Journal: JCI Insight

Article Title: Cystic fibrosis–related diabetes is caused by islet loss and inflammation

doi: 10.1172/jci.insight.98240

Figure Lengend Snippet: (A) Murine models created to excise Cftr exon 11 from β cells in an inducible fashion (β Δ11) or from the pancreas with constitutive Cre action (Panc Δ11). (B) Experimental timeline of murine models. Analyses included oral glucose tolerance testing (OGTT) on conscious animals, insulin secretion assays, and RNA sequencing of whole islets. OGTT of (C) male β cell–specific/inducible mice prior to (Pre-Tx; n = 46) and after treatment with vehicle (V; n = 17) or tamoxifen (β Δ11; n = 23) and (D) pancreatic/constitutive mice homozygous for the Cftr wt allele (Panc wt; n = 8) and mice homozygous for the Cftr FL11 allele (Panc Δ11; n = 11). Insulin secretion from isolated islets incubated in medium containing (E and H) 5.6 mM glucose (5.6 G), 16.7 mM glucose (16.7 G), or (F and I) 16.7 mM glucose and 100 μM 3-isobutyl-1-methylxanthine (16.7 G + IBMX), and (G and J) islet insulin content from β cell–specific/inducible mice (E–G: V, n = 8, 5 male, 3 female; β Δ11, n = 9, 6 male, 3 female) and pancreatic/constitutive mice (H–J: Panc wt, n = 13, 6 male, 7 female; Panc Δ11, n = 20, 10 male, 10 female). We observed slight differences in glucose-stimulated insulin secretion, cAMP-potentiated GSIS, and islet insulin content between the control animals of the β Δ11 and Panc Δ11 models. However, the control animals were individualized for each model and differ in the type of Cre recombinase expressed as well as the expression promotor (A). Red represents the β cell–specific/inducible model (β Δ11), blue the pancreatic constitutive model (Panc Δ11). Data represent mean ± SEM. No statistical significance (P < 0.05) was observed in OGTT AUC, insulin secretion, or insulin content in either model. Statistical data were calculated with 1-way ANOVA (C and D) or unpaired 2-tailed Student’s t test (E–J).

Article Snippet: For recording of β cell currents, fire-polished patch pipettes (Sutter Instruments) with a tip resistance of 4–7 MΩ were used for patch clamp recordings in the perforated-patch configuration, using a EPC10 amplifier and Patchmaster software (HEKA Electronik).

Techniques: RNA Sequencing, Isolation, Incubation, Control, Expressing

Journal: JCI Insight

Article Title: Cystic fibrosis–related diabetes is caused by islet loss and inflammation

doi: 10.1172/jci.insight.98240

Figure Lengend Snippet: Expression of CFTR and select β cell–related transcripts from published islet cell transcriptomes: (A) 270 single human β cells from 6 healthy and 4 diabetic donors (reads per kilobase of transcript per million mapped reads [RPKM], Segerstolpe and Palasantza et al., ref. 38) and (B) sorted β cells from 7 healthy adult donors (transcript per kilobase million [TPM], Blodgett et al., refs. 39). Note: Individual expression values are not presented in A, as the log2 of the mean expression value of 270 β cells was calculated to account for the approximately 85% of β cells in this data set that do not express CFTR; individual CFTR expression values are presented in Supplemental Figure 3E. Green bar, insulin; blue bars, key islet transcription factors; pink, islet hormone secretion related genes; red, CFTR. (C) Representative immunohistochemical labeling of CFTR (red), insulin (green), and glucagon (purple) in a pancreas from 3-month-old male donor. Insets depict the islet border and interior. (D) CFTR (red) channel alone (note: CFTR ductal localization and islet absence). Scale bars: 50 μm (C and D); 10 μm (insets). (E) Representative patch clamp recording of a human β cell and a INS832/13 + wtCFTR cell (n = 5 donors, 35 β cells; Supplemental Figure 5B). (F) Insulin secretion from human islets (n = 4 donors) in medium containing 1 mM glucose (1 G), 16.7 mM glucose (16.7 G), or 16.7 G plus 100 μM forskolin (16.7 G + Fsk) and no drug (white), 1 μM VX770 (blue, ivacaftor), 5 μM VX661 (green), or 5 μM VX809 (red, lumacaftor). 1 G, n = 22–24 replicates; 16.7 G, n = 11–12 replicates; 16.7 + Fsk, n = 10–12 replicates. VX770 is a selective CFTR potentiator that increases CFTR activity at the membrane and VX661, and VX809 are CFTR channel correctors that increase membrane channel density. Data represent mean ± SEM. No statistical significance (P < 0.05) was observed in in vitro human islet insulin secretion when comparing secretory responses at 1 G, 16.7 G, and 16.7 G + Fsk in the presence of absence of CFTR modulators. One-way ANOVA was used for statistical analysis.

