRNA processing in white and brown adipose tissue determination and provide a potential link between this process and HIV-related lipodystrophy.
- Sep 2013
For brown adipose tissue (BAT) to be effective at consuming calories, its blood flow must increase enough to provide sufficient fuel to sustain energy expenditure and also transfer the heat created to avoid thermal injury. Here we used a combination of human and rodent models to assess changes in BAT blood flow and glucose utilization. (99m)Tc-methoxyisobutylisonitrile (MIBI) SPECT (n = 7) and SPECT/CT (n = 74) scans done in adult humans for parathyroid imaging were reviewed for uptake in regions consistent with human BAT. Site-directed biopsies of subcutaneous and deep neck fat were obtained for electron microscopy and gene expression profiling. In mice, tissue perfusion was measured with (99m)Tc-MIBI (n = 16) and glucose uptake with (18)F-FDG (n = 16). Animals were kept fasting overnight, anesthetized with pentobarbital, and given intraperitoneally either the β3-adrenergic receptor agonist CL-316,243, 1 mg/kg (n = 8), or saline (n = 8) followed by radiotracer injection 5 min later. After 120 min, the mice were imaged using SPECT/CT or PET/CT. Vital signs were recorded over 30 min during the imaging. BAT, white adipose tissue (WAT), muscle, liver, and heart were resected, and tissue uptake of both (99m)Tc-MIBI and (18)F-FDG was quantified by percentage injected dose per gram of tissue and normalized to total body weight. In 5.4% of patients (4/74), (99m)Tc-MIBI SPECT/CT showed increased retention in cervical and supraclavicular fat that displayed multilocular lipid droplets, dense capillary investment, and a high concentration of ovoid mitochondria. Expression levels of the tissue-specific uncoupling protein-1 were 180 times higher in BAT than in subcutaneous WAT (P < 0.001). In mice, BAT tissue perfusion increased by 61% (P < 0.01), with no significant changes in blood flow to WAT, muscle, heart, or liver. CL-316,243 increased glucose uptake in BAT even more, by 440% (P < 0.01). Pharmacologic activation of BAT requires increased blood flow to deliver glucose and oxygen for thermogenesis. However, the glucose consumption far exceeds the vascular response. These findings demonstrate that activated BAT increases glucose uptake beyond what might occur by increased blood flow alone and suggest that activated BAT likely uses glucose for nonthermogenic purposes.
The aim of this study was to assess the volume and function of human brown adipose tissue (BAT) in vivo using MR imaging. BAT volumes under thermoneutral conditions in the cervical areas were assessed via water-fat contrast using the Dixon method and via water-saturation efficiency using fast spin-echo and T2-weighted images. The existence of cervical BAT was also assessed by (18)F-FDG PET/CT scans in the same subjects. BAT functionality was assessed via functional MR imaging (fMRI) blood oxygenation level-dependent (BOLD) signal changes in response to a mild cold challenge. Under thermoneutral conditions, we were able to distinguish BAT from white adipose tissue in the cervical and supraclavicular fat. BAT showed higher water-to-fat contrast and higher water-saturation efficiency in MR imaging scans. The location and volume of BAT assessed by MR imaging were comparable to the measurements by (18)F-FDG PET/CT scans. During mild cold challenge, BOLD fMRI signal increased in BAT by 10.7% ± 1.8% (P < 0.01). We demonstrated the feasibility of using MR imaging and fMRI to assess BAT volume and BAT responses to mild cold stimulation in the cervical areas of human subjects.
The imbalance between energy intake and expenditure is the underlying cause of the current obesity and diabetes pandemics. Central to these pathologies is the fat depot: white adipose tissue (WAT) stores excess calories, and brown adipose tissue (BAT) consumes fuel for thermogenesis using tissue-specific uncoupling protein 1 (UCP1). BAT was once thought to have a functional role in rodents and human infants only, but it has been recently shown that in response to mild cold exposure, adult human BAT consumes more glucose per gram than any other tissue. In addition to this nonshivering thermogenesis, human BAT may also combat weight gain by becoming more active in the setting of increased whole-body energy intake. This phenomenon of BAT-mediated diet-induced thermogenesis has been observed in rodents and suggests that activation of human BAT could be used as a safe treatment for obesity and metabolic dysregulation. In this study, we isolated anatomically defined neck fat from adult human volunteers and compared its gene expression, differentiation capacity and basal oxygen consumption to different mouse adipose depots. Although the properties of human neck fat vary substantially between individuals, some human samples share many similarities with classical, also called constitutive, rodent BAT.
