Dietary fat may modulate adipose tissue homeostasis through the processes of autophagy and apoptosis.

PURPOSE: Obesity increases the risk of cardiovascular disease, type 2 diabetes mellitus and cancer development. Autophagy and apoptosis are critical processes for development and homeostasis in multicellular organisms and have been linked to a variety of disorders. We aimed to investigate whether the quantity and quality of dietary fat can influence these processes in the adipose tissue of obese people. METHODS: A randomized, controlled trial within the LIPGENE study assigned 39 obese people with metabolic syndrome to 1 of 4 diets: (a) a high-saturated fatty acid diet, (b) a high-monounsaturated fatty acid (HMUFA) diet, and (c, d) two low-fat, high-complex carbohydrate diets supplemented with long-chain n-3 polyunsaturated fatty acids (LFHCC n-3) or placebo (LFHCC), for 12 weeks each. RESULTS: We found an increase in the expression of autophagy-related BECN1 and ATG7 genes after the long-term consumption of the HMUFA diet (p = 0.001 and p = 0.004, respectively) and an increase in the expression of the apoptosis-related CASP3 gene after the long-term consumption of the LFHCC and LFHCC n-3 diets (p = 0.001 and p = 0.029, respectively). CASP3 and CASP7 gene expression changes correlated with HOMA index. CONCLUSION: Our results suggest that the processes of autophagy and apoptosis in adipose tissue may be modified by diet and that the consumption of a diet rich in monounsaturated fat may contribute to adipose tissue homeostasis by increasing autophagy. They also reinforce the notion that apoptosis in adipose tissue is linked to insulin resistance. CLINICAL TRIAL REGISTRATION NUMBER: ClinicalTrials.gov NCT00429195.

[In vitro dynamics of parathyroid hormone secretion regulated by calcium and effects on the cell cycle: parathyroid hyperplasia versus adenoma]. / Dinámica in vitro de la secreción de hormona paratiroidea regulada por calcio y efecto sobre el ciclo celular: adenoma frente a hiperplasia paratiroidea.

AIM: To compare the dynamics of calcium-regulated PTH secretion in vitro from adenomatous versus hyperplastic glands and to investigate the relationship between the parathyroid cell cycle and the calcium-regulated PTH secretion in these glands. MATERIALS AND METHODS: A total of 31 parathyroid glands (8 adenomatous and 23 hyperplastic) from 8 patients with primary hyperparathyroidism and 7 with secondary hyperparathyroidism respectively were studied. For the evaluation of calcium-regulated PTH secretion, small parathyroid pieces of 1 mm were sequentially transferred to wells with varying Ca concentrations: 0.4, 0.6, 0.8, 1, 1.25 and 1.35 or 1.5 mM. PTH concentrations were determined in the medium. For the parathyroid cell cycle studies, parathyroid cells were isolated without the use of enzymes and cell cycle was analyzed using the method described by Vindelov. The nuclei were acquired by flow cytometer and analyzed using the CELLFIT software. RESULTS: In parathyroid tissues from hyperplastic glands, the increase in extracellular calcium produced a decrease in PTH secretion which was apparent with a calcium level as low as 0.8 mM and the maximal inhibition of PTH secretion was obtained with a calcium of 1.25 mM, by the contrary, adenomatous glands required a calcium of 1.2 mM to produce a minimal decrease in PTH secretion. In hyperplastic parathyroid glands but not in parathyroid adenomas there was a significant correlation between the percentage of cells in G0/G1 phase with the set point (r = 0.914; P < 0.005) and the basal serum Ca (r = 0.862; P < 0.02). CONCLUSIONS: The control of the extracellular calcium-PTH release in vitro is less sensitive in parathyroid adenomas than hyperplasic parathyroid glands. In parathyroid hyperplasia the cell proliferation may be regulated by the extracellular calcium concentration (higher calcemia less proliferation).

(99m)Tc-sestamibi scintigraphy and cell cycle in parathyroid glands of secondary hyperparathyroidism.

