Intermittent fasting preserves beta-cell mass in obesity-induced diabetes via the autophagy-lysosome pathway

Abstract

Obesity-induced diabetes is characterized by hyperglycemia, insulin resistance, and progressive beta cell failure. In islets of mice with obesity-induced diabetes, we observe increased beta cell death and impaired autophagic flux. We hypothesized that intermittent fasting, a clinically sustainable therapeutic strategy, stimulates autophagic flux to ameliorate obesity-induced diabetes. Our data show that despite continued high-fat intake, intermittent fasting restores autophagic flux in islets and improves glucose tolerance by enhancing glucose-stimulated insulin secretion, beta cell survival, and nuclear expression of NEUROG3, a marker of pancreatic regeneration. In contrast, intermittent fasting does not rescue beta-cell death or induce NEUROG3 expression in obese mice with lysosomal dysfunction secondary to deficiency of the lysosomal membrane protein, LAMP2 or haplo-insufficiency of BECN1/Beclin 1, a protein critical for autophagosome formation. Moreover, intermittent fasting is sufficient to provoke beta cell death in nonobese lamp2 null mice, attesting to a critical role for lysosome function in beta cell homeostasis under fasting conditions. Beta cells in intermittently-fasted LAMP2- or BECN1-deficient mice exhibit markers of autophagic failure with accumulation of damaged mitochondria and upregulation of oxidative stress. Thus, intermittent fasting preserves organelle quality via the autophagy-lysosome pathway to enhance beta cell survival and stimulates markers of regeneration in obesity-induced diabetes.

Keywords: autophagy; beta cells; diabetes; intermittent fasting; lysosomes.

Figures

Figure 1.

Intermittent fasting improves glucose regulation and preserves beta cell mass and function in mice with diet-induced obesity and diabetes. (A) Weight gain on high-fat diet (HFD, open red boxes, solid red line) as compared with chow feeding (open blue circles, solid blue line) in adult male C57BL/6 mice from 8 wk to 20 wk of age (n = 15 per group; ***P < 0.001 for HFD vs. chow). (B) Body weight after 6 wk of intermittent fasting (solid circles or boxes with dotted lines) in both chow (n = 15 per group) and high-fat feeding groups (n = 23 or 24/group, ***P < 0.001 for WT HFD-AL vs. WT chow-AL, ###P < 0.001 HFD-IF vs. HFD-AL). (C) Average cumulative caloric intake in mice treated as in (B) (***P < 0.001 for chow-IF vs. chow-AL, ###P < 0.001 HFD-IF vs. HFD-AL). (D, E) Glucose tolerance tests (GTT, D, *P < 0.05 for HFD-AL vs. chow-AL, #P < 0.05 HFD-IF vs. HFD-AL, $P < 0.05 for chow-IF vs. chow-AL), area under the curve for glucose measurement (E). (F) Insulin levels in response to glucose administration as in D (F, #P < 0.05 HFD-IF vs. HFD-AL, *P < 0.05 HFD-AL vs. chow-AL) in mice treated as in (B), n = 10 to 28/group. (G) Representative images of islets from mice treated as in (B); (n = 3 or 4/group) with immunostaining of beta cells (anti-insulin, red) and alpha cells (anti-glucagon, green); nuclei are blue (DAPI). (H) Quantification of beta cell area in pancreata from (G). (I) Insulin content per mg pancreas (n = 5 to 7/group; J) and in islets (n = 30 islets/mouse from n = 3 or 4 mice/group) from mice treated as in (B). (K) Glucose stimulated insulin release in isolated islets (n = 30 islets/mouse from n = 4 or 5 mice per group) as in (J). *P < 0.05 and **P < 0.001 for (H–K).

Figure 2.

Intermittent fasting prevents beta cell loss in mice with diet-induced obesity. (A) Representative immunofluorescence images of pancreatic islets from mice fed HFD or chow diets for 12 wk and then subjected to intermittent fasting for 6 wk, for TUNEL (red, see arrows) and beta cells (green, anti-insulin) in (A). (B) Quantification of TUNEL-positive beta cell nuclei (n = 30 to 40 islets/group; 3 or 4 mice/group). (C) Representative images for MKI67 (MKI67, green), insulin (red) and nuclei (blue, DAPI) in mice treated as in A; (D) Quantification of MKI67(+) beta cells as a percentage of total beta cells. (E–G) Assessment of autophagix flux in islets by immunoblotting for LC3B and SQSTM1 in mice modeled as in (A) and injected with chloroquine (CQ, 60 mg/kg) or saline 4 h prior to sacrifice (E), with quantification of LC3B-II (F) and SQSTM1 (G); n = 3 mice/group. (H) Tfeb transcripts in islets from mice treated as in (A); n = 3 or 4 per group. *P < 0.05; **P < 0.001 and ***P < 0.0001.

