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.

Although dietary intervention with the potential to ameliorate insulin resistance and type II diabetes has been studied extensively for decades, whether this has the potential to promote a lineage-reprogramming reminiscent of that achieved by iPSCs-based engineering remains unknown. We previously showed that cycles of prolonged fasting (2–3 days) can protect mice and humans from toxicity associated with chemotherapy and can promote hematopoietic stem-cell-dependent regeneration (). In consideration of the challenges and side effects associated with prolonged fasting in humans, we developed a low-calorie, low-protein and low-carbohydrate but high-fat 4-day fasting mimicking diet (FMD) that causes changes in the levels of specific growth factors, glucose, and ketone bodies similar to those caused by water-only fasting () (see also Figure S1 for metabolic cage studies). Here, we examine whether cycles of the FMD are able to promote the generation of insulin-producing β cells and investigate the mechanisms responsible for these effects.

(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.

The ability of animals to survive food deprivation is an adaptive response accompanied by the atrophy of many tissues and organs to minimize energy expenditure. This atrophy and its reversal following the return to a normal diet involve stem-cell-based regeneration in the hematopoietic and nervous systems (). However, whether prolonged fasting and refeeding can also cause pancreatic regeneration and/or cellular reprogramming leading to functional lineage development is unknown. β cells residing in pancreatic islets are among the most sensitive to nutrient availability. Whereas type 1 and type 2 diabetes (T1D and T2D) are characterized by β-cell dedifferentiation and trans-differentiation (), β-cell reprogramming, proliferation and/or stepwise re-differentiation from pluripotent cells are proposed as therapeutic interventions (), suggesting that lineage conversion is critical in both diabetes pathogenesis and therapy ().

In both healthy and T1D human islets, STS medium significantly reduced the activity of PKA, an effect reversed by IGF-1 treatment ( Figure 6 H). It also dampened the activity of mTOR, which is a key mediator of amino acid signaling ( Figure 6 I). To further investigate the role of these nutrient-sensing signaling pathways in regulating the expression of lineage markers (i.e., Sox2 and Ngn3), we tested the role of the mTOR-S6K and PKA pathways, which function downstream of IGF-1, in the reprogramming of pancreatic cells. Human pancreatic islets cultured in standard medium were treated with rapamycin, which inhibits mTOR, and H89, which inhibits PKA. mTOR and PKA were implicated by our group and others in the regeneration of other cell types (). We found that, in human islets from T1D subjects (T1DI), expression of the essential lineage markers Sox2 and Ngn3 was not induced by inhibition of either mTOR or PKA but was significantly induced when both mTOR and PKA were inhibited ( Figures 6 J and 6K). Interestingly, the constitutive mTOR, but not PKA, activity is trending higher in HI compared to T1D1 cells ( Figure 6 I, lane 1 for both sets for mTOR activity and Figure 6 H for PKA activity), which may explain the overall differences between HI and T1DI in Sox2 and Ngn3 expression shown in Figure 6 J. Taken together, these results indicate that fasting cycles may be effective in promoting lineage reprogramming and insulin generation in pancreatic islet cells, in part by reducing IGF-1 and inhibiting both mTOR and PKA signaling. Pancreatic cells from T1D subjects displayed constitutively elevated activity of mTOR-S6K and PKA, which points to the potential for inhibitors of both pathways in the induction of Ngn3-mediated lineage reprogramming. These results raise the possibility that the effect of the FMD on pancreatic regeneration in T1D subjects could be mimicked or enhanced by pharmacological inhibition of these pathways.

