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

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By IMPC

Published 25th June 2020

Article image: Brown fat cell rich in mitochondria and having lipid droplets scattered throughout. https://www.scientificanimations.com/ / 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). https://doi.org/10.1038/emm.2016.5

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.

Source:

Yoneshiro, T., Wang, Q., Tajima, K. et al. BCAA catabolism in brown fat controls energy homeostasis through SLC25A44. Nature 572, 614–619 (2019). https://doi.org/10.1038/s41586-019-1503-x

A study investigating the role of Arf1 shows promising results for new therapeutic strategies involving DAMP-mediated anti-tumour immunity to target cancer stem cells. This method of inducting an anti-tumour response could be another frontier in anti-tumour immune therapy.

“[This could be] the first demonstration that endogenous metabolic stress elicits anti-tumour immune responses by inducing DAMPs in rodent tumor models.”

Cancer stem cells (CSCs) are found in tumours and, like other stem cell types, have the ability to give rise to many different cell types. Their affinity for self-renewal, differentiation, tumourigenicity and their possible resistance to traditional treatments like chemotherapy suggests they could be responsible for treatment resistance, tumour metastasis, reoccurrence, evasion and patient death. CSCs, therefore, prove a good target for future tumour therapies.

CSCs have a unique metabolism. Leukaemia stem cells have been shown to rely on amino acids for oxidative phosphorylation and some CSC-enriched disseminated tumour cells obtain energy from fatty acids. The researchers’ previous work with drosophila found that the COPI/Arf1-mediated lipolysis pathway selectively sustains and transforms stem cells and that targeting this pathway by knocking out Arf1 kills stems cells via necrosis.

In this study, the researchers investigated the Arf1-mediated lipid metabolism pathway’s role in sustaining CSCs in mice and how disrupting this pathway would affect tumour growth and number. IMPC Arf1 embryonic stem cells were used in this study.

Does the Arf1 Lipolysis Pathway Sustain CSCs?

The researchers used the Lgr5-CreERT2/Apcf/f (Lgr5/Apc) mouse model, one known to be a good model for CSCs, and knocked out Arf1 to produce Lgr5-CreERT2/Arf1f/f/Apcf/f mice. They found a dramatic reduction in stem cell tumour numbers and a significant increase in lifespan for the Lgr4/Arf1/Apc mice. After examining intestinal cells, they found abnormal mitochondria, poor cristae, vacuoles, degeneration and necrosis.

Please see source scientific journal article for figure caption.

They then took mouse colon cancer CT26 cells and human liver cancer Huh-7 cells and treated them with Arf1 inhibitors, BFA and GCA. The treatment dramatically increased lipid droplet accumulation in both cell types.

Deletion of Arf1 from the ventral foregut endoderm with Foxa3-Cre resulted in significantly underdeveloped livers and lethality at embryonic day 15.5. Further study found that it also caused a significant reduction in hepatoblast markers Hnf4α and Hnf6, lipid droplet accumulation and necrotic death of hepatoblasts.

“The Arf1-regulated lipolysis pathway selectively sustains stem cells, progenitors and cells enriched with CSCs in mice and that disrupting the pathway in these cells results in lipid droplet accumulation, mitochondrial defects and cell necrosis.”

Arf1-KO’s Effect on Intestinal CSCs

Arf1 depleted mice showed accumulation of CD3+, CD8+ and CD4+, evidence of a T-cell immune response, with dendritic cells (DCs) and inflammatory DCs also being significantly increased. Upon examining the population of lamina propria lymphocytes within the GI tract, they found that certain T-cells (CD8/4+ with and without IFN-γ+) were all significantly increased. These changes were not seen in other areas within the GI tract nor were these changes observed in other key immune cell sites, including the spleen, draining lymph nodes and inguinal lymph nodes.

T-cell-related chemokines (CCL5, CXCL10 and CCL22) were upregulated in the Lgr5/Arf1/Apc model. Real-time PCR showed elevated expressions of IFN-γ, perforin, granzyme A and B and IL-1β. The accumulated evidence suggested that:

“knocking out Arf1 in Lgr5+ stem cells triggers T cell infiltration and activation, leading to CSC death and prolonged survival of Lgr5/Arf1/Apc mice.”

