Down-Regulation of ZnT8 Expression in INS-1 Rat Pancreatic Beta Cells Reduces Insulin Content and Glucose-Inducible Insulin Secretion

The SLC30A8 gene codes for a pancreatic beta-cell-expressed zinc transporter, ZnT8. A polymorphism in the SLC30A8 gene is associated with susceptibility to type 2 diabetes, although the molecular mechanism through which this phenotype is manifest is incompletely understood. Such polymorphisms may exert their effect via impacting expression level of the gene product. We used an shRNA-mediated approach to reproducibly downregulate ZnT8 mRNA expression by >90% in the INS-1 pancreatic beta cell line. The ZnT8-downregulated cells exhibited diminished uptake of exogenous zinc, as determined using the zinc-sensitive reporter dye, zinquin. ZnT8-downregulated cells showed reduced insulin content and decreased insulin secretion (expressed as percent of total insulin content) in response to hyperglycemic stimulus, as determined by insulin immunoassay. ZnT8-depleted cells also showed fewer dense-core vesicles via electron microscopy. These data indicate that reduced ZnT8 expression in cultured pancreatic beta cells gives rise to a reduced insulin response to hyperglycemia. In addition, although we provide no direct evidence, these data suggest that an SLC30A8 expression-level polymorphism could affect insulin secretion and the glycemic response in vivo

Effects of Subthalamic Nucleus Lesions and Stimulation upon Corticostriatal Afferents in the 6-Hydroxydopamine-Lesioned Rat

Abnormalities of striatal glutamate neurotransmission may play a role in the pathophysiology of Parkinson's disease and may respond to neurosurgical interventions, specifically stimulation or lesioning of the subthalamic nucleus (STN). The major glutamatergic afferent pathways to the striatum are from the cortex and thalamus, and are thus likely to be sources of striatal neuronally-released glutamate. Corticostriatal terminals can be distinguished within the striatum at the electron microscopic level as their synaptic vesicles contain the vesicular glutamate transporter, VGLUT1. The majority of terminals which are immunolabeled for glutamate but are not VGLUT1 positive are likely to be thalamostriatal afferents. We compared the effects of short term, high frequency, STN stimulation and lesioning in 6-hydroxydopamine (6OHDA)-lesioned rats upon striatal terminals immunolabeled for both presynaptic glutamate and VGLUT1. 6OHDA lesions resulted in a small but significant increase in the proportions of VGLUT1-labeled terminals making synapses on dendritic shafts rather than spines. STN stimulation for one hour, but not STN lesions, increased the proportion of synapses upon spines. The density of presynaptic glutamate immuno-gold labeling was unchanged in both VGLUT1-labeled and -unlabeled terminals in 6OHDA-lesioned rats compared to controls. Rats with 6OHDA lesions+STN stimulation showed a decrease in nerve terminal glutamate immuno-gold labeling in both VGLUT1-labeled and -unlabeled terminals. STN lesions resulted in a significant decrease in the density of presynaptic immuno-gold-labeled glutamate only in VGLUT1-labeled terminals. STN interventions may achieve at least part of their therapeutic effect in PD by normalizing the location of corticostriatal glutamatergic terminals and by altering striatal glutamatergic neurotransmission

Density of immuno-gold-labeled glutamate particles in post-synaptic spines in which presynaptic terminals were VGLUT1-labeled or –unlabeled.

<p>Mean ± standard deviation of number of particles/µm².</p>*<p>significantly different from 6OHDA alone p = 0.05.</p

Immuno-gold labeled glutamate particles in VGLUT1-labeled and -unlabeled terminals.

<p>(A) <b>Control</b>. Electron photomicrographs of VGLUT1-labeled (L-NT) and -unlabeled (UN-NT) nerve terminals making asymmetric synaptic contacts (arrow) onto dendritic spines (SP). The 10 nm gold particles within the nerve terminals (see arrowhead in the L-NT) indicate the location of an antibody against glutamate. The inserts show labeled details of a terminal containing VGLUT1-immunoreactive synaptic vesicles (above) and a terminal containing unlabeled synaptic vesicles (below). (B) <b>6-OHDA lesion</b>. L-NT and UN-NT both make asymmetric synaptic contacts (arrows) with SPs. The density of immuno-gold labeling within these terminals is similar to that of the control group (A). (C) <b>6-OHDA/STN lesion</b>: L-NT and UN-NT make asymmetric synaptic contacts (arrows) with SPs. The density of nerve terminal immuno-gold labeling is significantly reduced compared to the control (A) and 6OHDA (B) tissue. (D) <b>6-OHDA/STN stimulation</b>. Both L-NT and UN-NT make asymmetrical synaptic contacts (arrows) with SPs. The density of nerve terminal glutamate immuno-gold labeling is similar to that seen in the 6-OHDA/STN lesion group (C). (E) <b>6-OHDA/Sham-Stim</b>. The L-NT makes an asymmetric synaptic contact with the SP, and the UN-NT makes an asymmetric synaptic contact with a dendritic shaft (DEND). The density of nerve terminal glutamate immuno-gold labeling is greater than in the 6-OHDA/STN lesion (C) and 6-OHDA/STN stimulation (D) tissue. Calibration bar: 0.25 microns.</p

Summary of findings.

