Journal: Experimental & Molecular Medicine

Article Title: BCAT1 promotes osteoclast maturation by regulating branched-chain amino acid metabolism

doi: 10.1038/s12276-022-00775-3

Figure Lengend Snippet: a , b Effect of osteoclast differentiation on BCAT expression. BMMs were stimulated with M-CSF (30 ng/ml) and RANKL (100 ng/ml) for the indicated times. The cells were harvested and their lysates were subjected to ( a ) western blot or ( b ) RT-qPCR analysis of the indicated molecules. c – g Effect of gabapentin on osteoclast differentiation ( d , e ) and associated NFATc1 and ATP6v0d2 expression ( f , g ). BMMs were stimulated with M-CSF (30 ng/ml) and gabapentin at doses ranging from 0 to 20 mM for 3 days, after which the MTT cell viability assay was conducted ( c ). BMMs were incubated with M-CSF (30 ng/ml) and RANKL (100 ng/ml) with or without gabapentin at the indicated doses for 3 days. The cells were fixed, stained for TRAP ( d ) and the TRAP + cells with a diameter of ≥200 μm were counted ( e ). Scale bar, 500 μm. Alternatively, the cells were subjected to western blot ( f ) or RT-qPCR ( g ) analysis of NFATc1 and ATP6v0d2 expression. β-actin antibodies served as the control in ( f ). h , i BMMs were incubated with M-CSF (30 ng/ml) and RANKL (100 ng/ml) for 2 days ( h ) and 4 days ( i ) to respectively generate pre-osteoclasts and mature osteoclasts. The cells were then treated with gabapentin (5 mM) for 1 day, harvested, lysed and were subjected to RT-qPCR analysis of the indicated molecules. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001 versus control ( n = 4).

Article Snippet: Gabapentin was purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Expressing, Western Blot, Quantitative RT-PCR, Viability Assay, Incubation, Staining, Control

Journal: Experimental & Molecular Medicine

Article Title: BCAT1 promotes osteoclast maturation by regulating branched-chain amino acid metabolism

doi: 10.1038/s12276-022-00775-3

Figure Lengend Snippet: As depicted schematically ( a ), the calvaria of mice were injected twice with PBS, LPS (12.5 mg/kg of body weight) alone, or LPS with 30 or 100 mg/kg body-weight gabapentin. Three days after the last injection, the mice were sacrificed and their calvaria was sectioned and stained for TRAP ( b ). The amount of bone that was eroded was determined by histology and OsteoMeasucreXP analysis and was expressed as % eroded surface (ES)/bone surface (BS) ( c ). The number of osteoclasts per bone perimeter (N.Oc/B.Pm, per mm 2 ) was also determined ( d ). n = 3, 5, 5 and 4 in the PBS, LPS alone, LPS + 30 mg/kg gabapentin, and LPS + 100 mg/kg gabapentin groups, respectively. ** p ≤ 0.01. Scale bar, 50 μm.

Article Snippet: Gabapentin was purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Injection, Staining

Journal: Biomolecules

Article Title: Gabapentin Disrupts Binding of Perlecan to the α 2 δ 1 Voltage Sensitive Calcium Channel Subunit and Impairs Skeletal Mechanosensation

doi: 10.3390/biom12121857

Figure Lengend Snippet: Structure of voltage sensitive calcium channels. The channel complex is composed of the α 1 pore-forming subunit with auxiliary β, γ, and α 2 δ subunits bound to the pore, positioned to alter gating kinetics of the channel [ , , ]. The α 2 δ subunit is anchored in the membrane via the δ portion, with the α 2 region positioned extracellularly. In the extracellular portion (α 2 ) of the α 2 δ subunit, the von Willebrand Factor A domain (vWFA) sequence and the Arg-Arg-Arg (RRR) motif for Gabapentin binding are indicated. Reprinted/adapted with permission from Ref. , 2022, Springer Nature.