Maintenance of body temperature is essential for the survival of homeotherms. Brown adipose tissue (BAT) is a specialized fat tissue that is dedicated to thermoregulation. Owing to its remarkable capacity to dissipate stored energy and its demonstrated presence in adult humans, BAT holds great promise for the treatment of obesity and metabolic syndrome. Rodent data suggest the existence of two types of brown fat cells: constitutive BAT (cBAT), which is of embryonic origin and anatomically located in the interscapular region of mice; and recruitable BAT (rBAT), which resides within white adipose tissue (WAT) and skeletal muscle, and has alternatively been called beige, brite or inducible BAT. Bone morphogenetic proteins (BMPs) regulate the formation and thermogenic activity of BAT. Here we use mouse models to provide evidence for a systemically active regulatory mechanism that controls whole-body BAT activity for thermoregulation and energy homeostasis. Genetic ablation of the type 1A BMP receptor (Bmpr1a) in brown adipogenic progenitor cells leads to a severe paucity of cBAT. This in turn increases sympathetic input to WAT, thereby promoting the formation of rBAT within white fat depots. This previously unknown compensatory mechanism, aimed at restoring total brown-fat-mediated thermogenic capacity in the body, is sufficient to maintain normal temperature homeostasis and resistance to diet-induced obesity. These data suggest an important physiological cross-talk between constitutive and recruitable brown fat cells. This sophisticated regulatory mechanism of body temperature may participate in the control of energy balance and metabolic disease.
- Oct 2012
Background: HIV lipodystrophy - characterized by peripheral lipoatrophy, with or without central fat accumulation - confers increased metabolic risk. However, the functional activity of HIV lipodystrophic tissue in relation to metabolic risk has yet to be fully explored in vivo through the use of non-invasive imaging techniques. This study assesses the relationship between FDG uptake in various fat depots and metabolic/immune parameters among subjects with HIV lipodystrophy. Methods: Lipodystrophic men on antiretroviral therapy underwent whole-body (18)F-fluorodeoxyglucose positron emission tomography (FDG-PET)/computed tomography scans and detailed metabolic/immune phenotyping. Results: FDG uptake in the subcutaneous adipose tissue (SAT) of the extremities (mean standardized uptake value [SUV] of the arm and leg SAT) was found to correlate with the degree of peripheral lipoatrophy (r=0.7; P=0.01). Extremity SAT FDG uptake was positively associated with homeostasis model assessment of insulin resistance (HOMA-IR; r=0.6; P=0.02) and fasting hyperinsulinaemia (r=0.7; P=0.01), while fat percentage of extremities was not. Furthermore, extremity SAT FDG uptake was significantly associated with CD4(+) T-cell count (r=0.6; P=0.05). In multivariate modelling for HOMA-IR, extremity SAT FDG uptake remained significant after controlling for body mass index and tumour necrosis factor-α (R(2) for model =0.71, P=0.02; SUV in the extremity SAT β-estimate 12.3, P=0.009). Conclusions: In HIV lipodystrophic patients, extremity SAT FDG uptake is increased in association with reduced extremity fat and may contribute to insulin resistance. Non-invasive assessments of in situ inflammation using FDG-PET may usefully complement histological and gene expression analyses of metabolic dysregulation in peripheral fat among HIV-positive patients.
As potential activators of brown adipose tissue (BAT), mild cold exposure and sympathomimetic drugs have been considered as treatments for obesity and diabetes, but whether they activate the same pathways is unknown. In 10 healthy human volunteers, we found that the sympathomimetic ephedrine raised blood pressure, heart rate, and energy expenditure, and increased multiple circulating metabolites, including glucose, insulin, and thyroid hormones. Cold exposure also increased blood pressure and energy expenditure, but decreased heart rate and had little effect on metabolites. Importantly, cold increased BAT activity as measured by (18)F-fluorodeoxyglucose PET-CT in every volunteer, whereas ephedrine failed to stimulate BAT. Thus, at doses leading to broad activation of the sympathetic nervous system, ephedrine does not stimulate BAT in humans. In contrast, mild cold exposure stimulates BAT energy expenditure with fewer other systemic effects, suggesting that cold activates specific sympathetic pathways. Agents that mimic cold activation of BAT could provide a promising approach to treating obesity while minimizing systemic effects.
- Feb 2012
Brown adipose tissue (BAT) is the primary tissue responsible for nonshivering thermogenesis in mammals. The amount of BAT and its level of activation help regulate the utilization of excessive calories for thermogenesis as opposed to storage in white adipose tissue (WAT) which would lead to weight gain. Over the past several years, BAT activity in vivo has been primarily assessed by positron emission tomography-computed tomography (PET-CT) scan using 2-[18F]-fluoro-2-deoxy-D-glucose (18F-FDG) to measure glucose utilization associated with BAT mitochondrial respiration. In this study, we demonstrate the feasibility of mapping and estimating BAT volume and metabolic function in vivo in rats at a 9.4T magnetic resonance imaging (MRI) scanner using sequences available from clinical MR scanners. Based on the morphological characteristics of BAT, we measured the volume distribution of BAT with MRI sequences that have strong fat-water contrast. We also investigated BAT volume by utilizing spin-echo MRI sequences. The in vivo MRI-estimated BAT volumes were correlated with direct measurement of BAT mass from dissected samples. Using MRI, we also were able to map hemodynamic responses to changes in BAT metabolism induced pharmacologically by β3-adrenergic receptor agonist, CL-316,243 and compare this to BAT activity in response to CL-316,243 assessed by PET 18F-FDG. In conclusion, we demonstrate the feasibility of measuring BAT volume and function in vivo using routine MRI sequences. The MRI measurement of BAT volume is consistent with quantitative measurement of the tissue ex vivo.