We then applied the low-glucose and low-serum fasting mimicking medium (STS) to the cultured pancreatic islets and found that it significantly stimulated the secretion of insulin in both HI and T1DI ( Figure 6 C). We further investigated the expression of lineage-reprogramming markers, which we found to be upregulated in mice as a result of the FMD-treatment (i.e., Nanog, Sox17, Sox2, Ngn3, and Ins). The results indicate that the fasting mimicking conditions had strong effects in inducing the expression of Sox2, Ngn3, and insulin in human pancreatic islets from healthy (healthy islets, HI) and T1D subjects (T1D islets, T1DI) ( Figures 6 D–6F). In cells from normal human subjects, these effects were reversed by IGF-1 treatment ( Figure 6 G). Notably, in human T1D cells, IGF-1 reversed the increased insulin and Sox 2 gene expression, but not that of Ngn3 expression caused by the STS medium ( Figure 6 G versus Figures 6 D and 6E). Future studies are warranted to further investigate the role of circulating IGF-1 in the expression of lineage-reprogramming markers and pancreatic islet cells regeneration in vivo.

To investigate how the fasting mimicking conditions affect Ngn3 expression and β-cell function in human pancreatic cells, we performed ex vivo experiments using primary human pancreatic islets ( Figure 6 A). Briefly, the pancreatic islets from healthy and T1D subjects (HI and T1DI, respectively) were cultured according to the manufacturer’s instructions. The cultured islets were then treated with serum from subjects enrolled in a clinical trial testing the effects of a low-protein and low-calorie FMD lasting 5 days (NCT02158897). Serum samples were collected at baseline and at day 5 of the fasting mimicking diet in five subjects. We then measured IGF-1, glucose, and ketone bodies and treated human pancreatic islets with the subject-derived serum ( Figure 6 B and Table S1 ). In both healthy islets and T1D islets exposed to the serum of FMD-treated subjects, we observed a trend for glucose-dependent induction in the expression of Sox2 and Ngn3 ( Figure S6 A).

(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.

(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.

(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.

(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.

(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.

(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.

In both mouse and humans, Ngn3 expression occurs right before and during endocrine cell generation. Ngn3 mRNA expression in the developing mouse pancreas peaks around E15.5, which is roughly equivalent to week 7–8 (Carnegie stages 21–22) in human development. Expression of Ngn3 in adult mouse islets, although rare, has been demonstrated by rigorous lineage reporter analysis (). In agreement with results from others, our data ( Figures 5 and S5 ) indicate that Ngn3+ cells in adult pancreas islets are important for β-cell regeneration in mice. On the other hand, the role of Ngn3 in human islet development and β-cell regeneration in adulthood remains poorly understood ().

To confirm the contribution of FMD-induced Ngn3 lineages in reconstituting insulin-secreting β cells, we generated another mouse model (Ngn3-CreER/LSL-R26R) and performed lineage-ablation experiments in both wild-type non-diabetic mice and STZ-treated mice ( Figure 5 D). The results indicate that ablation of Ngn3+ lineage reverses FMD-induced β-cell regeneration and its effects on fasting glucose levels ( Figures 5 E and 5F and S5) and glucose clearance capacities (IPGTT assay) in STZ-treated diabetic mice ( Figure 5 G), confirming that the FMD-induced β-cell regeneration is Ngn3 dependent and suggesting a critical role for this in glucose homeostasis.

Ngn3cells within the pancreatic islets have been previously described as progenitor cells able to generate all lineages of endocrine cells, including the insulin-producing β cells, although the role of Ngn3 in adult β-cell regeneration remains unclear (). To investigate whether the FMD causes de novo expression of Ngn3 and whether Ngn3cells may contribute to FMD-induced β-cell regeneration, we generated Ngn3-CreER;tdTomato-reporter mice to trace the lineage of putative Ngn3-expressing cells and their progeny in the adult mice treated with the FMD ( Figure 5 A). To initiate the loxP recombination for lineage tracing, low-dose tamoxifen injections (2 mg per day for 3 days) inducing the recombination (maximized at 48 hr and minimized within a week) were given to mice before or after the FMD and to mice fed ad libitum (AL control) ( Figure 5 A). Tissue collection time points are relative to the time of injection and to that of FMD treatments ( Figure 5 A). Results indicate that the FMD induces the expansion of the Ngn3-derived lineages ( Figure 5 B and 5C). Characterization of tdTomato+ cells by immunostaining indicates that tdTomato+ cells contributed 50.8% ± 8.3% of the overall β-cell pool following the FMD ( Figure 5 C, group C).

