Effects of combined calcium and vitamin D supplementation on insulin secretion, insulin sensitivity and β-cell function in multi-ethnic vitamin D-deficient adults at risk for type 2 diabetes: A pilot randomized, placebo-controlled trial

Objectives: To examine whether combined vitamin D and calcium supplementation improves insulin sensitivity, insulin secretion, β-cell function, inflammation and metabolic markers.

Short-chain fatty acids increase expression and secretion of stromal cell-derived factor-1 in mouse and human pre-adipocytes

Objective: Stromal cell-derived factor-1 (SDF-1) is expressed in pre-adipocytes but its role is unknown. We investigated butyrate (a histone deacetylase inhibitor - HDACi) and other short-chain fatty acids (SCFA) in the regulation of SDF-1. We further investigated whether effects of SCFA were signalled through G protein-coupled receptors FFA2 and FFA3. Design and Results: SDF-1 mRNA expression and protein secretion were studied in 3T3-L1 cells and human pre-adipocytes. SDF-1 was abundant, with mRNA and protein levels increased by butyrate. This was replicated with acetate and propionate, but not with trichostatin or valproate. Trichostatin inhibited SDF-1 secretion. Pertussis toxin blocked stimulation by butyrate. The order of potency of SCFA in stimulating SDF-1 (C3 > C4 > C2) is consistent with action through FFA3. Silencing the FFA3 gene abolished butyrate-stimulated SDF-1 expression and secretion. FFA3 was expressed in both pre-adipocytes and adipocytes, while FFA2 was expressed in adipocytes only. SDF-1 expression was low in murine macrophage J774.2 cells, while the SDF-1 receptor CXCR4 was absent from 3T3-L1 cells but abundant in J774.2 macrophages. In human pre-adipocytes, FFA3 was also expressed and SCFA increased SDF-1 secretion. Conclusions: SDF-1 and CXCR4 may mediate the interaction between adipose stromal cells and macrophages. Effects of SCFA are mediated through FFA3, but not histone deacetylase inhibition.

Cytokine expression and secretion by skeletal muscle cells: regulatory mechanisms and exercise effects

Cytokines are important mediators of various aspects of health and disease, including appetite, glucose and lipid metabolism, insulin sensitivity, skeletal muscle hypertrophy and atrophy. Over the past decade or so, considerable attention has focused on the potential for regular exercise to counteract a range of disease states by modulating cytokine production. Exercise stimulates moderate to large increases in the circulating concentrations of interleukin (IL)-6, IL-8, IL- 10, IL-1 receptor antagonist, granulocyte-colony stimulating factor, and smaller increases in tumor necrosis factor-α, monocyte chemotactic protein-1, IL-1β, brain-derived neurotrophic factor, IL-12p35/p40 and IL-15. Although many of these cytokines are also expressed in skeletal muscle, not all are released from skeletal muscle into the circulation during exercise. Conversely, some cytokines that are present in the circulation are not expressed in skeletal muscle after exercise. The reasons for these discrepant cytokine responses to exercise are unclear. In this review, we address these uncertainties by summarizing the capacity of skeletal muscle cells to produce cytokines, analyzing other potential cellular sources of circulating cytokines during exercise, and discussing the soluble factors and intracellular signaling pathways that regulate cytokine synthesis (e.g., RNA-binding proteins, microRNAs, suppressor of cytokine signaling proteins, soluble receptors).

Altered expression of two zinc transporters, SLC30A5 and SLC30A6, underlies a mammary gland disorder of reduced zinc secretion into milk

Two cases of zinc deficiency in breastfed neonates were investigated where zinc levels in the mothers' milk were reduced by more than 75 % compared to normal. The objective of this study was to find the molecular basis of the maternal zinc deficiency condition. Significant reductions in mRNA expression and protein levels of the zinc transporters SLC30A5 and SLC30A6 were found in maternal tissue, suggesting a causal link to the zinc-deficient milk. Novel splice variants of the SLC30A6 transcript were detected. No modifications were found in coding regions, or in transcription binding sites of promoter regions or in 5' and 3' untranslated regions of both transporters in lymphoblasts and fibroblasts isolated from both mothers. Altered DNA methylation in SLC30A5 at two CpG sites was detected and may account for the reduced levels of SLC30A5 mRNA and protein in lymphoblasts. Reduced SLC30A6 mRNA and protein levels in lymphoblasts may be secondary to reduced SLC30A5 expression, as they function as a heterodimer in zinc transport. In conclusion, two cases of zinc deficiency are linked to low levels of the SLC30A5 and SLC30A6 zinc transporters. These two zinc transporters have not been previously associated with zinc deficiency in milk.

