GLP-2 Regulation of Dietary Fat Absorption and Intestinal Chylomicron Production via Neuronal Nitric Oxide Synthase (nNOS) Signaling

Insulin-resistant diseases, such as obesity and type 2 diabetes (T2D), continue to be a global epidemic in both adults and children (1). Affected individuals present with dyslipidemia, characterized by excess triglyceride (TG)–rich lipoproteins (TRL) in circulation, such as chylomicrons (CM) and VLDL (2). TRL overproduction and reduced clearance result in hypertriglyceridemia, excess atherogenic CM remnants, and an increased risk of cardiovascular disease, the leading cause of death in individuals with diabetes (2,3). Insulin resistance is also associated with postprandial dyslipidemia, a condition involving excess production of apolipoprotein B48 (apoB48)–containing CMs from the intestine after a meal (2). Elucidating the underlying mechanisms of CM overproduction is therefore crucial for the treatment of these clinical disorders.

The gut has been linked to lipid and lipoprotein abnormalities through the dysregulation of intestinal hormone glucagon-like peptide 2 (GLP-2) (4). GLP-2 is cosecreted with GLP-1 in equimolar amounts from enteroendocrine l cells in the small intestine upon nutrient sensing (5). Our laboratory was the first to demonstrate that GLP-2 increases postprandial CM production following lipid ingestion in a Syrian golden hamster model, an observation later confirmed in humans (6,7). The actions of GLP-2 also predominate in insulin-resistant conditions, resulting in an increased rate of dietary fat absorption and excess CM production (8), thus contributing to postprandial dyslipidemia. However, the mechanisms by which GLP-2 enhances CM production remain unclear.

Further studies in our laboratory have revealed that GLP-2 may stimulate lipid absorption into the enterocyte via nitric oxide (NO) signaling (9). When GLP-2–treated hamsters were administered the pan-specific NO synthase (NOS) inhibitor l-N^G-nitroarginine methyl ester, intestinal apoB48 secretion was blocked, and TG content in the TRL fraction was significantly reduced in the postprandial state. Additionally, endothelial NOS (eNOS)–deficient mice were resistant to GLP-2 stimulation; meanwhile, i.p. injection of the NO donor S-nitroso-l-glutathione was able to rescue the effect of GLP-2. These findings indicate that NO generation is critical for GLP-2–stimulated CM secretion in the postprandial state.

Although NO is an important secondary messenger involved in several major gastrointestinal processes (10–12), the proabsorptive mechanisms of NO are not yet fully understood. Interestingly, GLP-2 receptor (GLP-2R) expression has been found on enteric neurons, and receptor stimulation increases neuronal NOS (nNOS) expression in enteric neurons (13). Given that GLP-2Rs are lacking on enterocytes (14,15), it is possible that GLP-2 may bind its receptor on an intermediary cell to stimulate NO secretion, which will act directly on enterocytes.

Therefore, the aim of the current study was to examine how NO synthesized from nNOS mediates the stimulatory effects of GLP-2 on dietary lipid absorption and CM secretion. We used C57BL/6J (wild-type [WT]) mice, nNOS^−/− mice, and Syrian golden hamsters to examine the effect of GLP-2 on postprandial lipid and apoB48-containing CM accumulation after intraduodenal and oral fat administration. nNOS inhibition in hamsters was achieved using N^ω-propyl-l-arginine hydrochloride (NPA), and protein kinase G (PKG) was inhibited in WT mice using KT5823. We also characterized the metabolic phenotype of nNOS^−/− mice to understand the role of nNOS in intestinal lipid absorption and metabolism. Collectively, these findings reveal a novel role for nNOS in GLP-2–mediated stimulation of dietary lipid absorption and CM secretion via an NO–cyclic guanosine monophosphate (cGMP)-PKG–dependent pathway, with implications for understanding the action of GLP-2 in the context of postprandial dyslipidemia.

