清野 祐介

Background/Objectives: Carbohydrate and protein restriction are associated with sarcopenia and osteopenia, but the underlying mechanisms remain unclear. We aimed to determine whether mild protein restriction affects muscle and bone function in wild-type (WT) and homozygous carbohydrate response element binding protein (Chrebp) knockout (KO) mice. Methods: Eighteen-week-old male wild-type and homozygous carbohydrate response element binding protein (Chrebp) knockout (KO) mice were fed a control diet (20% protein) or a low-protein diet (15% protein) for 12 weeks. We estimated the muscle weight and limb grip strength as well as the bone mineral density, bone structure, and bone morphometry. Results: Chrebp deletion and a low-protein diet additively decreased body weight (WT control-KO low-protein: mean difference with 95% CI, 8.7 [6.3, 11.0], p < 0.0001) and epidydimal fat weight (1.0 [0.7, 1.2], p < 0.0001). Chrebp deletion and a low-protein diet additively decreased tibialis anterior muscle weight (0.03 [0.01, 0.05], p = 0.002) and limb grip strength (63.9 [37.4, 90.5], p < 0.0001) due to a decrease in insulin/insulin-like growth factor 1 mRNA and an increase in myostatin mRNA. In contrast, Chrebp deletion increased bone mineral density (BMD) (WT control-KO control: -6.1 [-1.0, -2.3], p = 0.0009), stiffness (-21.4 [-38.8, -4.1], p = 0.011), cancellous bone BV/TV (-6.517 [-10.99, -2.040], p = 0.003), and the number of trabeculae (-1.1 [-1.8, -0.5], p = 0.0008). However, in KO mice, protein restriction additively decreased BMD (KO control-KO low-protein: 8.1 [4.3, 11.9], p < 0.0001), bone stiffness (38.0 [21.3, 54.7], p < 0.0001), cancellous bone BV/TV (7.7 [3.3, 12.2], p = 0.006), and the number of trabeculae (1.2 [0.6, 1.9], p = 0.0004). The effects of mild protein restriction on bone formation parameters (osteoid volume (WT control-WT low-protein: -1.7 [-2.7, -0.7], p = 0.001) and the osteoid surface (-11.2 [-20.8, -1.5], p = 0.02) were observed only in wild-type (WT) mice. The levels of bone resorption markers, such as the number of osteoclasts on the surface, the number of osteoclasts, and surface erosion, did not differ between the groups. Conclusions: Both Chrebp deletion and protein restriction led to a decrease in muscle and bone function; therefore, an adequate intake of carbohydrates and proteins is important for maintaining muscle and bone mass and function. Further studies will be needed to elucidate the mechanisms by which ChREBP deletion and a low-protein diet cause osteosarcopenia.

OBJECTIVES: Phosphate (Pi) induces differentiation of arterial smooth muscle cells to the osteoblastic phenotype by inducing the type III Na-dependent Pi transporter Pit-1/solute carrier family member 1. This induction can contribute to arterial calcification, but precisely how Pi stress acts on the vascular wall remains unclear. We investigated the role of extracellular Pi in inducing microstructural changes in the arterial wall. METHODS: Aortae of Pit-1-overexpressing transgenic (TG) rats and their wild-type (WT) littermates were obtained at 8 weeks after birth. The thoracic descending aorta from WT and TG rats was used for the measurement of wall thickness and uniaxial tensile testing. Structural and ultrastructural analyses were performed using light microscopy and transmission electron microscopy. Gene expression of connective tissue components in the aorta was quantified by quantitative real-time polymerase chain reaction. RESULTS: Aortic wall thickness in TG rats was the same as that in WT rats. Uniaxial tensile testing showed that the circumferential breaking stress in TG rats was significantly lower than that in WT rats (p<0.05), although the longitudinal breaking stress, breaking strain, and elastic moduli in both directions in TG rats were unchanged. Transmission electron microscopy analysis of the aorta from TG rats showed damaged formation of elastic fibers in the aortic wall. Fibrillin-1 gene expression levels in the aorta were significantly lower in TG rats than in WT rats (p<0.05). CONCLUSIONS: Pi overload acting via the arterial wall Pit-1 transporter weakens circumferential strength by causing elastic fiber malformation, probably via decreased fibrillin-1 expression.

Hyponatremia is the most common clinical electrolyte disorder. Chronic hyponatremia has been recently reported to be associated with falls, fracture, osteoporosis, neurocognitive impairment, and mental manifestations. In the treatment of chronic hyponatremia, overly rapid correction of hyponatremia can cause osmotic demyelination syndrome (ODS), a central demyelinating disease that is also associated with neurological morbidity and mortality. Using a rat model, we have previously shown that microglia play a critical role in the pathogenesis of ODS. However, the direct effect of rapid correction of hyponatremia on microglia is unknown. Furthermore, the effect of chronic hyponatremia on microglia remains elusive. Using microglial cell lines BV-2 and 6-3, we show here that low extracellular sodium concentrations (36 mmol/L decrease; LS) suppress Nos2 mRNA expression and nitric oxide (NO) production of microglia. On rapid correction of low sodium concentrations, NO production was significantly increased in both cells, suggesting that acute correction of hyponatremia partly directly contributes to increased Nos2 mRNA expression and NO release in ODS pathophysiology. LS also suppressed expression and nuclear translocation of nuclear factor of activated T cells-5 (NFAT5), a transcription factor that regulates the expression of genes involved in osmotic stress. Furthermore, overexpression of NFAT5 significantly increased Nos2 mRNA expression and NO production in BV-2 cells. Expressions of Nos2 and Nfat5 mRNA were also modulated in microglia isolated from cerebral cortex in chronic hyponatremia model mice. These data indicate that LS modulates microglial NO production dependent on NFAT5 and suggest that microglia contribute to hyponatremia-induced neuronal dysfunctions.