(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.

(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.

(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.

(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.

(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.

(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.

These results suggest that, as a result of the FMD and re-feeding cycle, the pancreatic islets contain an elevated number of cells with features of progenitor cells, which may differentiate and generate insulin-producing cells.

To determine whether stepwise β-cell conversion from the dedifferentiated cells occurs during early refeeding, we performed double-staining for the targeted developmental markers (i.e., Sox17, Pdx-1, Ngn3) across the time points of FMD treatment. We also measured the expression of Oct4 (Pou5f1), which has been previously reported to be expressed in the nucleus of adult pancreatic islets in association with Foxo-1-related diabetic β-cell dedifferentiation (). Oct4 (Pou5f1) mRNA expression showed a trend for an increase in mice on the FMD, which is not significant ( Figure 4 C, p > 0.05). Results of immunostaining indicate that Oct4 (Pou5f1) and Sox17 may only be co-expressed very transiently after overnight re-feeding ( Figure S5 B, RF12hr) followed by robust expansion of Sox17Pdx1and then Pdx1Ngn3cells at RF1d ( Figure 4 D and see also Figure S5 B for all time points). Although Ngn3+ cells were also detectable in AL mice, they were mainly located outside or on the edge of the islets, in agreement with what was reported in previous studies ( Figure 4 D). The number of Ngn3+ cells was increased both inside and outside of the islets during the FMD and re-feeding ( Figure 4 D).

To identify the genes that may mediate the FMD-induced pancreatic regeneration, we measured gene expression in pancreatic islets at the end of the FMD and post-FMD re-feeding. At both time points, we observed a transient upregulation of Foxo1 (6.9-fold at FMD, 5.3-fold at RF1d,p < 0.05 comparing to AL) and of a set of genes that have been previously identified as dual regulators for both fat metabolism and fate determination in mammalian cells () ( Figure 4 A), in agreement with the metabolic changes found in mice receiving the FMD ( Figure S1 ). We further examined whether the metabolic reprogramming caused by the FMD affects lineage determination in pancreatic islets. In Figure 4 B, the expression of lineage markers was determined by the mRNA expression of purified islets from mice fed ad libitum (AL) or the FMD. Results from the qPCR array indicate that upregulation of the following genes was statistically significant (p < 0.05 comparing to AL, Figure 4 B; see also Figure S5 ): (1) pluripotency markers (e.g., Lefty1, 3.0-fold during FMD, 7.0-fold at RF1d; Podx1, 3.9-fold during FMD, 9.8-fold at RF1d; Nanog, 2.6-fold during FMD and 5.4-fold RF1d, and Dnmt3b, 31.6-fold during FMD and 18.3-fold RF1d), (2) embryonic development markers (e.g., Sox17, 3.4-fold during FMD and Gata6 3.1-fold during FMD and 2.7-fold at RF1d), (3) pancreatic fetal-stage markers, and (4) β-cell reprogramming markers (e.g., Mafa, 4.7-fold at RF1d; Pdx-1 3-fold during FMD, 5.07-fold at RF1d; and Ngn3, 21.5-fold during FMD, 45.6-fold at RF1d) ( Figure 4 B;). These changes in gene expression suggest that the FMD causes either: (1) a de-differentiation of pancreatic cells toward pluripotency at the end of the diet followed by re-differentiation to pancreatic β-cell lineage during early re-feeding (RF1d) or (2) recruitment of cells with these features from outside of the pancreatic islets. The assessment of protein expression of cells within the islets was also carried out by immunostaining for key proteins associated with pancreatic development ( Figures 4 C and 4D). In agreement with the results of qPCR array ( Figure 4 B), we found that protein levels of Sox17, as the early lineage marker, were elevated at the end of the FMD (FMD-4d) and protein levels of Ngn3, a marker for endocrine progenitors, were transiently upregulated during early re-feeding (FMD-4d to RF1d) ( Figure 4 C).

(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.

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.

(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.

(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 RT 2 PCR array indicating a fold regulation ranging from 77 (max, red) to −4 (min, green).

