Nicotinamide riboside kinase 1 protects against diet and age-induced pancreatic β-cell failure

Abstract

Objective: Disturbances in NAD⁺ metabolism have been described as a hallmark for multiple metabolic and age-related diseases, including type 2 diabetes. While alterations in pancreatic β-cell function are critical determinants of whole-body glucose homeostasis, the role of NAD⁺ metabolism in the endocrine pancreas remains poorly explored. Here, we aimed to evaluate the role of nicotinamide riboside (NR) metabolism in maintaining NAD⁺ levels and pancreatic β-cell function in pathophysiological conditions.

Methods: Whole body and pancreatic β-cell-specific NRK1 knockout (KO) mice were metabolically phenotyped in situations of high-fat feeding and aging. We also analyzed pancreatic β-cell function, β-cell mass and gene expression.

Results: We first demonstrate that NRK1, the essential enzyme for the utilization of NR, is abundantly expressed in pancreatic β-cells. While NR treatment did not alter glucose-stimulated insulin secretion in pancreatic islets from young healthy mice, NRK1 knockout mice displayed glucose intolerance and compromised β-cells response to a glucose challenge upon high-fat feeding or aging. Interestingly, β cell dysfunction stemmed from the functional failure of other organs, such as liver and kidney, and the associated changes in circulating peptides and hormones, as mice lacking NRK1 exclusively in β-cells did not show altered glucose homeostasis.

Conclusions: This work unveils a new physiological role for NR metabolism in the maintenance of glucose tolerance and pancreatic β-cell function in high-fat feeding or aging conditions.

Keywords: Metabolic disease; NAD(+); Nicotinamide riboside; Nicotinamide riboside kinase 1 (NRK1).

Figure 1

NR acts as a NAD⁺ precursor in pancreatic β-cells. (A) NAD⁺ biosynthesis paths used by nicotinic acid (NA), nicotinamide (NAM) and nicotinamide riboside (NR). In the figure, NAMN: NA mononucleotide; NAAD: NA adenine dinucleotide; NMN: NAM mononucleotide; NAPRT: NA phosphoribosyltransfersase; NMNAT: NMN adenylyltransferase; NADSYN: NAD synthase; NAMPT: Nicotinamide phosphoribosyltransferase; NRK: NR kinase. (B) Tissue homogenates from livers, pancreas and islets from ∼-20 week old wild type (WT) and NRK1 KO (KO) mice were used for western blotting, together with protein homogenates from INS-1E cells. A representative image is shown. (C) Total RNA was extracted from WT and NRK1 KO islets and the expression of different NAD⁺ metabolism enzymes was evaluated by real-time quantitative PCR. (D) Gene expression of NAD⁺ metabolic enzymes in islets from control (Nmrk1 floxed mice; white bars) and β-cell specific NRK1 KO mice (NRK1 BKO; grey bars). (E) Pancreatic islets from WT and NRK1 KO mice were collected and incubated with PBS (as vehicle) or NR (0.5 mM) for 2 h. Then acidic extracts were obtained to measure NAD⁺ levels. (F) NAD⁺ levels in pancreatic islets from either control or NRK1 BKO mice treated with PBS (as vehicle), NR (0.5 mM) or NAM (5 mM) for 2 h. (G) INS-1E cells were either left untreated (Ctrl) or transfected with empty vector (EV) or FLAG-NRK1 (NRK1) for 48 h prior to being treated with PBS (as vehicle) or NR (0.5 mM, 2 h). Then, acidic extracts were obtained to measure NAD⁺ levels. (H) As in (G), but 48 h after transfection, protein homogenates were obtained to evaluate the efficacy of our transfection by western blot. (I–J) Islets from WT mice were collected and incubated with either PBS (as vehicle) or NR (0.5 mM, 2 h) prior the evaluation of glucosestimulated insulin secretion (GSIS). Insulin secreted to the media (I) and cellular insulin content (J) were then analyzed. All values are expressed as mean +/-SEM of n = 5 per genotype (C–D), or n = 8 WT mice and n = 4 NRK1 KO mice (E), or n = 4 per genotype (F), or n = 4 independent experiments (G–J). ∗ indicates p < 0.05 vs. the respective WT or control group (C–F) or vs the vehicle group (E–G). ^# indicates p < 0.05 vs the vehicle or EV group within the same treatment (E–G).