The pathogenesis and function of dorsocervical sc adipose tissue (DSAT) accumulation in HIV-infected patients is not known. Previous investigations using either UCP-1 expression or positron emission tomography have been inconclusive as to whether this depot represents brown adipose tissue (BAT). We investigated DSAT gene expression, including DIO2, a deiodinase that contributes to increased thermogenesis in brown fat, and simultaneously determined [¹⁸F]fluorodeoxyglucose ([¹⁸F]FDG) uptake in lipodystrophic HIV and healthy control subjects. Thirteen HIV-infected and three non-HIV-infected men were recruited. HIV-infected subjects had evidence of significant lipodystrophy, including fat atrophy of the face, arms, and legs, and/or fat accumulation of the neck and abdomen. Subjects were cooled, followed by [¹⁸F]FDG positron emission tomography/computed tomography, fat biopsy of DSAT, and measurement of resting energy expenditure (REE). HIV-infected subjects were characterized as lipohypertrophic and lipoatrophic and compared. Mean standardized uptake value of [¹⁸F]FDG and UCP-1 expression were not significantly different in DSAT among the groups. However, lipohypertrophic subjects demonstrated increased expression of DIO2 in DSAT compared with lipoatrophic subjects (P = 0.03). Among HIV-infected patients, DIO2 expression was strongly related to REE (r = 0.78, P = 0.002) and was a predictor of REE in multivariate modeling controlling for age, TSH, and lean body mass (r² = 0.79, P = 0.008). One control subject demonstrated typical BAT in the supraclavicular area. Adipose tissue accumulating in the dorsocervical area in HIV lipodystrophy does not appear to be classical BAT. However, DIO2 expression is increased in DSAT among patients with HIV lipodystrophy, particularly those with increased visceral adiposity, and is positively associated with energy expenditure.
- Sep 2011
Islet β-cells express both insulin receptors and insulin signaling proteins. Recent studies suggest insulin signaling is physiologically important for glucose sensing. Preexposure to insulin enhances glucose-stimulated insulin secretion (GSIS) in healthy humans. We evaluated whether the effect of insulin to potentiate GSIS is modulated through regulation of free fatty acids (FFA). Subjects were studied on three occasions in this single-site study at an academic institution clinical research center. Subjects included nine healthy volunteers. Glucose-induced insulin response was assessed on three occasions after 4 h saline (low insulin/sham) or isoglycemic-hyperinsulinemic (high insulin) clamps with or without intralipid and heparin infusion, using B28 Asp-insulin that could be distinguished from endogenous insulin immunologically. During the last 80 min of all three clamps, additional glucose was administered to stimulate insulin secretion (GSIS) with glucose concentrations maintained at similar concentrations during all studies. β-Cell response to glucose stimulation was assessed. Preexposure to exogenous insulin increased the endogenous insulin-secretory response to glucose by 32% compared with sham clamp (P = 0.001). This was accompanied by a drop in FFA during hyperinsulinemic clamp compared with the sham clamp (0.06 ± 0.02 vs. 0.60 ± 0.09 mEq/liter, respectively), which was prevented during the hyperinsulinemic clamp with intralipid/heparin infusion (1.27 ± 0.17 mEq/liter). After preexposure to insulin with intralipid/heparin infusion to maintain FFA concentration, GSIS was 21% higher compared with sham clamp (P < 0.04) and similar to preexposure to insulin without intralipid/heparin (P = 0.2). Insulin potentiates glucose-stimulated insulin response independent of FFA concentrations in healthy humans.
