Glucosamine-induced endoplasmic reticulum stress affects GLUT4 expression via activating transcription factor 6 in rat and human skeletal muscle cells

AIMS/HYPOTHESIS: Glucosamine, generated during hyperglycaemia, causes insulin resistance in different cells. Here we sought to evaluate the possible role of endoplasmic reticulum (ER) stress in the induction of insulin resistance by glucosamine in skeletal muscle cells. METHODS: Real-time RT-PCR analysis, 2-deoxy-D: -glucose (2-DG) uptake and western blot analysis were carried out in rat and human muscle cell lines. RESULTS: In both rat and human myotubes, glucosamine treatment caused a significant increase in the expression of the ER stress markers immunoglobulin heavy chain-binding protein/glucose-regulated protein 78 kDa (BIP/GRP78 [also known as HSPA5]), X-box binding protein-1 (XBP1) and activating transcription factor 6 (ATF6). In addition, glucosamine impaired insulin-stimulated 2-DG uptake in both rat and human myotubes. Interestingly, pretreatment of both rat and human myotubes with the chemical chaperones 4-phenylbutyric acid (PBA) or tauroursodeoxycholic acid (TUDCA), completely prevented the effect of glucosamine on both ER stress induction and insulin-induced glucose uptake. In both rat and human myotubes, glucosamine treatment reduced mRNA and protein levels of the gene encoding GLUT4 and mRNA levels of the main regulators of the gene encoding GLUT4 (myocyte enhancer factor 2 a [MEF2A] and peroxisome proliferator-activated receptor-gamma coactivator 1alpha [PGC1alpha]). Again, PBA or TUDCA pretreatment prevented glucosamine-induced inhibition of GLUT4 (also known as SLC2A4), MEF2A and PGC1alpha (also known as PPARGC1A). Finally, we showed that overproduction of ATF6 is sufficient to inhibit the expression of genes GLUT4, MEF2A and PGC1alpha and that ATF6 silencing with a specific small interfering RNA is sufficient to completely prevent glucosamine-induced inhibition of GLUT4, MEF2A and PGC1alpha in skeletal muscle cells. CONCLUSIONS/INTERPRETATION: In this work we show that glucosamine-induced ER stress causes insulin resistance in both human and rat myotubes and impairs GLUT4 production and insulin-induced glucose uptake via an ATF6-dependent decrease of the GLUT4 regulators MEF2A and PGC1alpha

RESPOND – A patient-centred program to prevent secondary falls in older people presenting to the emergency department with a fall: Protocol for a multi-centre randomised controlled trial

Introduction: Participation in falls prevention activities by older people following presentation to the Emergency Department (ED) with a fall is suboptimal. This randomised controlled trial (RCT) will test the RESPOND program which is designed to improve older persons’ participation in falls prevention activities through delivery of patient-centred education and behaviour change strategies. Design and setting: An RCT at two tertiary referral EDs in Melbourne and Perth, Australia. Participants: Five-hundred and twenty eight community-dwelling people aged 60-90 years presenting to the ED with a fall and discharged home will be recruited. People who: require an interpreter or hands-on assistance to walk; live in residential aged care or >50 kilometres from the trial hospital; have terminal illness, cognitive impairment, documented aggressive behaviour or history of psychosis; are receiving palliative care; or are unable to use a telephone will be excluded. Methods: Participants will be randomly allocated to the RESPOND intervention or standard care control group. RESPOND incorporates: (1) home-based risk factor assessment; (2) education, coaching, goal setting, and follow-up telephone support for management of one or more of four risk factors with evidence of effective intervention; and (3) healthcare provider communication and community linkage delivered over six months. Primary outcomes are falls and fall injuries per-person-year. Discussion: RESPOND builds on prior falls prevention learnings and aims to help individuals make guided decisions about how they will manage their falls risk. Patient-centred models have been successfully trialled in chronic and cardiovascular disease however evidence to support this approach in falls prevention is limited. Trial registration. The protocol for this study is registered with the Australian New Zealand Clinical Trials Registry (ACTRN12614000336684)

Impairments in Site-Specific AS160 Phosphorylation and Effects of Exercise Training

The purpose of this study was to determine if site-specific phosphorylation at the level of Akt substrate of 160 kDa (AS160) is altered in skeletal muscle from sedentary humans across a wide range of the adult life span (18–84 years of age) and if endurance- and/or strength-oriented exercise training could rescue decrements in insulin action and skeletal muscle AS160 phosphorylation. A euglycemic-hyperinsulinemic clamp and skeletal muscle biopsies were performed in 73 individuals encompassing a wide age range (18–84 years of age), and insulin-stimulated AS160 phosphorylation was determined. Decrements in whole-body insulin action were associated with impairments in insulin-induced phosphorylation of skeletal muscle AS160 on sites Ser-588, Thr-642, Ser-666, and phospho-Akt substrate, but not Ser-318 or Ser-751. Twelve weeks of endurance- or strength-oriented exercise training increased whole-body insulin action and reversed impairments in AS160 phosphorylation evident in insulin-resistant aged individuals. These findings suggest that a dampening of insulin-induced phosphorylation of AS160 on specific sites in skeletal muscle contributes to the insulin resistance evident in a sedentary aging population and that exercise training is an effective intervention for treating these impairments

