Adipsin and adipocyte-derived C3aR1 regulate thermogenic fat in a sex-dependent fashion.

Thermogenesis in beige/brown adipose tissues can be leveraged to combat metabolic disorders such as type 2 diabetes and obesity. The complement system plays pleiotropic roles in metabolic homeostasis and organismal energy balance with canonical effects on immune cells and noncanonical effects on nonimmune cells. The adipsin/C3a/C3a receptor 1 (C3aR1) pathway stimulates insulin secretion and sustains pancreatic ß cell mass. However, its role in adipose thermogenesis has not been defined. Here, we show that male Adipsin/Cfd-knockout mice exhibited increased energy expenditure and white adipose tissue (WAT) browning. In addition, male adipocyte-specific C3aR1-knockout mice exhibited enhanced WAT thermogenesis and increased respiration. In stark contrast, female adipocyte-specific C3aR1-knockout mice displayed decreased brown fat thermogenesis and were cold intolerant. Female mice expressed lower levels of Adipsin in thermogenic adipocytes and adipose tissues than males. C3aR1 was also lower in female subcutaneous adipose tissue than in males. Collectively, these results reveal sexual dimorphism in the adipsin/C3a/C3aR1 axis in regulating adipose thermogenesis and defense against cold stress. Our findings establish a potentially new role of the alternative complement pathway in adaptive thermogenesis and highlight sex-specific considerations in potential therapeutic targets for metabolic diseases.

A beta cell subset with enhanced insulin secretion and glucose metabolism is reduced in type 2 diabetes.

The pancreatic islets are composed of discrete hormone-producing cells that orchestrate systemic glucose homeostasis. Here we identify subsets of beta cells using a single-cell transcriptomic approach. One subset of beta cells marked by high CD63 expression is enriched for the expression of mitochondrial metabolism genes and exhibits higher mitochondrial respiration compared with CD63lo beta cells. Human and murine pseudo-islets derived from CD63hi beta cells demonstrate enhanced glucose-stimulated insulin secretion compared with pseudo-islets from CD63lo beta cells. We show that CD63hi beta cells are diminished in mouse models of and in humans with type 2 diabetes. Finally, transplantation of pseudo-islets generated from CD63hi but not CD63lo beta cells into diabetic mice restores glucose homeostasis. These findings suggest that loss of a specific subset of beta cells may lead to diabetes. Strategies to reconstitute or maintain CD63hi beta cells may represent a potential anti-diabetic therapy.

Cross-species single-cell comparison of systemic and cardiac inflammatory responses after cardiac injury.

The immune system coordinates the response to cardiac injury and is known to control regenerative and fibrotic scar outcomes in the heart and subsequent chronic low-grade inflammation associated with heart failure. Here we profiled the inflammatory response to heart injury using single cell transcriptomics to compare and contrast two experimental models with disparate outcomes. We used adult mice, which like humans lack the ability to fully recover and zebrafish which spontaneously regenerate after heart injury. The extracardiac reaction to cardiomyocyte necrosis was also interrogated to assess the specific peripheral tissue and immune cell reaction to chronic stress. Cardiac macrophages are known to play a critical role in determining tissue homeostasis by healing versus scarring. We identified distinct transcriptional clusters of monocytes/macrophages in each species and found analogous pairs in zebrafish and mice. However, the reaction to myocardial injury was largely disparate between mice and zebrafish. The dichotomous response to heart damage between the mammalian and zebrafish monocytes/macrophages may underlie the impaired regenerative process in mice, representing a future therapeutic target.

The voltage-gated sodium channel ß2 subunit associates with lipid rafts by S-palmitoylation.

The voltage-gated sodium channel is critical for cardiomyocyte function. It consists of a protein complex comprising a pore-forming α subunit and associated ß subunits. In polarized Madin-Darby canine kidney cells, we show evidence by acyl-biotin exchange that ß2 is S-acylated at Cys-182. Interestingly, we found that palmitoylation increases ß2 association with detergent-resistant membranes. ß2 localizes exclusively to the apical surface. However, depletion of plasma membrane cholesterol, or blocking intracellular cholesterol transport, caused mislocalization of ß2, as well as of the non-palmitoylable C182S mutant, to the basolateral domain. Apical ß2 did not undergo endocytosis and displayed limited diffusion within the plane of the membrane; such behavior suggests that, at least in part, it is cytoskeleton anchored. Upon acute cholesterol depletion, its mobility was greatly reduced, and a slight reduction was also measured as a result of lack of palmitoylation, supporting ß2 association with cholesterol-rich lipid rafts. Indeed, lipid raft labeling confirmed a partial overlap with apical ß2. Although ß2 palmitoylation was not required to promote surface localization of the α subunit, our data suggest that it is likely implicated in lipid raft association and the polarized localization of ß2.

Resveratrol targets PD-L1 glycosylation and dimerization to enhance antitumor T-cell immunity.

