GLP-1 receptor agonism ameliorates hepatic VLDL overproduction and de novo lipogenesis in insulin resistance

Abstract

Background/objectives: Fasting dyslipidemia is commonly observed in insulin resistant states and mechanistically linked to hepatic overproduction of very low density lipoprotein (VLDL). Recently, the incretin hormone glucagon-like peptide-1 (GLP-1) has been implicated in ameliorating dyslipidemia associated with insulin resistance and reducing hepatic lipid stores. Given that hepatic VLDL production is a key determinant of circulating lipid levels, we investigated the role of both peripheral and central GLP-1 receptor (GLP-1R) agonism in regulation of VLDL production.

Methods: The fructose-fed Syrian golden hamster was employed as a model of diet-induced insulin resistance and VLDL overproduction. Hamsters were treated with the GLP-1R agonist exendin-4 by intraperitoneal (ip) injection for peripheral studies or by intracerebroventricular (ICV) administration into the 3rd ventricle for central studies. Peripheral studies were repeated in vagotomised hamsters.

Results: Short term (7-10 day) peripheral exendin-4 enhanced satiety and also prevented fructose-induced fasting dyslipidemia and hyperinsulinemia. These changes were accompanied by decreased fasting plasma glucose levels, reduced hepatic lipid content and decreased levels of VLDL-TG and -apoB100 in plasma. The observed changes in fasting dyslipidemia could be partially explained by reduced respiratory exchange ratio (RER) thereby indicating a switch in energy utilization from carbohydrate to lipid. Additionally, exendin-4 reduced mRNA markers associated with hepatic de novo lipogenesis and inflammation. Despite these observations, GLP-1R activity could not be detected in primary hamster hepatocytes, thus leading to the investigation of a potential brain-liver axis functioning to regulate lipid metabolism. Short term (4 day) central administration of exendin-4 decreased body weight and food consumption and further prevented fructose-induced hypertriglyceridemia. Additionally, the peripheral lipid-lowering effects of exendin-4 were negated in vagotomised hamsters implicating the involvement of parasympathetic signaling.

Conclusion: Exendin-4 prevents fructose-induced dyslipidemia and hepatic VLDL overproduction in insulin resistance through an indirect mechanism involving altered energy utilization, decreased hepatic lipid synthesis and also requires an intact parasympathetic signaling pathway.

Keywords: FFA, free fatty acid; Fasting dyslipidemia; GLP-1, glucagon-like peptide-1; GLP-1R, GLP-1 receptor; Glucagon-like peptide-1 (GLP-1); Hepatic steatosis; ICV, intracerebroventricular; Incretin; Insulin resistance; RER, respiratory exchange ratio; T2D, type 2 diabetes; VLDL, very low density lipoprotein; Very low density lipoprotein (VLDL); apoB100, apolipoproteinB100; ip, intraperitoneal.

Figure S1

Peripheral exendin-4 does not alter liver or fat pad weight in an intervention treatment strategy. Hamsters underwent the intervention study and plasma and tissue was collected from vehicle and exendin-4 hamsters after a 5 h fast. (A) Liver and (B) epididymal fat pad weight. (C) Plasma AST and (D) ALT following a 5 h fast. n = 5–7. Analyzed by Student's unpaired t-test.

Figure S2

Peripheral exendin-4 decreases liver weight and lipid content in a preventative treatment strategy. Hamsters underwent the intervention study and plasma and tissue was collected from vehicle and exendin-4 hamsters after a 5 h fast. (A) Liver weight and (B) epididymal fat pad weight. (C) Hepatic triglyceride and (D) cholesterol content following the preventative study. (E) Plasma AST and (F) ALT levels following a 5 h fast. n = 6; **p < 0.01, ***p < 0.001 as analyzed by Student's unpaired t-test.

Figure S3

Peripheral exendin-4 decreases hyperinsulinemia and fasting plasma glucose. Following the intervention (A,B) or preventative study (C), vehicle and exendin-4 hamsters were fasted for 5 h and plasma was collected over 6 h. (A,C) Fasting plasma glucose and (B) insulin levels. Intervention n = 5–7; Preventative n = 6; **p < 0.01, ***p < 0.001 as analyzed by two-way ANOVA with Bonferroni post hoc test.

