Inhibition of c-Kit Is Not Required for Reversal of Hyperglycemia by Imatinib in NOD Mice

<div><p>(1) Aim/Hypothesis</p><p>Recent studies indicate that tyrosine kinase inhibitors, including imatinib, can reverse hyperglycemia in non-obese diabetic (NOD) mice, a model of type 1 diabetes (T1D). Imatinib inhibits c-Abl, c-Kit, and PDGFRs. Next-generation tyrosine kinase inhibitors for T1D treatment should maintain activities required for efficacy while sparing inhibition of targets that might otherwise lead to adverse events. In this study, we investigated the contribution of c-Kit inhibition by imatinib in reversal of hyperglycemia in NOD mice.</p><p>(2) Methods</p><p>The T670I mutation in c-Kit, which confers imatinib resistance, was engineered into the mouse genome and bred onto the NOD background. Hematopoietic stem cells (HSCs) from NOD.c-Kit^T670I mice and NOD.c-Kit^wt littermates were expanded in the presence or absence of imatinib to verify imatinib resistance of the c-Kit^T670I allele. Diabetic mice were treated with imatinib at the onset of hyperglycemia for three weeks, and blood glucose was monitored.</p><p>(3 )Results</p><p><i>In vitro</i> expansion of HSCs from NOD.c-Kit^wt mice was sensitive to imatinib, while expansion of HSCs from NOD.c-Kit^T670I mice was insensitive to imatinib. However, <i>in vivo</i> treatment with imatinib lowered blood glucose levels in both strains of mice.</p><p>(4) Conclusions/Interpretation</p><p>The HSC experiment confirmed that, in NOD.c-Kit^T670I mice, c-Kit is resistant to imatinib. As both NOD.c-Kit^T670I and NOD.c-Kit^wt mice responded comparably to imatinib, c-Kit inhibition does not substantially contribute to the efficacy of imatinib in T1D. Thus, we conclude that inhibition of c-Kit is not required in next-generation tyrosine kinase inhibitors for T1D treatment, and may be selected against to improve the safety profile.</p></div

Diabetic NOD.c-Kit^T670I mice are sensitive to imatinib treatment.

<p>Blood glucose values for individual diabetic NOD.c-Kit^wt mice treated with either PBS (<b>A</b>) or imatinib (<b>B</b>), in comparison to diabetic NOD.c-Kit^T670I mice treated with either PBS (<b>C</b>) or imatinib (<b>D</b>) for 21 consecutive days. <b>E.</b> Average blood glucose values in diabetic NOD.c-Kit^wt or NOD.c-Kit^T670I mice treated with either PBS ((thick, solid line) NOD.c-Kit^wt; (thin, solid line) NOD.c-Kit^T670I) or imatinib ((thick, dashed line) NOD.c-Kit^wt; (thin, dashed line) NOD.c-Kit^T670I) for 21 consecutive days (n = 5–10 mice/group; *** p<0.0001 between PBS vs. imatinib within each mouse group for entire data set, as determined by one-way ANOVA). Note there was no statistically significant difference between NOD.c-Kit^wt and NOD.c-Kit^T670I within imatinib treatment groups. <b>F.</b> Area under curve for diabetes progression over 21 days comparing PBS ((open, white square) NOD.c-Kitwt; (closed, black square) NOD.c-Kit^T670I) or imatinib ((grey, slashed square) NOD.c-Kit^wt; (grey, closed square) NOD.c-Kit^T670I) treated mice. (* p≤0.01 between PBS vs. imatinib within each mouse group, as determined by one-way ANOVA).</p

NOD.c-Kit^T670I mice are imatinib resistant and develop diabetes.

<p>A. Targeting strategy for generation of NOD.c-Kit^T670I mice. PCR genotyping (<b>B</b>) and sequence traces (<b>C</b>) of wild-type (wt) and mutant (mut) mice using primers F and R to generate fragments of 795 bp and 967 bp for wt and mut alleles, respectively. The T670I codon change and accompanying introduction of <i>Bgl-II</i> restriction site are highlighted. <b>D.</b> Expansion of c-Kit⁺/Sca-1⁺ murine HSCs from either NOD.c-Kit^T670I (black bar) or NOD.c-Kit^wt (white bar) littermates in the presence or absence of 5 µM imatinib. (*** p = 0.0002, as determined by two-way ANOVA). <b>E.</b> NOD.c-Kit^T670I (black square) mice develop diabetes comparably to NOD.c-Kit^wt (white circle) littermates (n = 41–45 mice/group; average age of diabetes onset = 14.5 weeks in NOD.c-Kit^wt mice and 15.5 weeks in NOD.c-Kit^T670I mice).</p

Loss of Alms1 Does Not Affect Transcriptional Changes during Ciliogenesis but Causes Impairment in Flow-Induced Mechanosensation

