The Good Fat: Understanding Adipogenesis and Function of Brown Fat

Journal Articles

Sheng Bi

Bi S. Dorsomedial hypothalamic NPY modulation of ad iposity and thermogenesis. Physiol Behav. 2013. [Epub ahead of print]

Chao PT, Yang L, Aja S, et al. Knockdown of NPY expression in the dorsomedial hypothalamus promotes development of brown adipoxytes and prevents diet-induced obesity. Cell Metab. 2011;13(5):573–83.

Zheng F, Kim YJ, Chao PT, Bi S. Overexpression of neuropeptide Y in the dorsomedial hypothalamus causes hyperphagia and obesity in rats. Obesity (Silver Spring). 2013. [Epub ahead of print]

Sheila Collins

Bordicchia M, Liu D, Amri EZ, et al. Cardiac natriuretic peptides act via p38 MAPK to induce the brown fat thermogenic program in mouse and human adipocytes. J Clin Invest. 2012;122(3):1022–36.

Abdul G. Dulloo

Becattini B, Marone R, Zani F, et al. PI3Ky within a nonhematopoietic cell type negatively regulates diet-induced thermogenesis and promotes obesity and insulin resistance. Proc Natl Acad Sci U S A. 2011;108(42):E854–63.

Dulloo AG. Suppressed thermogenesis as a cause for resistance to slimming and obesity rebound: adaptation or illusion? Int J Obes (Lond). 2007;31(2):201–3.

Dulloo AG. The search for compounds that stimulate thermogenesis in obesity management: from pharmaceuticals to functional food ingredients. Obesity Rev. 2011;12(10):866–83.

Dulloo AG, Jacquet J, Montani JP, Schutz Y. Adaptive thermogenesis in human body weight regulation: more of a concept than a measurable entity? Obes Rev. 2012;13 Suppl 2:105–21.

Hursel R, Riechtbauer W, Dulloo AG, et al. The effects of catechin rich teas and caffeine on energy expenditure and fat oxidation: a meta-analysis. Obes Rev. 2011;12(7):e573–81.

Vicente Gilsanz

Chalfant JS, Smith ML, Hu HH, et al. Inverse association between brown adipose tissue activation and white adipose tissue accumulation in successfully treated pediatric malignancy. Am J Clin Nutr. 2012;95(5):1144–9.

Gilsanz V, Chung SA, Jackson H, et al. Functional brown adipose tissue is related to muscle volume in children and adolescents. J Pediatr. 2011;158(5):722–6.

Gilsanz V, Hu HH, Kajimura S. Relevance of brown adipose tissue in infancy and adolescence. Pediatr Res. 2013;73(1):3–9.

Gilsanz V, Hu HH, Smith ML, et al. The depiction of brown adipose tissue is related to disease status in pediatric patients with lymphoma. Am J Roentgenol. 2012;198(4):909–13.

Gilsanz V, Smith ML, Goodarzian F, et al. Changes in brown adipose tissue in boys and girls during childhood and puberty. J Pediatr. 2012;160(4):604–9.

Hu HH, Gilsanz V. Developments in the imaging of brown adipose tissue and its associations with muscle, puberty, and health in children. Front Endocrinol (Lausanne). 2011;2:33.

Hu HH, Perkins TG, Chia JM, Gilsanz V. Characterization of human brown adipose tissue by chemical-shift water-fat MRI. Am J Roentgenol. 2013;200(1):177–83.

Hu HH, Yin L, Aggabao PC, et al. Comparison of brown and white adipose tissues in infants and children with chemical-shift-encoded water-fat MRI. J Magn Reson Imaging. 2013. [Epub ahead of print]

Katzmarzyk PT, Shen W, Baxter-Jones A, et al. Adiposity in children and adolescents: correlates and clinical consequences of fat stored in specific body depots. Pediatr Obes. 2012;7(5):e42–61.

Ponrartana S, Aggabao PC, Hu HH, et al. Brown adipose tissue and its relationship to bone structure in pediatric patients. J Clin Endocrinol Metab. 2012;97(8):2693–8.

Sharp LZ, Shinoda K, Ohno H, et al. Human BAT possesses molecular signatures that resemble beige/brite cells. PLoS One. 2012;7(11):e4942.

Andrew C. Larner

Derecka M, Gornicka A, Koralov SB, et al. Tyk2 and Stat3 regulate brown adipose tissue differentiation and obesity. Cell Metab. 2012;16(6):814–24.

Devanjan Sikder

Sellayah D, Bharaj P, Sikder D. Orexin is required for brown adipose tissue development, differentiation, and function. Cell Metab. 2011;14(4):478–90.

J. Enrique Silva

Marsili A, Aguayo-Mazzucato C, Chen T, et al. Mice with a targeted deletion of the type 2 deiodinase are insulin resistant and susceptible to diet induced obesity. PLoS One. 2011;6(6):e20832.

Silva JE. Physiological importance and control of non-shivering facultative thermogenesis. Front Biosci (Schol Ed). 2011;3:352–71.

