DualAG decreases VLDL secretion and associated hepatic gene/ protein expression in DIO mice.

<p><b>A.</b> 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>B.</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). <b>C.</b> Western blots for Abca1, Abcg1, Abcg5 were quantified and plotted after normalizing to tubulin expression. <b>D.</b> Hepatic mRNA expression of Abcg5, Abcg8 and Abcg4 determined by RT-PCR.</p

Glp1/Gcgr dual agonist (DualAG) improved glucose and insulin levels in DIO mice.

<p>Mice were fasted for 2hrs followed by injection of vehicle, Liraglutide (25nmol/kg) or DualAG (25nmol/kg). <b>A.</b> Blood glucose levels after 6 hrs of treatment injection. <b>B.</b> DualAG induced blood glucose level decrease over the course of 6 hrs after injection. <b>C.</b> Plasma insulin levels after 6 hrs of treatment injection. <b>D.</b> Plasma insulin levels monitored at multiple time-points for vehicle and DualAG treated groups. <b>E.</b> Bioavailability of the DualAG peptide during the course of the study.</p

Plasma ketone bodies, non-esterified fatty acids (NEFA), glycerol and hepatic lipid oxidation target expression in DIO mice treated with DualAG.

<p>After fasting for 2 hrs, mice were injected vehicle, Liraglutide (25nmol/kg) or DualAG (25nmol/kg) subcutaneously. <b>A.</b> Plasma β-hydroxybutyrate (βHBA) levels in mice after 6 hrs of treatment injection. <b>B.</b> βHBA levels monitored over the period of 6 hrs after treatment with DualAG. <b>C.</b> Plasma NEFA levels after 6 hrs; <b>D.</b> Plasma NEFA levels from 0 to 6hrs and <b>E.</b> Plasma glycerol levels after 6 hrs of treatment. Hepatic mRNA expression of peroxisome proliferator activated receptor (Ppar) α <b>(F)</b>, acyl CoA oxidase (Acox) 1 <b>(G)</b>, carnitine palmitoyl transferase (Cpt) 1α <b>(H)</b>. Cpt2 <b>(I)</b> determined by RT-PCR.</p

DualAG reduced <i>de novo</i> lipogenesis in DIO mice.

<p><b>A.</b> Timeline for <i>de novo</i> 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>B.</b> <i>De novo</i> palmitate synthesis as determined from plasma fraction. <b>C.</b> <i>De novo</i> palmitate synthesis as determined from liver tissue. <b>D.</b> <i>De novo</i> synthesized cholesterol in plasma. <b>E.</b> <i>De novo</i> synthesized cholesterol in liver tissue.</p

DualAG decreased TG synthesis in DIO mice.

<p><b>A.</b> 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>B.</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). <b>C.</b> Timeline for <i>de novo</i> 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). <b>D.</b> Plasma concentration of newly made TG that incorporated ¹³C₁₈-oleate, which is an indicator of <i>de novo</i> TG synthesis and TG release from liver to blood. <b>E.</b> Overall ¹³C₁₈-oleate enrichment in plasma monoacyl glycerol, diacylglycerol and TG, indicator of <i>de novo</i> 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). <b>F.</b> Plasma TG levels over 30 minutes of experiment, suggesting clearance of unlabeled TG.</p

DualAG suppressed mRNA expression of key lipogenic transcription factors and enzymes in livers of DIO mice.

<p>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).</p

DualAG induced elevation of LDL receptor (LDLr) expression in livers of DIO mice.

<p>Mice were fasted for 2hrs followed by injection of vehicle, Liraglutide (25nmol/kg) or DualAG (25nmol/kg). <b>A.</b> Hepatic protein expression of LDLr and Pcsk9 after 6 hrs of treatment injection, as determined by western blots. <b>B.</b> Blot intensity for LDLr and Pcsk9 was quantified by ImageJ and plotted after normalizing to tubulin expression. <b>C.</b> Hepatic LDLr protein expression after 2, 3, and 6 hrs of treatment injection. <b>D.</b> Blot quantification for LDLr at 2, 3, and 6 hrs after treatment injection. <b>E.</b> Relative mRNA expression of LDLr in liver. <b>F.</b> Relative mRNA expression of Pcsk9 in liver. <b>G.</b> Plasma apolipoprotein (Apo) B100 levels over the course of 6 hrs after injection of DualAG. <b>H.</b> 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.</p