Joseph S. Dillon

Severe Metabolic Bone Disease as a Long-Term Complication of Obesity Surgery

Background: Metabolic bone disease is a well-documented long-term complication of obesity surgery. It is often undiagnosed, or misdiagnosed, because of lack of physician and patient awareness. Abnormalities in calcium and vitamin D metabolism begin shortly after gastrointestinal bypass operations; however, clinical and biochemical evidence of metabolic bone disease may not be detected until many years later. Case Report: A 57-year-old woman presented with severe hypocalcemia, vitamin D deficiency,and radiographic evidence of osteomalacia, 17 years after vertical banded gastroplasty and Roux-en-Y gastric bypass. Following these operations, she was diagnosed with a variety of medical disorders based on symptoms that, in retrospect, could have been attributed to metabolic bone disease. Additionally, she had serum metabolic abnormalities that were consistent with metabolic bone disease years before this presentation. Radiographic evidence of osteomalacia at the time of presentation suggests that her condition was advanced, and went undiagnosed for many years. These symptoms and laboratory and radiographic abnormalities most likely were a result of the long-term malabsorptive effects of gastric bypass, food intake restriction, or a combination of the two. Conclusion: This case illustrates not only the importance of informed consent in patients undergoing obesity operations, but also the importance of adequate follow-up for patients who have undergone these procedures. A thorough history and physical examination, a high index of clinical suspicion, and careful long-term follow-up, with specific laboratory testing, are needed to detect early metabolic bone disease in these patients.

Effects of Adenoviral Overexpression of Uncoupling Protein-2 and -3 on Mitochondrial Respiration in Insulinoma Cells1

The brown adipose tissue uncoupling protein 1 (UCP1) catalyzes proton reentry without ATP synthesis, thereby dissipating energy as heat. In contrast, the function(s) of the recently described homologs, UCP2 and UCP3, are less clear. The aim of the present study was to determine whether overexpressed UCP subtypes affect mitochondrial respiration and substrate oxidation in cultured insulin-secreting INS-1 insulinoma cells. Adenoviral overexpression of UCP2 significantly decreased the ADP/O ratio by 31% and 39% in comparison to β-galactosidase (β-gal) or the mitochondrial protein manganese superoxide dismutase (MnSOD), respectively, and increased state 4 respiration in the presence of succinate and oligomycin by 52% and 59% in comparison toβ -gal or MnSOD, respectively. Adenoviral overexpression of UCP3 also decreased the ADP/O ratio by 18% (nonsignificant) and increased state 4 respiration by 24% (nonsignificant) in comparison to β-gal and significantly decreased the ADP/O ratio by 32% and increased state 4...

Effects of dehydroepiandrosterone on proliferation, migration, and death of breast cancer cells

Cancer invasion and metastasis are the leading causes of mortality in patients with breast cancer. Dehydroepiandrosterone (DHEA) has a protective role against cancer, however, the mechanism by which DHEA has this effect remains poorly understood. The present study was aimed at investigating the actions of DHEA on the proliferation, cell cycle, death and migration of breast cancer cell lines. We used MCF-7 cells (estrogen receptors positive) and MDA-MB-231 and Hs578T cells (estrogen receptors negative) for these studies. Cell proliferation was evaluated by crystal violet staining, cell cycle by flow cytometry, and cell death by the carboxyfluorescein FLICA analysis of caspase activation. Migration was evaluated by transwell cell migration and wound assay. We also determined the expression of ECM-1 protein by western blotting and RT-PCR in real time. DHEA inhibited the proliferation of all breast cancer cell lines. This was associated with an arrest in the G1 phase of the cell cycle and death in MCF-7 cells. There was no alteration in any of the cell cycle phases or death in DHEA treated MDA-MB-231 or Hs578T cells. DHEA also suppressed the migration of all breast cancer cell lines, independently of the presence of estrogen receptors and decreased the expression of ECM-1 protein in Hs578T cells. These results suggest that the mechanism of DHEA actions against breast cancer involves the inhibition of cell proliferation and the suppression of migration, indicating that DHEA could be useful in the treatment of breast cancer.