Figure 3.

Intermittent fasting does not improve glucose tolerance in Lamp2 heterozygous null mice with obesity-induced diabetes. (A) Body weight in WT female mice fed HFD (red, squares) or chow (blue, circles) for 12 wk, during the subsequent 6 wk of intermittent fasting (closed squares or circles with dotted lines) or ad-lib feeding (open squares or circles with solid lines) while being continued on the respective diets (n = 5/ group for chow feeding, n = 10/group for HFD, *P < 0.05 HFD-AL vs. chow-AL). (B) Body weight in Lamp2 heterozygous null mice fed HFD (green, squares) or chow diets (black, circles) for 12 wk during the subsequent 6 wk of IF (closed squares or circles with dotted lines) or ad-lib feeding (open squares or circles with solid lines) while being continued on the respective diets (n = 6/group for HF diet and n = 3 or 4/group for chow diet; *P < 0.05 HFD-AL vs. chow-AL). (C and D) Caloric intake in mice treated as in (A and B), respectively (*P < 0.05 for HFD-AL vs. chow-AL, #P < 0.05 HFD-IF vs. HFD-AL). (E and F) GTTs in WT females (E, n = 10/ group, *P<0.05 for HFD-AL vs. chow-AL, #P < 0.05 HFD-IF vs. HFD-AL, $P < 0.05 for chow-IF vs. chow-AL) and Lamp2 heterozygous null mice (F, n = 3 to 6 per group, *P < 0.05 for HFD-AL vs. chow-AL) fed HFD or chow diets followed by IF or ad-lib access to respective diets. (G and H) AUC measurements from GTTs performed in (E and F). (I and J) Insulin levels upon glucose challenge in obese WT (I, n = 5/group) and Lamp2 heterozygous null mice (J, n = 3 or 4/group) after IF or ad-lib feeding on HFD for 6 wk. *P < 0.05; **P < 0.001 and ***P < 0.0001.

Figure 4.

Intermittent fasting worsens glucose tolerance in lamp2 null males fed a chow diet. (A) GTT performed in lamp2 male null mice after 6 wk IF (closed triangle, dotted line) or ad-lib feeding (open triangle, solid line) on a chow diet (n = 5/group); (B) AUC measurement from GTT in (A); *P < 0.05 for IF vs. AL for (A and B). (C) Representative images of islets from male lamp2 null mice showing beta cells (red, anti-insulin) and alpha-cells (green, anti-glucagon) for mice treated as in (A); nuclei are blue (DAPI). (D) Quantification of beta cell area from (C; n = 3 or 4 mice/group). (E) TUNEL staining (red) in islets costained with anti-insulin (green) and DAPI for mice treated as in (A; see arrows). (F) Quantification of TUNEL-positive beta cell nuclei in ad-lib versus IF treated mice fed a chow diet (n = 3 or 4 mice per group). *P < 0.05; **P < 0.001 and ***P < 0.0001.

Figure 5.

Intermittent fasting provokes beta cell loss in Lamp2 heterozygous null mice with obesity-induced diabetes. (A and B) Insulin content of whole pancreas in WT females (A) and Lamp2 heterozygous null female mice (B) after 12 wk HF diet or chow feeding followed by 6 wk of IF or ad-lib access to original dietary regimen (n = 4 to 6/ group). (C) Representative images of islets of WT female mice showing beta cells (red, anti-insulin) and alpha cells (green, anti-glucagon); (D) Quantification of beta cell area from (C; n = 3 or 4 mice/group); (E) TUNEL staining (red, arrows) in islets costained with anti-insulin (green) and DAPI. (F) Quantification of TUNEL-positive beta cell nuclei in WT females treated as in A (n = 3 or 4 mice/group); (G) Representative images of islets of Lamp2 heterozygous null female mice with staining for beta cells (red, anti-insulin) and alpha cells (green, anti-glucagon). (H) Quantification of beta cell area from (G; n = 3 or 4 mice/group); (I) TUNEL staining (red, arrows) in islets costained with anti-insulin (green) and DAPI in mice treated as in (B). and (J) Quantification of TUNEL-positive beta cell nuclei in in Lamp2 heterozygous null female mice in I (n = 3 or 4 mice/ group). *P < 0.05; **P < 0.001 and ***P < 0.0001.