Silva JE. Thermogenic mechanisms and their hormonal regulation. Physiol Rev. 2006;86(2):435–64.

Silva JE, Bianco SD. Thyroid-adrenergic interactions: physiological and clinical implications. Thyroid. 2008;18(2):157–65.

Bruce M. Spiegelman

Bostrom P, Wu J, Jedrychowski MP, et al. A PGC1-α-dependent myokine that drives brown-fat-like development of white fat and thermogenesis. Nature. 2012;481(7382):463–8.

Fisher FM, Kleiner S, Douris N, et al. FGF21 regulates PGC-1α and browning of white adipose tissues in adaptive thermogenesis. Genes Dev. 2012;26(3):271–81.

Ohno H, Shinoda K, Spiegelman BM, Kajimura S. PPARγ agonists induce a white-to-brown fat conversion through stabilization of PRDM16 protein. Cell Metab. 2012;15(3):395–404.

Ruas JL, White JP, Rao RR, et al. A PGC-1α isoform induced by resistance training regulates skeletal muscle hypertrophy. Cell. 2012;151(6):1319–31.

Seale P, Conroe HM, Estall J, et al. Prdm16 determines the thermogenic program of subcutaneous white adipose tissue in mice. J Clin Invest. 2011;121(1):96–105.

Wu J, Bostrom P, Sparks LM, et al. Beige adipocytes are a distinct type of thermogenic fat cell in mouse and human. Cell. 2012;150(2):366–76.

Wu J, Cohen P, Spiegelman BM. Adaptive thermogenesis in adipocytes: is beige the new brown? Genes Dev. 2013;27(3):234–50.

Anne-Ulrike Trendelenburg

Fournier B, Murray B, Gutzwiller S, et al. Blockade of the activin receptor IIB activates functional brown adipogenesis and thermogenesis by inducing mitochondrial oxidative metabolism. Mol Cell Biol. 2012;32(14):2871–9.

Yu-Hua Tseng

Schultz TJ, Huang P, Huang TL, et al. Brown-fat paucity due to impaired BMP signaling induces compensatory browning of white fat. Nature. 2013;495(7441):379–83.

Tseng YH, Kokkotou E, Schulz TJ, et al. New role of bone morphogenetic protein 7 in brown adipogenesis and energy expenditure. Nature. 2008;454(7207):1000–4.

Zhang H, Schulz TJ, Espinoza DO, et al. Cross talk between insulin and bone morphogenetic protein signaling systems in brown adipogenesis. Mol Cell Biol. 2010;30(17):4224–33.

Richard L. Veech

Clarke K, Tchabanenko K, Pawlosky R, et al. Kinetics, safety and tolerability of (R)-3-hydroxybutyl (R)-3-hydroxybutyrate in healthy adult subjects. Regul Toxicol Pharmacol. 2012;63(3):401–8.

Kashiwaya Y, Pawlosky R, Markis W, et al. A ketone diet increases brain malonyl-CoA and Uncoupling proteins 4 and 5 while decreasing good intake in the normal Wistar Rat. J Biol Chem. 2010;285(34):25950–6.

Srivastava S, Baxa U, Niu G, et al. A ketogenic diet increasese brown adipose tissue mitochondrial proteins and UCP1 levels in mice. Int Union Biochem Mol Biol. 2013;65(1):58–66.

Srivastava S, Kashiwaya Y, King MT, et al. Mitochondrial biogenesis and increased uncoupling protein 1 in brown adipose tissue of mice fed a ketone ester diet. FASEB J. 2012;26(6):2351–62.

Veech RL. The therapeutic implications of ketone bodies: the effects of ketone bodies in pathological conditions: ketosis, ketogenic diet, redox states, insulin resistance, and mitochondrial metabolism. Prostaglandins Leukot Essent Fatty Acids. 2004;70(3):309–19.

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Not all fat is created equal. Anatomists recognized two types of fat—white adipose tissue (WAT) and brown adipose tissue (BAT)—at least 100 years ago. The two fats have opposing roles in metabolism: WAT stores energy from calories in food and BAT uses energy. In rodents, WAT is associated with obesity, and a substantial level of BAT is found in lean animals. Recently, researchers discovered that adult humans also have functional brown fat depots and, as in rodents, it is associated with leanness. This has raised hopes that identifying safe ways to increase this tissue will help combat obesity.

White fat—the adipose tissue that increases with weight gain—stores energy in the form of triglycerides. Each white adipocyte holds a single large lipid droplet and just a few mitochondria. In contrast, brown adipocytes are specialized for energy expenditure. They develop from the skeletal muscle progenitor cell Myf-5, contain several small lipid droplets, and are densely populated with large mitochondria and blood vessels. They are the only cells that commonly express functional levels of uncoupling protein-1 (UCP1). Located on the inner mitochondrial membrane, this key protein alters the movement of protons to uncouple oxygen consumption from the final step in ATP production, thereby releasing this energy as heat. Cold environments and some dietary changes stimulate this process, called nonshivering thermogenesis. This is distinct from the heat produced by muscle tissue (via shivering thermogenesis). BAT can also use both glucose and fatty acids in mitochondrial metabolism.

