Exocyst inactivation in urothelial cells disrupts autophagy and activates non-canonical NF-κB signaling.

Ureter obstruction is a highly prevalent event during embryonic development and is a major cause of pediatric kidney disease. We have previously reported that ureteric bud-specific ablation of the gene expressing the exocyst subunit EXOC5 in late murine gestation results in failure of urothelial stratification, cell death and complete ureter obstruction. However, the mechanistic connection between disrupted exocyst activity, urothelial cell death and subsequent ureter obstruction was unclear. Here, we report that inhibited urothelial stratification does not drive cell death during ureter development. Instead, we demonstrate that the exocyst plays a critical role in autophagy in urothelial cells, and that disruption of autophagy activates a urothelial NF-κB stress response. Impaired autophagy first provokes canonical NF-κB activity, which is progressively followed by increasing levels of non-canonical NF-κB activity and cell death if the stress remains unresolved. Furthermore, we demonstrate that ureter obstructions can be completely rescued in Exoc5 conditional knockout mice by administering a single dose of the pan-caspase inhibitor z-VAD-FMK at embryonic day 16.5 prior to urothelial cell death. Taken together, ablation of Exoc5 disrupts autophagic stress response and activates progressive NF-κB signaling, which promotes obstructive uropathy.

Disrupted glucose homeostasis and skeletal-muscle-specific glucose uptake in an exocyst knockout mouse model.

Skeletal muscle is responsible for the majority of glucose disposal following meals, and this is achieved by insulin-mediated trafficking of glucose transporter type 4 (GLUT4) to the cell membrane. The eight-protein exocyst trafficking complex facilitates targeted docking of membrane-bound vesicles, a process underlying the regulated delivery of fuel transporters. We previously demonstrated the role of exocyst subunit EXOC5 in insulin-stimulated GLUT4 exocytosis and glucose uptake in cultured rat skeletal myoblasts. However, the in vivo role of EXOC5 in skeletal muscle remains unclear. Using mice with inducible, skeletal-muscle-specific knockout of exocyst subunit EXOC5 (Exoc5-SMKO), we examined how muscle-specific disruption of the exocyst would affect glucose homeostasis in vivo. We found that both male and female Exoc5-SMKO mice displayed elevated fasting glucose levels. Additionally, male Exoc5-SMKO mice had impaired glucose tolerance and lower serum insulin levels. Using indirect calorimetry, we observed that male Exoc5-SMKO mice have a reduced respiratory exchange ratio during the light period and lower energy expenditure. Using the hyperinsulinemic-euglycemic clamp method, we further showed that insulin-stimulated skeletal muscle glucose uptake is reduced in Exoc5-SMKO males compared with wild-type controls. Overall, our findings indicate that EXOC5 and the exocyst are necessary for insulin-stimulated glucose uptake in skeletal muscle and regulate glucose homeostasis in vivo.

The exocyst complex regulates insulin-stimulated glucose uptake of skeletal muscle cells.

Skeletal muscle handles ~80-90% of the insulin-induced glucose uptake. In skeletal muscle, insulin binding to its cell surface receptor triggers redistribution of intracellular glucose transporter GLUT4 protein to the cell surface, enabling facilitated glucose uptake. In adipocytes, the eight-protein exocyst complex is an indispensable constituent in insulin-induced glucose uptake, as it is responsible for the targeted trafficking and plasma membrane-delivery of GLUT4. However, the role of the exocyst in skeletal muscle glucose uptake has never been investigated. Here we demonstrate that the exocyst is a necessary factor in insulin-induced glucose uptake in skeletal muscle cells as well. The exocyst complex colocalizes with GLUT4 storage vesicles in L6-GLUT4myc myoblasts at a basal state and associates with these vesicles during their translocation to the plasma membrane after insulin signaling. Moreover, we show that the exocyst inhibitor endosidin-2 and a heterozygous knockout of Exoc5 in skeletal myoblast cells both lead to impaired GLUT4 trafficking to the plasma membrane and hinder glucose uptake in response to an insulin stimulus. Our research is the first to establish that the exocyst complex regulates insulin-induced GLUT4 exocytosis and glucose metabolism in muscle cells. A deeper knowledge of the role of the exocyst complex in skeletal muscle tissue may help our understanding of insulin resistance in type 2 diabetes.

Fibroproliferative response to urothelial failure obliterates the ureter lumen in a mouse model of prenatal congenital obstructive nephropathy.

Congenital obstructive nephropathy (CON) is the most prevalent cause of pediatric chronic kidney disease and end-stage renal disease. The ureteropelvic junction (UPJ) region, where the renal pelvis transitions to the ureter, is the most commonly obstructed site in CON. The underlying causes of congenital UPJ obstructions remain poorly understood, especially when they occur in utero, in part due to the lack of genetic animal models. We previously showed that conditional inactivation of Sec10, a central subunit of the exocyst complex, in the epithelial cells of the ureter and renal collecting system resulted in late gestational bilateral UPJ obstructions with neonatal anuria and death. In this study, we show that without Sec10, the urothelial progenitor cells that line the ureter fail to differentiate into superficial cells, which are responsible for producing uroplakin plaques on the luminal surface. These Sec10-knockout urothelial cells undergo cell death by E17.5 and the urothelial barrier becomes leaky to luminal fluid. Also at E17.5, we measured increased expression of TGFß1 and genes associated with myofibroblast activation, with evidence of stromal remodeling. Our findings support the model that a defective urothelial barrier allows urine to induce a fibrotic wound healing mechanism, which may contribute to human prenatal UPJ obstructions.

Growth hormone alters lipid composition and increases the abundance of casein and lactalbumin mRNA in the MAC-T cell line.

The MAC-T cell line has been used extensively to investigate bovine mammary epithelial cell function. A lactogenic phenotype is generally induced in this cell line by a combination of dexamethasone, insulin and prolactin and has typically been assessed by milk protein production. Few studies have focused on identifying other factors that may affect milk protein synthesis in the MAC-T cell line, and none have considered the lipid class distribution of MAC-T cells as a component of the lactogenic phenotype. Growth hormone (GH) has been shown to increase milk protein synthesis both in vivo and in mammary cell models, and has been shown to alter the lipogenic profile of mammary explant models. We tested the hypothesis that MAC-T cells would respond directly to GH and that the response would include alterations to the lipid class distribution as well as to milk protein gene expression, leading to a more appropriate model for mammary cell function than treatment with dexamethasone, insulin and prolactin alone. Differentiated cells expressed GH receptor mRNA, and addition of GH to the differentiation medium significantly induced production of alpha-s1 casein and alpha-lactalbumin mRNA. GH also significantly affected the proportion of triacylglycerol and sphingomyelin. These results indicate that GH is an important factor in inducing a lactogenic phenotype in the MAC-T cell line, and support the possibility of a direct effect of GH on milk synthesis in vivo.