Figure S4

Peripheral exendin-4 decreases plasma TG, cholesterol and apoB100 accumulation. Following the intervention (A–C) or preventative (D–F) study, hamsters were fasted for 5 h and received an intraperitoneal (ip) injection of vehicle or exendin-4 (20 μg/kg) followed by an ip injection of poloxamer (0.5 g/kg). (A,D) Plasma triglyceride, (B,E) cholesterol, (C,F) and apoB100 accumulation. Intervention n = 5–7; Preventative n = 6; *p < 0.05, **p < 0.01, ***p < 0.001 as analyzed by two-way ANOVA with Bonferroni post hoc test.

Figure S5

Liraglutide reduces VLDL-cholesterol without changes in weight. (A) C57BL/6J mice on a chow diet + 4% fructose water for 16 weeks were given a once daily intraperitoneal (ip) injection of vehicle or liraglutide (20 μg/kg) during the final 4 weeks. On the final day, an ip injection of poloxamer (0.5 g/kg) was administered following a 5 h fast. (B) Body weight was measured for the duration of the study (16 weeks). (C) VLDL-cholesterol levels 6 h after poloxamer. n = 3. Analyzed by two-way ANOVA with Bonferroni post hoc test (B) or by Student's unpaired t-test (C).

Figure S6

Exendin-4 acutely reduces RER and CO2 production. Vehicle, pair-fed and exendin-4 treated hamsters underwent the intervention study and placed into CLAMS metabolic cages for the final 48 h. Dashed lines indicate injection timepoints. (A) RER and (B) CO₂ production of hamsters in the cages over time. (C) O₂ consumption, (D) heat production and (E) total movement average 3 h postinjection. Dashed lines indicate injection time. n = 10–11. *p < 0.05, **p < 0.01, ***p < 0.001. *Significance between vehicle and exendin-4; #Significance between pair-fed and exendin-4. Analyzed by two-way ANOVA with Bonferroni post hoc test (A,B) or by one-way ANOVA (C–E).

Figure S7

Primary hamster hepatocytes do not contain the GLP-1 receptor. Primary hamster hepatocytes were treated ex vivo with or without oleic acid (0.7 mM) for 16 h followed by 6 h of vehicle or exendin-4. (A) Cellular and (B) secreted TG levels in primary hamster hepatocytes. n = 3 in triplicate. Analyzed by two-way ANOVA with Bonferroni post hoc test.

Figure S8

Central exendin-4 prevents fructose–induced hyperinsulinemia. (A,B) Daily weight and food consumption in hamsters that underwent the ICV study (C) Fasting plasma insulin levels before (day 0) and after (Day 14) the study. (D,E) Fasting plasma AST and ALT levels. n = 8. *p < 0.05, ***p < 0.001. (A–C) Analyzed by two-way ANOVA with Bonferroni post hoc test (A–C) or by unpaired Student's t-test (D,E).

Figure S9

Vagotomy prevents exendin-4 mediated reductions in fasting VLDL-apoB100. (A) VLDL-ApoB100 blots in vagotomized or sham hamsters treated peripherally with vehicle or exendin-4. (B,C) Fasting plasma AST and ALT. n = 3.

Figure 1

Short term peripheral exendin-4 decreases food consumption and body weight. Fructose-fed hamsters received vehicle or exendin-4 (20 μg/kg) for 7 (A–C; Intervention study) or 10 days (D–F; Preventative study). Fasting plasma was collected following poloxamer (0.5 g/kg). Average daily food consumption (B,E) and body weight (C,F) during intervention (B,C) and preventative (D,F) studies. Intervention n = 5–7; Preventative n = 6; *p < 0.05, **p < 0.01, ***p < 0.001 as analyzed by two-way ANOVA with Bonferroni post hoc test.

Figure 2

Peripheral exendin-4 prevents fructose-induced dyslipidemia. Plasma from vehicle or exendin-4 hamsters on Day 0 and after the intervention (Day 15; A–C) or preventative study (Day 11; D–F). (A,D) Plasma triglyceride (TG), (B,E) cholesterol and (C,F) glucose. Intervention n = 5–7; Preventative n = 6; *p < 0.05, **p < 0.01, ***p < 0.001 as analyzed by two-way ANOVA with Bonferroni post hoc test.

