White-to-brown metabolic conversion of human adipocytes

 

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INTRODUCTION

The remarkable increase in the number of cases involving obesity and related disorders, such as diabetes and heart diseases driven by ready access to high-calorie food and an increasingly sedentary way of life, left an urgent need to try discovering pharmacotherapies targeting adipose tissue. How could that be effectively done? The main idea came from the existence and the potential conversion of fat-storing cells into metabolically active thermogenic cells.

Mammals possess two different kinds of adipose tissue:

  • White adipose tissue (WAT), which stores excess energy and also has a role in regulating satiety through leptin secretion.
  • Brown adipose tissue (BAT), which releases energy in the form of heat by uncoupling the respiratory chain (UCP1), so that body temperature may be maintained  through non-shivering thermogenesis. Its activation has been shown to ameliorate insulin resistance and to protect against obesity.

Fig 1: Brown adipose tissue contains lots of tiny lipid (fat) droplets, compared to white adipose tissue, which contains a single drop of lipid. BAT also contain a lot more mitochondria than WAT. These mitochondria store iron, thereby making BAT appear brown. WAT are the fats that we are referring to when we talk about how fat we are. BAT, however, are the “good” fats that play a role in generating and controlling body heat.

 

Uncoupling Proteins (UCP) generate heat by permitting proton influx without ATP synthesis. Brown adipocytes express thermogenin (UCP-1), which catalyzes the re-entry  of protons into the matrix, uncoupling the mitochondrial respiratory chain, and consequently reducing the ATP synthesis and generating heat.

Fig 2: Uncoupling Proteins (UCP) generate heat by permitting proton influx without ATP synthesis. Brown adipocytes express thermogenin (UCP-1), which catalyzes the re-entry of protons into the matrix, uncoupling the mitochondrial respiratory chain, and consequently reducing the ATP synthesis and generating heat.

A basic, but often misunderstood, concept is that weight gain is caused by a fundamental energy imbalance, when energy intake from food chronically exceeds energy expended by physical activity and metabolic processes. Humans have evolved efficient biological mechanisms to acquire and defend their energy stores. A therapy for weight loss must, therefore, involve a decrease in food intake and/or an increase in energy expenditure, in this case, provided by converting  fat-storing cells into metabolically active thermogenic cells.

BAT contributes to energy expenditure. Weight gain and obesity are caused by chronic periods of positive energy balance. Energy intake comes from food consumption, whereas the major contributors to expenditure are exercise and basic metabolic processes. The studies reviewed here suggest that BAT activity could impact daily energy expenditure. BAT dissipates energy as heat and can thus counteract weight gain. Interindividual variability in the amount or function of this tissue may impact body weight. In addition, therapeutic expansion/activation of this tissue may prove to be an effective therapy for obesity. WAT, white adipose tissue.

Fig 3: BAT contributes to energy expenditure.  Energy intake comes from food consumption, whereas the major contributors to expenditure are exercise and basic metabolic processes.  BAT dissipates energy as heat and can thus counteract weight gain. Interindividual variability in the amount or function of this tissue may impact body weight. In addition, therapeutic expansion/activation of this tissue may prove to be an effective therapy for obesity.

RATIONALE, METHODS AND RESULTS

Browning is the phenomenon referred as the white-to-brown metabolic conversion in human adipocytes, in which white adipocytes are conferred brown-like metabolic activity: elevated UCP1 expression and increased mitochondrial activity, therefore being a huge therapeutic target to treat metabolic disorders.

A screening platform for adipocyte browning was set to identify the induction of UCP1 activation, using a human pluripotent stem cell (PSC)-derived adipocyte model. As a browning index, UCP1 expression was monitored by UCP1 mRNA capture plates followed by branched DNA (bDNA) amplification, and expression of fatty acid binding protein 4 (FABP4), an adipocyte-specific gene served as an internal control to eliminate anti- and pro-adipogenic compounds not specific to UCP1.

As a result, two inhibitors of JAK3 and SYK showed highest UCP1/FABP4 ratio and lipid droplet morphology changes, typical of brown-like adipocytes:

  • Tofacitinib – JAK3 inhibitor
  • R406 – SYK inhibitor
Fig 4 (c) Each data point represents the average of two biological replicates per compound, normalized to the ​DMSO control. x axis: ​UCP1 mRNA level as an indicator of adipocyte browning, y axis: ​FABP4 mRNA as an indicator of general adipogenesis. (d) Validation of browning hits by bDNA analysis showing that ​JAK3 inhibitors, ​SYK inhibitors and ​THRB agonists scored as the best ​UCP1/​FABP4 inducers.