Male and female nNOS^−/− mice (B6.129S4-Nos1^tm1Plh/J; JAX stock Catalog no. 002986), generated on a C57BL/6J background (16) and C57BL/6J mice (JAX stock Catalog no. 000664) were acquired from The Jackson Laboratory. Male Syrian golden hamsters (Mesocricetus auratus) were acquired from Envigo. All animals were bred and maintained in-house at the Peter Gilgan Centre for Research and Learning. The 8–12-week-old mice and 10–12-week-old hamsters were housed under a 12 h light/dark cycle with access to a standard chow diet and water ad libitum. Age- and sex-matched animals were randomly assigned to experimental groups. All procedures were approved by The Hospital for Sick Children Animal Care Committee.

Fasting of age- and sex-matched WT and nNOS^−/− mice occurred during the light/fasting cycle, 5 h prior to baseline blood draw (Fig. 3A). Mice were anesthetized with isoflurane, and a midabdominal incision was done to expose the duodenum. A total of 200 μL of olive oil (Sigma-Aldrich) was injected directly into the proximal duodenum (time 0). The method of intraduodenal fat administration removes any confounding effects of gastric emptying on lipid transit (20), as previous studies have indicated that nNOS^−/− mouse strains present with achalasia, hypertrophic pyloric stenosis, and gastroparesis (21,22). The 0.25 mg/kg human GLP-2^1-33 (Bachem) or vehicle (PBS) was infused into the abdominal cavity, followed by 500 mg/kg Pluronic F-127 (Sigma-Aldrich) to inhibit peripheral TRL catabolism. The incision was sutured closed, and mice were conscious during blood collection time points.

Fasting of age- and sex-matched WT and nNOS^−/− mice occurred in the light/fasting cycle, 5 h prior to baseline blood draw (Fig. 3H). Mice were orally gavaged with 200 μL of olive oil at time 0 and immediately treated with 0.25 mg/kg human GLP-2^1-33 or vehicle (PBS) i.p., followed by 500 mg/kg Pluronic F-127 i.p.

Fasting of male hamsters occurred in the dark/feeding cycle, 16 h prior to baseline blood draw (Fig. 4A). Hamsters required an extended fasting period, as the intestines were not entirely emptied from previous food intake when excised after a 5 h fast. In comparison, 5 h is sufficient to activate the fasting state in mice, while fasting for 16 h induces unnecessary stress. A total of 2 mg/kg NPA (Cayman Chemical) or vehicle (saline) was administered i.p., 15 min prior to an oral gavage of olive oil (200 μL) at time 0. Hamsters were then treated with 0.25 mg/kg human GLP-2^1-33 or vehicle (PBS) i.p., followed by 500 mg/kg Pluronic F-127 i.p.

Fasting of male WT mice occurred in the light/fasting cycle, 5 h prior to baseline blood draw and treatment with 1 mg/kg KT5823 (Cayman Chemical) or vehicle (DMSO) i.p. (Fig. 5A). One hour later, mice were orally gavaged with 200 μL of olive oil at time 0 and immediately treated with 0.25 mg/kg human GLP-2^1-33 or vehicle (PBS) i.p., followed by 500 mg/kg Pluronic F-127 i.p.

Blood samples were collected via the tail vein for mice and retro-orbitally for hamsters at 30, 60, and 90 min post–fat administration. A cardiac puncture was conducted at 120 min, and animals were euthanized via exsanguination. Blood samples were collected into heparinized tubes for isolation of plasma and stored at −80°C.

The plasma layer was separated by centrifuging blood samples at 6,000 rpm, 4°C for 10 min, and supplemented with a cocktail of protease inhibitors (Sigma-Aldrich). To isolate the TRL fraction, 100 μL of plasma from mice and 150 μL of plasma from hamsters were layered under 1.006 g/mL potassium bromide in Microfuge tubes (Beckman Coulter) as previously described (17). Tubes were centrifuged at 35,000 rpm, 10°C, for 70 min in an SW 55 Ti rotor (Beckman Coulter). The top 500 μL of the tube volume was collected for mice and the top 300 μL for hamsters. TG and cholesterol levels from the plasma and TRL fractions were determined using an enzymatic-based colorimetric assay (Randox Laboratories).

