Positional cloning of Sorcs1, a type 2 diabetes quantitative trait locus.

We previously mapped the type 2 diabetes mellitus-2 locus (T2dm2), which affects fasting insulin levels, to distal chromosome 19 in a leptin-deficient obese F2 intercross derived from C57BL/6 (B6) and BTBR T+ tf/J (BTBR) mice. Introgression of a 7-Mb segment of the B6 chromosome 19 into the BTBR background (strain 1339A) replicated the reduced insulin linked to T2dm2. The 1339A mice have markedly impaired insulin secretion in vivo and disrupted islet morphology. We used subcongenic strains derived from 1339A to localize the T2dm2 quantitative trait locus (QTL) to a 242-kb segment comprising the promoter, first exon and most of the first intron of the Sorcs1 gene. This was the only gene in the 1339A strain for which we detected amino acid substitutions and expression level differences between mice carrying B6 and BTBR alleles of this insert, thereby identifying variation within the Sorcs1 gene as underlying the phenotype associated with the T2dm2 locus. SorCS1 binds platelet-derived growth factor, a growth factor crucial for pericyte recruitment to the microvasculature, and may thus have a role in expanding or maintaining the islet vasculature. Our identification of the Sorcs1 gene provides insight into the pathway underlying the pathophysiology of obesity-induced type 2 diabetes mellitus.

Deletion of vacuolar proton-translocating ATPase V(o)a isoforms clarifies the role of vacuolar pH as a determinant of virulence-associated traits in Candida albicans.

Vacuolar proton-translocating ATPase (V-ATPase) is a central regulator of cellular pH homeostasis, and inactivation of all V-ATPase function has been shown to prevent infectivity in Candida albicans. V-ATPase subunit a of the Vo domain (Voa) is present as two fungal isoforms: Stv1p (Golgi) and Vph1p (vacuole). To delineate the individual contribution of Stv1p and Vph1p to C. albicans physiology, we created stv1Δ/Δ and vph1Δ/Δ mutants and compared them to the corresponding reintegrant strains (stv1Δ/ΔR and vph1Δ/ΔR). V-ATPase activity, vacuolar physiology, and in vitro virulence-related phenotypes were unaffected in the stv1Δ/Δ mutant. The vph1Δ/Δ mutant exhibited defective V1Vo assembly and a 90% reduction in concanamycin A-sensitive ATPase activity and proton transport in purified vacuolar membranes, suggesting that the Vph1p isoform is essential for vacuolar V-ATPase activity in C. albicans. The vph1Δ/Δ cells also had abnormal endocytosis and vacuolar morphology and an alkalinized vacuolar lumen (pHvph1Δ/Δ = 6.8 versus pHvph1Δ/ΔR = 5.8) in both yeast cells and hyphae. Secreted protease and lipase activities were significantly reduced, and M199-induced filamentation was impaired in the vph1Δ/Δ mutant. However, the vph1Δ/Δ cells remained competent for filamentation induced by Spider media and YPD, 10% FCS, and biofilm formation and macrophage killing were unaffected in vitro. These studies suggest that different virulence mechanisms differentially rely on acidified vacuoles and that the loss of both vacuolar (Vph1p) and non-vacuolar (Stv1p) V-ATPase activity is necessary to affect in vitro virulence-related phenotypes. As a determinant of C. albicans pathogenesis, vacuolar pH alone may prove less critical than originally assumed.

Candida albicans VMA3 is necessary for V-ATPase assembly and function and contributes to secretion and filamentation.

The vacuolar membrane ATPase (V-ATPase) is a protein complex that utilizes ATP hydrolysis to drive protons from the cytosol into the vacuolar lumen, acidifying the vacuole and modulating several key cellular response systems in Saccharomyces cerevisiae. To study the contribution of V-ATPase to the biology and virulence attributes of the opportunistic fungal pathogen Candida albicans, we created a conditional mutant in which VMA3 was placed under the control of a tetracycline-regulated promoter (tetR-VMA3 strain). Repression of VMA3 in the tetR-VMA3 strain prevents V-ATPase assembly at the vacuolar membrane and reduces concanamycin A-sensitive ATPase-specific activity and proton transport by more than 90%. Loss of C. albicans V-ATPase activity alkalinizes the vacuolar lumen and has pleiotropic effects, including pH-dependent growth, calcium sensitivity, and cold sensitivity. The tetR-VMA3 strain also displays abnormal vacuolar morphology, indicative of defective vacuolar membrane fission. The tetR-VMA3 strain has impaired aspartyl protease and lipase secretion, as well as attenuated virulence in an in vitro macrophage killing model. Repression of VMA3 suppresses filamentation, and V-ATPase-dependent filamentation defects are not rescued by overexpression of RIM8, MDS3, EFG1, CST20, or UME6, which encode positive regulators of filamentation. Specific chemical inhibition of Vma3p function also results in defective filamentation. These findings suggest either that V-ATPase functions downstream of these transcriptional regulators or that V-ATPase function during filamentation involves independent mechanisms and alternative signaling pathways. Taken together, these data indicate that V-ATPase activity is a fundamental requirement for several key virulence-associated traits in C. albicans.

Inhibitors of V-ATPase proton transport reveal uncoupling functions of tether linking cytosolic and membrane domains of V0 subunit a (Vph1p).

