Gene Found for BCAA Clearance in Brown Fat Protects Against Obesity & Diabetes



Published 25th June 2020

Article image: Brown fat cell rich in mitochondria and having lipid droplets scattered throughout. / CC BY-SA

Branched-chain amino acids (BCAAs) are amino acids that have a central carbon atom with a branch of three or more other carbon atoms (otherwise known as aliphatic side chains.) BCAAs includes valine, leucine and isoleucine. They play several key roles in metabolism, such as promoting protein synthesis, neurotransmitter synthesis and the production of energy through glycolysis.

Increased circulating levels of BCAA are linked to obesity, insulin resistance and type 2 diabetes. This link is still unclear – higher BCAA levels should correlate with more energy expenditure which is usually correlated with weight loss.

Morphological differences between WAT, beige and brown adipose tissue (BAT) adipocytes as shown by cartoon and hematoxylin/eosin staining (× 40 magnification). CREDIT:
Kwok, K., Lam, K. & Xu, A. Heterogeneity of white adipose tissue: molecular basis and clinical implications. Exp Mol Med 48, e215 (2016).

A recent paper using Pparg-floxed mice supplied by our consortium member the Jackson Laboratory explored BCAA activity in brown fat. Brown adipose tissue (BAT) is well known to be a thermogenic (heat-producing and energy using) organ that helps remove excess glucose from our systems. The thermogenic function of BAT is crucial for survival and metabolic health. Humans usually have the highest amount of BAT during infancy. This is because BAT produces heat and helps infants keep warm whilst their muscles are still underused. As humans grow older and gain more mobility, this thermogenic role is taken over by skeletal muscle and we lose most of our BAT. In adults, BAT helps with cold acclimatisation, not only producing heat but also stimulating the uptake of glucose, lipoproteins and fatty acids.

Yoneshiro et al simulated cold exposure to look at how temperature affected BAT and BCAA levels.

How does Cold Stimulus Affect BCAA uptake?

The researchers performed a metabolite analysis on healthy male human subjects, dividing them into those with high BAT activity and low BAT activity. They used a cold stimulus of 19 Celsius, a temperature point that stimulates BAT thermogenesis without triggering skeletal muscle shivering.

Fig 1 excerpt: “d, Correlation between cold-induced amino acid changes and BAT activity (y axis) against the degree of BAT-dependent amino acid changes (x axis) in a. e, Cold-induced changes in plasma amino acids in diet-induced obese mice at 30 °C (thermoneutral (TN), n = 5) or 15 °C (cold, n = 6).”

The cold exposure stimulated lipolysis (the breakdown of fat) and led to a significant increase in circulating fatty acids but did not change blood glucose levels. Valine (Val) levels were significantly reduced in the high BAT subjects but not in the low BAT subjects. As Val concentration dropped, BAT activity increased. A similar response was also seen for Leucine (Leu) concentration but not for any other amino acids. Val, Leu and Isoleucine (Ile) reduction after cold exposure was also seen in obese mice.

Yoneshiro et al then tracked Leu uptake in various tissue types. After cold acclimatisation, they found a robust increase in BAT and a modest increase in inguinal white adipose tissue (WAT) in mice. Uptake was also seen in liver and heart tissues but no significant change was seen in these organs. BAT also displayed high Val oxidation compared to different types of WAT in both mice and humans.

BCAA oxidation can increase fatty acid oxidation which can reduce the risk of obesity. It is the oxidation of BCAAs that promote energy production and protein synthesis, particularly in muscle tissues. In BAT, BCAA is primarily oxidised in the mitochondria, the organelle within cells that produces energy for all cell functions. How BCAAs are transported into the mitochondria, and the transporter that facilitates this, is still unknown, as well as how BAT exactly utilises BCAAs.

How does BCAA Uptake in BAT Affect Metabolism?

As BAT is already known for the metabolic clearance of glucose, Yoneshiro et al investigated whether BAT also contributes to the clearance of BCAAs. They generated a mouse model without functioning (ablated) BAT and compared them to controls. After cold exposure, they found that the BCAA concentration was significantly reduced in control mice but not in the BAT-ablated mice, suggesting BAT plays a role in clearing BCAAs.

The researchers next examined how BCAA catabolism functioned within BAT after clearance, as well as how this affected energy homeostasis.

