Date Published: May 19, 2014
Publisher: Public Library of Science
Author(s): Lesya V. Zelenchuk, Anne-Marie Hedge, Peter S. N. Rowe, Dominique Heymann.
PHEX or DMP1 mutations cause hypophosphatemic-rickets and altered energy metabolism. PHEX binds to DMP1-ASARM-motif to form a complex with α5β3 integrin that suppresses FGF23 expression. ASARM-peptides increase FGF23 by disrupting the PHEX-DMP1-Integrin complex. We used a 4.2 kDa peptide (SPR4) that binds to ASARM-peptide/motif to study the DMP1-PHEX interaction and to assess SPR4 for the treatment of energy metabolism defects in HYP and potentially other bone-mineral disorders.
Subcutaneously transplanted osmotic pumps were used to infuse SPR4-peptide or vehicle (VE) into wild-type mice (WT) and HYP-mice (PHEX mutation) for 4 weeks.
SPR4 partially corrected HYP mice hypophosphatemia and increased serum 1.25(OH)2D3. Serum FGF23 remained high and PTH was unaffected. WT-SPR4 mice developed hypophosphatemia and hypercalcemia with increased PTH, FGF23 and 1.25(OH)2D3. SPR4 increased GAPDH HYP-bone expression 60× and corrected HYP-mice hyperglycemia and hypoinsulinemia. HYP-VE serum uric-acid (UA) levels were reduced and SPR4 infusion suppressed UA levels in WT-mice but not HYP-mice. SPR4 altered leptin, adiponectin, and sympathetic-tone and increased the fat mass/weight ratio for HYP and WT mice. Expression of perlipin-2 a gene involved in obesity was reduced in HYP-VE and WT-SPR4 mice but increased in HYP-SPR4 mice. Also, increased expression of two genes that inhibit insulin-signaling, ENPP1 and ESP, occurred with HYP-VE mice. In contrast, SPR4 reduced expression of both ENPP1 and ESP in WT mice and suppressed ENPP1 in HYP mice. Increased expression of FAM20C and sclerostin occurred with HYP-VE mice. SPR4 suppressed expression of FAM20C and sclerostin in HYP and WT mice.
ASARM peptides and motifs are physiological substrates for PHEX and modulate osteocyte PHEX-DMP1-α5β3-integrin interactions and thereby FGF23 expression. These interactions also provide a nexus that regulates bone and energy metabolism. SPR4 suppression of sclerostin and/or sequestration of ASARM-peptides improves energy metabolism and may have utility for treating familial rickets, osteoporosis, obesity and diabetes.
Studies carried out by the Centers for Disease Control (CDC) confirm that approximately 70% of US adults were obese or overweight from 2009 to 2010. Of these nearly 40% were classed as overtly obese. Osteoporosis, a component of the metabolic syndrome that is associated with dyslipidemia and obesity , is also a major health issue for more than 44 million Americans. The cost for osteoporosis and related fractures total $14 billion per year. The social and financial cost to society is incalculable and growing at an exponential rate. Several genome wide association studies have confirmed MEPE as a major gene locus for bone mineral density and osteoporosis –. Also, serum levels of MEPE in normal humans correlates with serum phosphorus, parathyroid hormone and bone mineral density (BMD) ,  Recent research has begun to unravel the intricacies of the molecular and physiological pathways linking energy metabolism and bone-renal mineral metabolism. This novel approach has exploited transgenic mice models and the new paradigm has been heralded as integrative physiology . Thus far, three key physiological pathways have emerged. First, bone formation and resorption are proposed to regulate blood glucose via a feedback loop controlled by insulin and a bone matrix protein osteocalcin. Second, this pathway is also impacted by an adipokine leptin that can traverse the blood brain barrier and regulate biosynthesis of a neurotransmitter serotonin. The serotonergic signaling in the brainstem is proposed to affect sympathetic tone in the arcuate nucleus (AN) and ventrolateral medial nucleus (VMN) of the hypothalamus. Thus, AN serotonergic signaling increases appetite and VMH serotonergic-signaling decreases “bone resorption” and increases “bone formation” via sympathetic activation of osteoblast β-adrenergic receptors. Third, additional complexity has arisen with the discovery that circulating gut derived serotonin whose biosynthesis is regulated by Lrp5 negatively regulates bone formation. Serotonin does not cross the blood-brain barrier so the two distinct central and peripheral pools of serotonin are proposed to have opposite effects on bone turnover. Other neuropeptides also play key roles. For example brain expressed Cocaine Amphetamine Related Transcript (CART) is an inhibitor of bone resorption. The evidence for these pathways is compelling and the experimental science used to formulate the hypotheses elegant –. However, recent equally compelling and scientifically rigorous studies contradict all three pathways and so the models remain controversial –.
