Deficiency of glucocorticoid receptor in bone marrow adipocytes has mild effects on bone and hematopoiesis but does not influence expansion of marrow adiposity with caloric restriction.

Introduction: Unlike white adipose tissue depots, bone marrow adipose tissue (BMAT) expands during caloric restriction (CR). Although mechanisms for BMAT expansion remain unclear, prior research suggested an intermediary role for increased circulating glucocorticoids. Methods: In this study, we utilized a recently described mouse model (BMAd-Cre) to exclusively target bone marrow adipocytes (BMAds) for elimination of the glucocorticoid receptor (GR) (i.e. Nr3c1) whilst maintaining GR expression in other adipose depots. Results: Mice lacking GR in BMAds (BMAd-Nr3c1 -/-) and control mice (BMAd-Nr3c1 +/+) were fed ad libitum or placed on a 30% CR diet for six weeks. On a normal chow diet, tibiae of female BMAd-Nr3c1-/- mice had slightly elevated proximal trabecular metaphyseal bone volume fraction and thickness. Both control and BMAd-Nr3c1-/- mice had increased circulating glucocorticoids and elevated numbers of BMAds in the proximal tibia following CR. However, no significant differences in trabecular and cortical bone were observed, and quantification with osmium tetroxide and µCT revealed no difference in BMAT accumulation between control or BMAd-Nr3c1 -/- mice. Differences in BMAd size were not observed between BMAd-Nr3c1-/- and control mice. Interestingly, BMAd-Nr3c1-/- mice had decreased circulating white blood cell counts 4 h into the light cycle. Discussion: In conclusion, our data suggest that eliminating GR from BMAd has minor effects on bone and hematopoiesis, and does not impair BMAT accumulation during CR.

Lipolysis of bone marrow adipocytes is required to fuel bone and the marrow niche during energy deficits.

To investigate roles for bone marrow adipocyte (BMAd) lipolysis in bone homeostasis, we created a BMAd-specific Cre mouse model in which we knocked out adipose triglyceride lipase (ATGL, Pnpla2 gene). BMAd-Pnpla2-/- mice have impaired BMAd lipolysis, and increased size and number of BMAds at baseline. Although energy from BMAd lipid stores is largely dispensable when mice are fed ad libitum, BMAd lipolysis is necessary to maintain myelopoiesis and bone mass under caloric restriction. BMAd-specific Pnpla2 deficiency compounds the effects of caloric restriction on loss of trabecular bone in male mice, likely due to impaired osteoblast expression of collagen genes and reduced osteoid synthesis. RNA sequencing analysis of bone marrow adipose tissue reveals that caloric restriction induces dramatic elevations in extracellular matrix organization and skeletal development genes, and energy from BMAd is required for these adaptations. BMAd-derived energy supply is also required for bone regeneration upon injury, and maintenance of bone mass with cold exposure.

BAd-CRISPR: Inducible gene knockout in interscapular brown adipose tissue of adult mice.

CRISPR/Cas9 has enabled inducible gene knockout in numerous tissues; however, its use has not been reported in brown adipose tissue (BAT). Here, we developed the brown adipocyte CRISPR (BAd-CRISPR) methodology to rapidly interrogate the function of one or multiple genes. With BAd-CRISPR, an adeno-associated virus (AAV8) expressing a single guide RNA (sgRNA) is administered directly to BAT of mice expressing Cas9 in brown adipocytes. We show that the local administration of AAV8-sgRNA to interscapular BAT of adult mice robustly transduced brown adipocytes and ablated expression of adiponectin, adipose triglyceride lipase, fatty acid synthase, perilipin 1, or stearoyl-CoA desaturase 1 by >90%. Administration of multiple AAV8 sgRNAs led to simultaneous knockout of up to three genes. BAd-CRISPR induced frameshift mutations and suppressed target gene mRNA expression but did not lead to substantial accumulation of off-target mutations in BAT. We used BAd-CRISPR to create an inducible uncoupling protein 1 (Ucp1) knockout mouse to assess the effects of UCP1 loss on adaptive thermogenesis in adult mice. Inducible Ucp1 knockout did not alter core body temperature; however, BAd-CRISPR Ucp1 mice had elevated circulating concentrations of fibroblast growth factor 21 and changes in BAT gene expression consistent with heat production through increased peroxisomal lipid oxidation. Other molecular adaptations predict additional cellular inefficiencies with an increase in both protein synthesis and turnover, and mitochondria with reduced reliance on mitochondrial-encoded gene expression and increased expression of nuclear-encoded mitochondrial genes. These data suggest that BAd-CRISPR is an efficient tool to speed discoveries in adipose tissue biology.

