Targeting brown adipose tissue (BAT) content or activity has therapeutic potential for treating o... more 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 therm...
Brown adipose tissue (BAT) acts in mammals as a natural defense system against hypothermia, and i... more 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 mole...
As part of a current worldwide effort to understand the physiology of human BAT (hBAT) and whethe... more As part of a current worldwide effort to understand the physiology of human BAT (hBAT) and whether its thermogenic activity can be manipulated to treat obesity, the workshop "Exploring the Roles of Brown Fat in Humans" was convened at the National Institutes of Health on February 25-26, 2014. Presentations and discussion indicated that hBAT and its physiological roles are highly complex, and research is needed to understand the health impact of hBAT beyond thermogenesis and body weight regulation, and to define its interactions with core physiological processes like glucose homeostasis, cachexia, physical activity, bone structure, sleep, and circadian rhythms.
Proceedings of The National Academy of Sciences, 2001
We prepared a stable cell line expressing the glucagon receptor to characterize the effect of Gs-... more We prepared a stable cell line expressing the glucagon receptor to characterize the effect of Gs-coupled receptor stimulation on extracellular signal-regulated protein kinase 1͞2 (ERK1͞2) 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 kinase͞ ERK kinase (MEK1͞2) and ERK1͞2. Inhibition of either PKA or MEK1͞2 blocked ERK1͞2 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 ERK1͞2 activation. In transient transfection experiments, glucagon receptor mutants that bound glucagon but failed to increase intracellular cAMP and calcium concentrations showed no glucagon-stimulated ERK1͞2 phosphorylation. We conclude that glucagon-induced MEK1͞2 and ERK1͞2 activation is mediated by PKA and that an increase in intracellular calcium concentration is required for maximal ERK activation.
Proceedings of the National Academy of Sciences of the United States of America, Jan 19, 2012
As potential activators of brown adipose tissue (BAT), mild cold exposure and sympathomimetic dru... more 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 sy...
White, beige, and brown adipocytes are developmentally and functionally distinct but often occur ... more 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.
Proceedings of the National Academy of Sciences, 1996
Polyclonal antibodies were prepared against synthetic peptides corresponding to four different ex... more 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 Cterminal 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 1251-labeled glucagon to its receptor in
Proceedings of the National Academy of Sciences, 2010
Islet β-cells express both insulin receptors and insulin-signaling proteins. Recent evidence from... more Islet β-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 β-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 β-cell function. Preexposure to exogenous insulin increased the endogenous insulin secretory response to glucose by ≈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 β-cell secretory response when using these methods during prolonged dynamic testing. beta cell | type 2 diabetes mellitus | insulin resistance | insulin clearance |
BACKGROUND-Obesity results from an imbalance between energy intake and expenditure. In rodents an... more BACKGROUND-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.
The insulin/IGF-1 (insulin-like growth factor 1) signalling pathway promotes adipocyte differenti... more 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 γ (PPARγ) 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.
To evaluate the prevalence and factors affecting the detection of active brown adipose tissue (BA... more 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.
For brown adipose tissue (BAT) to be effective at consuming calories, its blood flow must increas... more 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. Methods: 99m Tc-methoxyisobutylisonitrile (MIBI) SPECT (n 5 7) and SPECT/CT (n 5 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 5 16) and glucose uptake with 18 F-FDG (n 5 16). Animals were kept fasting overnight, anesthetized with pentobarbital, and given intraperitoneally either the b 3adrenergic receptor agonist CL-316,243, 1 mg/kg (n 5 8), or saline (n 5 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. Results: 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). Conclusion: 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 glucagon receptor is a member of a distinct class of G protein-coupled receptors (GPCRs) shar... more 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.
G protein-coupled receptors (GPCRs) are seven-transmembrane (TM) helical proteins that bind extra... more 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 i R 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 R coupling.
Brown fat can reduce obesity through the dissipation of calories as heat. Control of thermogenic ... more 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-1a. 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-1a and PRDM16 and interacts with PGC-1a, driving Ucp1 expression. Finally, cold, b-agonists, or forced expression of PGC-1a 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.
