Mitochondrial Adaptation in Skeletal Muscle: Impact of Obesity, Caloric Restriction, and Dietary Compounds.

PURPOSE OF REVIEW: The global obesity epidemic has become a major public health concern, necessitating comprehensive research into its adverse effects on various tissues within the human body. Among these tissues, skeletal muscle has gained attention due to its susceptibility to obesity-related alterations. Mitochondria are primary source of energy production in the skeletal muscle. Healthy skeletal muscle maintains constant mitochondrial content through continuous cycle of synthesis and degradation. However, obesity has been shown to disrupt this intricate balance. This review summarizes recent findings on the impact of obesity on skeletal muscle mitochondria structure and function. In addition, we summarize the molecular mechanism of mitochondrial quality control systems and how obesity impacts these systems. RECENT FINDINGS: Recent findings show various interventions aimed at mitigating mitochondrial dysfunction in obese model, encompassing strategies including caloric restriction and various dietary compounds. Obesity has deleterious effect on skeletal muscle mitochondria by disrupting mitochondrial biogenesis and dynamics. Caloric restriction, omega-3 fatty acids, resveratrol, and other dietary compounds enhance mitochondrial function and present promising therapeutic opportunities.

SHU9119 and MBP10 are biased ligands at the human melanocortin-4 receptor.

The melanocortin-4 receptor (MC4R), a G protein-coupled receptor, is critically involved in regulating energy homeostasis as well as modulation of reproduction and sexual function. Two peptide antagonists (SHU9119 and MBP10) were derived from the endogenous agonist α-melanocyte stimulating hormone. But their pharmacology at human MC4R is not fully understood. Herein, we performed detailed pharmacological studies of SHU9119 and MBP10 on wild-type (WT) and six naturally occurring constitutively active MC4Rs. Both ligands had no or negligible agonist activity in Gαs-cAMP signaling on WT MC4R, but stimulated extracellular signal-regulated kinases 1 and 2 (ERK1/2) activation on WT and mutant MC4Rs. Mechanistic studies revealed that SHU9119 and MBP10 stimulated ERK1/2 signaling of MC4R by different mechanisms, with SHU9119-stimulated ERK1/2 signaling mediated by phosphatidylinositol 3-kinase (PI3K) and MBP10-initiated ERK1/2 activation through PI3K and ß-arrestin. In summary, our studies demonstrated that SHU9119 and MBP10 were biased ligands for MC4R, preferentially activating ERK1/2 signaling through different mechanisms. SHU9119 acted as a biased ligand and MBP10 behaved as a biased allosteric modulator.

Molecular Cloning, Tissue Distribution, and Pharmacological Characterization of GPR84 in Grass Carp (Ctenopharyngodon Idella).

The G-protein-coupled receptor GPR84, activated by medium-chain fatty acids, primarily expressed in macrophages and microglia, is involved in inflammatory responses and retinal development in mammals and amphibians. However, our understanding of its structure, function, tissue expression, and signaling pathways in fish is limited. In this study, we cloned and characterized the coding sequence of GPR84 (ciGPR84) in grass carp. A phylogenetic analysis revealed its close relationship with bony fishes. High expression levels of GPR84 were observed in the liver and spleen. The transfection of HEK293T cells with ciGPR84 demonstrated its responsiveness to medium-chain fatty acids and diindolylmethane (DIM). Capric acid, undecanoic acid, and lauric acid activated ERK and inhibited cAMP signaling. Lauric acid showed the highest efficiency in activating the ERK pathway, while capric acid was the most effective in inhibiting cAMP signaling. Notably, DIM did not activate GPR84 in grass carp, unlike in mammals. These findings provide valuable insights for mitigating chronic inflammation in grass carp farming and warrant further exploration of the role of medium-chain fatty acids in inflammation regulation in this species.

Divergent Pharmacology and Biased Signaling of the Four Melanocortin-4 Receptor Isoforms in Rainbow Trout (Oncorhynchus mykiss).

