Metabolic syndrome (MetS) or insulin resistance syndrome, is a cluster of conditions that raises the risk of heart disease, type II diabetes, and stroke. These conditions include central obesity (i.e., excess body fat at the waist), hypertension, hyperglycemia, high blood triglycerides, and low HDL cholesterol. Those with three or more of these conditions may be diagnosed with MetS. MetS affects approximately one-third of adults in the United States. Pathogenesis of MetS is still not fully understood, though a variety of genetic, environmental, and lifestyle factors are thought to be involved.
Rho GTPases are a family of signaling proteins that play a key role in regulating the cytoskeleton. Activation of RhoA by GTP binding results in the activation of downstream effector proteins that mediate actin polymerization and stabilization and myosin II function1. RhoA is involved in essential cellular processes, such as cytokinesis2, stress fiber formation3, and smooth muscle cell contractility4, as well as several pathological processes, including tumor cell migration and invasion5 and cardiovascular diseases4. RhoA and its downstream effector proteins, Rho-associated protein kinases (ROCK1 and ROCK2), are also critical in MetS pathogenesis6. Elevated ROCK activity has been demonstrated in models of MetS conditions, including obesity, insulin resistance, elevated triglycerides, and hypertension4,7. In the PBMCs of patients with type 2 diabetes mellitus, ROCK activity is elevated 420-570% compared to healthy controls8. Serum ROCK activity is also elevated in those diagnosed with MetS and correlated with waist circumference, fasting glucose, and triglyceride levels9. As a result, RhoA and ROCK have become attractive therapeutic targets for MetS treatment10,11. One randomized controlled trial revealed that atorvastatin inhibits ROCK activity, which they suggest contributes to the anti-atherosclerotic benefit of statins12. This newsletter will focus on recently identified roles for RhoA and ROCK signaling in the biological processes involved in MetS conditions.
RhoA/ROCK signaling mediates actin cytoskeleton dynamics in adipogenesis
There are three main types of adipocytes. White adipocytes store excess lipids as triglycerides and release free fatty acids during times of high energy expenditure13. Brown adipocytes are thermogenic, that is, they convert energy into heat14. Brown adipocytes decrease with age, while overall adiposity, particularly white adipocyte accumulation, increases with age. Adipocytes with intermediate characteristics are referred to as beige adipocytes and can switch between the functionality of white and brown adipocytes based on current energy conditions13,14. Brown and beige adipocytes are critical for maintaining energy homeostasis and their decline is associated with an increased risk of obesity, diabetes, and other metabolic disorders15. For this reason, identifying signaling components that can modulate the adipogenesis program to favor thermogenic adipocytes is a promising therapeutic strategy for MetS treatment.
RhoA and ROCK play a key role in regulating actin cytoskeleton dynamics in all metabolic tissues and during adipogenesis, the process of creating new fat cells6,16. RhoA/ROCK activity inhibits the differentiation of white and beige adipocytes as well as the transdifferentiation of white adipocytes to beige6. Rho and ROCK activity further promote obesity by shifting away from thermogenic brown and beige adipogenesis in favor of myogenesis and cardiomyocyte differentiation, respectively6. Thus, targeting RhoA/ROCK activity represents a means to shift toward brown and beige adipogenesis by affecting multiple points of the adipogenesis program. Indeed, ROCK2 inhibition in mice enhances the thermogenic program in both white and brown adipocytes17. Another study demonstrated that mice deficient in the transcription factor MRTFA, which lies downstream of ROCK signaling, were protected from diet-induced obesity and demonstrated “beiging” of white adipose tissue18
Figure 1: Schematic of how distinct GPCRs induce RhoA activation resulting in opposing metabolic regulatory mechanisms
Gα12/13/RhoA affect obesity, whole-body energy metabolism, insulin sensitivity, and liver steatosis
Upstream of RhoA, heterotrimeric G-proteins containing Gα12/13 subunits transduce signals from a wide range of G protein-coupled receptors (GPCRs), including sphingosine 1-phosphate (S1P) receptors, thrombin receptors, and purinergic receptors19. Significant evidence indicates that GPCR signaling through Gα12/13 influences a wide-range of metabolic disorders19,20,21. Though both Gα12 and Gα13 lead to the activation of RhoA, they appear to exert their effects in a tissue-specific manner and serve opposing roles in the regulation of pathways involved in energy homeostasis in certain tissues19 (Figure 1).
In mice, exercise reduces Gα13 levels in skeletal muscle while high-fat diet (HFD)-fed mice have elevated Gα13 levels. Similarly, Gα13 is elevated in the skeletal muscle of patients with type II diabetes mellitus19. Skeletal muscle-specific ablation of Gα13 in mice leads to higher levels of whole-body metabolism and increased insulin sensitivity22. This effect is dependent on Gα13/RhoA/ROCK2-mediated suppression of nuclear factor of activated T cells 1 (NFATc1) by phosphorylation at Ser24322. Phosphorylation of NFATc1 at this site results in its inactivation and is elevated in obese mice and reduced following exercise23. While Gα13-mediated activation of RhoA in skeletal muscle leads to ROCK-mediated diet-induced adiposity, Gα12-mediated activation of RhoA leads to HIF-1α-dependent fatty acid oxidation19,22,23. Mice fed a HFD and patients with liver steatosis have lower levels of liver Gα12 while expression is upregulated under fasting conditions23. Ablation of Gα12 in mice leads to higher rates of lipid accumulation23. Mechanistically, this role for Gα12 in the regulation of fatty acid oxidation and mitochondrial respiration is mediated by the SIRT1/PPARα pathway23,24. Thus, in skeletal muscle, Gα12/RhoA and Gα13/RhoA serve opposing roles and together maintain energy homeostasis.
This dual regulatory program is absent in the liver where NFATc1 is not expressed. In contrast to skeletal muscle expression levels, hepatic Gα13 levels are lower in HFD-fed or genetically obese mice and patients with diabetes19. Additionally, hyperglycemia in mice leads to decreased hepatic Gα13 levels, contributing to glucose intolerance and insulin resistance25. In the liver, Gα12 contributes to hepatic fibrosis by promoting JNK-dependent autophagy in hepatic stellate cells (HSCs)19.
Summary and Future Perspective: Gα12/13/RhoA/ROCK as therapeutic targets in MetS
Though clinical trials to evaluate the pharmacological modulation of these signaling components for MetS are not yet underway, many preclinical animal studies have underscored the opportunities that exist. In one such example, the inhibition of ROCK suppressed obesity, hypercholesterolemia, and glucose intolerance in mice through the activation of the LKB1/AMPK pathway26. Because RhoA downstream effectors and subsequent cellular processes are tissue-specific, targeted therapeutic approaches such as liver-specific ROCK1 inhibition, have been evaluated in preclinical mouse models, resulting in increased energy expenditure, greater insulin sensitivity, and reduced lipid accumulation27. The multifaceted roles of Gα12/13/RhoA/ROCK signaling in metabolic syndrome continue to be uncovered. Recent evidence suggests this signaling axis represents a novel therapeutic target for MetS treatment based on several preclinical studies that have investigated RhoA signaling components as therapeutic targets.