A role for PAK1 mediated phosphorylation of ß-catenin Ser552 in the regulation of insulin secretion.

The presence of adherens junctions and the associated protein ß-catenin are requirements for the development of glucose-stimulated insulin secretion (GSIS) in ß-cells. Evidence indicates that modulation of ß-catenin function in response to changes in glucose levels can modulate the levels of insulin secretion from ß-cells but the role of ß-catenin phosphorylation in this process has not been established. We find that a Ser552Ala version of ß-catenin attenuates glucose-stimulated insulin secretion indicating a functional role for Ser552 phosphorylation of ß-catenin in insulin secretion. This is associated with alterations F/G actin ratio but not the transcriptional activity of ß-catenin. Both glucose and GLP-1 stimulated phosphorylation of the serine 552 residue on ß-catenin. We investigated the possibility that an EPAC-PAK1 pathway might be involved in this phosphorylation event. We find that reduction in PAK1 levels using siRNA attenuates both glucose and GLP-1 stimulated phosphorylation of ß-catenin Ser552 and the effects of these on insulin secretion in ß-cell models. Furthermore, both the EPAC inhibitor ESI-09 and the PAK1 inhibitor IPA3 do the same in both ß-cell models and mouse islets. Together this identifies phosphorylation of ß-catenin at Ser552 as part of a cell signalling mechanism linking nutrient and hormonal regulation of ß-catenin to modulation of insulin secretory capacity of ß-cells and indicates this phosphorylation event is regulated downstream of EPAC and PAK1 in ß-cells.

α-catenin isoforms are regulated by glucose and involved in regulating insulin secretion in rat clonal ß-cell models.

The recent finding that ß-catenin levels play an important rate-limiting role in processes regulating insulin secretion lead us to investigate whether its binding partner α-catenin also plays a role in this process. We find that levels of both α-E-catenin and α-N-catenin are rapidly up-regulated as levels of glucose are increased in rat clonal ß-cell models INS-1E and INS-832/3. Lowering in levels of either α-catenin isoform using siRNA resulted in significant increases in glucose stimulated insulin secretion (GSIS) and this effect was attenuated when ß-catenin levels were lowered indicating these proteins have opposing effects on insulin release. This effect of α-catenin knockdown on GSIS was not due to increases in insulin expression but was associated with increases in calcium influx into cells. Moreover, simultaneous depletion of α-E catenin and α-N catenin decreased the actin polymerisation to a similar degree as latrunculin treatment and inhibition of ARP 2/3 mediated actin branching with CK666 attenuated the α-catenin depletion effect on GSIS. This suggests α-catenin mediated actin remodelling may be involved in the regulation of insulin secretion. Together this indicates that α-catenin and ß-catenin can play opposing roles in regulating insulin secretion, with some degree of functional redundancy in roles of α-E-catenin and α-N-catenin. The finding that, at least in ß-cell models, the levels of each can be regulated in the longer term by glucose also provides a potential mechanism by which sustained changes in glucose levels might impact on the magnitude of GSIS.

ß-catenin is important for the development of an insulin responsive pool of GLUT4 glucose transporters in 3T3-L1 adipocytes.

GLUT4 is unique among specialized glucose transporters in being exclusively expressed in muscle and adipocytes. In the absence of insulin the distribution of GLUT4 is preferentially intracellular and insulin stimulation results in the movement of GLUT4 containing vesicles to the plasma membrane. This process is responsible for the insulin stimulation of glucose uptake in muscle and fat. While signalling pathways triggering the translocation of GLUT4 are well understood, the mechanisms regulating the intracellular retention of GLUT4 are less well understood. Here we report a role for ß-catenin in this process. In 3T3-L1 adipocytes in which ß-catenin is depleted, the levels of GLUT4 at and near the plasma membrane rise in unstimulated cells while the subsequent increase in GLUT4 at the plasma membrane upon insulin stimulation is reduced. Small molecule approaches to acutely activate or inhibit ß-catenin give results that support the results obtained with siRNA and these changes are accompanied by matching changes in glucose transport into these cells. Together these results indicate that ß-catenin is a previously unrecognized regulator of the mechanisms that control the insulin sensitive pool of GLUT4 transporters inside these adipocyte cells.

The putative leucine sensor Sestrin2 is hyperphosphorylated by acute resistance exercise but not protein ingestion in human skeletal muscle.