Double-phase parathyroid MIBI ((99m)Tc-sestamibi) was performed in 27 patients with secondary hyperparathyroidism (SPT). Focal areas of increased uptake were scored for intensity on a three-point scale. All patients underwent subtotal parathyroidectomy (SPTx), and a total of 78 glands were removed at operation. Blood was obtained from the jugular vein before and after SPTx to measure the parathyroid hormone (PTH) levels. The volume and weight of the glands were calculated. The tissue was divided, with one aliquot being used for cell cycle analysis. The nuclei were acquired by flow cytometry and analyzed using CELLEIT software. Cell viability was assessed by flow cytometry and analyzed with LYSIS II software. Positive MIBI uptake was observed in 88.8% of patients. Focal MIBI uptake of one, two, or three glands was observed in 6, 11, and 8 patients, respectively. All patients experienced an 86% decrease in PTH blood level after SPTx compared to that before excision. A correlation was found between the volume of glands and the blood levels of intact PTH (iPTH) (r = 0.5, p < 0.05). A positive correlation was observed between MIBI uptake and the iPTH levels before SPTx (p < 0.01) and between the uptake of MIBI in the parathyroid glands and the cell cycle phases; low-grade uptake correlated with the G(0) phase and higher uptake with G(2)+S phase (r = 7, p < 0.01). No correlation was observed between MIBI uptake and the weight of the glands. MIBI scintigraphy accurately reflects the functional status of the hyperplastic parathyroid glands: Higher uptake grades correlated with the active growth phase. MIBI uptake does not reveal parathyroid enlargement; rather, it identifies the presence of hyperfunctioning autonomous glands. SPTx and total parathyroidectomy with autografting (TPTx+A) are the most widely accepted surgical approaches for patients with SPT. Reoperation for recurrence is necessary in 6% to 15% of cases. MIBI is now considered to be the radionuclide of reference for parathyroid gland scanning, although it is widely accepted that it produces poor results when trying to detect hyperplastic glands.

PTHrP enhances the secretory response of PTH to a hypocalcemic stimulus in rat parathyroid glands.

BACKGROUND: The secretion of parathyroid hormone (PTH) from the parathyroid glands might be regulated by autocrine/paracrine factors, and a feedback regulatory mechanism of PTH on the secretion of PTH has been suggested. Because of the existence of a common receptor between PTH and PTH-related peptide (PTHrP), the aim of the present study was to examine the possible effects of PTHrP 1-40 and 1-86 on PTH secretion in rats. METHODS: In vivo, the effect of PTHrP on Ca++-regulated PTH secretion was examined by the induction of hypocalcemia and hypercalcemia by an infusion of EGTA and Ca++, with and without PTHrP. The eventual effects of PTHrP on the peripheral metabolism of PTH were examined by infusion of human PTH (hPTH) with and without PTHrP. hPTH was measured by an intact hPTH assay not cross reacting with rat PTH or PTHrP. To examine whether near physiological levels of circulating PTH have an autoregulatory effect in vivo on PTH secretion from the parathyroid gland, an acute reduction of the circulating PTH was induced by an acute unilateral parathyroidectomy (UPTX). PTH secretion from the remaining parathyroid gland was followed in response to EGTA-induced hypocalcemia. In vitro investigations on the effect of PTHrP 1-40 on PTH secretion from whole rat parathyroid glands incubated in media containing a calcium concentration of 0.6 or 1.35 mmol/L were performed to confirm whether the effect of PTHrP was directly on the gland. The rat PTH assay was examined for cross reaction with PTHrP. RESULTS: In vivo, the same rate of decrease of plasma Ca++ was induced in the experimental groups. The maximal response of PTH to hypocalcemia (218 +/- 16 pg/mL, N = 6) was significantly enhanced by PTHrP 1-40 (525 +/- 79 pg/mL, N = 6) and by PTHrP 1-86 (465 +/- 29 pg/mL, N = 6, P < 0.001). No effect of PTHrP on PTH secretion was found during normocalcemia or hypercalcemia. UPTX resulted in a 50% reduction of PTH secretion, and no compensatory increase of PTH was observed. PTHrP had no effect on the metabolism of PTH. In vitro, low-Ca++-induced PTH secretion was significantly augmented by 300% (P < 0.01) when the medium contained PTHrP 1-40. PTHrP did not cross react with the PTH assay. CONCLUSIONS: PTHrP significantly enhanced the low-Ca++-stimulated PTH secretion in vivo and in vitro. An autocrine/paracrine role of PTHrP in the parathyroid glands is suggested. An autoregulatory effect of circulating PTH on the PTH secretion from parathyroid glands seems unlikely. The "maximal secretory capacity" of the parathyroid glands induced by hypocalcemia in vivo and in vitro is not the maximum, as PTH secretion can be increased even further, by several-fold.

