Dana Sabine Hutchinson

The glucagon-like peptide-1 receptor (GLP-1R) and glucose-dependent insulinotropic polypeptide receptor (GIPR) are important incretin receptors that are therapeutic targets for the treatment of type 2 diabetes and obesity. This study extensively characterised the metabolic phenotype of mice with global deletion of either the GLP-1R or GIPR side by side under identical conditions. Age-matched male wild-type (WT) C57Bl6NTac, GLP-1RKO or GIPRKO mice were placed on a high-fat or chow diet for 12 weeks, and a range of in vivo (weight gain, food intake, glucose tolerance, insulin tolerance, and whole-body energy metabolism) and ex vivo (white adipocyte lipolysis, brown adipose tissue and liver mitochondrial function, adipocyte and islet size, and hepatic steatosis) parameters were measured. While both WT and GLP-1RKO mice gained weight similarly on a HFD, obese high-fat-fed GLP-1RKO mice had altered glucose and insulin tolerance, and exhibited hepatic steatosis, highlighting the physiological importance of the GLP-1R in the regulation of blood glucose and lipid homoeostasis. In contrast, GIPRKO mice were partially resistant to diet-induced obesity compared to the WT mice, which was associated with a small reduction in food intake and intact epididymal and subcutaneous white adipocyte β-adrenoceptor-mediated lipolysis. Similarly, WT mice treated with a GIPR antagonist prevented weight gain due to a reduction in food intake on a HFD. These findings provide further support that the GLP-1R is important for normal glycaemic control, whereas the GIPR may play a role in the regulation of body weight.

Background and purpose: Patients with amyotrophic lateral sclerosis (ALS) are prescribed many medications for symptomatic relief. However, how potential alterations to the blood–brain barrier (BBB) affect the brain exposure of drugs in ALS remains under‐investigated. Experimental approach: We used high‐dimensional proteomic analysis, cellular metabolism, and mitochondrial functional assays to characterise isolated brain microvascular endothelial cells (BMECs) from wildtype and SOD1 ^G93A transgenic mice, a mouse model of familial ALS, at a late‐symptomatic age (P115–120), together with a transcardiac brain perfusion technique to assess BBB function in situ . Key results: The BBB of the SOD1 ^G93A transgenic (TG) mice was significantly altered, including a 1.3‐fold decrease in apparent brain microvascular volume, and decreased BBB transport of ³ H‐diazepam (1.4‐fold) and ³ H‐2‐deoxy‐D‐glucose (1.2‐fold). BMEC proteomic analysis revealed multiple changes in TG mice including altered transmembrane activity, metabolism, and mitochondrial function, as revealed by gene set enrichment analysis, alongside altered glucose transporter (Glut1) abundance. These proteomic findings supported the identified increase in mitochondrial basal/ATP‐linked respiration, mitochondrial action potential, ATP production, and intracellular ATP levels in SOD1 ^G93A mouse BMECs. The BBB transport of ³ H‐digoxin, a specific ATP‐binding cassette efflux P‐glycoprotein (P‐gp) substrate, was reduced by 11.0% in SOD1 ^G93A mice. This was confirmed by a 46.7% reduction in BMEC uptake of ³ H‐digoxin, an observation that was reversed by resolving the hypermetabolic state of SOD1 ^G93A BMECs. Conclusion and implications: These findings open possible therapeutic avenues that could be exploited to overcome P‐gp‐mediated CNS drug resistance in ALS.

Truncation of the C‐terminal tail of the β₂‐AR, transfection of βARKct or over‐expression of a kinase‐dead GRK mutant reduces isoprenaline‐stimulated glucose uptake, indicating that GRK is important for this response. We explored whether phosphorylation of the β₂‐AR by GRK2 has a role in glucose uptake or if this response is related to the role of GRK2 as a scaffolding protein. CHO‐GLUT4myc cells expressing wild‐type and mutant β₂‐ARs were generated and receptor affinity for [³H]‐CGP12177A and density of binding sites determined together with the affinity of isoprenaline and BRL37344. Following receptor activation by β₂‐AR agonists, cAMP accumulation, GLUT4 translocation, [³H]‐2‐deoxyglucose uptake, and β₂‐AR internalization were measured. Bioluminescence resonance energy transfer was used to investigate interactions between β₂‐AR and β‐arrestin2 or between β₂‐AR and GRK2. Glucose uptake after siRNA knockdown or GRK inhibitors was measured in response to β₂‐AR agonists. BRL37344 was a poor partial agonist for cAMP generation but displayed similar potency and efficacy to isoprenaline for glucose uptake and GLUT4 translocation. These responses to β₂‐AR agonists occurred in CHO‐GLUT4myc cells expressing β₂‐ARs lacking GRK or GRK/PKA phosphorylation sites as well as in cells expressing the wild‐type β₂‐AR. However, β₂‐ARs lacking phosphorylation sites failed to recruit β‐arrestin2 and did not internalize. GRK2 knock‐down or GRK2 inhibitors decreased isoprenaline‐stimulated glucose uptake in rat L6 skeletal muscle cells. Thus, GRK phosphorylation of the β₂‐AR is not associated with isoprenaline‐ or BRL37344‐stimulated glucose uptake. However, GRKs acting as scaffold proteins are important for glucose uptake as GRK2 knock‐down or GRK2 inhibition reduces isoprenaline‐stimulated glucose uptake.