These PET/CT scans show the activation of BAT at cold temperatures. Left is a lean subject under thermoneutral conditions (1 hour at 22°C) and right is a lean subject under mild cold exposure (2 hours at 16°C). The lack of metabolic activity under warm conditions makes the BAT undetectable. (Image courtesy of Vicente Gilsanz)

Rodents express BAT in interscapular depots between their shoulders. When the animal's central nervous system senses chronic cold, it sends a signal to brown fat via the catecholamine norepinephrine, which, assisted by the thyroid hormone, stimulates BAT to expand and become thermogenically active.

A rodent's white fat is found in both subcutaneous and visceral areas, or depots. There are different types of white fat in these depots. Subcutaneous white fat (sWAT), which is not associated with the insulin resistance and other negative metabolic consequences that are observed with visceral/intra-abdominal obesity, also contains a small number of cells that express UCP1. These cells enable sWAT depots to produce brown-like fat cells—called beige or brite cells—in response to cold or catecholamine exposure when BAT stores are inadequate or missing.

BAT also has anti-obesity properties. BAT is stimulated when rodents are overfed, an apparent physiological effort to curtail weight gain and maintain homeostasis. Mice that are genetically altered to eliminate brown fat production gain weight. When genetic modification eliminates UCP1 production, mice gain weight in a thermoneutral environment. A lack of sWAT depots also correlates with obesity. Conversely, genetically altered mice with an increased amount or activation of brown fat are leaner and healthier, with less weight gain, more insulin sensitivity, lower levels of serum fatty acids, and protection from diabetes and other metabolic sequelae of obesity.

Secreted factors that promote BAT differentiation and function. (Image courtesy of Yu-Hua Tseng)

Interest in brown fat and its potential role in humans has alternated between periods of great excitement and periods in which it has been considered irrelevant. Scientists knew that human newborns have BAT deposits—perhaps to help them adapt to cold exposure outside the womb—but it was assumed that this tissue quickly regressed after birth and played no role in adult humans. Although other BAT depots had been found in adult humans, they were believed to have negligible impact on physiology.

This position was overturned by advances in medical imaging in the early 2000s, when physicians used a combination of positron emission tomography and computed tomography (PET/CT) to assess metastases in cancer patients and detected brown fat deposits. Because these data were published in highly specialized journals, the results went unnoticed by brown fat researchers and did not gain attention until 2007, when research on brown fat in humans began. More recently, technologies enabling much faster and less costly DNA sequencing and analysis, combined with higher-resolution imaging, have yielded an increasingly complex road map of molecules that influence the development and activation of brown fat. Much of the initial work in brown fat research was done by the lab of Bruce M. Spiegelman.

It is now estimated that as little as 50 g of BAT—less than 0.1% of an adult human's body weight—could utilize up to 20% of basal caloric needs if it were maximally stimulated. Transplantation of only 0.1–0.4 g of BAT into the visceral cavity of mice prevented weight gain and improved glucose homeostasis in diet-induced obese mice, and BAT also seemed to enhance resistance from infiltrating pro-inflammatory macrophages. This conference featured speakers from interdisciplinary fields, who reviewed recent discoveries and presented new data highlighting the potential for altering brown fat, with the goal of developing novel therapeutics for treating human obesity and metabolic disease.

Speakers:
Bruce M. Spiegelman, Dana-Farber Cancer Institute, Harvard Medical School
Vicente Gilsanz, Children's Hospital Los Angeles

Highlights

• Human brown fat resembles the beige fat found in rodents.
• A new form of PGC1 protein upregulates the expression of UCP1 and increases thermogenesis.
• Brown fat is prominent in adolescents.
• Chemical-shift-encoded water–fat MRI uses tissue composition differences to detect brown fat and can safely be used to study healthy human subjects because it does not deliver radiation.

Transcriptional control of brown and beige fat

"The interest in my lab for many years has been the regulation, especially the transcription, of the lineage for both white and brown fat cells," said Bruce M. Spiegelman from the Dana-Farber Cancer Institute at Harvard Medical School. His lab elucidated the molecular basis and muscle-like lineage of brown adipose tissue. Nuclear regulatory protein PPARγ is the master regulator of brown fat development. In combination with the PGC1 and PRDM16 proteins, PPARγ turns fibroblasts into brown fat cells. The cells' lineage initially follows the same path as muscle cells, but then diverges in response to PRDM16 signaling. A molecule called UCP1, unique to brown fat, enables the mitochondria of these cells to eliminate their final energy-producing metabolic step and generate heat instead. After summarizing some of this earlier work, Spiegelman discussed his lab's progress in understanding brown-like fat cells called beige or brite cells and concluded by describing their exploration of PGC1α4, a recently identified polypeptide that may promote BAT and its benefits.

