Glycine Normalizes Hepatic Triglyceride-Rich VLDL Secretion by Triggering the CNS in High-Fat Fed RatsNovelty and Significance by Jessica T.Y. Yue, Patricia I. Mighiu, Mark Naples, Khosrow Adeli, and Tony K.T. Lam Circulation Research Volume 110(10):1345-1354 May 11, 2012 Copyright © American Heart Association, Inc. All rights reserved.

A, Schematic representation of working hypothesis: glycine, a coagonist of the NR1 subunit of the N-methyl-d-aspartate (NMDA) receptor, potentiates the activation of NMDA receptors in the dorsal vagal complex (DVC) and lowers the secretion of hepatic triglyceride-rich very low-density lipoproteins (VLDL-TG). A, Schematic representation of working hypothesis: glycine, a coagonist of the NR1 subunit of the N-methyl-d-aspartate (NMDA) receptor, potentiates the activation of NMDA receptors in the dorsal vagal complex (DVC) and lowers the secretion of hepatic triglyceride-rich very low-density lipoproteins (VLDL-TG). NMDA receptor ion channel blocker, MK-801, negates the ability of glycine to lower hepatic VLDL-TG. B, Experimental protocol. Day 0: Stereotaxic placement of cannulae in the DVC, targeting the NTS, in male Sprague Dawley rats (≈280–300 g). Day 7: Indwelling catheters were surgically implanted in the left carotid artery and right jugular vein for blood sampling and infusion, respectively. Day 12: Rats were given 5 days to recover from vascular surgery prior to tyloxapol experiments. During tyloxapol experiments, DVC infusions at 0.33 μL/h of saline (n=5), MK-801 (0.06 ng/min; n=7) glycine (10 μmol/L; n=12), or glycine (10 μmol/L)+MK-801 (0.06 ng/min; n=7) were commenced at t=−10 minutes after basal blood samples were obtained. Time 0 minutes blood samples were obtained after 10 minutes of DVC infusions, followed by an intravenous injection of tyloxapol (600 mg/kg). Blood samples were subsequently obtained every 15 minutes until the end of the experiment at t=150 minutes. C, DVC infusion of 10 μmol/L glycine for 150 minutes resulted in an increase of DVC tissue glycine levels as compared with DVC saline infusion. Peripheral (iv=intravessel) injection of 50 nmol/kg glycine (n=7) increased: (D) plasma glycine levels and (E) DVC tissue glycine levels 160 minutes postinjection as compared with iv saline (n=6). Following tyloxapol administration, DVC glycine: (F) lowered plasma triglyceride (TG) and (G) decreased the rate of appearance of VLDL-TG in plasma compared with saline. Coinfusion of NMDA receptor blocker MK-801 fully reversed the effects of glycine. H, DVC glycine reduced the hepatic mRNA expression of stearoyl-CoA denaturase-1 (scd-1) as compared with vehicle, and simultaneous administration of DVC MK-801 reversed glycine's effect to lower the expression of this gene. White (□), saline; light gray (), MK-801 only; black (■), glycine; dark gray (), glycine+MK-801. Crossed-hatched bars represent iv saline or glycine administration. White with light gray diagonal stripes represents a vehicle control group (n=7). Data are presented as mean±SEM. *P<0.05 glycine vs all other treatment groups. Jessica T.Y. Yue et al. Circ Res. 2012;110:1345-1354 Copyright © American Heart Association, Inc. All rights reserved.

A, Schematic representation of working hypothesis: dorsal vagal complex (DVC) administration of 7-chlorokyurenic acid (7CKNA), an inhibitor of the NR1 subunit of the N-methyl-d-aspartate (NMDA) receptor, negates the effect of DVC glycine to lower hepatic triglyceride-rich very low-density lipoproteins (VLDL-TG) secretion. A, Schematic representation of working hypothesis: dorsal vagal complex (DVC) administration of 7-chlorokyurenic acid (7CKNA), an inhibitor of the NR1 subunit of the N-methyl-d-aspartate (NMDA) receptor, negates the effect of DVC glycine to lower hepatic triglyceride-rich very low-density lipoproteins (VLDL-TG) secretion. B, Experimental protocol. Surgical timeline and methods as in Figure 1. DVC infusions of 7CKNA (30 μmol/L; n=5), glycine (10 μmol/L; n=12), or glycine (10 μmol/L)+7CKNA (30 μmol/L; n=11) were used. Following tyloxapol administration, DVC coinfusion of 7CKNA with glycine reversed the effect of glycine to: (C) lower plasma triglyceride (TG) and (D) decrease the rate of VLDL-TG appearance in plasma. E, DVC glycine reduced the hepatic mRNA expression of stearoyl-CoA denaturase-1 (scd-1) as compared with 7CKNA only, and simultaneous administration of DVC 7CKNA reversed glycine's effect to lower the expression of this gene. White (□), 7CKNA; black (■), glycine; gray (), glycine+7CKNA. Data are presented as mean±SEM. *P<0.05 glycine vs glycine+7CKNA. Jessica T.Y. Yue et al. Circ Res. 2012;110:1345-1354 Copyright © American Heart Association, Inc. All rights reserved.