Plasma samples were diluted to blot for apoB (1:20) and albumin (1:32,000). Plasma proteins were run on a 5% gel for apoB and 10% gel for albumin at 100 V. For apoB immunoblotting in the TRL fraction, samples were run on a 5% gel at 35 V overnight at 4°C. Separated proteins were transferred to a polyvinylidene difluoride membrane at 35 V overnight at 4°C. Membranes were incubated with primary antibodies (1:1,000 dilution; anti-human apoB and anti-human albumin; Nittobo America Inc.) overnight at 4°C and then incubated with secondary antibody (1:80,000 dilution; anti-goat IgG; Sigma-Aldrich) for 1 h at room temperature. Membranes were visualized using Western Lightning ECL Detection Reagent (PerkinElmer), exposed to film, and developed. Plasma apoB intensities were normalized to albumin.

Lipids were extracted from flash-frozen liver tissue and dried fecal samples as previously described (18) using a 2:1 chloroform-methanol mixture (Folch method). Fecal samples were collected per cage (three to four mice per cage) over a 24-h period. TG and cholesterol content were determined using an enzymatic-based colorimetric assay (Randox Laboratories).

Mice were fasted for 5 h prior to an oral gavage of 2 g/kg glucose (MilliporeSigma). A blood sample was drawn from the tail vein at 0, 15, 30, 60, 90, and 120 min after glucose administration. Blood glucose concentrations were measured using the OneTouch Ultra 2 glucometer.

RNA was extracted from liver and jejunum tissue samples using TRIzol Reagent (Thermo Fisher Scientific) according to the manufacturer’s instructions. cDNA was synthesized using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems) on a Mastercycler gradient (Eppendorf). The resultant cDNA was used to assess mRNA expression by real-time quantitative PCR with Power SYBR Green PCR Master Mix (Applied Biosystems). The forward and reverse primers used are listed in Supplementary Table 1. The ΔΔ cycle threshold method (19) was used to quantify transcript levels, and all genes were normalized to 18 s.

Statistical analyses were performed using Prism version 8.2.1 software (GraphPad, San Diego, CA), and results are reported as mean ± SEM. Statistical comparisons were performed using two-tailed Student unpaired t test, one-way ANOVA, and two-way ANOVA with the Bonferroni post hoc test.

Data set and resources are available upon request.

To understand the importance of nNOS in intestinal lipid absorption and metabolism, we first characterized the basal metabolic profile of the nNOS^−/− mouse model. Body weight gain of chow-fed nNOS^−/− and C57BL/6J (WT) control mice was monitored weekly from 3 to 8 weeks of age. Male and female nNOS^−/− mice displayed significantly lower body weights at each week than their WT counterparts (P < 0.0001) (Fig. 1A and Supplementary Fig. 1A). At 8 weeks of age, total food intake was measured. Over the course of this representative week, male and female nNOS^−/− mice consumed less food than WT mice (P < 0.0001) (Fig. 1B and Supplementary Fig. 1B).

Fasting plasma lipid and blood glucose analyses were conducted at 8–12 weeks of age. While male nNOS^−/− mice did not display a significant difference in fasting plasma TG compared with WT control mice (Fig. 1C), fasting plasma cholesterol concentration was significantly reduced in nNOS^−/− mice (P < 0.05) (Fig. 1D). In contrast, female nNOS^−/− mice exhibited significantly increased fasting plasma TG concentration (P < 0.001) and no difference in fasting plasma cholesterol levels compared with WT control mice (Supplementary Fig. 1C and D). No significant differences were observed in fasting blood glucose concentration in both sexes compared with control mice (Fig. 1D and Supplementary Fig. 1D).