Fig 4: (c) Each data point represents the average of two biological replicates per compound, normalized to the ​DMSO control. x axis: ​UCP1 mRNA level as an indicator of adipocyte browning, y axis: ​FABP4 mRNA as an indicator of general adipogenesis. (d) Validation of browning hits by bDNA analysis showing that ​JAK3 inhibitors, ​SYK inhibitors and ​THRB agonists scored as the best ​UCP1/​FABP4 inducers.

(a) bDNA analysis of dose–response with ​tofacitinib and ​R406. At high doses, ​R406 increases both ​UCP1 and ​FABP4 expression but ​UCP1/​FABP4 remains above 2. Values represent the mean of two biological replicates. (b) Western blot analysis showing that upregulation of ​UCP1 and ​PRDM16 protein levels correlates with upregulation of ​UCP1 mRNA by ​tofacitinib and ​R406. (c) bDNA analysis showing that ​tofacitinib (​tofa.) and ​R406 increase ​UCP1 expression in human primary adipocytes. ADSC: adipose tissue-derived stromal cells. (d) Bright-field images showing that ​tofacitinib (​tofa.) and ​R406 induce brown-like lipid morphology (arrows) in human primary adipocytes more prominently than ​BMP7. RT–PCR analysis of ​UCP1 gene expression in mouse subcutaneous WAT explants following 7 days of treatment with the indicated compound.

Fig 5:  (a) bDNA analysis of dose–response with ​tofacitinib and ​R406. At high doses, ​R406 increases both ​UCP1 and ​FABP4 expression but ​UCP1/​FABP4 remains above 2.  (b) Western blot analysis showing that upregulation of ​UCP1 and ​PRDM16 protein levels correlates with upregulation of ​UCP1 mRNA by ​tofacitinib and ​R406. (c) bDNA analysis showing that ​tofacitinib (​tofa.) and ​R406 increase ​UCP1 expression in human primary adipocytes. ADSC: adipose tissue-derived stromal cells. (d) Bright-field images showing that ​tofacitinib (​tofa.) and ​R406 induce brown-like lipid morphology (arrows) in human primary adipocytes more prominently than ​BMP7. (e) RT–PCR analysis of ​UCP1 gene expression in mouse subcutaneous WAT explants following 7 days of treatment with the indicated compound.

 

Both were also shown to block tyrosine phosphorylation of the JAK-STAT1/3 pathway during adipocyte browning, mediators of pro-inflammatory pathways. Noticeably, STAT1 protein level decreased after treatment with tofacitinib and R406. In addition, it was observed that they also had the ability to antagonize Tumor necrosis factor alpha (TNF-alpha), previously observed to repress UCP1.

Fig 6: (a) Transcript abundance in RPKM (reads per kilobase transcript per million reads) of known targets of ​tofacitinib and ​R406 indicating that the JAK kinases are predominantly represented in PSC-WAs. (b) Western blot analyses of STATs, AKT and MAPKs in PSC-WAs previously treated with ​DMSO, ​tofacitinib and ​R406 showing a pronounced inhibition of STAT phosphorylation by ​tofacitinib and ​R406 at both time points. ​R406 also significantly decreased phosphorylation levels of AKT and ​ERK1/2. (c) Western blot analysis of a dose–response with ​tofacitinib and ​R406 showing the correlation between ​UCP1 accumulation and inhibition of ​STAT3 phosphorylation. (d) negative effect of ​TNFα on ​UCP1 expression is rescued by ​tofacitinib and ​R406. (e) ​Tofacitinib (​tofa.) and ​R406 do not synergize during adipocyte browning. (f) The ​JAK1/2 inhibitor ​ruxolitinib positively modulates ​UCP1 expression (graph) and inhibits ​STAT1/3 phosphorylation (right panels) in PSC-WAs.

 

Cytokine Signaling by the JAK/STAT Pathway

 

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Progressive and stable conversion of adipocytes by JAK inhibition

If a stable white to brown-like conversion is achieved through JAK inhibition, the brown-like phenotype should persist on removal of tofacitinib and R406 and be stable. Results indicate that JAK inhibition leads to stable acquisition of brown-like metabolic properties (high mitochondrial content as well as high metabolic activity) through functionally remodelling in human adipocytes, since all cells pre-treated with JAK inhibitors exhibited high levels of UCP1 mRNA and reduced lipid droplet size.

progressive accumulation of ​UCP1 induced by ​tofacitinib and ​R406 contrasts with the acute effect of ​THRB agonists.

