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Functional characterization of retromer in GLUT4 storage vesicle formation and adipocyte differentiation [Research Communication]

Insulin-stimulated translocation of glucose transporter 4 (GLUT4) storage vesicles (GSVs), the specialized intracellular compartments within mature adipocytes, to the plasma membrane (PM) is a fundamental cellular process for maintaining glucose homeostasis. Using 2 independent adipocyte cell line models, human primary Simpson-Golabi-Behmel syndrome and mouse 3T3-L1 fibroblast cell lines, we demonstrate that the endosome-associated protein-sorting complex retromer colocalizes with GLUT4 on the GSVs by confocal microscopy in mature adipocytes. By use of both confocal microscopy and differential ultracentrifugation techniques, retromer is redistributed to the PM of mature adipocytes upon insulin stimulation. Furthermore, stable knockdown of the retromer subunit-vacuolar protein-sorting 35, or the retromer-associated protein sorting nexin 27, by lentivirus-delivered small hairpin RNA impaired the adipogenesis process when compared to nonsilence control. The knockdown of retromer decreased peroxisome proliferator activated receptor expression during differentiation, generating adipocytes with decreased levels of GSVs, lipid droplet accumulation, and insulin-stimulated glucose uptake. In conclusion, our study demonstrates a role for retromer in the GSV formation and adipogenesis.—Yang, Z., Hong, L. K., Follett, J., Wabitsch, M., Hamilton, N. A., Collins, B. M., Bugarcic, A., Teasdale, R. D. Functional characterization of retromer in GLUT4 storage vesicle formation and adipocyte differentiation.

The prototoxin LYPD6B modulates heteromeric {alpha}3{beta}4-containing nicotinic acetylcholine receptors, but not {alpha}7 homomers [Research Communication]

Prototoxins are a diverse family of membrane-tethered molecules expressed in the nervous system that modulate nicotinic cholinergic signaling, but their functions and specificity have yet to be completely explored. We tested the selectivity and efficacy of leukocyte antigen, PLAUR (plasminogen activator, urokinase receptor) domain-containing (LYPD)-6B on α3β4-, α3α5β4-, and α7-containing nicotinic acetylcholine receptors (nAChRs). To constrain stoichiometry, fusion proteins encoding concatemers of human α3, β4, and α5 (D and N variants) subunits were expressed in Xenopus laevis oocytes and tested with or without LYPD6B. We used the 2-electrode voltage-clamp method to quantify responses to acetylcholine (ACh): agonist sensitivity (EC50), maximal agonist-induced current (Imax), and time constant () of desensitization. For β4–α3–α3–β4–α3 and β4–α3–β4–α3–α3, LYPD6B decreased EC50 from 631 to 79 μM, reduced Imax by at least 59%, and decreased . For β4–α3–α5D–β4–α3 and β4–α3–β4–α–α5D, LYPD6B decreased Imax by 63 and 32%, respectively. Thus, LYPD6B acted only on (α3)3(β4)2 and (α3)2(α5D)(β4)2 and did not affect the properties of (α3)2(β4)3, α7, or (α3)2(α5N)(β4)2 nAChRs. Therefore, LYPD6B acts as a mixed modulator that enhances the sensitivity of (α3)3(β4)2 nAChRs to ACh while reducing ACh-induced whole-cell currents. LYPD6B also negatively modulates α3β4 nAChRs that include the α5D common human variant, but not the N variant associated with nicotine dependence.—Ochoa, V., George, A. A., Nishi, R., Whiteaker, P. The prototoxin LYPD6B modulates heteromeric α3β4-containing nicotinic acetylcholine receptors, but not α7 homomers.

