Adipose tissue NAD+ biosynthesis is required for regulating adaptive thermogenesis and whole-body energy homeostasis in mice

Abstract

Nicotinamide adenine dinucleotide (NAD⁺) is a critical coenzyme for cellular energy metabolism. The aim of the present study was to determine the importance of brown and white adipose tissue (BAT and WAT) NAD⁺ metabolism in regulating whole-body thermogenesis and energy metabolism. Accordingly, we generated and analyzed adipocyte-specific nicotinamide phosphoribosyltransferase (Nampt) knockout (ANKO) and brown adipocyte-specific Nampt knockout (BANKO) mice because NAMPT is the rate-limiting NAD⁺ biosynthetic enzyme. We found ANKO mice, which lack NAMPT in both BAT and WAT, had impaired gene programs involved in thermogenesis and mitochondrial function in BAT and a blunted thermogenic (rectal temperature, BAT temperature, and whole-body oxygen consumption) response to acute cold exposure, prolonged fasting, and administration of β-adrenergic agonists (norepinephrine and CL-316243). In addition, the absence of NAMPT in WAT markedly reduced adrenergic-mediated lipolytic activity, likely through inactivation of the NAD⁺-SIRT1-caveolin-1 axis, which limits an important fuel source fatty acid for BAT thermogenesis. These metabolic abnormalities were rescued by treatment with nicotinamide mononucleotide (NMN), which bypasses the block in NAD⁺ synthesis induced by NAMPT deficiency. Although BANKO mice, which lack NAMPT in BAT only, had BAT cellular alterations similar to the ANKO mice, BANKO mice had normal thermogenic and lipolytic responses. We also found NAMPT expression in supraclavicular adipose tissue (where human BAT is localized) obtained from human subjects increased during cold exposure, suggesting our finding in rodents could apply to people. These results demonstrate that adipose NAMPT-mediated NAD⁺ biosynthesis is essential for regulating adaptive thermogenesis, lipolysis, and whole-body energy metabolism.

Keywords: NAD; adipose tissue; energy metabolism; lipolysis; thermogenesis.

Fig. 1.

NAMPT-mediated NAD⁺ biosynthesis is associated with BAT activity in mice and humans. (A) In mammals, NAMPT functions as a key enzyme in the salvage biosynthetic pathway and converts nicotinamide into NMN. NaMN, nicotinic acid mononucleotide; NaAD⁺, nicotinic acid adenine dinucleotide. The levels of NAD⁺ and key intermediates (B), gene expression of Ucp1 and Nampt, and NAMPT protein expression (C) were measured in BAT obtained from mice kept under thermoneutrality (TN, 30 °C) and cold exposure (CE, 4 °C) (n = 4 to 5 per group). (D) Gene expression of UCP1 and NAMPT was determined in BAT obtained from human subjects under the thermoneutral (TN, 26–28 °C) and cold (CE, ∼22 °C) conditions. *Value significantly different from the TN value (P < 0.05). Values are means ± SEM.

Fig. 2.

ANKO mice have severe cold intolerance. The levels of NAMPT protein (A) and NAD⁺ metabolites (B) were determined in BAT (n = 3 to 5 per group). (C) BAT weight normalized by body weight (n = 5 per group). (D) Images of hematoxylin and eosin (H&E) staining of BAT. (E) Heat map of DEGs induced by Nampt knockout in BAT (n = 4 per group). Z-scores were calculated from the log-transformed fragments per kilobase of transcript per million fragments mapped values in RNA-seq data. (F) Enriched pathways of the down-regulated DEGs. (G) Gene expression of BAT-selective markers and proteins involved in thermogenesis and FFA metabolism and lipogenic enzymes (n = 4 per group). (H) BAT gene expression of mitochondrially encoded proteins and mitochondrial DNA (mtDNA) contents normalized to nuclear DNA (nucDNA) contents (n = 4 to 5 per group). (I) ETC complex-I and -IV activities (n = 3 to 6 per group) and ATP contents (n = 2 to 3 per group) in isolated mitochondria. Rectal temperature during cold exposure after acclimation to thermoneutrality (J) or room temperature (K) (n = 6 to 7 per group). Mice were fasted during cold exposure. (L) BAT temperature before and after 5 h cold exposure (n = 5 per group). (M) Representative EMG and the rms traces and quantification of the rms during 1 h cold exposure (n = 5 per group). ^#Repeated measures ANOVA revealed a significant group × time interaction (P < 0.05). *Value significantly different from the control value (P < 0.05). Values are means ± SEM.

