Transmission electron microscopy reveals autophagy within <i>Lepr^flox/flox RIP-Cre</i> β-cells.

<p>Pancreas sections from <i>Lepr^flox/flox</i> (A) and <i>Lepr^flox/flox RIP-Cre</i> (B and D) mice were analyzed by transmission electron microscopy (magnification 9300X and 11000X) and quantified (C). Multigranular bodies were numerous in <i>Lepr^flox/flox RIP-Cre</i> β-cells compared to <i>Lepr^flox/flox</i> β-cells (white squares). Events of macroautophagy (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071075#pone-0071075-g004" target="_blank">Figure 4D</a>, upper inset) and microautophagy (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071075#pone-0071075-g004" target="_blank">Figure 4D</a>, bottom inset) were captured in <i>Lepr^flox/flox RIP-Cre</i> β-cells. Scale bar = 2 µm (A and B) and 0.5 µm (D). Micrographs are representative of 3 pancreata analyzed per group. Data are expressed as mean ± SEM. Statistical analysis was performed using Student t test, ** p<0.01.</p

Decreased amplitude in intracellular Ca²⁺ responses to tolbutamide in <i>Lepr^flox/flox RIP-Cre</i> adult islets.

<p>A: Representative recordings from a control <i>Lepr^flox/flox</i> islet (solid line) and a <i>Lepr^flox/flox RIP-Cre</i> islet (dotted line) in response to tolbutamide. B: Graph plotting ΔFmax-Fmin of each peak in response to tolbutamide in the population of islets that showed two peaks. Data are expressed as mean ± SEM. Statistical analysis was performed using Student t test, *** p<0.0001. Responses are representative of 22 islets from 4 mice per group.</p

Insulin granule patterns during postnatal development.

<p>The number per beta cell area of: A) total insulin granules (filled, immature and empty); B) immature insulin secretory granules (containing pale-staining proinsulin); and C) filled insulin granules (containing dense-core mature insulin). D) Representative electron microscopy photographs of saline and nicotine-exposed beta cells at 26 weeks of age illustrate both the typical insulin granule patterns (immature insulin granules are indicated by solid black arrows and mature insulin granules by striped arrows), and mitochondrial structures (indicated by white boxes); N = nucleus. All data are presented as mean±SEM. Values with an asterisk are significantly (p<0.05) different from saline controls.</p

<i>Lepr^+/+ RIP-Cre</i> mice present impaired β-cell Ca²⁺ signaling in response to glucose and tolbutamide.

<p>A: Representative [Ca²⁺]_i recordings of a <i>Lepr^+/+</i> islet (left panel) and <i>Lepr^+/+ RIP-Cre</i> islet (right panel) in response to increasing glucose (G) concentrations and potassium chloride (KCl) from 5–6 week old mice. B: Representative [Ca²⁺]_i recordings of a <i>Lepr^+/+</i> islet (solid line) and <i>Lepr^+/+ RIP-Cre</i> islet (dotted line) in response to tolbutamide. C: Graph plotting the AUC of the Ca²⁺ transients during the stimulation with tolbutamide. Data are expressed as mean ± SEM. Statistical analysis was performed using Student t test. *** p<0.0001. Graphs are representative of 18–20 islets from 3 mice per group (in response to glucose) and 16–27 islets from 2–3 mice per group (in response to tolbutamide).</p

Mitochondrial morphology during postnatal development.

<p>A) The number of mitochondria per beta cell area; B) individual mitochondrion area; and C) percentage of mitochondria with blebbing and/or merging with a neighboring mitochondria from offspring at 3, 15 and 26 weeks of age following exposure to either saline or nicotine during fetal and neonatal development. Representative electron microscopy photographs are provided to illustrate: D) typical mitochondrial structure (indicated by striped arrows) in the beta cells of saline and nicotine-exposed offspring during postnatal development, and E) examples of mitochondrial blebbing and merging (indicated by solid black arrows); N = nucleus. All data are presented as the mean±SEM. Values with an asterisk are significantly different from the saline control (p<0.05).</p

Oxidative stress at 26 weeks of age.

<p>Reactive oxygen species (ROS) production and the incidence of protein carbonyl groups (an indication of oxidative damage to protein) in islets isolated from the pancreas of saline- (white bar) and nicotine-exposed (black bar) offspring at 26 weeks of age. All data are expressed as a percentage of the average saline control value and are presented as the mean±SEM. Values with an asterisk are significantly (p<0.05) different from saline controls.</p

Similar glucose metabolic rates in islets from <i>Lepr^flox/flox</i> and <i>Lepr^flox/flox RIP-Cre</i> adult mice.

<p>A: Two [NAD(P)H]_i representative recordings of a <i>Lepr^flox/flox</i> islet (solid line) and a <i>Lepr^flox/flox RIP-Cre</i> islet (dotted line) in response to glucose (G) and sodium azide (NaN₃). B: Graph plotting percentage of AUC/min in response to different glucose concentrations and normalized to the maximum reduction level obtained with 3 mM NaN₃. Data are expressed as mean ± SEM. Statistical analysis was performed using Student t test. Graphs are representative of 32–34 islets from 3 mice per group.</p

Stages of mitochondrial health during postnatal development.

<p>The percentage of mitochondria within beta cells at: A) stage 1, B) stage 2, C) stage 3, D) stage 4, and E) stage 5. F) Electron microscopy images with examples of mitochondria at each of the five morphological stages. δ indicates that statistical analysis was not performed on these data due to lack of variability in the saline treatment group replicates. All data are presented as mean±SEM.</p

Oral glucose tolerance tests during postnatal development.

<p>Area under the curve (AUC) for the total glucose response to an oral glucose challenge at 4, 15 and 26 weeks of age in saline (closed circles) and nicotine-exposed (open circles) animals. All data are presented as mean±SEM. Values with an asterisk are significantly (p<0.05) different from saline controls.</p

Mitochondrial enzyme activity during postnatal development.

<p>A) Complex IV enzyme activity (an indicator of mitochondrial electron transport chain function) and B) citrate synthase enzyme activity (an indicator of mitochondrial mass) in the pancreas of saline- and nicotine-exposed offspring at 3, 15 and 26 weeks of age. All data are presented as mean±SEM. Values with an asterisk are significantly (p<0.05) different from saline controls.</p