Hematopoietic cell– versus enterocyte-derived dipeptidyl peptidase-4 differentially regulates triglyceride excursion in mice

Postprandial triglycerides (TGs) are elevated in people with type 2 diabetes (T2D). Glucose-lowering agents, such as glucagon-like peptide-1 (GLP-1) receptor agonists and dipeptidyl peptidase-4 (DPP-4) inhibitors, also reduce postprandial TG excursion. Although the glucose-lowering mechanisms of DPP-4 have been extensively studied, how the reduction of DPP-4 activity improves lipid tolerance remains unclear. Here, we demonstrate that gut-selective and systemic inhibition of DPP-4 activity reduces postprandial TG excursion in young mice. Genetic inactivation of Dpp4 simultaneously within endothelial cells and hematopoietic cells using Tie2-Cre reduced intestinal lipoprotein secretion under regular chow diet conditions. Bone marrow transplantation revealed a key role for hematopoietic cells in modulation of lipid responses arising from genetic reduction of DPP-4 activity. Unexpectedly, deletion of Dpp4 in enterocytes increased TG excursion in high-fat diet–fed (HFD-fed) mice. Moreover, chemical reduction of DPP-4 activity and increased levels of GLP-1 were uncoupled from TG excursion in older or HFD-fed mice, yet lipid tolerance remained improved in older Dpp4–/– and Dpp4EC–/– mice. Taken together, this study defines roles for specific DPP-4 compartments, age, and diet as modifiers of DPP-4 activity linked to control of gut lipid metabolism.


Introduction
Elevated concentrations of postprandial triglyceride-rich lipoproteins (TRLs) are associated with an increased incidence of cardiovascular disease (1), and therapies that lower triglycerides (TGs) have demonstrated cardiovascular benefit (2). TRLs, including very-low-density lipoproteins and chylomicrons, are elevated in the setting of insulin resistance and type 2 diabetes (T2D), reflecting impaired clearance and dysregulated TG secretion from both the liver and intestine (3). These findings have translational relevance, as elevated levels of plasma TGs may contribute to the excess burden of cardiovascular risk in people with T2D (4).
DPP4i lower glucose in part through activation of the GLP-1R in mice and humans (11)(12)(13). These drugs also lower postprandial TGs and apoB48 in humans (5,14,15) and in mice through mechanisms requiring the GLP-1R (9). Interestingly, intracerebroventricular administration of a DPP4i produces an acute TG-lowering effect in hamsters (16). Nevertheless, the mechanisms by which peripherally administered DPP4i inhibits enterocyte chylomicron secretion have not been determined.
DPP-4 is a ubiquitous cell membrane-associated serine protease expressed in many tissues, including the gut (17,18); it also circulates as a catalytically active soluble form. Here, we studied the cellular sites of DPP-4 expression required for control of lipid metabolism in mice using gut-selective versus systemic Postprandial triglycerides (TGs) are elevated in people with type 2 diabetes (T2D). Glucoselowering agents, such as glucagon-like peptide-1 (GLP-1) receptor agonists and dipeptidyl peptidase-4 (DPP-4) inhibitors, also reduce postprandial TG excursion. Although the glucoselowering mechanisms of DPP-4 have been extensively studied, how the reduction of DPP-4 activity improves lipid tolerance remains unclear. Here, we demonstrate that gut-selective and systemic inhibition of DPP-4 activity reduces postprandial TG excursion in young mice. Genetic inactivation of Dpp4 simultaneously within endothelial cells and hematopoietic cells using Tie2-Cre reduced intestinal lipoprotein secretion under regular chow diet conditions. Bone marrow transplantation revealed a key role for hematopoietic cells in modulation of lipid responses arising from genetic reduction of DPP-4 activity. Unexpectedly, deletion of Dpp4 in enterocytes increased TG excursion in high-fat diet-fed (HFD-fed) mice. Moreover, chemical reduction of DPP-4 activity and increased levels of GLP-1 were uncoupled from TG excursion in older or HFD-fed mice, yet lipid tolerance remained improved in older Dpp4 -/and Dpp4 EC-/mice. Taken together, this study defines roles for specific DPP-4 compartments, age, and diet as modifiers of DPP-4 activity linked to control of gut lipid metabolism.
