Pharmacological inhibition of G protein-coupled receptor kinase 5 decreases high-fat diet-induced hepatic steatosis in mice

Abstract

G protein-coupled receptor kinase 5 (GRK5) is implicated in the pathogenesis of obesity in both humans and rodent models. Our previous work demonstrated that genetic deletion or pharmacological inhibition of GRK5 suppresses 3T3-L1 adipocyte differentiation. Here, we assessed the small-molecule GRK5 inhibitor, GRK5-IN-2, for its effects on metabolic tissues and therapeutic potential in a diet-induced obesity mouse model. Mice were fed a high-fat diet for 8 weeks to induce obesity, followed by continued a high-fat diet with oral administration of GRK5-IN-2 (25 or 50 mg/kg) or water vehicle, five days per week for an additional 16 weeks. GRK5-IN-2 treatment had no effect on body weight, fat/lean mass, insulin tolerance, food intake, or energy expenditure but significantly reduced hepatic triglyceride accumulation and de novo lipogenesis. A follow-up study using 25 mg/kg of GRK5-IN-2 confirmed no effect on adiposity but reduced hepatic triglycerides. GRK5-IN-2 treatment decreased expression of the lipogenic gene Acc2 while upregulating lipid utilization proteins COXIV and ACSL1 in the liver, likely contributing to lower triglyceride levels. Together, these findings suggest that GRK5 inhibition selectively modulates hepatic lipid metabolism without altering systemic metabolic parameters, highlighting GRK5 as a potential therapeutic target for fatty liver disease.

Keywords: Adiposity; G protein-coupled receptor kinase 5; Hepatic steatosis; High fat diet; Obesity.

Figure 1.. Effects of GRK5-IN-2 on adiposity and metabolic health in diet-induced obese mice.

(A) Study design showing weeks of age and treatment. Six-week-old male C57Bl/6J mice were fed a high fat diet (HFD; 45% fat, D12451, Research Diets Inc) for 8 weeks to induce obesity prior to starting water control (open square), 25 mg/kg (closed square) or 50 mg/kg (closed circle) GRK5-IN-2 treatment. After 9 weeks of treatment, metabolic phenotyping started including EchoMRI, intraperitoneal insulin and glucose tolerance tests (IPITT and IPGTT), TSE PhenoMaster chambers, and a lipogenesis functional study. Mice were euthanized after 16 weeks of treatment (Sac). (B) Body weight was measured weekly (n=10/group). (C) Body composition was measured after 9 weeks of treatment (n=10/group). (D-E) After 11–12 weeks of treatment, mice (n=4/group) were used for indirect calorimetry (TSE PhenoMaster System). Results analyzed using the CalR Web Application (Version 1.3). Gray shading indicates a dark cycle. (F-G) Mice (n=10/group) were fasted for 16 and 4 hours in preparation for an IPGTT and IPITT, respectively. Blood glucose was measured at 0, 15, 30, 60, 90, and 120-minutes post injection. Area under the curve (AUC) was calculated to assess glucose and insulin sensitivity. All results are mean ± SEM. Statistical significance was assessed using one-way or two-way ANOVA.

Figure 2.. Effects of GRK5-IN-2 on visceral adipose tissue functionality in diet-induced obese mice.

De novo and fatty acid uptake were measured using radiolabeled [¹⁴C]-acetate and [³H]-oleate. Control and GRK5-IN-2 treated mice (n=3/group) were injected with 5 μCi per mouse of [1,2-¹⁴C]-acetic acid (A-B) or 50 μCi per mouse of [9,10-³H(N)]-oleic acid (C-D). Tissues were collected for 3 hours post injection. Lipids were extracted and separated using thin layer chromatography. [¹⁴C]-triglycerides (TG), [¹⁴C]-cholesteryl esters (CE), [¹⁴C]-free cholesterols (FC), [¹⁴C]-phospholipids (PL), [³H]-oleic acids, [³H]-TG, [³H]-CE, and [³H]-PL were quantified by liquid scintillation counting. All results are mean ± SEM. Statistical significance was assessed using one-way ANOVA.