Arf1-KO’s Effect on Liver CSCs

The researchers induced liver tumours using the Tet-o-MYC/LAP-tTA system before adding the Arf1 inhibitors GCA or BFA to mouse food, dramatically reducing the number of liver tumours and extending the lifespan of the mice. Selective depletion of Arf1 in Axin2+ liver cells or CSCs also produced this effect.

DCs, B cells and both CD4+ and CD8+ T-cells were significantly increased in the liver, as were many T-cell-related cytokines (CXCL10, CXCL11 and CCL22.) Real-time PCR showed elevated expression levels for the same molecules as in the intestine. Real-time PCR also, however, showed a significant decrease in PD-L1 expression in several mouse models and cell types.

“Arf1 inhibition may down-regulate PD-L1 through reducing the AP-1/C-Jun pathway…PD-L1 reduction could be partially responsible for the activation of infiltrated T cells that enhances the anti-tumour effect of Arf1 ablation”

Defining the Effect: T-cell Activation, DC Infiltration and DAMPs

In order to confirm that the effect relied on an adaptive immune response, they used anti-CD4 and anti-CD8 antibodies to deplete T-cells. In response, tumour numbers dramatically increased and lifespans were shortened. They then additionally knocked out either Rag1 or IFN-γ in the Lgr5/Arf1/Apc mice. Both caused survival times to significantly decrease. They concluded that the anti-tumour effects were due to an induction of a T-cell-dependant immune response.

Knocking out Arf1 had an effect on other aspects of the immune system. They found Arf1 inhibition may also induce inflammasome-mediated cell necrosis/pyroptosis. Arf1 inhibition or ablation also induced key DAMPS like Calr, HMGBI, ERp46 and LAMP1, as well as ER-stress markers. It was concluded that Arf1 inhibition triggers ER stress and, as a result, induces DAMPS and DC infiltration, enhancing the T-cell infiltration.


a–b Huh-7 cells were treated with control DMSO or GCA and then injected into athymic nude mice…Calr, HMGB1, ERp46, and the lysosome protein LAMP1 were dramatically induced in the GCA-treated tumours (a and b). Nuclear HMGB1 moved to the extracellular space (b).”

Through tests with a series of inhibitors, including the ATPase inhibitor ARL, it was concluded that the anti-tumour effect worked via ATP and HMGBI. Arf1 knockdown was not directly cytotoxic to tumour cells and, together with the above conclusions, the anti-tumour effect was DCs-ATP-IFN-γ-mediated T-cell immunity. Knockdown also prevented tumour metastasis.

Using Arf1-KO Cells for Vaccination

Vaccination with knockout Arf1 cells also proved successful in protecting animals from developing tumours. The vaccination was effective not only against melanoma but also against a variety of histologic types like colon cancer and breast carcinomas. Arf1 inhibition triggered a localised T-cell immune response that then attacked cancer throughout the body.

Please see source scientific journal article for figure caption.

Simultaneous inhibition of T-cell checkpoint receptor PD-1 with Arf1 ablation had a synergistic effect and further reduced tumour numbers.

Upon analysing public datasets on human cancer, the researchers found Arf1 was amplified or overexpressed in the majority of cancer types examined. The cases with lower expression levels of Arf1 had significantly better survival probabilities for a number of cancer types, suggesting Arf1 is a negative prognostic factor.

How it All Happens

The researchers proposed a model:

“a small number of active anti-CSC T cells penetrate the tumor and attack…; as a result…the CTLs produce cytokines and re-stimulate the local environment to convert an immunosuppressive to an immunostimulatory environment;…[reawakening] the pre-existing inactive anti-tumor T cells [and recruiting] new anti-tumor T cells; these additional T cells are directed against tumour antigens other than the DAMPs to amplify the effects for destroying the bulk tumors; these active T cells may then migrate to other tumor sites and become memory T cells to produce widespread and durable antitumor effects.”

“Proposed model depicting how Arf1 knockdown promotes metabolic stress, the induction of DAMPs, and immune cell infiltration and activation to attack tumours”

If results like the above are reproducible in humans, DAMP-mediated anti-tumour immune therapy could be a breakthrough for the treatment of reoccurring tumours, particularly those with high Arf1 expression. When used in conjunction with other already effective treatments or with the addition of successful checkpoint blockades, this method could prove even more effective.

