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. 2018 Oct;224(2):e13085.
doi: 10.1111/apha.13085. Epub 2018 May 22.

Decreased Levels of Keratin 8 Sensitize Mice to Streptozotocin-Induced Diabetes

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Free PMC article

Decreased Levels of Keratin 8 Sensitize Mice to Streptozotocin-Induced Diabetes

C M Alam et al. Acta Physiol (Oxf). .
Free PMC article

Abstract

Aim: Diabetes is a result of an interplay between genetic, environmental and lifestyle factors. Keratin intermediate filaments are stress proteins in epithelial cells, and keratin mutations predispose to several human diseases. However, the involvement of keratins in diabetes is not well known. K8 and its partner K18 are the main β-cell keratins, and knockout of K8 (K8-/- ) in mice causes mislocalization of glucose transporter 2, mitochondrial defects, reduced insulin content and altered systemic glucose/insulin control. We hypothesize that K8/K18 offer protection during β-cell stress and that decreased K8 levels contribute to diabetes susceptibility.

Methods: K8-heterozygous knockout (K8+/- ) and wild-type (K8+/+ ) mice were used to evaluate the influence of keratin levels on endocrine pancreatic function and diabetes development under basal conditions and after T1D streptozotocin (STZ)-induced β-cell stress and T2D high-fat diet (HFD).

Results: Murine K8+/- endocrine islets express ~50% less K8/K18 compared with K8+/+ . The decreased keratin levels have little impact on basal systemic glucose/insulin regulation, β-cell health or insulin levels. Diabetes incidence and blood glucose levels are significantly higher in K8+/- mice after low-dose/chronic STZ treatment, and STZ causes more β-cell damage and polyuria in K8+/- compared with K8+/+ . K8 appears upregulated 5 weeks after STZ treatment in K8+/+ islets but not in K8+/- . K8+/- mice showed no major susceptibility risk to HFD compared to K8+/+ .

Conclusion: Partial K8 deficiency reduces β-cell stress tolerance and aggravates diabetes development in response to STZ, while there is no major susceptibility to HFD.

Keywords: cytoskeleton; diabetes; glucose-stimulated insulin secretion; high-fat diet; keratin intermediate filaments; streptozotocin; β-cell.