Article Snippet: At 16 weeks of age, mice were randomly assigned into 2 groups to receive daily intraperitoneal injections of saline (vehicle, VEH) or gabapentin (GBP, 300 mg/kg BW; 50 mg/mL stock diluted in saline) (Acros Organics AC458020050, ThermoFisher Scientific, MA, USA) for 4 weeks ( n = 9 mice/treatment).

Techniques: Membrane, Sequencing, Binding Assay

Journal: Biomolecules

Article Title: Gabapentin Disrupts Binding of Perlecan to the α 2 δ 1 Voltage Sensitive Calcium Channel Subunit and Impairs Skeletal Mechanosensation

doi: 10.3390/biom12121857

Figure Lengend Snippet: LSPR-based interactions among α 2 -functionalized sensors, perlecan and gabapentin.

Article Snippet: At 16 weeks of age, mice were randomly assigned into 2 groups to receive daily intraperitoneal injections of saline (vehicle, VEH) or gabapentin (GBP, 300 mg/kg BW; 50 mg/mL stock diluted in saline) (Acros Organics AC458020050, ThermoFisher Scientific, MA, USA) for 4 weeks ( n = 9 mice/treatment).

Techniques:

Journal: Biomolecules

Article Title: Gabapentin Disrupts Binding of Perlecan to the α 2 δ 1 Voltage Sensitive Calcium Channel Subunit and Impairs Skeletal Mechanosensation

doi: 10.3390/biom12121857

Figure Lengend Snippet: Ulna changes in response to mechanical loading in vehicle and gabapentin treated mice.

Article Snippet: At 16 weeks of age, mice were randomly assigned into 2 groups to receive daily intraperitoneal injections of saline (vehicle, VEH) or gabapentin (GBP, 300 mg/kg BW; 50 mg/mL stock diluted in saline) (Acros Organics AC458020050, ThermoFisher Scientific, MA, USA) for 4 weeks ( n = 9 mice/treatment).

Techniques:

Journal: Biomolecules

Article Title: Gabapentin Disrupts Binding of Perlecan to the α 2 δ 1 Voltage Sensitive Calcium Channel Subunit and Impairs Skeletal Mechanosensation

doi: 10.3390/biom12121857

Figure Lengend Snippet: Gabapentin treatment impairs bone mechanosensitivity and load-induced bone formation. Male C57BL/6J mice were injected daily with saline (vehicle, VEH) (n = 9) or Gabapentin (GBP, 300 mg/kg BW) (n = 7) for 4 weeks while undergoing axial ulnar loading. ( a ) Representative images of control (non-loaded) and loaded ulnas from VEH and GBP treated mice. To monitor load-induced bone formation, calcein (green) and alizarin (red) fluorochromes were injected intraperitoneally 8 and 20 days after the first loading day, respectively. Changes in ( b ) mineralizing surface (MS/BS), mineral apposition rate (MAR), and bone formation rate (BFR/BS) in response to mechanical loading were assessed in VEH and GBP treated mice were analyzed by ANOVA and Fisher’s LSD test for pairwise comparisons. ( c ) The percent difference between a loaded and non-loaded limbs within each animal [%Δ = ((loaded limb − control limb)/control limb) × 100%)] for MS/BS, MAR and BFR/BS in GBP and VEH groups was calculated analyzed separately by unpaired Student’s t -tests. Each dot represents one mouse. Values are shown as Mean ± SEM; (*) p ≤ 0.05.

Article Snippet: At 16 weeks of age, mice were randomly assigned into 2 groups to receive daily intraperitoneal injections of saline (vehicle, VEH) or gabapentin (GBP, 300 mg/kg BW; 50 mg/mL stock diluted in saline) (Acros Organics AC458020050, ThermoFisher Scientific, MA, USA) for 4 weeks ( n = 9 mice/treatment).