Source:

Wang, G., Xu, J., Zhao, J. et al. Arf1-mediated lipid metabolism sustains cancer cells and its ablation induces anti-tumor immune responses in mice. Nat Commun11, 220 (2020). https://doi.org/10.1038/s41467-019-14046-9

What is SARS-CoV-2 and How Can Mice Help?

Viruses are infective agents that cause disease. They are different from other pathogens, such as bacteria, fungi and parasites, because most scientists don’t technically classify them as alive. They’re not 100% an organism as they cannot grow and reproduce by themselves. They’re made of the same building blocks of life (DNA, RNA and nucleic acids) as other organisms (such as bacteria or our own cells) but are unable to read and use this information. They reproduce by infecting host cells and hijacking the host’s processes. By inserting its genetic information into a cell’s DNA, when the cell begins to produce proteins it reads the viral DNA instead of its own, producing viral components that assemble into a new virion which can then infect a new cell.

Scientists classify viruses in several ways because they vary so widely, such as whether they have DNA or RNA or by their size and shape. Coronaviruses have single-stranded RNA genetic material encased in a viral envelope – a layer of protein usually made up of host cell and viral proteins that allows a virion to bind to a new host cell and fuse with it, starting the viral replication process. Coronaviruses have many club-shaped viral spike proteins attached to its envelope that creates a crown or corona-like appearance when viewed under a microscope.

Transmission Electron Microscope image of SARS-CoV with its characteristic corona. Credit: CDC/ Dr. John Hierholzer

There are a number of different coronaviruses that infect different animal species and cause widely varying severity of respiratory disease. There are seven known human coronavirus species (HCoVs), including the recent MERS-CoV and SARS-CoV. ‘COVID-19’ actually refers to the disease caused, not the virus that causes the disease, and stands for ‘coronavirus disease 2019’. COVID-19 is caused by the most recent novel coronavirus, first known as 2019-nCoV and now known as SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2.) SARS-CoV-2 is more infective than SARS-CoV and MERS-CoV and therefore spreads more easily but is less fatal(1).

It is SARS-CoV-2’s spike protein that allows it to bind to human cells via the ACE2 (angiotensin converting enzyme 2) receptor(2). This receptor is found on lung, artery, heart, kidney and intestine cells. Normally, human angiotensin, a protein, will attach to the ACE2 receptor, causing a lowering of blood pressure. The viral spike protein’s shape also fits ACE2 like angiotensin does, allowing a virion to bind to the receptor and enter cells.

SARS-CoV-2 and spike protein 3D models. Credit: NIAID / CC BY (https://creativecommons.org/licenses/by/2.0)

Mice also have an ACE2 receptor but its shape is different from the human version and SARS-CoV-2 doesn’t bind to it as easily. This means mice can be naturally infected with the virus but the severity of the disease is less than in humans(3). A mammalian model is vital for studying the virus and the disease it causes, as well as validating possible treatments and vaccines. Institutions such as our consortium member The Jackson Laboratory (JAX) are breeding a mouse colony with humanised ACE2 receptors (K18-hACE2 transgenic mouse model(3).) By genetically altering the mice to produce human ACE2 instead of mouse ACE2 we can create a much more applicable mouse model for the study of SARS-CoV-2. This will enable scientists to complete the research needed to treat the outbreak.

Main image credit: Felipe Esquivel Reed / CC BY-SA (https://creativecommons.org/licenses/by-sa/4.0)

Sources

Singhal, T. A Review of Coronavirus Disease-2019 (COVID-19). Indian J Pediatr 87, 281–286 (2020). https://doi.org/10.1007/s12098-020-03263-6

Letko, M., Marzi, A. & Munster, V. Functional assessment of cell entry and receptor usage for SARS-CoV-2 and other lineage B betacoronaviruses. Nat Microbiol 5, 562–569 (2020). https://doi.org/10.1038/s41564-020-0688-y

McCray PB Jr, Pewe L, Wohlford-Lenane C, et al. Lethal infection of K18-hACE2 mice infected with severe acute respiratory syndrome coronavirus. J Virol. 81(2), 813–821 (2007.) https://doi:10.1128/JVI.02012-06

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By IMPC

Published 2nd April 2020

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