Arthur D. Conigrave

Osteoblasts play key roles in the mechanisms of action of strontium ranelate

Background and purpose:  Strontium ranelate reduces fracture risk in postmenopausal women with osteoporosis. Evidence from non‐clinical studies and analyses of bone markers in phase III trials indicate that this is due to an increase in osteoblast formation and a decrease of osteoclastic resorption. The aim of this work was to investigate, in human cells, the mechanisms by which strontium ranelate is able to influence the activities of osteoblasts and osteoclasts.

Inhibition of glutamine transport depletes glutamate and GABA neurotransmitter pools: further evidence for metabolic compartmentation

The role of glutamine and alanine transport in the recycling of neurotransmitter glutamate was investigated in Guinea pig brain cortical tissue slices and prisms, and in cultured neuroblastoma and astrocyte cell lines. The ability of exogenous (2 mm) glutamine to displace 13C label supplied as [3‐13C]pyruvate, [2‐13C]acetate, l‐[3‐13C]lactate, or d‐[1‐13C]glucose was investigated using NMR spectroscopy. Glutamine transport was inhibited in slices under quiescent or depolarising conditions using histidine, which shares most transport routes with glutamine, or 2‐(methylamino)isobutyric acid (MeAIB), a specific inhibitor of the neuronal system A. Glutamine mainly entered a large, slow turnover pool, probably located in neurons, which did not interact with the glutamate/glutamine neurotransmitter cycle. This uptake was inhibited by MeAIB. When [1‐13C]glucose was used as substrate, glutamate/glutamine cycle turnover was inhibited by histidine but not MeAIB, suggesting that neuronal system A may not play a prominent role in neurotransmitter cycling. When transport was blocked by histidine under depolarising conditions, neurotransmitter pools were depleted, showing that glutamine transport is essential for maintenance of glutamate, GABA and alanine pools. Alanine labelling and release were decreased by histidine, showing that alanine was released from neurons and returned to astrocytes. The resultant implications for metabolic compartmentation and regulation of metabolism by transport processes are discussed.

Testing protein leverage in lean humans: a randomised controlled experimental study

A significant contributor to the rising rates of human obesity is an increase in energy intake. The ‘protein leverage hypothesis’ proposes that a dominant appetite for protein in conjunction with a decline in the ratio of protein to fat and carbohydrate in the diet drives excess energy intake and could therefore promote the development of obesity. Our aim was to test the ‘protein leverage hypothesis’ in lean humans by disguising the macronutrient composition of foods offered to subjects under ad libitum feeding conditions. Energy intakes and hunger ratings were measured for 22 lean subjects studied over three 4-day periods of in-house dietary manipulation. Subjects were restricted to fixed menus in random order comprising 28 foods designed to be similar in palatability, availability, variety and sensory quality and providing 10%, 15% or 25% energy as protein. Nutrient and energy intake was calculated as the product of the amount of each food eaten and its composition. Lowering the percent protein of the diet from 15% to 10% resulted in higher (+12±4.5%, p = 0.02) total energy intake, predominantly from savoury-flavoured foods available between meals. This increased energy intake was not sufficient to maintain protein intake constant, indicating that protein leverage is incomplete. Urinary urea on the 10% and 15% protein diets did not differ statistically, nor did they differ from habitual values prior to the study. In contrast, increasing protein from 15% to 25% did not alter energy intake. On the fourth day of the trial, however, there was a greater increase in the hunger score between 1–2 h after the 10% protein breakfast versus the 25% protein breakfast (1.6±0.4 vs 25%: 0.5±0.3, p = 0.005). In our study population a change in the nutritional environment that dilutes dietary protein with carbohydrate and fat promotes overconsumption, enhancing the risk for potential weight gain.