Heart failure remains a leading cause of morbidity and mortality worldwide. Current treatment for patients with heart failure include drugs targeting G protein‐coupled receptors such as β‐adrenoceptor antagonists (β‐blockers) and angiotensin II type 1 receptor antagonists (or angiotensin II receptor blockers). However, many patients progress to advanced heart failure with persistent symptoms, despite treatment with available therapeutics that have been shown to reduce mortality and mortality. GPCR targets currently being explored for the development of novel heart failure therapeutics include adenosine receptor, formyl peptide receptor, relaxin/insulin‐like family peptide receptor, vasopressin receptor, endothelin receptor and the glucagon‐like peptide 1 receptor. Many GPCR drug candidates are limited by insufficient efficacy and/or dose‐limiting unwanted effects. Understanding the current challenges hindering successful clinical translation and the potential to overcome existing limitations will facilitate the future development of novel heart failure therapeutics.LINKED ARTICLES: This article is part of a themed issue Therapeutic Targeting of G Protein‐Coupled Receptors: hot topics from the Australasian Society of Clinical and Experimental Pharmacologists and Toxicologists 2021 Virtual Annual Scientific Meeting. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v181.14/issuetoc

Diet-induced obesity (DIO) is a contributor to co-morbidities, resulting in alterations in hormones, lipids, and low-grade inflammation, with the cannabinoid type 2 receptor (CB₂) contributing to the inflammatory response. The effects of modulating CB₂ with pharmacological treatments on inflammation and adaptations to the obese state are not known. Therefore, we aimed to investigate the molecular mechanisms in adipose tissue of CB₂ agonism and CB₂ antagonism treatment in a DIO model. Male Sprague Dawley rats were placed on a high-fat diet (HFD) (21% fat) for 9 weeks, then received daily intraperitoneal injections with a vehicle, AM630 (0.3 mg/kg), or AM1241 (3 mg/kg), for a further 6 weeks. AM630 or AM1241 treatment in DIO rats did not alter their body weight, food intake, or liver weight, and it had no effect on their numerous circulating cytokines or peri-renal fat pad mass. AM1241 decreased heart weight and BAT weight; both treatments (AM630 or AM1241) decreased plasma leptin levels, while AM630 also decreased plasma ghrelin and GLP-1 levels. Both treatments decreased Adrb3 and TNF-α mRNA levels in eWAT and TNF-α levels in pWAT. AM630 treatment also decreased the mRNA levels of Cnr2, leptin, and Slc2a4 in eWAT. In BAT, both treatments decreased leptin, UCP1, and Slc2a4 mRNA levels, with AM1241 also decreasing Adrb3, IL1β, and PRDM16 mRNA levels, and AM630 increasing IL6 mRNA levels. In DIO, CB₂ agonist and CB₂ antagonist treatment reduces circulating leptin in the absence of weight loss and modulates the mRNA responsible for thermogenesis.

Diet-induced obesity (DIO) reduces fatty acid oxidation in skeletal muscle and decreases circulating levels of adiponectin. Endocannabinoid signaling is overactive in obesity, with some effects abated by antagonism of cannabinoid receptor 1 (CB₁). This research aimed to determine if treatment with the global CB₁ antagonist/inverse agonist, AM251, in high-fat diet (HFD) fed rats influenced adiponectin signaling in skeletal muscle and a “browning” of white adipose tissue (WAT) defined by UCP1 expression levels. Male Sprague Dawley rats consumed an HFD (21% fat) for 9 weeks before receiving daily intraperitoneal injections with vehicle or AM251 (3 mg/kg) for 6 weeks. mRNA expression of genes involved in metabolic functions were measured in skeletal muscle and adipose tissue, and blood was harvested for the measurement of hormones and cytokines. Muscle citrate synthase activity was also measured. AM251 treatment decreased fat pad weight (epididymal, peri-renal, brown), and plasma levels of leptin, glucagon, ghrelin, and GLP-1, and increased PAI-1 along with a range of pro-inflammatory and anti-inflammatory cytokines; however, AM251 did not alter plasma adiponectin levels, skeletal muscle citrate synthase activity or mRNA expression of the genes measured in muscle. AM251 treatment had no effect on white fat UCP1 expression levels. AM251 decreased fat pad mass, altered plasma hormone levels, but did not induce browning of WAT defined by UCP1 mRNA levels or alter gene expression in muscle treated acutely with adiponectin, demonstrating the complexity of the endocannabinoid system and metabolism. The CB₁ ligand AM251 increased systemic inflammation suggesting limitations on its use in metabolic disorders.