Glucagon-like peptide 1 and fatty acids amplify pulsatile insulin secretion from perifused rat islets

Glucose-induced insulin secretion from isolated, perifused rat islets is pulsatile with a period of about 5-10 min, similar to the insulin oscillations that are seen in healthy humans but which are impaired in Type II diabetes. We evaluated the pattern of enhancement by the potent incretin, glucagon-like peptide 1 (GLP-1). GLP-1 increased the amplitude of pulses and the magnitude of insulin secretion from the perifused islets, without affecting the average time interval between pulses. Forskolin and the phosphodiesterase inhibitor isobutylmethylxanthine had the same effect, suggesting that the effect was due to elevated cAMP levels. The possibility that cAMP might enhance the amplitude of pulses by reducing phosphofructo-2-kinase (PFK-2) activity was eliminated when the liver isoform of PFK-2 was shown to be absent from beta-cells. The possibility that cAMP enhanced pulsatile secretion, at least in part, by stimulating lipolysis was supported by the observations that added oleate had a similar effect on secretion, and that the incretin effect of GLP-1 was inhibited by the lipase inhibitor orlistat. These data show that the physiological incretin GLP-1 preserves and enhances normal pulsatile insulin secretion, which may be essential in proposed therapeutic uses of GLP-1 or its analogues.

The North American Neuroendocrine Tumor Society Consensus Guidelines for Surveillance and Medical Management of Midgut Neuroendocrine Tumors

Abstract There have been significant developments in diagnostic and therapeutic options for patients with neuroendocrine tumors (NETs). Key phase 3 studies include the CLARINET trial, which evaluated lanreotide in patients with nonfunctioning enteropancreatic NETs; the RADIANT-2 and RADIANT-4 studies, which evaluated everolimus in functioning and nonfunctioning NETs of the gastrointestinal tract and lungs; the TELESTAR study, which evaluated telotristat ethyl in patients with refractory carcinoid syndrome; and the NETTER-1 trial, which evaluated 177Lu-DOTATATE in NETs of the small intestine and proximal colon (midgut). Based on these and other advances, the North American Neuroendocrine Tumor Society convened a multidisciplinary panel of experts with the goal of updating consensus-based guidelines for evaluation and treatment of midgut NETs. The medical aspects of these guidelines (focusing on systemic treatment, nonsurgical liver-directed therapy, and postoperative surveillance) are summarized in this article. Surgical guidelines are described in a companion article.

Dehydroepiandrosterone inhibits intracellular calcium release in β-cells by a plasma membrane-dependent mechanism

Both dehydroepiandrosterone (DHEA) and DHEA sulfate (DHEAS) affect glucose stimulated insulin secretion, though their cellular mechanisms of action are not well characterized. We tested the hypothesis that human physiological concentrations of DHEA alter insulin secretion by an action initiated at the plasma membrane of beta-cells. DHEA alone had no effect on intracellular calcium concentration ([Ca(2+)](i)) in a rat beta-cell line (INS-1). However, it caused an immediate and dose-dependent inhibition of carbachol-induced Ca(2+) release from intracellular stores, with a 25% inhibition at zero. One nanometer DHEA. DHEA also inhibited the Ca(2+) mobilizing effect of bombesin (29% decrease), but did not inhibit the influx of extracellular Ca(2+) evoked by glyburide (100 microM) or glucose (15 mM). The steroids (androstenedione, 17-alpha-hydroxypregnenolone, and DHEAS) had no inhibitory effect on carbachol-induced intracellular Ca(2+) release. The action of DHEA depended on a signal initiated at the plasma membrane, since membrane impermeant DHEA-BSA complexes also inhibited the carbachol effect on [Ca(2+)](i) (39% decrease). The inhibition of carbachol-induced Ca(2+) release by DHEA was blocked by pertussis toxin (PTX). DHEA also inhibited the carbachol induction of phosphoinositide generation, with a maximal inhibition at 0.1 nM DHEA. Furthermore, DHEA inhibited insulin secretion induced by carbachol in INS-1 cells by 25%, and in human pancreatic islets by 53%. Taken together, this is the first report showing that human physiological concentrations of DHEA decrease agonist-induced Ca(2+) release by a rapid, non-genomic mechanism in INS-1 cells. Furthermore, these data provide evidence consistent with the existence of a specific plasma membrane DHEA receptor, mediating this signal transduction pathway by pertussis toxin-sensitive G-proteins.

Cyclic changes in glycemia assessed by continuous glucose monitoring system during multiple complete menstrual cycles in women with type 1 diabetes.