Brown adipose tissue plays an important role in obesity, insulin resistance, and diabetes. We have previously shown that the transition from brown preadipocytes to mature adipocytes is mediated in part by insulin receptor substrate (IRS)-1 and the cell cycle regulator protein necdin. In this study, we used pharmacological inhibitors and adenoviral dominant negative constructs to demonstrate that this transition involves IRS-1 activation of Ras and ERK1/2, resulting in phosphorylation of cAMP response element-binding protein (CREB) and suppression of necdin expression. This signaling did not include an elevation of intracellular calcium. A constitutively active form of CREB expressed in IRS-1 knockout cells decreased necdin promoter activity, necdin mRNA, and necdin protein levels, leading to a partial restoration of differentiation. By contrast, forkhead box protein (Fox)O1, which is regulated by the phosphoinositide 3 kinase-Akt pathway, increased necdin promoter activity. Based on reporter gene assays using truncations of the necdin promoter and chromatin immunoprecipitation studies, we demonstrated that CREB and FoxO1 are recruited to the necdin promoter, likely interacting with specific consensus sequences in the proximal region. Based on these results, we propose that insulin/IGF-I act through IRS-1 phosphorylation to stimulate differentiation of brown preadipocytes via two complementary pathways: 1) the Ras-ERK1/2 pathway to activate CREB and 2) the phosphoinositide 3 kinase-Akt pathway to deactivate FoxO1. These two pathways combine to decrease necdin levels and permit the clonal expansion and coordinated gene expression necessary to complete brown adipocyte differentiation.
- Aug 2011
To evaluate the prevalence and factors affecting the detection of active brown adipose tissue (BAT) in children and adolescents using (18)F-fluorodeoxyglucose positron emission tomography. A total of 385 positron emission tomography scans performed for various oncologic indications in 172 patients aged 5-21 years were reviewed. BAT activity was detected by visual inspection as present or absent in the neck, thorax, and abdomen based on its well-characterized and typical appearance and then quantified by comparing the (18)F-fluorodeoxyglucose activity in the cervical-supraclavicular depots with that measured in the liver. Clinical indices were recorded. The BAT detection rate was not significantly different between boys and girls (43.3% vs 45.3%). BAT activity was found most often in the cervical-supraclavicular depots. The highest percentage of patients with detectable BAT and the highest BAT/liver activity were in the 13- to 14.99-year age group in both males and females (P = .005). Body mass index percentile correlated inversely with BAT activity (P = .012). BAT activity did not correlate with outdoor temperature or clinical diagnosis. Under typical clinical imaging conditions, BAT is detected more frequently in children than in adults. BAT activity increases from childhood into adolescence, when it is detected in almost half of patients, and it correlates inversely with obesity, suggesting that BAT may play a prominent role in pediatric metabolism.
- Jun 2010
Obesity develops when energy intake exceeds energy expenditure. Although most current obesity therapies are focused on reducing calorific intake, recent data suggest that increasing cellular energy expenditure (bioenergetics) may be an attractive alternative approach. This is especially true for adaptive thermogenesis - the physiological process whereby energy is dissipated in mitochondria of brown fat and skeletal muscle in the form of heat in response to external stimuli. There have been significant recent advances in identifying the factors that control the development and function of these tissues, and in techniques to measure brown fat in human adults. In this article, we integrate these developments in relation to the classical understandings of cellular bioenergetics to explore the potential for developing novel anti-obesity therapies that target cellular energy expenditure.
- May 2010
Children and adults have two major types of adipocytes, which represent the predominant cells in white adipose tissue, which is involved in energy storage, and brown adipose tissue (BAT), which is responsible for thermogenesis and energy expenditure. This review discusses BAT physiology and evaluates the recent discoveries regarding its development, identification, and function. Last year, multiple independent research teams using combined PET and computed tomography imaging, immunohistochemistry, and gene and protein expression have proven conclusively that adult humans have functional BAT. In parallel, basic studies defined BAT origins, its transcriptional regulation, and the role of hormones in BAT growth and activation. These methods have begun to be applied to children to understand pediatric BAT anatomy and physiology. Adult humans have functional BAT, which plays a role in energy balance. BAT is more prevalent in children, suggesting an even greater physiological role than that seen in adults. Future studies will identify safe ways to quantify BAT mass and activity and which interventions might be used to increase BAT mass, thermogenesis, or both to treat obesity.
- Feb 2010
Human fat consists of white and brown adipose tissue (WAT and BAT). Though most fat is energy-storing WAT, the thermogenic capacity of even small amounts of BAT makes it an attractive therapeutic target for inducing weight loss through energy expenditure. This review evaluates the recent discoveries regarding the identification of functional BAT in adult humans and its potential as a therapy for obesity and diabetes. Over the past year, several independent research teams used a combination of positron-emission tomography and computed tomography (PET/CT) imaging, immunohistochemistry, and gene and protein expression assays to prove conclusively that adult humans have functional BAT. This has occurred against a backdrop of basic studies defining the origins of BAT, new components of its transcriptional regulation, and the role of hormones in stimulation of BAT growth and differentiation. Adult humans have functional BAT, a new target for antiobesity and antidiabetes therapies focusing on increasing energy expenditure. Future studies will refine the methodologies used to measure BAT mass and activity, expand our knowledge of critical-control points in BAT regulation, and focus on testing pharmacological agents that increase BAT thermogenesis and help achieve long-lasting weight loss and an improved metabolic profile.