The effect of repeated boar exposure on cortisol secretion and reproduction in gilts

It has been proposed that short-term activation of the hypothalamo-pituitary adrenal axis, with a consequent increase in the secretion of cortisol, amy disrupt the endocrine events prior to ovulation and thereby impair reproduction in females. We investigated this concept in gilts in which oestrus was detected by introduction to boars, where intense physical contact is possible, or by applying pressure to the back of gilts (back-pressure test) during fence-line exposure to boars, where intense physical contact is prohibited. We expected that there would be a greater release of cortisol and that reproduction would be inhibited in gilts introduced to boars compared to gilts in which the back-pressure test was used. As expected, introduction of gilts to boars resulted in a significant transient increase in plasma concentrations of cortisol while there was no significant effect of using the back-pressure test on plasma cortisol. Nevertheless, introduction of gilts to boars did not impair reproduction and there was no effect of method of detecting oestrus on duration of oestrus, sexual receptivity, fertility or fecundity. The length of the oestrous cycle was decreased and ovulation rate increased in gilts that were introduced to boars compared to gilts that underwent the back-pressure test, indicating that introduction of gilts to boars may have stimulated these aspects of reproduction. These stimulatory effects may have been due to an increased exposure of gilts to sexual behaviour and stimuli from boars when introduced to boars and/or to stimulatory effects of the hypothalamo-pituitary adrenal axis on some aspects of reproduction.

Inhibition of the secretion of LH in ovariectomised pigs by sustained but not repeated acute elevation of cortisol in the absence but not the presence of oestradiol

Prolonged stress is known to impair reproduction. It has been proposed that reproduction will also be impaired when a severe acute stress occurs during a period of elevated plasma concentrations of oestradiol, such as during the follicular phase of the oestrous cycle. In this experiment, we hypothesised that repeated acute and sustained elevation of cortisol would suppress the secretion of LH in ovariectomised pigs and that these effects would be enhanced in the presence of oestradiol negative feedback. Cortisol (or vehicle) was administered 12 hourly to ovariectomised pigs (n=6/treatment) for 8 days in the absence of oestradiol treatment and for a further 8 days during treatment with oestradiol. Vehicle was administered to 'control' pigs, 10 or 20 mg cortisol was administered i.v. to pigs to produce 'repeated acute' elevation of cortisol and 250 mg cortisol was administered i.m. to pigs to give a 'sustained' elevation of cortisol. Both before and during treatment with oestradiol, plasma concentrations of LH were monitored on the day before treatment, on the 4th and 8th days of treatment and following an i.v. injection of GnRH at the end of the 8th day of treatment. The repeated acute elevation of cortisol did not impair any parameters of LH secretion (i.e. mean plasma concentrations of LH, pulse amplitude or frequency, pre-LH pulse nadir or the LH response to GnRH) in the absence or in the presence of oestradiol. In contrast, when the elevation of cortisol was sustained, the mean plasma concentrations of LH and the pre-LH pulse nadir were significantly (P<0.05) lower on the 8th day of treatment than on the day before treatment and on the 4th day of treatment. Nevertheless, no other parameters of LH secretion were affected and these effects only occurred in the absence (not in the presence) of oestradiol. In conclusion, cortisol needed to be elevated for more than 4 days to impair the secretion of LH, and oestradiol did not enhance the impact of cortisol on LH secretion in ovariectomised pigs.

Progesterone and testosterone in combination act in the hypothalamus of castrated rams to regulate the secretion of LH

We tested the hypotheses that progesterone enhances the negative feedback actions of testosterone in rams and that this occurs through actions at the hypothalamus. In the first part of this study, blood samples were collected every 10 min for 12 h before and after 7 days of treatment (i.m.) of castrated Romney Marsh rams (n=5 per group) with vehicle, progesterone (4 mg/12 h), testosterone (4 mg/12 h) or a combination of progesterone (4 mg/12 h) and testosterone (4 mg/12 h). In the second part of this study the brains of four gonad-intact Romney Marsh rams were collected, the hypothalamus was sectioned and in situ hybridisation of mRNA for progesterone receptors conducted. After 7 days of treatment with vehicle or progesterone or testosterone alone, there were no changes in the secretion of LH. In contrast, treatment with a combination of progesterone and testosterone resulted in a significant (P<0.01, repeated measures ANOVA) decrease in mean plasma concentrations of LH, the number of LH pulses per hour and the pre-LH pulse nadir and a significant (P<0.01) increase in the inter-LH pulse interval. We found cells containing mRNA for progesterone receptors throughout the hypothalamus, including the preoptic area (where most GnRH neurons are located in sheep), the periventricular, ventromedial and arcuate nuclei and the bed nucleus of the stria terminalis. This study shows that progesterone is capable of acting centrally with testosterone to suppress the secretion of LH in castrated rams and that cells containing mRNA for progesterone receptors are located in the hypothalamus of rams in the vicinity of GnRH neurons.