Oral glucose tolerance tests were performed on 5 h fasted WT and nNOS^−/− mice. Male nNOS^−/− mice displayed significantly elevated blood glucose concentrations at the 60-, 90-, and 120-min time points compared with WT mice (P < 0.001 and P < 0.0001) (Fig. 1F). Similar results were observed in female nNOS^−/− mice at the 60- and 90-min time points (P < 0.0001) (Supplementary Fig. 1F).

Next, we assessed measurements of liver and intestinal size. No significant difference was observed in liver weight as a percentage of body weight in male nNOS^−/− mice compared with WT control mice (Fig. 2A). However, male nNOS^−/− mice displayed significantly decreased small intestinal weight as a percentage of total body weight (P < 0.05) (Fig. 2B) and intestinal weight per centimeter (P < 0.001) (Fig. 2C). In contrast, female nNOS^−/− mice did not exhibit differences in these measurements (Supplementary Fig. 2A–C).

To evaluate lipid storage and output, we analyzed hepatic and fecal lipid content in these mice. Hepatic TG and cholesterol content was significantly reduced in both male (P < 0.05) (Fig. 2D and E) and female (P < 0.001 and P < 0.0001) (Supplementary Fig. 2D and E) nNOS^−/− mice compared with WT controls. Comparatively, female nNOS^−/− mice exhibited lower fecal TG and cholesterol content (P < 0.0001) (Supplementary Fig. 2F and G). Male mice showed no differences in fecal lipid content (Fig. 2F and G).

Finally, levels of hepatic and jejunal mRNA transcripts encoding lipid metabolic proteins were analyzed in mice following oral fat administration. Hepatic mRNA levels for Cd36 and Acaca were significantly reduced in male nNOS^−/− mice compared with WT mice, while hepatic mRNA levels for Fasn were significantly elevated (P < 0.05) (Fig. 2H). In contrast, hepatic mRNA levels for Nos2, Fasn, and Scarb1 were significantly elevated in female nNOS^−/− mice compared with WT mice, while hepatic mRNA levels for Cd36 were significantly reduced (P < 0.05) (Supplementary Fig. 2H). Interestingly, jejunal mRNA levels for Nos2 were significantly elevated in male nNOS^−/− mice compared with WT mice (P < 0.01) (Fig. 2H), while mRNA levels of Nos3 were significantly increased in female nNOS^−/− mice compared with WT mice (P < 0.05) (Supplementary Fig. 2H).

In vivo studies in nNOS^−/− and WT control mice were conducted to determine if nNOS-generated NO is required for GLP-2–mediated lipid absorption and CM secretion. The experimental timeline in Fig. 3A shows that fasted mice received an intraduodenal injection of olive oil and were treated with GLP-2 or vehicle, followed by Pluronic F-127. As previously shown (6), GLP-2–treated WT mice exhibited significant increases in both plasma TG and cholesterol 90 and 120 min after olive oil administration compared with WT control mice (P < 0.0001) (Fig. 3B and C). Interestingly, the effect of GLP-2 was abolished in nNOS^−/− mice, as no significant differences were observed in plasma lipid accumulation between nNOS^−/− mice treated with GLP-2 or vehicle.

TRL lipid content isolated from plasma collected at the 120-min time point also showed significant increases in TG and cholesterol content in GLP-2–treated WT mice (P < 0.01 and P < 0.001) (Fig. 3D and E). However, the effect of GLP-2 on increased TRL lipid accumulation was abolished in nNOS^−/− mice despite GLP-2 treatment. Consistent with its known effect, GLP-2 administration in WT mice also resulted in significant increases in circulating levels of plasma and TRL apoB48 (P < 0.01 and P < 0.0001) (Fig. 3F and G). Once again, the effect of GLP-2 on apoB48 was abolished in nNOS^−/− mice despite GLP-2 treatment. Similarly, GLP-2 treatment in female nNOS^−/− mice did not increase plasma and TRL lipid accumulation after intraduodenal olive oil administration (Supplementary Fig. 3B–E).

