Fasting-Mimicking Diet Promotes Ngn3-Driven β-Cell Regeneration to Reverse Diabetes

Abstract

Stem-cell-based therapies can potentially reverse organ dysfunction and diseases, but the removal of impaired tissue and activation of a program leading to organ regeneration pose major challenges. In mice, a 4-day fasting mimicking diet (FMD) induces a stepwise expression of Sox17 and Pdx-1, followed by Ngn3-driven generation of insulin-producing β cells, resembling that observed during pancreatic development. FMD cycles restore insulin secretion and glucose homeostasis in both type 2 and type 1 diabetes mouse models. In human type 1 diabetes pancreatic islets, fasting conditions reduce PKA and mTOR activity and induce Sox2 and Ngn3 expression and insulin production. The effects of the FMD are reversed by IGF-1 treatment and recapitulated by PKA and mTOR inhibition. These results indicate that a FMD promotes the reprogramming of pancreatic cells to restore insulin generation in islets from T1D patients and reverse both T1D and T2D phenotypes in mouse models. PAPERCLIP.

Figure 1. FMD cycles promote β-cell regeneration and reverse β-cell failure in T2D

(A) Experimental scheme to determine effects of the periodic FMD on T2D in the leptin receptor deficient (Lepr^db/db) mice. Mice were monitored for hyperglycemia and insulinemia for two weeks (baseline, BL) and then assigned to the dietary groups. Each FMD cycle entails 4-day FMD and up to 10 days of refeeding (RF). During refeeding, mice received a regular chow identical to that given prior to the FMD and that given to the ad libitum (AL) controls. (B) Plasma glucose levels and (C) Plasma insulin levels; vertical dashed lines indicate each cycle of the FMD and horizontal lines indicate the range of glucose levels (mean ± s.e.m) in age-matched healthy wild-type littermates. Blood samples were collected at the last refeeding day/1^st day of the indicated cycles. Mice were fasted for 6 hours (morning fasting) for blood glucose measurements. (D) Homeostatic model assessment (HOMA) of insulin resistance (IR) and steady state β-cell function (%B) at indicated time points. HOMA-B = (20 x Fasting Insulin)/(Fasting Glucose-3.5) %. (E) Glucose tolerance test and Insulin tolerance test at day 60. (F) Survival curve. mean ± s.e.m,*p<0.05, Log-rank (Mantel-Cox) Test for trend. n ≥ 16 mice per group; for (B–E), each point represents the mean ± s.e.m; *p<0.05, ** p<0.01, ***p<0.005, two-way ANOVA. (G) Proportion of β cells per islet (H) Proliferative proportion of β cells per islet. For (G to I), mean ± s.e.m,*p<0.05, ** p<0.01, ***p<0.005, one-way ANOVA. (I) Immunostaining of pancreatic sections from Lepr^db/db mice and their wild-type littermates at the indicated time points. Arrow in the 8x-enlarged example image indicates a typical proliferative β cell (PCNA⁺Insulin⁺). Scale bar represents 50um. (J) Representative images for size-matched islets isolated from AL-dbdb and FMD-dbdb mice and results of glucose-stimulated insulin secretion (GSIS) test in islets isolated from Lepr^db/db mice on FMD or fed ad libitum. Scale bar represents 50um. Mice are of the C57BL/6J background, of the age indicated. In (a) mice received no additional treatments other than the indicated diet. For (F–J), each point represents the mean ± s.e.m; *p<0.05, ** p<0.01, ***p<0.005, two-way ANOVA. n ≥ 6 mice per group, n ≥ 15 islets per sample;

Figure 2. FMD cycles reverse STZ-induced β-cell depletion and restore glucose homeostasis

