Comments on 'FNIP1 regulates adipocyte browning and systemic glucose homeostasis in mice by shaping intracellular calcium dynamics'.

Owing to its remarkable benefits on metabolic health and its demonstrated presence in adult humans, beige or ‘brite’ adipocytes hold great promise to combat obesity and metabolic diseases. Delineation of the mechanisms involved in adipocyte ‘beiging’ or ‘browning’ is thus of particular interest. Previous studies of the molecular regulatory pathways that drive the beiging of white adipose tissue (WAT) have centered on the cyclic adenosine monophosphate (cAMP)-related signaling (Wang and Seale, 2016; Cohen and Kajimura, 2021). Acting via the cAMP–protein kinase A pathway, norepinephrine dually stimulates adipocyte lipolysis and the expression of thermogenic genes, such as uncoupling protein 1 (Ucp1) and peroxisome proliferator-activated receptor-gamma coactivator-1alpha (Ppargc1a) (Inagaki et al., 2016). While the second messenger calcium (Ca2+) is fundamental to a wide variety of cellular processes including, but not limited to, muscle contraction, gene transcription, exocytosis, and cellular metabolism in virtually all eukaryotic cells (Arruda and Hotamisligil, 2015), whether and how Ca2+ signals orchestrate WAT beiging remains largely unclear, in part because of a limited understanding of the regulatory mechanism that controls intracellular Ca2+ dynamics in adipocytes.

In our recent work (Yin et al., 2022), we discovered a previously unrecognized WAT beiging mechanism that targets the folliculin (FLCN) interacting protein 1 (FNIP1)–sarco/endoplasmic reticulum Ca2+-ATPase (SERCA)–intracellular Ca2+ axis in adipocytes (Figure 1; commented in Bunk and Kazak, 2022). Using multiple Ca2+ imaging approaches, together with gain- and loss-of-function strategies, we provided clear data demonstrating that FNIP1 is a crucial regulator of intracellular Ca2+ dynamics and negatively regulates WAT beiging. We also uncovered the working mechanisms that FNIP1 binds to and promotes the activity of SERCA, a main Ca2+ pump responsible for cytosolic Ca2+ removal, thereby dampening Ca2+-dependent thermogenic programs. Furthermore, we showed that adipocyte-specific ablation of FNIP1 leads to a broad thermogenic remodeling of WAT with strong effects on whole-body glucose homeostasis and hepatic steatosis.

Figure 1

The model of FNIP1 in control of beige adipocyte thermogenesis and metabolic health through modulating SERCA. FNIP1 binds to SERCA to enhance its Ca2+ pump activity. This limits intracellular Ca2+ dynamics and dampens Ca2+-dependent thermogenic programs. Mice with adipocyte-specific ablation of FNIP1 showed enhanced thermogenic remodeling of WAT and were protected against HFD-induced insulin resistance and hepatic steatosis.

In this study, we set to investigate the intracellular Ca2+ dynamics in association with WAT beiging by the employment of GCaMP6f reporter mice, a mouse model that allowed us to monitor Ca2+ activity in adipose tissue in vivo. We found that dynamic changes of intracellular Ca2+ are required for the thermogenic gene program in white adipocytes upon thermogenic stimuli. The endoplasmic reticulum (ER) serves as the main Ca2+ store that determines intracellular Ca2+ homeostasis. Among the ER membrane-localized Ca2+ channels, SERCA is the solely Ca2+ influx pump that has been shown to fine-tune intercellular Ca2+ dynamics. By performing immunoprecipitation and mass spectrometry analysis, we identified that SERCA2 is able to interact and precipitate with FNIP1, an adaptor protein originally identified through its interaction with FLCN and AMP-activated protein kinase (AMPK) (Baba et al., 2006). We then performed a series of cellular assays to demonstrate that FNIP1 acts as a positive regulator of SERCA in adipocytes, and binding of FNIP1 promotes both ATP hydrolysis and Ca2+ transport of the SERCA pump, leading to a faster removal of cytosol Ca2+. As such, in FNIP1 knockout (KO) adipocytes, the reuptake of cytosolic Ca2+ into the ER lumen mediated by SERCA is reduced, resulting in elevated Ca2+ levels and an increased duration of Ca2+ transients in the cytosol upon external stimuli. More importantly, we showed that, functionally, loss of adipocyte FNIP1 in mice results in enhanced thermogenic remodeling in WAT. This improves systemic glucose homeostasis and prevents hepatic steatosis when mice are fed a high-fat diet (HFD).