The branched-chain α-keto acid dehydrogenase (BCKDH) complex is found in the mitochondria and catalyses key oxidation reactions involved in energy production. To examine the extent to which BCAA catabolism in BAT regulates energy homeostasis, Yoneshiro et al produced another mouse model in which BCAA oxidation is impaired specifically in BAT. They deleted the Bckdha gene which produces a subunit of BCKDH, impairing BCKDH’s function. In this model (BckdhaUCP1-KO), they saw that there was no difference in BAT mass and thermogenic gene expression but the core-body temperature was significantly lower after cold exposure compared to controls. Thermogenesis in BAT was impaired (but not in other tissues) and the mice had higher circulating BCAA levels. This suggests BCAA oxidation is vital for both BAT thermogenesis and BCAA clearance.

Further examination on the use of BCAA within BAT was performed using Leu tracing, stimulation of brown adipocytes with noradrenaline and cold exposure. The researchers found that acute cold exposure activates BCAA oxidation in the TCA cycle, whereas chronic cold promotes other lipogenesis (fat production) processes.

Yoneshiro et al also wanted to find to what extent the BCAA oxidation impairment affected whole-body metabolism for the BckdhaUCP1-KO mice. When fed a high-fat diet, the impaired mice gained significantly more body weight compared to controls, particularly in terms of adipose tissue and liver mass. The impaired mice “exhibited increased systemic glucose intolerance and insulin resistance” as well as reduced glucose oxidation and fatty oxidation.

How Does the Mitochondria Take Up BCAAs?

The big question now was: how do cells take up BCAAs into the mitochondria? The researchers found that members of the SLC25A protein family would be promising candidates due to including many mitochondrial amino acid transporters. Transcriptome analysis found high expression levels for the already known SLC25A20 and SLC25A22 in mouse and human BAT but also two uncharacterised members: SLC25A39 and SLC25A44. Only SLC25A44 had increased mRNA expression after cold exposure and also showed positive correlations with UCP1 and BCKDHA mRNA expression. SLC25A44 was localised to the mitochondria and was more highly expressed in BAT than other metabolic tissues.

The researchers needed to determine the function of SLC25A44 so they generated brown adipocytes with ablated SLC25A44 (Slc25a44-KO.) Val and Leu uptake was significantly reduced, unlike other amino acids. This response was also found using shRNAs to deplete SLC25A44 whereas ectopic expression in Neuro2a cells restored Val and Leu uptake. Similar responses were also found in cell-free systems.

Fig 3 excerpt: ” a, Expression profile of SLC25A family members in human supraclavicular BAT and abdominal subcutaneous WAT from the same individual at 27 °C and 19 °C (ref. 5). FPKM, fragments per kilobase of transcript per million mapped reads.d, e, Mitochondrial uptake of indicated molecules in control and Slc25a44-KO brown adipocytes (d) or in Neuro2a cells expressing Slc25a44 or an empty vector (e) “

To determine the role of SLC25A44 in BCAA catabolism, the researchers selectively knocked down (reduction in the expression) SLC25A44 in BAT in mice (Slc25a44BAT-KD.) The KD mice had larger lipid droplets in their brown adipocytes and impaired BAT thermogenesis. SLC25A44-deficient mice (Slc25a44-KD) mirrored this response as well as higher levels of triglycerides and lower Val oxidation in BAT. Core body temperature was also significantly lower compared to controls after cold exposure but plasma BCAA levels were not lowered. Together, this suggested that “SLC25A44 is the primary BCAA transporter in BAT [and] is required for cold-stimulated BAT thermogenesis and systemic BCAA clearance in vivo.”

Click here for the full figure (4) and caption

Further tests also found that SLC25A44 depletion did not cause a general mitochondrial defect and SLC25A44 depleted adipocytes displayed active mitochondrial respiration.

What Does This Mean for Obesity and Diabetes?

The researchers proposed the following model:

“in addition to glucose and fatty acids, cold stimuli potently increase mitochondrial BCAA uptake and oxidation in BAT, leading to enhanced BCAA clearance in the circulation. This process requires SLC25A44, a mitochondrial BCAA transporter in brown adipocytes. In turn, defective BCAA catabolism in BAT results in impaired BCAA clearance and thermogenesis, leading to the development of diet-induced obesity and glucose intolerance.”