Beige fat is the third type of fat cell in rodents, which emerges in subcutaneous white depots that express UCP1. Beige cells—first described in a 2008 paper from Spiegelman's lab—generate heat like "classical" BAT, but are from a different cell lineage. A different lineage means different regulatory proteins, sensitivities, and receptors, which may offer novel therapeutic targets. Spiegelman and his group have discovered two beige cell identities. In their basal (unstimulated) state, beige cells resemble white fat cells. Given a stimulus, such as the signaling molecule cAMP, they suddenly change to resemble brown fat cells. After examining biopsies of adult human brown fat, Spiegelman and his colleagues concluded that "it is much more similar to the rodent's beige fat than to the classical brown fat."

Mice engineered to lack the PRDM16 gene have normal interscapular brown fat depots and visceral white fat depots, but no sWAT and thus no beige fat. Although excessive visceral fat is associated with metabolic disease in rodents and humans, sWAT does not carry this risk and the emergence of beige fat here protects against excessive white fat. Loss of beige fat capability in this knockout mouse resulted in mild weight gain, severe insulin resistance, and deficient glucose uptake. Spiegelman said that "PRDM16 in subcutaneous fat not only plays an important role in the thermogenic program, but is probably a direct suppressor for a pro-inflammatory gene program [involved in metabolic disease]."

He also discussed his lab's work on PGC1α4, a new form of PGC1 they recently discovered. PGC1α4 causes muscle hypertrophy, and elevating PGC1α4 levels significantly upregulates the expression of UCP1 and thus increases thermogenesis. (This observation is in line with the recent discovery that resistance exercise stimulates brown fat development.) These changes hinge on a polypeptide Spiegelman has named meteorin-like because of its striking similarity to the neurite-growth-inducing protein meteorin expressed in the brain. He noted that inducing a 6-fold elevation of the meteorin-like polypeptide in the blood initiates the broadest thermogenic program he has ever seen, and thus he hopes it holds therapeutic potential.

Brown adipose tissue in childhood and adolescence

Vicente Gilsanz from Children's Hospital Los Angeles is a pediatric radiology expert who was intrigued when BAT was unexpectedly discovered in adult humans during PET/CT scans for tumor assessment in cancer patients. Gilsanz and his colleagues soon found "the presence of activated BAT extremely common during puberty." First, these researchers studied 31 children—17 boys and 14 girls—with lymphoma to determine whether BAT in pediatric patients is related to disease status. Only 10% of patients showed BAT at diagnosis, compared to 77% four months later, after successful cancer treatment, regardless of sex or lymphoma type. Those who went from BAT-negative to BAT-positive gained significantly less weight and adipose tissue and had six times less visceral fat than those who remained BAT-negative. Next, Gilsanz retrospectively studied PET/CT scans that showed functionally active BAT in other successfully treated patients. The most important predictor of BAT levels was the amount of muscle development. Muscle tissue more than doubles during puberty, explaining the marked presence of brown fat during that period. Children with the most BAT also had the thickest bones and the lowest adiposity.

BAT contains fat and water, but WAT contains only fat. This makes MRI an accurate and radiation-free technique for imaging brown fat in humans, since the fat and water components of MRI signals can be separated, as this image demonstrates. (Image courtesy of Vincente Gilsanz)

The amount of radiation delivered by PET and CT scans has prevented researchers from studying BAT in healthy children and adults. Gilsanz addressed this barrier with a new MRI-based technique that utilizes the different magnetic resonance frequencies of fat and water. White adipocytes contain only fat while brown adipocytes also contain water, and the MRI detects these differences via chemical shift imaging. Results correlate tightly with PET/CT results, "and we now can assess the fat fractions in infants and very young children," Gilsanz said. An early surprise was the discovery of brown fat in the infant head, in the orbits and the buccal fat pad. Doppler assessment of the BAT in infants identifies it as a highly vascular tissue.

Speakers:
Devanjan Sikder, Sanford-Burnham Medical Research Institute
Yu-Hua Tseng, Harvard Medical School
Andrew C. Larner, Virginia Commonwealth University
Anne-Ulrike Trendelenburg, Novartis Institutes for BioMedical Research

Highlights

• The neuropeptide orexin, which regulates the sleep cycle and hunger, enables PRDM16 protein function and promotes differentiation of brown adipocytes.
• The BMP7 gene is essential for brown adipocyte differentiation.
• The non-kinase portion of the phosphorylating enzyme Tyk2 apparently stabilizes PRDM16 and enables differentiation of brown adipocytes.
• Inhibiting activin A activity promotes skeletal muscle and BAT and reduces visceral fat.

