GIPR agonism mediates weight-independent insulin sensitization by tirzepatide in obese mice

Abstract

Tirzepatide (LY3298176), a dual GIP and GLP-1 receptor (GLP-1R) agonist, delivered superior glycemic control and weight loss compared with GLP-1R agonism in patients with type 2 diabetes. However, the mechanism by which tirzepatide improves efficacy and how GIP receptor (GIPR) agonism contributes is not fully understood. Here, we show that tirzepatide is an effective insulin sensitizer, improving insulin sensitivity in obese mice to a greater extent than GLP-1R agonism. To determine whether GIPR agonism contributes, we compared the effect of tirzepatide in obese WT and Glp-1r-null mice. In the absence of GLP-1R-induced weight loss, tirzepatide improved insulin sensitivity by enhancing glucose disposal in white adipose tissue (WAT). In support of this, a long-acting GIPR agonist (LAGIPRA) was found to enhance insulin sensitivity by augmenting glucose disposal in WAT. Interestingly, the effect of tirzepatide and LAGIPRA on insulin sensitivity was associated with reduced branched-chain amino acids (BCAAs) and ketoacids in the circulation. Insulin sensitization was associated with upregulation of genes associated with the catabolism of glucose, lipid, and BCAAs in brown adipose tissue. Together, our studies show that tirzepatide improved insulin sensitivity in a weight-dependent and -independent manner. These results highlight how GIPR agonism contributes to the therapeutic profile of dual-receptor agonism, offering mechanistic insights into the clinical efficacy of tirzepatide.

Keywords: Diabetes; Metabolism; Therapeutics.

Figure 1. Chronic treatment with tirzepatide enhanced insulin tolerance in obese mice.

Obese insulin-resistant mice were dosed once daily for 14 days with vehicle or tirzepatide (TZP, 10 nmol/kg; n = 5–8 per group). (A) Daily body weight and food intake. (B) Tissue weights, (C) plasma, (D) leptin triglycerides and free fatty acids (FFA), and (E) liver triglyceride after 14 days of treatment. (F) Fed and (G) fasted blood glucose and plasma insulin. (H) Fed adiponectin and insulin-like growth factor binding protein 2 (IGFBP2). (I) Insulin tolerance test. Data are presented as mean ± SEM. *P < 0.05 compared with vehicle. Statistical analyses performed included (A) 2-way ANOVA, (B–H) Student’s unpaired t test, and (I) 1-way ANOVA followed by Dunnett’s multiple comparisons test, where appropriate.

Figure 2. Chronic treatment with tirzepatide ameliorates insulin resistance in mice.

Obese insulin-resistant mice were dosed once daily for 14 days with vehicle or tirzepatide (TZP, 10 nmol/kg; n = 13–15 per group). Following 14 days of treatment, insulin sensitivity was assessed via a hyperinsulinemic-euglycemic clamp. (A) Average glucose infusion rates (GIR) throughout and GIR during the final 30 minutes of the clamp. (B) Endogenous glucose production (EGP). Insulin-stimulated glucose disposal in (C) white gastrocnemius, (D) red gastrocnemius, (E) soleus skeletal muscle, (F) epididymal (eWAT), and (G) inguinal (iWAT) white adipose tissue. Data are presented as mean ± SEM. *P < 0.05 compared with vehicle. Statistical analyses performed included Kruskal-Wallis test followed by Bonferroni multiple comparisons test, where appropriate.

Figure 3. Tirzepatide enhances insulin sensitivity in a weight-dependent and -independent manner.

Obese insulin-resistant mice dosed once daily with vehicle, semaglutide, or tirzepatide for 14 days (n = 14–15 per group). (A) Daily body weight and food intake. Following 14 days of treatment, insulin sensitivity was assessed via a hyperinsulinemic-euglycemic clamp. (B) Average glucose infusion rates (GIR) throughout and during the final 30 minutes of the clamp. (C) Average GIR fold change when comparing TZP to that of weight-matched groups during the final 30 minutes of the clamp. (D) Endogenous glucose production (EGP). Insulin-stimulated glucose disposal in (E–G) skeletal muscle, (H and I) white adipose tissue (epididymal and inguinal adipose tissue [eWAT and iWAT]), and (J) brown adipose tissue (interscapular brown adipose tissue [iBAT]). Data are presented as mean ± SEM. *P < 0.05 compared with vehicle, ^#P < 0.05 compared with semaglutide, and ^§P < 0.05 compared with pair fed. Statistical analyses performed included 1-way ANOVA or Kruskal-Wallis test followed by Bonferroni multiple comparisons test, where appropriate.

