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.
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.
A decline in stress tolerance is a hallmark of aging. For instance, older organisms showed extensive hepatic damage, along with increased morbidity and mortality, after environmental heating. We hypothesized that hyperthermic challenge would produce exaggerated oxidative stress in old animals, leading to increased hepatic injury. After a heat-stress protocol, time-course changes in reactive oxygen species (ROS) levels, oxidative damage markers, glutathione (GSH)/glutathione disulfide (GSSG) ratios, and activation of stress-response transcription factors (AP-1 and NF-kappaB) were measured in young and old rats. A small, transient increase in hepatic oxidative damage, with minimal injury, was observed in young rats. However, old rats showed widespread hepatic injury that was manifested over a 24 h period after heating. This pathology was preceded by elevated steady-state levels of ROS, along with large increases in lipid peroxidation products, prolonged hepatic DNA oxidation damage, aberrant GSH/GSSG profiles, and altered activation patterns for AP-1. These data indicate that young animals have an effective oxidation-reduction buffering system in the liver that provides protection from oxidative damage to intracellular macromolecules under stress conditions. In sharp contrast, an environmental challenge in older animals produces exaggerated oxidative stress and alterations in signal transduction pathways, which can contribute to cellular dysfunction and age-related reductions in stress tolerance.
Hemophilia A is a clinically important coagulation disorder caused by the lack or abnormality of plasma coagulation factor VIII (FVIII). Gene transfer of the FVIII cDNA to hepatocytes using lentiviral vectors is a potential therapeutic approach. We investigated the efficacy of feline immunodeficiency virus (FIV)-based vectors in targeting hepatocytes and correcting FVIII deficiency in a hemophilia A mouse model. Several viral envelope glycoproteins were screened for efficient FIV vector pseudotyping and hepatocyte transduction. The GP64 glycoprotein from baculovirus Autographa californica multinuclear polyhedrosis virus pseudotyped FIV efficiently and showed excellent hepatocyte tropism. The GP64-pseudotyped vector was stable in the presence of human or mouse complement. Inclusion of a hybrid liver-specific promoter (murine albumin enhancer/human ␣1-antitrypsin promoter) further enhanced transgene expression in hepatocytes. We generated a GP64-pseudotyped FIV vector encoding the B domaindeleted human FVIII coding region driven by the liver-specific promoter, with 2 ben- IntroductionHemophilia A is an X-linked disorder caused by a deficiency of plasma factor VIII (FVIII), affecting 1 in 5000 to 10 000 males 1,2 and characterized by an abnormal bleeding tendency. In its severe form (less than 1% of the normal FVIII level) the disease may be fatal, 2,3 and spontaneous hemorrhage into joints and muscles leading to permanent disability remains a significant clinical problem. Intravenous injections of FVIII concentrates purified from human plasma or produced by recombinant technology are effective in controlling bleeding episodes. However, prophylactic treatment of hemophilia A is problematic due to limited availability and high cost. Venous access and the risk of blood-borne transmissible diseases such as AIDS and hepatitis are also a concern. Moreover, the development of inhibitory antibodies can significantly reduce the efficacy of replacement therapy in about 20% of patients. 4 Gene transfer provides an alternative therapeutic approach for long-term correction of FVIII deficiency. Transferring a functional FVIII gene to somatic cells, particularly hepatocytes, could provide sustained production of FVIII. Increasingly, prophylactic FVIII replacement therapy to maintain plasma levels above 1% is recommended in selected children and young adults by most hemophilia treatment centers in the United States and Europe. 5,6 A stable level of FVIII in the bloodstream may prevent breakthrough bleeds and protect against spontaneous bleeding and chronic joint injury. Gene therapy is particularly suitable for hemophilia A, 7,8 because as little as 5 ng/mL of plasma FVIII (normal levels about 200 ng/mL), equivalent to a production rate of 30 g per 10 9 cells per day, is sufficient to convert severe hemophilia A to a mild form. 7 Indeed, recent reports from many laboratories including ours have demonstrated the potential feasibility of retroviral gene therapy for hemophilia A in animal models. 9,10 While an efficacious therapy h...
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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