PURPOSE: Dietary protein and resistance exercise (RE) are both potent stimuli of the mammalian target of rapamycin complex 1 (mTORC1). Sestrins1, 2, 3 are multifunctional proteins that regulate mTORC1, stimulate autophagy and alleviate oxidative stress. Of this family, Sestrin2 is a putative leucine sensor implicated in mTORC1 and AMP-dependent protein kinase (AMPK) regulation. There is currently no data examining the responsiveness of Sestrin2 to dietary protein ingestion, with or without RE. METHODS: In Study 1, 16 males ingested either 10 or 20 g of milk protein concentrate (MPC) with muscle biopsies collected pre, 90 and 210 min post-beverage consumption. In Study 2, 20 males performed a bout of RE immediately followed by the consumption of 9 g of MPC or carbohydrate placebo. Analysis of Sestrins, AMPK and antioxidant responses was examined. RESULTS: Dietary protein ingestion did not result in Sestrin2 mobility shift. After RE, Sestrin2 phosphorylation state was significantly altered and was not further modified by post-exercise protein or carbohydrate ingestion. With RE, AMPK phosphorylation remained stable, while the mRNA expressions of several antioxidants were upregulated. CONCLUSIONS: Dietary protein ingestion did not affect the signalling by the family of Sestrins. With RE, Sestrin2 was hyperphosphorylated, with no further evidence of a relationship to AMPK signalling.

A Critical Role for ß-Catenin in Modulating Levels of Insulin Secretion from ß-Cells by Regulating Actin Cytoskeleton and Insulin Vesicle Localization.

The processes regulating glucose-stimulated insulin secretion (GSIS) and its modulation by incretins in pancreatic ß-cells are only partly understood. Here we investigate the involvement of ß-catenin in these processes. Reducing ß-catenin levels using siRNA knockdown attenuated GSIS in a range of ß-cell models and blocked the ability of GLP-1 agonists and the depolarizing agent KCl to potentiate this. This could be mimicked in both ß-cell models and isolated islets by short-term exposure to the ß-catenin inhibitory drug pyrvinium. In addition, short-term treatment with a drug that increases ß-catenin levels results in an increase in insulin secretion. The timing of these effects suggests that ß-catenin is required for the processes regulating trafficking and/or release of pre-existing insulin granules rather than for those regulated by gene expression. This was supported by the finding that the overexpression of the transcriptional co-activator of ß-catenin, transcription factor 7-like 2 (TCF7L2), attenuated insulin secretion, consistent with the extra TCF7L2 translocating ß-catenin from the plasma membrane pool to the nucleus. We show that ß-catenin depletion disrupts the intracellular actin cytoskeleton, and by using total internal reflectance fluorescence (TIRF) microscopy, we found that ß-catenin is required for the glucose- and incretin-induced depletion of insulin vesicles from near the plasma membrane. In conclusion, we find that ß-catenin levels modulate Ca2+-dependent insulin exocytosis under conditions of glucose, GLP-1, or KCl stimulation through a role in modulating insulin secretory vesicle localization and/or fusion via actin remodeling. These findings also provide insights as to how the overexpression of TCF7L2 may attenuate insulin secretion.

Functional rescue of mutant ABCA1 proteins by sodium 4-phenylbutyrate.

Mutations in the ATP-binding cassette transporter A1 (ABCA1) are a major cause of decreased HDL cholesterol (HDL-C), which infers an increased risk of cardiovascular disease (CVD). Many ABCA1 mutants show impaired localization to the plasma membrane. The aim of this study was to investigate whether the chemical chaperone, sodium 4-phenylbutyrate (4-PBA) could improve cellular localization and function of ABCA1 mutants. Nine different ABCA1 mutants (p.A594T, p.I659V, p.R1068H, p.T1512M, p.Y1767D, p.N1800H, p.R2004K, p.A2028V, p.Q2239N) expressed in HEK293 cells, displaying different degrees of mislocalization to the plasma membrane and discrete impacts on cholesterol efflux, were subject to treatment with 4-PBA. Treatment restored localization to the plasma membrane and increased cholesterol efflux function for the majority of mutants. Treatment with 4-PBA also increased ABCA1 protein expression in all transfected cell lines. In fibroblast cells obtained from low HDL-C subjects expressing two of the ABCA1 mutants (p.R1068H and p.N1800H), 4-PBA increased cholesterol efflux without any increase in ABCA1 expression. Our study is the first to investigate the effect of the chemical chaperone, 4-PBA on ABCA1 and shows that it is capable of restoring plasma membrane localization and enhancing the cholesterol efflux function of mutant ABCA1s both in vitro and ex vivo. These results suggest 4-PBA may warrant further investigation as a potential therapy for increasing cholesterol efflux and HDL-C levels.

An ABCA1 truncation shows no dominant negative effect in a familial hypoalphalipoproteinemia pedigree with three ABCA1 mutations.