Fig. 3.

BANKO mice have normal cold tolerance. NAMPT protein expression (A) and NAD⁺ concentrations (B) in key metabolic organs (n = 5 to 8 per group). eWAT, epididymal WAT; scWAT, subcutaneous WAT; SkM, skeletal muscle. The levels of NAMPT protein (C) and NAD⁺ intermediates (D) were determined in BAT (n = 4 to 5 per group). (E) BAT weight normalized by body weight (n = 7 to 11 per group). (F) Images of H&E staining of BAT. (G) Gene expression of BAT-selective markers, proteins involved in thermogenesis and FFA metabolism, and lipogenic enzymes (n = 7 to 9 per group). (H) BAT gene expression of mitochondrially encoded proteins and mtDNA (n = 5 to 9 per group). (I) ETC complex activities (n = 3 to 6 per group) and ATP contents (n = 3 per group) in BAT mitochondria. (J) Rectal temperature during cold exposure (n = 6 to 7 per group). Mice were fasted during cold exposure. *Value significantly different from the control value (P < 0.05). Values are means ± SEM.

Fig. 4.

ANKO mice have impaired thermogenic responses to fasting and adrenergic stimulation. (A) Rectal temperature before and after 48 h fasting (n = 5 to 8 per group) and VO₂ during the 12 h dark period under fasting conditions (n = 4 per group). VO₂ (B) and BAT temperature (C) before and after a subcutaneous injection of NE (1 mg/kg) (n = 5 per group) or vehicle (n = 3 per group). n.s., not significant. (D) VO₂ values before and after an intraperitoneal injection of CL-316243 (0.6 mg/kg) (n = 3 to 4 per group). ^#Repeated measures ANOVA revealed a significant group × time interaction (P < 0.05). *Value significantly different from the control value (P < 0.05). Values are means ± SEM.

Fig. 5.

Loss of NAMPT impairs adrenergic-mediated lipolytic activity through inactivation of caveolin-1 in WAT. (A) Plasma concentrations of glycerol and FFA and blood glucose concentrations before and after an injection of CL-316243 (1 mg/kg) (n = 4 to 8 per group). (B) WAT depots were incubated in the presence or absence of ISO for 2 h (n = 5 to 8 per group). (C) Western blot analysis of CAV1 and IRβ in WAT (n = 4 to 5 per group). (D) Western blot analysis of phospho-PLIN1 (Ser522), PLIN1, phospho-HSL (Ser563), and HSL in WAT after CL-316243 injection (n = 4 per group). NAD⁺ concentrations (E) and Cav1 gene expression (F) in OP9 adipocytes in the presence or absence of FK866, NMN, and Ex527 (n = 4 per group). (G) ISO-stimulated glycerol release from GFP (G) or Cav1 siRNA-treated OP9 adipocytes (n = 4 per group). ^#Repeated measures ANOVA revealed a significant group × time interaction (P < 0.05). Data were analyzed by Student’s unpaired t test (A–D) and one-way ANOVA with Tukey’s post hoc test (E–G). *P < 0.05. Values are means ± SEM.

Fig. 6.

NMN administration restores thermogenesis in ANKO mice. NMN was administered in drinking water to ANKO mice (500 to 1,000 mg/kg). BAT NAD⁺ metabolites (A), tissue weight (B), H&E staining (C), gene expression of BAT-selective genes and key proteins involved in thermogenesis and FFA metabolism (D), cold tolerance (E), plasma glycerol concentrations before and after administration of CL-316243 (1 mg/kg) (F), and WAT gene expression of Cav1 (G) were determined in ANKO and NMN-treated ANKO mice (n = 3 to 5 per group). (H) A schematic overview of the proposed thermoregulation mediated by adipose tissue NAD⁺ biosynthesis. ^#Repeated measures ANOVA revealed a significant group × time interaction (P < 0.05). *Value significantly different from the control value (P < 0.05). Values are means ± SEM.