Ultradian Cortisol Pulsatility Encodes a Distinct, Biologically Important Signal

Cortisol is released in ultradian pulses. The biological relevance of the resulting fluctuating cortisol concentration has not been explored.Determination of the biological consequences of ultradian cortisol pulsatility.A novel flow through cell culture system was developed to deliver ultradian pulsed or continuous cortisol to cells. The effects of cortisol dynamics on cell proliferation and survival, and on gene expression were determined. In addition, effects on glucocorticoid receptor (GR) expression levels and phosphorylation, as a potential mediator, were measured.Pulsatile cortisol caused a significant reduction in cell survival compared to continuous exposure of the same cumulative dose, due to increased apoptosis. Comprehensive analysis of the transcriptome response by microarray identified genes with a differential response to pulsatile versus continuous glucocorticoid delivery. These were confirmed with qRT-PCR. Several transcription factor binding sites were enriched in these differentially regulated target genes, including CCAAT-displacement protein (CDP). A CDP regulated reporter gene (MMTV-luc) was, as predicted, also differentially regulated by pulsatile compared to continuous cortisol delivery. Importantly there was no effect of cortisol delivery kinetics on either GR expression, or activation (GR phosphoSer(211)).Cortisol oscillations exert important effects on target cell gene expression, and phenotype. This is not due to differences in cumulative cortisol exposure, or either expression, or activation of the GR. This suggests a novel means to regulate GR function

Amplification of pulsatile glucagon counterregulation by switch-off of α-cell-suppressing signals in streptozotocin-treated rats

Glucagon counterregulation (GCR) is a key protection against hypoglycemia that is compromised in diabetes via an unknown mechanism. To test the hypothesis that α-cell-inhibiting signals that are switched off during hypoglycemia amplify GCR, we studied streptozotocin (STZ)-treated male Wistar rats and estimated the effect on GCR of intrapancreatic infusion and termination during hypoglycemia of saline, insulin, and somatostatin. Times 10 min before and 45 min after the switch-off were analyzed. Insulin and somatostatin, but not saline, switch-off significantly increased the glucagon levels (P = 0.03), and the fold increases relative to baseline were significantly higher (P < 0.05) in the insulin and somatostatin groups vs. the saline group. The peak concentrations were also higher in the insulin (368 pg/ml) and somatostatin (228 pg/ml) groups vs. the saline (114 pg/ml) group (P < 0.05). GCR was pulsatile in most animals, indicating a feedback regulation. After the switch-off, the number of secretory events and the total pulsatile production were lower in the saline group vs. the insulin and somatostatin groups (P < 0.05), indicating enhancement of glucagon pulsatile activity by insulin and somatostatin compared with saline. Network modeling analysis demonstrates that reciprocal interactions between α- and δ-cells can explain the amplification by interpreting the GCR as a rebound response to the switch-off. The model justifies experimental designs to further study the intrapancreatic network in relation to the switch-off phenomenon. The results of this proof-of-concept interdisciplinary study support the hypothesis that GCR develops as a rebound pulsatile response of the intrapancreatic endocrine feedback network to switch-off of α-cell-inhibiting islet signals

Mutant Gene Found in Pancreatic Juice Activated Chromatin From Peri-ampullary Adenocarcinomas

