Mechanical Regulation of Glycogen Synthase Kinase 3β (GSK3β) in Mesenchymal Stem Cells Is Dependent on Akt Protein Serine 473 Phosphorylation via mTORC2 Protein

Mechanical signals can inactivate glycogen synthase kinase 3β (GSK3β), resulting in stabilization of β-catenin. This signaling cascade is necessary for the inhibition of adipogenesis in mesenchymal stem cells (MSC) that is produced by a daily strain regimen. We investigated whether Akt is the mechanically activated kinase responsible for phosphorylation and inactivation of GSK3β in MSC. Mechanical strain (2% magnitude, 0.17 Hz) induced phosphorylation of Akt at Ser-473 and Thr-308 in parallel with phosphorylation of GSK3β at Ser-9. Inhibiting Akt (Akt1/2 kinase inhibitor treatment or Akt knockdown) prevented strain-induced phosphorylation of GSK3β at Ser-9. Inhibition of PI3K prevented Thr-308 phosphorylation, but strain-induced Ser-473 phosphorylation was measurable and induced phosphorylation of GSK3β, suggesting that Ser-473 phosphorylation is sufficient for the downstream mechanoresponse. As Rictor/mTORC2 (mammalian target of rapamycin complex 2) is known to transduce phosphorylation of Akt at Ser-473 by insulin, we investigated whether it contributes to strain-induced Ser-473 phosphorylation. Phosphorylation of Ser-473 by both mechanical and insulin treatment in MSC was prevented by the mTOR inhibitor KU0063794. When mTORC2 was blocked, mechanical GSK3β inactivation was prevented, whereas insulin inhibition of GSK3β was still measured in the absence of Ser-473 phosphorylation, presumably through phosphorylation of Akt at Thr-308. In sum, mechanical input initiates a signaling cascade that is uniquely dependent on mTORC2 activation and phosphorylation of Akt at Ser-473, an effect sufficient to cause inactivation of GSK3β. Thus, mechanical regulation of GSK3β downstream of Akt is dependent on phosphorylation of Akt at Ser-473 in a manner distinct from that of growth factors. As such, Akt reveals itself to be a pleiotropic signaling molecule whose downstream targets are differentially regulated depending upon the nature of the activating input

Forces during cellular uptake of viruses and nanoparticles at the ventral side

Many intracellular pathogens, such as mammalian reovirus, mimic extracellular matrix motifs to specifically interact with the host membrane. Whether and how cell-matrix interactions influence virus particle uptake is unknown, as it is usually studied from the dorsal side. Here we show that the forces exerted at the ventral side of adherent cells during reovirus uptake exceed the binding strength of biotin-neutravidin anchoring viruses to a biofunctionalized substrate. Analysis of virus dissociation kinetics using the Bell model revealed mean forces higher than 30 pN per virus, preferentially applied in the cell periphery where close matrix contacts form. Utilizing 100 nm-sized nanoparticles decorated with integrin adhesion motifs, we demonstrate that the uptake forces scale with the adhesion energy, while actin/myosin inhibitions strongly reduce the uptake frequency, but not uptake kinetics. We hypothesize that particle adhesion and the push by the substrate provide the main driving forces for uptake

Bone marrow fat accumulation accelerated by high fat diet is suppressed by exercise

Marrow adipose tissue (MAT), associated with skeletal fragility and hematologic insufficiency, remains poorly understood and difficult to quantify. We tested the response of MAT to high fat diet (HFD) and exercise using a novel volumetric analysis, and compared it to measures of bone quantity. We hypothesized that HFD would increase MAT and diminish bone quantity, while exercise would slow MAT acquisition and promote bone formation. Eight week-old female C57BL/6 mice were fed a regular (RD) or HFD, and exercise groups were provided voluntary access to running wheels (RD-E, HFD-E). Femoral MAT was assessed by μCT (lipid binder osmium) using a semi-automated approach employing rigid co-alignment, regional bone masks and was normalized for total femoral volume (TV) of the bone compartment. MAT was 2.6-fold higher in HFD relative to RD mice. Exercise suppressed MAT in RD-E mice by more than half compared with RD. Running similarly inhibited MAT acquisition in HFD mice. Exercise significantly increased bone quantity in both diet groups. Thus, HFD caused significant accumulation of MAT; importantly running exercise limited MAT acquisition while promoting bone formation during both diets. That MAT is exquisitely responsive to diet and exercise, and its regulation by exercise appears to be inversely proportional to effects on exercise induced bone formation, is relevant for an aging and sedentary population