Positive and Negative Allosteric Modulators Promote Biased Signaling at the Calcium-Sensing Receptor

The calcium-sensing receptor (CaSR) is a G protein-coupled receptor whose function can be allosterically modulated in a positive or negative manner by calcimimetics or calcilytics, respectively. Indeed, the second-generation calcimimetic, cinacalcet, has proven clinically useful in the treatment of chronic kidney disease patients with secondary hyperparathyroidism but is not widely used in earlier stages of renal disease due to the potential to predispose such patients to hypocalcaemia and hyperphosphatemia. The development of a biased CaSR ligand that is more selective for specific signaling pathway(s) leading only to beneficial effects may overcome this limitation. The detection of such stimulus-bias at a G protein-coupled receptor requires investigation across multiple signaling pathways and the development of methods to quantify the effects of allosteric ligands on orthosteric ligand affinity and cooperativity at each pathway. In the current study, we determined the effects of the calcimimetics, NPS-R568 or cinacalcet, and the calcilytic, NPS-2143, on Ca(o)(2+)-mediated intracellular Ca(2+) mobilization, ERK1/2 phosphorylation, and plasma membrane ruffling in a stably transfected human embryonic kidney 293-TREx c-myc-CaSR cell line and applied a novel analytical model to quantify these modulator effects. We present quantitative evidence for the generation of stimulus bias by both positive and negative allosteric modulators of the CaSR, manifested as greater allosteric modulation of intracellular Ca(2+) mobilization relative to ERK1/2 phosphorylation, and a higher affinity of the modulators for the state of the CaSR mediating plasma membrane ruffling relative to the other two pathways. Our findings provide the first evidence that an allosteric modulator used in clinical practice exhibits stimulus bias.

L-Amino acid sensing by the calcium-sensing receptor: a general mechanism for coupling protein and calcium metabolism?

Cellular sensing of L-amino acids is widespread and controls diverse cellular responses regulating, for example, rates of hormone secretion, amino acid uptake, protein synthesis and protein degradation (autophagy). However, the nature of the sensing mechanisms involved has been elusive. One important sensing mechanism is selective for branched chain amino acids, acts via mTOR (mammalian target of rapamycin) and regulates the rates of insulin and IGF-1 secretion as well as hepatic, and possibly muscle, autophagy. A second sensing mechanism is selective for aromatic L-amino acids and regulates the rate of gastric acid secretion and other responses in the gastro-intestinal tract. Interactions between calcium and protein metabolism, including accelerated urinary calcium excretion in subjects consuming high-protein diets and secondary hyperparathyroidism in subjects consuming low-protein diets, suggest an additional amino acid sensing mechanism linked to the control of urinary calcium excretion and parathyroid hormone (PTH) release. New data demonstrating L-amino acid-dependent activation of the calcium-sensing receptor (CaR), which regulates PTH secretion and urinary calcium excretion, suggests an unexpected explanation for these links between calcium and protein metabolism. Furthermore, expression of the CaR in gastrin-secreting G-cells and acid-secreting parietal cells, together with data indicating that the CaR exhibits selectivity for aromatic amino acids, would appear to provide a molecular explanation for amino acid sensing in the gastrointestinal tract. This review examines what is known about the CaR as a gene, a receptor, a physiological regulator and, now, as an amino acid sensor. Possible new roles for the CaR are also considered.

Vitamin D Deficiency Promotes Human Breast Cancer Growth in a Murine Model of Bone Metastasis

Vitamin D exerts antiproliferative, prodifferentiation, and proapoptotic effects on nonclassic target tissues such as breast. Blood levels of 25-hydroxyvitamin D [25(OH)D], the most sensitive indicator of vitamin D status, are inversely correlated with breast cancer risk; however, a causal relationship between vitamin D deficiency and breast cancer growth in bone has not been assessed. We examined the effect of vitamin D deficiency on the intraskeletal growth of the human breast cancer cell line MDA-MB-231-TxSA in a murine model of malignant bone lesions. Subsets of mice were treated concurrently with osteoprotegerin (OPG) to abrogate bone resorption. Outcomes were assessed by repeated radiographic and end-point micro-computed tomography and histologic analyses. Mice weaned onto a vitamin D-free diet developed vitamin D deficiency within 4 weeks [mean +/- SE serum 25(OH)D: 11.5 +/- 0.5 nmol/L], which was sustained throughout the study and was associated with secondary hyperparathyroidism and accelerated bone turnover. Osteolytic lesions appeared earlier and were significantly larger in vitamin D-deficient than in vitamin D-sufficient mice after 2 weeks (radiographic osteolysis: +121.5%; histologic tumor area: +314%; P < 0.05). Although OPG treatment reduced the size of radiographic osteolyses and tumor area in both groups, tumors remained larger in OPG-treated vitamin D-deficient compared with OPG-treated vitamin D-sufficient mice (0.53 +/- 0.05 mm(2) versus 0.19 +/- 0.05 mm2; P < 0.05). We conclude that vitamin D deficiency promotes the growth of human breast cancer cells in the bones of nude mice. These effects are partly mediated through secondary changes in the bone microenvironment, along with direct effects of vitamin D on tumor growth.

