Quantitative analysis of light-phase restricted feeding reveals metabolic dyssynchrony in mice.

BACKGROUND: Considerable evidence suggests that the time of day at which calories are consumed markedly impacts body weight gain and adiposity. However, a precise quantification of energy balance parameters during controlled animal studies enforcing time-of-day-restricted feeding is currently lacking in the absence of direct human interaction. OBJECTIVE: The purpose of the present study was therefore to quantify the effects of restricted feeding during the light (sleep)-phase in a fully-automated, computer-controlled comprehensive laboratory animal monitoring system (CLAMS) designed to modulate food access in a time-of-day-dependent manner. Energy balance, gene expression (within metabolically relevant tissues), humoral factors and body weight were assessed. RESULTS: We report that relative to mice fed only during the dark (active)-phase, light (sleep)-phase fed mice: (1) consume a large meal upon initiation of food availability; (2) consume greater total calories per day; (3) exhibit a higher respiratory exchange ratio (indicative of decreased reliance on lipid/fatty acid oxidation); (4) exhibit tissue-specific alterations in the phases and amplitudes of circadian clock and metabolic genes in metabolically active tissues (greatest phase differences observed in the liver and diminution of amplitudes in epididymal fat, gastrocnemius muscle and heart); (5) exhibit diminished amplitude in humoral factor diurnal variations (for example, corticosterone); and (6) exhibit greater weight gain within 9 days of restricted feeding. CONCLUSIONS: Collectively, these data suggest that weight gain following light (sleep)-phase restricted feeding is associated with significant alterations in energy balance, as well as dyssynchrony between metabolically active organs.

Measurement of urinary collagen cross-links indicate response to therapy in patients with breast cancer and bone metastases.

Objective assessment of response in bone metastases from breast cancer using radiological techniques takes up to 6 months of treatment to be certain of a response, and sclerotic metastases are not evaluable. Standard serum and urinary tumour markers may not always be utilized to predict response, as they may not be elevated, and therefore may not change on treatment. The development of the urinary pyridinoline cross-link assays which measure mature bone breakdown products have been shown to be highly sensitive and specific as a measure of bone change in osteoporosis. We have measured pyridinoline (Pyr) and deoxypyridinoline (Dpyr) cross-links sequentially in 36 breast cancer patients with bone metastases, to determine if the measurement of these analytes predicts response at an earlier stage than radiological assessment. Response was assessed by UICC criteria. Seventeen women responded to hormonal therapy, whilst 19 developed progressive disease. Both Pyr and Dpyr increased sequentially in women with progressive disease with changes becoming apparent by 8 weeks (P<0.03). In responding women, cross-link levels did not change significantly. Pyr and Dpyr were more sensitive and specific than the standard serum tumour marker CA 15-3. Urinary cross-link measurements provide a novel objective method of assessing response to treatment in women with bone metastases. Initial elevated urinary cross-link markers identify patients who tend not to respond to changes in hormonal therapy.

Serum 1,25-dihydroxyvitamin D may be related inversely to disease activity in breast cancer patients with bone metastases.

1,25-dihydroxyvitamin D (1,25-(OH)2D) stimulates differentiation and controls proliferation in breast cancer cells. The role of endogenous 1,25-(OH)2D and its relation to PTH related protein (PTHrP) during the progression of breast cancer is not known; we therefore investigated these hormones in two studies. In a cross-sectional study of patients with breast cancer at different stages of disease, serum 1,25-(OH)2D levels (mean +/- SE) were highest in early disease (102 +/- 3.7 pmol/L), fell in normocalemic patients with bone metastases (52 +/- 5.3 pmol/L; P < 0.01), and were lowest in hypercalcemic patients (33 +/- 5.6 pmol/L; P < 0.001). PTHrP was detectable in the serum of only one normocalcemic patient with progressive metastases but was present in 11 of the 12 hypercalcemic patients, thus PTHrP did not stimulate 1,25-(OH)2D synthesis. In a 6-month longitudinal study of normocalcemic patients with bone metastases undergoing hormonal therapy, serum 1,25-(OH)2D concentrations fell in patients whose disease progressed (P = 0.0056), but remained constant in those who were stable or responded to treatment. These changes in 1,25-(OH)2D preceded clinical signs of progression and predicted disease response. In the progressive group, five of whom died during the study, 1,25-(OH)2D decreased between the initial and final samples, PTH fell significantly from 24.8 to 13.5 ng/L (P = 0.025), serum calcium rose from 2.27 to 2.39 mmol/L (P = 0.017), and the urinary calcium/creatinine ratio rose from 0.37 to 0.68 (P = 0.046). PTH and 1,25-(OH)2D were significantly correlated in the final samples from this group, Spearman's rank correlation = 0.80, P = 0.022. The results indicate that normocalcemia in these patients is maintained, at the expense of suppressing PTH and 1,25-(OH)2D, in the face of increased calcium released from lytic lesions in bone. Loss of the antiproliferative effects of 1,25-(OH)2D may then permit more rapid secondary growth of the tumor.