DOORS syndrome and a recurrent truncating ATP6V1B2 variant

PURPOSE: Biallelic variants in TBC1D24, which encodes a protein that regulates vesicular transport, are frequently identified in patients with DOORS (deafness, onychodystrophy, osteodystrophy, intellectual disability [previously referred to as mental retardation], and seizures) syndrome. The aim of the study was to identify a genetic cause in families with DOORS syndrome and without a TBC1D24 variant. METHODS: Exome or Sanger sequencing was performed in individuals with a clinical diagnosis of DOORS syndrome without TBC1D24 variants. RESULTS: We identified the same truncating variant in ATP6V1B2 (NM_001693.4:c.1516C>T; p.Arg506*) in nine individuals from eight unrelated families with DOORS syndrome. This variant was already reported in individuals with dominant deafness onychodystrophy (DDOD) syndrome. Deafness was present in all individuals, along with onychodystrophy and abnormal fingers and/or toes. All families but one had developmental delay or intellectual disability and five individuals had epilepsy. We also describe two additional families with DDOD syndrome in whom the same variant was found. CONCLUSION: We expand the phenotype associated with ATP6V1B2 and propose another causal gene for DOORS syndrome. This finding suggests that DDOD and DOORS syndromes might lie on a spectrum of clinically and molecularly related conditions

Massive endocytosis driven by lipidic forces originating in the outer plasmalemmal monolayer: a new approach to membrane recycling and lipid domains

The roles that lipids play in endocytosis are the subject of debate. Using electrical and imaging methods, we describe massive endocytosis (MEND) in baby hamster kidney (BHK) and HEK293 cells when the outer plasma membrane monolayer is perturbed by the nonionic detergents, Triton X-100 (TX100) and NP-40. Some alkane detergents, the amphipathic drugs, edelfosine and tamoxifen, and the phospholipase inhibitor, U73122, are also effective. Uptake of the membrane tracer, FM 4–64, into vesicles and loss of reversible FM 4–64 binding confirm that 40–75% of the cell surface is internalized. Ongoing MEND stops in 2–4 s when amphipaths are removed, and amphipaths are without effect from the cytoplasmic side. Thus, expansion of the outer monolayer is critical. As found for Ca-activated MEND, vesicles formed are <100 nm in diameter, membrane ruffles are lost, and β-cyclodextrin treatments are inhibitory. However, amphipath-activated MEND does not require Ca transients, adenosine triphosphate (ATP) hydrolysis, G protein cycling, dynamins, or actin cytoskeleton remodeling. With elevated cytoplasmic ATP (>5 mM), MEND can reverse completely and be repeated multiple times in BHK and HEK293 cells, but not cardiac myocytes. Reversal is blocked by N-ethylmaleimide and a nitric oxide donor, nitroprusside. Constitutively expressed Na/Ca exchangers internalize roughly in proportion to surface membrane, whereas Na/K pump activities decrease over-proportionally. Sodium dodecyl sulfate and dodecylglucoside do not cause MEND during their application, but MEND occurs rapidly when they are removed. As monitored capacitively, the binding of these detergents decreases with MEND, whereas TX100 binding does not decrease. In summary, nonionic detergents can fractionate the plasma membrane in vivo, and vesicles formed connect immediately to physiological membrane-trafficking mechanisms. We suggest that lateral and transbilayer inhomogeneities of the plasma membrane provide potential energies that, when unbridled by triggers, can drive endocytosis by lipidic forces

A pilot study of transrectal endoscopic ultrasound elastography in inflammatory bowel disease

BACKGROUND: Using standard diagnostic algorithms it is not always possible to establish the correct phenotype of inflammatory bowel disease which is essential for therapeutical decisions. Endoscopic ultrasound elastography is a new endoscopic procedure which can differentiate the stiffness of normal and pathological tissue by ultrasound. Therefore, we aimed to investigate the role of transrectal ultrasound elastography in distiction between Crohn's disease and ulcerative colitis. ----- METHODS: A total 30 Crohn's disease, 25 ulcerative colitis, and 28 non-inflammatory bowel disease controls were included. Transrectal ultrasound elastography was performed in all patients and controls. In all ulcerative coltis patients and 80% of Crohn's disease patients endoscopy was performed to assess disease activity in the rectum. ----- RESULTS: Significant difference in rectal wall thickness and strain ratio was detected between patients with Crohn's disease and controls (p = 0.0001). CD patients with active disease had higher strain ratio than patients in remission (p = 0.02). In ulcerative colitis group a significant difference in rectal wall thickness was found between controls and patients with active disease (p = 0.03). A significant difference in rectal wall thickness (p = 0.02) and strain ratio (p = 0.0001) was detected between Crohn's disease and ulcerative colitis patient group. Crohn's disease patients with active disease had a significantly higher strain ratio compared to ulcerative colitis patients with active disease (p = 0.0001). ----- CONCLUSION: Transrectal ultrasound elastography seems to be a promising new diagnostic tool in the field of inflammatory bowel disease. Further study on a larger cohort of patients is needed to definitely assess the role of transrectal ultrasound elastography in inflammatory bowel disease