FRD-fed mice display a pro-inflammatory phenotype with elevated blood glucose
FRD-fed mice developed a pro-inflammatory phenotype, with raised Il1b mRNA in WAT, BAT and islets (Fig. 1a–c), and increased Tnfa (also known as Tnfa) expression in WAT and BAT, but not in islets (Fig. 1d–f). Development of a pro-inflammatory phenotype was associated with raised fasting levels of plasma glucose and insulin in FRD-fed mice compared with control (Fig. 1g, h) and raised fed plasma glucose levels (Fig. 1i). Taken together, mice on an FRD for 16 weeks displayed characteristics of type 2 diabetes, including fasting hyperglycaemia and chronic inflammation, and thus are an attractive model for study of impaired islet function.
FRD increases iNAMPT abundance in adipose tissue while suppressing circulating eNAMPT concentrations
We next assessed whether increased inflammation and raised plasma glucose were associated with altered iNAMPT abundance in BAT and WAT (predominant sites of synthesis of iNAMPT and release of eNAMPT), or with changes in plasma eNAMPT in FRD-fed mice. Abundance of iNAMPT and expression of Nampt were increased in BAT (Fig. 2a, b) and WAT (Fig. 2c, d) of FRD mice, but despite raised iNAMPT levels, plasma levels of eNAMPT (Fig. 2e) were markedly decreased in FRD-fed mice compared with control.
Insulin secretion is suppressed in FRD-fed mice
We next assessed whether decreased eNAMPT was associated with impaired islet function. To assess the direct effects of FRD on islet function, insulin secretion in response to glucose or leucine was measured ex vivo in isolated islets. GSIS was markedly reduced by 75 ± 3% (mean±SEM; p < 0.05) (Fig. 3a) in islets isolated from FRD-fed mice compared with control mice. Similarly, LSIS was suppressed (91 ± 7%; p < 0.01) in FRD-fed mice relative to control mice (Fig. 3b). Basal insulin secretion (at 3 mmol/l glucose and 2 mmol/l leucine) was unchanged in FRD-fed mice (Fig. 3a, b). Taken together, decreased eNAMPT in FRD-fed mice was associated with suppressed islet function and increased inflammation.
NMN administration protects against islet dysfunction in FRD-fed mice
+/− mice show impaired islet function , we reasoned that the suppressed eNAMPT levels observed in FRD-fed mice might play a role in the onset of islet dysfunction in these mice. To further examine this, we investigated whether the reaction product of eNAMPT, NMN, provided protection in vivo against beta cell dysfunction in FRD-fed mice. NMN (500 mg/kg body weight; i.p.) [9, 14] was administered to FRD-fed mice 16 h prior to islet isolation. The effects of NMN administration in vivo on insulin secretion ex vivo were first examined with islets isolated from control mice maintained on a standard diet (CON+NMN group). Islets isolated from CON+NMN mice displayed elevated GSIS (twofold; p < 0.01) (Fig. 3c) compared with CON+V mice (which had been injected with saline). Similarly, LSIS was also modestly but significantly increased in CON+NMN mice (37 ± 8%) compared with CON+V mice (Fig. 3d). NMN had no effect on basal rates of insulin secretion measured at 3 mmol/l glucose or 2 mmol/l leucine (Fig. 3c, d). Significantly, in vivo administration of NMN abolished the suppressive effects of FRD on GSIS and LSIS (Fig. 3e, f). Whereas NMN administration to FRD-fed mice completely restored LSIS ex vivo (Fig. 3f), GSIS was significantly elevated above rates seen in CON+V mice (Fig. 3e) and also greatly exceeded those of CON+NMN mice.
NMN protects against pro-inflammatory cytokine-mediated islet dysfunction
FRD-fed mice developed a pro-inflammatory phenotype. Chronic inflammation through exposure to pro-inflammatory cytokines impairs beta cell function [5, 6]. We hypothesised that the beneficial effects of NMN on FRD-mediated islet dysfunction may occur in part through protection from the effects of pro-inflammatory cytokines. Therefore we investigated whether NMN protected against cytokine-mediated islet dysfunction in islets isolated from mice on a standard diet (control). We first assessed whether NMN affected GSIS and LSIS ex vivo. Culture of islets with NMN (100 μmol/l; 48 h) increased GSIS by 24% (p < 0.05) (Fig. 4a) and greatly enhanced LSIS by 2.7-fold (p < 0.001) (Fig. 4a). To investigate the effects of NMN on pro-inflammatory cytokine-mediated beta cell dysfunction, islets isolated from control mice were incubated with IL1β (5 ng/ml) and TNFα (10 ng/ml), or cultured with IL1β/TNFα plus NMN (100 μmol/l) for 48 h. In islets exposed to IL1β/TNFα, insulin secretion was significantly impaired in response to incubation with 17 mmol/l glucose (39%; p < 0.001) (Fig. 4c) and 20 mmol/l leucine (34%; p < 0.05) (Fig. 4d). However, co-incubation of control islets with NMN completely blocked the effects of IL1β/TNFα, restoring GSIS and LSIS (Fig. 4c, d). IL1β reportedly exerts auto-stimulatory effects, whereby it is able to induce its own production in beta cells, as well as that of TNFα . In agreement with this, Il1b mRNA and the corresponding protein levels, as well as Tnfa mRNA, were increased in islets exposed to IL1β/TNFα (Fig. 4e, f).
