Studies of the association of dietary protein intake and various components of the calcium economy lead to conflicting conclusions, some suggesting that protein would negatively affect calcium retention and others that it would have a positive effect. Most of the apparent conflict can be explained by two factors: the interdependence of the two nutrients for bone health and probable delayed adaptation to altered intake, such that short-term studies may fail to capture the steady-state relationship between protein intake and calcium balance. At the whole body level, most studies show that high protein intake is osteoprotective, but only if calcium intake is adequate; similarly, metabolic evidence indicates that the protective effect of calcium for the skeleton is evident only when protein intake is relatively high.
Protein makes up roughly 50% the volume of bone. Both collagen, which constitutes the bulk of bone protein, and the various non-collagenous proteins undergo extensive post-translational modification after their synthesis. As a result, when bone is remodeled and old volumes of are bone resorbed, many of the constituent amino acids released by proteolysis cannot be reutilized. Hence, not only does the primary building of bone during growth require a fresh dietary supply of protein in order to ensure both an adequate mass of protein and an adequate suite of essential amino acids, but the maintenance of bone during adult remodeling does as well. Thus, one would expect a high protein intake to promote skeletal health. There is, in fact, a relatively large body of evidence indicating that that expectation is correct. However, the effects of protein on the calcium economy are complex.
(Effects Of Protein On The Calcium Economy, 2007)
A molecular link between protein and calcium metabolism: L-Amino acid-sensing by calcium-sensing receptors
Low levels of dietary protein intake ( <0.8 g/kg body weight per day) are associated with reduced levels of bone mineral density. Furthermore, moderate to high dietary protein intakes are associated with a reduced fracture risk. The origins of these effects are uncertain although moderate–high dietary protein intake has been reported to promote the levels of serum IGF-1, a recognized growth factor for bone, and variations in dietary protein intake modulate whole body calcium metabolism. In particular, elevations in dietary protein intake stimulate urinary calcium excretion in humans and reductions in dietary protein intake to around 0.8 g kg−1 body weight per day in humans have been reported to induce a state of secondary hyperparathyroidism in the absence of changes in serum calcium concentration.
How does proteins have these effects on our body?
Proteins are made up of different amino acid sequences. It seems doubtful, therefore, that proteins per se have any impact on whole body macronutrient or mineral metabolism. After ingestion, however, proteins are digested to release short peptides and free amino acids, than absorbed by small intestine epithelial cells. Generally, short peptides are not released into blood, but rather are broken down intracellulary by the action of peptidases to free amino acids, that are transferred into the blood: because of this, protein derived chemical signals are likely to have amino acids form.
Amino acids sensors are located either intracellularly or extracellularly: intracellularly form is based on amino acyl-free tRNAs; on the other hand, extracellular amino acid – sensing mechanisms appear to be based either on surface membrane receptors or transporters and provide information on extracellular level of free amino acids. Receptors belong to a broad – spectrum amino acids sensing G protein coupled recently identified. They are encoded by approximately 20 genes and, like other GPRs (family C) they are typified by a seven transmembrane domain – signaling motif for the binding and activation of heterotrimeric G – proteins, but are unusual in exhibiting a markedly N – terminus of about 400/ 600 amino acids; the extreme N – Terminus is recognized as a distinct structural domain of approximately 400 – 500 amino acids that is bilobed and related to bacterial periplasmic – binding proteins that mediate biochemical interactions between nutrients and transmembrane proteins involved in transport and/ or intracellular signaling. This domain is now known as the Venus Fly – Trap (VFT) domain in recognition of its bilobed, nutrient – trapping structure. Class 3 GPCRs are thus eukaryotic descendants of an ancient nutrient – sensing system with different application in human biology.
The first recognized members of this class are the metabotropic glutamate receptors and γ aminobutyrate (GABA) B receptors: they don’t have a defined in role in nutrient sensing, but Glutamate in an acidic amino acid and GABA is a glutamate analog derived; the most interesting members of this class, as regards our work, are the extracellular Calcium – sensing receptors (CaRs), that, in addition to their agonist binding sites for Ca2+, exhibit an allosteric site for aromatic, aliphatic and polar amino acids in its VTF domain.
The CaR is expressed in tissues and cell types with recognized roles in amino acid sensing as well as calcium sensing, such as enzyme and hormone – secreting cells of stomach and small intestine, liver, pancreas and endocrine cells of the anterior pituituary.