Combination of small RNAs for skeletal muscle regeneration [Research Communication]

Selectively controlling the expression of the target genes through RNA interference (RNAi) has significant therapeutic potential for injuries or diseases of tissues. We used this strategy to accelerate and enhance skeletal muscle regeneration for the treatment of muscular atrophy. In this study, we used myostatin small interfering (si)RNA (siGDF-8), a major inhibitory factor in the development and postnatal regeneration of skeletal muscle and muscle-specific microRNAs (miR-1 and -206) to further accelerate muscle regeneration. This combination of 3 small RNAs significantly improved the gene expression of myogenic regulatory factors in vitro, suggesting myogenic activation. Moreover, cell proliferation and myotube formation improved without compromising each other, which indicates the myogenic potential of this combination of small RNAs. The recovery of chemically injured tibialis anterior muscles in rats was significantly accelerated, both functionally and structurally. This novel combination of siRNA and miRNAs has promising therapeutic potential to improve in situ skeletal muscle regeneration.—Kim, N., Yoo, J. J., Atala, A., Lee, S. J. Combination of small RNAs for skeletal muscle regeneration.

The alternative complement pathway aids in vascular regression during the early stages of a murine model of proliferative retinopathy [Research Communication]

Proliferative retinopathic diseases often progress in 2 phases: initial regression of retinal vasculature (phase 1) followed by subsequent neovascularization (NV) (phase 2). The immune system has been shown to aid in vascular pruning in such retinopathies; however, little is known about the role of the alternative complement pathway in the initial vascular regression phase. Using a mouse model of oxygen-induced retinopathy (OIR), we observed that alternative complement pathway–deficient mice (Fb–/–) exhibited a mild decrease in vascular loss at postnatal day (P)8 compared with age- and strain-matched controls (P = 0.035). Laser capture microdissection was used to isolate the retinal blood vessels. Expression of the complement inhibitors Cd55 and Cd59 was significantly decreased in blood vessels isolated from hyperoxic retinas compared with those from normoxic control mice. Vegf expression was measured at P8 and found to be significantly lower in OIR mice than in normoxic control mice (P = 0.0048). Further examination of specific Vegf isoform expression revealed a significant decrease in Vegf120 (P = 0.00032) and Vegf188 (P = 0.0092). In conjunction with the major modulating effects of Vegf during early retinal vascular development, our data suggest a modest involvement of the alternative complement pathway in targeting vessels for regression in the initial vaso-obliteration stage of OIR.—Kim, C., Smith, K. E., Castillejos, A., Diaz-Aguilar, D., Saint-Geniez, M., Connor, K. M. The alternative complement pathway aids in vascular regression during the early stages of a murine model of proliferative retinopathy.

Genetic ablation of lymphocytes and cytokine signaling in nonobese diabetic mice prevents diet-induced obesity and insulin resistance [Research Communication]

Obesity is characterized by a dysregulated immune system, which may causally associate with insulin resistance and type 2 diabetes. Despite widespread use of nonobese diabetic (NOD) mice, NOD with severe combined immunodeficiency (scid) mutation (SCID) mice, and SCID bearing a null mutation in the IL-2 common chain receptor (NSG) mice as animal models of human diseases including type 1 diabetes, the underlying metabolic effects of a genetically altered immune system are poorly understood. For this, we performed a comprehensive metabolic characterization of these mice fed chow or after 6 wk of a high-fat diet. We found that NOD mice had ~50% less fat mass and were 2-fold more insulin sensitive, as measured by hyperinsulinemic-euglycemic clamp, than C57BL/6 wild-type mice. SCID mice were also more insulin sensitive with increased muscle glucose metabolism and resistant to diet-induced obesity due to increased energy expenditure (~10%) and physical activity (~40%) as measured by metabolic cages. NSG mice were completely protected from diet-induced obesity and insulin resistance with significant increases in glucose metabolism in peripheral organs. Our findings demonstrate an important role of genetic background, lymphocytes, and cytokine signaling in diet-induced obesity and insulin resistance.—Friedline, R. H., Ko, H. J., Jung, D. Y., Lee, Y., Bortell, R., Dagdeviren, S., Patel, P. R., Hu, X., Inashima, K., Kearns, C., Tsitsilianos, N., Shafiq, U., Shultz, L. D., Lee, K. W., Greiner, D. L., Kim, J. K. Genetic ablation of lymphocytes and cytokine signaling in nonobese diabetic mice prevents diet-induced obesity and insulin resistance.