Figure 3

Peripheral exendin-4 decreases VLDL associated-TG, -cholesterol and apoB100 accumulation. VLDL isolated from vehicle or exendin-4 hamsters in the intervention (A–C) or preventative (D–F) study. (A,D) VLDL-triglyceride, (B,E) cholesterol, (C,F) and apoB100 accumulation with area under the curve (AUC) for VLDL-apoB100 (C). Intervention n = 5–7; Preventative n = 6; *p < 0.05, **p < 0.01, ***p < 0.001 as analyzed by two-way ANOVA with Bonferroni post hoc test. AUC analyzed by Mann–Whitney U test.

Figure 4

Exendin-4 decreases dyslipidemia and VLDL–TG independent of changes in food intake. Fasting plasma from pair-fed or exendin-4 hamsters on Day 0 or following treatment (Day 15). (A) Plasma triglyceride (TG), (B) cholesterol and (C) FFA levels. FPLC (D) TG and (E) cholesterol. n = 10–11. (D,E). *p < 0.05, **p < 0.01, ***p < 0.001 as analyzed by two-way ANOVA with Bonferroni post hoc test.

Figure 5

Liraglutide reduces dyslipidemia and hepatic steatosis in mice independent of changes in weight or food consumption. C57BL/6J mice receiving 4% fructose were treated with or liraglutide (20 μg/kg) for 4 weeks. Fasting plasma was collected following poloxamer (0.5 g/kg). (A) Glucose tolerance 3 weeks posttreatment. (B) Plasma triglyceride (TG) accumulation (C) VLDL-TG. (D,E) Hepatic TG and cholesterol. (F) Hepatic oil Red O staining. n = 3; *p < 0.05, **p < 0.01, ***p < 0.001. Analyzed by two-way ANOVA with Bonferroni post hoc test (A,B) or by Student's unpaired t-test (C–E).

Figure 6

Exendin-4 increases lipid utilization and decreases hepatic de novo lipogenesis via an indirect mechanism. Metabolic cage analysis for vehicle, pair-fed and exendin-4 hamsters from the intervention study. (A) Body weight change. (B) RER and (C) CO₂ averages 3 h postinjection. n = 10–11. Livers were excised for PCR analysis. (D) mRNA expression for hepatic inflammation and lipid homeostasis. n = 8. (E) cAMP levels of primary hamster hepatocytes treated ex vivo with vehicle, exendin-4 (100 nM) or forskolin (50 nM). n = 3. *p < 0.05, **p < 0.01, ***p < 0.001. Analyzed by two-way ANOVA with Bonferroni post hoc test (A) or by one way ANOVA (C–E).

Figure 7

Central exendin-4 decreases weight and food consumption and decreases both fasting hypertriglyceridemia and VLDL-associated lipid and apoB100 levels. (A) Fructose-fed hamsters received intracerebroventricular (ICV) vehicle or exendin-4 (250 μg) and FPLC analysis was carried out. (B) Total change in weight. (C,D) Changes in food consumption and fasting plasma TG levels before (Day 0) and after (Day 14) the diet/treatment. (E–G) Triglyceride (TG), cholesterol and apoB100. n = 8; *p < 0.05, ***p < 0.001. Analyzed by two-way ANOVA with Bonferroni post hoc test (C,D) or by Student's unpaired t-test (B).

Figure 8

Exendin-4 requires vagal signaling to reduce fasting dyslipidemia. Hamsters underwent a subdiaphragmatic truncal vagotomy/sham surgery and allowed 1 week recovery followed by the intervention study. (A) Total weight loss (B) Changes in food consumption on fructose (week 2) and during treatment (week 3). (C) Changes in fasted plasma TG and (D) cholesterol levels before (Day 0) and after the study (Day 21). (E,F) Changes in VLDL-TG and VLDL-cholesterol up to 2 h posttriton. n = 3. *p < 0.05, **p < 0.01, ***p < 0.001. Analyzed by one way (A) or two-way ANOVA (B–F) with Bonferroni post hoc test.