We previously transformed the C41-DE3 laboratory strain of E. coli (Ec) with At1g78690, an N-acyltransferase from Arabidopsis thaliana that catalyzes the synthesis of NAPEs. This markedly increased NAPE levels in these bacteria (pNAPE-Ec) compared with those transformed with empty vector (pEc) (32, 33). To determine whether NAPEs synthesized by intestinal E. coli could markedly alter food intake, we administered a daily bolus of 1011 CFU pNAPE-Ec or pEc bacteria by gavage to lean male C57BL/6J mice for 7 days. Additional groups of mice were administered either vehicle or pNAPE-Ec bacteria that had been killed by treatment with kanamycin prior to gavage. Cumulative food intake did not differ in mice receiving pEc compared with those receiving vehicle, but was reduced by 15% in mice receiving living pNAPE-Ec (Supplemental Figure 1; supplemental material available online with this article; doi:10.1172/JCI72517DS1). pNAPE-Ec are sensitive to kanamycin, and pNAPE-Ec pretreated with kanamycin prior to gavage did not reduce food intake to the same extent as untreated pNAPE-Ec, indicating that viable bacteria expressing NAPEs are needed for maximum effectiveness. Our follow-up studies revealed that administration of 1011 pNAPE-Ec bacteria once a day for 7 consecutive days resulted in an approximately 2-fold increase in NAPE levels in the colon (Supplemental Figure 2A). NAPE levels were not as markedly increased in other gastrointestinal tissues such as stomach, small intestine, and cecum, nor were they increased in plasma (Supplemental Figure 2, B–E).

To examine the effect of persistent NAPE expression by gut bacteria on food intake and obesity, we transformed the probiotic wild-type strain of E. coli, Nissle 1917 (EcN), with At1g78690 (pNAPE-EcN) (Supplemental Figure 3). To facilitate monitoring of colonization by transformed EcN, we also inserted the P. luminescens luciferase operon (Lux) into the recA gene of the EcN chromosome. We analyzed phospholipid extracts from both pNAPE-EcN and pEcN by negative ion mass spectrometry (Supplemental Figure 4), and the identified NAPE species were consistent with N-acylation of the major bacterial PE species (Supplemental Table 1). We observed that pNAPE-EcN had markedly higher levels of saturated and monounsaturated NAPEs expected to exert anorexigenic effects (Supplemental Figure 5). In addition to NAPEs, several species with m/z ions consistent with O-acyl-phosphatidylglycerols (acyl-PG) species (34) were also enriched in pNAPE-EcN (Supplemental Table 2).

We then tested whether incorporating pNAPE-EcN into the gut microbiota would protect against the development of obesity, insulin resistance, and liver steatosis that occurs in C57BL6 mice fed an ad libitum high-fat diet (60% of calories from fat). For these experiments, we administered pNAPE-EcN via the drinking water at 5 × 109 CFU bacteria/ml with 0.125% gelatin added to the water to keep the bacteria suspended and viable for at least 48 hours (Supplemental Figure 6). Supplementation of drinking water with pNAPE-EcN resulted in bioluminescence that could be readily detected in the intestinal tract (Supplemental Figure 7). Bacteria were administered for a total of 8 weeks (Supplemental Figure 8), and food intake, weight gain, and body composition were monitored. We also measured consumption of kaolin (pica), a measure of gastrointestinal distress in rodents (35), to assess whether bacterial administration had an adverse effect on the mice. At the end of the bacterial treatment period, we performed an oral glucose tolerance test. Mice were continued on the high-fat diet for an additional 4 weeks to determine the persistence of the altered gut microbiota and the resulting changes in food intake and obesity.

During the 8-week treatment period, pNAPE-EcN–treated mice gained less body weight (Figure 1A) and accumulated less fat mass (Figure 1B) compared with control mice. Consistent with their reduced adiposity, pNAPE-EcN mice also had lower plasma leptin and insulin levels (Table 1). In contrast to the striking differences in adiposity, we observed no significant differences in lean body mass between the groups (Figure 1C). One of the major bioactivities of the NAE metabolites of NAPEs is the reduction of food intake, and pNAPE-EcN–treated mice had markedly lower cumulative food intake than those treated with standard water, vehicle, or pEcN (Figure 1D).

