Cholecystokinin is up-regulated in obese mouse islets and expands beta-cell mass by increasing beta-cell survival

Abstract

An absolute or functional deficit in beta-cell mass is a key factor in the pathogenesis of diabetes. We model obesity-driven beta-cell mass expansion by studying the diabetes-resistant C57BL/6-Leptin(ob/ob) mouse. We previously reported that cholecystokinin (Cck) was the most up-regulated gene in obese pancreatic islets. We now show that islet cholecystokinin (CCK) is up-regulated 500-fold by obesity and expressed in both alpha- and beta-cells. We bred a null Cck allele into the C57BL/6-Leptin(ob/ob) background and investigated beta-cell mass and metabolic parameters of Cck-deficient obese mice. Loss of CCK resulted in decreased islet size and reduced beta-cell mass through increased beta-cell death. CCK deficiency and decreased beta-cell mass exacerbated fasting hyperglycemia and reduced hyperinsulinemia. We further investigated whether CCK can directly affect beta-cell death in cell culture and isolated islets. CCK was able to directly reduce cytokine- and endoplasmic reticulum stress-induced cell death. In summary, CCK is up-regulated by islet cells during obesity and functions as a paracrine or autocrine factor to increase beta-cell survival and expand beta-cell mass to compensate for obesity-induced insulin resistance.

Figure 1

CCK is up-regulated in pancreatic islets of ob/ob mice. A, Cck mRNA abundance in 18-d-, 4-wk-, and 14-wk-old islets (n = 3–5). Comparisons were made by ANOVA followed by Bonferroni-corrected Student’s unpaired t tests. B and C, CCK protein was measured by RIA analysis; B, total CCK levels were measured in 14-wk islets from lean and ob/ob islets (n = 4); C, islets from ob/ob mice were fractionated by HPLC, and RIA was performed on each fraction to determine CCK species. Antibodies for amidated and sulfated CCK are shown. Sulfated and amidated standards were used and labeled to help identify the different species. Nonamidated and nonsulfated antibodies were used, and no immunoreactivity was detected. D, Cckar and Cckbr mRNA abundance in 14-wk-old islets (n = 5 for each). E, Cck mRNA abundance in brain and intestinal tissue from 14-wk-old mice (n = 4–5 for each). F and G, Plasma insulin (F) and Cck mRNA (G) abundance in 16-wk-old agouti mice (n = 3). For all quantitative RT-PCR, TaqMan cycle threshold (Ct) values were normalized to β-actin levels to generate ΔCt values. Plasma insulin comparisons were made using log₁₀-transformed values. All comparisons were made by Student’s unpaired t test unless otherwise stated.

Figure 2

CCK is up-regulated and expressed in α- and β-cells of ob/ob pancreatic islets. Immunofluorescence images of lean (A–C) and ob/ob (D–L) islets from Cck-eGFP transgenic mice. Insulin is stained red, nuclei are blue using 4′,6-diamidino-2-phenylindole (DAPI) stain, and GFP is green by autofluorescence. Each picture of a lean islet (A–C) is representative of an individual mouse (n = 3). Each ob/ob islet is separated into its insulin and DAPI (D, G, and J), GFP and DAPI (E, H, and K), and merged layers (F, I, and L) and is representative of an individual mouse (n = 3). Examples of β-cells costaining with GFP are indicated by yellow arrows. Examples of β-cells not costaining for GFP are indicated by white arrows. Examples of non-β-cells staining for GFP are indicated by white arrowheads.

Figure 3

CCK deficiency results in reduced islet size and fractional β-cell area. Islet size and β-cell fractional area analysis from 10-wk-old (A–C) male Cck^WT-ob/ob (n = 4) and Cck^lacZ-ob/ob (n = 4) mice and 14-wk-old (D–F) male Cck^WT-ob/ob (n = 5) and Cck^lacZ-ob/ob mice (n = 4). A and D, Mean islet size of all islets analyzed (n > 225 for each genotype). Comparisons were made by Student’s unpaired t test. Histogram shape analysis was also performed by Kolmogorov-Smirnov test and found to be marginally different (P < 0.07). B and E, Total islet area contribution per mouse based upon islet size. Comparisons were made by ANOVA followed by Bonferroni-corrected Student’s unpaired t tests. C and F, Fractional β-cell (C) or islet (F) area as a percentage of total pancreatic sectional area. No difference in β-cell size or pancreatic wet weight was detected. Comparisons were made by Student’s unpaired t tests.

Figure 4

Loss of CCK causes a diabetogenic phenotype. Fasting plasma glucose (A) and plasma insulin (B) levels of male and female 6-wk-old (n = 161–163), 10-wk-old (n = 155–168), and 14-wk-old (n = 75–105) Cck^WT-ob/ob and Cck^lacZ-ob/ob mice. Glucose and insulin values were log₁₀ transformed. Comparisons were made by nested ANOVA adjusting for sex, genotype, time, and their interactions. For fasting glucose (A), no interactions of sex or time with genotype existed. For fasting insulin (B), genotype and sex interactions were not significant, but time by genotype interactions were significant (P = 0.012).

Figure 5

Cck^lacZ-ob/ob mice have increased β-cell death with no change in proliferation. A, Measurement of 2^H incorporation into islet DNA of 8- to 10-wk-old Cck^WT-ob/ob and Cck^lacZ-ob/ob mice (n = 7 for each). B, Percentage of PCNA-positive β-cells in 10-wk-old male Cck^WT-ob/ob and Cck^lacZ-ob/ob mice (n = 4 for each). C, Percentage of TUNEL-positive β-cells in 10-wk-old male Cck^WT-ob/ob and Cck^lacZ-ob/ob mice (n = 4 for each). D, Propidium iodide-positive cells per total islet area of freshly isolated islets from 14-wk-old male and female Cck^WT-ob/ob (n = 5) and Cck^lacZ-ob/ob (n = 4) mice. Comparisons were made by Student’s unpaired t tests.

Figure 6

CCK promotes β-cell survival. A, MIN-6 cells were reverse transfected with si-Scr negative control or si-Cck oligonucleotides (n = 3). MIN-6 were then incubated for 48 h before being treated for 24 h with IL-1β and TNF-α cytokine cocktail and cell death measured. Comparisons were made by repeated-measures ANOVA followed by Bonferroni-corrected Student’s paired t test. B, Isolated islets from Cck^WT-ob/ob (n = 9) and Cck^lacZ-ob/ob (n = 17) mice were dispersed and treated with 10 μm thapsigargin. Islet cells were incubated for 24 h, and cell death was measured. Comparisons were made by repeated-measures ANOVA followed by Bonferroni-corrected Student’s paired t test. C, Islets from Cck^lacZ-ob/ob (n = 8–11) mice were isolated, dispersed, and treated with 10 μm thapsigargin in the presence of increasing doses of sulfated CCK-8 peptide or vehicle control. Data were analyzed by ANOVA blocking on sample (P < 0.005). Using the vehicle as a covariate, CCK dose followed a log-linear trend to reduce islet cell death (P < 0.05).