Self-Assembly and Biogenesis of the Cellular Membrane are Dictated by Membrane Stretch and Composition.

The cell plasma membrane is a highly dynamic organelle governing a wide range of cellular activities including ion transport, secretion, cell division, growth, and development. The fundamental process involved in the addition of new membranes to pre-existing plasma membranes, however, is unclear. Here, we report, using biophysical, morphological, biochemical, and molecular dynamic simulations, the selective incorporation of proteins and lipids from the cytosol into the cell plasma membrane dictated by membrane stretch and composition. Stretching of the cell membrane as a consequence of volume increase following incubation in a hypotonic solution and results in the incorporation of cytosolic proteins and lipids into the existing plasma membrane. Molecular dynamic simulations further confirm that increased membrane stretch results in the rapid insertion of lipids into the existing plasma membrane. Similarly, depletion of cholesterol from the cell plasma membrane selectively alters the incorporation of lipids into the membrane.

G-CSF partially mediates effects of sleeve gastrectomy on the bone marrow niche.

Bariatric surgeries are integral to the management of obesity and its metabolic complications. However, these surgeries cause bone loss and increase fracture risk through poorly understood mechanisms. In a mouse model, vertical sleeve gastrectomy (VSG) caused trabecular and cortical bone loss that was independent of sex, body weight, and diet, and this loss was characterized by impaired osteoid mineralization and bone formation. VSG had a profound effect on the bone marrow niche, with rapid loss of marrow adipose tissue, and expansion of myeloid cellularity, leading to increased circulating neutrophils. Following VSG, circulating granulocyte-colony stimulating factor (G-CSF) was increased in mice, and was transiently elevated in a longitudinal study of humans. Elevation of G-CSF was found to recapitulate many effects of VSG on bone and the marrow niche. In addition to stimulatory effects of G-CSF on myelopoiesis, endogenous G-CSF suppressed development of marrow adipocytes and hindered accrual of peak cortical and trabecular bone. Effects of VSG on induction of neutrophils and depletion of marrow adiposity were reduced in mice deficient for G-CSF; however, bone mass was not influenced. Although not a primary mechanism for bone loss with VSG, G-CSF plays an intermediary role for effects of VSG on the bone marrow niche.

Secretion induces cell pH dynamics impacting assembly-disassembly of the fusion protein complex: A combined fluorescence and atomic force microscopy study.

A wide range of cellular activities including protein folding and cell secretion, such as neurotransmission or insulin release, are all governed by intracellular pH homeostasis, underscoring the importance of pH on critical life processes. Nano- scale pH measurements of cells and biomolecules therefore hold great promise in understanding a plethora of cellular functions, in addition to disease detection and therapy. In the current study, a novel approach using cadmium telluride quantum dots (CdTeQDs) as pH sensors, combined with fluorescent imaging, spectrofluorimetry, atomic force microscopy (AFM), and Western blot analysis, enabled the study of intracellular pH dynamics at 1 milli-pH sensitivity and 80nm pixel resolution, during insulin secretion. Additionally, the pH-dependent interaction between membrane fusion proteins, also called the soluble N-ethylmaleimide-sensitive factor activating protein receptor (SNARE), was determined. Glucose stimulation of CdTeQD-loaded insulin secreting Min-6 mouse insulinoma cell line demonstrated the initial (5-6min) intracellular acidification reflected as a loss in QD fluorescence, followed by alkalization and a return to resting pH in 10min. Analysis of the SNARE complex in insulin secreting Min-6 cells demonstrated an initial gain followed by loss of complexed SNAREs in 10min. Stabilization of the SNARE complex at low intracellular pH is further supported by results from studies utilizing both native and AFM measurements of liposome-reconstituted recombinant neuronal SNAREs, providing a molecular understanding of the role of pH during cell secretion.

Human Platelet Vesicles Exhibit Distinct Size and Proteome.

