Increased intracellular Ca2+ concentrations prevent membrane localization of PH domains through the formation of Ca2+-phosphoinositides.

Insulin resistance, a key etiological factor in metabolic syndrome, is closely linked to ectopic lipid accumulation and increased intracellular Ca2+ concentrations in muscle and liver. However, the mechanism by which dysregulated intracellular Ca2+ homeostasis causes insulin resistance remains elusive. Here, we show that increased intracellular Ca2+ acts as a negative regulator of insulin signaling. Chronic intracellular Ca2+ overload in hepatocytes during obesity and hyperlipidemia attenuates the phosphorylation of protein kinase B (Akt) and its key downstream signaling molecules by inhibiting membrane localization of pleckstrin homology (PH) domains. Pharmacological approaches showed that elevated intracellular Ca2+ inhibits insulin-stimulated Akt phosphorylation and abrogates membrane localization of various PH domain proteins such as phospholipase Cδ and insulin receptor substrate 1, suggesting a common mechanism inhibiting the membrane targeting of PH domains. PH domain-lipid overlay assays confirmed that Ca2+ abolishes the binding of various PH domains to phosphoinositides (PIPs) with two adjacent phosphate groups, such as PI(3,4)P2, PI(4,5)P2, and PI(3,4,5)P3 Finally, thermodynamic analysis of the binding interaction showed that Ca2+-mediated inhibition of targeting PH domains to the membrane resulted from the tight binding of Ca2+ rather than PH domains to PIPs forming Ca2+-PIPs. Thus, Ca2+-PIPs prevent the recognition of PIPs by PH domains, potentially due to electrostatic repulsion between positively charged side chains in PH domains and the Ca2+-PIPs. Our findings provide a mechanistic link between intracellular Ca2+ dysregulation and Akt inactivation in insulin resistance.

Externalized phosphatidylinositides on apoptotic cells are eat-me signals recognized by CD14.

Apoptotic cells are rapidly engulfed and removed by phagocytes after displaying cell surface eat-me signals. Among many phospholipids, only phosphatidylserine (PS) is known to act as an eat-me signal on apoptotic cells. Using unbiased proteomics, we identified externalized phosphatidylinositides (PIPs) as apoptotic eat-me signals recognized by CD14+ phagocytes. Exofacial PIPs on the surfaces of early and late-apoptotic cells were observed in patches and blebs using anti-PI(3,4,5)P3 antibody, AKT- and PLCδ PH-domains, and CD14 protein. Phagocytosis of apoptotic cells was blocked either by masking exofacial PIPs or by CD14 knockout in phagocytes. We further confirmed that exofacial PIP+ thymocytes increased dramatically after in vivo irradiation and that exofacial PIP+ cells represented more significant populations in tissues of Cd14-/- than WT mice, especially after induction of apoptosis. Our findings reveal exofacial PIPs to be previously unknown cell death signals recognized by CD14+ phagocytes.

High-phytate/low-calcium diet is a risk factor for crystal nephropathies, renal phosphate wasting, and bone loss.

Phosphate overload contributes to mineral bone disorders that are associated with crystal nephropathies. Phytate, the major form of phosphorus in plant seeds, is known as an indigestible and of negligible nutritional value in humans. However, the mechanism and adverse effects of high-phytate intake on Ca2+ and phosphate absorption and homeostasis are unknown. Here, we show that excessive intake of phytate along with a low-Ca2+ diet fed to rats contributed to the development of crystal nephropathies, renal phosphate wasting, and bone loss through tubular dysfunction secondary to dysregulation of intestinal calcium and phosphate absorption. Moreover, Ca2+ supplementation alleviated the detrimental effects of excess dietary phytate on bone and kidney through excretion of undigested Ca2+-phytate, which prevented a vicious cycle of intestinal phosphate overload and renal phosphate wasting while improving intestinal Ca2+ bioavailability. Thus, we demonstrate that phytate is digestible without a high-Ca2+ diet and is a risk factor for phosphate overloading and for the development of crystal nephropathies and bone disease.

Assessment of bone quality using finite element analysis based upon micro-CT images.

BACKGROUND: To evaluate the feasibility of a micro-image based finite element model to determine the efficacy of sequential treatments on the bone quality in a rat osteoporosis model. METHODS: Rat osteoporosis and treated osteoporosis models were established with the bone loss, restore and maintain concept. Thirty Sprague-Dawley rats were used in this study. A sham operation or ovariectomy was performed at 20 weeks after birth, which was followed by the respective sequential trials as follows: (1) sham-operation only, (2) ovariectomy only, (3) ovariectomized rats with parathyroid hormone maintenance, (4) ovariectomized rats treated with PTH for 5 weeks and then withdrawal, (5) ovariectomized rats treated with PTH for 5 weeks and then with 17 beta-estradiol, and (6) ovariectomized rats treated with parathyroid hormone for 5 weeks and then treated with zoledronate. The histomorphometry indices were determined using the micro-images from a micro-computed tomogram. Finite element analysis was carried out to determine the mechanical properties (Stiffness and Young's modulus) of the vertebra bodies. The differences in properties between the groups were compared using ANOVA and a Bonferroni's multiple group comparison procedure. RESULTS: The histomorphometry and mechanical properties were significantly better in groups (3) and (6) than in the groups (1) and (2) (p < 0.05). The stiffness (sigma(s)) and Young's modulus (E) was highest in group (3) following by group (6). CONCLUSIONS: Finite element analysis based on micro-images provides a useful tool that reflects the changes in micro-structural and mechanical properties of a rat vertebral body with the bone loss, restore and maintain concept.