We observed that FNIP1 deficiency in adipocytes triggers a robust UCP1+ beige adipocyte formation and mitochondrial biogenesis in the absence of cold exposure or β-adrenergic stimulation. Therefore, FNIP1 acts as an endogenous ‘brake’ to inhibit adipocyte beiging. Intriguingly, our results also indicate that FNIP1 suppression of thermogenic programs occurs in a depot-selective manner, the reason for which is not clear. This could reflect the intrinsic differences between beige adipocytes and brown adipocytes that affect the adaptive range of thermogenic remodeling.

While we have demonstrated previously that FNIP1 acts through AMPK but not mammalian target of rapamycin (mTOR) complex 1 signaling to regulate mitochondrial function in the skeletal muscle (Xiao et al., 2021), interestingly, we did not observe a change in either AMPK or mTOR signaling in both FNIP1 KO primary adipocytes and FNIP1 adipocyte-specific KO (AKO) adipose tissues. These results suggest that the regulations of metabolic regulators by FNIP1 are context-specific or cell type-specific. We clearly showed that FNIP1 binds to and promotes the activity of SERCA. We also examined the endogenous FNIP1 and SERCA interaction in adipose tissue after adrenergic stimulation. The results were interesting and consistent with the proposed mechanistic model. We found that CL316,243 stimulation significantly reduced the interaction between endogenous FNIP1 and SERCA in WAT.

Previously published studies have revealed that FNIP1 could actually act as a co-chaperone that regulates the chaperone function of Hsp90 (Woodford et al., 2016). We found that the N-terminal region of FNIP1 binds to the ATP hydrolysis site of the SERCA pump. Moreover, we observed that SERCA protein was stabilized in cells expressing FNIP1. Based on our data and the published literatures, it is possible that FNIP1 also functions as a co-chaperone of Hsp90 that regulates SERCA protein stability in adipocytes.

Notably, a recent study has shown that enhanced Ca2+ cycling in beige fat via the SERCA2–RyR pathway can promote a UCP1-independent thermogenesis (Ikeda et al., 2017). Whereas UCP1 and mitochondria were strongly induced by FNIP1 deletion, thus suggesting a UCP1-dependent thermogenesis in FNIP1 AKO inguinal WAT, our results did not determine whether this FNIP1 regulatory pathway is involved in UCP1-dependent or UCP1-independent beige adipocyte thermogenesis. Obviously, these are not mutually exclusive roles.

In summary, our recent work suggests that FNIP1 acts as a negative regulator of beige adipocyte thermogenesis to control systemic glucose homeostasis through modulating SERCA (Yin et al., 2022). Linking intracellular Ca2+ dynamics to adipocyte beiging is of interest, given that it could be an appealing strategy for the treatment of metabolic disorders. This raises several interesting questions. (i) What are the mechanisms by which the intracellular Ca2+ signals activate the thermogenic program? (ii) How is the interaction between FNIP1 and SERCA regulated under the physiological setting? (iii) Does this FNIP1–SERCA–intracellular Ca2+ pathway represent a broader metabolic regulatory network in other organs? (iv) Will it be possible to exploit such FNIP1–intracellular Ca2+ signals as treatment strategies?

[This work was supported by grants from the National Natural Science Foundation of China (31922033, 91857105, 32071136, 32100922, and 32100942), the Natural Science Foundation of Jiangsu Province (BK20170014 and SWYY-002), the China Postdoctoral Science Foundation (2021M691524), and the Fundamental Research Funds for the Central Universities (021414380511).]