To complement these results, WT and nNOS^−/− mice were challenged with an oral fat load followed by treatment with GLP-2 or vehicle, as shown in the experimental timeline in Fig. 3H. GLP-2 treatment in WT mice led to characteristic rises in plasma TG and cholesterol at 90 and 120 min (P < 0.05, P < 0.01, and P < 0.0001) (Fig. 3I and J). As previously observed, GLP-2–treated nNOS^−/− mice did not show increases in plasma lipids compared with control mice. Furthermore, GLP-2–treated WT mice exhibited significant increases in TRL TG and cholesterol (P < 0.01 and P < 0.001) (Fig. 3K and L), while GLP-2–treated nNOS^−/− mice failed to respond to GLP-2. Similarly, GLP-2 treatment in female nNOS^−/− mice did not increase plasma or TRL lipid accumulation after oral olive oil administration (Supplementary Fig. 3G–J).

Next, to determine if GLP-2 has a direct effect through nNOS, nNOS-specific inhibitor NPA (23,24) was administered to Syrian golden hamsters. A hamster model was used as its lipoprotein metabolism closely resembles that of humans (25–27). Mice produce and secrete apoB48-containing CMs from their liver and small intestine, while hamsters and humans only secrete apoB48-containing CMs from their small intestine (28), allowing for the determination of intestinal contributions to circulating lipoproteins. Treatment with NPA or vehicle occurred 15 min prior to an oral fat load. Hamsters were then treated with GLP-2 or vehicle, followed by Pluronic F-127. As expected, GLP-2–treated hamsters exhibited significant increases in plasma TG and cholesterol at 60, 90, and 120 min compared with control hamsters (P < 0.05, P < 0.01, and P < 0.0001) (Fig. 4B and C). Similar to the outcomes observed in GLP-2–treated nNOS^−/− mice, NPA administration abolished the effect of GLP-2 in hamsters cotreated with NPA and GLP-2. Comparatively, GLP-2–mediated increases in TRL TG content were also abrogated in hamsters cotreated with NPA and GLP-2 (Fig. 4D). Furthermore, GLP-2 administration resulted in significant increases in circulating levels of plasma apoB48 by 90 and 120 min (P < 0.001 and P < 0.0001) (Fig. 4E). Circulating levels of TRL apoB48 were also dramatically increased by 90 and 120 min (P < 0.0001) (Fig. 4F). Once again, GLP-2–mediated increases in postprandial lipoprotein secretion were completely abolished in hamsters cotreated with NPA and GLP-2.

To elucidate a mechanism of action for the role of nNOS in intestinal CM production and release, the involvement of PKG was explored as a potential downstream effector in the GLP-2–nNOS signaling pathway. nNOS-generated NO is known to activate PKG through cGMP and guanylyl cyclase (GC) signaling (29,30). Therefore, we sought to determine if nNOS is acting through a cGMP-GC-PKG–dependent pathway. PKG-specific inhibitor KT5823 was used to pretreat male WT mice, as previously described (31,32), followed by an oral fat load and treatment with GLP-2 or vehicle (Fig. 5A). Notably, GLP-2–mediated increases in postprandial plasma and TRL lipid content were observed in GLP-2–treated mice versus controls (P < 0.05, P < 0.001, and P < 0.0001) (Fig. 5B–E). In comparison, the effect of GLP-2 is lost in mice cotreated with KT5823 and GLP-2 compared with mice treated solely with KT5823.

Previous studies from our laboratory have demonstrated that GLP-2 augments intestinal lipoprotein production through enhanced CD36 glycosylation, leading to increased CM assembly and secretion (6,9,28). These findings were reported in mouse and hamster models and have been confirmed in human studies (7). While the stimulatory effects of GLP-2 on postprandial lipid absorption and secretion are well documented, the precise mechanism of action remains unclear. Interestingly, the GLP-2R is not expressed in the enterocyte; thus, GLP-2R–dependent stimulation on lipid absorption likely requires the activation of downstream mediators. GLP-2 action has been previously linked to nNOS activity in rats, in which GLP-2R stimulation increased nNOS mRNA expression in enteric neurons ex vivo (13). Therefore, it is likely that GLP-2 exerts its effects on an intermediary cell type, such as enteric neurons, to stimulate NO production, which in turn will regulate nutrient absorption by enterocytes. The current study examined how nNOS-synthesized NO mediates the stimulatory effects of GLP-2 on dietary lipid absorption and CM secretion.

