Peripheral-specific Y1 receptor antagonism increases thermogenesis and protects against diet-induced obesity

The study addresses how peripheral neuropeptide Y (NPY) Y1 receptor (Y1R) signaling regulates energy expenditure and thermogenesis, and whether selectively blocking peripheral Y1R can mitigate diet-induced obesity (DIO) without affecting central nervous system pathways that influence mood and anxiety. NPY centrally increases food intake and reduces EE by inhibiting sympathetic output, suppressing brown adipose tissue (BAT) thermogenesis and white adipose tissue (WAT) browning. However, peripheral NPY/Y-receptors are expressed in metabolic organs (adipose tissue, pancreas, liver, muscle), suggesting local roles in energy and glucose homeostasis. Prior work shows Y1R inhibits insulin secretion in β-cells and peripheral Y1R knockdown benefits fat mass and lipid oxidation. The authors hypothesize that peripheral Y1R blockade will protect against DIO by increasing EE/thermogenesis and improving glucose metabolism, and test this using a peripherally restricted Y1R antagonist (BIBO3304) in mice along with human adipose tissue analyses.

Background literature highlights: (1) Central NPY drives hyperphagia and reduces EE via sympathetic inhibition, affecting BAT and WAT thermogenesis; central targeting has psychiatric side effects. (2) Peripheral NPY/Y-receptors are present in adipose tissue and other organs; Y1R signaling inhibits insulin secretion in β-cells and its partial peripheral knockdown improves lipid oxidation. (3) Genetic association between NPY variants (Leu7/Pro) and obesity suggests elevated circulating NPY contributes to adiposity. (4) Browning of WAT and activation of BAT thermogenesis improve EE and glucose tolerance. These studies motivated targeting peripheral Y1R to enhance thermogenesis while avoiding central adverse effects.

- Animals and diets: Male C57BL/6JAusb mice housed at 22°C, 12-h light cycle, fed chow (8% kcal fat) or high-fat diet (HFD; 43% kcal fat). Additional models: global Y1R knockout (Y1R−/−), UCP1−/− mice, and inducible adipocyte-specific Y1R knockout (AdipoTMCre/+;Y1lox/lox) using AdipoQ-CreERT2 activated by tamoxifen gavage. - Pharmacology: Daily oral administration via flavored gelatin jelly of peripherally restricted Y1R antagonist BIBO3304 (non–brain-penetrant) or vehicle for 7–8 weeks starting at 8 weeks of age. For human adipocyte experiments, BIBO3304 and Y1R-preferring agonist [Leu31,Pro34]NPY used ex vivo. - Phenotyping: Body weight weekly; body composition by DEXA at baseline and 2/4/6 weeks; dissections of WAT depots (inguinal WATi, epididymal WATe, mesenteric WATm, retroperitoneal WATr) and BAT at cull. - Energy balance: Food intake (spontaneous daily and cumulative; fasting-induced refeeding) and fecal output; indirect calorimetry (VO2, VCO2, EE normalized to metabolically active tissue mass = lean mass + 0.2×fat mass), RER, and locomotor activity. - Thermography: Non-invasive infrared imaging of interscapular BAT, lumbar back (core), and tail temperatures (FLIR T640), averaged over repeated measures. - Glucose homeostasis: Glucose tolerance tests (GTT; 1.0 g/kg i.p. glucose) with concurrent insulin measurements; insulin tolerance tests (ITT; 1 IU/kg insulin). - Molecular analyses: qPCR of Y-receptor and thermogenic/adipogenic genes in BAT, WATi, WATe; western blot for UCP1, PGC1α, CPT1, phosphorylated ACC, OXPHOS complexes, p-CREB, p-ERK (14-3-3 loading control); UCP1 immunohistochemistry in WATi. - Adipocyte morphology: H&E staining of WATi and WATe; image analysis (ImageJ/FIJI) for adipocyte size distributions. - Human studies: SAT and VAT biopsies from lean (BMI < 24.9) and obese (BMI > 30) adults (surgery patients) with exclusions (no diabetes, CVD, etc.). qPCR profiling for Y-receptors and thermogenic/adipogenic markers. SVF from SAT of obese subjects differentiated to adipocytes and treated ±BIBO3304 and/or [Leu31,Pro34]NPY for 48 h followed by qPCR. - Intravital signaling imaging: Akt-FRET biosensor mice implanted with optical windows over BAT. Multiphoton fluorescence lifetime imaging (mTurquoise2 lifetime) in HFD-fed mice ±4 weeks BIBO3304, before and after i.p. glucose bolus, to track dynamic Akt activity; ex vivo validation with mTOR1/2 inhibitor AZD2014. - Statistics: Data shown as mean ± s.e.m.; comparisons by one-/two-way ANOVA (with Tukey/Sidak), repeated measures ANOVA, or two-tailed t tests; significance p < 0.05.