Figures

Figure 1
Figure 1
K8+/− mouse islet cells express less K8 and K18 than K8+/+ islets. A, Western blot for K8, K18 and Hsc70 (loading control) of protein lysate from pancreatic islets isolated from K8+/+ and K8+/− mice (lane 1‐3, K8+/+; lane 4‐6, K8+/−). B, Quantification of immunoblotting showed a statistically significant fivefold decrease in K8 protein levels in K8+/−‐isolated pancreatic islets compared to K8+/+ islets when normalized to Hsc70 levels. *P < .05, error bars represent mean ± SEM. C, Quantification of immunoblotting showed a significant twofold decrease in K18 protein levels in K8+/−‐isolated pancreatic islets compared to K8+/+ islets when normalized to Hsc70 levels. †P < .001, error bars represent mean ± SEM. D, Immunostaining of K8 or K18 (green), insulin (red, used as a marker for islets) and nuclei (blue, colours seen in the merged images) of pancreatic sections from K8+/+ and K8+/− mice shows a less dense islet keratin network in K8+/−.mice compared to K8+/+ mice. Scale bar: 100 μm
Figure 2
Figure 2
K8+/− mice exhibit no significant changes in blood glucose and insulin regulation compared to K8+/+ mice. A, Blood glucose levels after overnight fasting in 3‐ to 4‐mo‐old K8+/+ mice and K8+/− mice showed no significant differences using t test. Bars represent mean ± SEM, n  = 10 for K8+/+ and n = 8 for K8+/− mice. B, K8+/+ and K8+/− mice were subjected to a glucose tolerance test. Mice were fasted overnight and then challenged with 2 g/kg d‐glucose administered i.p. No changes in blood glucose levels were observed between K8+/+ and K8+/− mice as evaluated using repeated measures two‐way ANOVA. Bars represent mean ± SEM, n = 10 for K8+/+ and n = 8 for K8+/− mice. C, K8+/+ and K8+/− mice showed no significant changes in blood glucose levels, using repeated measures two‐way ANOVA, when subjected to an insulin tolerance test by 0.75 U/kg i.p. insulin after 4‐h fasting. Bars represent mean ± SEM, n  = 7 for K8+/+ and n = 9 for K8+/− mice. D, Glucose‐stimulated insulin secretion (GSIS) was measured 10 and 30 min (') after i.p. glucose 2 g/kg of body weight in K8+/+ mice and K8+/− mice. A significant increase in fasting versus 10 min GSIS can be seen for K8+/+ mice, analysed by one‐way ANOVA and Bonferroni's correction, but no overall differences in GSIS responses between the genotypes were observed when compared using two‐way ANOVA. Plot shows mean ± SEM, n  =  8‐10 mice/group for fasting insulin and 4‐5 mice/group for glucose‐stimulated insulin secretion. E, Relative pancreatic insulin content as determined by enzyme‐linked immunosorbent assay showed no differences between K8+/+ mice and K8−/− mice. Bars indicate mean ± SEM, n = 5 for K8+/+ mice and n = 7 for K8+/− mice. F, Immunoblotting of K8+/+ (n = 3, lanes 1‐3) and K8+/− (n = 3, lanes 4‐6) isolated pancreatic islets showed no changes in the protein amount of insulin between the genotypes
Figure 3
Figure 3
K8+/− mice exhibit increased diabetes incidence and aggravated diabetes after chronic low‐dose STZ treatment when compared to K8+/+ mice. A, A significantly higher diabetes incidence was seen for K8+/− compared to K8+/+ mice after daily low‐dose STZ i.p. injections during the first 5 d. n  =  13 for K8+/+ and n = 11 for K8+/− mice. P = .016, calculated with log‐rank test. B, K8+/− mice showed higher blood glucose levels 2 wk after low‐dose STZ injections compared to K8+/+ mice. Bars show mean ± SEM, n  =  17 for K8+/+ mice and n = 18 for K8+/− mice. *P < .05 calculated with two‐way ANOVA and Bonferroni's post hoc test. C, Urine secretion for basal untreated and STZ‐treated diabetic (collected 34 d after treatment) K8+/+ and K8+/− mice showed significantly larger urine volume for K8+/− mice compared to K8+/+ mice. *P < .05 calculated with two‐way ANOVA
Figure 4
Figure 4
Increased islet damage in K8+/− mice and more persistent keratin upregulation in K8+/+ than in K8+/− mice after STZ treatment. A, Scoring of islet cell damage in STZ‐treated (2 or 5 wk) K8+/+ and K8+/− haematoxylin‐eosin–stained pancreata showed more islet damage in K8+/− mice compared to K8+/+ mice. n = 6 for K8+/+ and K8+/− mice (2 wk STZ treatment); n = 3 for K8+/+ and n = 4 for K8+/− mice (5‐wk STZ treatment). *P < .05, † P < .001. B, Immunostaining for K8 (green) and nuclei (blue) of pancreatic sections from STZ‐induced diabetic K8+/+ and K8+/− mice 2 wk and 5 wk after start of the STZ treatment shows increased K8 levels after 2 wk of STZ treatment in both genotypes. At 5 wk, K8 was increased in islet cells compared to untreated control mice (see Figure S2 for keratin/insulin staining of these samples). Islets are indicated with white broken lines. Scale bar: 100 μm
Figure 5
Figure 5
MFN2 protein levels and GLUT2 protein levels and localization are unaltered in K8+/− mice. A, Western blot analysis for MFN2 and Hsc70 (loading control) of protein lysate from pancreatic islets isolated from K8+/+ and K8+/− mice (lanes 1‐3, K8+/+; lanes 4‐6, K8+/−) showed no changes in the protein levels of MFN2 between the genotypes. B, Quantification of MFN2 immunoblotting from (A) using Hsc70 as loading control showed no significant changes in protein levels of MFN2 between K8+/+ and K8+/− mice. C, Immunostaining of pancreatic sections from K8+/+ and K8+/− mice for GLUT2 (red) and nuclei (blue) showed equal intensities of GLUT2 staining on the β‐cell membranes. Scale bar: 5 μm. D, Western blot for GLUT2 and Hsc70 (loading control) of isolated islet lysates showed no differences in GLUT2 amounts in total lysates of K8+/+ and K8+/− pancreas (lanes 1‐3, K8+/+; lanes 4‐6, K8+/−). (E) Quantification of GLUT2 immunoblotting from (D) balanced to loading control Hsc70 showed no significant changes between the genotypes
Figure 6
Figure 6
High‐fat diet induces no increased T2D susceptibility risk for K8+/− mice. Four‐ to 6‐mo‐old male K8+/+ (n = 9) and K8+/− mice (n = 7) were treated for a HFD regimen for 16 wk. A, K8+/+ and K8+/− mice show similarly increased body weight in response to HFD, calculated by two‐way ANOVA. B, Fat % of K8+/+ and K8+/− mice (measured by EchoMRI) similarly increased in response to HFD (two‐way ANOVA). C, Analysed by two‐way ANOVA, K8+/− mice showed no statistical difference in non‐fasting blood glucose values in response to 8‐10 wk of HFD (wk 8, P = .06; wk 9, P = .15; wk 10, P = .06) compared to K8+/+. D, Glucose tolerance test of K8+/+ and K8+/− mice after HFD showed no changes in fasting glucose levels before and after 2 g/kg i. p. glucose stimulation analysed by two‐way ANOVA

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