Characterization of an nNOS^−/− mouse model clearly demonstrated the importance of nNOS signaling in the maintenance of basal metabolic health. Interestingly, nNOS^−/− mice displayed sex-specific differences in fasting plasma lipids, with male nNOS^−/− mice exhibiting significantly reduced plasma cholesterol levels with no change in TG, while female nNOS^−/− mice only exhibited significantly increased plasma TG levels. This is surprising, as previous studies in male NOS knockout (KO) models, either deficient in all three NOS isoforms (nNOS/inducible NOS/eNOS^−/− mice) or singly NOS-deficient mice, demonstrated hypertriglyceridemia (33). However, nNOS KO was sufficient to incite significant impairments in glucose tolerance, consistent with previous findings implicating NO as a regulator of insulin sensitivity (33,34).

Analysis of hepatic lipid content revealed that nNOS^−/− mice exhibit significant reductions in hepatic TG and cholesterol content compared with WT mice. This decreased hepatic lipid storage is consistent with findings from Schild et al. (35), which revealed that lipid droplets are absent from the cytosol of hepatocytes in nNOS^−/− mice. In line with this, hepatic mRNA levels for Cd36 and Acaca were significantly reduced in male nNOS^−/− mice. Finally, jejunal inducible NOS mRNA was significantly upregulated in male nNOS^−/− mice, and eNOS mRNA was significantly upregulated in female nNOS^−/− mice. Given that nNOS is the predominant NOS isoform in the small intestine that provides constant, low levels of NO (36), these results may indicate that male and female nNOS^−/− mice may preferentially upregulate different NOS isoforms to compensate for the loss of nNOS in the jejunum.

Following the characterization of the nNOS^−/− mice, we investigated whether nNOS-generated NO is required for GLP-2–mediated postprandial lipid absorption and CM secretion. In this study, we demonstrate, for the first time, that GLP-2–mediated increases on postprandial plasma and TRL lipid accumulation and circulating apoB48 protein levels are abolished in nNOS^−/− mice following an intraduodenal or oral fat load. Importantly, while the effect of GLP-2 on lipid absorption and CM secretion is only partially lost in eNOS KO mice (9), the lipemic effects of GLP-2 are completely abrogated in our nNOS^−/− mouse model. The relative importance of nNOS over eNOS in lipid metabolism can be attributed to the fact that nNOS is the predominant NOS isoform in the small intestine, with nNOS contributing to >90% of all intestinal NOS isoforms (36). Furthermore, nNOS and the GLP-2R have been shown to be colocalized on enteric neurons, which are in close proximity to enterocytes (13). While it has been previously suggested that GLP-2 may increase nutrient absorption via increases in mesenteric blood flow (37), recent studies have shown that the absorptive effects of GLP-2 are independent of blood flow (38,39). It is also important to consider the possibility that nNOS may be a global regulator of CM secretion; however, in this study, we report no baseline differences in circulating apoB48 protein levels between WT and nNOS^−/− mice, and jejunal gene expression analysis displayed similar apoB and MTP levels between groups, suggesting nNOS^−/− mice have similar CM secretion pathways to WT mice.