inhibitory concentrations of the DPP4i sitagliptin (19,20). We also determined the relative importance of Dpp4 expression within endothelial, hematopoietic, and gut epithelial cells for control of lipid metabolism in mice fed regular chow (RC) or high-fat diets (HFD). Finally, we assessed the relative importance of Tie2-sensitive versus Vil1-Cre-targeted DPP-4 + cell populations for transducing the lipid-lowering actions of gut-selective versus systemically effective doses of DPP4i in mice under RC and HFD conditions.

Results
Systemic and gut-selective inhibition of DPP-4 activity improves lipid tolerance and reduces TG production in young RC-fed mice. Studies of glucose metabolism have shown that low doses of DPP4i that reduce DPP-4 activity within the gut, but not meaningfully in the systemic circulation, are sufficient to improve glucose tolerance (19,20). Accordingly, we used low and high doses of sitagliptin, with or without tyloxapol, to assess how acute DPP-4 inhibition modulates lipid tolerance in mice ( Figure 1A). Consistent with our previous findings (20), oral gavage of systemically active doses of sitagliptin (10 mg/kg) inhibited plasma DPP-4 activity by approximately 85% when assessed 30 minutes after oral administration ( Figure 1B). DPP-4 activity remained suppressed at 210 minutes after gavage of sitagliptin (180 minutes after olive oil gavage) ( Figure  1B). In contrast, plasma DPP-4 activity was modestly and transiently reduced by lower doses (14 μg/ mouse) of sitagliptin ( Figure 1B), shown to selectively target DPP-4 activity within the intestinal tract (20).
In WT C57BL/6 male mice, systemic inhibition of DPP-4 activity with sitagliptin reduced TG excursion following oral olive oil gavage during a lipid tolerance test (LTT) ( Figure 1C). Circulating levels of active GLP-1 were increased approximately 4-fold in response to 10 mg/kg sitagliptin ( Figure 1C). Plasma levels of active GLP-1 were also induced, albeit to a lesser extent, with lower gut-selective doses of sitagliptin (~2.5-fold vs. 4-fold higher, for gut-selective vs. systemic doses of sitagliptin, respectively) ( Figure 1C). Nevertheless, the improvement in lipid tolerance was similar in mice receiving the 2 different doses of sitagliptin ( Figure 1C), highlighting the importance of DPP-4 + cells within the gut for control of lipid tolerance.
We next used tyloxapol to determine whether the decrease in TG excursion observed with sitagliptin was secondary to a reduction in TRL production or an increase in clearance (21). Plasma TG levels were similarly reduced by the 2 different doses of sitagliptin in the presence of tyloxapol ( Figure 1D). Moreover, sitagliptin, at doses that inhibited systemic DPP-4 activity, reduced levels of plasma apoB48, without affecting plasma apoB100 concentrations, consistent with a reduction in intestinal TRL secretion (Figure 1, E and F). In contrast, levels of apoB48 were not reduced by the gut-selective dose of sitagliptin ( Figure 1, E and F).
In contrast to findings of improved glycemia in Dpp4 -/mice, glucose tolerance was not enhanced in Dpp4 EC-/and Dpp4 EC-/-(BMT) mice, despite reductions in DPP-4 activity of approximately 50% and approximately 25%, respectively (Figure 2, C and E, and Supplemental Figure 1, C-F). Similarly, body weight and plasma TG were not different in Dpp4 EC-/mice, despite several-fold increases in fasting plasma GLP-1 levels ( Figure 2C). However, TG excursion was reduced in Dpp4 EC-/mice, in association with elevated GLP-1 levels, following olive oil administration ( Figure 2D).