Figure 6.

Intermittent fasting provokes autophagosome accumulation with mitochondrial ultrastructural abnormalities in Lamp2 heterozygous null beta cells with obesity-induced diabetes. (A) Representative images of WT or Lamp2 heterozygous null mice fed a chow diet ad-lib or subjected to 6 wk intermittent fasting with coimmunostaining for LC3B and SQSTM1 (anti-insulin, green; anti-LC3B, red; anti-SQSTM1, gray). (B) Quantification of LC3B puncta per beta cell nucleus (n = 3 or 4 mice per group). (C) Quantification of SQSTM1 puncta per beta cell nucleus (n = 3 or 4 mice per group). (D) Representative TEM images of beta cells from mice treated as in (A) with autophagic structures (white arrows) and mitochondria (white arrowheads). Scale bar: 500 nm. *P < 0.05; **P < 0.001 and ***P < 0.0001.

Figure 7.

Intermittent fasting worsens autophagy impairment and provoke apoptosis in beta cells of lamp2 null male mice. (A) Representative images demonstrating expression of autophagy markers, LC3B and SQSTM1, in the islets from lamp2 null mice and wild-type controls fed a chow diet with ad-lib access to food or subjected to intermittent fasting for 6 wk (anti-insulin, green; anti-LC3B, red; anti-SQSTM1, gray). (B) Quantification of LC3B puncta from (A); n = 3 or 4 mice per group. (C) Quantification of SQSTM1 puncta from (A); n = 3 or 4 mice per group. (D) Representative electron micrographs of islets from mice treated as in (A). Top row demonstrates islet architecture. White arrow points to an apoptotic nucleus. Scale bar: 2 µm. Bottom row demonstrates organelle characteristics. Black arrows point to autophagic structures. White arrows point to mitochondria. Scale bar: 500 nm. **P < 0.001, ***P < 0.0001.

Figure 8.

Intermittent fasting exacerbates glucose intolerance in Becn1+/− mice. (A) GTT in Becn1+/− mice fed a HFD for 12 wk followed by ad-lib feeding or intermittent fasting for 6 wk (n = 3/group). (B) AUC from GTT as in (A). (C) Representative images of islets from obese Becn1+/− mice after ad-lib feeding or intermittent fasting as in (A) with staining for beta cells (red, anti-insulin) and alpha cells (green, anti-glucagon). (D) Quantification of beta cell area from C (n = 3 mice/group). (E) TUNEL staining (red, arrows) in islets costained for insulin (green) with DAPI. (F) Quantification of TUNEL-positive beta cell nuclei in mice as in E (n = 3 mice/group). (G) Representative images of islets from Becn1+/− mice as in A with coimmunostaining for LC3B (red) with insulin (green); nuclei are blue (DAPI). (H) Quantification of LC3B puncta from (G; n = 3/group). (I) Representative images of islets from Becn1+/− mice as in (A) with coimmunostaining for SQSTM1 (gray) with insulin (green); nuclei are blue (DAPI). (J) Quantification of SQSTM1 puncta (n = 3/group). (K) Representative TEM images of beta cells from mice treated as in (A) with black arrowheads indicating mitochondria. Scale bar: 500 nm. (L) Glucose stimulated insulin release in isolated islets from Becn1+/− mice fed a HFD for 12 wk followed by ad-lib feeding or intermittent fasting for 6 wk, or Becn1+/− and littermate wild-type (Becn1+/+) mice fed chow ad-lib as controls (n = 30 islets/mouse from n = 3 or 4 mice per group) as in (J). Age matched Becn1+/− mice of both sexes was employed for the above analyses. *P < 0.05; **P < 0.001 and ***P < 0.0001.

Figure 9.

Intermittent fasting increases NEUROG3 expression in high-fat fed mice. (A) Representative images showing immunohistochemical staining for the transcription factor NEUROG3 (anti-NEUROG3, red), insulin (anti-insulin, green) in (A) chow-fed wild-type male and female mice subjected to ad-libitum feeding or intermittent fasting; (B) HFD-fed wild-type male and female mice subjected to ad-libitum feeding or intermittent fasting; and (C and D) HFD-fed Lamp2 heterozygous null female mice (C) or and Becn1+/− mice (D) subjected to ad-libitum feeding or intermittent fasting. Representative from n = 3 mice/group. (E and F) Neurog3 (E) and Neurod1 (F) transcripts in islets from chow and HFD-fed male mice subjected to ad-libitum feeding or intermittent fasting; N = 6 or 7/group. * indicates P < 0.05 by post-hoc test.