To determine if GLP-2 exerts its effects directly through nNOS, an nNOS-specific inhibitor, NPA, was administered to male hamsters. Overall, the effect of GLP-2–mediated increases on postprandial lipemia and apoB48 secretion was abolished in hamsters cotreated with NPA. Our results are consistent with previous studies in Syrian golden hamsters in which administration of NOS inhibitor l-N^G-nitroarginine methyl ester blocked the effect of GLP-2 on enhanced intestinal apoB48 secretion and TRL TG accumulation (9). In contrast, findings from Xiao et al. (39) suggested that GLP-2 mobilizes lipids from the intestine of healthy human males by a systemic NO-independent mechanism. However, Xiao et al. (39) examined the effect of NO on TG mobilization from preformed intestinal lipid storage pools 7 h after ingestion of a high-fat load, while the current study demonstrated a requirement for nNOS-generated NO on postprandial lipid absorption and CM secretion immediately after high-fat ingestion. The intestine can store dietary TG in cytoplasmic lipid droplets within the enterocyte well beyond the postprandial period (40), with the gut contributing to CM secretion for up to 16 h after a previous meal (41,42). Furthermore, the production of NO may be low 7 h after ingestion of dietary lipids; thus, NOS inhibition may not have a robust effect on NO production in the postabsorptive state. Given the conflicting findings between these studies, further investigation into the involvement of NO in nutrient handling within the enterocyte is warranted. Nonetheless, our study points to downstream PKG signaling.

Finally, to elucidate the mechanism by which nNOS regulates GLP-2–stimulated intestinal CM production and release, PKG was explored, as nNOS-generated NO activation of PKG through cGMP and GC signaling has been well characterized (43,44). In this study, we show that PKG inhibition in male WT mice using KT5823 blocks GLP-2–stimulated postprandial lipid accumulation, suggesting an important role for PKG signaling in intestinal lipoprotein metabolism. The involvement of protein kinases in the action of GLP-2 is not uncommon, as GLP-2R activation results in cAMP accumulation and protein kinase A activation (45). Therefore, we used a highly specific, cell-permeable inhibitor that selectively inhibits PKG over protein kinase A (46). Future studies are required to assess the cellular targets of PKG within the enterocyte to elucidate how PKG is modulating lipid handling. While the conclusions of this study are based on the presented data, it is possible that additional modulators of the CM secretory pathway may be involved in these initial findings.

In conclusion, our data indicate a novel role for nNOS-generated NO in the stimulatory effects of GLP-2 on dietary lipid absorption and CM secretion. We also demonstrate the role of PKG as a potential downstream signaling molecule in intestinal lipoprotein metabolism. Taken together, our findings begin to uncover a novel mechanism by which the actions of GLP-2 are mediated by a NO-cGMP-PKG–dependent pathway. Our findings may have translational relevance for understanding GLP-2–induced postprandial dyslipidemia in insulin-resistant conditions such as obesity and diabetes. Elevated plasma GLP-2 levels have been significantly correlated with increases in hemoglobin A_1c and insulin resistance in obese adult subjects (47,48). Additionally, elevated levels of circulating GLP-2 have been reported in streptozotocin-induced diabetic rats compared with nondiabetic controls (49), indicating that GLP-2 may also be associated with diabetic dyslipidemia. Given that patients with T2D display excessive CM secretion (50), further studies are required to understand the exact role GLP-2 plays in the development of insulin resistance and the associated dyslipidemia. It is possible that increased GLP-2 secretion may contribute to insulin resistance through the enhanced absorption of nutrients such as fatty acids, a key factor in insulin resistance (51). Elucidation of the mechanistic actions of GLP-2 may provide insight for future therapeutic interventions to treat postprandial dyslipidemia in insulin-resistant conditions such as obesity and T2D.

Funding. This study was supported by a Canadian Institutes of Health Research (CIHR) Foundation grant (6210100485) to K.A. E.M.G. is supported by a Physiology Graduate Fellowship award from the University of Toronto. F.R. is supported by a CIHR doctoral award.

Duality of Interest. No potential conflicts of interest relevant to this article were reported.

Author Contributions. E.M.G. designed the study, executed the experiments, analyzed the data, and wrote the manuscript. F.R. designed the study, executed experiments, and edited the manuscript. S.H. executed experiments and edited the manuscript. K.A. designed the study and edited the manuscript. E.M.G. and K.A. are the guarantors of this work and, as such, had full access to all of the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis.

Prior Presentation. This work was presented in poster form at the 80th Scientific Sessions of the American Diabetes Association, 12–16 June 2020.