External pancreatic duct stents inserted after resection of pancreatic head tumors provide unique access to pancreatic juice analysis of genetic and metabolic components that may be associated with peri-ampullary tumor progression. For this pilot study, portal venous blood and pancreatic juice samples were collected from 17 patients who underwent pancreaticoduodenectomy for peri-ampullary tumors. Portal vein circulating tumor cells (CTC) were isolated by high-speed fluorescence-activated cell sorting (FACS) and analyzed by quantitative reverse transcription polymerase chain reaction (RT-PCR) for K-RAS exon 12 mutant gene expression ( K-RASmut ). DNA, chromatin, and histone acetylated active chromatin were isolated from pancreatic juice samples by chromatin immunoprecipitation (ChIP) and the presence of K-RASmut and other cancer-related gene sequences detected by quantitative polymerase chain reaction (PCR) and ChIP-Seq. Mutated K-RAS gene was detectable in activated chromatin in pancreatic juice secreted after surgical resection of pancreatic, ampullary and bile duct carcinomas and directly correlated with the number of CTC found in the portal venous blood ( P  = .0453). ChIP and ChIP-Seq detected acetylated chromatin in peri-ampullary cancer patient juice containing candidate chromatin loci, including RET proto-oncogene, not found in similar analysis of pancreatic juice from non-malignant ampullary adenoma. The presence of active tumor cell chromatin in pancreatic juice after surgical removal of the primary tumor suggests that viable cancer cells either remain or re-emerge from the remnant pancreatic duct, providing a potential source for tumor recurrence and cancer relapse. Therefore, epigenetic analysis for active chromatin in pancreatic juice and portal venous blood CTC may be useful for prognostic risk stratification and potential identification of molecular targets in peri-ampullary cancers

Assessment of endogenous growth hormone pulsatility in gelded yearling horses using deconvolution analysis

Hypothesis/objectives: Defining normal Growth Hormone (GH) secretory dynamics in the horse is necessary to understand altered GH dynamics related to issues like welfare and disease. Animals and methods: Twelve healthy yearlings and two mature Standardbreds were used to quantify GH secretion. Endogenous GH half-life was determined after administration of 1.0 mg/kg BW GH releasing hormone (GHRH). Exogenous GH half-life was determined after administration of 20 mg/kg BW recombinant equine GH (reGH) with and without suppression of endogenous GH secretion by somatostatin infusion (50 mg/m 2/h). Pulse detection algorithm (Cluster) as well as deconvolution analysis was used to quantify GH secretory dynamics based on GH concentration-time series sampled every 5 min from 22:00 till 06:00 h. In addition, reproducibility, impact of sampling frequency and influence of altering initial GH half-life on parameter estimates were studied. Results: Mean endogenous GH half-life of 17.7±4.4 (SD) min and mean exogenous half-life of 26.0±2.9 min were found. The mean number of GH secretion peaks in 8 h was 12±3.2. Ninety-nine percent of the total amount of GH secreted occurred in pulses, basal secretion was 0.012±0.014 mg/L/min and half-life was 8.9±2.6 min. Compared with a 5-min sampling frequency, 20-and 30-min sampling underestimated the number of secretory events by 45% and 100%, respectively. Conclusions: The deconvolution model used was valid to GH time series in Standardbreds. As in man, the equine pituitary gland secretes GH in volleys consisting of multiple secretory bursts, without measurable intervening tonic secretion. The required GH sampling frequency for the horse should be around 3 min. Clinical relevance: Defining normal GH secretory dynamics in the horse will make it possible to detect alterations in the GH axis due to pathophysiologic mechanisms as well as abuse of reGH

T1R2 receptor-mediated glucose sensing in the upper intestine potentiates glucose absorption through activation of local regulatory pathways

Objective: Beyond the taste buds, sweet taste receptors (STRs; T1R2/T1R3) are also expressed on enteroendocrine cells, where they regulate gut peptide secretion but their regulatory function within the intestine is largely unknown. Methods: Using T1R2-knock out (KO) mice we evaluated the role of STRs in the regulation of glucose absorption in vivo and in intact intestinal preparations ex vivo. Results: STR signaling enhances the rate of intestinal glucose absorption specifically in response to the ingestion of a glucose-rich meal. These effects were mediated specifically by the regulation of GLUT2 transporter trafficking to the apical membrane of enterocytes. GLUT2 translocation and glucose transport was dependent and specific to glucagon-like peptide 2 (GLP-2) secretion and subsequent intestinal neuronal activation. Finally, high-sucrose feeding in wild-type mice induced rapid downregulation of STRs in the gut, leading to reduced glucose absorption. Conclusions: Our studies demonstrate that STRs have evolved to modulate glucose absorption via the regulation of its transport and to prevent the development of exacerbated hyperglycemia due to the ingestion of high levels of sugars. Keywords: Glucose homeostasis, Sweet taste receptors, GLP-2, GLUT2, T1R