Structure-dynamic basis of splicing-dependent regulation in tissue-specific variants of the sodium-calcium exchanger [Research Communication]

Tissue-specific splice variants of Na+/Ca2+ exchangers contain 2 Ca2+-binding regulatory domains (CBDs), CBD1 and CBD2. Ca2+ interaction with CBD1 activates sodium-calcium exchangers (NCXs), and Ca2+ binding to CBD2 alleviates Na+-dependent inactivation. A combination of mutually exclusive (A, B) and cassette (C–F) exons in CBD2 raises functionally diverse splice variants through unknown mechanisms. Here, the effect of exons on CBDs backbone dynamics were investigated in the 2-domain tandem (CBD12) of the brain, kidney, and cardiac splice variants by using hydrogen-deuterium exchange mass spectrometry and stopped-flow techniques. Mutually exclusive exons stabilize interdomain interactions in the apoprotein, which primarily predefines the extent of responses to Ca2+ binding. Deuterium uptake levels were up to 20% lower in the cardiac vs. the brain CBD12, reveling that elongation of the CBD2 FG loop by cassette exons rigidifies the interdomain Ca2+ salt bridge at the 2-domain interface, which secondarily modulates the Ca2+-bound states. In matching splice variants, the extent of Ca2+-induced rigidification correlates with decreased (up to 10-fold) Ca2+ off rates, where the cardiac CBD12 exhibits the slowest Ca2+ off rates. Collectively, structurally disordered/dynamic segments at mutually exclusive and cassette exons have local and distant effects on the folded structures nearby the Ca2+ binding sites, which may serve as a structure-dynamic basis for splicing-dependent regulation of NCX.—Lee, S. Y., Giladi, M., Bohbot, H., Hiller, R., Chung, K. Y., Khananshvili, D. Structure-dynamic basis of splicing-dependent regulation in tissue-specific variants of the sodium-calcium exchanger.

Adivasis, tribes and other neologisms for erasing precolonial pasts: An example from Northeast India

This essay engages Dipesh Chakrabarty, Sumit Guha and James C. Scott by arguing that all of them overlook a historically well-evidenced set of subject positions and concepts of space. This was monastic governmentality. Lay and ordained populations attached to monastic teachers and lineages moved and interacted across a vast network of societies till the eighteenth century. The arrival of colonial European armies in the terrain marked by monastic geographicity led to the creation of sites set apart as ‘Nepal’, ‘colonial Assam’, ‘Burma’ and so on. Hitherto pastoralist but Bon-Buddhist monastic subjects were separated from their ‘brothers’ in monastic subjecthood. Such physical separation was reinforced by historical writing as well. In the twentieth century, colonially educated native scribes embraced both geographical and epistemic projects enthusiastically. Bhuyan, the foremost practitioner of this mode of history writing, thus failed to recognise the Buddhist and Bon Tantric cohabitants of the Brahmaputra river valley.
In order to establish what was known of these people before Bhuyan’s time of writing, this essay has been organised in three parts. The first contains a short discussion of monastic governmentality and subjecthood. A second provides a truncated narrative of events that brought Mughal armies in the seventeenth century to the same terrain. The third surveys moments from the Company and British military accounts of 1794–1928 to explain the modern postcolonial Indian historian’s aporia towards Buddhist and Bon Tantric populations living alongside Muslim and Hindu groups in the Brahmaputra valley and plains.

Diagnostic accuracy study of anorectal manometry for diagnosis of dyssynergic defecation

ObjectiveThe diagnostic accuracy of anorectal manometry (AM), which is necessary to diagnose functional defecatory disorders (FDD), is unknown. Using blinded analysis and standardised reporting of diagnostic accuracy, we evaluated whether AM could discriminate between asymptomatic controls and patients with functional constipation (FC).
DesignDerived line plots of anorectal pressure profiles during simulated defecation were independently analysed in random order by three expert observers blinded to health status in 85 women with FC and 85 age-matched asymptomatic healthy volunteers (HV). Using accepted criteria, these pressure profiles were characterised as normal (ie, increased rectal pressure coordinated with anal relaxation) or types I–IV dyssynergia. Interobserver agreement and diagnostic accuracy were determined.
ResultsBlinded consensus-based assessment disclosed a normal pattern in 16/170 (9%) of all participants and only 11/85 (13%) HV. The combined frequency of dyssynergic patterns (I–IV) was very similar in FC (80/85 (94%)) and HV (74/85 (87%)). Type I dyssynergia (‘paradoxical’ contraction) was less prevalent in FC (17/85 (20%) than in HV (31/85 (36.5%), p=0.03). After statistical correction, only type IV dyssynergia was moderately useful for discriminating between FC (39/85 (46%)) and HV (17/85 (20%)) (p=0.001, positive predictive value=70.0%, positive likelihood ratio=2.3). Interobserver agreement was substantial or moderate for identifying a normal pattern, dyssynergia types I and IV, and FDD, and fair for types II and III.
ConclusionsWhile the interpretation of AM patterns is reproducible, nearly 90% of HV have a pattern that is currently regarded as ‘abnormal’ by AM. Hence, AM is of limited utility for distinguishing between FC and HV.