To elucidate the contributions of hematopoietic versus endothelial cells to the control of TG excursion in Dpp4 EC-/mice, we reanalyzed these parameters after BMT. BMT itself increased plasma active GLP-1 levels in control animals (Supplemental Figure 2, B and D). Dpp4 EC-/-(BMT) mice exhibited an approximately 25% decrease in plasma DPP-4 activity and an approximately 2.5-fold increase in plasma active GLP-1, without differences in body weight or levels of fasting TG, consistent with the importance of DPP-4 expression in endothelial cells ( Figure 2E). However, postprandial TG excursion and plasma levels of active GLP-1 were not different between Dpp4 EC-/-(BMT) and Dpp4 EC+/+ (BMT) mice ( Figure 2F). Unexpectedly, these findings reveal a key role for DPP-4 within hematopoietic cells in the control of postprandial lipid excursion.
As gut enterocytes represent a major source of DPP-4 within the gut mucosa, and the key intestinal cell type responsible for lipid uptake and TRL secretion, we studied lipid tolerance in Dpp4 Gut-/mice (20). Plasma DPP-4 activity, glucose tolerance, body weight, plasma active GLP-1, and TG levels were not different in RC-fed Dpp4 Gut-/mice, whether examined in the fasting state or following olive oil gavage ( [LTT] in B and C) or given an i.v. injection of 0.5 g/kg tyloxapol just before olive oil (for TG and ApoB production in D-F). (B and C) Plasma DPP-4 activity just before (time -30 minutes), and at 30 (time 0), 60, 120, and 180 minutes after oral gavage or water of sitagliptin, as indicated (B, n = 6-8/group). (C) Plasma TG and AUC over 3 hours (left and middle) and plasma active GLP-1 levels 30 minutes before and 10 minutes after oral gavage of olive oil (right, n = 23-29/group) during a LTT. (D-F) Plasma TG and AUC over 180 minutes (D), and apoB48 and apoB100 protein levels measured by Western blot (WB) (E and F, n = 6-8/group) after oral gavage of water or sitagliptin, followed by i.v. injection of tyloxapol and oral gavage of olive oil, as described in A. Data are presented as mean ± SEM. Each n represents a biological replicate from 4 independent cohorts (C) and 1 group (B and D-F) of sex-and age-matched animals. (B-D) *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, using 1-way ANOVA with Tukey's correction for multiple comparisons for indicated groups. (E) *P < 0.05 vs. water, # P < 0.05 and ## P < 0.01 vs. gut-selective dose of sitagliptin using 1-way ANOVA with Tukey's correction for multiple comparisons for indicated groups.
Identification of cell types required for DPP4i to control lipid tolerance. We next assessed the specific DPP-4 + cell types essential for transducing the inhibitory actions of DPP4i in control of lipid tolerance. Plasma DPP-4 activity and TG excursion were reduced, and plasma active GLP-1 was increased, in sitagliptin-treated Dpp4 +/+ mice, but sitagliptin had no effect in Dpp4 -/mice ( Figure 1 We next examined the importance of intestinal DPP-4 as a target for the lipid-regulating activity of sitagliptin by studying Dpp4 Gut-/mice. Plasma DPP-4 activity and TG excursion after olive oil administration was reduced, and plasma active GLP-1 was increased to a greater extent after administration of systemically active versus gut-selective doses of sitagliptin in Dpp4 Gut+/+ mice ( Figure 4, A and B). , and Dpp4 Gut-/vs. Dpp4 Gut+/+ (G and H, n = 33/group for TG, n = 7-11 for other groups) mice fed a regular chow (RC) diet. Data are presented as mean ± SEM. Each n represents a biological replicate from 7 (A and B), 10 (C and D), and 2 (E and F) independent cohorts of sex-and age-matched animals. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, using Student's t test (A, C, E, and G and left panels in B, D, F, and H) or 1-way ANOVA with Tukey's correction for multiple comparisons (right panels in B, D, F, and H), for the indicated groups. BMT, bone marrow transplantation.