- Y1R expression increases with obesity: In HFD-fed mice, Y1R mRNA is upregulated in BAT and WATi (not WATe), and in muscle; low in liver. In humans, Y1R mRNA is elevated in SAT and VAT of obese vs lean individuals; SAT Y1R correlates positively with BMI (r^2 = 0.198, p = 0.019). - Peripheral Y1R antagonism reduces weight gain via fat loss: Daily BIBO3304 reduces body weight gain in HFD-fed WT mice (no change on chow), driven by decreased fat mass (DEXA and reduced weights of WAT depots); lean mass unchanged. - No change in energy intake or activity: Spontaneous and fasting-induced food intake and locomotor activity are unchanged by BIBO3304 on chow or HFD; fecal output unchanged, supporting increased EE as the cause of reduced adiposity. - Energy expenditure increases and fuel preference shifts: EE (normalized to metabolically active tissue) is significantly higher in HFD+BIBO3304 vs HFD control; RER decreases, indicating increased lipid oxidation (notably in both light and dark phases on HFD). - Thermogenesis rises: Infrared imaging shows higher lumbar (core) and BAT surface temperatures with BIBO3304 on both diets; BAT weight unaffected. Tail temperature increases on HFD with BIBO3304, suggesting increased heat loss accompanies higher thermogenesis. - BAT activation: In BAT, BIBO3304 elevates thermogenic gene expression. On chow: Ucp1 mRNA increased; on HFD: DIO2, PRDM16, PGC1α, CIDEA, EVA1, PDK4, NRG4, and β3-AR mRNAs are increased. Protein levels of UCP1 and PGC1α are up in BAT (significantly on HFD). CPT1 protein increases under both diets; p-ACC increases, implying reduced lipogenesis. OXPHOS complexes I–III proteins are elevated, consistent with enhanced mitochondrial capacity. - WAT browning (WATi): On chow, BIBO3304 upregulates beige markers (CD137, Tmem26, Klhl13, Slc27a1) and PGC1α, PPARγ, adiponectin mRNAs. On HFD, BIBO3304 reverses suppression of thermogenic genes, significantly increasing Ucp1, DIO2, CIDEA, COX7a1, Tbx1, PDK4, PGC1α, Acot2, CD137 and adiponectin; UCP1 protein and PGC1α trend upward; UCP1 IHC shows induction in WATi. Y1R−/− mice have higher WATi thermogenic gene expression than WT; BIBO3304 fails to induce thermogenic genes in Y1R−/− WATi, confirming Y1R dependence. - Adipocyte size reduction: In HFD-fed mice, BIBO3304 reduces mean adipocyte size and shifts distributions toward smaller adipocytes in both WATi and WATe; WATe shows increased PPARγ and adiponectin mRNAs, though limited beige gene induction. - UCP1 dependency: In UCP1−/− mice on HFD, BIBO3304 does not reduce body weight gain, fat mass, or WAT weights; EE, RER, BAT/core temperatures, and glucose tolerance are unchanged, demonstrating UCP1 is required for the anti-obesity and thermogenic effects. - Adipocyte Y1R is critical: Inducible adipocyte-specific Y1R deletion (AdipoTMCre/+;Y1lox/lox) markedly reduces Y1R mRNA in adipose depots. Tamoxifen alone increases body weight and fat mass in Cre-negative controls on chow and HFD, but this obesogenic effect is prevented in adipocyte Y1R-deleted mice; fat mass is reduced (chow) or normalized (HFD). Glucose intolerance and worsened insulin responsiveness induced by tamoxifen in controls are absent in adipocyte Y1R-deleted mice. - Glucose metabolism improves: In HFD-fed WT mice, BIBO3304 improves GTT responses at 2 and 6 weeks without increasing insulin levels; at 6 weeks, ITT shows improved insulin tolerance to chow-like AUC. - Human translation: Obese human SAT and VAT show reduced UCP1, PGC1α, CIDEA, CD137, TMEM26, adiponectin, and PPARγ mRNAs vs lean. In primary human SAT adipocytes from obese subjects, BIBO3304 upregulates UCP1, CD137, TMEM26 mRNAs; Y1R agonist [Leu31,Pro34]NPY suppresses PGC1α, which is rescued by co-treatment with BIBO3304. - Signaling mechanisms: BIBO3304 increases p-CREB in BAT (chow and HFD) and WATi (chow), and increases p-ERK in BAT (chow) and WATi (chow and HFD), indicating cAMP-CREB and MAPK-ERK pathway engagement in a depot- and diet-dependent manner. Intravital Akt-FRET imaging in BAT shows higher Akt activity after glucose challenge in HFD+BIBO3304 vs control, indicating enhanced insulin signaling; ex vivo AZD2014 inhibits Akt signal as control.

