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, 111 (8), 1147-60

Increased Islet Apoptosis in Pdx1+/- Mice

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Increased Islet Apoptosis in Pdx1+/- Mice

James D Johnson et al. J Clin Invest.

Abstract

Mice with 50% Pdx1, a homeobox gene critical for pancreatic development, had worsening glucose tolerance with age and reduced insulin release in response to glucose, KCl, and arginine from the perfused pancreas. Surprisingly, insulin secretion in perifusion or static incubation experiments in response to glucose and other secretagogues was similar in islets isolated from Pdx1(+/-) mice compared with Pdx1(+/+) littermate controls. Glucose sensing and islet Ca(2+) responses were also normal. Depolarization-evoked exocytosis and Ca(2+) currents in single Pdx1(+/-) cells were not different from controls, arguing against a ubiquitous beta cell stimulus-secretion coupling defect. However, isolated Pdx1(+/-) islets and dispersed beta cells were significantly more susceptible to apoptosis at basal glucose concentrations than Pdx1(+/+) islets. Bcl(XL) and Bcl-2 expression were reduced in Pdx1(+/-) islets. In vivo, increased apoptosis was associated with abnormal islet architecture, positive TUNEL, active caspase-3, and lymphocyte infiltration. Although similar in young mice, both beta cell mass and islet number failed to increase with age and were approximately 50% less than controls by one year. These results suggest that an increase in apoptosis, with abnormal regulation of islet number and beta cell mass, represents a key mechanism whereby partial PDX1 deficiency leads to an organ-level defect in insulin secretion and diabetes.