Influence of sex and gonadal status of sheep on cortisol secretion in response to ACTH and on cortisol and LH secretion in response to stress: importance of different stressors

There are sex differences in the response to stress and in the influence of stress on reproduction which may be due to gonadal steroids but the nature of these differences and the role of the gonads are not understood. We tested the hypotheses that sex and the presence/absence of gonads (gonadal status) will influence the cortisol response to injection of ACTH, insulin-induced hypoglycaemia and isolation/restraint stress, and that sex and gonadal status will influence the secretion of LH in response to isolation/restraint stress. Four groups of sheep were used in each of three experiments: gonad-intact rams, gonadectomised rams, gonad-intact ewes in the mid-luteal phase of the oestrous cycle and gonadectomised ewes. In Experiment 1 (n=4/group), jugular blood samples were collected every 10 min for 6 h; after 3 h, two animals in each group were injected (i.v.) with ACTH and the remaining two animals were injected (i.v.) with saline. Treatments were reversed 5 days later so that every animal received both treatments. Experiment 2 (n=4/group) used a similar schedule except that insulin was injected (i.v.) instead of ACTH. In Experiment 3 (n=5/group), blood samples were collected every 10 min for 16 h on a control day and again 2 weeks later when, after 8 h of sampling, all sheep were isolated and restrained for 8 h. Plasma cortisol was significantly (P<0.05) elevated following injection of ACTH or insulin and during isolation/restraint stress. There were no significant differences between the sexes in the cortisol response to ACTH. Rams had a greater (P<0.05) cortisol response to insulin-induced hypoglycaemia than ewes while ewes had a greater (P<0.05) cortisol response to isolation/restraint stress than rams. There was no effect of gonadal status on these parameters. Plasma LH was suppressed (P<0.05) in gonadectomised animals during isolation/restraint stress but was not affected in gonad-intact animals, and there were no differences between the sexes. Our results show that the sex that has the greater cortisol response to a stressor depends on the stressor imposed and that these sex differences are likely to be at the level of the hypothalamo-pituitary unit rather than at the adrenal gland. Since there was a sex difference in the cortisol response to isolation/restraint, the lack of a sex difference in the response of LH to this stress suggests that glucocorticoids are unlikely to be a major mediator of the stress-induced suppression of LH secretion.

Sulfate transporters involved in sulfate secretion in the kidney are localized in the renal proximal tubule II of the elephant fish (Callorhinchus milii)

Most vertebrates, including cartilaginous fishes, maintain their plasma SO4 (2-) concentration ([SO4 (2-)]) within a narrow range of 0.2-1 mM. As seawater has a [SO4 (2-)] about 40 times higher than that of the plasma, SO4 (2-) excretion is the major role of kidneys in marine teleost fishes. It has been suggested that cartilaginous fishes also excrete excess SO4 (2-) via the kidney. However, little is known about the underlying mechanisms for SO4 (2-) transport in cartilaginous fish, largely due to the extraordinarily elaborate four-loop configuration of the nephron, which consists of at least 10 morphologically distinguishable segments. In the present study, we determined cDNA sequences from the kidney of holocephalan elephant fish (Callorhinchus milii) that encoded solute carrier family 26 member 1 (Slc26a1) and member 6 (Slc26a6), which are SO4 (2-) transporters that are expressed in mammalian and teleost kidneys. Elephant fish Slc26a1 (cmSlc26a1) and cmSlc26a6 mRNAs were coexpressed in the proximal II (PII) segment of the nephron, which comprises the second loop in the sinus zone. Functional analyses using Xenopus oocytes and the results of immunohistochemistry revealed that cmSlc26a1 is a basolaterally located electroneutral SO4 (2-) transporter, while cmSlc26a6 is an apically located, electrogenic Cl(-)/SO4 (2-) exchanger. In addition, we found that both cmSlc26a1 and cmSlc26a6 were abundantly expressed in the kidney of embryos; SO4 (2-) was concentrated in a bladder-like structure of elephant fish embryos. Our results demonstrated that the PII segment of the nephron contributes to the secretion of excess SO4 (2-) by the kidney of elephant fish. Possible mechanisms for SO4 (2-) secretion in the PII segment are discussed.