Figure 2

NRK1 KO mice display alterations in lipid oxidation rates upon high-fat feeding. Eight week old wild type (WT) and NRK1 KO mice were placed on either a low-fat diet (LFD) or a high-fat diet (HFD). (A) Body weight of WT and NRK1 KO mice 16 weeks after the initiation of the LFD or HFD treatments. (B–C) Food intake (B) and daily activity (C) were evaluated 8 weeks after the initiation of the regimes, using a comprehensive laboratory animal monitoring system (CLAMS, Columbus instruments). (D–E) Tissues weight from LFD (D) or HFD (E) fed animals were determined 16 weeks after the initiation of the diets. (F–H) WT and NRK1 KO mice submitted to a HFD were used for indirect calorimetry experiments using a CLAMS systems 8 weeks after the initiation of the dietary intervention. VO₂ values (F), respiratory exchange ratios (RER; VCO₂/VO₂) (G) and lipid oxidation rates (H) were then estimated. Through the figure, white bars represent WT mice, while black bars represent NRK1 KO mice. All values are expressed as mean +/-− SEM of n = 10 mice for the WT group and n = 11 mice for the NRK1 KO group. ∗ indicates p < 0.05 vs. the respective WT group.

Figure 3

NRK1 KO mice under HFD exhibit pancreatic β-cell dysfunction. (A–B) intraperitoneal glucose tolerance tests (ipGTT) were performed on HFD-fed wild type (WT) and NRK1 KO mice 10 weeks after the initiation of the dietary regime. Glucose excursions (A) and circulating insulin levels (B) were then evaluated. (C) Intraperitoneal insulin tolerance tests (ipITT) were performed on HFD-fed WT and NRK1 KO mice 12 weeks after the initiation of the dietary regime. (D–H) Histological evaluation and quantifications for pancreatic islets characteristics in HFD-fed WT and NRK1 KO mice. The scale bar on (D) is set at 500 μm. (I–J) Pancreatic islets from WT and NRK1 KO mice submitted to a HFD for 16 weeks were used for ex vivo glucose-stimulated insulin secretion (GSIS) assays. In (I), % of insulin secretion in the media is shown, while (J) displays total insulin content in islets. (K) Total RNA was isolated from WT and NRK1 KO pancreatic islets, 16 weeks after the initiation of the diet, and multiple markers were analyzed by real time quantitative PCR. Through the figure, white bars represent WT mice, while black bars represent NRK1 KO mice. All values are expressed as mean +/-− SEM of n = 10 WT mice and n = 11 for NRK1 KO mice (A–C). For histology image analysis, all values are expressed as mean +/-−SEM of entire pancreas sections from n = 4 per mice genotype (E–H). For the work on isolated islets, values are expressed as mean +/-− SEM of n = 5 mice per genotype (I–J) or n = 5 WT mice and n = 4 NRK1 KO mice (K). ∗ indicates p < 0.05 vs. the respective WT group.

Figure 4

Metabolic abnormalities in aged NRK1 KO mice. (A–B) Body (A) and tissue (B) weights of wild type (WT) and NRK1 KO mice at 24 months of age. (C–E) WT and NRK1 mice, around 23 months of age were used for indirect calorimetry experiments using a CLAMS systems. VO₂ values (C), respiratory exchange ratios (RER; VCO₂/VO₂) (D) and lipid oxidation rates (E) were then estimated. (F–G) Intraperitoneal glucose tolerance tests were performed on ∼23 month old WT and NRK1 KO mice. Glucose excursions (F) and circulating insulin levels (G) were then evaluated. (H–I) 22 month old WT and NRK1 mice were fasted for 24 h and the refed for 2 h. Blood glucose (H) and insulin (I) levels were analyzed in the fasted and refed state. (J) WT and NRK1 KO at 23–24 months of age were challenged with 1 U/kg of insulin after a 6 h fast. Then, blood glucose levels were evaluated for 2 h. Through the figure, white bars represent WT mice, while black bars represent NRK1 KO mice. All values are expressed as mean +/-− SEM of n = 12 for WT mice and n = 11 for NRK1 KO mice. ∗ indicates p < 0.05 vs. the respective WT group.