New strategies to block the immune evasion activity of programmed death ligand-1 (PD-L1) are urgently needed. When exploring the PD-L1-targeted effects of mechanistically diverse metabolism-targeting drugs, exposure to the dietary polyphenol resveratrol (RSV) revealed its differential capacity to generate a distinct PD-L1 electrophoretic migration pattern. Using biochemical assays, computer-aided docking/molecular dynamics simulations, and fluorescence microscopy, we found that RSV can operate as a direct inhibitor of glyco-PD-L1-processing enzymes (α-glucosidase/α-mannosidase) that modulate N-linked glycan decoration of PD-L1, thereby promoting the endoplasmic reticulum retention of a mannose-rich, abnormally glycosylated form of PD-L1. RSV was also predicted to interact with the inner surface of PD-L1 involved in the interaction with PD-1, almost perfectly occupying the target space of the small compound BMS-202 that binds to and induces dimerization of PD-L1. The ability of RSV to directly target PD-L1 interferes with its stability and trafficking, ultimately impeding its targeting to the cancer cell plasma membrane. Impedance-based real-time cell analysis (xCELLigence) showed that cytotoxic T-lymphocyte activity was notably exacerbated when cancer cells were previously exposed to RSV. This unforeseen immunomodulating mechanism of RSV might illuminate new approaches to restore T-cell function by targeting the PD-1/PD-L1 immunologic checkpoint with natural polyphenols.

Trafficking and Function of the Voltage-Gated Sodium Channel ß2 Subunit.

The voltage-gated sodium channel is vital for cardiomyocyte function, and consists of a protein complex containing a pore-forming α subunit and two associated ß subunits. A fundamental, yet unsolved, question is to define the precise function of ß subunits. While their location in vivo remains unclear, large evidence shows that they regulate localization of α and the biophysical properties of the channel. The current data support that one of these subunits, ß2, promotes cell surface expression of α. The main α isoform in an adult heart is NaV1.5, and mutations in SCN5A, the gene encoding NaV1.5, often lead to hereditary arrhythmias and sudden death. The association of ß2 with cardiac arrhythmias has also been described, which could be due to alterations in trafficking, anchoring, and localization of NaV1.5 at the cardiomyocyte surface. Here, we will discuss research dealing with mechanisms that regulate ß2 trafficking, and how ß2 could be pivotal for the correct localization of NaV1.5, which influences cellular excitability and electrical coupling of the heart. Moreover, ß2 may have yet to be discovered roles on cell adhesion and signaling, implying that diverse defects leading to human disease may arise due to ß2 mutations.

N-Glycosylation of the voltage-gated sodium channel ß2 subunit is required for efficient trafficking of NaV1.5/ß2 to the plasma membrane.

The voltage-gated sodium channel is critical for cardiomyocyte function and consists of a protein complex comprising a pore-forming α subunit and two associated ß subunits. It has been shown previously that the associated ß2 subunits promote cell surface expression of the α subunit. The major α isoform in the adult human heart is NaV1.5, and germline mutations in the NaV1.5-encoding gene, sodium voltage-gated channel α subunit 5 (SCN5A), often cause inherited arrhythmias. Here, we investigated the mechanisms that regulate ß2 trafficking and how they may determine proper NaV1.5 cell surface localization. Using heterologous expression in polarized Madin-Darby canine kidney cells, we show that ß2 is N-glycosylated in vivo and in vitro at residues 42, 66, and 74, becoming sialylated only at Asn-42. We found that fully nonglycosylated ß2 was mostly retained in the endoplasmic reticulum, indicating that N-linked glycosylation is required for efficient ß2 trafficking to the apical plasma membrane. The nonglycosylated variant reached the cell surface by bypassing the Golgi compartment at a rate of only approximately one-third of that of WT ß2. YFP-tagged, nonglycosylated ß2 displayed mobility kinetics in the plane of the membrane similar to that of WT ß2. However, it was defective in promoting surface localization of NaV1.5. Interestingly, ß2 with a single intact glycosylation site was as effective as the WT in promoting NaV1.5 surface localization. In conclusion, our results indicate that N-linked glycosylation of ß2 is required for surface localization of NaV1.5, a property that is often defective in inherited cardiac arrhythmias.

Trafficking and localisation to the plasma membrane of Nav 1.5 promoted by the ß2 subunit is defective due to a ß2 mutation associated with Brugada syndrome.

BACKGROUND INFORMATION: Cardiac channelopathies arise by mutations in genes encoding ion channel subunits. One example is Brugada Syndrome (BrS), which causes arrhythmias and sudden death. BrS is often associated with mutations in SCN5A, encoding Nav 1.5, the α subunit of the major cardiac voltage-gated sodium channel. This channel forms a protein complex including one or two associated ß subunits as well as other proteins. RESULTS: We analysed regulation of Nav 1.5 localisation and trafficking by ß2, specifically, Nav 1.5 arrival to the cell surface. We used polarised Madin-Darby canine kidney (MDCK) cells and mouse atria-derived HL-1 cells, which retain phenotypic features of adult cardiomyocytes. In both, Nav 1.5 was found essentially intracellular, mainly in the endoplasmic reticulum, whereas ß2 localised to the plasma membrane, and was restricted to the apical surface in MDCK cells. A fraction of ß2 interacted with Nav 1.5, despite their limited overlap. Importantly, ß2 promoted Nav 1.5 localisation to the cell surface. Both ß2 WT and the BrS-associated mutation D211G (substitution of Asp for Gly) effectively reached the plasma membrane. Strikingly, however, ß2 D211G was defective in promoting Nav 1.5 surface localisation. CONCLUSIONS: Our data sustain that ß2 promotes surface localisation of Nav 1.5, which can be affected due to ß2 mutations associated with channelopathies. SIGNIFICANCE: Our findings add to the understanding of ß2 role in Nav 1.5 trafficking and localisation, which must influence cell excitability and electrical coupling in the heart. This study will contribute to knowledge on development of arrhythmias.