High phosphate level directly stimulates parathyroid hormone secretion and synthesis by human parathyroid tissue in vitro.

Phosphate retention plays an important role in the pathogenesis of secondary hyperparathyroidism in patients with renal failure. In in vitro studies, high extracellular phosphate levels directly stimulate PTH secretion in rat and bovine parathyroid tissue. The present study evaluates the effect of high phosphate levels on the secretion of PTH and the production of prepro PTH mRNA in human hyperplastic parathyroid glands. The study includes parathyroid glands obtained from patients with primary adenomas and from hemodialysis and kidney-transplant patients with diffuse and nodular secondary hyperplasia. The experiments were performed in vitro using small pieces of parathyroid tissue. The ability of high calcium levels to decrease PTH secretion was less in adenomas than in secondary hyperplasia; among the secondary hyperplasia, nodular was less responsive to an increase in calcium than diffuse hyperplasia. In diffuse hyperplasia, PTH secretion was increased in response to 3 and 4 mM phosphate compared with 2 mM phosphate, despite a high calcium concentration in the medium; prepro PTH mRNA levels increased after incubation in 4 mM phosphate. Similar results were obtained with nodular hyperplasia, except that the elevation of PTH secretion in response to 3 mM phosphate did not attain statistical significance. In adenomas, high calcium concentrations (1.5 mM) did not result in inhibition of PTH secretion, independent of the phosphate concentration, and the prepro PTH mRNA was not significantly increased by high phosphate levels. In conclusion, first, the PTH secretory response to an increase in calcium concentration is less in nodular than diffuse hyperplasia; second, high phosphate levels directly affect PTH secretion and gene expression in patients with advanced secondary hyperparathyroidism.

Relative effects of PTH and dietary phosphorus on calcitriol production in normal and azotemic rats.

In moderate renal failure, the serum calcitriol level is influenced by the stimulatory effect of high PTH and the inhibitory action of phosphorus retention. Our goal was to evaluate the relative effect that high PTH levels and increased dietary phosphorus had on calcitriol production in normal rats (N) and rats with moderate renal failure (Nx). Normal and Nx (3/4 nephrectomy) rats were divided into two groups: (1) rats with intact parathyroid glands (IPTG) and (2) parathyroidectomized rats in which PTH was replaced (PTHR) by the continuous infusion of rat 1-34 PTH, 0.022 microgram/hr/100 g body wt, using a miniosmotic Alzet pump. To test the effect of dietary phosphorus, rats received either a moderate (MPD, 0.6% P) or a high phosphorus (HPD, 1.2%) diet for 14 days. The experimental design included pair-fed N and Nx rats with either IPTG or PTHR. Serum calcitriol and PTH levels in N rats fed a MPD were 69 +/- 3 and 40 +/- 5 pg/ml, respectively. In Nx rats on a MPD, serum calcitriol levels decreased only if hyperparathyroidism was not allowed to occur (76 +/- 4 vs. 62 +/- 4 pg/ml in Nx-IPTG-MPD and Nx-PTHR-MPD groups respectively, P < 0.05). Even in N rats on a HPD, high PTH levels (67 +/- 8 pg/ml in the N-IPTG-HPD group) were required to maintain normal serum calcitriol levels (69 +/- 4 vs. 56 +/- 6 pg/ml in Nx-IPTG-HPD and Nx-PTHR-HPD groups, respectively; P < 0.05). In Nx rats on a HPD, the development of secondary hyperparathyroidism (286 +/- 19 pg/ml in the Nx-IPTG-HPD group) prevented a decrease in serum calcitriol levels (68 +/- 7 pg/ml). In contrast, serum calcitriol levels were low in the Nx-PTHR-HPD group (52 +/- 4 pg/ml, P < 0.05), which were deprived of the adaptative increase in endogenous PTH production. In conclusion, our results in rats indicate that in moderate renal failure, an elevated PTH level maintains calcitriol production and overcomes the inhibitory action of phosphorus retention.