Parathyroid hormone-related protein, bone metastases and hypercalcaemia of malignancy.

Breast cancer patients frequently develop bone metastases. A newly discovered peptide, Parathyroid Hormone Related Protein (PTHrP), causes humoral hypercalcaemia of malignancy. We have studied whether production of this protein by breast cancers leads to the development of bone metastases or hypercalcaemia. PTHrP was produced by nearly 60% of early breast cancers and its production by the tumours was associated with malignant mammographic microcalcification, and the development of both bone metastases and hypercalcaemia. The hypercalcaemia associated with bone metastases has been shown to have a significant humoral component and measurement of plasma PTHrP in patients with hypercalcaemia is diagnostically useful. Potential mechanisms of preventing bone metastasis are discussed.

In vitro production of parathyroid hormone-related protein by cholesteatoma and normal skin.

Severe inflammation and infection of the middle ear occasionally results in clinical evidence of bone resorption but with the addition of the cholesteatoma epithelium it becomes inevitable. This study examined production by the cholesteatoma epithelium of a bone resorbing factor, namely parathyroid hormone-related protein (PTH-rP), which would not be expected in inflammatory states alone. PTH-rP was detected in the conditioned medium of primary and secondary cell cultures derived from 12 cholesteatoma biopsies. The levels of PTH-rP were significantly greater than in control cultures of cells derived from normal scalp tissue. Production by the cholesteatoma epithelium may explain the increased incidence of bone resorption in this disease, particularly in cases where inflammation is minimal.

Parathyroid hormone related protein in oral squamous cell carcinomas invading the mandible.

AIM: To assess parathyroid hormone related protein (PTHrP) as a candidate biochemical marker of invasion of the mandible by oral squamous cell carcinoma. METHODS: Tumour PTHrP concentrations were quantitated by immunoassay, and PTHrP was detected by immunohistochemistry, in a cohort of 24 primary squamous cell carcinomas of the mandible. RESULTS: PTHrP was identified in all tumours examined, but no correlation was found between scores of the intensity and/or consistency of staining or tumour PTHrP concentrations and the histological classification of tumour invasion. CONCLUSION: Although PTHrP was present in all squamous tumours studied, there was no correlation between PTHrP expression and pattern of tumour invasion. However, tumour derived PTHrP may act locally to influence tumour growth and differentiation and resorption of bone.

Survival in hypercalcaemic patients with cancer and co-existing primary hyperparathyroidism.

Hypercalcaemia associated with malignancy is generally thought to carry a poor prognosis. Of 47 consecutive patients with hypercalcaemia and malignancy, serum parathyroid hormone (PTH) was elevated in seven, consistent with co-existing hyperparathyroidism. Median survival from onset of hypercalcaemia in these seven patients was 817 days; compared to 33 days in the remaining 40 patients with hypercalcaemia of malignancy, in whom PTH was suppressed (p = 0.007). Among patients with hypercalcaemia of malignancy, plasma PTH-related protein (PTHrP) concentration showed no correlation with survival (r2 = 2.1%), but one patient with increased levels of both PTH and PTHrP survived only nine days after the onset of hypercalcaemia. A raised PTH had a positive predictive value of 86% for survival > 100 days, and of 71% for survival > 1 year. A raised plasma PTHrP predicted death within 100 days with a positive predictive value of 69%. We conclude that measurement of serum PTH is indicated in patients with hypercalcaemia and malignancy to identify the 15% with hyperparathyroidism, since this is associated with prolonged survival. In patients with hyperparathyroidism, assay of plasma PTHrP may indicate concurrent hypercalcaemia of malignancy, with an associated poor prognosis.

Parathyroid hormone-related protein (PTHrP) in breast cancer and benign breast tissue.

Parathyroid hormone-related protein (PTHrP) 1-86 was quantified by immunoassay in extracts of 132 breast cancers, 27 samples of normal breast tissue and four fibroadenomas. PTHrP 1-86, was detected in 68% of primary tumours (range 40-302,000 fmol/g), 33% of normal breast tissues (range 100-1800 fmol/g), and all four fibroadenomas (range 110-11,600 fmol/g). PTHrP displayed molecular heterogeneity on gel filtration chromatography, and 1-86, 1-34 and 37-67 immunoreactivity eluted as 25-27 kDa together with a peak of 19-21 kDA containing only 37-67 activity. Tumour PTHrP 1-86 levels correlated inversely with age (P < 0.05) and were higher in premenopausal women (P = 0.05). The proportion of tumours containing PTHrP was higher in axillary node positive premenopausal women (P < 0.05). These data suggest that oestrogen may regulate expression of PTHrP in breast cancer and that production of PTHrP may be linked to development of axillary node metastases.

