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

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 (Cnop et al., 2005, Dor and Glaser, 2013). 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 (Brandhorst et al., 2015; 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 (Barnosky et al., 2014). 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.

Given that β cells replicate at an extremely low rate in the adult pancreas (Meier et al., 2008, Teta et al., 2005) and that β-cell neogenesis occurs rarely (Xiao et al., 2013), 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 (Fiorina et al., 2008, Kroon et al., 2008, Milanesi et al., 2012, Pipeleers et al., 2002, Villani et al., 2014). 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^db/db), 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 1A). The insulin levels first increased to compensate for insulin resistance but drastically declined after 2 weeks of severe hyperglycemia (Figures 1B and 1C) (Arakawa et al., 2001). 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 1A). FMD cycles caused a major reduction and return to nearly normal levels of blood glucose in db/db mice by day 60 (Figure 1B). The FMD cycles also reversed the decline in insulin secretion at day 30 and improved plasma insulin levels at day 90 (Figure 1C). A homeostasis model assessment (HOMA) was performed to estimate steady-state β-cell function (%B) and insulin sensitivity (%S), as previously described (Hsu et al., 2013, Matthews et al., 1985). The results indicate that the reversal of hyperglycemia was mainly caused by an induction of steady-state β-cell function (%B) (Figure 1D). Nevertheless, mice receiving the FMD showed improved glucose tolerance and insulin tolerance compared to the ad libitum (AL) fed controls (Figure 1E). 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 S2C). 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 1F, ^∗p < 0.05, log-rank test for trend).

Dedifferentiation of β cells, which results in increased non-hormone-producing cells within pancreatic islets, is a feature of diabetic β-cell failure (Dor and Glaser, 2013, Kim-Muller et al., 2014, Talchai et al., 2012). 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 1G 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 1H, 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 1G-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 1J). 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 1J). 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.

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 (Wu and Huan, 2008, Yin et al., 2006). 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 2A 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 2B, 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 2B 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 2D).

Levels of certain circulating cytokines have been used as indicators to determine islet pathological status in patients with recent-onset T1D (Baeyens et al., 2014, Grunnet et al., 2009, Lebastchi and Herold, 2012). 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 2E, day 30) (Dirice et al., 2014, Rabinovitch, 1998, Zhernakova et al., 2006).

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.

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 2F, STZ BL). The transient increase of non-α/β cells was reversed by day 30 in both groups (Figures S2D 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 2F–2H). In contrast, mice that received ad libitum access to regular chow remained depleted of β cells for >50 days (Figures 2F 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.

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 3A) and reduced the proportion of β cells by 35% (Figures 3B and 3C; see also Figure S4 for absolute numbers). These effects were reversed within 3 days of re-feeding (Figures 3A 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 3B and 3C). The expression of the proliferation marker PCNA was elevated in β cells, but not α cells, after re-feeding post FMD (Figures 3B 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 3D). 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 3E).

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 (Cook et al., 2015, Haeusler et al., 2014, Johnson et al., 2004, Kim-Muller et al., 2014, Mu et al., 2006, Stanger, 2008, Talchai et al., 2012, Talchai and Accili, 2015, Tonne et al., 2013) (Figure 4A), 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 4B, 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 4B; 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 4B; Zhou et al., 2008). 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 4C and 4D). In agreement with the results of qPCR array (Figure 4B), 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 4C).

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 (Talchai et al., 2012, Xiao et al., 2013). Oct4 (Pou5f1) mRNA expression showed a trend for an increase in mice on the FMD, which is not significant (Figure 4C, p > 0.05). Results of immunostaining indicate that Oct4 (Pou5f1) and Sox17 may only be co-expressed very transiently after overnight re-feeding (Figure S5B, RF12hr) followed by robust expansion of Sox17⁺Pdx1⁺ and then Pdx1⁺Ngn3⁺ cells at RF1d (Figure 4D and see also Figure S5B 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 (Baeyens et al., 2014, Gomez et al., 2015; Figure 4D). The number of Ngn3+ cells was increased both inside and outside of the islets during the FMD and re-feeding (Figure 4D).

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.

Ngn3⁺ cells 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 (Baeyens et al., 2014, Van de Casteele et al., 2013, Xu et al., 2008). To investigate whether the FMD causes de novo expression of Ngn3 and whether Ngn3⁺ cells may contribute to FMD-induced β-cell regeneration, we generated Ngn3-CreER;tdTomato^LSL-reporter mice to trace the lineage of putative Ngn3-expressing cells and their progeny in the adult mice treated with the FMD (Figure 5A). 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 5A). Tissue collection time points are relative to the time of injection and to that of FMD treatments (Figure 5A). Results indicate that the FMD induces the expansion of the Ngn3-derived lineages (Figure 5B 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 5C, group C).

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

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 (Wang et al., 2009). 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 (McKnight et al., 2010).

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 6A). 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 6B 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 S6A).

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 6C). 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 6D–6F). In cells from normal human subjects, these effects were reversed by IGF-1 treatment (Figure 6G). 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 6G versus Figures 6D 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.

In both healthy and T1D human islets, STS medium significantly reduced the activity of PKA, an effect reversed by IGF-1 treatment (Figure 6H). It also dampened the activity of mTOR, which is a key mediator of amino acid signaling (Figure 6I). 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 (Cheng et al., 2014, Yilmaz et al., 2012). 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 6J and 6K). Interestingly, the constitutive mTOR, but not PKA, activity is trending higher in HI compared to T1D1 cells (Figure 6I, lane 1 for both sets for mTOR activity and Figure 6H for PKA activity), which may explain the overall differences between HI and T1DI in Sox2 and Ngn3 expression shown in Figure 6J. 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.