Calcium-sensing receptor (CaSR): pharmacological properties and signaling pathways.

In this article we consider the mechanisms by which the calcium-sensing receptor (CaSR) induces its cellular responses via the control (activation or inhibition) of signaling pathways. We consider key features of CaSR-mediated signaling including its control of the heterotrimeric G-proteins Gq/11, Gi/o and G12/13 and the downstream consequences recognizing that very few CaSR-mediated cell phenomena have been fully described. We also consider the manner in which the CaSR contributes to the formation of specific signaling scaffolds via peptide recognition sequences in its intracellular C-terminal along with the origins of its high level of cooperativity, particularly for Ca(2+)o, and its remarkable resistance to desensitization. We also consider the nature of the mechanisms by which the CaSR controls oscillatory and sustained Ca(2+)i mobilizing responses and inhibits or elevates cyclic adenosine monophosphate (cAMP) levels dependent on the cellular and signaling context. Finally, we consider the diversity of the receptors ligands, ligand binding sites and broader compartment-dependent physiological roles leading to the identification of pronounced ligand-biased signaling for agonists including Sr(2+) and modulators including l-amino acids and the clinically effective calcimimetic cinacalcet. We note the implications of these findings for the development of new designer drugs that might target the CaSR in pathophysiological contexts beyond those established for the treatment of disorders of calcium metabolism.

Cooperative multi-modal sensing and therapeutic implications of the extracellular Ca2+ sensing receptor

The extracellular Ca(2+)-sensing receptor (CaR) is an unusual member of the diverse superfamily of seven-transmembrane domain G-protein-coupled receptors. Originally identified as the receptor providing the calciostat for extracellular ionized Ca(2+) ¿[Ca(2+)](o)¿, the CaR corrects small changes in [Ca(2+)](o) by regulating the secretion of the hormone that controls Ca(2+) fluxes between the blood and Ca(2+) stores in bone, and between blood and the urine. Now, research is beginning to reveal the structure and function of its unusually large N-terminal head. In addition to its role as a divalent and polyvalent cation sensor, recent studies indicate that the receptor also responds sensitively to changes in ionic strength and pH. Furthermore, new work indicates that the CaR is subject to allosteric activation by L-amino acids.

The gastrointestinal tract as a nutrient-balancing organ

Failure to provision tissues with an appropriate balance of nutrients engenders fitness costs. Maintaining nutrient balance can be achieved by adjusting the selection and consumption of foods, but this may not be possible when the nutritional environment is limiting. Under such circumstances, rebalancing of an imbalanced nutrient intake requires post-ingestive mechanisms. The first stage at which such post-ingestive rebalancing might occur is within the gastrointestinal tract (GIT), by differential release of digestive enzymes—releasing less of those enzymes for nutrients present in excess while maintaining or boosting levels of enzymes for nutrients in deficit. Here, we use an insect herbivore, the locust, to show for the first time that such compensatory responses occur within the GIT. Furthermore, we show that differential release of proteases and carbohydrases in response to nutritional state translate into differential extraction of macronutrients from host plants. The prevailing view is that physiological and structural plasticity in the GIT serves to maximize the rate of nutrient gain in relation to costs of maintaining the GIT; our findings show that GIT plasticity is integral to the maintenance of nutrient balance.

Dietary Protein and Bone Health: Roles of Amino Acid–Sensing Receptors in the Control of Calcium Metabolism and Bone Homeostasis

In this article, we review the evidence that dietary protein has a positive influence on bone health, reduces hip fracture risk, and promotes postfracture recovery, and we consider the molecular, cellular, and endocrine bases of the interactions that link protein and calcium metabolism, including effects via IGF-1 and PTH. In addition, we consider the roles of amino acid-sensing mechanisms in coupling dietary protein intake to metabolic change as well as the central role of calcium-sensing receptors (CaRs) in the control of calcium metabolism. Finally, we consider how recently identified broad-spectrum amino acid-sensing receptors from class 3 of the G-protein coupled receptor superfamily including, remarkably, the CaR itself may contribute to the impact of dietary protein on bone.