Figure 3.. Effects of GRK5-IN-2 on liver lipid metabolism in diet-induced obese mice.

(A-B) At study end point, mouse liver (n= 5–7/group) was collected, weighed, and normalized to total body weight. (C) Liver lipid content was extracted (n=15/group), and triglyceride content was measured using a colorimetric assay normalized to liver tissue weight. After 15 weeks of GRK5-IN-2 treatment, de novo lipogenesis and fatty acid uptake were measured using radiolabeled isotopes. Control and drug treated mice (n=3/group) were injected with 5 μCi per mouse of [1,2-¹⁴C]-acetic acid (D-E) and 50 μCi per mouse of [9,10-³H(N)]-oleic acid (F-G). Tissues were collected for 3 hours post injection. Lipids were extracted and separated using thin layer chromatography. [¹⁴C]-triglycerides (TG), [¹⁴C]-cholesteryl esters (CE), [¹⁴C]-free cholesterols (FC), [¹⁴C]-phospholipids (PL), [³H]-oleic acids, [³H]-TG, [³H]-CE, and [³H]-PL were quantified by liquid scintillation counting. All results are mean ± SEM. Statistical significance was assessed using one-way ANOVA.

Figure 4.. Effects of GRK5-IN-2 treatment on adipose and systemic metabolism in a follow-up study.

(A) Study design illustrating timeline and treatments. Six-week-old male C57Bl/6J mice were fed a high-fat diet (HFD; 45% fat, D12451, Research Diets Inc.) for 8 weeks to induce obesity before initiating GRK5-IN-2 treatment. After 9 weeks of treatment, metabolic phenotyping was conducted, including EchoMRI, intraperitoneal insulin (IPITT) and glucose tolerance tests (IPGTT), TSE PhenoMaster metabolic chambers, and a lipogenesis functional assay using radiolabeled tracer. Mice were euthanized after 13 weeks of treatment (Sac). (B) Body weight measured weekly (n=10/group). (C) Body composition assessed via EchoMRI after 9 weeks of treatment (n=10/group). (D) Gonadal white adipose tissue (gWAT) was collected, fixed in 10% formalin, and stained with hematoxylin and eosin (H&E) for histological analysis (n=10/group). Representative H&E-stained gWAT images from water- and GRK5-IN-2-treated mice. (E) Triglyceride (TG) content in gWAT lipids extracted and quantified using a colorimetric assay (n=10/group). (F) RNA was extracted from gWAT (n=10/group), reverse-transcribed to cDNA, and analyzed via real-time PCR to quantify adipogenic and inflammatory gene expression normalized to 18S rRNA (endogenous control). Results are expressed as fold change relative to the water-treated group. All data are presented as mean ± SEM. Statistical significance was assessed using two-tailed Student’s unpaired t test.

Figure 5.. Effects of GRK5-IN-2 on liver triglyceride content, function, and molecular profiles in a follow-up study.

(A) Hepatic lipids were extracted from liver tissue (n=10/group), and total triglyceride (TG) content and percent liver TG levels were quantified using a colorimetric assay. (B) Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were measured using a colorimetric assay to calculate the ALT/AST ratio (n=10/group). (C) RNA was extracted from liver tissue (n=10/group), reverse-transcribed to cDNA, and analyzed via real-time PCR to quantify expression of genes involved in lipogenesis, inflammation, fibrosis, fatty acid oxidation, and mitochondrial biogenesis, normalized to 18S rRNA (endogenous control). Results are expressed as fold change relative to the water-treated group. (D) Liver protein (n=10/group) was extracted and subject to Western blot using anti-ACSL1, anti-COXIV, and anti-α-Tubulin antibodies. All data are presented as mean ± SEM. Statistical significance was assessed using two-tailed Student’s unpaired t test.