Islet beta-cells express both insulin receptors and insulin-signaling proteins. Recent evidence from rodents in vivo and from islets isolated from rodents or humans suggests that the insulin signaling pathway is physiologically important for glucose sensing. We evaluated whether insulin regulates beta-cell function in healthy humans in vivo. Glucose-induced insulin secretion was assessed in healthy humans following 4-h saline (low insulin/sham clamp) or isoglycemic-hyperinsulinemic (high insulin) clamps using B28-Asp insulin that could be immunologically distinguished from endogenous insulin. Insulin and C-peptide clearance were evaluated to understand the impact of hyperinsulinemia on estimates of beta-cell function. Preexposure to exogenous insulin increased the endogenous insulin secretory response to glucose by approximately 40%. C-peptide response also increased, although not to the level predicted by insulin. Insulin clearance was not saturated at hyperinsulinemia, but metabolic clearance of C-peptide, assessed by infusion of stable isotope-labeled C-peptide, increased modestly during hyperinsulinemic clamp. These studies demonstrate that insulin potentiates glucose-stimulated insulin secretion in vivo in healthy humans. In addition, hyperinsulinemia increases C-peptide clearance, which may lead to modest underestimation of beta-cell secretory response when using these methods during prolonged dynamic testing.
Obesity results from an imbalance between energy intake and expenditure. In rodents and newborn humans, brown adipose tissue helps regulate energy expenditure by thermogenesis mediated by the expression of uncoupling protein 1 (UCP1), but brown adipose tissue has been considered to have no physiologic relevance in adult humans. We analyzed 3640 consecutive (18)F-fluorodeoxyglucose ((18)F-FDG) positron-emission tomographic and computed tomographic (PET-CT) scans performed for various diagnostic reasons in 1972 patients for the presence of substantial depots of putative brown adipose tissue. Such depots were defined as collections of tissue that were more than 4 mm in diameter, had the density of adipose tissue according to CT, and had maximal standardized uptake values of (18)F-FDG of at least 2.0 g per milliliter, indicating high metabolic activity. Clinical indexes were recorded and compared with those of date-matched controls. Immunostaining for UCP1 was performed on biopsy specimens from the neck and supraclavicular regions in patients undergoing surgery. Substantial depots of brown adipose tissue were identified by PET-CT in a region extending from the anterior neck to the thorax. Tissue from this region had UCP1-immunopositive, multilocular adipocytes indicating brown adipose tissue. Positive scans were seen in 76 of 1013 women (7.5%) and 30 of 959 men (3.1%), corresponding to a female:male ratio greater than 2:1 (P<0.001). Women also had a greater mass of brown adipose tissue and higher (18)F-FDG uptake activity. The probability of the detection of brown adipose tissue was inversely correlated with years of age (P<0.001), outdoor temperature at the time of the scan (P=0.02), beta-blocker use (P<0.001), and among older patients, body-mass index (P=0.007). Defined regions of functionally active brown adipose tissue are present in adult humans, are more frequent in women than in men, and may be quantified noninvasively with the use of (18)F-FDG PET-CT. Most important, the amount of brown adipose tissue is inversely correlated with body-mass index, especially in older people, suggesting a potential role of brown adipose tissue in adult human metabolism.
The insulin/IGF-1 (insulin-like growth factor 1) signalling pathway promotes adipocyte differentiation via complex signalling networks. Here, using microarray analysis of brown preadipocytes that are derived from wild-type and insulin receptor substrate (Irs) knockout animals that exhibit progressively impaired differentiation, we define 374 genes/expressed-sequence tags whose expression in preadipocytes correlates with the ultimate ability of the cells to differentiate. Many of these genes, including preadipocyte factor-1 (Pref-1) and multiple members of the Wnt signalling pathway, are related to early adipogenic events. Necdin is also markedly increased in Irs knockout cells that cannot differentiate, and knockdown of necdin restores brown adipogenesis with downregulation of Pref-1 and Wnt10a expression. Insulin receptor substrate proteins regulate a necdin-E2F4 interaction that represses peroxisome-proliferator-activated receptor gamma (PPARgamma) transcription via a cyclic AMP response element binding protein (CREB)-dependent pathway. Together these define a key signalling network that is involved in brown preadipocyte determination.
