Pyridoxal 5′ phosphate protects islets against streptozotocin-induced beta-cell dysfunction – in vitro and in vivo

Abstract

Administration of pyridoxal 5' phosphate (PLP) has demonstrated beneficial effects in the management of diabetes, albeit the mechanism(s) are not clearly understood. The present study addressed the islet-cell function(s) in streptozotocin (STZ)-induced diabetic mice both in vitro and in vivo. Primary islet cells primed with or without PLP (5 mmol/L) were treated with STZ (2 mmol/L) and were measured for cell viability, insulin secretion, free radicals and mRNA of Insulin and Pdx1. The specificity of PLP's response on insulin secretion was assessed with amino oxy acetic acid (AOAA)-PLP inhibitor. In vivo, the STZ (200 mg/kg b.w)-treated diabetic mice received 10 mmol/L PLP intraperitoneally a day before (PLP + STZ) or after (STZ + PLP) with three more doses once every 48 h. On 7, 14 and 21 d of STZ treatment, physiological parameters, islet morphology, insulin:glucagon, insulin:HSP104, and mRNA of Insulin, Glut2, Pdx1 and Reg1 were determined. In vitro, PLP protected islets against STZ-induced changes in viability, insulin secretion, prevented increase in free radical levels and normalized mRNA of Insulin and Pdx1. Further, AOAA inhibited PLP-induced insulin secretion in islets. In vivo, PLP treatment normalized STZ-induced changes in physiological parameters, circulating levels of PLP and insulin. Also, islet morphology, insulin:glucagon, insulin:HSP104 and mRNA levels of Insulin, Pdx1 and Glut2 were restored by 21 d. Although PLP treatment (pre- and post-STZ) prevented development of frank diabetes, STZ + PLP mice showed transient hyperglycemia, and increased mRNA for Reg1. The data suggest the cytoprotective vis-à-vis insulinotrophic effects of PLP against STZ-induced beta-cell dysfunction and underline its prophylactic use in the management of diabetes.

Islet adaptation to obesity and insulin resistance in WNIN/GR-Ob rats

“…14,15 In similar lines, islets of mutant obese rats showed increased lipid peroxidation levels as well as intracellular ROS levels with concomitant increase in the expression of TNFa by immunofluorescence and gene expression. In agreement, islets of both young and adult diet-induced obese groups.…”

“…Inflammation and oxidative stress associated with obesity contributes significantly to tissue damage. 13,14 Elevated lipid peroxidation levels are indirect indicator of oxidative stress, 15 and levels of all inflammatory and oxidative stress markers studied were significantly higher, both in plasma and tissues at all ages in the mutants compared to their corresponding lean and parental control groups. Although age dependent increment were observed for pancreatic TBARS, plasma IL-1b and islets ROS in both lean and parental control groups, the data were not significantly different at any age studied.…”

“…Oxidative stress parameters: ROS levels Intracellular ROS levels were measured in all age groups of mutants, lean littermates and parental controls, by method described by Kiran et al 15 Briefly, 150 islets were incubated in the presence of 10 mM dichlorofluorescein diacetate at 37 C for 30 min and the resulting fluorescence was measured with excitation at 495 nm and emission at 538 nm. Values were reported as fluorescence intensity unit (FIU).…”

“…15 Briefly, islets were washed thrice in Krebs Ringer bicarbonate (KRBH) buffer (with 1 mg/ ml bovine serum albumin (BSA), 1 mM glucose and 10 mM HEPES). The islets were then subsequently incubated at 37 C with KRBH buffer containing 5.5 mM and then 16.5 mM glucose for 1 hr each.…”

Optimization of condition(s) towards establishment of primary islet cell cultures from WNIN/Ob mutant rat

“…The functional response measurements were carried out in the islets by insulin secretion assay as per our recently published method (Kiran et al 2011). Briefly, 250 islets in triplicates from each group were placed in 6 well plate (Corning), containing 1 mL of KrebsRinger bicarbonate HEPES (KRBH) buffer (pH 7Á4), 10 mmol/L HEPES, 1 mg/mL BSA with 5.5 mM glucose (basal level), 16.5 mM glucose (high glucose challenge) followed by 1 h incubation at 37°C.…”