Similarly, both gut-selective and systemically active doses of sitagliptin increased plasma levels of active GLP-1 and reduced TG excursion after olive oil gavage in Dpp4 Gut-/mice ( Figure 4, C and D). Hence, enterocyte DPP-4 is dispensable for the actions of sitagliptin to reduce TG excursion.
HFD feeding attenuates the lipid-lowering actions of sitagliptin. To extend our findings to mice fed an energy-rich HFD, we studied lipid tolerance in C57BL/6 male mice after 6-9 weeks of 45% HFD feeding. In WT mice, systemic inhibition of DPP-4 activity, which inhibited plasma DPP-4 activity by about 90%, improved lipid tolerance and increased plasma active GLP-1 levels by approximately 4.5-fold in HFD-fed mice ( Figure 5). Using a gut-selective dose of sitagliptin, plasma DPP-4 activity was reduced by approximately 25% and plasma GLP-1 levels were increased ( Figure 5). Nevertheless, despite increased levels of GLP-1, TG excursion was not reduced, and was actually transiently increased, in HFD-fed mice after gut-selective sitagliptin ( Figure 5B).
lipid tolerance was not improved in HFD-fed Dpp4 -/-, Dpp4 EC-/-, and Dpp4 EC-/-(BMT) mice ( Figure 6, A-F). Surprisingly, plasma TG excursion was actually increased after olive oil administration in HFD-fed Dpp4 Gut-/mice, despite a lack of differences in fasting plasma DPP-4 activity, TG levels, and body weight and in fasting or olive oil-induced plasma active GLP-1 levels ( Figure 6, G and H).
To probe changes in gut gene expression potentially linked to the lipid phenotypes observed in these mice, we measured mRNA levels in the jejunum corresponding to a subset of genes involved in lipid metabolism. The majority of mRNA transcripts examined were not different in Dpp4 -/mice; levels of Apoe and Syb11 were reduced and Pcsk9 trended higher (Supplemental Figure 4A). Similarly, most mRNA transcripts were similar in Dpp4 EC-/versus Dpp4 EC+/+ mice, with higher levels of Apoa4 and Apob mRNAs (Supplemental Figure 4B). Gene expression profiles from the proximal jejunum of Dpp4 Gut-/mice were largely similar to profiles in Dpp4 Gut+/+ mice; however, Pcsk9 was higher and levels of Apoc3 and Srebf1c were reduced (Supplemental Figure 4C).
Consistent with the known specificity of action of selective DPP4i, sitagliptin had no effect on plasma TG excursion or GLP-1 levels in HFD-fed Dpp4 -/mice (Supplemental Figure 3, C and D). Moreover, the gut-selective dose of sitagliptin was unable to lower TG excursion in HFD-fed Dpp4 EC+/+ and Dpp4 EC-/mice, before and after BMT, despite an approximately 20% reduction in plasma DPP-4 activity and an increase in active GLP-1 (Figure 7). Systemic inhibition of DPP-4 activity reduced lipid excursion and increased active GLP-1 in HFD-fed Dpp4 EC+/+ mice, but not in Dpp4 EC-/mice (Figure 7, A-D). In contrast, both gut-selective and systemic inhibition of DPP-4 activity increased GLP-1 and lowered TG excursions in HFD-fed Dpp4 Gut-/mice ( Figure 8). Hence, intestinal enterocyte-derived DPP-4 is not required for the TG-reducing actions of sitagliptin under RC-or HFD-fed conditions.
