Salt-inducible kinase inhibition promotes the adipocyte thermogenic program and adipose tissue browning

by Selleckchem | Nov 26 2023

Objective: Norepinephrine stimulates the adipose tissue thermogenic program through a β-adrenergic receptor (βAR)-cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA) signaling cascade. We discovered that a noncanonical activation of the mechanistic target of rapamycin complex 1 (mTORC1) by PKA is required for the βAR-stimulation of adipose tissue browning. However, the downstream events triggered by PKA-phosphorylated mTORC1 activation that drive this thermogenic response are not well understood.

Methods: We used a proteomic approach of Stable Isotope Labeling by/with Amino acids in Cell culture (SILAC) to characterize the global protein phosphorylation profile in brown adipocytes treated with the βAR agonist. We identified salt-inducible kinase 3 (SIK3) as a candidate mTORC1 substrate and further tested the effect of SIK3 deficiency or SIK inhibition on the thermogenic gene expression program in brown adipocytes and in mouse adipose tissue.

Results: SIK3 interacts with RAPTOR, the defining component of the mTORC1 complex, and is phosphorylated at Ser884 in a rapamycin-sensitive manner. Pharmacological SIK inhibition by a pan-SIK inhibitor (HG-9-91-01) in brown adipocytes increases basal Ucp1 gene expression and restores its expression upon blockade of either mTORC1 or PKA. Short-hairpin RNA (shRNA) knockdown of Sik3 augments, while overexpression of SIK3 suppresses, Ucp1 gene expression in brown adipocytes. The regulatory PKA phosphorylation domain of SIK3 is essential for its inhibition. CRISPR-mediated Sik3 deletion in brown adipocytes increases type IIa histone deacetylase (HDAC) activity and enhances the expression of genes involved in thermogenesis such as Ucp1, Pgc1α, and mitochondrial OXPHOS complex protein. We further show that HDAC4 interacts with PGC1α after βAR stimulation and reduces lysine acetylation in PGC1α. Finally, a SIK inhibitor well-tolerated in vivo (YKL-05-099) can stimulate the expression of thermogenesis-related genes and browning of mouse subcutaneous adipose tissue.

Conclusions: Taken together, our data reveal that SIK3, with the possible contribution of other SIKs, functions as a phosphorylation switch for β-adrenergic activation to drive the adipose tissue thermogenic program and indicates that more work to understand the role of the SIKs is warranted. Our findings also suggest that maneuvers targeting SIKs could be beneficial for obesity and related cardiometabolic disease.

Comments:

This research investigates the molecular mechanisms underlying the stimulation of adipose tissue thermogenesis through the β-adrenergic receptor (βAR)-cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA) signaling cascade. The study identifies salt-inducible kinase 3 (SIK3) as a crucial player in this process. Here's a summary of the key findings and conclusions from the study:

### **Objective:**
Norepinephrine stimulates adipose tissue thermogenesis via the βAR-cAMP-PKA pathway. The study aims to elucidate the downstream events triggered by PKA-phosphorylated mechanistic target of rapamycin complex 1 (mTORC1) activation that drive the thermogenic response. SIK3 is identified as a candidate mTORC1 substrate, and its role in adipose tissue browning is investigated.

### **Methods:**
- **Proteomic Analysis:** Global protein phosphorylation profile in brown adipocytes treated with a βAR agonist is characterized using Stable Isotope Labeling by/with Amino acids in Cell culture (SILAC).
- **SIK3 Identification:** SIK3 is identified as a substrate of mTORC1 and is studied using pharmacological inhibition (HG-9-91-01), shRNA knockdown, and CRISPR-mediated deletion techniques.
- **HDAC4 Interaction:** SIK3 inhibition leads to increased activity of type IIa histone deacetylase (HDAC), affecting the expression of thermogenic genes such as Ucp1, Pgc1α, and mitochondrial OXPHOS complex proteins.
- **In Vivo Study:** A SIK inhibitor (YKL-05-099) stimulates thermogenesis-related gene expression and browning of mouse subcutaneous adipose tissue.

### **Results:**
- **SIK3 Phosphorylation:** SIK3 is phosphorylated at Ser884 in a rapamycin-sensitive manner and interacts with RAPTOR, a component of mTORC1.
- **Effect of SIK Inhibition:** Pharmacological SIK inhibition increases basal Ucp1 gene expression and restores its expression upon blockade of mTORC1 or PKA. SIK3 deficiency augments, while SIK3 overexpression suppresses, Ucp1 gene expression in brown adipocytes.
- **Role of HDAC4:** SIK3 inhibition increases type IIa HDAC activity, enhancing the expression of thermogenic genes. HDAC4 interacts with PGC1α after βAR stimulation and reduces lysine acetylation in PGC1α.
- **In Vivo Efficacy:** The SIK inhibitor (YKL-05-099) stimulates thermogenesis-related genes and browning of mouse subcutaneous adipose tissue.

### **Conclusions:**
- **SIK3 Function:** SIK3 acts as a phosphorylation switch for β-adrenergic activation, driving the adipose tissue thermogenic program. Other SIKs might also contribute to this process.
- **Therapeutic Implications:** Targeting SIKs could be beneficial for obesity and related cardiometabolic diseases, suggesting potential therapeutic avenues for further exploration.

In summary, this study uncovers a novel regulatory mechanism involving SIK3 in the adipose tissue thermogenic program, shedding light on potential therapeutic strategies for conditions related to metabolic dysregulation. Further research is warranted to comprehensively understand the roles of SIK family members and to explore the therapeutic potential of targeting SIKs in obesity and related disorders.