- Nov 2001
The modulation of glucagon receptor (GR) expression and biological response was investigated in human embryonic kidney cell (HEK-293) clones permanently expressing the GR with different densities. The GR mRNA expression level in these clones was upregulated by cellular cAMP accumulation and presented a good correlation with both the protein expression level and the maximum number of glucagon binding sites. However, the determination of glucagon-induced cAMP accumulation in these cell lines revealed that the enhancement of receptor expression did not lead to a proportional increase in cAMP formation. Under these conditions, the maximum cAMP production induced by NaF and forskolin was not significantly different among selected clones, regardless of the receptor expression level. High receptor-expressing clones showed the greatest susceptibility for agonist-induced desensitization compared with clones with lower GR expression levels. The results of the present study suggest that the GR can recruit non-GR-specific desensitization mechanism(s). Furthermore, the partial inhibition or alteration of the overall cAMP synthesis pathway at the receptor level may be a necessary adaptive step for a cell in response to a massive increase in membrane receptor expression level.
We prepared a stable cell line expressing the glucagon receptor to characterize the effect of G(s)-coupled receptor stimulation on extracellular signal-regulated protein kinase 1/2 (ERK1/2) activity. Glucagon treatment of the cell line caused a dose-dependent increase in cAMP concentration, activation of cAMP-dependent protein kinase (PKA), and transient release of intracellular calcium. Glucagon treatment also caused rapid dose-dependent phosphorylation and activation of mitogen-activated protein kinase kinase/ERK kinase (MEK1/2) and ERK1/2. Inhibition of either PKA or MEK1/2 blocked ERK1/2 activation by glucagon. However, no significant activation of several upstream activators of MEK, including Ras, Rap1, and Raf, was observed in response to glucagon treatment. In addition, chelation of intracellular calcium reduced glucagon-mediated ERK1/2 activation. In transient transfection experiments, glucagon receptor mutants that bound glucagon but failed to increase intracellular cAMP and calcium concentrations showed no glucagon-stimulated ERK1/2 phosphorylation. We conclude that glucagon-induced MEK1/2 and ERK1/2 activation is mediated by PKA and that an increase in intracellular calcium concentration is required for maximal ERK activation.
- May 2000
G protein-coupled receptors (GPCRs) are seven-transmembrane (TM) helical proteins that bind extracellular molecules and transduce signals by coupling to heterotrimeric G proteins in the cytoplasm. The human D4 dopamine receptor is a particularly interesting GPCR because the polypeptide loop linking TM helices 5 and 6 (loop i3) may contain from 2 to 10 similar direct hexadecapeptide repeats. The precise role of loop i3 in D4 receptor function is not known. To clarify the role of loop i3 in G protein coupling, we constructed synthetic genes for the three main D4 receptor variants. D4-2, D4-4, and D4-7 receptors contain 2, 4, and 7 imperfect hexadecapeptide repeats in loop i3, respectively. We expressed and characterized the synthetic genes and found no significant effect of the D4 receptor polymorphisms on antagonist or agonist binding. We developed a cell-based assay where activated D4 receptors coupled to a Pertussis toxin-sensitive pathway to increase intracellular calcium concentration. Studies using receptor mutants showed that the regions of loop i3 near TM helices 5 and 6 were required for G protein coupling. The hexadecapeptide repeats were not required for G protein-mediated calcium flux. Cell membranes containing expressed D4 receptors and receptor mutants were reconstituted with purified recombinant G protein alpha subunits. The results show that each D4 receptor variant is capable of coupling to several G(i)alpha subtypes. Furthermore, there is no evidence of any quantitative difference in G protein coupling related to the number of hexadecapeptide repeats in loop i3. Thus, loop i3 is required for D4 receptors to activate G proteins. However, the polymorphic region of the loop does not appear to affect the specificity or efficiency of G(i)alpha coupling.
These studies were performed to establish whether functional receptors for calcitonin gene-related peptide (CGRP) are present on human dendritic cells (DCs) and to investigate potential immunomodulatory effects of CGRP on DCs other than Langerhans cells. Reverse transcriptase-PCR revealed expression of mRNA for a type 1 CGRP receptor by mature and immature blood-derived DCs. Sequence analysis confirmed the identity of the type 1 CGRP receptor (CGRP-R1). Addition of CGRP (10-7 M) to mature and immature DCs resulted in mobilization of intracellular calcium. Treatment of immature DCs with CGRP (10-7 M), before and after maturation in monocyte-conditioned medium, resulted in decreased cell surface expression of HLA-DR MHC class II and the costimulatory molecule, CD86. Treatment of immature DCs with CGRP (10-7 M) also resulted in decreased expression of CD86, but expression of HLA-DR was unchanged. When CGRP-treated mature DCs were used to stimulate allogeneic T cells, proliferative responses were dampened (approximately 50%), especially at low DC:T cell ratios (1:360). This effect was not observed with CGRP-treated, immature DCs. In contrast, CGRP-treated mature or immature DCs were no less efficient than untreated DCs in driving syngeneic T cell-proliferative responses to staphylococcal enterotoxin B. We conclude that mature and immature DCs express type 1 CGRP receptors and that signaling through these receptors may dampen mature DC-driven T cell proliferation most likely via down-regulation of CD86 and HLA-DR.