The melanocortin-4 receptor (MC4R) is essential for the modulation of energy balance and reproduction in both fish and mammals. Rainbow trout (Oncorhynchus mykiss) has been extensively studied in various fields and provides a unique opportunity to investigate divergent physiological roles of paralogues. Herein we identified four trout mc4r (mc4ra1, mc4ra2, mc4rb1, and mc4rb2) genes. Four trout Mc4rs (omMc4rs) were homologous to those of teleost and mammalian MC4Rs. Multiple sequence alignments, a phylogenetic tree, chromosomal synteny analyses, and pharmacological studies showed that trout mc4r genes may have undergone different evolutionary processes. All four trout Mc4rs bound to two peptide agonists and elevated intracellular cAMP levels dose-dependently. High basal cAMP levels were observed at two omMc4rs, which were decreased by Agouti-related peptide. Only omMc4rb2 was constitutively active in the ERK1/2 signaling pathway. Ipsen 5i, ML00253764, and MCL0020 were biased allosteric modulators of omMc4rb1 with selective activation upon ERK1/2 signaling. ML00253764 behaved as an allosteric agonist in Gs-cAMP signaling of omMc4rb2. This study will lay the foundation for future physiological studies of various mc4r paralogs and reveal the evolution of MC4R in vertebrates.

Pharmacology of orange-spotted grouper (Epinephelus coioides) melanocortin-5 receptor and its modulation by Mrap2.

The mammalian melanocortin-5 receptors (MC5Rs) are involved in various functions, including exocrine gland secretion, glucose uptake, adipocyte lipolysis, and immunity. However, the physiological role of fish Mc5r is rarely studied. Melanocortin-2 receptor accessory protein 2 (MRAP2) modulates pharmacological properties of melanocortin receptors. Herein, to lay the foundation for future physiological studies, we cloned the orange-spotted grouper (Epinephelus coioides) mc5r, with a 1008 bp open reading frame and a predicted protein of 334 amino acids. Grouper mc5r had abundant expression in the brain, skin, and kidney. Four ligands could bind to grouper Mc5r and dose-dependently increase intracellular cAMP levels. Grouper Mrap2 did not affect binding affinity or potency of Mc5r; however, grouper Mrap2 decreased cell surface expression and maximal binding of Mc5r. Mrap2 also significantly decreased the maximal response to a superpotent agonist but not the endogenous agonist. This study provided new data on fish Mc5r pharmacology and its regulation by Mrap2.

Modulation of Canine Melanocortin-3 and -4 Receptors by Melanocortin-2 Receptor Accessory Protein 1 and 2.

The neural melanocortin receptors (MCRs), melanocortin-3 and -4 receptors (MC3R and MC4R), have crucial roles in regulating energy homeostasis. The melanocortin-2 receptor accessory proteins (MRAPs, MRAP1 and MRAP2) have been shown to regulate neural MCRs in a species-specific manner. The potential effects of MRAP1 and MRAP2 on canine neural MCRs have not been investigated before. Herein, we cloned canine (c) MC3R and identified one canine MRAP2 splice variant, MRAP2b, with N-terminal extension of cMRAP2a. Canine MC3R showed higher maximal responses to five agonists than those of human MC3R. We further investigated the modulation of cMRAP1, cMRAP2a, and cMRAP2b, on cMC3R and cMC4R pharmacology. For the cMC3R, all MRAPs had no effect on trafficking; cMRAP1 significantly decreased Bmax whereas cMRAP2a and cMRAP2b significantly increased Bmax. Both MRAP1 and MRAP2a decreased Rmaxs in response to α-MSH and ACTH; MRAP2b only decreased α-MSH-stimulated cAMP generation. For the MC4R, MRAP1 and MRAP2a increased cell surface expression, and MRAP1 and MRAP2a increased Bmaxs. All MRAPs had increased affinities to α-MSH and ACTH. MRAP2a increased ACTH-induced cAMP levels, whereas MRAP2b decreased α-MSH- and ACTH-stimulated cAMP production. These findings may lead to a better understanding of the regulation of neural MCRs by MRAP1 and MRAP2s.

Ligands for Melanocortin Receptors: Beyond Melanocyte-Stimulating Hormones and Adrenocorticotropin.

The discovery of melanocortins in 1916 has resulted in more than 100 years of research focused on these peptides. Extensive studies have elucidated well-established functions of melanocortins mediated by cell surface receptors, including MSHR (melanocyte-stimulating hormone receptor) and ACTHR (adrenocorticotropin receptor). Subsequently, three additional melanocortin receptors (MCRs) were identified. Among these five MCRs, MC3R and MC4R are expressed primarily in the central nervous system, and are therefore referred to as the neural MCRs. Since the central melanocortin system plays important roles in regulating energy homeostasis, targeting neural MCRs is emerging as a therapeutic approach for treating metabolic conditions such as obesity and cachexia. Early efforts modifying endogenous ligands resulted in the development of many potent and selective ligands. This review focuses on the ligands for neural MCRs, including classical ligands (MSH and agouti-related peptide), nonclassical ligands (lipocalin 2, ß-defensin, small molecules, and pharmacoperones), and clinically approved ligands (ACTH, setmelanotide, bremelanotide, and several repurposed drugs).

