Urocortins (UCNs) are members of the corticotropin releasing hormone (CRH) family, which includes UCN1, UCN2, and UCN3. All three are established neuroendocrine signaling peptides regulating physiological responses to stress via the hypothalamic-pituitary-adrenal axis (HPA). More recently, these peptides have been found to have diverse roles outside of the central nervous system, including mediating apoptosis, regulating inflammation, improving heart function, and inhibiting lipolysis. UCNs carry out their signaling through binding to the G protein-coupled receptors (GPCRs), Corticotropin Releasing Hormone Receptor 1 (CRHR1) and 2 (CRHR2). UCN1 binds to both CRHR1 and CRHR2, while UCN2 and UCN3 bind exclusively to CRHR2. Unlike CRHR1, CRHR2 is expressed in the central nervous system (CNS) as well as in several peripheral tissues.
UCN2 and UCN3 have been implicated in various aspects of energy balance and metabolism. Intracerebroventricular injection of UCN2 and UCN3 suppresses motor activity and food intake. UCN3-overexpressing mice are protected from diet-induced obesity (DIO) and have enhanced insulin signaling and glucose sensitivity. Studies on the effects of UCN2 on insulin sensitivity, however, have been contradicting. Germline CRHR2 KO mice have improved glucose tolerance. Germline UCN2 KO animals have improved glucose tolerance that is worsened following UCN2 re-administration. Treatment of animals with CRHR2 antagonists improves glucose tolerance and humans in the upper quartile range of circulating UCN2 have increased risk of insulin resistance. These findings indicate a role for UCN2 as an insulin desensitizer. In contrast, animals overexpressing UCN2 while on high-fat diet and animals receiving a daily subcutaneous dose of PEGylated UCN2 have improved glucose tolerance, pointing to UCN2 as an insulin sensitizer.
Type-II GPCRs like CRHR2 typically transduce signal through activation of protein Gs, leading to increased intracellular cAMP concentrations and phosphorylation of downstream target proteins. Persistent GPCR stimulation can be deleterious, however, resulting in cellular toxicity or uncontrolled growth, and thus nature has developed mechanisms to regulate GPCR activation through homologous receptor desensitization. To initiate desensitization, G protein-coupled receptor kinases (GRKs) are recruited to the cell membrane. GRKs phosphorylate the intracellular domain of the ligand-bound receptor attracting β-Arrestins to compete with Gs for binding, sterically hindering downstream transduction of the signal. In cases of chronically sustained Type II GPCR activation, β-Arrestins can induce the internalization of the GPCR through clathrin-mediated endocytosis, insulating the receptor from extracellular signals. The regulation of Type II GPCRs through these diverse mechanisms can result in different downstream biological effects. Some previous studies have shown Gs-alternative signaling and potential internalization of CRHR2.
In this study we examined the effects of CRHR2 signal transduction on systemic and local insulin sensitivity. We monitored glucose homeostasis in response to CRHR2 activation by UCN2 and, strikingly, found that acute treatment of UCN2 induces glucose intolerance in mice, while chronic treatment of UCN2 improves glucose tolerance. This phenomenon persisted in ex vivo isolated peripheral tissues and in vitro cell culture systems. We observed that each human CRHR2 isoform effectively recruits Gs in response to low UCN2 concentrations. At high concentrations of UCN2, CRHR2 also engaged Gi/o and β-Arrestins. The engagement of inhibitory proteins and β-Arrestins, known to promote receptor desensitization, potentially serves as an explanation for the divergent effects of UCN2 on insulin signaling when treated either acutely or chronically. Overall, this study has important implications for the potential use of CRHR2 antagonists for treatment of insulin resistance and offers insights into the molecular relationship between insulin’s effects and GPCR signal transduction.
Given the published effects that UCN2 and UCN3 have on insulin signaling, we began by investigating circulating UCN2 and UCN3 levels in established genetic models of insulin resistance. Leptin-deficient (ob/ob) animals have elevated levels of circulating UCN2 and UCN3. Leptin receptor-deficient (db/db) animals, likewise, have elevated UCN2 and UCN3 serum levels. UCN2 serum levels were also elevated in diet-induced obese (DIO) WT mice fed a high-fat diet for 20 weeks, while UCN3 levels remained unchanged. Gene expression analysis in human tissue indicated that ucn2 is mostly produced in the brown fat, skeletal muscle, kidney, and smooth muscle, while ucn3 is produced mostly in the pancreas and smooth muscle.
Fig. 1: UCN2 gene expression in metabolic tissues and circulating UCN2 levels are associated with insulin resistance.
To determine the source of the UCNs in these insulin-resistant models, gene expression patterns were determined for ob/ob and db/db tissues. Ucn2 expression was increased in the white adipose tissue, brown adipose tissue, heart, and skeletal muscle. Ucn3 expression, on the other hand, was significantly increased in the pancreas and kidney in both insulin-resistant models. This indicates that the majority of the increased circulating UCN2 in obese animals is contributed by skeletal muscle and BAT.
To validate the direct effects of UCN2 on glucose metabolism, mice were treated acutely with a single intraperitoneal (IP) injection of 1 mg/kg human UCN2 recombinant protein. Twenty minutes after injection, circulating concentrations of UCN2 reached an average of 40 nM (−7.4 log[M]). Oral glucose tolerance tests (oGTTs) performed twenty minutes after UCN2 injection showed that mice treated with UCN2 displayed glucose intolerance, with highly elevated glucose levels two hours after the glucose bolus.