The researchers highlighted the implications these findings have for our understanding of obesity and diabetes. There is ongoing evidence that incompletely oxidised intermediates resulting from BCAA oxidation can cause insulin resistance. Lowering circulating BCAA levels in rats via inhibition of kinase BDK or overexpression of phosphatase PPM1K has also been found to improve glucose tolerance, regardless of body-weight. Reduced BCAA oxidation and the subsequent accumulation of BCAAs can cause inhibition of insulin signalling. This study suggests impaired BAT activity in obesity and diabetes reduces systemic BCAA clearance. Active and well-functioning BAT acts as a significant metabolic filter for circulating BCAA and protects against obesity and insulin resistance.

The researchers finally suggest that utilising SLC25A44 to enhance BCAA catabolism could improve BCAA clearance. This, in turn, could assist with glucose homeostasis and metabolic diseases such as obesity and diabetes.


Yoneshiro, T., Wang, Q., Tajima, K. et al. BCAA catabolism in brown fat controls energy homeostasis through SLC25A44. Nature 572, 614–619 (2019).

Obesity and its related ailments like type 2 diabetes and fatty liver disease pose a major global health burden, but researchers report in Nature Communications that blocking an RNA-silencing protein in the livers of mice keeps the animals from getting fat and diabetic conditions.

Takahisa Nakamura, and colleagues at Cincinnati Children’s Hospital Medical Center genetically deleted a protein called Argonaute 2 (Ago2) from the livers of mice. Ago2 controls the silencing of RNA in cells, affecting energy metabolism in the body, according to the study. When Ago2 silences RNA in the liver, it slows metabolism and liver’s ability to process a high-fat diet, the scientists report.

When they deleted Ago2 from the livers of mice, it was not toxic to the animals but it did stabilize energy metabolism. This helped stave off obesity and prevented the mice from developing diabetes and fatty liver disease, which can severely damage the vital organ–which helps rid the body of toxic substances.

“Although this is still basic science, we propose that there may be important translational implications for our findings for chronic metabolic disorders like diabetes, fatty liver diseases, and other obesity associated illnesses,” said Nakamura, senior investigator and a member of the Division of Endocrinology. “This allows us to explore the potential of finding a novel therapeutic approach that alters energy balance in obesity and modulates the associated diseases.”

The scientists caution the research is early stage. Their findings still need additional study and verification in laboratory models and the development of a practical therapeutic to inhibit Ago2 in a clinical setting for patients. But the current paper provides a solid basis for subsequent work.

Ago2 was identified after the researchers conducted a thorough screen and analysis of the activity of genes and their molecular targets in the liver, such as critical proteins. They analyzed wild type and genetically modified mice with high-fat diets by deleting certain proteins that are critical to liver metabolism–such as one called AMPK (AMP-activated protein kinase).

Nakamura said that identifying Ago2’s role in the process “connects the dots” between how proteins are translated in the liver, how energy is produced and consumed and the activity of AMPK in these processes. He pointed out that disruption of these events is already a common feature in obesity and its related illnesses.


Journal article: Hepatic Ago2-mediated RNA silencing controls energy metabolism linked to AMPK activation and obesity-associated pathophysiology

A team of researchers at the Harvard T. H. Chan School of Public Health has illuminated a critical player in cholesterol metabolism that acts as a molecular guardian in cells to help maintain cholesterol levels within a safe, narrow range. Known as Nrf1, it both senses and responds to excess cholesterol, and could represent a potential new therapeutic target in a multitude of diseases where cholesterol metabolism is disrupted.

The study is published in the journal Cell. To produce mice deficient in Nrf1 or Nrf2, the research used alleles from the International Mouse Phenotyping Consortium.

“We’ve uncovered a key missing piece in our understanding of how cells can precisely control their cholesterol levels,” said senior author Gökhan S. Hotamisligil, J.S. Simmons Professor of Genetics and Metabolism and head of the Sabri Ülker Center for Nutrient, Genetic, and Metabolic Research at Harvard Chan School. “That piece forms part of a molecular yin-yang that is critical for keeping cholesterol levels in proper balance and ensuring proper cellular function.”