(A) Experimental scheme of the periodic FMD’s effects on STZ-induced T1D; Baseline measurements (BL) were performed at day 5 after STZ treatment. (B) Fasting glucose levels and (C) Plasma insulin levels during and 55 days after the FMD cycles (d5 to d35); vertical dashed lines indicate each cycle of FMD; horizontal lines (125±12mg/dl) indicate levels of blood glucose in the naïve control mice. (D) Glucose tolerance test at d50. (E) Cytokine profile of mice treated with STZ or STZ+FMD at d30, compared to that in naïve controls. Pancreatic samples collected at indicated time points were analyzed for: (F) Proliferative proportion in β cells. (G) Proportion of insulin-producing β cells per islet and (H) Representative micrographs with immunostaining of insulin, glucagon and DAPI on pancreas sections of mice treated with STZ or STZ + FMD at the indicated time points. Scale bar represents 50um. Mice of the C57BL/6J background, age 3–6 months, received STZ treatments (150 mg/kg) as indicated in (A). For (B–G), each point represents the mean ± s.e.m and sample size (n) is indicated in parentheses and for (F and G), *p<0.05, **p<0.01, ***p<0.005, one-way ANOVA. Ctrl, STZ-untreated control; STZ BL, baseline level of STZ treated mice at day 5. n≥6 mice per group per time point, n≥15 islets per mouse.

Figure 3. FMD and post-FMD refeeding promote β-cell proliferation and regeneration

(A) Size and number of pancreatic islets per pancreatic section. (B) Proliferative proportion of β cells and proportion of β cells per islet. (C) Representative images of pancreatic islets with Insulin, glucagon and PCNA immuno-staining. (D) Transitional cell population co-expressing both the markers of α and β cells: Proportion of α cells and Pdx1⁺α cells. Arrows in the images with split channels indicating Pdx1⁺Gluc⁺ and Insulin⁺Glucagon⁺ cells. (E) Schematic of FMD- and post-FMD refeeding induced cellular changes in pancreatic islets. Mice of the C57BL/6J background, at age 3–6 months, received no additional treatments other than the indicated diet. Pancreatic samples were collected from mice fed ad libitum (AL) or the fasting mimicking diet (FMD) at indicated time points: the end of the 4d FMD (FMD), 1 day after re-feeding (RF1d) and 3 days after re-feeding (RF3d); for immunohistochemical and morphometric analysis (A to E): n ≥ 6 mice per group, n ≥ 30 islets per staining per time point. mean ± s.e.m,*p<0.05, ** p<0.01, ***p<0.005, one-way ANOVA.^†p<0.05, t-test.

Figure 4. Fasting mimicking diet (FMD) initiates metabolic reprogramming in pancreatic islets and stepwise redirect development of β cells in adult mice

(A) mRNA expression profile indicating changes of metabolic genes in pancreatic islets and (B) mRNA expression profile indicating changes in lineage markers in pancreatic islets, at the end of 4d FMD (FMD) and 1d after refeeding (RF1d), comparing the ad libitum (AL) control; *p<0.05. t-test. Heat-map generated by Qiagen RT² PCR array indicating a fold-regulation ranging from 77 (max, red) to −4 (min, green). (C) Quantification of protein expressing cells of lineage markers in pancreatic islets, from mice fed AL or on FMD at indicated time points. Protein expression was defined as a marker+ area/total islet area. See also Figure S5B. (D) Representative images of immunofluorescent staining indicating stepwise transition of Sox17/Pdx1 and Pdx1/Ngn3. Scale bar represents 50um. Mice of the C57Bl6J background, at age 3–6 months, received no additional treatments other than the indicated diet. Pancreatic samples were collected from mice fed ad libitum (AL) or on the fasting mimicking diet (FMD) at indicated time points: the end of 4d FMD (FMD), 1 day after re-feeding (RF1d) and 3 days after re-feeding (RF3d); n=6 mice per group, ≥ 30 islets per marker.