Fig 7: Progressive accumulation of ​UCP1 induced by ​tofacitinib and ​R406 contrasts with the acute effect of ​THRB agonists.

Fig 8: Bright-field images showing a reduced lipid vacuole size at day 28 for ​tofacitinib (​tofa.)- and ​R406-pre-treated adipocytes.

Fig 8: Bright-field images showing a reduced lipid vacuole size at day 28 for ​tofacitinib (​tofa.)- and ​R406-pre-treated adipocytes.

Fig 9: Mitochondrial content was assessed by determining the ratio of COX-I on ​SDH-A protein levels by immunoblots as shown in the upper panel for PSC-WAs. Quantification of immunoblots revealed upregulation of mitochondrial content in ​tofacitinib- and ​R406-treated PSC-WAs and ADSC adipocytes.

Fig 9: Quantification of immunoblots revealed upregulation of mitochondrial content in ​tofacitinib- and ​R406-treated PSC-WAs and ADSC adipocytes.

Down-regulation of IFN and activation of hedgehog signaling contribute to metabolic browning downstream of JAK inhibition.

In addition to  the use of Tofacitinib and ​R406 to inhibit the JAK–STAT pathway in human adipocytes – leading to downregulation of the interferon alpha, beta and gamma responses -, the persistent repression of IFN signaling relieves inhibition of the SHH pathway, contributes to the up regulation of UCP1 and promotes the metabolic browning of adipocytes, therefore being an extra browning indutor.

Meanwhile, by using Hedgehog activator Smoothened Agonist (SAG), previous studies show that activation of hedgehog blocks white, but not brown, adipocyte differentiation, promoting the acquisition of a brown-like metabolic phenotype.

Fig 10: Pharmacological inhibition of JAK by ​Tofacitinib and ​R406, which  ​acts as  through activation of Peroxisome proliferator-activated receptor gamma ​(PPARG), Bone morphogenetic proteins (BMPs) and Sterol regulatory element-binding transcription factor (SREBF) target genes. In Redt: negative regulator of browning; in green: positive regulator of browning; arrows: activation; flat lines: inhibition; dashed lines: hypothetical.

Fig 10: Pharmacological inhibition of JAK by ​Tofacitinib and ​R406, which ​acts as through activation of Peroxisome proliferator-activated receptor gamma ​(PPARG), Bone morphogenetic proteins (BMPs) and Sterol regulatory element-binding transcription factor (SREBF) target genes. In Redt: negative regulator of browning; in green: positive regulator of browning; arrows: activation; flat lines: inhibition; dashed lines: hypothetical.

 

In conclusion, the central question addressed is whether BAT function significantly impacts energy balance and human obesity, and how that can be reached through developmental biology and targeted molecular pathways. Although the idea of stimulating BAT activity to combat obesity is a rational approach, it is also conceivable that this would trigger counterregulatory mechanisms such as increased appetite to maintain energy homeostasis and preserve fuel reserves. (1)

The new human data, generated from research projects such the one presented above, have invigorated interest and excitement in the function and physiological relevance of BAT. Hopefully, these findings can be, in the time to come, translated into:

1) a better understanding of the mechanisms that work together to regulate body weight and;

2) a novel therapeutic interventions to reduce the burden of obesity in our society.

BROWN FAT IS GOOD!

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REFERENCES

Moisan et al, “White-to-brown metabolic conversion of human adipocytes by JAK inhibition” Nature Cell biology 17,  57–67 (2015) DOI: 10.1038/ncb3075

(1) http://diabetes.diabetesjournals.org/content/58/7/1482.full

Figure 1:  http://www.vivo.colostate.edu/hbooks/pathphys/misc_topics/brownfat.html

Figure 2: http://www.scielo.br/img/revistas/abem/v56n4/01f01.jpg

Figure 3: http://diabetes.diabetesjournals.org/content/58/7/1482/F1.expansion.html

Figure 4-10: http://www.nature.com/ncb/journal/v17/n1/full/ncb3075.html

Video 1: https://www.youtube.com/watch?v=G4K6IQZGHJc

Video 2: https://www.youtube.com/watch?v=JYZkJkHRsRY

Video 3: https://www.youtube.com/watch?v=KMbhBL3rhz0

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