The ATP binding cassette transporter (ABCA1) A1 is a key determinant of circulating high density lipoprotein cholesterol (HDL-C) levels. Mutations in ABCA1 are a major genetic contributor to low HDL-C levels within the general population. Following the finding of three different ABCA1 mutations, p.C978fsX988, p.T1512M and p.N1800H in a subject with hypoalphalipoproteinemia, we aimed to establish whether the p.C978fsX988 truncation exerted a dominant negative effect on the full-length ABCA1 alleles within family members as has been reported for other ABCA1 truncations. Characterisation of the p.C978fsX988 mutant in transfected HEK 293 cells showed it to be expressed as a GFP fusion protein but lacking in cholesterol efflux function. This was in keeping with results from cholesterol efflux assays in the fibroblasts of p.C978fsX988 carriers which also showed impaired efflux. Allele- specific quantification of p.C978fsX988 mRNA and analysis of ABCA1 protein levels in the fibroblasts of p.C978fsX988 heterozygotes showed negligible levels of mRNA and protein expression. There was no evidence of a dominant negative effect on wildtype or p.N1800H protein levels. We conclude that in the case of the p.C978fsX988 truncated mutant a lack of expression precludes it from having a dominant negative effect.

Glucose regulates expression of pro-inflammatory genes, IL-1ß and IL-12, through a mechanism involving hexosamine biosynthesis pathway-dependent regulation of α-E catenin.

High glucose levels are associated with changes in macrophage polarisation and evidence indicates that the sustained or even short-term high glucose levels modulate inflammatory responses in macrophages. However, the mechanism by which macrophages can sense the changes in glucose levels are not clearly understood. We find that high glucose levels rapidly increase the α-E catenin protein level in RAW264.7 macrophages. We also find an attenuation of glucose-induced increase in α-E catenin when hexosamine biosynthesis (HB) pathway is inhibited either with glutamine depletion or with the drugs azaserine and tunicamycin. This indicates the involvement of HB pathway in this process. Then, we investigated the potential role of α-E catenin in glucose-induced macrophage polarisation. We find that the reduction in α-E catenin level using siRNA attenuates the glucose-induced changes of both IL-1ß and IL-12 mRNA levels under LPS-stimulated condition but does not affect TNF-α expression. Together this indicates that α-E catenin can sense the changes in glucose levels in macrophages via HB pathway and also can modulate the glucose-induced gene expression of inflammatory markers such as IL-1ß and IL-12. This identifies a new part of the mechanism by which macrophages are able to respond to changes in glucose levels.

A gel-based method for purification of apolipoprotein A-I from small volumes of plasma.

We present here a gel-based method for rapid purification of apolipoprotein A-I (apoA-I) from small volumes of human plasma. After isolation of high density lipoprotein from plasma, the apoA-I protein was separated by electrophoresis and the apoA-I band excised from the gel. The apoA-I was then eluted from the gel strip, concentrated, and delipidated ready for use. The structure and function of the gel-purified apoA-I protein was compared against apoA-I purified by the traditional size-exclusion chromatography method. The α-helical content of the gel-purified apoA-I as determined by circular dichroism was similar to chromatography-purified apoA-I. The functional activity of gel-purified apoA-I, as determined by cholesterol efflux assays in primary human fibroblasts and RAW264.7 macrophages, was also comparable with chromatography-purified apoA-I. This method is a valid alternative for apoA-I purification with some advantages over traditional chromatography purification including a much reduced plasma volume requirement, less time and cost, and a higher percentage protein recovery. The method is particularly suitable for applications requiring the purification of apoA-I from multiple human or animal samples of interest.

ß-catenin regulates muscle glucose transport via actin remodelling and M-cadherin binding.