- Apr 2000
G protein-coupled receptors (GPCRs) are seven-transmembrane (TM) helical proteins that bind extracellular molecules and transduce signals by coupling to heterotrimeric G proteins in the cytoplasm. The human D4 dopamine receptor is a particularly interesting GPCR because the polypeptide loop linking TM helices 5 and 6 (loop i3) may contain from 2 to 10 similar direct hexadecapeptide repeats. The precise role of loop i3 in D4 receptor function is not known. To clarify the role of loop i3 in G protein coupling, we constructed synthetic genes for the three main D4 receptor variants. D4-2, D4-4, and D4-7 receptors contain 2, 4, and 7 imperfect hexadecapeptide repeats in loop i3, respectively. We expressed and characterized the synthetic genes and found no significant effect of the D4 receptor polymorphisms on antagonist or agonist binding. We developed a cell-based assay where activated D4 receptors coupled to a Pertussis toxin-sensitive pathway to increase intracellular calcium concentration. Studies using receptor mutants showed that the regions of loop i3 near TM helices 5 and 6 were required for G protein coupling. The hexadecapeptide repeats were not required for G protein-mediated calcium flux. Cell membranes containing expressed D4 receptors and receptor mutants were reconstituted with purified recombinant G protein R subunits. The results show that each D4 receptor variant is capable of coupling to several G iR subtypes. Furthermore, there is no evidence of any quantitative difference in G protein coupling related to the number of hexadecapeptide repeats in loop i3. Thus, loop i3 is required for D4 receptors to activate G proteins. However, the polymorphic region of the loop does not appear to affect the specificity or efficiency of GiR coupling.
The glucagon receptor is a member of a distinct class of G protein-coupled receptors (GPCRs) sharing little amino acid sequence homology with the larger rhodopsin-like GPCR family. To identify the components of the glucagon receptor necessary for G-protein coupling, we replaced sequentially all or part of each intracellular loop (i1, i2, and i3) and the C-terminal tail of the glucagon receptor with the 11 amino acids comprising the first intracellular loop of the D4 dopamine receptor. When expressed in transiently transfected COS-1 cells, the mutant receptors fell into two different groups with respect to hormone-mediated signaling. The first group included the loop i1 mutants, which bound glucagon and signaled normally. The second group comprised the loop i2 and i3 chimeras, which caused no detectable adenylyl cyclase activation in COS-1 cells. However, when expressed in HEK 293T cells, the loop i2 or i3 chimeras caused very small glucagon-mediated increases in cAMP levels and intracellular calcium concentrations, with EC50 values nearly 100-fold higher than those measured for wild-type receptor. Replacement of both loops i2 and i3 simultaneously was required to completely abolish G protein signaling as measured by both cAMP accumulation and calcium flux assays. These results show that the i2 and i3 loops play a role in glucagon receptor signaling, consistent with recent models for the mechanism of activation of G proteins by rhodopsin-like GPCRs.
Polyclonal antibodies were prepared against synthetic peptides corresponding to four different extramembrane segments of the rat glucagon receptor. The antibodies bound specifically to native glucagon receptor as judged by immunofluorescence microscopy of cultured cells expressing a synthetic gene for the receptor. Antibodies to peptides designated PR-15 and DK-12 were directed against amino acid residues 103-117 and 126-137, respectively, of the extracellular N-terminal tail. Antibody to peptide KD-14 was directed against residues 206-219 of the first extracellular loop, and antibody to peptide ST-18, against the intracellular C-terminal tail, residues 468-485. The DK-12 and KD-14 antibodies, but not the PR-15 and ST-18 antibodies, could effectively block binding of 125I-labeled glucagon to its receptor in liver membranes. Incubation of these antibodies with rat liver membranes resulted in both a decrease in the maximal hormonal binding capacity and an apparent decrease in glucagon affinity for its receptor. These effects were abolished in the presence of excess specific peptide antigen. In addition, DK-12 and KD-14 antibodies, but not PR-15 and ST-18 antibodies, interfered with glucagon-induced adenylyl cyclase activation in rat liver membranes and behaved as functional glucagon antagonists. These results demonstrate that DK-12 and KD-14 antibodies are pharmacologically active glucagon antagonists and strongly suggest that residues 126-137 of the N-terminal tail and residues 206-219 of the first extracellular loop contain determinants of ligand binding and may comprise the primary ligand-binding site on the glucagon receptor.