Coercing bad fat to work

Devanjan Sikder from the Diabetes and Obesity Research Center at the Sanford-Burnham Medical Research Institute discussed his work on the regulatory neuropeptide orexin (OX). The two OX variants, A and B, act primarily in the hypothalamus to regulate the sleep–wake cycle, hunger, and metabolism. High levels of OX produce wakefulness and hunger. Low-OX humans develop narcolepsy, a sleep disorder often associated with obesity. Disrupting OX signaling in mice to create a model of narcolepsy also made them hypophagic and obese. Sikder wanted to find out "how one can become obese despite consuming less," so he and his colleagues dissected the orexin signaling pathway and then addressed the anti-obesity properties of beige fat.

Mice fed a high-fat diet developed high levels of the beige fat-associated protein PRDM16 in back subcutaneous white adipose tissue (bsWAT), which comprises a very small fraction of the interscapular fat depot. This tissue also showed the presence of UCP1 and adipocytes with multiple fat globules. Exposing these mice to acute cold initiated thermogenesis in bsWAT adipocytes. White adipose tissue shrank as a result, and darker fat tissue appeared. Sikder found that the induction of thermogenesis he observed involved orexin signaling, specifically via its R2 receptor, in concert with PRDM16. Depleting PRDM16 blocked orexin from functioning, and mice lacking the R2 receptor could not express PRDM16 and became obese.

Next, Sikder began to explore orexin's therapeutic potential. After 17 weeks on a high-fat diet, animals were assigned to twice-weekly orexin or placebo injections for one month. The orexin-treated animals, despite an increased appetite and unchanged activity levels, lost 50% of their fat mass and showed improved glucose metabolism. They also produced substantially more heat when exposed to cold temperatures, reflecting brown fat activation. Histology confirmed increased bsWAT browning and lipolysis.

Bone morphogenetic proteins in brown fat biology

Yu-Hua Tseng from the Joslin Diabetes Center at Harvard Medical School studies obesity, a major risk factor for many chronic, debilitating human diseases. Because obesity develops when energy intake exceeds expenditure, her lab's primary focus is on understanding the regulation of energy homeostasis and applying this knowledge therapeutically. Maintenance of body temperature by brown adipose tissue-mediated energy release is an important component of energy homeostasis, and Tseng studies the regulatory role of bone morphogenetic proteins—especially BMP7—in brown fat. "We found that BMP is secreted locally by the supporting cells in the adipose tissue in both BAT and WAT, and we think it is very important in providing the signal to guide BAT differentiation," she said. Her lab has found that BAT development is severely impaired in BMP7-knockout mice, and that BMP7 acts on mature BAT to promote fatty acid uptake and oxidation. Tseng discussed the cross-talk between "classical" brown fat and beige fat.

Control (CON) and knockout (KO) animals were fed a high-fat diet and housed at room temperature and thermoneutral conditions for 6 weeks to eliminate cold stress and thus thermogenesis, enabling diet-induced obesity. CON animals became obese. In the KO group, however, deletion of the BMP receptor (BMPR1A) to restrict BAT production increased beige fat, with no increase in obesity. (Image courtesy of Yu-Hua Tseng)

Tseng and colleagues eliminated BMP signaling to impair the development of interscapular BAT and induce the production of beige fat cells to study. They eliminated BMP signaling specifically in Myf5-expressing cells—the progenitor cells for BAT—by knocking out the BMPR1A receptor in these cells. The loss of BMP7 signaling prevented Myf5 cells from developing into brown fat, and the newborn mice showed impaired thermal regulation. As beige cells increased in sWAT, their thermal regulation normalized. Although the subgroup of these mice fed a high-fat diet and maintained at room temperature became obese, the group given the same high-fat diet but subjected to chronic cold did not become obese. Their beige fat stores were activated by the cold and continuously released sufficient energy to maintain body temperature, showing that compensatory brown fat formation can maintain normal body weight and energy homeostasis. "The most important question is the mechanism enabling these two types of brown fat cells to talk to each other," Tseng said. She found that the answer involves stimulation of beige progenitor cells in sWAT by norepinephrine from the sympathetic nervous system.

TYK2 and STAT3 regulate brown adipose tissue differentiation and obesity

The long-term research focus of Andrew C. Larner from the Massey Cancer Center at Virginia Commonwealth University has been how interferons transmit their signal to the cell nucleus after binding with cell-surface receptors. This work led him to the Jak/Stat signaling pathway. The Jak phosphorylating enzymes, which include Tyk2, activate the respective receptor's intracellular tail, thus recruiting a Stat transcription factor that enables the signal to reach the cell nucleus and activate early-response genes. Larner began to research brown adipose tissue after discovering that his Tyk2-knockout mice had become obese as adults despite normal food intake. Their glucose clearance was also impaired and their insulin, free fatty acids, and cholesterol were elevated. He wanted to find out why.

Extensive testing showed only one abnormality: decreased energy expenditure. This led Larner and his lab to BAT, and their discovery that Tyk2-knockout mice were completely unable to adjust to cold temperature, and died as a result. Normal mice increased their Tyk2 expression during cold exposure and maintained the amount of brown fat needed for adequate thermogenesis. But when these normal mice were fed a high-fat diet, Tyk2 levels in their BAT depots dropped significantly. In humans, Tyk2 levels in skeletal muscle were normal in lean patients but significantly reduced in obese patients. When Tyk2 is absent or inadequate, brown fat preadipocytes are unable to differentiate to form BAT.