<p>Compared with control (A), in 6OHDA-lesioned rats, there were relatively more VGLUT-1 labeled terminals making synapses upon the shafts of dendrites (red circle; B). We hypothesise that this is due to fewer spines being available. There was no change in density of glutamate labeling. With STN lesions, there was decreased glutamate labeling in VGLUT1-labeled terminals, indicating increased release, indicated here by increased line thickness (C). With STN stimulation, there was decreased glutamate labeling, suggesting increased release in both types of terminals, and an increase in the proportion of VGLUT1-labeled terminals upon spines (red circle; D). We did not count spines, but postulate that there were more spines available for synapses due to plasticity. The relative numbers of VGLUT1-labeled and –unlabeled terminals and their locations are not accurate.</p

Effect of ZnT8-directed and irrelevant shRNA constructs upon ZnT8 expression in INS-1 cells.

<p>Effect of ZnT8-directed and irrelevant shRNA constructs upon ZnT8 expression in INS-1 cells.</p

Nigral and subthalamic lesions.

<p>(A) Depletion of TH-immunoreactive neurons in left substantia nigra pars compacta, as compared with the unlesioned right side (arrows). (B) Electrolytic ablation of left subthalamic nucleus (white arrow; outlined by black dotted line); results were only analyzed from animals in which at least 50% of the subthalamic nucleus was lesioned; the intact contralateral subthalamic nucleus is indicated on the right side (small black arrow; dashed outline). CP = cerebral peduncle, SNc = substantia nigra, pars compacta, VTA = ventral tegmental area.</p

Comparison of localization of VGLUT1-labeled and -unlabeled terminals between groups.

<p>Percentages of VGLUT1-labeled terminals synapsing upon dendritic spines (A) and shafts (B) in the 5 experimental groups; control (n = 8), 6OHDA-lesioned (n = 6), 6OHDA+STN lesion (n = 5), 6OHDA+STN stimulation (n = 4), and 6OHDA+ sham stimulation (electrode placement alone; n = 3). In 6OHDA-lesioned animals, there was a shift from synapses on spines to upon shafts (p<0.02), which was reversed by STN stimulation (p<0.05). Percentages of non-VGLUT1-labeled terminals synapsing upon dendritic spines (C) and shafts (D). There were no significant differences between any of the experimental groups for unlabeled terminals.</p

Location of VGLUT1-labeled and -unlabeled terminals for each experimental group.

<p>(A) control (n = 8), (B) 6OHDA-lesioned (n = 6), (C) 6OHDA+STN lesion (n = 5), (D) 6OHDA+STN stimulation (n = 4), (E) 6OHDA+ sham stimulation (electrode placement alone; n = 3).</p

Effect of ZnT8-specific shRNA upon ZnT8 mRNA level in cultured INS-1 cells.

<p>A. Relative ZnT8 mRNA abundance as determined via real-time PCR in INS-1 cells were stably transfected with shRNA directed against rat ZnT8 (ZnT8-shRNA) or with shRNA vector alone (VEC-shRNA). Of note, the ZnT8-directed shRNA was comprised of two separate inserts (ZnT8-3 and ZnT8-4; see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005679#pone-0005679-t001" target="_blank">Table 1</a>) cloned independently into expression vector and applied in combination. A. Stable cell lines were generated with these vectors on three separate occasions and the results from these three separate experiments are shown here (n = 3, with one determination per experiment). * denotes <i>p</i><0.0001 relative to VEC-shRNA. B. Specificity of ZnT8 downregulation. RNA prepared from cells treated as in A were assessed via real-time PCR (n = 3) for the presence of ZnT8, for the presence of additional beta cell-expressed ZnT family members (ZnT4 and ZnT5), and for the presence of the alpha-1C and alpha-1D subunits of the beta cell-expressed L-type voltage-gated calcium channel (i.e., CACNA1C and CACNA1D gene products). For each mRNA, expression data were normalized to VEC-shRNA cells.</p