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 from 10 weeks (baseline, BL) to 12 weeks and were 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 ± SEM) in age-matched healthy wild-type littermates. Blood samples were collected at the last refeeding day/first day of the indicated cycles. Mice were fasted for 6 hr (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 × fasting insulin)/(fasting glucose – 3.5)%.

(E) Glucose tolerance test and insulin tolerance test at day 60.

(B–E) Each point represents the mean ± SEM. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.005, two-way ANOVA.

(F) Survival curve. Mean ± SEM,^∗p < 0.05, log-rank (Mantel-Cox) test for trend. n ≥ 16 mice per group.

(G) Proportion of β cells per islet. n ≥ 6 mice per group, n ≥ 15 islets per sample.

(H) Proliferative proportion of β cells per islet.

(G–I) Mean ± SEM,^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.005, one-way ANOVA. n ≥ 6 mice per group, n ≥ 15 islets per sample.

(I) Immunostaining of pancreatic sections from Lepr^db/db mice and their wild-type littermates at the indicated time points. Arrow in the 8×-enlarged example image indicates a typical proliferative β cell (PCNA⁺Insulin⁺). Scale bar, 50 μm.

(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, 50 μm.

Mice are of the C57BL/6J background of the age indicated. In (A), mice received no additional treatments other than the indicated diet.

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 ± 12 mg/dl) indicate levels of blood glucose in the naive 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 naive controls.

Pancreatic samples collected at indicated time points were analyzed for:

(F) PCNA+ proliferating 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, 50 μm.

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 ± SEM, and sample size (n) is indicated in parentheses. (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) Proportion of PCNA+ proliferating β cells and of total β cells per islet.

(C) Representative images of pancreatic islets with insulin, glucagon, and PCNA immuno-staining. Scale bar, 50 μm.

(D) Transitional cell populations 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. Scale bar, 50 μm.

(E) Schematic of FMD and post-FMD refeeding induced cellular changes in pancreatic islets.

Mice of the C57BL/6J background at ages 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–E): n ≥ 6 mice per group, n ≥ 30 islets per staining per time point. Mean ± SEM,^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.005, one-way ANOVA. ^Ϯp < 0.05, t test.

Figure 4

FMD Promotes Expression of Genes in Pancreatic Islets Characteristic of Embryonic and Fetal Development

(A) mRNA expression profile indicating changes in metabolic genes in pancreatic islets and (B) mRNA expression profile indicating changes in lineage markers in pancreatic islets at the end of 4 days FMD (FMD) and 1 day after refeeding (RF1d), comparing the ad libitum (AL) control. ^∗p < 0.05, t test. Heatmap 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 S5 B. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.005. t test comparing to AL control.

(D) Representative images of immunofluorescent staining indicating stepwise transition of Sox17/Pdx1 and Pdx1/Ngn3. Scale bar, 50 μm.

Mice of the C57Bl6J background at ages 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 FMD at indicated time points: the end of 4 days 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 timeline 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. (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) Scale bar, 200 μm; (right) scale bar, 100 μm.

(C) Quantification of total tdTomato-labeled Ngn3 lineage cells per islet (top) and proportion of labeled insulin-producing β cells (ins+tdTomato+) (bottom). Mean ± SEM, ^∗∗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. Scale bar, 50 μm.

(F) Quantification of insulin-producing β cells from Ngn3-lineage ablated mice of indicated groups. Mean ± SEM, ^∗p < 0.05, ^∗∗p < 0.01 t test, (top) paired t test (bottom). n = 6 for TAM and STZ, n = 3 for vehicle controls.

(G) Glucose levels in homeostasis and intraperitoneal glucose tolerance tests (IPGTTs) for the indicated groups. Mean ± SEM, ^∗∗p < 0.01 t test, (top) paired t test (bottom). n = 6 for TAM and STZ, n = 3 for vehicle controls.