Working with immortalized Tyk2-null brown fat preadipocytes, Larner and his colleagues restored complete differentiation by adding in either a kinase-dead version of Tyk2 or a constitutively active Stat3. Wild-type preadipocytes missing Stat3 do not differentiate. The effects of constitutive Stat 3's absence and presence were confirmed in vivo in a Tyk2-knockout mouse. Examining the known transcription factors involved in BAT differentiation, Larner discovered that overexpression of PRDM16 also compensated for missing Tyk2. Further studies led him to conclude that the nonkinase portion of Tyk2 appears to stabilize the PRDM16 protein. Larner's team then found that restoring Tyk2 expression in BAT and skeletal muscle in Tyk2-knockout mice reverses the obesity phenotype from birth. They are studying the possibility that Tyk2's nonkinase portion acts in the cell nucleus on PRDM16 and perhaps other BAT transcription factors, but Larner says the role of Stat3 "is still an open question."

Activating functional brown adipogenesis and thermogenesis

Anne-Ulrike Trendelenburg from the Novartis Institutes for BioMedical Research became involved in brown fat research through her interest in understanding and treating muscle-wasting conditions, especially in aging and disuse. Myostatin, part of the TGFβ superfamily, inhibits muscle growth and regeneration. Trendelenburg described the complex series of studies that mapped the relevant pathways and interactions, ultimately determining that TGFβ is activated by TNF-α/II-1α. This occurs when TNF-α/II-1α binds with its ActRIIb receptor and induces activin A secretion. Transgenic mice lacking either myostatin or ActRIIb not only showed pronounced muscle development, but their white fat mass was also markedly decreased and their brown fat had increased. This finding made sense, because brown fat and skeletal muscle originate from a common precursor.

Trendelenburg and her colleagues blocked the activin receptor pharmacologically by treating normal mice with an ActRIIb monoclonal antibody for four weeks. Both muscle mass and BAT increased. UCP1 expression in BAT was unchanged. WAT was unaffected; no browning occurred. The brown fat induced by inhibiting ActRIIb functioned appropriately: it promoted oxidative metabolism, carried out thermogenesis in response to cold exposure, and did not increase energy expenditure in a thermoneutral environment. Trendelenburg is now working to identify the proteins that influence or interact with the ActRIIb-inhibition pathway. She and her group have also begun to take a more comprehensive view of age-related changes, and found that the phenotype of muscle wasting includes BAT wasting, which may also be linked with diabetes and obesity, among other chronic health problems.

Speakers:
Sheila Collins, Sanford-Burnham Medical Research Institute
J. Enrique Silva, Tufts University School of Medicine
Sheng Bi, Johns Hopkins University School of Medicine

Highlights

• High levels of heart-produced natriuretic peptides expand BAT and improve glucose tolerance.
• Because T3 is critical for maximum UCP1 expression, adequate expression of the enzyme that transforms thyroxin to T3 (the D2 form of deiodinase) is essential.
• Knocking down neuropeptide Y in the dorsomedial hypothalamus increases UCP1 expression and reduces or prevents diet-induced obesity.

The hormonal signals at the heart of brown adipocyte recruitment

Sheila Collins from the Diabetes and Obesity Research Center at Sanford-Burnham Medical Research Institute became interested in natriuretic peptides—which are produced in the heart—because of the metabolic links between the heart and adipose tissue. Both organs are regulated by the sympathetic nervous system and beta-adrenergic receptor activation. The two receptors for the natriuretic peptides, NPRA for signaling and NPRC for clearance, are expressed in adipose tissue as well as the heart, and the peptides have been observed to stimulate lipolysis in cultured human adipocytes. In humans with obesity and metabolic syndrome, circulating levels of these peptides are often reduced and the peptides themselves are less effective in controlling blood pressure. Collins found elevated levels of the clearance form of the receptor in the adipose tissue of these patients, as reported by others.

Using human adipocytes, Collins and her colleagues showed that cardiac-derived natriuretic peptides behave like catecholamines, activating cGMP and PKG to promote fat browning and activating all of the transcriptional machinery and UCP1. Adding an inhibitor of the p38 signaling protein eliminates all of these effects. Genetically altered mice lacking the signaling receptor tended to be somewhat obese; those lacking the clearance receptor were lean. Collins studied these NPRC-knockout mice because the lack of clearance produced very high levels of natriuretic peptides. The animals had extensive interscapular brown adipose tissue, minimal white adipose depots, and high UCP1 expression. Feeding these animals a high-fat diet did not produce obesity or poor glucose tolerance.