It has been accepted for decades that high cholesterol in the blood can set the stage for cardiovascular disease and other significant health problems. But elevated cholesterol is even more dangerous at the cellular level, leading to toxicity, inflammation, and eventually cell death. “The cell must guard against any rise in cholesterol — it cannot tolerate levels that are too high or too low,” said Hotamisligil. While there are well known cellular factors that send and receive signals when cholesterol is in short supply (orchestrated by a protein called SREBP2), it has been unclear how cells handle some crucial aspects of the converse problem of too much cholesterol.

To explore the mechanisms that defend cells against cholesterol, first author Scott Widenmaier, research fellow in the Department of Genetic and Complex Diseases and Sabri Ülker Center, and colleagues focused their attention on an area of the cell known as the endoplasmic reticulum or ER, which is surrounded by a membrane notoriously low in cholesterol — lower, in fact, than any other cellular membrane. “This would be a particularly vulnerable place in the cell, where a small increase in cholesterol would make a significant impact,” explained Hotamisligil. As an intracellular structure, the ER requires fluidity, and adding more cholesterol to its membrane makes it more brittle.

Based on their assumption, the scientists set out to find molecules that reside in the ER membrane and that might play a role in detecting or responding to cellular cholesterol levels; they homed in on a handful of likely suspects. In initial experiments, Nrf1 protein stood out because it responds to cholesterol — when cholesterol is added to cells, the levels of Nrf1 increase, indicating that the protein can react to high cholesterol. And when Nrf1 function is disrupted in mice, the liver becomes dramatically enlarged and overrun with excess cholesterol, suggesting that it normally acts to protect the liver from cholesterol accumulation.

To dig more deeply into Nrf1’s protective role in cholesterol metabolism, the researchers set out to determine how it works at the molecular level. They discovered that Nrf1 has the capacity to bind to cholesterol directly, and pinpointed specific regions of the protein that mediate this binding. Moreover, the binding of cholesterol triggers a cascade of molecular events that suppress inflammation and promote cholesterol removal from the cell.

Taken together, these findings highlight a novel program for responding to high cholesterol in the cell that operates alongside other molecular components that guard against low cholesterol.

“This discovery really teaches us a lot about how cells maintain cholesterol homeostasis,” said Hotamisligil. “Now, we demonstrate that there is a molecular yin-yang– formed by NRF1 and SREBP2 — that together keep cellular cholesterol within a safe, narrow range. That’s an exciting finding that could have broad, new therapeutic applications.”

Research article: NRF1 Is an ER Membrane Sensor that Is Central to Cholesterol Homeostasis

It has been known for decades that low temperatures can trigger specialized fat cells to burn energy to produce heat, but in a new study, UC San Francisco researchers have discovered a new heat-producing pathway in fat cells that works by burning excess blood glucose, suggesting a potential new approach to treating metabolic disorders such as obesity and type 2 diabetes.

The findings, published in Nature Medicine, represent a paradigm shift in scientists’ understanding of how mammals turn their fat reserves into heat, said senior investigator Shingo Kajimura, PhD, an associate professor of cell and tissue biology in UCSF’s School of Dentistry and a member of the UCSF Diabetes Center.

All mammals, including humans, use white fat cells to store energy, but can also have reserves of brown fat, which burns stored energy to produce heat. Human babies are born with brown fat as a natural defense against cold and hibernating animals such as bears build up large stores of brown fat for the same reason. However, adult humans do not usually have much brown fat.

In 2015, the Kajimura lab identified a new type of fat in adult humans — so-called “beige” fat — which exists in pockets within fat tissue and can convert white fat into brown fat in response to cold or other stresses. In follow-up studies, the lab showed that mice with more beige fat were protected from diabetes and diet-induced obesity — by burning calories to generate heat, the animals were more easily able to shed unhealthy excess white fat. However, the mechanism by which these cells turn energy into heat was a missing piece of the puzzle.

“Now we’ve found this new pathway by which beige fat cells make heat,” Kajimura said. “We’re very excited not only for the science, but also for its terrific therapeutic potential to treat obesity and type 2 diabetes.”


More than one way to make heat

When you are cold, your muscles initially shiver to produce heat, but with longer exposure to cold temperatures, your body needs other ways to keep you warm.