We investigated whether and how the FMD and the post-FMD re-feeding period could regulate the cell populations within the islets to promote β-cell regeneration independently of diabetes, with a focus on the non-α/β cells and proliferative β cells. To characterize cellular and hormonal changes, pancreatic samples and peripheral blood of wild-type C57Bl6 mice fed with the FMD for 4 days were collected before the diet (BL), at the end of the diet (day 4), and 1 or 3 days after mice returned to the normal diet (RF1d or RF3d). The FMD caused a trend of decrease in the number and size of pancreatic islets ( Figure 3 A) and reduced the proportion of β cells by 35% ( Figures 3 B and 3C; see also Figure S4 for absolute numbers). These effects were reversed within 3 days of re-feeding ( Figures 3 A and 3C). Non-α/β cells began to proliferate at the end of the FMD, and this proliferation persisted until 1 day after re-feeding (RF1d), leading to a 2.5-fold increase in non-α/β cells (proportion per islet) at RF1d ( Figure S4 ). By RF3d, the number of non-α/β cells had dropped and that of β cells returned to basal levels (BL), although β cells remained in a much more proliferative state in the FMD group ( Figure 3 B and 3C). The expression of the proliferation marker PCNA was elevated in β cells, but not α cells, after re-feeding post FMD ( Figures 3 B and 3C and S4 ). Despite the number of α cells per islet remaining the same, the transitional α-to-β or β-to-α cells that co-express both α (i.e., glucagon) and β cell (i.e., Pdx-1 or insulin) markers were increased in mice that received the FMD ( Figure 3 D). In summary, the FMD promotes a decrease in the numbers of differentiated or committed cells, followed by the induction of transitional cells and major increases in the proliferation and number of insulin-generating β cells ( Figure 3 E).

(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.

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. Ϯ p < 0.05, t test.

(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.

The characterization of pancreatic islet cells indicates a strong association between restored glucose homeostasis and the replenishment of pancreatic β cells in animals undergoing FMD cycles. STZ treatments resulted in an increase of non-α/β cells ( Figure S3 ) and an ∼85% depletion of insulin-secreting β cells ( Figure 2 F, STZ BL). The transient increase of non-α/β cells was reversed by day 30 in both groups ( Figures S2 D and S2E). Mice receiving weekly cycles of the FMD showed a major increase in proliferative β cells followed by a return to a nearly normal level of insulin-generating β cells by d50 ( Figures 2 F–2H). In contrast, mice that received ad libitum access to regular chow remained depleted of β cells for >50 days ( Figures 2 F and 2H). Overall, the increase of non-α/β prior to β-cell proliferation raises the possibility that weekly cycle of the FMD might mediate the fate conversion of non-α/β cells to β cells to reverse the STZ-induced β-cell depletion, although other scenarios are possible.

(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.

Taken together, these results indicate that FMD cycles reduce inflammation and promote changes in the levels of cytokines and other proteins, which may be beneficial for the restoration of insulin secretion and the reversal of hyperglycemia.

Levels of certain circulating cytokines have been used as indicators to determine islet pathological status in patients with recent-onset T1D (). We performed a 23-plex immunoassay to determine the effects of the FMD on inflammatory markers. We found that FMD cycles not only suppressed the circulating cytokines associated with β-cell damage (e.g., TNFα and IL-12), but also increased circulating cytokines associated with β-cell regeneration (e.g., IL-2 and IL-10) ( Figure 2 E, day 30) ().