In contrast to the diminished response to sitagliptin in older mice, basal TG and apoB48 levels, but not apoB100 levels, remained lower in the presence of tyloxapol in older Dpp4 -/and Dpp4 EC-/mice after olive oil administration (Figure 10, A-F). Consistent with findings in younger mice, plasma TG, apoB48, and apoB100 were not different in older Dpp4 Gut+/+ versus Dpp4 Gut-/mice after olive oil and tyloxapol administration ( Figure 10

Discussion
The mechanisms and cell types important for DPP-4-mediated control of glucose and islet function have been extensively studied in humans and mice. The available evidence supports the importance of GLP-1 and GIP as key DPP-4 substrates required for potentiating glucose-stimulated insulin secretion and reduction of glycemia following enteral glucose challenge (11)(12)(13). Moreover, studies using mouse genetics have identified the importance of endothelial cell DPP-4 in the physiological control of glucose homeostasis and as a key cellular target for the pharmacological glucoregulatory response to DPP-4 inhibition (20). In contrast, DPP-4 expressed within murine hepatocytes, adipocytes, or gut enterocytes is not required for physiological regulation of glucose tolerance and is dispensable for the glucoregulatory actions of DPP4i (20,(23)(24)(25).
DPP4i also regulate TG excursion following meals or oral lipid challenge in mice and humans (5,9,10,14). Although insulin is a powerful regulator of lipid metabolism, the acute lipid-lowering actions of GLP-1 have been shown to be mediated independent of changes in islet hormones and glucose and free fatty acid levels in mice and humans (9,26). However, the key tissues and cell types linking reduction of DPP-4 activity to control of nutrient-stimulated TG excursions have not been previously identified. Here, we show that both gut-selective and systemic inhibition of DPP-4 activity using sitagliptin improves lipid tolerance and systemic doses of sitagliptin robustly reduce intestinal TG production. Unexpectedly, the gut-selective dose of DPP4i failed to reduce apoB48 levels. Our studies also eliminate the possibility that enterocyte-derived DPP-4, accounting for about 90% of gut DPP-4, is the pool responsible for glucoseand lipid-lowering effects of either gut-selective or systemic doses of DPP4i. The results of our studies reveal that bone marrow-derived DPP-4 plays an unexpectedly important role in the lipid-lowering effects of DPP4i ( Figure 11).
DPP-4 is highly expressed within enterocytes, the major absorptive epithelial cell type, and genetic elimination of Dpp4 expression within cells targeted by Vil1-Cre reduces intestinal DPP-4 activity by up to approximately 90% (20). Although enterocytes are the major cell type responsible for lipid uptake, chylomicron assembly, and secretion of TRLs (31), loss of enterocyte DPP-4 in Dpp4 Gut-/mice does not perturb lipid tolerance in RC-fed mice. Surprisingly, HFD-fed Dpp4 Gut-/mice demonstrate increased TG excursion during a LTT, despite olive oil-stimulated increases in circulating levels of GLP-1. These findings raise the possibility that loss of enterocyte DPP-4 in the context of HFD feeding upregulates pathways converging on enhanced TRL secretion. It is possible that loss of GLP-1 responsivity, simultaneous with preservation of GLP-2 activity (32), may shift the enterocyte to a state favoring enhanced lipid secretion, a hypothesis that should be tested in future experiments.
Our molecular analysis of gut mRNA transcript abundance failed to reveal major differences in intestinal mRNA transcripts encoding proteins linked to lipoprotein secretion. Intriguingly, Pcsk9 mRNA trended higher in Dpp4 -/mice and was increased in Dpp4 Gut-/mice, whereas Pcsk9 -/mice exhibited reduced postprandial TG, attributed to reduced lymphatic chylomicron secretion and enhanced hepatic clearance (33). Although we detected reduced intestinal levels of Apoe mRNA in Dpp4 -/mice and Apoc3, Dgat2, and Srebf1c expression was decreased in Dpp4 Gut-/mice, the putative importance of these changes in the context of altered TRL secretion remains unclear. Data are presented as the mean ± SEM. Each n represents a biological replicate from 6 (A and B), 5 (C and D), 1 (E and F), and 4 (G and H) independent cohorts of sex-and age-matched animals. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, using Student's t test (A, C, E, and G and left panels in B, D, F, and H) or 1-way ANOVA with Tukey's correction for multiple comparisons (right panels in B, D, F, and H), for the indicated groups. BMT, bone marrow transplantation.
