Horacio L. Rilo

A high throughput live transparent animal bioassay to identify non-toxic small molecules or genes that regulate vertebrate fat metabolism for obesity drug development

BackgroundThe alarming rise in the obesity epidemic and growing concern for the pathologic consequences of the metabolic syndrome warrant great need for development of obesity-related pharmacotherapeutics. The search for such therapeutics is severely limited by the slow throughput of animal models of obesity. Amenable to placement into a 96 well plate, zebrafish larvae have emerged as one of the highest throughput vertebrate model organisms for performing small molecule screens. A method for visually identifying non-toxic molecular effectors of fat metabolism using a live transparent vertebrate was developed. Given that increased levels of nicotinamide adenine dinucleotide (NAD) via deletion of CD38 have been shown to prevent high fat diet induced obesity in mice in a SIRT-1 dependent fashion we explored the possibility of directly applying NAD to zebrafish.MethodsZebrafish larvae were incubated with daily refreshing of nile red containing media starting from a developmental stage of equivalent fat content among siblings (3 days post-fertilization, dpf) and continuing with daily refreshing until 7 dpf.ResultsPPAR activators, beta-adrenergic agonists, SIRT-1 activators, and nicotinic acid treatment all caused predicted changes in fat, cholesterol, and gene expression consistent with a high degree of evolutionary conservation of fat metabolism signal transduction extending from man to zebrafish larvae. All changes in fat content were visually quantifiable in a relative fashion using live zebrafish larvae nile red fluorescence microscopy. Resveratrol treatment caused the greatest and most consistent loss of fat content. The resveratrol tetramer Vaticanol B caused loss of fat equivalent in potency to resveratrol alone. Significantly, the direct administration of NAD decreased fat content in zebrafish. Results from knockdown of a zebrafish G-PCR ortholog previously determined to decrease fat content in C. elegans support that future GPR142 antagonists may be effective non-toxic anti-obesity therapeutics.ConclusionOwing to the apparently high level of evolutionary conservation of signal transduction pathways regulating lipid metabolism, the zebrafish can be useful for identifying non-toxic small molecules or pharmacological target gene products for developing molecular therapeutics for treating clinical obesity. Our results support the promising potential in applying NAD or resveratrol where the underlying target protein likely involves Sirtuin family member proteins. Furthermore data supports future studies focused on determining whether there is a high concentration window for resveratrol that is effective and non-toxic in high fat obesity murine models.

Long-term outcomes after total pancreatectomy and islet cell autotransplantation: is it a durable operation?

Objective:Total pancreatectomy and islet cell autotransplantation (TPIAT) has been increasingly utilized for the management of chronic pancreatitis (CP) with early success. However, the long-term durability of this operation remains unclear. Methods:All patients undergoing TPIAT for the treatment of CP with 5-year or greater follow-up were identified for inclusion in this single-center observational study. End points included narcotic requirements, glycemic control, islet function, quality of life (QOL), and survival. Results:Between 2000 and 2013, 166 patients underwent TPIAT; 112 of these patients had 5-year follow-up data to analyze. All patients underwent successful IAT with a mean of 6027 ± 595 islet equivalents per body weight. There was no perioperative mortality and actuarial survival at 5 years was 94.6%. The narcotic independence rate at 1 year was 55% and continued to improve to 73% at 5-year follow-up (P < 0.05). The insulin independence rate declined over time (38% at 1 year vs 27% at more than 5 years), but insulin requirements remained similar (21.4 vs 24.3 units per day, P = 0.6). All patients achieved stable glycemic control with a median hemoglobin A1C (HgA1C) of 6.9% (range: 5.85%–8.3%). The short form 36-item QOL assessment of a subset of patients available for contact demonstrated continued improvements in all tested modules in patients with at least 5-year follow-up. Two patients developed diabetic complications requiring whole organ pancreas transplant for salvage. Conclusions:This represents one of the largest series examining long-term outcomes after TPIAT. This operation produces durable pain relief and improvement in QOL parameters. Insulin independence rates decline over time, but most patients maintain stable glycemic control.