(A–C) Mice of ICR and B6;129S6 mixed background at ages 3–6 months received the diet and/or STZ treatments indicated in (A).

(D–G) Mice of ICR and B6;129S6 mixed background at ages 3–6 months received the diet and/or STZ treatments indicated in (D).

Figure 6

Ngn3 Expression and Insulin-Production in 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 were then exposed to fasting conditions (i.e., STS medium, mTOR and PKA inhibitors, and PKA siRNA) or control medium for 36 hr.

(B) Levels of hIGF-1, glucose, insulin, and ketone bodies in the serum from human subjects prior to (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 25 mM 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 (40 ng/ml). n = 6 per group.

(F) Immunostaining for Ngn3 protein expression in HI and T1DI. n = 5 per group. Scale bar, 100 μm.

(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 (40 ng/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 ± SEM,^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.005, unpaired t test.

Figure S1

Diet: FMD, Related STAR Methods

(A) Metabolic effects of FMD and short-term starvation (STS) on body weights with lean- and fat-mass ratio prior to, after STS or FMD and 3 days after refeeding. (B) Water intake, food intake (kcal/day), Total movement and VCO2/VO2 before, during and after STS and (C) after FMD. (D) Levels of circulating insulin and ketone body (β-HB) in mice on FMD and post-FMD refeeding, comparing to that of mice under prolonged fasting (24, 36 and 60 hr). ^∗p < 0.05, ^∗∗p < 0.01 and ^∗∗∗p < 0.005, t test.

Figure S2

Effects of FMD in Lepr^db/db Mice, Related to Figure 1 and STAR Methods

(A) Numbers of indicated cell type per islet, (B) Proliferation frequency of indicated cell type per islet, (C) Body weight and (D) Proliferation frequency and numbers and (E) example image of non-insulin/glucagon producing cells (non-α/b) and Pdx1⁺α cells. (F) Levels of circulating insulin during IPGTT. (G) illustration of pancreatic islet sampling. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.005, one-way ANOVA.

Figure S3

Effects of FMD Cycles on STZ-Treated Mice, Related to Figure 2

(A) body weight, one cycle of FMD (B) Numbers of indicated cell type per islet, (C) Proliferation frequency of indicated cell type per islet, (D) Proliferation frequency of α cells and number of Pdx1⁺α cells per islet and (E) Proliferation frequency and numbers of the non-insulin/glucagon producing cells (non-α/b) and (F) Levels of circulating cytokines. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.005, t test.

Figure S4

Effects of FMD and Post-FMD Refeeding on Non-diabetic Wild-Type Mice, Related to Figure 3

(A) Number and area of pancreatic islets per pancreas section. (B) Numbers of indicated cell type per islet. (C) Proportion and (D) number of Proliferation frequency of indicated cell type per islet. (E) Number of Pdx1+α transitional cells per islet, (F) Representative images of Pdx1+α transitional cells. (G) z stack confocal microscopy images of Gluc+ Ins+ cells (H) Proliferation frequency and numbers of the non-insulin/glucagon producing cells (non-α/b) in wild-type mice without STZ treatments. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.005, one-way ANOVA.

Figure S5

Effects of FMD on Expression of Developmental Markers of β-Cell in Adult Mice, Related to Figures 4 and 5

(A) Fold-regulation of genes significantly (^∗p < 0.05) up- or downregulated by FMD or RF1d comparing to AL. The p values are calculated based on a Student’s t test of the replicate 2^∧(- Delta Ct) values for each gene in the control group and treatment groups. (B) Immunostaining for proteins expression of lineage markers in pancreatic islets. (C) schematic time line and representative images of corn oil (vehicle control) treatments for Ngn3-lineage ablation experiments shown in Figure 5 F.

Figure S6

Related to Figure 6

(A) Gene expression and (B) insulin production of healthy pancreatic islets (HI) and T1DI treated with serum form subjects at indicated time points. ^∗∗∗∗p < 0.0001, t test.