Most experiments examining the metabolic actions of DPP-4 on glucose and lipid metabolism have been carried out in younger mice. Surprisingly, in contrast to the favorable metabolic activity of sitagliptin evident in younger mice, sitagliptin failed to improve lipid tolerance in older mice. The lipid-lowering actions of sitagliptin were also diminished in HFD-fed mice, despite simultaneous reductions in DPP-4 activity and increased levels of active GLP-1. Collectively, these findings are consistent with the development of relative "GLP-1 resistance" acquired in the context of aging or HFD feeding.
Although the concept of GLP-1 resistance in humans has not been extensively explored, substantial interindividual differences in β cell responses to GLP-1 infusion have been described in healthy humans (37), findings correlated with the degree of concomitant insulin resistance. Lack of responsiveness to GLP-1 has also been described in individuals with defective β cell function, including subjects harboring genetic variants of TCF7L2 (38). Indeed, attenuated clinical responses to DPP4i in humans with T2D are associated with the extent of insulin resistance (39). Renal GLP-1 resistance, associated with attenuated natriuretic responses to GLP-1 has been observed in subjects with T2D (40,41). Intriguingly, studies in rats have correlated the diminution of GLP-1-stimulated natriuresis in hypertensive animals with a reduction of GLP-1R expression in the kidney (42,43).
Our current findings in aging or HFD-fed mice with abrogation of improved lipid tolerance, despite reduced DPP-4 activity and increased GLP-1 levels, suggest that the concept of GLP-1 resistance should be further explored in studies examining the GLP-1-dependent control of TG excursion. DPP-4 not only cleaves and inactivates GLP-1, but also regulates the biological activities of GLP-2, a peptide known to enhance intestinal lipid absorption and/or chylomicron secretion (9,(44)(45)(46). Furthermore, the incretin hormone GIP enhances lipid uptake into adipose tissue (47), an action which appears impaired in subjects with obesity (48). Moreover, GIP levels are increased in older people (49), and in subjects with T2D and obesity, and correlate with levels of fasting and postprandial TGs (50,51). Hence, it remains possible that the lipid-lowering actions of GLP-1 are opposed by increased activity of 1 or more DPP-4 substrates that retain their activity in older mice or after HFD feeding.
An unexpected finding in our studies was the loss of improved lipid tolerance in Dpp4 EC-/mice, with or without coadministration of sitagliptin, following BMT, implying an interaction between bone marrow-derived cells and the metabolic actions emanating from the reduction of DPP-4 activity in endothelial cells.   19-week-old Dpp4 Gut+/+ (A and B, n = 9-14/group) and Dpp4 Gut-/-(C and D, n = 7-11/group) mice fed a 45% high-fat (HF) diet for 6-9 weeks. Data are presented as mean ± SEM. Each n represents a biological replicate from 4 independent cohorts of sex-and age-matched animals. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, using 2-way ANOVA with Tukey's correction for multiple comparisons for the indicated groups.
Consistent with these studies, our current findings, showing a notable increase in plasma active GLP-1 levels after BMT in control and Dpp4 EC-/mice, imply that BMT may somehow confer GLP-1 resistance in Dpp4 EC-/mice. Understanding which bone marrow cells are reconstituted and responsive to a DPP4i and how these cells may influence GLP-1 action and TG metabolism in the gut represents an important focus for further studies.
Our current findings have several limitations. First, we studied several lines of mice with germline inactivation of the Dpp4 gene, invoking the possibility of compensation that might modify the observed phenotypes. Second, the majority of our studies were acute in nature and carried out in mice without chronic hyperglycemia, limiting the extrapolation of our data to settings using chronic administration of DPP4i in diabetic mice. Third, we did not incorporate systemic analyses of hepatic lipid metabolism in the elucidation of TG excursion, thus excluding potential hepatic contributions to the interpretation of our data. Moreover, we did not prospectively collect stool samples for analysis of possible changes in the gut microbiome linked to mouse phenotypes. Taken together, our current data advance the understanding of the cellular sites of DPP-4 action linked to the control of TG excursion. These data also raise the possibility of tissue GLP-1 resistance as a key factor underlying the failure to improve lipid tolerance in mouse models characterized by reduced DPP-4 activity, elevated levels of GLP-1, and yet normal or deteriorated lipid tolerance.

