SUMMARYAdipocytes undergo considerable volumetric expansion in the setting of obesity. It has been proposed that such marked increases in adipocyte size may be sensed via adipocyte-autonomous mechanisms to mediate size-dependent intracellular signaling. Here, we show that SWELL1 (LRRC8a), a member of the Leucine Rich Repeat Containing protein family, is an essential component of a volume-sensitive ion channel (VRAC) in adipocytes. We find that SWELL1-mediated VRAC is augmented in hypertrophic murine and human adipocytes in the setting of obesity. SWELL1 regulates adipocyte insulin-PI3K-AKT2-GLUT4 signaling, glucose uptake and lipid content via SWELL1 C-terminal leucine-rich repeat domain interactions with GRB2/Cav1. Silencing GRB2 in SWELL1 KO adipocytes rescues insulin-pAKT2 signaling. In vivo, shRNA-mediated SWELL1 knock-down and adipose-targeted SWELL1 knock-out reduce adiposity and adipocyte size in obese mice while impairing systemic glycaemia and insulin-sensitivity. These studies identify SWELL1 as a cell-autonomous sensor of adipocyte size that regulates adipocyte growth, insulin sensitivity and glucose tolerance.
The coiled-coil domain of cartilage oligomeric matrix protein (COMPcc) assembles into a homopentamer that naturally recognizes the small molecule 1,25-dihydroxyvitamin D(3) (vit D). To identify the residues critical for the structure, stability, oligomerization, and binding to vit D as well as two other small molecules, all-trans-retinol (ATR) and curcumin (CCM), here we perform an alanine scanning mutagenesis study. Ten residues lining the hydrophobic pocket of COMPcc were mutated into alanine; of the mutated residues, the N-terminal aliphatic residues L37, L44, V47, and L51 are responsible for maintaining the structure and function. Furthermore, two polar residues, T40 and Q54, within the N-terminal region when converted into alanine improve the alpha-helical structure, stability, and self-assembly behavior. Helical stability, oligomerization, and binding appear to be linked in a manner in which mutations that abolish helical structure and assembly bind poorly to vit D, ATR, and CCM. These results provide not only insight into COMPcc and its functional role but also useful guidelines for the design of stable, pentameric coiled-coils capable of selectively storing and delivering various small molecules.
Insulin secretion is initiated by activation of voltage-gated Ca2+ channels (VGCC) to trigger Ca2+-mediated insulin vesicle fusion with the β-cell plasma membrane. The firing of VGCC requires β-cell membrane depolarization, which is regulated by a balance of depolarizing and hyperpolarizing ionic currents. Here, we show that SWELL1 mediates a swell-activated, depolarizing chloride current (ICl,SWELL) in both murine and human β-cells. Hypotonic and glucose-stimulated β-cell swelling activates SWELL1-mediated ICl,SWELL and this contributes to membrane depolarization and activation of VGCC-dependent intracellular calcium signaling. SWELL1 depletion in MIN6 cells and islets significantly impairs glucose-stimulated insulin secretion. Tamoxifen-inducible β-cell-targeted Swell1 KO mice have normal fasting serum glucose and insulin levels but impaired glucose-stimulated insulin secretion and glucose tolerance; and this is further exacerbated in mild obesity. Our results reveal that β-cell SWELL1 modulates insulin secretion and systemic glycaemia by linking glucose-mediated β-cell swelling to membrane depolarization and activation of VGCC-triggered calcium signaling.
Metal dependent protein‐based assemblies derived from the cartilage oligomeric matrix protein (C) coiled‐coil domain (His6‐C) and two variants with mutation at position 40 (His6‐T40A) and 44 (His6‐L44A) are explored. All proteins have an N‐terminal hexahistidine tag (His6) that interacts with divalent metal ions Zn(II) and Ni(II). Binding to Zn(II) confers enhanced helical structure and stability, while Ni(II) promotes aggregation. Surprisingly, His6‐L44A undergoes a conformational switch from unstructured to α‐helix in the presence of Zn(II). Both His6‐C and His6‐T40A further assemble into discrete nanofibers that appear to be stabilized by Zn(II) in which the fiber formation is dictated by the α‐helical content. Because Ni(II) promotes aggregation, the proteins visibly cluster, forming large fiber mats in the case of His6‐C and His6‐T40A or aggregated structures as observed for His6‐L44A. Due to the unique pentameric assembly of the proteins, recognition of a small molecule within the pore is assessed using curcumin as the guest molecule. In the presence of Zn(II), there is enhanced binding to curcumin, while the addition of Ni(II) causes a loss in binding. It is shown that metal binding serves as a trigger to control the conformation of the proteins, affecting the nanoscopic fibrous assemblies and small molecule recognition abilities.
Maintenance of skeletal muscle is beneficial in obesity and Type 2 diabetes. Mechanical stimulation can regulate skeletal muscle differentiation, growth and metabolism, however the molecular mechanosensor remains unknown. Here, we show that SWELL1 (Lrrc8a) functionally encodes a swell-activated anion channel that regulates PI3K-AKT, ERK1/2, mTOR signaling, muscle differentiation, myoblast fusion, cellular oxygen consumption, and glycolysis in skeletal muscle cells. LRRC8A over-expression in Lrrc8a KO myotubes boosts PI3K-AKT-mTOR signaling to supra-normal levels and fully rescues myotube formation. Skeletal muscle targeted Lrrc8a KO mice have smaller myofibers, generate less force ex vivo, and exhibit reduced exercise endurance, associated with increased adiposity under basal conditions, and glucose intolerance and insulin resistance when raised on a high-fat diet, compared to WT mice. These results reveal that the LRRC8 complex regulates insulin-PI3K-AKT-mTOR signalling in skeletal muscle to influence skeletal muscle differentiation in vitro and skeletal myofiber size, muscle function, adiposity and systemic metabolism in vivo.
The endothelium responds to numerous chemical and mechanical factors in regulating vascular tone, blood pressure and blood flow. The endothelial volume regulatory anion channel (VRAC) has been proposed to be mechano-sensitive and thereby sense fluid flow and hydrostatic pressure to regulate vascular function. Here, we show that the Leucine Rich Repeat Containing Protein 8a, LRRC8A (SWELL1) is required for VRAC in human umbilical vein endothelial cells (HUVECs). Endothelial LRRC8A regulates AKT-eNOS signaling under basal, stretch and shear-flow stimulation, forms a GRB2-Cav1-eNOS signaling complex, and is required for endothelial cell alignment to laminar shear flow. Endothelium-restricted Lrrc8a KO mice develop hypertension in response to chronic angiotensin-II infusion and exhibit impaired retinal blood flow with both diffuse and focal blood vessel narrowing in the setting of Type 2 diabetes (T2D). These data demonstrate that LRRC8A regulates AKT-eNOS in endothelium and is required for maintaining vascular function, particularly in the setting of T2D.
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