Development of Vitamin D Toxicity from Overcorrection of Vitamin D Deficiency: A Review of Case Reports

Over the past two decades, vitamin D level measurements have become some of the most frequently ordered tests in the laboratory. This increase is due to a growing awareness of widespread vitamin D deficiency and scientific data suggesting the beneficial effects of vitamin D in various diseases. A literature search was carried out in PubMed for cases reporting vitamin D intoxication and overdose. Thirteen articles were included in this review. Intoxication was severe in the reported cases. Patients presented with serum vitamin D concentrations ranging between 150 and 1220 ng/mL and serum calcium concentrations between 11.1 and 23.1 mg/dL. Most of the reported patients showed symptoms of vitamin D toxicity such as vomiting, dehydration, pain, and loss of appetite. The underlying causes included manufacturing errors, overdosing by patients or prescribers, and combinations of these factors. Our literature search highlights the fact that even though vitamin D intoxication is rare, it does occur and therefore patients and prescribers should be more cognizant of the potential dangers of vitamin D overdose

10 years of 25-hydroxyvitamin-D testing by LC-MS/MS-trends in vitamin-D deficiency and sufficiency

In early 2000's vitamin-D deficiency was shown to be prevalent in several countries including the United States (US). Studies exploring the role of vitamin-D metabolism in diverse disease pathways generated an increased demand for vitamin-D supplementation and an immense public interest in measurement of vitamin-D metabolite levels. In this report, we review the role of vitamin-D metabolism in disease processes, clinical utility of measuring vitamin-D metabolites including 25-hydroxyvitamin-D (25(OH)D), 1,25-dihydroxyvitamin-D and 24,25-dihydroxyvitamin-D and discuss vitamin-D assay methodologies including immunoassays and liquid chromatography mass spectrometry (LC-MS/MS) assays. We also provide examples of vitamin-D toxicity and insight into the trends in serum 25(OH)D levels in the US population based on 10 years of data from on serum 25(OH)D values from ~5,000,000 patients who were tested at the Mayo Medical Laboratories between February 2007–February 2017. Keywords: Vitamin D supplementation trends, LC-MS/MS, Vitamin D toxicit

Titin-Based Nanoparticle Tension Sensors Map High-Magnitude Integrin Forces within Focal Adhesions

Mechanical forces transmitted through integrin transmembrane receptors play important roles in a variety of cellular processes ranging from cell development to tumorigenesis. Despite the importance of mechanics in integrin function, the magnitude of integrin forces within adhesions remains unclear. Literature suggests a range from 1 to 50 pN, but the upper limit of integrin forces remains unknown. Herein we challenge integrins with the most mechanically stable molecular tension probe, which is comprised of the immunoglobulin 27th (I27) domain of cardiac titin flanked with a fluorophore and gold nanoparticle. Cell experiments show that integrin forces unfold the I27 domain, suggesting that integrin forces exceed ∼30–40 pN. The addition of a disulfide bridge within I27 “clamps” the probe and resists mechanical unfolding. Importantly, incubation with a reducing agent initiates SH exchange, thus unclamping I27 at a rate that is dependent on the applied force. By recording the rate of S–S reduction in clamped I27, we infer that integrins apply 110 ± 9 pN within focal adhesions of rat embryonic fibroblasts. The rates of S–S exchange are heterogeneous and integrin subtype-dependent. Nanoparticle titin tension sensors along with kinetic analysis of unfolding demonstrate that a subset of integrins apply tension many fold greater than previously reported