Increased GABA Transport Activity in Rat Calvarial Osteoblasts Cultured under Hyperglycemic Conditions

Abstract

Insulin-dependent diabetes mellitus (IDDM; Type I) is characterized with low or no insulin production, which sometimes leads to diabetic osteopenia and osteoporosis. 1,2) In the absence of insulin, insulin-sensitive cells exhibit marked reduction of glucose uptake activity, resulting in increased serum glucose levels and subsequent development of a variety of diabetic complications. Histological and bone marker assessments indicate a low turnover state in bone formation rate and decreased osteoblastic activity in rat models of type I diabetes.3) In addition, there is a report about the close relationship between the bone loss and the fasting blood glucose level.4) The occurrence of hyperglycemic bone loss is controversial because not only high glucose but also other factors including insulin deficiency could mediate bone loss in IDDM patients, however, while little is known about the direct effect of hyperglycemia on bone metabolism. By contrast, in vitro analysis reveals the induction by hyperglycemia of osteoblastic dysfunctions. In human osteosarcoma (MG-63) cells cultured under high glucose conditions (ϭ55 mM), for example, impaired response is seen to parathyroid hormone and to 1,25-dihydroxyvitamin D 3 , an active form of vitamin D 3 , required for the synthesis of the matured osteoblast marker protein osteocalcin. 5,6) Other reports using calvarial osteoblastic cell line MC3T3-E1 cells have demonstrated that high glucose (ϭ15.5 mM) inhibits Ca 2ϩ intake and bone mineralization with an increase in both cellular proliferation and alkaline phosphatase (ALP) activity.7) These findings indicate that elevated extracellular glucose concentrations would directly impair osteoblastic functions resulting in defective mineralization similar to clinical findings. Therefore, these in vitro analyses are useful for the better understanding of mechanisms relevant to osteoblastic malfunctions associated with diabetes mellitus.On the other hand, we have previously reported the possible functional expression of particular GABAergic signaling machineries in cultured rat calvarial osteoblasts. 8,9) GABA is known as one of the most abundant inhibitory amino acid neurotransmitters in the mammalian central nervous system (CNS). In the CNS, GABA is supposed to mediate inhibitory neurotransmission thorough different signaling machineries including GABA synthase, GABA receptors and GABA transporters.10-13) These GABAergic machineries are found not only in the CNS but also in some non-neuronal and peripheral organs such as heart, lung, kidney, adrenal, pancreas, liver, spleen and uterus.10) In addition to these peripheral tissues, the expression of GABA and particular GABAergic signaling molecules is found in bone cells such as osteoblasts 8,9) and chondrocytes. 14-16) Amongst different isoforms of GABA transporters required for signal termination, expression of betaine/GABA transporter-1 (BGT-1) is exclusively detected at mRNA and protein levels with a temperature-, sodium-and chloride-dependent activity of [ 3 H]GABA accumulation i...

Skeletal Site-specific Changes in Bone Mass in a Genetic Mouse Model for Human 15q11-13 Duplication Seen in Autism

“…GABA receptors mediate signals of the neurotransmitter GABA 39 . Osteoblasts constitutively express metabotropic GABA B receptor subunits and not the majority of the GABA A receptor subunits to which the Gabrα5 , Gabrγ3 , Gabrβ3 in the patDp /+ locus belong 39 , 40 . However, at least one of these genes, Gabrγ3 has been shown to be induced in expression when osteoblasts are exposed to external stimuli 41 .…”

Promotion of osteoblastic Ca2+ accumulation by Eucommia leaf extract

“…Throughout the experiments, -MEM containing 10% FBS, 50 mg/ml ascorbic acid (Sigma-Aldrich Co. LLC., MO, USA), 5 mM sodium β-glycerophosphate (Sigma-Aldrich), and 40 mM NaHCO 3 (Sigma-Aldrich) were used [5].…”

“…The assay buffer composed of 0.05 M 2-amino-2methylpropanol, 2 mM MgCl 2, and 10 mM p-nitrophenylphosphoric acid was added at a volume of 200 µl into 10 µl of cell suspensions, followed by a reaction for 30 min at 37°C and subsequent immediate determination of absorbance of p-nitrophenol at 405 nm in a SpectraMax M5 e microplate reader (Molecular Devices, Minneapolis, MN, USA). Simultaneously, the protein concentrations of cell suspension were determined by using the Bradford method, and the obtained alkaline phosphatase (ALP) activities were standardized by the protein concentration and the incubation time of the ALP assay [5].…”

“…Then, the cell suspensions were incubated for 16 to 24 hours at room temperature. Finally, the cell suspensions were centrifuged at 15,000 rpm for 5 min, and the Ca 2+ content was determined in the supernatant by using a calcium E-TEST kit (Wako Pure Chemical Industries, Ltd. Osaka, Japan) [5].…”