Impaired Proinsulin Processing is a Characteristic of Transplanted Islets

We sought to determine whether recipients of islet transplants have defective proinsulin processing. Individuals who had islet allo‐ or autotransplantation were compared to healthy nondiabetic subjects. Insulin (I), total proinsulin (TP), intact proinsulin and C‐peptide (CP) were measured in samples of fasting serum by immunoassay, and the ratios of TP/TP+I and TP/CP were calculated. Islet allotransplant recipients had elevated TP levels relative to nondiabetic controls (16.8 [5.5–28.8] vs. 8.4 [4.0–21.8] pmol/L; p < 0.05) and autologous transplant recipients (7.3 [0.3–82.3] pmol/L; p < 0.05). Islet autotransplant recipients had significantly higher TP/TP+I ratios relative to nondiabetic controls (35.9 ± 6.4 vs. 13.9 ± 1.4%; p < 0.001). Islet allotransplant recipients, some of whom were on insulin, tended to have higher TP/TP+I ratios. The TP/CP ratio was significantly higher in both islet autotransplant (8.9 [0.6–105.2]; p < 0.05) and allotransplant recipients (2.4 [0.8–8.8]; p < 0.001) relative to nondiabetic controls (1.4 [0.5–2.6]%). Consistent with these findings, TP/TP+I and TP/CP values in islet autotransplant recipients increased significantly by 1‐year posttransplant compared to preoperative levels (TP/CP: 3.8 ± 0.6 vs. 23.3 ± 7.9%; p < 0.05). Both allo‐ and autotransplant subjects who received <10 000 IE/kg had higher TP/CP ratios than those who received >10 000 IE/kg. Islet transplant recipients exhibit defects in the processing of proinsulin similar to that observed in subjects with type 2 diabetes manifest as higher levels of total proinsulin and increased TP/TP+I and TP/CP ratios.

Development and characterization of simulant pancreatic islets.

Insulin is stored in pancreatic islets as a zinc-insulin complex, and stimulating the islets results in the release of insulin and zinc. Simulant pancreatic islet beads have been developed using agarose beads (50-250 micro m diameter) derivatized with iminodiacetic acid that have been loaded with zinc. A qualitative comparison of the simulant beads with pancreatic islets has been made by staining with dithizone and a zinc-binding fluorescent dye, TSQ. The binding capacity of simulant beads was determined to be 34 micro mol Zn(2+)/g of dried beads using anodic stripping voltammetry. Hydrochloric acid was used to release zinc from beads to mimic the secretion of insulin from pancreatic islets and a release profile was established. The simulant beads can be used to optimize the islet isolation process and reduce the use of real islets in method development.

Remission of Digestive Insufficiency by Islet Transplantation to the Pancreas

Pancreatic digestive insufficiency is a common problem in both Type 1 and Type 2 diabetes and remains a serious consequence of diabetes in developing countries. The problem is not corrected by supportive therapies including exogenous insulin injections. It is our hypothesis that digestive insufficiency may be corrected or diminished by the transplantation of islets to the pancreas, thereby supplying islet hormones directly to acinar tissue analogous to the normal pancreas. Diabetic rats received 1000 syngeneic islets and dogs received 7600 autologous islets per kilogram as a transplant to the pancreas. Blood glucose and amylase concentrations were normalized in islet recipients in contrast with controls receiving no islets or islets transplanted to the renal capsule. These results suggest that diabetic digestive insufficiency may be corrected by intrapancreatic islet transplantation.

A Microfluidic Biosystem for Metabolic Monitoring of Human Islet Cells with Integrated Biosensors

This paper presents a microfluidic biosystem for metabolic monitoring of human islet cells with an integrated biosensor. The polymeric microfluidic system has been designed and developed for real time handling and monitoring of human islet cell metabolism with simultaneous glucose measurements using the integrated glucose sensor. The microfluidic system, integrated with a glucose sensor for simultaneous glucose measurements, entraps and sustains pancreatic islet cells in a micro reaction chamber for monitoring cell metabolism. A single cluster (group of cells) of Islets of Langherans was exposed to different concentrations of glucose for 8 hours and the secreted insulin were transported and collected for further investigation on diabetes.