To examine further the role of FMD cycles in stimulating β-cell regeneration, we applied FMD cycles on a T1D model in which high-dose streptozotocin (STZ) treatment causes the depletion of insulin-secreting β cells (). Starting 5 days after STZ treatment, which we refer to as baseline (STZ BL), hyperglycemia (>300 mg/dl) was observed in both mice fed AL and those subjected to multiple cycles of the 4-day FMD every 7 days (4 days of FMD followed by 3 days of re-feeding, every 7 days per cycle) ( Figures 2 A and 2B ). Levels of blood glucose continued to increase in STZ-treated mice receiving the AL diet and reached levels above 450 mg/dl at both days 30 and 50. On the other hand, in mice receiving FMD cycles, hyperglycemia and insulin deficiency were both significantly alleviated on day 30 ( Figure 2 B, sample size indicated in parentheses). Remarkably, the levels of these physiological parameters returned to a nearly normal range at days 50–60 after the FMD cycles ( Figures 2 B and 2C, sample size indicated in parentheses). Intraperitoneal glucose tolerance tests (IPGTTs) at day 50 confirmed that STZ-treated mice undergoing the FMD cycles have improved capacity to clear exogenous blood glucose (STZ+ FMD), compared to mice on the regular chow (STZ) ( Figure 2 D).

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.

(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.

(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.

Dedifferentiation of β cells, which results in increased non-hormone-producing cells within pancreatic islets, is a feature of diabetic β-cell failure (). Similar to what was previously reported by others, we found an increase in cells producing neither insulin nor glucagon (i.e., non-α/β) and a decrease in β-cell number in pancreatic islets of late-stage T2D mice, but not in age-matched WT controls ( Figures 1 G and S2 , db/db BL compared to WT/AL). We also found that β-cell proliferation was low in the late stage of the disease ( Figure 1 H, AL day 60 compared to BL). Whereas db/db mice fed ad libitum (db/db:AL) showed a 60%–80% reduction in β-cell count at day 60, db/db mice receiving FMD cycles (db/db:FMD) displayed a major improvement in the number and proliferation of insulin-generating β cells (comparing db/db BL, Figures 1 G-1I). Pancreatic islets collected from db/db mice treated with FMD cycles (day 60) displayed increased glucose-stimulated insulin secretion (GSIS), compared to that of islets from db/db:AL mice ( Figure 1 J). We also determined that a longer exposure time (time point 120) was necessary to distinguish between the functionality of islets from db/db:AL and db/db:FMD group mice ( Figure 1 J). Overall, these results suggest that, in addition to improving insulin sensitivity, FMD induced β-cell regeneration to reverse β-cell loss, which may alleviate late-stage T2D symptoms and mortality.

Given that β cells replicate at an extremely low rate in the adult pancreas () and that β-cell neogenesis occurs rarely (), depletion of β cells and the consequent loss of insulin secretion during late-stage diabetes have often been considered conditions whose reversals require islet and stem cell transplantation (). To determine whether the FMD could affect the β-cell deficiency associated with T2D, we studied its effect on mice with a point mutation in the leptin receptor gene (Lepr), which causes insulin resistance in the early stages and failure of β-cell function in the late stages. As reported by others, db/db mice developed hyperglycemia at 10 weeks of age, which we refer to as baseline (BL) ( Figure 1 A). The insulin levels first increased to compensate for insulin resistance but drastically declined after 2 weeks of severe hyperglycemia ( Figures 1 B and 1C) (). As a result, db/db mice began to die at around 4 months of age. We attempted to reverse these late-stage T2D phenotypes by treating 12-week-old mice (14 days after the hyperglycemia stabilized, baseline) with weekly cycles of the 4-day FMD ( Figure 1 A). FMD cycles caused a major reduction and return to nearly normal levels of blood glucose in db/db mice by day 60 ( Figure 1 B). The FMD cycles also reversed the decline in insulin secretion at day 30 and improved plasma insulin levels at day 90 ( Figure 1 C). A homeostasis model assessment (HOMA) was performed to estimate steady-state β-cell function (%B) and insulin sensitivity (%S), as previously described (). The results indicate that the reversal of hyperglycemia was mainly caused by an induction of steady-state β-cell function (%B) ( Figure 1 D). Nevertheless, mice receiving the FMD showed improved glucose tolerance and insulin tolerance compared to the ad libitum (AL) fed controls ( Figure 1 E). Notably, although db/db mice on the FMD diet gained less weight compared to those on the regular diet, they maintained a weight that was ∼22% higher than that of their healthy wild-type (WT) littermates during the entire experiment ( Figure S2 C). Altogether, these results indicate that FMD cycles rescued mice from late-stage T2D by restoring insulin secretion and reducing insulin resistance, leading to a major improvement in survival ( Figure 1 F,p < 0.05, log-rank test for trend).