Collins found that exposing normal mice to cold substantially increased their natriuretic peptide plasma levels. The cold also increased the animals' expression of NPRA and dramatically decreased their NPRC, especially in inguinal white adipose tissue. Infusing the human natriuretic peptide BNP into mice robustly increased UCP1 and PGC-1α expression in white and brown fat. These animals also showed increased energy expenditure while maintaining food intake and physical activity. In humans, Collins and her colleagues found higher levels of NPRA in the adipose tissue depots of lean subjects and higher levels of NPRC among obese subjects. NPRA levels correlated negatively with parameters linked to obesity and diabetes; NPRC levels correlated positively with these parameters. The group is preparing for an interventional clinical study. Collins emphasized that the parallel pathways initiated by natriuretic peptides and catecholamines can work effectively together.

Brown adipose tissue regulation by thyroid hormone

J. Enrique Silva from Tufts University School of Medicine discussed the leap forward in evolution from poikilothermy, in which internal body temperature varies considerably, to homeothermy, which enabled the rise of mammals but also created the challenge of warming the body. The crucial components in meeting this challenge are thyroid hormone, which acquired the new role of stimulating and supporting thermogenesis; brown fat, the most energy-efficient way to warm the body; and the autonomic nervous system. Facultative thermogenesis—the heat-generating process triggered by cold—has two parts. The slower response, nonshivering thermogenesis, involves brown fat and the abundant expression of uncoupling protein in the mitochondria. Thyroid hormone is critical in modulating this process, which is impaired in both hypothyroid and hyperthyroid conditions.

The most active form of thyroid, T3, is needed for producing effective levels of both brown fat and uncoupling protein. T3 is produced from thyroxin by a process called 5-deiodination. This process is catalyzed in brown fat by the type 2 form of the enzyme deiodinase. This form, called D2, is stimulated by norepinephrine and is very closely regulated. Its activation is essential for a full thermogenic response to cold.

Silva noted that normal mice have a very high level of D2 activity in red muscle tissue and that this activity level doubles in mice that cannot produce UCP1. He speculated that red muscle may provide an alternative form of thermogenesis when BAT is disabled, in addition to enabling the emergence of beige fat.

Promoting white to brown adipocyte transformation

Sheng Bi from the Department of Psychiatry and Behavioral Science at Johns Hopkins University School of Medicine is interested in the hypothalamic neural systems that underlie energy homeostasis. In particular, he studies neuropeptide Y (NPY), which has a modest presence in the dorsomedial hypothalamus (DMH), a structure important in thermoregulation. NPY is prominent in the arcuate nucleus, part of the mediobasal hypothalamus, where its appetite-stimulating properties are well known. Its function in the DMH, however, has been poorly understood. Bi initially worked with a rat model of type II diabetes and obesity, using virus-mediated RNA to minimize NPY levels specifically in DMH neurons. This knockdown immediately reduced the rats' hyperphagia, obesity, and diabetes. Because reduced food intake did not fully explain the weight loss, Bi and his colleagues turned their attention to adipogenesis.

The researchers fed both the NPY-knockdown and control rats a standard chow diet for five weeks, which slightly increased their body weight, then switched the animals to a high-fat diet for 12 weeks. After this diet switch, the control animals significantly increased their food intake and became obese, with expanded white adipose depots. The NPY-knockdown animals, on the other hand, briefly increased their food intake, then returned to normal and did not gain weight. Their white fat depots were much smaller, their inguinal fat had browned, and UCP1 expression was dramatically higher in both inguinal tissue and interscapular brown fat. Bi and his colleagues determined that this browning was under sympathetic nervous system (SNS) control when they repeated the high-fat diet after denervating one inguinal fat pad in each control and knockdown animal with a neurotoxin injection. The denervated fat pads did not brown, while norepinephrine levels in the functional fat pad were substantially higher in the NPY-knockdown animals. Additional experiments made it clear that knocking down the DMH NPY increased energy expenditure, enhanced the thermogenic response to a cold environment, increased locomotor activity, improved glucose homeostasis, enhanced insulin sensitivity, and reduced or prevented diet-induced obesity.

Wild-type rats (AAVshCTL) were compared with rats whose expression of the appetite-stimulating hormone neuropeptide Y (NPY) had been knocked down (AAVshNPY). Initially they were fed standard rat chow (RC). After they were switched to a high-fat diet, the knockdown rats gained significantly less weight, due not just to maintaining normal food intake but to substantial activation of BAT (shown by a marked increase in UCP1 expression) and to the appearance of beige fat. (Image courtesy of Sheng Bi)

Speakers:
Abdul G. Dulloo, University of Fribourg, Switzerland
Richard L. Veech, National Institute on Alcohol Abuse and Alcoholism, NIH

Panel Moderator:
John E. Hambor, Boehringer Ingelheim

Highlights

• Food-associated thermogenic compounds may be able to increase thermogenesis or inhibit limitations on it.
• Ketone bodies in the diet, in the form of D-β-hydroxybutyrate, increase brown fat, glucose tolerance, and energetics.

The search for thermogenic compounds in the management of obesity

Abdul G. Dulloo from the Department of Medicine and Physiology at the University of Fribourg became interested in brown fat through his work on nutritional energetics in the context of understanding how variations in metabolic efficiency—thermogenesis—regulate body weight and composition. He also studies the mechanisms by which altered thermogenesis influences the development of insulin resistance and metabolic syndrome. Dulloo summarized "the very long history of the search for thermogenic compounds," emphasizing the ideal approach of combining a thermogenic stimulant, which activates the SNS, with a compound that inhibits limitations on this response. He mentioned his earlier studies of green tea, which combines polyphenols with caffeine. Results were very modest, although obese patients did lose visceral fat preferentially and their metabolic profile improved.

Dulloo provided a long list of food-associated bioactive compounds currently under study: an assortment of phytochemicals derived from plants, fruits and seeds, vegetables, herbs, and spices; animal products; and specific fatty acids. Some have been tested in humans. A useful compound (or cocktail of compounds) should be capable of stimulating an energy expenditure of up to 400–500 kcal/day and, for safety, should not increase heart rate by more than 10 beats/minute. It is essential that increased thermogenesis is not accompanied by increased energy intake. To measure the results, researchers also need a meaningful way to measure the increase in human brown and beige adipocytes, and a mechanism to activate them.

Using diet to increase BAT and its activity. (Image courtesy of Abdul G. Dulloo)

Ketone esters, brown fat, and insulin resistance

Richard L. Veech from the National Institute on Alcohol Abuse and Alcoholism at the NIH studies mechanisms that maintain cellular energy homeostasis. An important component of this research involves the development of therapeutic ketones. Veech's team currently works with the ketone ester D-β-hydroxybutyrate. He noted that feeding ketone esters to rodents robustly amplifies their brown fat stores, which also show increased cellularity and mitochondria size along with decreased lipids. The concomitant higher expression of signaling proteins PPAR-γ, PGC1-α, and AMP kinase indicate that ketone esters act through the SNS. In addition to increasing brown fat, ketone intake increases the efficiency of muscle cells. Veech explained that adding ketone bodies to a perfused working rat heart increases cardiac output and hydraulic work level substantially more than adding insulin. Feeding ketones to elite competitive athletes improves their physical, physiological, and cognitive performance. The critical factor in this effect is ketones' marked upregulation of the multi-enzyme complex pyruvate dehydrogenase. This enzyme's expression is increased 15-fold by ketones, but only 8-fold by insulin. Veech explained the underlying biochemistry, and then noted his view that ketones resemble insulin in their actions: they not only amplify brown fat but also increase its mitochondrial energetics and glucose utilization.

Veech discussed D-β-hydroxybutyrate's similar effect in the brain. Adding amyloid to hippocampal cells decreases their glucose utilization and pyruvate dehydrogenase activity. Subsequently adding D-β-hydroxybutyrate overcomes those effects. Veech noted additional benefits observed in vitro, and then discussed the significantly decreased amyloid and P-tau in the brain and more effective maze navigation when β-hydroxybutyrate is added to the diet of a mouse model of Alzheimer's disease. A human study will begin shortly. The body naturally produces ketones when fasting, but Veech suggested "that we ought to think of them as a food, just as we think of carbohydrate, fat, and protein."

Panel discussion

A panel discussion moderated by John E. Hambor of Boehringer Ingelheim featured a question and answer session with audience members. One asked whether proliferation is involved in the browning or beigeing of white fat. Spiegelman first emphasized that the browning or beigeing of white fat does not involve transdifferentiation. The misconception that white fat cells themselves are transformed into brown fat arose from observations at the tissue level, in which it appears that white fat actually becomes brown. At the molecular level, however, it is clear that a differentiated white cell is unable to turn into a brown fat cell. Instead, sWAT depots contain precursor cells that are predisposed to become beige fat cells and thus to turn on a thermogenic program. Spiegelman noted that his lab found the population of these precursors to range from "~10% to 40% of the subcutaneous layer of white fat, a very substantial portion of these white fat depots." He added that the role of proliferation in browning has not yet been determined. If it does occur, however, it cannot be significant in the early phases of browning because beige fat appears too rapidly to reflect proliferation. Collins added that "any evidence of proliferation is scant."

Another participant asked about differences among the mitochondria in fat cells. Larner observed that a particularly interesting question is whether the proteomes of brown fat cell mitochondria and beige fat cell mitochondria are different. Spiegelman noted the tissue-specific nature of mitochondria, and that while all mitochondria share the majority of mitochondrial proteins, each mitochondrial tissue-specific subgroup also has a unique protein component. The question regarding brown vs. beige proteosomes is under study in Spiegelman's lab.

On a different note, an audience member pointed out that some people would benefit most from burning more glucose during thermogenesis and others would benefit most from burning more lipids, and asked whether this could be manipulated. Spiegelman explained that beige and brown fat cells can each use both carbohydrates and lipids, but that the different effects of these nutrients on the respiratory quotient indicate different "preferences." It appears from early data that brown fat may be more lipid-loving while beige fat may not have a preference. It is a question that needs to be explored.