Vacuolar ATPases (V-ATPases) are important for many cellular processes, as they regulate pH by pumping cytosolic protons into intracellular organelles. The cytoplasm is acidified when V-ATPase is inhibited; thus we conducted a high-throughput screen of a chemical library to search for compounds that acidify the yeast cytosol in vivo using pHluorin-based flow cytometry. Two inhibitors, alexidine dihydrochloride (EC(50) = 39 µM) and thonzonium bromide (EC(50) = 69 µM), prevented ATP-dependent proton transport in purified vacuolar membranes. They acidified the yeast cytosol and caused pH-sensitive growth defects typical of V-ATPase mutants (vma phenotype). At concentrations greater than 10 µM the inhibitors were cytotoxic, even at the permissive pH (pH 5.0). Membrane fractions treated with alexidine dihydrochloride and thonzonium bromide fully retained concanamycin A-sensitive ATPase activity despite the fact that proton translocation was inhibited by 80-90%, indicating that V-ATPases were uncoupled. Mutant V-ATPase membranes lacking residues 362-407 of the tether of Vph1p subunit a of V(0) were resistant to thonzonium bromide but not to alexidine dihydrochloride, suggesting that this conserved sequence confers uncoupling potential to V(1)V(0) complexes and that alexidine dihydrochloride uncouples the enzyme by a different mechanism. The inhibitors also uncoupled the Candida albicans enzyme and prevented cell growth, showing further specificity for V-ATPases. Thus, a new class of V-ATPase inhibitors (uncouplers), which are not simply ionophores, provided new insights into the enzyme mechanism and original evidence supporting the hypothesis that V-ATPases may not be optimally coupled in vivo. The consequences of uncoupling V-ATPases in vivo as potential drug targets are discussed.

Diabetes-associated SorCS1 regulates Alzheimer's amyloid-beta metabolism: evidence for involvement of SorL1 and the retromer complex.

SorCS1 and SorL1/SorLA/LR11 belong to the sortilin family of vacuolar protein sorting-10 (Vps10) domain-containing proteins. Both are genetically associated with Alzheimer's disease (AD), and SORL1 expression is decreased in the brains of patients suffering from AD. SORCS1 is also genetically associated with types 1 and 2 diabetes mellitus (T1DM, T2DM). We have undertaken a study of the possible role(s) for SorCS1 in metabolism of the Alzheimer's amyloid-ß peptide (Aß) and the Aß precursor protein (APP), to test the hypothesis that Sorcs1 deficiency might be a common genetic risk factor underlying the predisposition to AD that is associated with T2DM. Overexpression of SorCS1cß-myc in cultured cells caused a reduction (p = 0.002) in Aß generation. Conversely, endogenous murine Aß(40) and Aß(42) levels were increased (Aß(40), p = 0.044; Aß(42), p = 0.007) in the brains of female Sorcs1 hypomorphic mice, possibly paralleling the sexual dimorphism that is characteristic of the genetic associations of SORCS1 with AD and DM. Since SorL1 directly interacts with Vps35 to modulate APP metabolism, we investigated the possibility that SorCS1cß-myc interacts with APP, SorL1, and/or Vps35. We readily recovered SorCS1:APP, SorCS1:SorL1, and SorCS1:Vps35 complexes from nontransgenic mouse brain. Notably, total Vps35 protein levels were decreased by 49% (p = 0.009) and total SorL1 protein levels were decreased by 29% (p = 0.003) in the brains of female Sorcs1 hypomorphic mice. From these data, we propose that dysfunction of SorCS1 may contribute to both the APP/Aß disturbance underlying AD and the insulin/glucose disturbance underlying DM.

Loss of PDGF-B activity increases hepatic vascular permeability and enhances insulin sensitivity.

Hepatic vasculature is not thought to pose a permeability barrier for diffusion of macromolecules from the bloodstream to hepatocytes. In contrast, in extrahepatic tissues, the microvasculature is critically important for insulin action, because transport of insulin across the endothelial cell layer is rate limiting for insulin-stimulated glucose disposal. However, very little is known concerning the role in this process of pericytes, the mural cells lining the basolateral membrane of endothelial cells. PDGF-B is a growth factor involved in the recruitment and function of pericytes. We studied insulin action in mice expressing PDGF-B lacking the proteoglycan binding domain, producing a protein with a partial loss of function (PDGF-B(ret/ret)). Insulin action was assessed through measurements of insulin signaling and insulin and glucose tolerance tests. PDGF-B deficiency enhanced hepatic vascular transendothelial transport. One outcome of this change was an increase in hepatic insulin signaling. This correlated with enhanced whole body glucose homeostasis and increased insulin clearance from the circulation during an insulin tolerance test. In obese mice, PDGF-B deficiency was associated with an 80% reduction in fasting insulin and drastically reduced insulin secretion. These mice did not have significantly higher glucose levels, reflecting a dramatic increase in insulin action. Our findings show that, despite already having a high permeability, hepatic transendothelial transport can be further enhanced. To the best of our knowledge, this is the first study to connect PDGF-B-induced changes in hepatic sinusoidal transport to changes in insulin action, demonstrating a link between PDGF-B signaling and insulin sensitivity.

The role of blood vessels, endothelial cells, and vascular pericytes in insulin secretion and peripheral insulin action.

The pathogenesis of type 2 diabetes is intimately intertwined with the vasculature. Insulin must efficiently enter the bloodstream from pancreatic beta-cells, circulate throughout the body, and efficiently exit the bloodstream to reach target tissues and mediate its effects. Defects in the vasculature of pancreatic islets can lead to diabetic phenotypes. Similarly, insulin resistance is accompanied by defects in the vasculature of skeletal muscle, which ultimately reduce the ability of insulin and nutrients to reach myocytes. An underappreciated participant in these processes is the vascular pericyte. Pericytes, the smooth muscle-like cells lining the outsides of blood vessels throughout the body, have not been directly implicated in insulin secretion or peripheral insulin delivery. Here, we review the role of the vasculature in insulin secretion, islet function, and peripheral insulin delivery, and highlight a potential role for the vascular pericyte in these processes.