For many years, researchers interested in understanding how our bodies turn stored energy into heat have studied the function of a protein called uncoupling protein 1 (UCP1). This protein is present only in brown and beige fat cells, and works by redirecting energy flow in mitochondria, the power plants of our cells, so they produce heat rather than biologically available energy.

In many mammals, UCP1 is responsible for producing heat in response to environmental needs without muscle shivering: its activity shoots up with cold temperatures as well as after overfeeding. However, some species — such as pigs — don’t have a functional UCP1 protein, but are somehow still able to stay warm in cold environments. This led the Kajimura lab to wonder whether there might be another mechanism involved in beige and brown fat’s ability to generate heat.

In previous studies, the team deleted the UCP1 protein from mice with higher-than-normal levels of beige fat, and found the animals were still resistant to diabetes and diet-induced obesity, demonstrating that beige fat’s beneficial effects were completely independent of UCP1.

“This was conceptually very surprising to us and for the field, because UCP1 has been the only known thermogenic protein for over twenty years,” Kajimura said.

Now, study lead author Kenji Ikeda, MD, PhD, and colleagues have found a new mechanism that beige fat cells can use to generate heat when exposed to cold temperatures, which involves activating a pair of proteins called SERCA2b and ryanodine receptor 2 (RyR2).

Normally, these proteins are responsible for controlling the availability of calcium, a key ion within fat cells. When calcium levels are too high, SERCA2b can use some energy to pump the extra calcium into storage sites within the cell. Then, when calcium in the cell is low, RyR2 acts as a valve to release some of these stored calcium reserves. But in cold conditions, Kajimura’s team found, cells can activate both proteins at once. Like revving a car’s engine by hitting the gas and brake at the same time, the main consequence is to generate a lot of heat and burn a lot of fuel – in this case glucose.

In fact, the researchers found that this process can burn so much glucose that lowering SERCA2b activity in beige fat cells in mice impacted whole-body glucose usage, suggesting potential applications for the prevention of type 2 diabetes, which is thought to be triggered in part by long-term elevations in blood glucose levels.

“Now that we’ve found that beige fat burns glucose using SERCA2b, it explains many things,” Kajimura said. “This is why mice become diabetic when we reduce beige fat, but disrupting UCP1 does not cause diabetes, and this is why mice are protected from diabetes in the presence of more beige fat.”

An evolutionarily conserved mechanism

Experiments in isolated pig and human beige fat cells laboratory dishes showed that it isn’t just mice who can use SERCA2b to turn excess glucose into heat, the researchers found, though it isn’t yet clear whether this mechanism or UCB1-based thermogenesis dominate in human beige fat.

The discovery that beige fat has more than one mechanism to stave off the cold makes evolutionary sense, Kajimura explained: “Thermoregulation is so important to life that there must be multiple mechanisms — like in pigs that don’t have a functional UCP1.”

Interestingly, Kajimura said, there is a human disease called malignant hyperthermia, in which mutations in the gene for ryanodine receptor 1 — the form of ryanodine receptor used in muscles — cause hyperthermia in the skeletal muscle, suggesting a related mechanism may be involved.

Kajimura and Ikeda are excited by the possibility of using drugs — or even nutritional supplements — to activate SERCA2b in beige fat in hopes of improving the body’s ability to process glucose for patients with type 2 diabetes and obesity, and also to help patients with diseases such as malignant hyperthermia that disrupt normal temperature regulation.

For example, it is well known that eating ginger warms up our bodies, Kajimura said, but the mechanism is poorly understood. Intriguingly, there is some evidence that gingerol, or ginger extract, activates SERCA proteins, suggesting interesting future opportunities for the scientists to test gingerol as a way to trigger beige fat to burn more calories.

Research article: UCP1-independent signaling involving SERCA2b-mediated calcium cycling regulates beige fat thermogenesis and systemic glucose homeostasis

Glucose is the energy that fuels cells, and the body likes to store glucose for later use. But too much glucose can contribute to obesity, and scientists have long wanted to understand what happens within a cell to tip the balance.

To solve this riddle, researchers at UT Southwestern’s Cecil H. and Ida Green Center for Reproductive Biology Sciences examined specialized compartments inside the cell to reveal the role of a molecule termed NAD+ in turning on genes that make fat cells.