Targeting trafficking as a therapeutic avenue for misfolded GPCRs leading to endocrine diseases.

G protein-coupled receptors (GPCRs) are plasma membrane proteins associated with an array of functions. Mutations in these receptors lead to a number of genetic diseases, including diseases involving the endocrine system. A particular subset of loss-of-function mutant GPCRs are misfolded receptors unable to traffic to their site of function (i.e. the cell surface plasma membrane). Endocrine disorders in humans caused by GPCR misfolding include, among others, hypo- and hyper-gonadotropic hypogonadism, morbid obesity, familial hypocalciuric hypercalcemia and neonatal severe hyperparathyroidism, X-linked nephrogenic diabetes insipidus, congenital hypothyroidism, and familial glucocorticoid resistance. Several in vitro and in vivo experimental approaches have been employed to restore function of some misfolded GPCRs linked to endocrine disfunction. The most promising approach is by employing pharmacological chaperones or pharmacoperones, which assist abnormally and incompletely folded proteins to refold correctly and adopt a more stable configuration to pass the scrutiny of the cell's quality control system, thereby correcting misrouting. This review covers the most important aspects that regulate folding and traffic of newly synthesized proteins, as well as the experimental approaches targeted to overcome protein misfolding, with special focus on GPCRs involved in endocrine diseases.

Melanocortin-5 Receptor: Pharmacology and Its Regulation of Energy Metabolism.

As the most recent melanocortin receptor (MCR) identified, melanocortin-5 receptor (MC5R) has unique tissue expression patterns, pharmacological properties, and physiological functions. Different from the other four MCR subtypes, MC5R is widely distributed in both the central nervous system and peripheral tissues and is associated with multiple functions. MC5R in sebaceous and preputial glands regulates lipid production and sexual behavior, respectively. MC5R expressed in immune cells is involved in immunomodulation. Among the five MCRs, MC5R is the predominant subtype expressed in skeletal muscle and white adipose tissue, tissues critical for energy metabolism. Activated MC5R triggers lipid mobilization in adipocytes and glucose uptake in skeletal muscle. Therefore, MC5R is a potential target for treating patients with obesity and diabetes mellitus. Melanocortin-2 receptor accessory proteins can modulate the cell surface expression, dimerization, and pharmacology of MC5R. This minireview summarizes the molecular and pharmacological properties of MC5R and highlights the progress made on MC5R in energy metabolism. We poInt. out knowledge gaps that need to be explored in the future.

Early transcriptomic response of mouse adrenal gland and Y-1 cells to dexamethasone.

Glucocorticoids have short- and long-term effects on adrenal gland function and development. RNA sequencing (RNA-seq) was performed to identify early transcriptomic responses to the synthetic glucocorticoid, dexamethasone (Dex), in vitro and in vivo. In total, 1711 genes were differentially expressed in the adrenal glands of the 1-h Dex-treated mice. Among them, only 113 were also considered differentially expressed genes (DEGs) in murine adrenocortical Y-1 cells treated with Dex for 1 h. Gene ontology analysis showed that the upregulated DEGs in the adrenal gland of the 1-h Dex-treated mice were highly associated with the development of neuronal cells, suggesting the adrenal medulla had a rapid response to Dex. Interestingly, only 4.3% of Dex-responsive genes in the Y-1 cell line under Dex treatment for 1 h were differentially expressed under Dex treatment for 24 h. The heatmaps revealed that most early responsive DEGs in Y-1 cells during 1 h of treatment exhibited a transient response. The expression of these genes under treatment for 24 h returned to basal levels similar to that during control treatment. In summary, this research compared the rapid transcriptomic effects of Dex stimulation in vivo and in vitro. Notably, adrenocortical Y-1 cells had a transient early response to Dex treatment. Furthermore, the DEGs had a minimal overlap in the 1-h Dex-treated group in vivo and in vitro.