Glucolipotoxicity, which results from elevated circulating levels of glucose and NEFA such as palmitate, can also lead to suppression of islet insulin secretion [26–28], in part through induction of Il1b expression [5, 6, 29]. Consistent with this, exposure of islets to palmitate (100 μmol/l) and glucose (20 mmol/l) in combination for 48 h induced Il1b mRNA (Fig. 4g), and suppressed GSIS and LSIS (Fig. 4h, i). Similarly to the effects seen in cytokine-exposed islets, the effects of palmitate/glucose were reversed by co-incubation with NMN (Fig. 4h, i).
NMN reverses FRD and pro-inflammatory cytokine-mediated changes in expression of genes encoding islet markers
Suppressed GSIS and LSIS in FRD+V mouse islets occurred in parallel with changes in expression of genes encoding key islet markers. Pdx1, which encodes a transcription factor essential for islet beta cell differentiation , was suppressed in FRD+V islets compared with CON+V (−21%; p < 0.05) (Fig. 5a). Similarly, expression of the glucose transporter Glut2 (also known as Slc2a2) and glucokinase (Gk, also known as Gck), the latter of which initiates glycolysis following glucose uptake into the islet, and both of which are under the transcriptional control of PDX1 , was suppressed by 51% (p < 0.01) and 17% (p < 0.05), respectively (Fig. 5b, c). Similarly to effects on GSIS and LSIS, FRD-mediated changes in gene expression were reversed in FRD-fed mice administered NMN, indicating that NMN improves islet function in part through beneficial changes in expression of several genes essential for glucose sensing and beta cell differentiation. FRD also led to elevated islet mRNA levels of inducible nitric oxide synthase (Inos [also known as Nos2]) (49%; p < 0.05) (Fig. 5d), a target (via nuclear factor κB [NFκB]) of IL1β ; inducible nitric oxide synthase induces cellular stress and cell death through production of reactive oxygen species. In addition, mRNA levels of the pro-apoptotic gene Bax (23%; p < 0.05) (Fig. 5e) were elevated in FRD+V mice. FRD-mediated induction of Inos and Bax expression was blocked by NMN (Fig. 5d, e). NMN administration also lowered increased Il1b expression to basal levels in FRD+NMN compared with FRD+V mice (p < 0.001) (Fig. 5f), indicating that a potential anti-inflammatory mechanism mediates the actions of NMN.
Similarly, isolated islets incubated with IL1β/TNFα displayed reduced expression of Pdx1 (72%; p < 0.001), Glut2 (90%; p < 0.01) and Gk (42%; p < 0.05), as well as increased expression of Inos (3.9-fold; p < 0.001) and Bax (twofold; p < 0.001). These changes in gene expression elicited by IL1β/TNFα were reversed by co-incubation with NMN (ESM Fig. 1a–e). Moreover, the IL1β/TNFα-mediated induction of Il1b gene expression and production of the corresponding protein that are described above were also suppressed by NMN (ESM Fig. 1f), supporting the notion of an anti-inflammatory mechanism of NMN action. Expression of two other islet transcription factors, Tfam and Hnf1a, were unchanged by IL1β/TNFα and/or NMN (data not shown). These changes in gene expression in response to pro-inflammatory cytokines and NMN are reminiscent of those observed in islets isolated from FRD-fed mice.
Taken together, these data indicate that NMN improves islet function in FRD-fed mice in association with beneficial changes in expression of genes involved in glucometabolic, anti-inflammatory and apoptotic processes.
NMN-mediated induction of insulin secretion involves sirtuin 1
We next assessed a possible role for sirtuin 1 as a target mediating the actions of NMN in islets. Consistent with a potential role for sirtuin 1 in mediating the effects of NMN, expression of Sirt1 was suppressed by 65% in FRD+V mice (p < 0.05) (Fig. 5h). In addition, FRD+V mice displayed decreased mRNA levels of the mitochondrial sirtuin, Sirt3 (29%; p < 0.05) (Fig. 5i). The role of sirtuin 3 in islet function is not yet known, but it is known to positively regulate mitochondrial ATP production [33–35], which is important for nutrient-stimulated insulin secretion, and to be induced by NAMPT [36, 37]. These effects were reversed by NMN in FRD+NMN mice (Fig. 5h, i). Similarly, IL1β/TNFα treatment led to a 46% (p < 0.01) reduction in Sirt1 mRNA and a 54% (p < 0.001) decrease in Sirt3 mRNA. Co-incubation of NMN with TNFα/IL1β restored Sirt1 and Sirt3 expression to control levels (ESM Fig. 2a, b). Consistent with the notion of a mechanistic role for sirtuin 1 in mediating the effects of NMN, co-incubation of islets with the specific sirtuin 1 inhibitor EX-527 blocked the enhancing effects of NMN alone upon insulin secretion (ESM Fig. 2c). We next investigated whether EX-527 could inhibit the effect of NMN on cytokine-mediated islet dysfunction. The effects of NMN in restoring TNFα/IL1β-mediated suppression of GSIS were partially blocked by EX-527 (45%) (ESM Fig. 2d), suggesting that sirtuin 1 mediates approximately 55% of the protective effects of NMN against cytokine-mediated impairment of GSIS.