Figure 5. FMD promotes Ngn3-dependent lineage reprogramming to generate insulin-producing β cells

(A) Genetic strategy used to perform lineage tracing (tdTomato) of NGN3-expressing cells in pancreas and schematic time line of tamoxifen (TAM) treatments for lineage tracing experiments. A, mice fed ad libitum were treated with TAM; B, mice receiving FMD 3 days after TAM injection; C, mice receiving TAM and FMD concurrently and D, mice receiving FMD and vehicle (corn oil) concurrently. Pancreatic tissues were collected 11 days after TAM injection to analyze the effects of FMD on Ngn3-lineage generation. Tdtomato+ cells (red, arrows) are Ngn3-derived cells; n=6 for each group. (B) Representative images of the labeled Ngn3-lineage cells (red, tdTomato) and Insulin-producing β cells (green, Ins) at the indicated time-points in pancreatic islets. Left panel, scale bar represents 150um; right panel, scale bar represents 50um. (C) Quantification of total tdTomato labeled Ngn3-lineage cells per islet (top) and proportion of labeled insulin-producing β cells (ins+tdTomato+) (bottom). mean ± s.e.m, **p<0.01, ***p<0.005, t-test. (D) Genetic strategy used to perform diphtheria toxin gene A chain (DTA)-mediated Ngn3-lineage ablation in pancreas and schematic time line of tamoxifen (TAM) treatments for lineage ablation experiments (left) and results of glucose homeostasis (right). Mice were injected with TAM prior to and after the FMD, to ablate Ngn3 lineage developed and/or expanded during FMD and early refeeding (RF3d). Alternatively, mice were given additional STZ injection and then assigned to the indicated dietary groups (i.e. AL+STZ or FMD+STZ), to analyze the contribution of FMD-induced β-cell conversion to glucose homeostasis. (E) Representative images of pancreatic islets with insulin and Pdx1 immunostaining for β cells, DAPI for nuclei. See also Figure S5 for the images of vehicle controls. (F) Quantification of insulin-producing β cells from Ngn3-lineage ablated mice of indicated groups. mean ± s.e.m,*p<0.05, **p<0.01 t-test, (top) paired t-test (bottom). n=6 for TAM and STZ, n=3 for vehicle controls. Scale bar represents 50um. (G) Glucose levels in homeostasis and intraperitoneal glucose tolerance tests (IPGTTs) for the indicated groups. mean ± s.e.m,**p<0.01 t-test, (top) paired t-test (bottom). n=6 for TAM and STZ, n=3 for vehicle controls. For (A) to (C), mice of ICR and B6;129S6 mixed background, at age 3–6 months, received the diet and/or STZ treatments indicated in (B). For (D) to (G), mice of ICR and B6;129S6 mixed background, at age 3–6 months, received the diet and/or STZ treatments indicated in (F).

Figure 6. Ngn3 expression and insulin producing function of human pancreatic islets in response to fasting conditions

(A) Experimental scheme for fasting conditioning treatments on human pancreatic islet. Pancreatic islets from healthy human subjects (HI) or from T1D subjects (T1DI) were cultured separately based on manufacturer’s instructions and then exposed to fasting conditions (i.e. STS media, mTOR and PKA inhibitors and PKA siRNA) or control media for 36hr. (B) Levels of hIGF-1, glucose, insulin and ketone bodies in the serum from human subjects, prior (baseline) and after receiving the FMD (FMD). n=5 per group. (C) Insulin secretion capacity of HI and T1DI pre-treated with short-term starvation (STS) conditioned medium (2% FBS and 0.5 g/L glucose) and then induced with 25mM glucose, compared to that of islets cultured in standard medium (STD). n=3 per group. (D) Sox2 and (E) Ngn3 expression in HI and T1DI pre-treated with STS-conditioned medium with or without administration of IGF-1 (40ng/ml). n=6 per group. (F) Immunostaining for Ngn3 protein expression in HI and T1DI. n=5 per group. Scale bar represents 100um. (G) Insulin gene expression, (H) PKA activity and (I) mTOR activity in HI and T1DI pretreated with STS-conditioned medium with or without administration of IGF-1 (40ng/ml); phosphorylated versus total p70S6K ratio was used as an indicator of mTOR activity, which was normalized to the levels of STD (standard medium); n=6 per group. (J and K) expression of lineage markers (Sox2 and Ngn3) in HI and T1DI treated with inhibitors dampening IGF-1 signaling; rapamycin, mTOR inhibitor; H89, PKA inhibitor and PKA siRNA. mean ± s.e.m,*p<0.05, ** p<0.01, ***p<0.005, unpaired t-test.