Mutations in rhodopsin, endothelin B receptor, and CC chemokine receptor 5 in large animals: Modeling human diseases.

G protein-coupled receptors (GPCRs) are the largest family of cell membrane receptors involved in modulating almost all physiological processes by transducing extracellular signals into the cytoplasm. Dysfunctions of GPCR-regulated signaling result in diverse human diseases, making GPCRs the most popular drug targets for human medicine. Large animals share higher similarities (in physiology and metabolism) with humans than rodents. Similar to findings in human genetics, diverse diseases caused by mutations in GPCR genes have also been discovered in large animals. Rhodopsin, endothelin B receptor, and CC chemokine receptor type 5 have been shown to be involved in human retinitis pigmentosa, Hirschsprung disease, and HIV infection/AIDS, respectively, and several mutations of these GPCRs have also been identified from large animals. The large animals with naturally occurring mutations of these GPCRs provide an opportunity to gain a better understanding of the pathogenesis of human diseases, and can be used for preclinical trials of therapies for human diseases. In this review, we aim to summarize the naturally occurring mutations of these three GPCRs in large animals and humans.

Mutations in melanocortin-4 receptor: From fish to men.

Melanocortin-4 receptor (MC4R), expressed abundantly in the hypothalamus, is a critical regulator of energy homeostasis, including both food intake and energy expenditure. Shortly after the publication in 1997 of the Mc4r knockout phenotypes in mice, including increased food intake and severe obesity, the first mutations in MC4R were reported in humans in 1998. Studies in the subsequent two decades have established MC4R mutation as the most common monogenic form of obesity, especially in early-onset severe obesity. Studies in animals, from fish to mammals, have established the conserved physiological roles of MC4R in all vertebrates in regulating energy balance. Drug targeting MC4R has been recently approved for treating morbid genetic obesity. How the MC4R can be exploited for animal production is highly worthy of active investigation.

Melanocortin-1 receptor mutations and pigmentation: Insights from large animals.

The melanocortin-1 receptor (MC1R) is a G protein-coupled receptor expressed in cutaneous and hair follicle melanocytes, and plays a central role in coat color determination in vertebrates. Numerous MC1R variants have been identified in diverse species. Some of these variants have been associated with specific hair and skin color phenotypes in humans as well as coat color in animals. Gain-of-function mutations of the MC1R gene cause dominant or partially dominant black/dark coat color, and loss-of-function mutations of the MC1R gene cause recessive or partially recessive red/yellow/pale coat color phenotypes. These have been well documented in a large number of mammals, including human, dog, cattle, horse, sheep, pig, and fox. Higher similarities between large mammals and humans makes them better models to understand pathogenesis of human diseases caused by MC1R mutations. High identities in MC1Rs and similar variants identified in both humans and large mammals also provide an opportunity for receptor structure and function study. In this review, we aim to summarize the naturally occurring mutations of MC1R in humans and large animals.

Development of a Therapeutic Peptide for Cachexia Suggests a Platform Approach for Drug-like Peptides.

During the development of a melanocortin (MC) peptide drug to treat the condition of cachexia (a hypermetabolic state producing lean body mass wasting), we were confronted with the need for peptide transport across the blood-brain barrier (BBB): the MC-4 receptors (MC4Rs) for metabolic rate control are located in the hypothalamus, i.e., behind the BBB. Using the term "peptides with BBB transport", we screened the medical literature like a peptide library. This revealed numerous "hits"-peptides with BBB transport and/or oral activity. We noted several features common to most peptides in this class, including a dipeptide sequence of nonpolar residues, primary structure cyclization (whole or partial), and a Pro-aromatic motif usually within the cyclized region. Based on this, we designed an MC4R antagonist peptide, TCMCB07, that successfully treated many forms of cachexia. As part of our pharmacokinetic characterization of TCMCB07, we discovered that hepatobiliary extraction from blood accounted for a majority of the circulating peptide's excretion. Further screening of the literature revealed that TCMCB07 is a member of a long-forgotten peptide class, showing active transport by a multi-specific bile salt carrier. Bile salt transport peptides have predictable pharmacokinetics, including BBB transport, but rapid hepatic clearance inhibited their development as drugs. TCMCB07 shares the general characteristics of the bile salt peptide class but with a much longer half-life of hours, not minutes. A change in its C-terminal amino acid sequence slows hepatic clearance. This modification is transferable to other peptides in this class, suggesting a platform approach for producing drug-like peptides.