Techniques: Injection, Saline, Control

Journal: Biomolecules

Article Title: Gabapentin Disrupts Binding of Perlecan to the α 2 δ 1 Voltage Sensitive Calcium Channel Subunit and Impairs Skeletal Mechanosensation

doi: 10.3390/biom12121857

Figure Lengend Snippet: Summary of results. In this work we found that ( a ) the α 2 δ 1 subunit of voltage sensitive Ca 2+ channels binds perlecan (PLN) creating a mechanosensory complex that enables connection between the mineralized matrix and the osteocyte cell membrane. ( b ) We also demonstrated that gabapentin (GBP) interferes with binding of PLN and α 2 δ 1 in vitro. As the PLN/α 2 δ 1 complex is necessary for mechanotransduction, GBP uncoupling of the complex results in impaired osteocyte mechanosensation in vivo, which may account for the deleterious skeletal effects observed with chronic use of this drug.

Article Snippet: At 16 weeks of age, mice were randomly assigned into 2 groups to receive daily intraperitoneal injections of saline (vehicle, VEH) or gabapentin (GBP, 300 mg/kg BW; 50 mg/mL stock diluted in saline) (Acros Organics AC458020050, ThermoFisher Scientific, MA, USA) for 4 weeks ( n = 9 mice/treatment).

Techniques: Membrane, Binding Assay, In Vitro, In Vivo

Journal: European Journal of Pain (London, England)

Article Title: Impaired chronic pain‐like behaviour and altered opioidergic system in the TASTPM mouse model of Alzheimer's disease

doi: 10.1002/ejp.1288

Figure Lengend Snippet: Altered analgesic responses in TASTPM Mice. Mechanical withdrawal thresholds of MIA ‐injected mice were assessed prior to and at specified intervals post‐analgesic or vehicle administration. Both wild‐type ( WT : three males and three females per treatment group) and TASTPM (four males and four females per treatment group) mice exhibited increase in mechanical thresholds 1 h post‐oral (p.o.) administration of celecoxib (30 mg/kg) on 6–7 days post‐ MIA injection compared to their respective vehicle (methyl cellulose) controls (* p < 0.05) (A). Administration of morphine (6 mg/kg; s.c.), an opioid agonist, on 28 days after MIA injection resulted in significant reversal of mechanical hypersensitivity in both WT (4 males and 4 females per treatment group) and TASTPM (five males and five females per treatment group) mice (1 h post‐injection; ** p < 0.01; *** p < 0.001) compared to their respective vehicle (saline) controls, with greater degree of analgesia observed in TASTPM compared to WT mice ( # p < 0.05) (B). Gabapentin (60 mg/kg; p.o.; 22–24 days post‐ MIA ) was only effective in reversing MIA ‐induced mechanical hypersensitivity in WT (five males and three females per treatment group) mice (** p < 0.01; 3 h post‐injection), with no effect detected in TASTPM (five males and four females per treatment group) ( p > 0.05), compared to their respective vehicle (dH 2 O) controls. The analgesic effect of gabapentin was significantly greater in WT mice compared to TASTPM ( ## p < 0.01) (C). Paracetamol (300 mg/kg; p.o.) was ineffective in alleviating MIA ‐induced mechanical hypersensitivity ( p > 0.05) in both WT (Vehicle: three males and three females; Paracetamol: three male and two females) and TASTPM (Vehicle: two male and three females: Paracetamol: five males and four females) mice when administrated 28 days after MIA injection (30 min post‐injection), compared to their respective vehicle (saline) control group (D). Statistical analysis was conducted using two‐way ANOVA followed by the Student–Newman–Keuls post hoc test. Data are presented as difference between post‐dose and pre‐dose 50% PWT values that are illustrated as mean ± SEM ( n = 5–10 mice per experimental group).

Article Snippet: Gabapentin (60 mg/kg) was supplied by LKT Laboratories inc. and was dissolved in dH 2 O.

Techniques: Injection, Saline, Control