<div><p>(A) Confocal microscopic analysis of cilia biogenesis in mIMCD3 cells. Short cilia can be detected at day 3 after transfection. mIMCD3 cells transfected with Alms1a siRNA have stunted cilia at days 3 and 5. Cells were stained with anti-acetylated tubulin (yellow, cilia) and TO-PRO-3 (red, nuclei).</p><p>(B) Suppression of Alms1 does not affect the upregulation of <i>Bbs4</i> and <i>Ttc10</i> during ciliogenesis. N, negative siRNA; 1a, Alms1a siRNA; 1b, Alms1b siRNA.</p><p>(C<b>)</b> Heat map representation of microarray analysis of mIMCD3 during ciliogenesis. 98 genes with the most dramatic changes in expression showed approximately equivalent expression changes in the presence of a scrambled siRNA control, or in the presence of specific siRNAs that decreased <i>Alms1</i> mRNA levels and blocked cilia formation.</p><p>(D) Stunted cilia formed in the presence of Alms1a siRNA (red) lack flow-induced Ca2+ influx in mIMCD3 cells, compared with a negative control siRNA (blue). Representative data are shown for cytosolic calcium change of individual cells in response to mechanical flow. Arrow points to the start of flow.</p></div

Kidney Abnormalities in <i>Alms1</i> Mutant Mice

<div><p>(A) H&E-stained kidney sections of a 6-mo-old <i>Alms1^{L2131X/L2131X}</i> mouse showing dilated cortex tubules compared with an age-matched wild-type control. Lack of kidney cilia is observed in some tubules in the cortex of <i>Alms1^{L2131X/L2131X}</i> kidney, whereas cilia in the medulla appear normal. H&E, hematoxylin-eosin; Acet, acetylated.</p><p>(B) Cortex cilia count comparison between <i>Alms1^{L2131X/L2131X}</i> and controls. 300–400 kidney nuclei were examined for each of six fields of <i>Alms1^{L2131X/L2131X}</i> and wild-type controls. The bar chart represents the average and standard deviations of cilia count per 100 kidney cortex epithelial cells from eight mice per group.</p><p>(C) In the cortex of <i>Alms1^{L2131X/L2131X}</i> kidney, cilia are lost selectively in LTA-labeled tubules but not in aquaporin-2-expressing tubules.</p><p>(D) Upper panel: clusters of Ki67-positive proliferating epithelial cells in the <i>Alms1^{L2131X/L2131X}</i> kidney, potentially lining the same convoluted tubule. Lower panel: TUNEL staining reveals apoptotic cells in <i>Alms1^{L2131X/L2131X}</i> kidneys but rarely in a wild-type control. Arrow, whole tubule cross sections were labeled by TUNEL, suggesting progression of nephropathy in <i>Alms1^{L2131X/L2131X}</i> mutant kidneys. WT, wild-type.</p><p>(E) Urinalysis of 3- to 6-mo-old <i>Alms1^{L2131X/L2131X}</i> mice and age-matched littermate controls. Urine from <i>Alms1^{L2131X/L2131X}</i> mice showed slight elevation of protein levels, <i>p</i> = 0.007. Scale bars, 50 μm.</p></div

<i>Alms1^{L2131X/L2131X}</i> Mice Recapitulate Human Alström Syndrome

<div><p>(A) <i>Alms1^{L2131X/L2131X}</i> mice gain more fat mass than heterozygote or wild-type controls but equivalent lean mass.</p><p>(B) Histological examination of <i>Alms1^{L2131X/L2131X}</i> mice and wild-type littermate control. Insets show oil red O staining of frozen liver sections.</p><p>(C) Testis H&E sections show degeneration of seminiferous tubules (arrow), which have reduced numbers of germinal cells. Reduced numbers of sperm flagella with decreased length are observed in the epididymus of <i>Alms1^{L2131X/L2131X}</i> mice (anti-acetylated tubulin, green). H&E, hematoxylin-eosin.</p><p>(D) Rhodopsin staining in the outer nuclear layer cell bodies is seen in rare cells in the <i>Alms1^{L2131X/L2131X}</i> animals (arrows) but not in wild-type littermate controls. Insets illustrate higher magnification images. ONL, outer nuclear layer; IS, inner segment; OS; outer segment. Scale bars, 50 μm.</p></div

Suppression of <i>Alms1</i> Expression Alters Primary Cilium Formation in Kidney Epithelial Cells

<div><p>(A) Elongated cilia, visualized with staining of acetylated tubulin (green), form normally in mIMCD3 cells after mock-transfection, transfection with a negative control siRNA, or transfection with two inactive siRNAs directed against Alms1 (Alms1c and Alms1d). Focal staining of acetylated tubulin without axoneme extension is seen after transfection with two active siRNAs targeting <i>Alms1</i> (Alms1a and Alms1b).</p><p>(B) Real-time PCR analysis with two mouse <i>Alms1</i> probes recognizing the junctions of exons 1 and 2 and exons 12 and 13, respectively: Alms1a and Alms1b siRNAs both cause 70%–80% knockdown of <i>Alms1</i> mRNA; no effect on <i>Alms1</i> mRNA was seen with the three siRNAs that were inactive in the ciliogenesis assay.</p><p>(C) Alms1a siRNA-treated cells lose endogenous Alms1 protein expression. Acet, acetylated. Scale bars, 10 μm.</p></div