The study utilised IMPC resources for the generation of Nmnat1 conditional knockout mice. Frozen Nmnat1tm1a(EUCOMM)Wtsi embryos on a C57BL/6N background were obtained and recovered at UT Southwestern’s Transgenic Core Facility. The research is reported in the journal Science.

NAD+ is found in every cell of the body and some scientists believe that boosting its production may be tied to better health and to the slowing down of the aging process.

What is NAD+?

NAD+ stands for nicotinamide adenine dinucleotide. It’s a molecule found inside cells in the body that helps transfer energy between molecules.

Why is it important?

NAD+ is believed to play important roles in longevity, aging, and diseases ranging from neurodegenerative disorders to cancer.

UT Southwestern biologists examined individual compartments inside cells that house NAD+ molecules to determine how they control genes that are essential to the fat-storing process – knowledge that could help in a wide range of ailments, including metabolic disorders, neurodegenerative diseases, inflammation and aging, and cancer.

“This compartmentalization ends up having profound effects on gene expression in the nucleus, as well as metabolism in the cytoplasm,” (the jellylike substance outside the cell’s nucleus), said Dr. W. Lee Kraus, Director of the Green Center and senior author on the research. “We found that these processes play key roles in fat cell differentiation and in cancer cells.”

“The previous thinking in the field was that NAD+ was evenly distributed throughout cells and moved freely between different subcellular compartments,” said Dr. Kraus, Professor of Obstetrics and Gynecology and Pharmacology. “We showed that NAD+ is actually compartmentalized – there are separate nuclear and cytoplasmic pools of NAD+ whose levels change under certain cellular conditions.”

Accounting for the levels of NAD+ biosynthesis separately rather than in their totality helped increase the understanding of the biology involved, said first author Dr. Keun Ryu, a postdoctoral researcher in Obstetrics and Gynecology.

“Our study provides a new understanding of NAD+ biology,” he said.

Research article:

Scientists have identified a network of genes that could play an important role in the development of metabolic diseases such as diabetes. A research team from the Helmholtz Center Munich and the International Mouse Phenotyping Consortium (IMPC) led the work that is published in ‘Nature Communications‘.

The development of metabolic diseases like diabetes is a complex process. As well as lifestyle and environmental factors, many different genes are responsible for the pathogenesis of both type 1 and type 2 diabetes. These genes encode information on how to assemble individual proteins that function in glucose metabolism.

Many genes that play an important role in the development of human diseases are still unknown. It is only by deciphering the causal genetic links that we can understand diseases, develop therapeutic interventions, or even prevent an outbreak. Thus, the new diabetes genes discovered in this study could be used, for example, as biomarkers for individual risk prediction, early diagnosis of the disease, or personalised approaches for treatment.

Twenty-three new candidate genes for diabetes in humans

As part of the IMPC, knockout mice – each lacking a specific gene – were examined for metabolic dysfunction. Using this method, researchers are trying to establish whether the missing gene is involved in important metabolic processes and can be linked to human diseases.

“Our analysis of the phenotyping data has identified a total of 974 genes whose loss has strong effects on glucose and lipid metabolism,” said Martin Hrabě de Angelis, who led the study and is the Chair of Experimental Genetics at the Technical University of Munich. “For more than a third of the genes no connection to metabolism was known previously.”

In addition, the researchers that teamed up with first author Dr. Jan Rozman, report that the functions of 51 of the discovered metabolic genes in the mouse were hitherto completely unknown. When compared with genome data collected in humans, they found that 23 of these also appear to play a role in human diabetes. “They are new candidate genes, and mice that lack these genes may be important models to investigate impaired glucose metabolism and diabetes,” explains Rozman, who coordinates the metabolic phenotyping at the German Mouse Clinic as part of the IMPC. One of these genes is C4orf22, which appears to be involved in insulin action in participants of the diabetes study “Tübingen Family Study (TÜF)”.

Interestingly, according to the bioinformatician and co-author Dr. Thomas Werner, these genes were also similar in their structure – many had common genetic elements. The scientists therefore assume that these genes belong to a network. In the future, they want to investigate these new regulatory structures and to explore to what extent they allow the prediction of gene functions of so-far unknown genes.

Rozman et al. Nature Communications 2018

Further links…

To see the data, click here



Published 5th April 2018

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