Parathyroid hormone and parathyroid hormone-related protein in the investigation of hypercalcaemia in two hospital populations.

BACKGROUND AND OBJECTIVE: Parathyroid hormone-related protein (PTHrP) produced by cancers has a central role as a humoral mediator of hypercalcaemia in patients with malignancy. Since the prevalences of hypercalcaemia of malignancy and parathyroid disease reflect the population studied, hypercalcaemic patients from a district general hospital (DGH) and an oncology centre (OC) were studied in order to assess the diagnostic role of assays for serum PTH and plasma PTHrP in different clinical settings. DESIGN: A prospective study of consecutive patients presenting with their first episode of hypercalcaemia during an 18-month period. PATIENTS: A total of 123 patients (DGH, n = 69; OC, n = 54) had corrected serum calcium concentrations > 2.65 mmol/l. MEASUREMENTS: PTH, PTHrP, calcium and albumin were measured together with urine calcium and creatinine, enabling fractional calcium excretion to be assessed. Urine cyclic adenosine monophosphate was also measured. RESULTS: Hypercalcaemia was attributed to malignancy alone in 72 patients (DGH, n = 20; OC, n = 52), benign causes in 42 (DGH, n = 42) and parathyroid disease coexisting with malignancy in 9 (DGH, n = 7; OC, n = 2). Plasma PTHrP levels were increased in 59/72 (82%) patients with hypercalcaemia due to malignancy. Measurements of both analytes contributed to a change in the final diagnosis in 12 patients (10%). Thus serum PTH was increased in seven patients with parathyroid disease coexisting with malignancy, and plasma PTHrP was increased in five patients with previous undiagnosed malignancy. Median survival for patients with parathyroid disease and coexisting malignancy was 13 months compared with 3 months for those with hypercalcaemia due to malignancy alone (P < 0.02). CONCLUSIONS: Since hypercalcaemia was attributable to parathyroid disease in 10% of all patients with malignancy we advise measurement of serum PTH at the initial presentation of hypercalcaemia in all patients, while plasma PTHrP may be useful to identify those patients in whom malignancy may not be clinically apparent, or coexist with primary hyperparathyroidism.

Immunohistochemical localization of parathyroid hormone-related protein (PTHrP) in human term placenta and membranes.

Parathyroid hormone-related protein (PTHrP), the major factor responsible for hypercalcaemia of malignancy, is widely expressed in normal adult and fetal tissues. In this study, the distribution of PTHrP was examined in human term placenta and membranes by immunohistochemistry using antisera to PTHrP 1-34 and 37-67. PTHrP was detected in cuboidal epithelial cells of amnion and in cytotrophoblastic cells of chorionic laeve and adherent maternal decidua. In placenta, PTHrP 1-34 was detected in the syncytiotrophoblast, while PTHrP 37-67 activity was mainly present in the brush border of the syncytiotrophoblast. This study also identified PTHrP 37-67 associated with fetal vessels of placental villi. These findings may reflect the cellular distribution of intact PTHrP or sub-fragments derived by post-translational processing. Postulated actions of PTHrP in the uteroplacental unit include transport of calcium across the placenta, stretch of membranes, inhibition of uterine contractility, growth and differentiation, and vasoregulation.

Parathyroid hormone-related protein in human term placenta and membranes.

Parathyroid hormone-related protein (PTHrP), the hypercalcaemia of malignancy factor, is expressed in the tissues of the human uteroplacental unit, including the placenta, amnion and chorion. We have used three region-specific immunoassays to quantitate and compare the distribution of PTHrP in tissues obtained at term following spontaneous labour and vaginal delivery or elective Caesarean section. In non-labouring women highest PTHrP(1-86) and (37-67) immunoreactivity was found in amnion covering the placenta, rather than the decidua parietalis of the uterus (reflected amnion) (median 1020 vs 451 fmol/g; 2181 vs 1444 fmol/g respectively). In labouring women, the PTHrP(1-86) concentration in reflected amnion was inversely correlated with the interval between rupture of the membranes and delivery. Tissue PTHrP(1-86) concentrations were lower in placenta than in chorion and amnion (medians 12, 109 and 664 fmol/g respectively) and, in all tissues, PTHrP(1-34) and (37-67) concentrations were significantly higher than that of PTHrP(1-86). Bioactive PTHrP(1-34) was detected in placenta, chorion and amnion using the ROS cell bioassay. The PTHrP(1-86) concentration (mean +/- S.E.M. = 41.4 +/- 4.5 pmol/l) was high in amniotic fluid at term, although in maternal and cord plasma levels were only modestly increased. The molecular forms of PTHrP present in tissues and amniotic fluid were investigated by column chromatography which confirmed its molecular heterogeneity and suggested that processing is tissue-specific and occurs at both amino- and carboxy-terminals of the peptide.

