Cross talk of non-canonical Gαi2 and Gαi3 signaling and endothelial nitric oxide synthase in adipocytes: implications for metabolic homeostasis

Adipose tissue plays a central role in whole-body energy homeostasis through regulation of lipid storage, thermogenesis, and endocrine signaling. Dysfunction of adipose tissue is a key driver of metabolic disease, yet the molecular mechanisms governing adipocyte function remain incompletely understood. Among the signaling pathways that regulate adipocyte biology, G protein-dependent signaling plays a critical role in mediating responses to hormones, neurotransmitters, and metabolic signals. However, the specific contributions of individual Gα protein isoforms to adipocyte function and systemic metabolism have only recently begun to be elucidated.

The inhibitory G proteins Gαi2 and Gαi3 belong to the Gαi/o family and share 88% amino acid sequence identity, yet emerging evidence suggests they exert distinct, non-redundant functions in a cell-type- and context-specific manner. Recent work from Veronika Leiss demonstrated that adipocyte-specific deletion of Gαi2 protects mice from high-fat diet (HFD)-induced obesity, insulin resistance, and adipose tissue inflammation 36 (Leiss et al., 2020). This finding established Gαi2 as a critical driver of diet-induced metabolic dysfunction.

In striking contrast, unpublished data from Veronika Leiss reveal that adipocyte-specific deletion of Gαi3 (Gnai3ako) results in a markedly different phenotype. Gnai3ako mice fed with control diet exhibit reduced brown adipose tissue mass, decreased energy expenditure and improved glucose tolerance. However, these metabolic improvements are completely abolished when mice are fed a high-fat diet (Figure 1). These findings highlight that Gαi3-mediated regulation of adipocyte metabolism is highly diet-dependent, which reveals an unexpected plasticity in isoform-specific signaling under different nutritional states, see also (Köhler et al., 2024; Leiss et al., 2025; Leiss et al., 2020; Nürnberg et al., 2024). However, the molecular mechanisms underlying the Gnai3ako metabolic phenotype remain unknown.

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Canonical Gαi signaling involves inhibition of adenylyl cyclase, leading to reduced cAMP production and decreased protein kinase A (PKA) activity. However, the phenotype of Gnai3ako mice suggests involvement of alternative, non-canonical effector pathways. Accumulating evidence from non-adipocyte cell types demonstrates that Gαi proteins can activate the phosphatidylinositol 3-kinase (PI3K)-Akt signaling axis through released Gβγ subunits (Lambert, 2008); for Review see (Nürnberg & Beer-Hammer, 2019). Specifically, Gβγ dimers released upon GPCR activation can directly bind to and activate PI3Kγ, leading to phosphorylation and activation of the serine/threonine kinase Akt (Blesen et al., 1995). Activated Akt, in turn, phosphorylates endothelial nitric oxide synthase (eNOS) at serine 1177, resulting in increased enzymatic activity and enhanced nitric oxide (NO) production (Dimmeler et al., 1999). This Gαi-PI3K-Akt-eNOS signaling cascade has been well characterized in endothelial cells and cardiomyocytes but to the best of our knowledge has never been investigated in adipocytes.

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Of relevance, it was shown that eNOS is expressed in both white and brown adipocytes  (Nisoli et al., 2003; Sansbury et al., 2012; Xia et al., 2016). Accumulating evidence show that eNOS-derived NO modulates adipocyte differentiation, mitochondrial biogenesis, insulin sensitivity, and their thermogenic capacity  (Engeli et al., 2004; Nisoli et al., 2003). In vivo studies demonstrated that lack of eNOS in global eNOS knockout mice reduced energy expenditure and promote diet-induced obesity, while eNOS overexpression or nitrate supplementation protect the mice against metabolic dysfunction (Carlström et al., 2010). These findings establish eNOS as a key regulator of adipocyte metabolism and systemic energy homeostasis. However, the upstream signaling pathways that regulate eNOS activity in adipocytes remain poorly defined.

Therefore taking into consideration the established role of Gβγ-PI3K-Akt signaling in eNOS activation and the critical function of eNOS-derived NO in adipocyte metabolism, we here hypothesize that Gai3 regulates adipocyte metabolic function through a non-canonical PI3K-Akt-eNOS signaling axis.

Testing this hypothesis requires definitive genetic and biochemical evidence establishing the Gαi3-eNOS signaling axis in adipocytes. Miriam Cortese-Krott has established cell specific conditional eNOS knockout (eNOSflox/flox) and knock-in (eNOSinv/inv) mouse models  (Leo et al., 2021; LoBue et al., 2024), which can be used to generate adipocyte-specific eNOS models using Adipoq-Cre or UCP1-Cre driver lines. These tools will enable rigorous genetic experiments to test whether eNOS mediates the metabolic effects of Gαi3 in adipocytes. Such experiments represent a gold-standard approach for establishing causal pathway relationships in vivo.