In the past 50 years, isolated blood platelets have had restricted use in wound healing, cancer therapy, and organ and tissue transplant, to name a few. The major obstacle for its unrestricted use has been, among others, the presence of ultrahigh concentrations of growth factors and the presence of both pro-angiogenic and anti-angiogenic proteins. To overcome this problem requires the isolation and separation of the membrane bound secretory vesicles containing the different factors. In the current study, high-resolution imaging of isolated secretory vesicles from human platelets using atomic force microscopy (AFM) and mass spectrometry enabled characterization of the remaining vesicles size and composition following their immunoseparation. The remaining vesicles obtained following osmotic lysis, when subjected to immunoseparation employing antibody to different vesicle-associated membrane proteins (VAMPs), demonstrate for the first time that VAMP-3-, VAMP-7-, and VAMP-8-specific vesicles each possesses distinct size range and composition. These results provide a window into our understanding of the heterogeneous population of vesicles in human platelets and their stability following both physical manipulation using AFM and osmotic lysis of the platelet. This study further provides a platform for isolation and the detailed characterization of platelet granules, with promise for their future use in therapy. Additionally, results from the study demonstrate that secretory vesicles of different size found in cells reflect their unique and specialized composition and function.

Functionalized nanoparticles enable tracking the rapid entry and release of doxorubicin in human pancreatic cancer cells.

Efficient drug delivery is critical to therapy. Using electron microscopy, X-ray, and light microscopy, we have characterized functionalized superparamagnetic iron oxide (SPIO) nanoparticles, and determined their ability for rapid entry and release of the cancer drug doxorubicin in human pancreatic cancer cells. Dextran-coated SPIO nanoparticle ferrofluid, functionalized with the red-autofluorescing doxorubicin and the green-fluorescent dye fluorescein isothiocyanate as a reporter, enables tracking the intracellular nanoparticle transport and drug release. This engineered nanoparticle enables a >20 fold rapid entry and release of the drug in human pancreatic cancer cells, holding therapeutic potential as an advanced drug delivery and imaging platform. The low extracellular pH of most tumors precluding the entry of a number of weakly basic drugs such as doxorubicin, conferring drug resistance, can now be overcome.

Functional Reconstitution of the Insulin-Secreting Porosome Complex in Live Cells.

Supramolecular cup-shaped lipoprotein structures called porosomes embedded in the cell plasma membrane mediate fractional release of intravesicular contents from cells during secretion. The presence of porosomes, have been documented in many cell types including neurons, acinar cells of the exocrine pancreas, GH-secreting cells of the pituitary, and insulin-secreting pancreatic ß-cells. Functional reconstitution of porosomes into artificial lipid membranes, have also been accomplished. Earlier studies on mouse insulin-secreting Min6 cells report 100-nm porosome complexes composed of nearly 30 proteins. In the current study, porosomes have been functionally reconstituted for the first time in live cells. Isolated Min6 porosomes reconstituted into live Min6 cells demonstrate augmented levels of porosome proteins and a consequent increase in the potency and efficacy of glucose-stimulated insulin release. Elevated glucose-stimulated insulin secretion 48 hours after reconstitution, reflects on the remarkable stability and viability of reconstituted porosomes, documenting the functional reconstitution of native porosomes in live cells. These results, establish a new paradigm in porosome-mediated insulin secretion in ß-cells.

Proteome of the porosome complex in human airway epithelia: interaction with the cystic fibrosis transmembrane conductance regulator (CFTR).

The surface of the airways is coated with a thin film of mucus composed primarily of mucin, which is under continuous motion via ciliary action. Mucin not only serves to lubricate the airways epithelia, but also functions as a trap for foreign particles and pathogens, thereby assisting in keeping the airways clean and free of particulate matter and infections. Altered mucin secretion especially increased mucin viscosity, results in mucin stagnation due to the inability of the cilia to propel them, leading to infections and diseases such as cystic fibrosis (CF). Since porosomes have been demonstrated to be the secretory portals at the cell plasma membrane in cells, their presence, structure, and composition in the mucin-secreting human airway epithelial cell line Calu-3 expressing CF transmembrane receptor (CFTR), were investigated. Atomic force microscopy (AFM) of Calu-3 cells demonstrates the presence of approximately 100nm in diameter porosome openings at the plasma membrane surface. Electron microscopy confirms the AFM results, and tandem mass spectrometry and immunoanalysis performed on isolated Calu-3 porosomes, reveal the association of CFTR with the porosome complex. These new findings will facilitate understanding of CFTR-porosome interactions influencing mucous secretion, and provide critical insights into the etiology of CF disease. BIOLOGICAL SIGNIFICANCE: In the present study, the porosome proteome in human airway epithelia has been determined. The interaction between the cystic fibrosis transmembrane conductance regulator (CFTR) and the porosome complex in the human airway epithelia is further demonstrated. The possible regulation by CFTR on the quality of mucus secretion via the porosome complex at the cell plasma membrane is hypothesized. These new findings will facilitate understanding of CFTR-porosome interactions influencing mucous secretion, and provide critical insights into the etiology of CF disease.