Local control of parathyroid hormone-related protein secretion by the mammary gland of the goat.

In late-pregnant goats, daily removal of secretion from one mammary gland stimulated an early secretion of fluid by that gland in five of the six animals studied. This early secretion of fluid was accompanied by an early increase of parathyroid hormone-related protein (PTHrP) 1-86 concentration in the secretion of that gland alone. When lactation was established, glands emptied thrice daily secreted PTHrP (1-86) in greater quantities and concentrations than glands emptied once daily concurrently in the same animals. Toward the end of lactation, abrupt cessation of emptying one gland caused levels of PTHrP (1-86) to decline in the fluid in that gland but not the contralateral, emptied gland. We conclude that, in the goat, PTHrP (1-86) secretion into milk is linked to autocrine factor(s) that stimulate milk secretion when fluid is removed from the gland.

Parathyroid hormone-related protein gene expression in invasive cervical tumors.

BACKGROUND: Parathyroid hormone-related protein (PTHrP) is the main tumor-derived factor responsible for the hypercalcemia of malignancy. METHODS: Using a polyclonal serum to the 37-67 region of PTHrP, and 35S-labeled riboprobes, the authors investigated the cellular expression of PTHrP mRNA and peptide in formalin fixed, paraffin embedded sections from 16 invasive cervical tumors. In addition, the relationship among the histologic cell type, degree of differentiation, pattern of invasion, and tumor expression of PTHrP were examined. RESULTS: PTHrP mRNA and peptide were identified in 10 of 10 and 16 of 16 tumors examined, respectively. Overall strong mRNA expression with moderate to intense intracellular staining for peptide was associated, with adenosquamous carcinoma displaying a spray pattern of invasion. CONCLUSION: PTHrP mRNA and peptide were observed in all cervical tumors studied. Despite their high frequency of expression of PTHrP, cervical tumors seldom give rise to humoral hypercalcemia of malignancy, but the autocrine/paracrine effects of PTHrP may be important in the growth and dedifferentiation of the malignant cell population.

Response to intravenous bisphosphonate therapy in hypercalcaemic patients with and without bone metastases: the role of parathyroid hormone-related protein.

Plasma parathyroid hormone related-protein (PTHrP) may inhibit the calcium-lowering effect of bisphosphonate therapy. In this prospective study we examined the relationship between plasma PTHrP levels, renal tubular markers of calcium reabsorption, and the effectiveness of intravenous bisphosphonate therapy (IVBPT) in lowering serum calcium in patients with hypercalcaemia of malignancy (HM), with and without bone metastases. Thirty-five symptomatic hypercalcaemic patients (17 without and 18 with bone metastases) were treated with IVBPT (pamidronate 30-60 mg or BM21.0955 2-6 mg). Normocalcaemia was achieved in 24/35 (71%) patients with a mean fall in serum calcium of 0.85 mmol l-1 (range 0.11-1.93, P < 0.001). In the 35 patients studied, serum calcium levels reached a nadir between days 3 and 7, and this was accompanied by a small but significant reduction in plasma PTHrP levels (median reduction 0.77 pmol l-1, P = 0.007). Patients who responded to bisphosphonate therapy by becoming normocalcaemic had significantly lower basal plasma PTHrP levels, mean 4.06 vs 8.2 pmol l-1 (P < 0.04). A significant reduction in urinary calcium excretion was seen (mean 106 mumol l-1, P < 0.02) in patients with bone metastases, and urinary cAMP (mean 170 mmol l-1, P < 0.01) fell in all patients. Patients without demonstrable bone metastases had significantly higher plasma PTHrP levels (P < 0.002), required more doses of IVBPT, and had a poorer reduction in serum calcium compared with patients with bone metastases, only one of whom required more than one dose. We conclude that circulating PTHrP has an important role in increasing renal tubular reabsorption of calcium in HM, thus reducing the effectiveness of bisphosphonate therapy, particularly in patients with humoral HM and no bone metastases.

The role of parathyroid hormone-related protein in calcium homeostasis in the fetal pig.

The concentrations of plasma parathyroid hormone-like bioactivity and parathyroid hormone-related protein (1-86) (PTHrP) immunoreactivity were both higher in fetal pigs than in their mothers during the last 3 weeks of gestation. Both activities changed inversely with alterations in the plasma ionized calcium concentration. The data suggest that PTHrP may have a role in calcium homeostasis in the fetal pig, similar to its postulated role in sheep in the stimulation of calcium transport across the placenta.