Many women with diabetes notice changes in glucose control perimenstrually. To describe the pattern of changes in glucose control throughout the complete menstrual cycle, and the reproducibility of these changes, we performed a pilot study evaluating glycemic profiles continuously for three cycles in four women with type 1 diabetes. All participants had hemoglobin A1c <7.5% and regular menstrual periods off oral contraceptives. They used Medtronic MiniMed (Northridge, CA) Continuous Glucose Monitoring System (CGMS) devices continuously for three complete menstrual cycles, checked capillary glucose measurements six times daily, changed their own sensors every 3 days, and were seen seven times per menstrual cycle to download data and draw blood. Prolonged monitoring was safely carried out over three consecutive menstrual cycles. We observed two different patterns of glycemic control in relation to the menstrual cycle in these women. The first pattern, seen in two women, was characterized by increased frequency of hyperglycemia in the luteal phase. One of these women also had a hyperglycemic peak in the follicular phase. In the other two women, no characteristic cycle-related pattern was noted. The glucose profiles appeared reproducible between cycles in all women, but varied between women. Thus the menstrual cycle has a reproducible effect on glucose control in a subset of women with type 1 diabetes. Prolonged use of continuous glucose monitoring was safe in the subjects studied, and is the first method clinically available to monitor glucose control over prolonged periods in individuals with diabetes.

Evaluation of a novel photoactive and biotinylated dehydroepiandrosterone analog.

To characterize the cell surface receptor for dehydroepiandrosterone (DHEA), we synthesized a DHEA analog containing biotin and benzophenone groups (DHEA-BP-Bt). DHEA-BP-Bt was equipotent with DHEA in competing with [(3)H]DHEA for binding to solubilized plasma membranes of bovine aortic endothelial cells (BAEC). Additionally, DHEA-BP-Bt pre-conjugated to avidin and immobilized on agarose, also inhibited plasma membrane binding of [(3)H]DHEA. Furthermore, DHEA-BP-Bt activated endothelial nitric oxide synthase, similar to DHEA. Confocal micrographs showed that, upon photoirradiation, DHEA-BP-Bt bound to sites on the cell surface of BAEC in a DHEA inhibitable manner. Finally, DHEA-BP-Bt bound specifically to proteins of approximately 55 kDa and 80 kDa, either when live cells were UV irradiated with the analog and plasma membrane proteins separated by SDS-PAGE or in a ligand blot analysis. These data confirm the successful synthesis of a photoactive, biotinylated DHEA analog which is capable of cross-linking to and identifying plasma membrane DHEA binding sites and which will allow us to further purify this receptor.

Synthesis and application of a photoaffinity analog of dehydroepiandrosterone (DHEA).

We have synthesized an analog of dehydroepiandrosterone (DHEA, 1) containing both a benzophenone (BP) and a biotin (Bt) group (DHEA-BP-Bt, 8). Compound 8 was prepared by functionalization on C-17 of 1. Biocytin was reacted with 4-benzoylbenzoic acid and the product was condensed with 1 containing a diamine-hexane linker. We detected specific protein bands of approximately 55, 80, and 150 kDa by SDS-PAGE analysis of vascular endothelial cell plasma membranes which had been photoirradiated in the presence of 8.

Hydrogen peroxide signaling mediates DHEA-induced vascular endothelial cell proliferation.

Dehydroepiandrosterone (DHEA) activates a putative plasma membrane G(i)-protein coupled receptor to induce vascular endothelial proliferation. We now test the hypothesis that hydrogen peroxide (H(2)O(2)) signaling mediates this effect. Incubation of EA.hy926 cells, a human vascular endothelial cell line, with DHEA for 5 min produced a significant increase in H(2)O(2) production, measured by oxidation of either p-hydroxyphenylacetate or dichlorodihydrofluorescein. The DHEA effect on H(2)O(2) production was maximal at 1 nM DHEA, was evident within the first minute of incubation, and remained for 10 min. Similar results were present in primary bovine aortic endothelial cells. The induction of H(2)O(2) in EA.hy926 cells was mimicked by a membrane-impermeable albumin-conjugated DHEA and was inhibited by either catalase or pertussis toxin. Incubation of endothelial cells with DHEA for 5 min resulted in a 2-fold increase of cyclin D1 mRNA and protein expression at 4h. These effects were abolished by co-incubation with catalase. DHEA induced a 50 ± 7% increase in cell proliferation over 24h, measured as cellular Ki-67 immunoreactivity. This proliferative effect was abolished by either catalase or pertussis toxin co-incubation, indicating an H(2)O(2) and G(i)-protein-dependent effect. We conclude that H(2)O(2) is a key signaling molecule mediating the proliferative effects of DHEA in vascular endothelial cells, possibly by up-regulating cell-cycle associated genes, such as cyclin D1.