Targeting brown adipose tissue (BAT) content or activity has therapeutic potential for treating o... more 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 therm...
Brown adipose tissue (BAT) acts in mammals as a natural defense system against hypothermia, and i... more 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 mole...
As part of a current worldwide effort to understand the physiology of human BAT (hBAT) and whethe... more As part of a current worldwide effort to understand the physiology of human BAT (hBAT) and whether its thermogenic activity can be manipulated to treat obesity, the workshop "Exploring the Roles of Brown Fat in Humans" was convened at the National Institutes of Health on February 25-26, 2014. Presentations and discussion indicated that hBAT and its physiological roles are highly complex, and research is needed to understand the health impact of hBAT beyond thermogenesis and body weight regulation, and to define its interactions with core physiological processes like glucose homeostasis, cachexia, physical activity, bone structure, sleep, and circadian rhythms.
Proceedings of The National Academy of Sciences, 2001
We prepared a stable cell line expressing the glucagon receptor to characterize the effect of Gs-... more We prepared a stable cell line expressing the glucagon receptor to characterize the effect of Gs-coupled receptor stimulation on extracellular signal-regulated protein kinase 1͞2 (ERK1͞2) 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 kinase͞ ERK kinase (MEK1͞2) and ERK1͞2. Inhibition of either PKA or MEK1͞2 blocked ERK1͞2 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 ERK1͞2 activation. In transient transfection experiments, glucagon receptor mutants that bound glucagon but failed to increase intracellular cAMP and calcium concentrations showed no glucagon-stimulated ERK1͞2 phosphorylation. We conclude that glucagon-induced MEK1͞2 and ERK1͞2 activation is mediated by PKA and that an increase in intracellular calcium concentration is required for maximal ERK activation.
Proceedings of the National Academy of Sciences of the United States of America, Jan 19, 2012
As potential activators of brown adipose tissue (BAT), mild cold exposure and sympathomimetic dru... more 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 sy...
White, beige, and brown adipocytes are developmentally and functionally distinct but often occur ... more 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.
Proceedings of the National Academy of Sciences, 1996
Polyclonal antibodies were prepared against synthetic peptides corresponding to four different ex... more 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 Cterminal 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 1251-labeled glucagon to its receptor in
Proceedings of the National Academy of Sciences, 2010
Islet β-cells express both insulin receptors and insulin-signaling proteins. Recent evidence from... more Islet β-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 β-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 β-cell function. Preexposure to exogenous insulin increased the endogenous insulin secretory response to glucose by ≈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 β-cell secretory response when using these methods during prolonged dynamic testing. beta cell | type 2 diabetes mellitus | insulin resistance | insulin clearance |
BACKGROUND-Obesity results from an imbalance between energy intake and expenditure. In rodents an... more BACKGROUND-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.
The insulin/IGF-1 (insulin-like growth factor 1) signalling pathway promotes adipocyte differenti... more 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 γ (PPARγ) 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.
To evaluate the prevalence and factors affecting the detection of active brown adipose tissue (BA... more 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.
For brown adipose tissue (BAT) to be effective at consuming calories, its blood flow must increas... more 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. Methods: 99m Tc-methoxyisobutylisonitrile (MIBI) SPECT (n 5 7) and SPECT/CT (n 5 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 5 16) and glucose uptake with 18 F-FDG (n 5 16). Animals were kept fasting overnight, anesthetized with pentobarbital, and given intraperitoneally either the b 3adrenergic receptor agonist CL-316,243, 1 mg/kg (n 5 8), or saline (n 5 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. Results: 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). Conclusion: 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 glucagon receptor is a member of a distinct class of G protein-coupled receptors (GPCRs) shar... more 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.
G protein-coupled receptors (GPCRs) are seven-transmembrane (TM) helical proteins that bind extra... more 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 i R 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 R coupling.
Brown fat can reduce obesity through the dissipation of calories as heat. Control of thermogenic ... more 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-1a. 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-1a and PRDM16 and interacts with PGC-1a, driving Ucp1 expression. Finally, cold, b-agonists, or forced expression of PGC-1a 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.
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Papers by Aaron Cypess