Figures

Figure 1
Figure 1
Glucose tolerance and secretagogue-induced pancreatic insulin release in Pdx1+/– mice. (a) IPGTTs are shown for males (n = 9 Pdx1+/+; n = 8 Pdx1+/–) and females (n = 3 Pdx1+/+; n = 3 Pdx1+/–). (b) Insulin release from perfused pancreata of male Pdx1+/+ (n = 5) and Pdx1+/– (n = 5) mice in response to stepwise increases to 20 mM glucose (black bar) and 20 mM KCl (white bar). (c) Insulin release from perfused pancreata of Pdx1+/+ (n = 3) and Pdx1+/– (n = 3) mice in response to a linear elevation from 2 mM to 26 mM glucose. Insulin secretion in response to 20 mM arginine (arg) was assayed in the continued presence of 26 mM glucose (G) (note the different scale). Data are from mice 8–10 weeks of age.
Figure 2
Figure 2
Evoked insulin release from populations of size-matched isolated islets is not altered in Pdx1+/– mice. (a) Perifused islets were exposed to 20 mM glucose (black bar) and 20 mM KCl (white bar) in the same experiments. Islets from three mice of each genotype were compared. Thirty to fifty islets/column were used. Insulin secretion is normalized to pretreatment values to allow pooling of columns with different basal insulin release. Basal insulin release was approximately 1.5 μU/ml/h. (b) The response of Pdx1+/+ and Pdx1+/– islets to a ramp increase to 26 mM glucose is shown (n = 4). In the presence of 26 mM glucose, islets were challenged with 20 mM arginine (right). Note the different scale. (c) Groups of five physically similar islets from Pdx1+/– mice or littermate controls were incubated for 1 hour in 2 mM glucose (G) (n = 33), 5 mM glucose (n = 12), 20 mM glucose (n = 33), or 2mM glucose plus either 20 mM KCl (n = 23), 5 μM forskolin (n = 12), 250 μM carbachol (n = 6), or 30 μM mastoparan (n = 15). Unless otherwise indicated, 2 mM glucose was used. forsk, forskolin; Cch, carbachol; masto, mastoparan.
Figure 3
Figure 3
Estimation of intracellular Ca2+ signals in islets and single β cells. (a) No differences were seen in Ca2+ signals from whole islets loaded with Fura-2 in response to 20 mM glucose (n = 20), 10 mM glyceraldehyde (Glycer; n = 12), 10 mM α-ketoisocaproic acid (KIC; n = 12), 20 mM KCl (n = 50), or carbachol (Cch; in the presence or absence of 2 mM EGTA; n = 8 for each). Average area under the curve (arbitrary units [AU]) was calculated from raw traces consisting of unitless ratio values (340/380). (b) Average NADH autofluorescence (AU) was measured from groups of individual islets (n = 8). Calibrated Ca2+ traces are shown for single, large Fura-4F–loaded Pdx1+/+ cells (c and d) or Pdx1+/– cells (e and f) exposed to 15 mM glucose (black bars), 15 mM arginine (gray bars), or 30 mM KCl (white bars). Representative traces from 66 Pdx1+/+ cells and 35 Pdx1+/– cells are shown. Similar results were seen with 20 mM glucose (not shown). See Table 1 for a full quantification of single-cell Ca2+ signals. arg, arginine.
Figure 4
Figure 4
Single-cell exocytosis and voltage-gated Ca2+ currents are not altered in β cells from PDX1 transgenic mice. (a) A representative I/V curve made from consecutive (same-day) recordings of two Pdx1+/– and two Pdx1+/+ cells is shown. Analysis of voltage-gated Ca channels was performed on cells dispersed from six Pdx1+/+ and six Pdx1+/– mice. VC, voltage. (b) Stimulated exocytosis is intact in Pdx1+/– β cells. Results from both standard and TEA-treated conditions were pooled, and these results are summarized (n = 33 for Pdx1+/+; n = 30 for Pdx1+/–). Cm, membrane capacitance. Examples of changes in capacitance evoked by a train of 20 depolarizations (200 ms) from a holding potential of –70 mV to +10 mV in patch-clamped cells from Pdx1+/+ (c, top panel) and Pdx1+/– (d, top panel) islets. Recordings were made using a Cs+ pipette, with 30 mM TEA in the bath to simultaneously measure voltage-gated Ca2+ currents. Integrated Ca2+ charge entry is shown for each depolarization (bottom panels). Ca2+ current traces are shown for the first and 20th depolarizations (insets). Q(pC); charge entry; fF, femto Ferad.
Figure 5
Figure 5
Pdx1+/– islets and cells are prone to apoptosis when cultured in low glucose concentrations. (a) Apoptosis, measured by PCR-enhanced DNA laddering, was compared in groups of five islets cultured in RPMI with 5 mM, 10 mM, and 25 mM glucose for 72 hours (n = 10 for each genotype). Islets cultured in 10 μM thapsigargin (Tg), a known inducer of islet apoptosis, were used as a positive control. Apoptotic calf-thymus DNA served as an additional reference control, independent of our cultures. DNA-ladder bands were quantified by densitometry and pooled as described in Methods. (b) A representative gel is shown. (c) The average percentage of apoptotic cells measured in dispersed islet cells cultured overnight in 5 mM glucose, measured by cell uptake of a specific dye (Methods). Cells were counted manually in phenol red–free RPMI. Shown are pooled results from three coverslips of islets dispersed from three mice of each genotype. Asterisks denote significant differences.
Figure 6
Figure 6
RT-PCR analysis of gene expression. (a) The densitometric quantification of four apoptosis-related genes — BclXL, caspase-3 (Casp), BAX, and Bcl-2 — relative to GAPDH in cultured islets (cultured as in Figure 5) is quantified by densitometry (above). Data are pooled from four gels (representative example shown below). White bars over gel images denote Pdx1+/+ islets, while black bars denote Pdx1+/– islets. Each RT-PCR sample was pooled from the cultured islets of three mice. (bd) Relative abundance of PDX1, insulin-1 (Ins-1), insulin-2 (Ins-2), and glucagon (Gluca) mRNA are quantified from the same samples. Single asterisks denote significant differences between Pdx1+/– and Pdx1+/+ islets. Double asterisks denote significant differences between different treatments to the same type of islet. Insulin content per islet protein was also not reduced in Pdx1+/– islets (not shown).
Figure 7
Figure 7
β cell and α cell architecture in Pdx1+/– islets. Insulin (a, c, e, and g) and glucagon (b, d, f, and h) are stained red in adjacent 5-μm-thick sections. Pdx1+/– islets from 3-month-old mice show striking disruption of islet architecture compared with littermate controls. Note the presence of cells stained for insulin and glucagon in e and f (arrowheads). Localization of α cells to the periphery was reduced in 1-year-old mice from both groups.
Figure 8
Figure 8
Immunohistochemistry for proapoptotic active caspase-3 and TUNEL. (ad) Pdx1+/– islets showed modest activation of caspase-3 in vivo (red staining) compared with Pdx1+/+ islets. The difference was most prominent in older animals and varied between islets from the same pancreas section. (eh) TUNEL-positive cells (red) are seen in Pdx1+/– islets at all ages studied but are extremely rare in Pdx1+/+ islets. Note the loss of intact islet structure and the striking increase in vascularization in Pdx1+/– islets (arrow in h points to one of many red blood cells within the islet). Islet structure and TUNEL staining was also different in Pdx1+/– islets at 5 months of age (not shown).
Figure 9
Figure 9
Evidence of enhanced pancreatic immune activity in Pdx1+/– mice. Infiltration of lymphocytes, some positive for TUNEL (a), were seen within or around islets (arrow) in old mice. Immunofluorescent localization of Ki67 nuclear antigen (b) reveals proliferation of lymphocytes within Pdx1+/– islets (arrows in b and c point to the same islet, also delineated by higher-density nuclear DAPI staining (c). Infiltrating lymphocytes are distinguished from islet cells by their small size and low-intensity nuclear staining with DAPI.
Figure 10
Figure 10
Reduced β cell mass and islet number in Pdx1+/– mice. (a) Islet cell area at 3, 5, and 12 months was estimated using insulin immunoreactivity as described in Methods and normalized to the total pancreatic cross-sectional area. Four sections were analyzed from each animal. (b) The number of islets was counted in complete pancreatic sections at low magnification (1.25× objective). Asterisks denote significant difference from wild type. Number of mice studied at 3 months, 5 months, and 12 months, respectively: Pdx1+/+, n = 4, 2, and 5; Pdx1+/–, n = 4, 2, and 4.

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