Gpr75 in glutamatergic neurons regulates body weight. Wyler et al. 2026
Description
The G-protein coupled receptor 75 (GPR75) emerged as a promising therapeutic target for treating diet-induced-obesity. Loss-of-function mutations of GPR75 in humans are associated with reduced body mass index (BMI). Also, Gpr75 deficient mice are protected from diet induced obesity (DIO). Here, we generated genetically-modified mice that enables us to selectively delete or reactivate Gpr75 in a Cre-dependent manner. Loss of Gpr75 in vGlut2+ glutamatergic neurons (Gpr75vGlut2-KO) results in a protection against high fat diet (HFD)-induced weight gain, whereas loss of Gpr75 in GABAergic neurons show no protection against DIO. Furthermore, male Gpr75vGlut2-KO mice have reduced food intake on HFD without a change in energy expenditure. Reactivation of Gpr75 only in vGlut2-expressing cells in a Gpr75 null mouse (Gpr75TB) completely rescues the HFD-induced weight gain, whereas reactivation in GABAergic cells has no effect on body weight or adiposity. These complementary results demonstrate the importance of glutamatergic neurons in GPR75’s regulation of food intake and protection from obesity.
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RNAScopeTM in situ hybridization (ISH) was done using a multiplex fluorescent kit (cat# 323110) Probes were as follows: The Gpr75 probe (cat# 318281-c1) the Slc17a6 probe (cat#319171-c2) or Gad1 (cat#400951-C2) probe was applied at 40°C for 2 h. Amplification steps were carried out using either Opal570 (cat# FP1488001KT; Akoya Biosciences, Japan) or Opal520 (cat# FP1487001KT). Sections were imaged on a Leica DM6B with a Leica DFC9000T camera. ImageJ (NIH, Bethesda, MD, USA) was used to uniformly adjust the resolution and contrast of all our digital images. For Immuno precipitation Tandem mass spectrometry, brains were lysed for 30 minutes in sample lysis buffer (Lauryl Maltose Neopentyl Glycol (0.5%), CHS (0.05%, 100 mM Tris pH 7, 300 mM NaCl supplemented with 1x protease/phosphatase inhibitor cocktail (78429, ThermoFisher). Lysate was incubated with 3 μg of GPR75 Antibody (NBP1-59406, Novus Biologicals) then precipitated with protein A/G beads following the manufacturer’s protocol (88803, ThermoFisher). Samples were digested on bead with 1 μg of Trypsin/LysC (V5072, Promega) in 100 μl of digestion buffer containing n-dodecyl β-D-maltoside (DDM) (ThermoFisher, BN2005) (0.01%) and 100 mM ABC pH 8 for 1 h at 37 °C. Peptides were removed from the beads, reduced with Tris(2-carboxyethyl)phosphine hydrochloride (5 mM), alkylated with 2-chloroacetamide (10 mM), and allowed to digest overnight at 37 °C. The next morning formic acid was added to 1% to stop the reaction. To validate the results, a synthetic stable isotope labeled peptide of Gpr75 (NINQMPIPSA(R10), AQUA QuantProHeavy, ThermoFisher) was spiked from 0.001-1 fmol/uL. Approximately 1 μg of peptide was loaded onto a PicoChip® C18 column heated to 50 °C with a Thermo Easy-nLC 1200. Buffer A was 0.1% formic acid in water, and buffer B was 0.1% formic acid in 80% acetonitrile. Peptides were separated with a 45 min gradient from 5-40% B then a 5 min gradient from 40%-90% B. Peptides were eluted from the column and electrosprayed into a Orbitrap Exploris™ 480 Mass Spectrometer with a distal voltage of 2.2 kV. MS1 spectra were collected with a mass range 350-1400 m/z using a resolving power of 120,000 and an ion target of 300%. MS2 spectra were acquired from 145-1450 m/z in data independent acquisition mode using 59 10 m/z windows with 1 m/z overlap at a resolving power of 15,000. Higher energy collisional dissociation (HCD) was set to 27% NCE (default charge state of 3), the ion target was 1000%, and a maximum injection time was set to 25 ms. All raw data files were processed using DIA-NN v1.8.1 for peptide identification and quantification. For Metabolic phenotyping, a magnetic-resonance whole-body composition analyzer (EchoMRI, TX) was used to analyze the fat and lean mass of mice. Food intake, locomotor activity, oxygen consumption rate (vO2), and carbon dioxide production rate (vCO2) were quantified using an indirect colorimetric system (PhenoMaster; TSE Systems Inc).