Although phosphate levels are not the sole mediator of mineralization defects in familial hypophosphatemic disorders it is well documented that hypophosphatemia or systemic phosphate status correlates with changes in glucose production, energy metabolism and oxygen consumption , , –. More recently, the familial hypophosphatemic rickets disorders all show changes in glucose, insulin sensitivity and fat metabolism –, –, –. Hypophosphatemia is also associated with metabolic syndrome and because phosphate is involved in carbohydrate metabolism low serum phosphate compromises utilization of glucose, increases insulin resistance and induces hyperinsulinemia , , . Also, patients with primary hyperparathyroidism have impaired glucose-tolerance, hyperglycemia and reduced insulin sensitivity . Our studies and others show HYP mice have increased serum PTH with hyperglycemia and hypoinsulinemia –, . Also, consistent with the HYP mice hypoinsulinemia and hyperglycemia a complete loss of insulin with hyperglycemia in diabetes type 1 patients (DM1) is associated with a loss of bone mineral density . Of relevance, a recent microarray study showed major up-regulation of genes belonging to the PPAR-γ family (notably adiponectin, a marker of insulin resistance ) and PHEX during mineralization of osteoblast cultures over 27 days . Adiponectin stimulates the proliferation, differentiation, and mineralization of osteoblasts via the AdipoR1 and AMP kinase signaling pathways in autocrine and/or paracrine fashions . Thus the down regulation of adiponectin in HYP-mice shown in this study may also contribute to the abnormal bone phenotype. Moreover, there is an association of low serum phosphate levels with glucose intolerance, insulin sensitivity and insulin secretion in non-diabetic healthy-subjects  and a phosphate deplete diet impairs rat insulin secretion (markedly reduced) by pancreatic islets ex vivo. HYP mice also have increased hepatic glucose-6-phosphatase activity  and rats fed a phosphate deplete diet up-regulate expression and activity of this enzyme , . Also, overexpression of glucose-6-phosphatase in rats induces glucose intolerance, hyperglycemia with changes in circulating free fatty acids and triglycerides . Remarkably, targeted deletion of the renal proximal-tubule insulin-receptor in mice promotes hyperglycemia, up regulation of glucose 6 phosphatase and gluconeogenesis . This is of interest since the renal proximal tubule contains the Na-dependent phosphate cotransporters (NPT2a and NPT2c) and hypophosphatemia negatively regulates insulin synthesis and sensitivity , , , , , , , . Although the liver is traditionally thought to be the major organ involved in glucose homeostasis the kidney is now also well recognized as a major player –. There are also strong correlations with FGF23, obesity and insulin resistance , . Indeed, cardiovascular disease (CVD) and non-insulin-dependent diabetes mellitus (NIDDM) in obese patients have been proposed to be directly caused by hypophosphatemia . Specifically, low serum phosphate adversely affects glucose metabolism resulting in hyperglycemia, with increased risk of NIDDM, hypertension and increased risk of stroke . In obese individuals, a major role for phosphate in regulatory thermogenesis and dysregulation of the basal metabolic rate occurs , . Also, alterations in red cell glycolytic intermediates and oxygen transport due to a striking increase in red cell oxygen affinity occur in hypophosphatemic subjects . These changes are accompanied by defective ATP synthesis and reduced renal cortical ribonucleoside triphosphate pools , . The alterations in oxygen-affinity and glucose-metabolism in hypophosphatemic subjects is due primarily to regulatory abnormalities at the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) step . In line with this, defective ATP synthesis and impaired thermoregulatory regulation with increased metabolic rate and oxygen consumption also occurs with X-linked hypophosphatemic rickets mice (HYP) , . Also, our study shows that there is a major reduction in HYP renal and bone GAPDH expression.