Lithium chloride modulates chondrocyte primary cilia and inhibits Hedgehog signaling [Research Communication]

Lithium chloride (LiCl) exhibits significant therapeutic potential as a treatment for osteoarthritis. Hedgehog signaling is activated in osteoarthritis, where it promotes chondrocyte hypertrophy and cartilage matrix catabolism. Hedgehog signaling requires the primary cilium such that maintenance of this compartment is essential for pathway activity. Here we report that LiCl (50 mM) inhibits Hedgehog signaling in bovine articular chondrocytes such that the induction of GLI1 and PTCH1 expression is reduced by 71 and 55%, respectively. Pathway inhibition is associated with a 97% increase in primary cilia length from 2.09 ± 0.7 μm in untreated cells to 4.06 ± 0.9 μm in LiCl-treated cells. We show that cilia elongation disrupts trafficking within the axoneme with a 38% reduction in Arl13b ciliary localization at the distal region of the cilium, consistent with the role of Arl13b in modulating Hedgehog signaling. In addition, we demonstrate similar increases in cilia length in human chondrocytes in vitro and after administration of dietary lithium to Wistar rats in vivo. Our data provide new insights into the effects of LiCl on chondrocyte primary cilia and Hedgehog signaling and shows for the first time that pharmaceutical targeting of the primary cilium may have therapeutic benefits in the treatment of osteoarthritis.—Thompson, C. L., Wiles, A., Poole, C. A., Knight, M. M. Lithium chloride modulates chondrocyte primary cilia and inhibits Hedgehog signaling.

Training intensity modulates changes in PGC-1{alpha} and p53 protein content and mitochondrial respiration, but not markers of mitochondrial content in human skeletal muscle [Research Communication]

Exercise training has been associated with increased mitochondrial content and respiration. However, no study to date has compared in parallel how training at different intensities affects mitochondrial respiration and markers of mitochondrial biogenesis. Twenty-nine healthy men performed 4 wk (12 cycling sessions) of either sprint interval training [SIT; 4–10 x 30-s all-out bouts at ~200% of peak power output (WPeak)], high-intensity interval training (HIIT; 4–7 x 4-min intervals at ~90% WPeak), or sublactate threshold continuous training (STCT; 20–36 min at ~65% WPeak). The STCT and HIIT groups were matched for total work. Resting biopsy samples (vastus lateralis) were obtained before and after training. The maximal mitochondrial respiration in permeabilized muscle fibers increased significantly only after SIT (25%). Similarly, the protein content of peroxisome proliferator-activated receptor coactivator (PGC)-1α, p53, and plant homeodomain finger–containing protein 20 (PHF20) increased only after SIT (60–90%). Conversely, citrate synthase activity, and the protein content of TFAM and subunits of the electron transport system complexes remained unchanged throughout. Our findings suggest that training intensity is an important factor that regulates training-induced changes in mitochondrial respiration and that there is an apparent dissociation between training-induced changes in mitochondrial respiration and mitochondrial content. Moreover, changes in the protein content of PGC-1α, p53, and PHF20 are more strongly associated with training-induced changes in mitochondrial respiration than mitochondrial content.—Granata, C., Oliveira, R. S. F., Little, J. P., Renner, K., Bishop, D. J. Training intensity modulates changes in PGC-1α and p53 protein content and mitochondrial respiration, but not markers of mitochondrial content in human skeletal muscle.