Figure 5

Pancreatic defects in aged NRK1 KO mice. (A–E) Pancreas from 24 month old wild type (WT) and NRK1 KO mice were used for histological evaluation and the quantification of islet counts (B), islet size (C) and the respective contribution of α or β-cells to overall pancreatic mass (D–E). The scale bar in (A) stands for 500 μm. (F–G) Total pancreas homogenates from 24 month-old WT and NRK1 KO mice were used to analyze insulin (F) and glucagon (G) content. (H–I)- Pancreatic islets from 24 months-old WT and NRK1 KO mice were used for glucose-stimulated insulin secretion (GSIS) assays. In (H), % of insulin secretion to the media is shown, while (I) shows total insulin content in islets. (J) Total RNA was extracted from pancreatic islets of 24 months-old WT and NRK1 KO mice and multiple markers were analyzed by real time quantitative PCR. (K–L) Representative sirius red stating of pancreas sections from aged WT and NRK1 KO mice (K) and quantification of fibrotic depots from the different samples (L). Through the figure, white bars represent WT mice, while black bars represent NRK1 KO mice. For histology image analysis, all values are expressed as mean +/-− SEM of entire pancreas sections from n = 4 per genotype (B-E; L). For all other analyses, values are expressed as mean +/-− SEM of n = 7 WT mice and n = 8 NRK1 KO mice (F–G) or n = 4 per genotype (H–I) or n = 6 WT mice and n = 7 NRK1 KO mice (J). ∗ indicates p < 0.05 vs. the respective WT group.

Figure 6

NRK1 deletion prompts age-related fibrosis in multiple tissues. (A–C) Pancreas, liver and kidney from 3 months-old (Young) and 24 months-old (Old) wild type (WT) or NRK1 KO mice were used to analyzed total NAD⁺ content. (D–E) Total mRNA was extracted from liver (D) or kidney (E) from 24 month-old WT and NRK1 KO mice and fibrosis markers were evaluated by real time quantitative PCR. (F–G) Liver (F) and kidney (G) homogenates from 24 month old WT or NRK1 KO mice were used for high-resolution respirometry studies. Samples were treated with malate, pyruvate and glutamate to stimulate complex I in the absence of ADP (Leak), then ADP was added to evaluate coupled CI activity (CI). Next, succinate was added to stimulate Complex II and evaluate CI + CII driven respiration (CI + CII). FCCP was added to evaluate maximal electron transport system capacity (ETS). Rotenone was used then to evaluate the contribution of CII to ETS (ETC CII). (H) The levels of different circulating factors were evaluated in plasma from young (3 month-old) or old (24 month-old) WT and NRK1 KO mice by dot-blot analyses. (I) The levels of active GLP-1 in plasma were measured in young (3 month old) and old (24 month old) WT and NRK1 KO mice after a 16 h fast and 45 min after refeeding, using a commercial immunoassay (Meso Scale Discovery). Through the figure, white bars represent WT mice, while black bars represent NRK1 KO mice. All values are expressed as mean +/-− SEM of n = 12 WT mice and n = 11 NRK1 KO mice (A–C), or n = 8 per genotype (D–G) or n = 4 mice per genotype (H) or n = 6 mice per genotype (I). ∗ indicates p < 0.05 vs. the respective WT group.

Figure 7

Summary of diet and age-related pancreatic β-cell defects in NRK1 KO mice. NRK1 KO mice cannot utilize nicotinamide riboside (NR) as a NAD⁺ precursor, but can use other common NAD⁺ precursors, such as nicotinamide (NAM) or nicotinic acid (NA). When submitted to a high-fat diet, NRK1 KO mice displayed exacerbated glucose intolerance and impaired glucosestimulated insulin secretion. A similar phenotype is observed in aged NRK1 KO mice. While impaired pancreatic function in obese NRK1 KO seems to stem from dysfunctional β-cells and altered islet gene expression, the impairments in aging rather seem to stem from reduced β--cell mass and pancreatic fibrosis. Our research also suggests that a common factor that could contribute to impaired glucose stimulated insulin secretion in both scenarios (obesity and aging) is an increase in circulating DPP-IV levels. The figure was created with BioRender.com.