Figure 4. GIPR agonism contributes to the weight-independent insulin sensitization action of tirzepatide.

WT (C57BL/6J) and germline, whole-body, glucagon-like peptide 1 receptor–KO mice (Glp-1r^–/– mice) were individually housed in a temperature-controlled (27˚C) environment with a 12-hour light/12-hour dark cycle and fed a high-fat (60% of calories from fat) diet for 12 weeks. (A) Weekly body weight and food intake. (B) Fat and lean mass after 12 weeks of high-fat feeding. Obese insulin-resistant Glp-1r^–/– mice, dosed once daily with either vehicle (n = 12–15) or tirzepatide (TZP, 10 nmol/kg) for 14 days. (C) Daily body weight and food intake. (D) Fasting blood glucose and plasma insulin following 14 days of treatment. Following 14 days of treatment, insulin sensitivity was assessed via a hyperinsulinemic-euglycemic clamp. (E) Average glucose infusion rate (GIR) and that during the final 30 minutes of the clamp. (F) Endogenous glucose production (EGP). Insulin-stimulated glucose disposal in (G) soleus, (H) red, and (I) white gastrocnemius skeletal muscle as well as (J) epididymal white adipose tissue (eWAT) and (K) inguinal white adipose tissue (iWAT). Data are presented as mean ± SEM. *P < 0.05 compared with vehicle. Statistical analyses performed included Student’s unpaired t test, 2-way ANOVA, where appropriate.

Figure 5. Development and characterization of a long-acting GIPR agonist.

(A) Structure schematic of a long-acting glucose-dependent insulinotropic polypeptide receptor agonist (LAGIPRA). Intraperitoneal glucose tolerance tests in (B) WT and (C) germline whole-body glucagon-like peptide 1 receptor–KO mice (Glp-1r^–/– mice) and (D) glucose-insulinotropic polypeptide receptor–null mice (Gipr^–/– mice) dosed s.c. with vehicle (LAGIPRA, 1000 nmol/kg, n = 5) or long-acting glucagon-like peptide 1 receptor agonist (LAGLP-1RA; semaglutide [30 nmol/kg], n = 5) 16 hours prior to assay. Data are presented as mean ± SEM. *P < 0.05 compared with vehicle. Statistical analyses performed included a 1-way ANOVA, followed by Dunnett’s multiple comparisons test, where appropriate.

Figure 6. LAGIPRA improves insulin sensitivity in obese insulin-resistant mice.

High-fat diet–fed obese insulin-resistant mice dosed daily with either vehicle (n = 8–14) or a long-acting glucose-dependent insulinotropic polypeptide receptor agonist (LAGIPRA, n = 8–14) for 14 days. (A) Daily body weight and food intake. (B) Fasting blood glucose and plasma insulin following 14 days of treatment. Hyperinsulinemic-euglycemic clamp following 14 days of treatment. (C) Average glucose infusion rates throughout and during the final 30 minutes of clamp (GIR). (D) Endogenous glucose production (EGP). Insulin-stimulated glucose disposal in (E) soleus, (F) red, and (G) white gastrocnemius skeletal muscle and (H) epididymal white adipose tissue (eWAT) and (I) inguinal white adipose tissue (iWAT). Data are presented as mean ± SEM. *P < 0.05 compared with vehicle. Statistical analyses performed included Student’s unpaired t test, 2-way ANOVA, or Kruskal-Wallis test, where appropriate.

Figure 7. Tirzepatide and GIPR agonism induced BCAA catabolic gene expression in BAT in obese IR mice.

High-fat diet–fed obese insulin-resistant mice (C57BL/6J) were dosed once daily with vehicle (saline, n = 6), tirzepatide (TZP, n = 6), a long-acting glucose-dependent insulinotropic polypeptide receptor agonist (LAGIPRA, n = 6), or saline (pair fed, n = 6). Following 14 days of treatment, tissue samples were collected for metabolic and molecular analyses. (A) Venn diagram of differentially (up- and downregulated gene expression) expressed genes (FDR < 0.05). (B) Heatmap of RNA-Seq expression Z-scores computed for genes associated with the branched-chain amino acid (BCAA) pathway in brown adipose tissue (BAT). *P < 0.05 compared with vehicle and ^#P < 0.05 compared with pair fed. Statistical analyses was performed using 1-way ANOVA, followed by FDR correction, where appropriate.