Methods
Animals. C57BL/6 male mice were housed (2-5 mice per cage) under a 12-hour-light/dark cycle in the Toronto Centre for Phenogenomics (TCP) facility. WT control mice originated from an in-house mouse colony. and 90 minutes after oral gavage of water or sitagliptin as indicated (B, n = 9-14/group), plasma TG and AUC over 3 hours (C, n = 9-14/group), and apoB48 and apoB100 protein levels measured by Western blot (WB) (D and E, n = 3-5/group) after oral gavage of water or sitagliptin, followed by i.v. injection of 0.5 g/kg tyloxapol and oral gavage of 200 μl olive oil, as described in Figure 1A. Data are presented as mean ± SEM. Each n represents a biological replicate from 2 independent cohorts of sex-and age-matched animals. ***P < 0.001, ****P < 0.0001, using Student's t test for the indicated groups.
Whenever possible, we carried out experiments in all groups of mice and age-matched littermate controls. Due to the lack of phenotypic differences in metabolic parameters within the control lines (20), we present data for control mice (aggregate of mice expressing Cre (Vil1-Cre for Dpp4 Gut-/and Tie2-Cre for Dpp4 EC-/-) and Dpp4 fl/fl mice (littermate controls from the corresponding line) versus mice with the 2 different targeted Dpp4 deletions. Mice were fasted for 5 hours before metabolic studies and sacrifice.
LTTs and TG production measurement. After a 5-hour fast, mice were given water or sitagliptin (Merck Laboratories) by oral gavage at 14 μg/mouse (for intestinal-selective inhibition of DPP-4, ref. 20) or 10 mg/kg (for systemic inhibition of DPP-4). Thirty minutes later, mice were gavaged with 200 μL olive oil (MilliporeSigma). For TG production measurement only, mice were fasted for 4 hours (bled the day before for baseline t0 values to avoid bleeding during the i.v. injection) and administered tyloxapol (0.5 g/kg body weight from a 15% solution [3.3 times body weight]) by i.v. injection immediately before olive oil gavage. Blood was collected from the tail vein up to 3 hours after gavage to measure TG levels and other metabolic parameters (40-75 μl per time point). As TGs accumulate in the circulation after tyloxapol injection, these experiments were terminal, and mice were sacrificed after the 3-hour time point. For LTT experiments, each mouse received all 3 treatments (water, gut-selective DPP4 dose, or systemic DPP4 dose), separated from each other by 6-10 days. For experiments using tyloxapol, mice were randomized to treatment group based on genotype, sex, age, and body weight before the experiment.
Analysis of lipoprotein particles by FPLC. Mice (18-24 weeks old) were fasted for 5 hours. Two hundred μl of olive oil was given orally at time 0, and approximately 100 μl of blood was collected from the tail vein into EDTA-coated capillary tubes 60 minutes later. Blood was centrifuged at 18,000g for 5 minutes at 4°C, and plasma was collected. Plasma lipoproteins were separated by FPLC, and collected fractions were assayed for TG and cholesterol as previously described (55).
Oral glucose tolerance tests. After a 5-hour fast, mice were administered water or sitagliptin at 14 μg/ mouse (gut-selective dose) or 10 mg/kg (systemic DPP-4 inhibition) by gavage. Thirty minutes later, RC-fed mice were gavaged with 20% glucose in PBS (2 g/kg of body weight) and HFD-fed mice were gavaged with 50% glucose in PBS (2 g/kg of body weight). Blood samples for glucose measurements (Contour glucometer, Bayer Healthcare) were taken from the tail vein up to 120 minutes after injection.
Blood and tissue collection and metabolic assays. For metabolic studies, blood was taken via the tail vein into heparin-coated capillary tubes. For DPP-4 activity assays, blood was collected before and 30 minutes