OBJECTIVE: Skeletal muscle glucose disposal following a meal is mediated through insulin-stimulated movement of the GLUT4-containing vesicles to the cell surface. The highly conserved scaffold-protein ß-catenin is an emerging regulator of vesicle trafficking in other tissues. Here, we investigated the involvement of ß-catenin in skeletal muscle insulin-stimulated glucose transport. METHODS: Glucose homeostasis and transport was investigated in inducible muscle specific ß-catenin knockout (BCAT-mKO) mice. The effect of ß-catenin deletion and mutation of ß-catenin serine 552 on signal transduction, glucose uptake and protein-protein interactions were determined in L6-G4-myc cells, and ß-catenin insulin-responsive binding partners were identified via immunoprecipitation coupled to label-free proteomics. RESULTS: Skeletal muscle specific deletion of ß-catenin impaired whole-body insulin sensitivity and insulin-stimulated glucose uptake into muscle independent of canonical Wnt signalling. In response to insulin, ß-catenin was phosphorylated at serine 552 in an Akt-dependent manner, and in L6-G4-myc cells, mutation of ß-cateninS552 impaired insulin-induced actin-polymerisation, resulting in attenuated insulin-induced glucose transport and GLUT4 translocation. ß-catenin was found to interact with M-cadherin in an insulin-dependent ß-cateninS552-phosphorylation dependent manner, and loss of M-cadherin in L6-G4-myc cells attenuated insulin-induced actin-polymerisation and glucose transport. CONCLUSIONS: Our data suggest that ß-catenin is a novel mediator of glucose transport in skeletal muscle and may contribute to insulin-induced actin-cytoskeleton remodelling to support GLUT4 translocation.

The role of adherens junction proteins in the regulation of insulin secretion.

In healthy individuals, any rise in blood glucose levels is rapidly countered by the release of insulin from the ß-cells of the pancreas which in turn promotes the uptake and storage of the glucose in peripheral tissues. The ß-cells possess exquisite mechanisms regulating the secretion of insulin to ensure that the correct amount of insulin is released. These mechanisms involve tight control of the movement of insulin containing secretory vesicles within the ß-cells, initially preventing most vesicles being able to move to the plasma membrane. Elevated glucose levels trigger an influx of Ca2+ that allows fusion of the small number of insulin containing vesicles that are pre-docked at the plasma membrane but glucose also stimulates processes that allow other insulin containing vesicles located further in the cell to move to and fuse with the plasma membrane. The mechanisms controlling these processes are complex and not fully understood but it is clear that the interaction of the ß-cells with other ß-cells in the islets is very important for their ability to develop the appropriate machinery for proper regulation of insulin secretion. Emerging evidence indicates one factor that is key for this is the formation of homotypic cadherin mediated adherens junctions between ß-cells. Here, we review the evidence for this and discuss the mechanisms by which these adherens junctions might regulate insulin vesicle trafficking as well as the implications this has for understanding the dysregulation of insulin secretion seen in pathogenic states.

A Simple Bioreactor-Based Method to Generate Kidney Organoids from Pluripotent Stem Cells.

Kidney organoids made from pluripotent stem cells have the potential to revolutionize how kidney development, disease, and injury are studied. Current protocols are technically complex, suffer from poor reproducibility, and have high reagent costs that restrict scalability. To overcome some of these issues, we have established a simple, inexpensive, and robust method to grow kidney organoids in bulk from human induced pluripotent stem cells. Our organoids develop tubular structures by day 8 and show optimal tissue morphology at day 14. A comparison with fetal human kidneys suggests that day-14 organoid tissue most closely resembles late capillary loop stage nephrons. We show that deletion of HNF1B, a transcription factor linked to congenital kidney defects, interferes with tubulogenesis, validating our experimental system for studying renal developmental biology. Taken together, our protocol provides a fast, efficient, and cost-effective method for generating large quantities of human fetal kidney tissue, enabling the study of normal and aberrant kidney development.

Homology modeling and functional testing of an ABCA1 mutation causing Tangier disease.

OBJECTIVE: To investigate the impact of the p.R1068H mutation on the structure and function of the ATP-binding cassette A1 (ABCA1) protein. METHODS: A homology model of the nucleotide binding domains of ABCA1 was constructed to identify the three-dimensional orientation of R1068. Cholesterol efflux assays were performed on fibroblasts obtained from members of a Tangier disease (TD) family carrying the p.R1068H mutation and in HEK293 cells transfected with a p.R1068H mutant cDNA vector. Confocal microscopy was used to investigate the localisation of the wildtype and mutant p.R1068H protein in HEK293 cells. RESULTS: Sequence alignments and modeling indicated residue R1068 to be located in an α-helix downstream of the Walker B motif in the first nucleotide binding domain (NBD-1), in a position to form ionic interactions with D1092 and E1093. Cholesterol efflux studies showed the efflux from TD fibroblasts and HEK293 cells expressing the mutant p.R1068H protein to be markedly reduced compared to wildtype. Localisation of the mutant p.R1068H protein in HEK293 cells showed intracellular retention of the protein indicating a defect in trafficking to the plasma membrane. CONCLUSION: Homology modeling of the ABCA1 protein showed that the p.R1068H mutation would likely disrupt the conformation of NBD-1. Functional studies of p.R1068H showed a lack of cholesterol efflux function due to defective trafficking to the plasma membrane, most likely caused by impaired oligomerisation.