N-Terminal Alms1 Protein Can Support Cilia Formation

<div><p>(A) Cotransfection of Alms1a siRNA-treated cells with a 5′ <i>Alms1</i> cDNA construct rescues primary cilia formation in mIMCD3 cells.</p><p>(B) Real-time PCR analysis of Alms1a siRNA and N-terminal Alms1-transfected cells. Upper panel: over-expression of the 5′ cDNA does not affect knockdown of endogenous <i>Alms1</i> mRNA with Alms1a siRNA. Lower panel: knockdown of endogenous <i>Alms1</i> mRNA does not affect overexpression of the 5′ cDNA. N, negative control siRNA; cDNA, 5′ <i>Alms1</i> cDNA; 1a, Alms1a siRNA.</p><p>(C) Stable expression of <i>Alms1</i> mRNA from the <i>Alms1^{L2131X/L2131X}</i> allele. Real-time PCR analysis of <i>Alms1</i> gene expression in an <i>Alms1^{L2131X/L2131X}</i> mouse and a wild-type littermate control.</p><p>(D) The N-terminal mouse Alms1 antibody detects Alms1 mutant protein at the ciliary basal body in primary kidney cells from the <i>Alms1^{L2131X/L2131X}</i> mouse. Shown are low and high magnifications of the ciliated cells. Arrowheads point out the base of cilia.</p><p>(E) Normal appearance of primary cilia in primary fibroblasts (MEF) and primary kidney cells (PKC) from the <i>Alms1^{L2131X/L2131X}</i> mouse strain.</p><p>(F) Inhibition of cilia formation in Alms1a siRNA-treated <i>Alms1^{L2131X/L2131X}</i> primary fibroblasts. Scale bars, 10 μm.</p></div

IL-2 Immunotherapy Reveals Potential for Innate Beta Cell Regeneration in the Non-Obese Diabetic Mouse Model of Autoimmune Diabetes

<div><p>Type-1 diabetes (T1D) is an autoimmune disease targeting insulin-producing beta cells, resulting in dependence on exogenous insulin. To date, significant efforts have been invested to develop immune-modulatory therapies for T1D treatment. Previously, IL-2 immunotherapy was demonstrated to prevent and reverse T1D at onset in the non-obese diabetic (NOD) mouse model, revealing potential as a therapy in early disease stage in humans. In the NOD model, IL-2 deficiency contributes to a loss of regulatory T cell function. This deficiency can be augmented with IL-2 or antibody bound to IL-2 (Ab/IL-2) therapy, resulting in regulatory T cell expansion and potentiation. However, an understanding of the mechanism by which reconstituted regulatory T cell function allows for reversal of diabetes after onset is not clearly understood. Here, we describe that Ab/IL-2 immunotherapy treatment, given at the time of diabetes onset in NOD mice, not only correlated with reversal of diabetes and expansion of Treg cells, but also demonstrated the ability to significantly increase beta cell proliferation. Proliferation appeared specific to Ab/IL-2 immunotherapy, as anti-CD3 therapy did not have a similar effect. Furthermore, to assess the effect of Ab/IL-2 immunotherapy well after the development of diabetes, we tested the effect of delaying treatment for 4 weeks after diabetes onset, when beta cells were virtually absent. At this late stage after diabetes onset, Ab/IL-2 treatment was not sufficient to reverse hyperglycemia. However, it did promote survival in the absence of exogenous insulin. Proliferation of beta cells could not account for this improvement as few beta cells remained. Rather, abnormal insulin and glucagon dual-expressing cells were the only insulin-expressing cells observed in islets from mice with established disease. Thus, these data suggest that in diabetic NOD mice, beta cells have an innate capacity for regeneration both early and late in disease, which is revealed through IL-2 immunotherapy. </p> </div

Insulin+/glucagon+ cells do not reflect mature endocrine cells.

<p>Recent onset diabetic NOD pancreata treated with Ab/IL-2 were processed and stained for insulin (red), glucagon (green), DAPI (blue) and mature endocrine markers, Brn4, Pdx1, or Nkx6.1 (white). (<b>A</b>) Staining for Brn4, a transcription factor expressed in mature alpha cells, showed expression in glucagon+ (arrowhead) and insulin+/glucagon+ (arrow) cells. (<b>B</b>) Staining for Pdx1, a beta cell specific transcription factor, showed weak expression in insulin+/glucagon+ (arrow) cells, as well some hormone negative cells (arrowhead). (<b>C</b>) Staining for Nkx6.1 showed only weak or absent nuclear staining (arrows) in insulin+/glucagon+ cells, as well as abnormal cytoplasmic staining (arrowhead). (<b>D</b>) Graph represents quantitation of the percent of insulin+/glucagon+ cells that also express nuclear Brn4, Pdx1 or Nkx6.1 (n=97+ insulin+/glucagon+ cells analyzed per transcription factor, from n≥4 individual samples).</p