CaR expressing tissues operate in two distinct ways:
(a) sensing and normalizing inappropriate fluctuations in Ca2+ and adjusting the serum inorganic phosphate level
(b) supporting the development, growth, maintenance, and/or turnover of the skeleton.
The Calcium sensing receptor regulates whole body calcium metabolism. Its physiological role is underscored by the phenotype of some mice without this receptor that show severe hyperparathyroidism at birth due to a failure of extracellular Ca2+dependent negative feedback on the secretion of parathyroid hormone (PTH). In addition, well known familiar disorders of human calcium metabolism arise from heterozygous mutations of the CaR that either impair receptor function, such as familiar hypocalciuric hypercalcemia, or inappropriately sensitize the receptor to the extracellular Ca2+ concentration, such as autosomal dominant hypocalcemia
The finding that the extracellular Ca2+sensing receptor (CaR) is modulated by L-amino acids suggests one mechanism by which protein and calcium metabolism might be linked. Recent experiments indicate that amino acids that activate the CaR have acute suppressive effects on PTH secretion from human parathyroid cells, promote renal calcium excretion and stimulate gastric acid secretion, a process that facilitates intestinal calcium absorption by solubilizing calcium ions.
L-Amino acids have no effect on CaR-expressing human HEK293 embryonic kidney cells at basal levels of extracellular Ca2+ concentration (0.5–1.0 mM). However, in the presence of elevated but submaximal Ca2+ concentrations (1.0–3.0 mM), various L-amino acids activate the CaR and enhance its sensitivity to extracellular Ca2+ concentration. This behaviour suggests that amino acids act as allosteric activators of the CaR and indicates, remarkably, that the CaR responds to two distinct classes of nutrients: amino acids and calcium ions. Several sub-classes are effective including aromatic, aliphatic and polar amino acids.
The effects are stereoselective: D – amino acids are much less effective than L – amino acids. Besides, at Ca2+ concentration of 2.5mmol/l in CaR – expressing HEK293 cells, aromatic amino acids were clearly more effective than other amino acids. Thus, the order of effectiveness was: aromatics > aliphatics, polars > acidic basic, branch – chain amino acids. In human parathyroid cells which express the CaR endogenously, the aromatic amino acids L – Phe and L – Trp exhibited higher potencies than other amino acids
(Allosteric activation of the extracellular Ca2+-sensing receptor by L-amino acids enhances ERK1/2 phosphorylation, 2007)
Protein intake and urinary calcium excretion
It has been recognized for many years that increased protein intake often leads to an increase in urinary calcium loss. The rise in urine calcium reported for protein intake probably has at least three bases: an increase in glomerular filtration rate, a lowering of the renal calcium threshold and binding by certain of the absorbed amino acids to calcium-sensing receptors in the renal tubule (CaRs).
While this increase in calciuria might be due in part, or at least offset by, greater absorption of dietary calcium, several other observation make it clear that the issue is more complex.
CaRs are widely expressed in the kidney and control renal calcium and water excretion. In the rat kidney, the CaR is expressed throughout the nephron, with highest expression on the basolateral surfaces of cortical thick ascending limb (CTAL) and distal convoluted tubule cells, which support PHT regulated reabsorption of divalent cations and respond to hypercalcemia with suppressed Ca2+ reabsortions. The CaR is also expressed in the proximal tubule, where it attenuates the phosphaturic effect of PTH and lowers serum calcitriol levels at least in part by increasing vitamin D receptor expression. Interestingly, CaR expression in the proximal tubule is under the inhibitory control of dietary phosphate and PTH.
We can hypothesized that dietary protein induced elevations in renal calcium excretion might arise from the amino acid-dependent activation of CaRs in the CTAL, which is a major site of regulated calcium excretion. To test this hypothesis, intravenous infusions of CaR-active amino acids were performed on anaesthetized rats. The CaR-active amino acids, L-Phe and L-Ala activated renal calcium excretion, as indicated in the diagram below
(l-Amino acid-sensing by calcium-sensing receptors: A molecular link between protein and calcium metabolism, 2007):
Protein intake and Calcium absorption
Recent studies indicate that a partial or a total gastrectomy, as well as proton pump inhibitors, interfere with the absorption of Ca2+ ions from some calcium salts. Besides, some studies demonstrate that there is a drop in the solubility of Calcium salts when the duodenum pH is above 6, the approximate pH of this organ following the entry of gastric acid. Since calcium and phosphate absorption occurs primarly in the duodenum, these findings indicate that gastric acid production plays a significant role in the release and solubilization of Ca2+ ions from ingested food.
Gastric acid production is stimulated not only by the activity of the parasympathetic nervous system but also by chemical signals including gastrin and its local effector histamine, nutrients including Ca2+ and some amino acids.
Gastric acid secretion is generally divided into three phases:
(a) a first cephalic phase mediated by the vagus nerve and activated by the thought of food and by his odor, appearance or taste
(b) a second gastric phase mediated by stretch receptors in the stomach walls and by the protein content of ingested food, specifically by hydrolyzed proteins (the effects of intact proteins are weak). This phase accounts for about half of the total acid secretory response to a meal.
© a third phase, the intestinal phase, that accounts for about 5% of the total acid secretory response to a meal, mediated by gastrin released into the circulation in the duodenum and also by direct effects of absorbed amino acids on the parietal cells.
In humans, intravenous infusion of L – amino acids has long been known to stimulate gastric acid secretion. Both basal and stimulated secretion is greater in men than in women, presumably because men have a greater mass of parietal cells and because women have a blunted sensitivity of parietal cells to endogenous stimulation with gastrin. Basal and stimulated secretion has a circadian rhythm, with a peak at around midnight and a nadir approximately 7 a.m. this rhythm is independent of the endocrine pathway and mediated by the neurocrine pathway.
Calcium stimulates gastric acid secretion in humans under circumstances: several studies confirmed that gastric acid secretion doubled after ingestion of calcium carbonate and it is possible that the effect of calcium on stomach secretion is mediated at least in part by calcium – induced activation of the CaRs.
The most important determinant of calcium absorption are:
(a) calcium intake: this is the most important determinant of absorbed calcium. Absorbed calcium increases with increasing intake, however the relationship between the two is not linear.
Absorption occurs by active and passive transport across the intestinal mucosa.
(b) vitamin D: the active metabolite of this vitamin ( 1,25 – dihydroxyvitamin D) stimulate active calcium transport
© age: calcium absorption declines with age in both men and women. Several reasons for this have been proposed, for example an age – related resistance to the action of vitamin D due to a decline in the VDR concentration in duodedal mucosal (actually there is a rise in the vitamin D blood levels with age) or an age – related decrease in renal production of 1, 25 (OH)2D.
(d) gastric acid secretion: calcium is released from food more efficiently at low pH and calcium carbonate is more soluble in an acidic than in an alkaline environment.
The effects of Calcium and amino acids are mediated, at least in part, by the CaR, expressed on gastric antral G – cells, controlling the release of gastrin, and parietal cells, providing a mechanism by which food rich of calcium can directly stimulate acid secretion.
In rat whole stomachs studied ex vivo, Phenylalanine activated CaRs located on parietal cells and significantly lowered gastric luminal pH, whereas the branched-chain amino acid Leucine did not. The effect of the amino acids occurred by enhancing H+, K+-ATPase activity and was independent of the hormonal status of the tissue. Increasing Ca2+ in the absence of secretagogues also lowered gastric pH and this effect was amplified by the addition of the amino acid phenylalanine. These findings are consistent with the authors' conclusion that CaR-dependent acid secretion occurs by allosteric activation by amino acids.
Another potential effect of the amino acids is to activate CaRs on gastric G cells. These cells produce gastrin, the hormone that stimulates acid production by the parietal cell. However, in previous studies, amino acid stimulation of gastric acid did not increase gastrin levels.
(Dietary Protein and Bone Health: Roles of Amino Acid–Sensing Receptors in the Control of Calcium Metabolism and Bone Homeostasis, 2008)
Protein intake and PTH secretion
Both protein and calcium, even if they belong to different nutritional categories, are key structural elements of the skeleton and are found in important growth – promoting food, such as milk and meat. It is not a surprise, therefore, that variations in the level of dietary protein ingestion have a significant impact on whole body calcium metabolism or that positive effects of dietary protein intake on bone health appear to be dependent, at least in part, on calcium intake.
In addition to the effects on urinary calcium excretion and intestinal absorption mentioned above, protein dietary intake has also some effects on serum parathyroid hormone levels, which increases the calcium concentration in the blood: careful studies demonstrate that reduced dietary protein intake in humans promptly induces a state of secondary hyperparathyroidism over 2 – 3 days that is typified by normal serum total and ionized Ca2+ concentrations and elevated serum PTH levels in healthy young subjects consuming dietary protein at a level below 0.9g kg-1 day-1. Secondary hyperparathyroidism, when serum Ca2+ levels are normal, is commonly ignored in clinical practice provided renal function is normal and serum vitamin D is satisfactory, but may be dangerous due to prolonged skeleton exposure to elevated PTH levels.
According to this point of view, low dietary protein – induced elevations in serum PTH levels were associated with secondary elevations in serum calcitriol and urinary cAMP levels, demonstrating that type1 PTH/ PTHrP receptors had been activated in the kidney and other tissue, including osteoblasts in bone. Even if the molecular basis of these effects are unknown, these findings demonstrate that protein and calcium metabolism are coordinated and that calcium metabolism is modulated by variations in protein intake.
(Dietary Protein and Bone Health: Roles of Amino Acid–Sensing Receptors in the Control of Calcium Metabolism and Bone Homeostasis, 2008)
The table below shows a review of the impact of dietary protein on calcium metabolism:
Proposed Pathway for the impact of dietary protein on calcium absorbtion
(Protein intake and calcium absorption – Potential role of the calcium sensor receptor, 2006)
Protein and IGF1
Dietary amino acid intake promotes the release of insulin as well as the key growth factor IGF1. In addition, amino acids promote the secretion of GH from the anterior pituitary gland, which stimulates production and secretion of IGF1 from the liver and other tissue. Because of this, a reduced dietary protein intake is the cause of a smaller plasma IGF1 levels, which bring, at least in part, to a Hepatic resistance to GH action and enhanced metabolic clearance.
Why is IGF1 so important?
IGF1 is an essential factor for longitudinal bone growth and exerts anabolic effects on bone mass during adulthood.
Systemic IGF1, as said above, is synthesized primarily in the liver, in a GH dependent manner: pituitary gland derived GH acts in the liver, stimulating IGF1 synthesis and release. IGF1 than circulates to target organs, such as bone and cartilage, acting in an endocrine manner. Circulating IGF1 also inhibits the further release of GH from pituitary gland, completing a negative feedback loop. IGF1 is produced in some extrahepatic tissues too, where it has an autocrine/ paracrine action: it is thought that GH is also able to promote the formation of a complex which stabilizes serum IGF1. This second model incorporates GH – independent effects of IGF1 on embryonic growth and reproductive competence.
IGF1 has pluripotent effects on calcium and phosphate metabolism, including enhanced calcitriol synthesis and stimulated renal phosphate absorption. It also selectively stimulates the plasma membrane uptake of inorganic phosphate in osteoblastic cell lines, which promotes mineralization and based on analyses in knockout mice, both IGF-1 and expression of IGF-1 receptors on osteoblasts are required for the anabolic effect of acutely administered PTH.
In addition to its systemic production in the liver under GH control, IGF-1 is produced by osteoblastic cells in response to free amino acids including arginine (39), and in recently completed, short-term studies on elderly subjects, a protein supplement of 20 g day−1 significantly increased serum IGF-1 and IGF-binding protein-3 levels within a week.
In other experiments, adult male rats developed osteoporosis on low-protein diets in association with reductions in serum IGF-1 levels. Based on these observations in rats as well as the human studies referred to above, IGF-1 appears to play a prominent role in maintaining normal bone health, and reductions in IGF-1 levels appear to increase the risk of osteoporosis and associated fractures. Besides, IGF-1 levels respond sensitively and positively to changes in dietary protein and amino acid intake. Thus, it is evident that dietary protein intake and protein-derived amino acids modulate calcium metabolism and bone homeostasis via effects on calcium absorption and excretion as well as the hormonal and growth factor milieu.
(Anabolic effects of IGF-1 signaling on the skeleton, 2013)
Conclusion
Taken in its entirety, the body of evidence is compatible with the conclusion that protein is trophic for bone, both because it provides one of the essential building blocks of bone and because it elevates IGF-1. However, that protein-related benefit is dependent upon an adequate calcium intake. Similarly, the previously established benefit of high calcium intake for bone appears to be dependent upon having an adequate protein intake.
The mechanisms that underlie the effects of protein on whole body homeostasis are only now emerging. They include mechanisms that link changes in amino acid levels to the control of calcium absorption and excretion, effects on the hormonal milieu including elevated levels of IGF1 and suppressed levels of PTH, and effects on the fate and function of bone cells. One important group of amino acid sensors belongs to GPCR class 3, which includes the calcium-sensing receptor, a key regulator of calcium homeostasis.