The findings demonstrate that peripheral Y1R signaling restrains thermogenesis and promotes adiposity under positive energy balance. Selective peripheral Y1R antagonism (BIBO3304) enhances whole-body EE by activating BAT and inducing WAT browning, thereby reducing fat mass and protecting against DIO without altering food intake or activity. The effects are mediated through UCP1-dependent thermogenesis and are supported by increased mitochondrial function and lipid oxidation (lower RER, higher CPT1, OXPHOS). Improved glucose tolerance and insulin sensitivity likely arise from enhanced adipose tissue glucose and lipid utilization and increased adiponectin/PPARγ, with direct evidence of increased Akt activity in BAT. Depot- and diet-dependent signaling changes (cAMP-CREB and MAPK-ERK) explain differential gene induction patterns in BAT vs WAT and chow vs HFD states. Genetic ablation models confirm that adipocyte Y1R is necessary for obesity development and that BIBO3304’s benefits require UCP1 and Y1R in adipocytes. Concordant human data (elevated Y1R in obese adipose tissue, reduced thermogenic markers, and BIBO3304-induced browning gene upregulation in primary human adipocytes) support translational relevance. Together, peripherally restricted Y1R antagonism offers a strategy to boost thermogenesis and EE, providing a potential therapy for obesity and diabetes while avoiding central adverse effects.

Peripheral Y1R signaling acts as a negative regulator of adipose thermogenesis. Pharmacologic blockade with the peripherally restricted antagonist BIBO3304 increases EE, activates BAT and WAT browning, reduces fat mass, and improves glucose homeostasis in DIO. The benefits require UCP1 and adipocyte Y1R, and involve cAMP-CREB, MAPK-ERK, and enhanced Akt signaling in BAT. Human adipose tissue exhibits conserved patterns, and primary human adipocytes respond to Y1R antagonism with increased browning markers. These results highlight peripheral Y1R as a promising therapeutic target for obesity and type 2 diabetes. Future work should delineate tissue-specific contributions beyond adipose tissue, optimize Y1R antagonist pharmacology for clinical use, and assess long-term efficacy and safety, including cardiovascular and vascular effects.

- Potential contributions from non-adipose Y1R (e.g., vasculature, skeletal muscle, liver, bone) are not fully excluded; vascular Y1R blockade could alter blood flow and heat distribution. - Tamoxifen used to induce adipocyte-specific deletion has obesogenic effects that complicate interpretation; although deletion counteracted these effects, mechanisms remain unclear. - Diet- and depot-specific signaling responses (p-CREB/p-ERK patterns) indicate complexity that may affect generalizability across nutritional states and fat depots. - Most in vivo efficacy data are from male mice; sex-specific effects were not detailed. - Human evidence is ex vivo and correlative in tissue biopsies; clinical efficacy and safety of Y1R antagonism remain to be established. - BIBO3304 is a research compound; translational pharmacokinetics/dynamics and long-term safety are not addressed.