Heterogeneity of growth hormone (GH)-producing cells in aging male rats: ultrastructure and GH gene expression in somatotrope subpopulations.

Mammalian aging is characterized by a decline in the content and release of pituitary growth hormone (GH). However, few studies on the age-related changes in the population of GH-producing cells (somatotropes) have been carried out. We have investigated whether changes in number, ultrastructure and GH gene expression in subpopulations of somatotropes could explain the reduced GH release in aged rats. Three representative ages were studied: adult (5-month-old), old (19-month-old), and senescent (26-month-old) male rats. The total number of immunoreactive-GH cells per pituitary gland remained invariable to age. The separation of dispersed pituitary cells on a density gradient yielded two somatotrope subpopulations, of low density (LD) and high density (HD). Both subpopulations were equally represented in adults, whereas in old and senescent rats a predominance of LD-somatotropes was observed. Morphometric analysis showed that subpopulations exhibited storage and biosynthetic features inversely related. In LD-somatotropes, rough endoplasmic reticulum (RER) was more prominent but secretory granules (SG) were less abundant than in HD somatotropes. Concurrently, in situ hybridization for GH mRNA showed that GH gene expression was higher in LD-cells. Differences between subpopulations were essentially retained through the animals' lifespan, but small-sized SG, reduced RER, and low GH mRNA levels were inherent to aging both in LD- and in HD-somatotropes. The present findings demonstrate that the reduced content of pituitary GH in aged male rats is not due to a diminished number of GH-producing cells, but to the numerical predominance of scarcely granulated LD-somatotropes, combined with the decline in GH biosynthetic capacity observed in both subpopulations. In addition, age-related changes in ultrastructure and GH gene expression suggest a chronic inhibition of GH release and/or a weak stimulation of GH biosynthesis affecting both subpopulations.

Hormonal storage patterns and morphological heterogeneity of porcine gonadotrope cells during postnatal development.

Previous reports indicate that gonadotrope cells of the porcine pituitary gland can be separated into three subpopulations of low- (1.049 g/cm3), middle- (1.062 g/cm3) and high- (1.087 g/cm3) density in a continuous Percoll density gradient. The aim of this work was to study the hormonal storage patterns and morphological features of these subpopulations at three representative ages of the postnatal development: neonatals (30-day-old animals), prepubertals (5-6-month-old animals) and matures (16-18-month-old animals). The low-density subpopulation, present at the three ages studied, was mainly composed of bihormonal LH/FSH cells in neonatal and monohormonal LH cells in prepubertal and mature animals. On the other hand, middle- (only present in prepubertal and mature animals) and high-density subpopulations (only present in neonatal and prepubertal animals) were mainly composed of bihormonal LH/FSH gonadotropes. In ultrastructural terms, these subpopulations exhibit a correlation between density and morphology irrespective of the animal's age. The low-density subpopulation was composed of poorly granulated cells with highly developed biosynthetic machinery (rough endoplasmic reticulum and Golgi complex), while high-density cells were of opposite morphology, with a highly granulated cytoplasm and poorly developed rough endoplasmic reticulum and Golgi complex. The middle-density subpopulation was composed of poorly granulated cells with scarcely developed biosynthetic machinery. In conclusion, these results indicate that porcine gonadotrope cells during postnatal development are composed of three subpopulations of different hormonal storage patterns and morphology. The presence of these subpopulations at the different stages of postnatal development strongly suggests that their proportions may play a major role in the endocrine control process.