Glucagon receptor mutants were characterized with the aim of elucidating minimal structural requirements for proper biosynthesis, ligand binding, and adenylyl cyclase coupling. One N-terminal deletion mutant and five truncation mutants with progressively shorter C termini were expressed in transiently transfected monkey kidney (COS-1) cells. Each truncation mutant was designed so that the truncated C-terminal tail would remain on the cytoplasmic surface of the receptor. In order to characterize the cellular location of the expressed receptor mutants, a highly specific, high affinity antipeptide antibody was prepared against the extracellular, N-terminal tail of the receptor. Immunoblot analysis and immunofluorescence microscopy showed that the presence of all seven putative transmembrane segments, but not not an intact N-terminal tail, was required for cell surface expression of the receptor. Membranes from cells expressing receptor mutants lacking a large portion of the N-terminal tail or any of the seven putative transmembrane segments failed to bind glucagon. Membranes from cells expressing the C-terminal tail truncation mutants, which retained all seven transmembrane segments, bound glucagon with affinities similar to that of the native receptor and activated cellular adenylyl cyclase in response to glucagon. These results indicate that all seven helices are necessary for the proper folding and processing of the glucagon receptor. Glycosylation is not required for the receptor to reach the cell surface, and it may not be required for ligand binding. However, the N-terminal extracellular portion of the receptor is required for ligand binding. Most of the distal C-terminal tail is not necessary for ligand binding, and the absence of the tail may increase slightly the receptor binding affinity for glucagon. The C-terminal tail is also not necessary for adenylyl cyclase coupling and therefore does not play a direct role in G protein (GS) activation by the glucagon receptor.
Various first messengers linked to phospholipase C, including acetylcholine and interleukin 1, regulate the production both of the secreted form of the amyloid protein precursor (APP) and of amyloid beta-protein. We have now identified intracellular signals which are responsible for mediating these effects. We show that activation of phospholipase C may affect APP processing by either of two pathways, one involving an increase in protein kinase C and the other an increase in cytoplasmic calcium levels. The effects of calcium on APP processing appear to be independent of protein kinase C activation. The observed effects of calcium on APP processing may be of therapeutic utility.
Rhodopsin mutants responsible for autosomal dominant retinitis pigmentosa (ADRP) were prepared by site-directed mutagenesis and characterized. The aim was to evaluate ADRP mutations that occur at three locations on the cytoplasmic surface of rhodopsin: Thr-58 near the cytoplasmic border of helix A, the tetrapeptide Leu-68 to Pro-71 in the first cytoplasmic loop, and Arg-135 at the cytoplasmic border of helix C. It was hypothesized that amino acid changes at these sites would result in mutant rhodopsins with normal spectral properties but defects in their ability to interact with the rod outer segment G protein, transducin. A set of 12 mutant opsin genes was prepared. Four of the mutants were known to cause ADRP: Thr-58 replaced by Arg, a four-amino acid deletion (Leu-68/Arg-69/Thr-70/Pro-71), Arg-135 replaced by Leu, and Arg-135 replaced by Trp. Eight additional mutants were prepared to provide complementary structure-function information. The four-amino acid deletion mutant failed to bind 11-cis-retinal. However, each of the Thr-58 and Arg-135 mutants bound 11-cis-retinal to form a pigment with a visible absorbance maximum (lambda max) of 500 nm. Upon illumination, each pigment was converted to a metarhodopsin II-like spectral form (lambda max = 380 nm). However, each of these spectrally normal ADRP mutants was defective in activating guanine nucleotide exchange by transducin. These results identify a defect in the signal transduction pathway in spectrally normal mutant rhodopsins that cause ADRP.
- Aug 1991
An assay for the antileukaemic agent cytosine-beta-D-arabinoside (ara-C) has been developed using capillary zone electrophoresis. Solid-phase extraction and on-capillary peak concentration are used to improve the detection limit. The electrophoretic separation time is less than 5 min. The limit of detection for ara-C in plasma is 0.5 microM (signal-to-noise ratio = 3). The assay has been validated for the determination of ara-C in human plasma over the concentration range 1-10 microM. The calibration curve was linear with a correlation coefficient r2 = 0.996. At an ara-C concentration of 8 microM the intra-day coefficient of variation was 9.1% and the inter-day coefficient of variation was 12.3%. At an ara-C concentration of 2 microM the coefficients of variation were 15.2 and 12.0%, respectively.
Variation in skin-to-kidney center distance has been shown to have a significant influence on quantification of renal function with the gamma camera. Several techniques to compensate for this variability have been proposed in adults, yet it has been suggested that depth correction is not necessary for quantitative renography in children. Skin-to-kidney center distances were measured from computed tomograms in 53 supine pediatric patients. Nearly 40% of the kidneys examined varied more than 1 cm from the average renal depth, and 8% deviated more than 2 cm. Right kidney depth differed from left kidney depth by more than 1 cm in less than 10% of the patients. Measurements were in agreement with regression equations based on lateral scintigraphy in children, but were consistently underestimated by nomograms developed for skin-to-kidney center distance in adults. Failure to recognize interindividual variability in skin-to-kidney center distance can introduce significant errors in quantitative pediatric renography.