Article Snippet: For recording of β cell currents, fire-polished patch pipettes (Sutter Instruments) with a tip resistance of 4–7 MΩ were used for patch clamp recordings in the perforated-patch configuration, using a EPC10 amplifier and Patchmaster software (HEKA Electronik).

Techniques: Expressing, Immunohistochemical staining, Labeling, Patch Clamp, Activity Assay, Membrane, In Vitro

Journal: JCI Insight

Article Title: Cystic fibrosis–related diabetes is caused by islet loss and inflammation

doi: 10.1172/jci.insight.98240

Figure Lengend Snippet: Characteristic CF-related pancreatic pathology observed in (A) donor 1: islet aggregations (black arrowhead) with inter-islet fibrosis (blue arrowhead) and islets in adipose niches (yellow arrowhead). Pathology observed in (B) donor 2: ectopic adipose (white arrowhead) and fibrotic deposition (red arrowhead). (C) Pathology observed in donor 3: formation of fibrotic cyst-like structures with embedded dilated duct-like structures (red arrowhead) and islets in fibrotic niches (black arrowhead). The pancreata from all donors lacked discernible exocrine tissue. Scale bars: 500 μm. (D) β Cell area of CF donors (n = 7) compared with healthy pancreatic donors (n = 7). Examples of abnormal islet morphology: (E, donor 1) islet aggregations and (F, donor 2) scattering of islet cells and dilated structures within and around islets. Scale bars: 100 μm. (G) Percentage of β, α, and δ cells relative to all β, α, and δ cells in the CF pancreas (n = 7) compared with healthy donors (n = 5). Additional islet abnormalities observed in a subset of CF pancreata in (H) donor 6: β cell apoptosis (scale bar: 100 μm; 20 μm [insets]) quantified in Supplemental Figure 7 and (I) intraislet amyloid, as detected by Thioflavin S in 2 of 7 donors (scale bar: 100 μm; 15 μm [insets]). Data represent mean ± SEM. Statistical significance (P < 0.05) was observed in β cell area and α cell ratio where noted by the asterisk. Unpaired 2-tailed Student’s t test was used for statistical analysis. The squares and dots represent individual donors and are color coded according to CF donor (Table 1).

Article Snippet: For recording of β cell currents, fire-polished patch pipettes (Sutter Instruments) with a tip resistance of 4–7 MΩ were used for patch clamp recordings in the perforated-patch configuration, using a EPC10 amplifier and Patchmaster software (HEKA Electronik).

Techniques:

Journal: JCI Insight

Article Title: Cystic fibrosis–related diabetes is caused by islet loss and inflammation

doi: 10.1172/jci.insight.98240

Figure Lengend Snippet: Pancreatic autolysis and remodeling results in destruction of the islet niche and environment, β cell loss, and immune infiltration. Islet loss and inflammation combined with the numerous physiological derangements observed in CF, particularly those responsible for nutrient assimilation, lead to insulin insufficiency and CFRD.

Article Snippet: For recording of β cell currents, fire-polished patch pipettes (Sutter Instruments) with a tip resistance of 4–7 MΩ were used for patch clamp recordings in the perforated-patch configuration, using a EPC10 amplifier and Patchmaster software (HEKA Electronik).

Techniques: