Fig 1.
Glp1/Gcgr dual agonist (DualAG) improved glucose and insulin levels in DIO mice.
Mice were fasted for 2hrs followed by injection of vehicle, Liraglutide (25nmol/kg) or DualAG (25nmol/kg). A. Blood glucose levels after 6 hrs of treatment injection. B. DualAG induced blood glucose level decrease over the course of 6 hrs after injection. C. Plasma insulin levels after 6 hrs of treatment injection. D. Plasma insulin levels monitored at multiple time-points for vehicle and DualAG treated groups. E. Bioavailability of the DualAG peptide during the course of the study.
Fig 2.
Plasma and hepatic lipids in DIO mice treated with DualAG.
Mice were fasted for 2hrs followed by injection of vehicle, Liraglutide (25nmol/kg) or DualAG (25nmol/kg). A. Plasma TG after 6 hrs of treatment injection. B. Reduction in plasma TG levels by DualAG over the course of 6 hrs. C. Plasma total cholesterol levels at 6 hrs. D. Plasma cholesterol levels over the course of 6 hrs. E. Plasma LDL after 6 hrs of treatment injection. F. Plasma HDL after 6 hrs of treatment injection. G. TG levels in liver tissue after 6 hrs of treatment injection. H. Total cholesterol levels in liver tissue after 6 hrs of treatment injection.
Fig 3.
DualAG induced elevation of LDL receptor (LDLr) expression in livers of DIO mice.
Mice were fasted for 2hrs followed by injection of vehicle, Liraglutide (25nmol/kg) or DualAG (25nmol/kg). A. Hepatic protein expression of LDLr and Pcsk9 after 6 hrs of treatment injection, as determined by western blots. B. Blot intensity for LDLr and Pcsk9 was quantified by ImageJ and plotted after normalizing to tubulin expression. C. Hepatic LDLr protein expression after 2, 3, and 6 hrs of treatment injection. D. Blot quantification for LDLr at 2, 3, and 6 hrs after treatment injection. E. Relative mRNA expression of LDLr in liver. F. Relative mRNA expression of Pcsk9 in liver. G. Plasma apolipoprotein (Apo) B100 levels over the course of 6 hrs after injection of DualAG. H. Plasma ApoB48 levels over the course of 6 hrs after injection of DualAG]. I. Plasma levels of total ApoB in vehicle and Liraglutide treated groups at 6 hrs after injection.
Fig 4.
DualAG reduced de novo lipogenesis in DIO mice.
A. Timeline for de novo lipogenesis experiments. After fasting for 2 hrs, mice were injected with 20ml/kg i.p. deuterated water (D2O), simultaneously with s.c. injection of vehicle, Liraglutide (25nmol/kg) or DualAG (25nmol/kg). After 6 hrs, the plasma and tissues were collected for tracer analysis. B. De novo palmitate synthesis as determined from plasma fraction. C. De novo palmitate synthesis as determined from liver tissue. D. De novo synthesized cholesterol in plasma. E. De novo synthesized cholesterol in liver tissue.
Fig 5.
DualAG decreased TG synthesis in DIO mice.
A. Monoacylglycerol acyltransferase (Mgat) enzyme activity. Recombinant human Mgat2 was used as a positive control, and ratio of C14-diacylglycerol to TG was normalized to protein content from the livers of DIO mice treated with vehicle, Liraglutide (25nmol/kg) or DualAG (25nmol/kg). B. Diacylglycerol acyltransferase (Dgat) enzyme activity. Recombinant human Dgat1 was used as a positive control, and amount of C14-TG was normalized to protein content from the livers of DIO mice treated with vehicle, Liraglutide (25nmol/kg) or DualAG (25nmol/kg). C. Timeline for de novo TG synthesis and dynamic TG metabolism experiments. DIO mice were injected with vehicle, Recombinant human Mgat2 was used as a positive control, and ratio of C14-diacylglycerol to TG was normalized to protein content from the livers of DIO mice treated with vehicle, Liraglutide (25nmol/kg) or DualAG (25nmol/kg) s.c., followed by oral administration of Dgat2 and Mtp inhibitors. After one hour, mice were injected with intravenous 13C18-oleate in intralipids, which was followed by blood collection at 5, 10, 20 and 30 mins (n = 4/ time point). D. Plasma concentration of newly made TG that incorporated 13C18-oleate, which is an indicator of de novo TG synthesis and TG release from liver to blood. E. Overall 13C18-oleate enrichment in plasma monoacyl glycerol, diacylglycerol and TG, indicator of de novo synthesis. The percentage enrichment of 13C18-oleate tracer in plasma TG 52:2 was calculated as the ratio of labeled isotopologues (M18 = TG52:2 incorporating 1 equivalent of 13C18 and M36 = TG 52:2 incorporating 2 equivalents of 13C18) to total TG 52:2 (sum of all isotopologues, M0, M18 and M36). F. Plasma TG levels over 30 minutes of experiment, suggesting clearance of unlabeled TG.
Fig 6.
DualAG suppressed mRNA expression of key lipogenic transcription factors and enzymes in livers of DIO mice.
Sterol regulatory element binding protein (Srebp) 1c, Srebp2, monoacylglycerol acyltransferase (Mgat) 1, Mgat 2, glycerol-3-phosphate acetyltransferase (Gpat), peroxisome proliferator activated receptor (Ppar) γ, liver-X-receptor (Lxr) α, and Lxrβ mRNA expression was analyzed by quantitative real-time PCR (RT-PCR).
Fig 7.
DualAG decreases VLDL secretion and associated hepatic gene/ protein expression in DIO mice.
A. Plasma TG levels, as a measure of VLDL secretion. DIO mice were injected with poloxamer, followed by vehicle, Liraglutide or DualAG injection. The plasma was collected at 1, 2, and 4 hrs post treatment, and TG levels were determined. B. Hepatic protein levels of ATP-binding cassette transporters Abca1, Abcg1, and Abcg5 by western blot, after 6 hrs of treatment with vehicle, Liraglutide (25nmol/kg) or DualAG (25nmol/kg). C. Western blots for Abca1, Abcg1, Abcg5 were quantified and plotted after normalizing to tubulin expression. D. Hepatic mRNA expression of Abcg5, Abcg8 and Abcg4 determined by RT-PCR.
Fig 8.
Plasma ketone bodies, non-esterified fatty acids (NEFA), glycerol and hepatic lipid oxidation target expression in DIO mice treated with DualAG.
After fasting for 2 hrs, mice were injected vehicle, Liraglutide (25nmol/kg) or DualAG (25nmol/kg) subcutaneously. A. Plasma β-hydroxybutyrate (βHBA) levels in mice after 6 hrs of treatment injection. B. βHBA levels monitored over the period of 6 hrs after treatment with DualAG. C. Plasma NEFA levels after 6 hrs; D. Plasma NEFA levels from 0 to 6hrs and E. Plasma glycerol levels after 6 hrs of treatment. Hepatic mRNA expression of peroxisome proliferator activated receptor (Ppar) α (F), acyl CoA oxidase (Acox) 1 (G), carnitine palmitoyl transferase (Cpt) 1α (H). Cpt2 (I) determined by RT-PCR.