Demonstration of a Common DPhe7 to DNal(2')7 Peptide Ligand Antagonist Switch for Melanocortin-3 and Melanocortin-4 Receptors Identifies the Systematic Mischaracterization of the Pharmacological Properties of Melanocortin Peptides.

Melanocortin peptides containing a 3-(2-naphthyl)-d-alanine residue in position 7 (DNal(2')7), reported as melanocortin-3 receptor (MC3R) subtype-specific agonists in two separate publications, were found to lack significant MC3R agonist activity. The cell lines used at the University of Arizona for pharmacological characterization of these peptides, consisting of HEK293 cells stably transfected with human melanocortin receptor subtypes MC1R, MC3R, MC4R, or MC5R, were then obtained and characterized by quantitative polymerase chain reaction (PCR). While the MC1R cell line correctly expressed only hMCR1, the three other cell lines were mischaracterized with regard to receptor subtype expression. The demonstration that a 3-(2-naphthyl)-d-alanine residue in position 7, irrespective of the melanocortin peptide template, results primarily in the antagonism of MC3R and MC4R then allowed us to search the published literature for additional errors. The erroneously characterized DNal(2')7-containing peptides date back to 2003; thus, our analysis suggests that systematic mischaracterization of the pharmacological properties of melanocortin peptides occurred.

Regulation of Melanocortin-3 and -4 Receptors by Isoforms of Melanocortin-2 Receptor Accessory Protein 1 and 2.

The neural melanocortin receptors (MCRs), melanocortin-3 and -4 receptors (MC3R and MC4R), play essential non-redundant roles in the regulation of energy homeostasis. Interaction of neural MCRs and melanocortin-2 receptor accessory proteins (MRAPs, MRAP1 and MRAP2) is suggested to play pivotal roles in MC3R and MC4R signaling. In the present study, we identified two new human (h) MRAP2 splice variants, MRAP2b (465 bp open reading frame) and MRAP2c (381 bp open reading frame). Human MRAP2s are different in C-termini. We investigated the effects of five isoforms of MRAPs, hMRAP1a, hMRAP1b, hMRAP2a, hMRAP2b, and hMRAP2c, on MC3R and MC4R pharmacology. At the hMC3R, hMRAP1a and hMRAP2c increased and hMRAP1b decreased the cell surface expression. hMRAP1a increased affinity to ACTH. Four MRAPs (hMRAP1a, hMRAP1b, hMRAP2a, and hMRAP2c) decreased the maximal responses in response to α-MSH and ACTH. For hMC4R, hMRAP1a, hMRAP2a, and hMRAP2c increased the cell surface expression of hMC4R. Human MRAP1b significantly increased affinity to ACTH while MRAP2a decreased affinity to ACTH. Human MRAP1a increased ACTH potency. MRAPs also affected hMC4R basal activities, with hMRAP1s increasing and hMRAP2s decreasing the basal activities. In summary, the newly identified splicing variants, hMRAP2b and hMRAP2c, could regulate MC3R and MC4R pharmacology. The two MRAP1s and three MRAP2s had differential effects on MC3R and MC4R trafficking, binding, and signaling. These findings led to a better understanding of the regulation of neural MCRs by MRAP1s and MRAP2s.

Naturally occurring mutations in G protein-coupled receptors associated with obesity and type 2 diabetes mellitus.

G protein-coupled receptors (GPCRs) are the largest family of membrane receptors involved in the regulation of almost all known physiological processes. Dysfunctions of GPCR-mediated signaling have been shown to cause various diseases. The prevalence of obesity and type 2 diabetes mellitus (T2DM), two strongly associated disorders, is increasing worldwide, with tremendous economical and health burden. New safer and more efficacious drugs are required for successful weight reduction and T2DM treatment. Multiple GPCRs are involved in the regulation of energy and glucose homeostasis. Mutations in these GPCRs contribute to the development and progression of obesity and T2DM. Therefore, these receptors can be therapeutic targets for obesity and T2DM. Indeed some of these receptors, such as melanocortin-4 receptor and glucagon-like peptide 1 receptor, have provided important new drugs for treating obesity and T2DM. This review will focus on the naturally occurring mutations of several GPCRs associated with obesity and T2DM, especially incorporating recent large genomic data and insights from structure-function studies, providing leads for future investigations.