(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.

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

(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.

(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.

(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.

(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.

As a consequence of insulin resistance, the decrease in the number of functional insulin-producing β cells contributes to the pathophysiology of T2D by eventually leading to insulin deficiency (). Previously, we showed that a 4-day fasting mimicking diet (FMD) could induce metabolic changes similar to those caused by prolonged fasting and could reduce insulin and glucose levels while increasing ketone bodies and igfbp1 ( Figure S1 ). Although the role of periodic fasting and fasting mimicking diets on insulin secretion is unknown, the effects of intermittent fasting and chronic calorie restrictions (CR) on insulin sensitivity have been previously reported (). Here, our focus is on the putative effects of the FMD in promoting β-cell regeneration, although we have also investigated the effects of the FMD on insulin resistance.

Discussion

+ endocrine precursors give rise to all of the principal islet endocrine cells, including glucagon+ α cells and insulin+ β cells ( Arnes et al., 2012 Arnes L.

Hill J.T.

Gross S.

Magnuson M.A.

Sussel L. Ghrelin expression in the mouse pancreas defines a unique multipotent progenitor population. Arnes et al., 2012 Arnes L.

Hill J.T.

Gross S.

Magnuson M.A.

Sussel L. Ghrelin expression in the mouse pancreas defines a unique multipotent progenitor population. During mouse development, at embryonic day E8.5, pancreatic progenitor cells co-express the SRY-related HMG-box transcription factor Sox17 and the homeodomain transcription factor Pdx1. These multipotent pancreatic progenitors are then converted into bipotent epithelial cells that generate duct cells or a transient population of endocrine precursor cells expressing the bHLH factor Neurogenin3 (Ngn3). Ngn3endocrine precursors give rise to all of the principal islet endocrine cells, including glucagonα cells and insulinβ cells (). In mice, expression of Ngn3 in the developing pancreas is transient, detectable between E11.5 and E18 (). Whether developmental genes, including Sox17, Pdx-1, and Ngn3, could be activated to generate functional β cells in adults was previously unknown.

In this study, we discovered that a low-protein and low-sugar fasting mimicking diet (FMD) causes a temporary reduction in β-cell number followed by its return to normal levels after re-feeding, suggesting an in vivo lineage reprogramming. We show that the severe hyperglycemia and insulinemia in both the late-stage Leprdb/db T2 and the STZ-treated T1 mouse diabetes models were associated with severe β-cell deficiency in pancreatic islets. Six to eight cycles of the FMD and re-feeding were required to restore the β-cell mass and insulin secretion function and to return the 6-hr-fasting blood glucose to nearly normal levels. In non-diabetic wild-type mice, the portion of β cells per islet, as well as the total number of β cells per pancreas, were reduced at the end of a 4-day FMD, but their normal level was completely restored within 3–5 days post re-feeding. Also, insulin and blood glucose levels were reduced by 70% or more at the end of the FMD period but returned to normal levels within 24–36 hr of re-feeding. Interestingly, in diabetic mice, the majority of cells residing in the islets expressed neither insulin nor glucagon (i.e., non-α/β). This phenotype was also found in non-diabetic wild-type mice during the FMD and was accompanied by an increase of other transitional cell types (i.e., Pdx1+Glucagon+ cells and Insulin+glucagon+) followed by significant β-cell regeneration upon re-feeding. This suggests that the FMD alters the gene expression profile that normally suppresses the generation of β cells. More importantly, these results suggest that dietary-induced lineage conversion occurring prior to the β-cell proliferation may play an important role in β-cell regeneration across the diabetic and non-diabetic mouse models. One possibility is that glucagon and insulin expression are transiently suppressed in α and β cells during the FMD, followed by lineage reprograming in committed cells. Another possibility is that the FMD may cause cell death and then stimulate progenitor or other cells to regenerate β cells.