Background and purpose The physiological role of vascular β₃‐adrenoceptors (β₃‐ARs) is not fully understood. Recent evidence suggests cardiac β₃‐AR are functionally effective only after down‐regulation of β₁/β₂‐ARs. This study investigated the functional interaction between the β₃‐AR and other β‐AR subtypes in rat striated muscle arteries. Experimental approach Studies were performed in cremaster muscle arteries isolated from male Sprague‐Dawley rats. β‐AR expression was assessed through rt‐PCR and immunofluorescence (IF). Functional effects of β₃‐AR agonists and antagonists and other β‐AR ligands were measured with pressure myography. Key results All three β‐AR subtypes were present in the endothelium of the rat cremaster muscle artery. The β₃‐AR agonists mirabegron and CL316,243 had no effect on the diameter of rat pressurized (70 mmHg) cremaster muscle arterioles with myogenic tone, while the β₃‐AR agonist SR58611A and the non‐selective β‐AR agonist isoprenaline caused concentration‐dependent dilation. In the presence of β_1/2‐AR antagonists including nadolol (10 μM), atenolol (1 μM) and ICI 118,551 (0.1 μM), mirabegron and CL316,243 were effective in causing vasodilation and the potency of SR58611A was enhanced, while responses to isoprenaline were inhibited. The β₃‐AR antagonist L748,337 (1 μM) inhibited vasodilation caused by β₃‐AR agonists (in the presence of β_1/2‐AR blockade), but L748,337 had no effect on isoprenaline‐induced vasodilation. Conclusion and implications All three β‐AR subtypes were present in the endothelium of the rat cremaster muscle artery, but β₃‐AR‐mediated vasodilation was only evident after blockade of β_1/2‐ARs. This suggests constitutive β_1/2‐AR activity inhibits β₃‐AR function in the endothelium of skeletal muscle resistance arteries.

The β₃‐adrenoceptor agonist mirabegron is approved for use for overactive bladder and has been purported to be useful in the treatment of obesity‐related metabolic diseases in humans, including those involving disturbances of glucose homeostasis. We investigated the effect of mirabegron on glucose homeostasis with in vitro and in vivo models, focusing on its selectivity at β‐adrenoceptors, ability to cause browning of white adipocytes, and the role of UCP1 in glucose homeostasis. In mouse brown, white, and brite adipocytes, mirabegron‐mediated effects were examined on cyclic AMP, UCP1 mRNA, [³H]‐2‐deoxyglucose uptake, cellular glycolysis, and O₂ consumption. Mirabegron increased cyclic AMP levels, UCP1 mRNA content, glucose uptake, and cellular glycolysis in brown adipocytes, and these effects were either absent or reduced in white adipocytes. In brite adipocytes, mirabegron increased cyclic AMP levels and UCP1 mRNA content resulting in increased UCP1‐mediated oxygen consumption, glucose uptake, and cellular glycolysis. The metabolic effects of mirabegron in both brown and brite adipocytes were primarily due to actions at β₃‐adrenoceptors as they were largely absent in adipocytes derived from β₃‐adrenoceptor knockout mice. In vivo, mirabegron increased whole body oxygen consumption, glucose uptake into brown and inguinal white adipose tissue, and improved glucose tolerance, all effects that required the presence of the β₃‐adrenoceptor. Furthermore, in UCP1 knockout mice, the effects of mirabegron on glucose tolerance were attenuated. Thus, mirabegron had effects on cellular metabolism in adipocytes that improved glucose handling in vivo, and were primarily due to actions at the β₃‐adrenoceptor.

Activin-A inhibits NK cell effector functions and suppresses antimelanoma immunity.

The type 2 diabetes epidemic makes it important to find insulin-independent ways to improve glucose homeostasis. This study examines the mechanisms activated by a dual β₂-/β₃-adrenoceptor agonist, BRL37344, to increase glucose uptake in skeletal muscle and its effects on glucose homeostasis in vivo. We measured the effect of BRL37344 on glucose uptake, glucose transporter 4 (GLUT4) translocation, cAMP levels, β₂-adrenoceptor desensitization, β-arrestin recruitment, Akt, AMPK, and mammalian target of rapamycin (mTOR) phosphorylation using L6 skeletal muscle cells as a model. We further tested the ability of BRL37344 to modulate skeletal muscle glucose metabolism in animal models (glucose tolerance tests and in vivo and ex vivo skeletal muscle glucose uptake). In L6 cells, BRL37344 increased GLUT4 translocation and glucose uptake only by activation of β₂-adrenoceptors, with a similar potency and efficacy to that of the nonselective β-adrenoceptor agonist isoprenaline, despite being a partial agonist with respect to cAMP generation. GLUT4 translocation occurred independently of Akt and AMPK phosphorylation but was dependent on mTORC2. Furthermore, in contrast to isoprenaline, BRL37344 did not promote agonist-mediated desensitization and failed to recruit β-arrestin1/2 to the β₂-adrenoceptor. In conclusion, BRL37344 improved glucose tolerance and increased glucose uptake into skeletal muscle in vivo and ex vivo through a β₂-adrenoceptor-mediated mechanism independently of Akt. BRL37344 was a partial agonist with respect to cAMP, but a full agonist for glucose uptake, and importantly did not cause classical receptor desensitization or internalization of the receptor.