A, Schematic representation of working hypothesis: molecular knockdown of the NR1 subunit of the N-methyl-d-aspartate (NMDA) receptor in the dorsal vagal complex (DVC) abolishes the effect of DVC glycine to lower hepatic triglyceride-rich very low-density lipoproteins (VLDL-TG) secretion. A, Schematic representation of working hypothesis: molecular knockdown of the NR1 subunit of the N-methyl-d-aspartate (NMDA) receptor in the dorsal vagal complex (DVC) abolishes the effect of DVC glycine to lower hepatic triglyceride-rich very low-density lipoproteins (VLDL-TG) secretion. B, Experimental protocol. Day 0: Immediately following stereotaxic surgery, adenovirus containing short hairpin RNA (shRNA) to NR1 or a mismatch (MM) sequence was injected into the DVC (3 μL bilaterally; 4.0×1011 pfu/mL). Day 12: Five days following catheterization of the left carotid artery and right jugular vein (Day 7), NR1 shRNA (n=9) and mismatch sequence (MM) (n=6) adenovirus injected rats underwent tyloxapol experiments and received DVC glycine (10 μmol/L) infusions. Molecular knockdown of DVC NR1 negated the ability of DVC glycine to: (C) lower plasma triglyceride (TG) and (D) decrease the rate of VLDL-TG appearance in plasma as compared to rats that received DVC adenovirus expressing a MM sequence. Black (■), MM+glycine; gray (), NR1 shRNA+glycine. Data are presented as mean±SEM *P<0.05 MM+glycine vs NR1 shRNA+glycine. Jessica T.Y. Yue et al. Circ Res. 2012;110:1345-1354 Copyright © American Heart Association, Inc. All rights reserved.

A, Schematic representation of working hypothesis: The effect of dorsal vagal complex (DVC) glycine to lower hepatic triglyceride-rich very low-density lipoproteins (VLDL-TG) secretion is mediated via the hepatic vagus nerve. A, Schematic representation of working hypothesis: The effect of dorsal vagal complex (DVC) glycine to lower hepatic triglyceride-rich very low-density lipoproteins (VLDL-TG) secretion is mediated via the hepatic vagus nerve. Thus, transection of the hepatic vagus nerve (as indicated by the dotted lines) will negate the suppressive effect of DVC glycine on hepatic VLDL-TG secretion. B, Experimental protocol. Day 0: Stereotaxic surgery as in Figure 1. Day 7: Vascular catheterization of the left carotid artery and right jugular vein as in Figure 1 followed by hepatic vagotomy or sham-vagotomy surgery. Day 12: After 5 days of recovery, vagotomized or sham-operated rats underwent tyloxapol experiments and received DVC glycine (10 “micro”mol/L) or saline infusions. DVC administration of glycine in sham-operated rats with an intact vagus nerve (n=9) (C) lowered plasma triglycerides and (D) lowered the rate of appearance of VLDL-TG in the plasma as compared to vagotomised rats (n=8) and sham-operated rats given DVC saline (n=3). There was no difference between vagotomised rats given DVC saline (n=4) or DVC glycine. White (□), sham Vx+saline; light gray (), hep Vx+saline; black (■), sham Vx+glycine; dark gray (), hep Vx+glycine. Data are presented as mean±SEM *P<0.05 sham Vx+glycine vs sham Vx+saline, hep Vx+glycine. Jessica T.Y. Yue et al. Circ Res. 2012;110:1345-1354 Copyright © American Heart Association, Inc. All rights reserved.

A, Schematic representation of working hypothesis: overfed rats on a high-fat diet (HFD) have increased hepatic triglyceride-rich very low-density lipoproteins (VLDL-TG) secretion. A, Schematic representation of working hypothesis: overfed rats on a high-fat diet (HFD) have increased hepatic triglyceride-rich very low-density lipoproteins (VLDL-TG) secretion. Dorsal vagal complex (DVC) glycine will normalize hepatic secretion of VLDL-TG. B, Experimental protocol: Surgical timeline and methods as in Figure 1. Day 9: Three days prior to the experimental day, rats were either maintained on regular chow (reg chow) or introduced to a HFD. Day 12: During the tyloxapol experiment, rats fed with regular chow or HFD were infused with saline, glycine (10 μmol/L), or glycine (10 μmol/L)+MK-801 (0.06 ng/min). DVC glycine administered to rats given regular chow (n=12): (C) lowered plasma triglyceride (TG) and (D) decreased the rate of VLDL-TG appearance in the plasma as compared to rats given DVC saline (n=5). HFD increased plasma VLDL-TG levels and the rate of VLDL-TG secretion (n=9). DVC glycine normalized the HFD-induced increase in plasma VLDL-TG levels and rate of VLDL-TG appearance (n=8) to the level of regular chow controls. Coinfusion of MK-801 (n=4) blocked the ability of glycine to normalize plasma VLDL-TG levels and the rate of VLDL-TG appearance. White squares (□), reg chow+saline; black squares (■), reg chow+glycine; white triangles (▵), HFD+saline; black triangles (▴), HFD+glycine; gray triangles (), HFD+glycine+MK-801. Data are presented as mean±SEM *P<0.05 reg chow+saline vs reg chow+glycine. †P<0.05 reg chow+saline vs HFD+saline, HFD+glycine+MK-801. ‡P<0.05 HFD+glycine vs HFD+saline, HFD+glycine+MK-801. §P<0.05 HFD+glycine vs reg chow+glycine. Jessica T.Y. Yue et al. Circ Res. 2012;110:1345-1354 Copyright © American Heart Association, Inc. All rights reserved.