Figure 1 Treatment with pNAPE-EcN, but not pEcN, inhibits gain in body weight and adiposity. All values are the mean ± SEM (n = 10 mice per group). Solid bars indicate time points with significant differences between pNAPE-EcN and other groups (P < 0.05 by Bonferroni’s multiple comparison test). (A) Effect of treatments on gain in body weight from start of treatment (2-way RM ANOVA, for treatment P = 0.0073, for time P < 0.0001). (B) Effect of treatments on fat mass (2-way RM ANOVA, for treatment P = 0.0127, for time P < 0.0001). (C) Effect of treatments on lean body mass (2-way RM ANOVA, for treatment P = 0.8113, for time P < 0.001). (D) Effect of treatments on cumulative food intake from start of treatment (2-way RM ANOVA, for treatment P = 0.0035, for time P < 0.0001).

Table 1 Effects of NAPE-secreting bacteria on metabolic biomarkers during an 8-week treatment and a 4-week follow-up period

Importantly, we found no evidence that changes in body weight and adiposity were the result of an adverse health effect of treatment with pNAPE-EcN. For instance, there was no difference between treatment groups in the consumption of kaolin (Supplemental Figure 9), so bacterial administration did not produce gastrointestinal distress. Nor did bacterial overgrowth appear to be the cause of reduced food intake and adiposity in pNAPE-EcN–treated mice, as levels of EcN retained in the intestinal tract did not significantly differ between pNAPE-EcN– and pEcN-treated mice (Supplemental Figure 10). Scores on simple tests of muscle strength, coordination, and speed either did not differ or were improved with pNAPE-EcN treatment (Supplemental Table 3). Perhaps most importantly, since it is well established that endotoxemia impairs glucose tolerance and insulin sensitivity (12, 13), mice administered pNAPE-EcN had significantly improved glucose tolerance compared with that observed in the other groups (Table 1). Overall, these findings support the conclusion that the reduced adiposity found in high-fat diet–fed mice receiving pNAPE-EcN results from the action of NAPEs rather than from an adverse effect of bacterial administration.

Our larger goal was to incorporate NAPE-secreting bacteria into the gut microbiota in order to endow their host with persistent resistance to obesity. Mice initially treated with either pNAPE-EcN or pEcN excreted luminescent bacteria in their feces for at least 4 weeks after the cessation of bacterial administration (Supplemental Figure 11), consistent with persistence of pNAPE-EcN in the gut. We therefore investigated whether the beneficial effects of pNAPE-EcN on adiposity would persist after stopping its administration. Food intake, body weight, and fat mass during the follow-up period (experimental weeks 9–12) remained significantly lower in mice initially treated with pNAPE-EcN than was the case in the other groups (Table 1), consistent with the notion that incorporation of pNAPE-EcN in the gut lumen leads to long-lasting delivery of NAPEs and concomitant inhibition of obesity. Subsequent studies in a separate cohort of mice found that body weight was still significantly lower than that of vehicle- or pEcN-treated mice even 12 weeks after ending administration of pNAPE-EcN (Figure 2). The difference in body weight between vehicle- and pNAPE-EcN–treated mice continued to increase in magnitude up until 6 weeks after cessation of administration. Bioluminescence was no longer detectable in feces at this time, suggesting that the loss of inhibited weight gain at this time was the result of loss of pNAPE-EcN from the gut.

Figure 2 Effects of pNAPE-EcN persist for more than 6 weeks after ending administration. Groups of mice were fed a high-fat diet for a total of 20 weeks (n = 10 mice per group). For the first 8 weeks, mice were administered either vehicle (0.125% gelatin), 5 × 109 CFU pEcN/ml, or 5 × 109 CFU pNAPE-EcN/ml. For the remaining 12 weeks, all mice received standard drinking water. (A) Effect of treatment on total body weight. (B) Body weight of group treated with pNAPE-EcN normalized to the vehicle-treated group. (C) Percentage of body fat for all 3 groups. (D) Percentage of body fat of pNAPE-EcN mice normalized to the vehicle-treated mice. (E) Daily average food intake for each group as a 2-week rolling average. A rolling average was used to minimize day-to-day variations to better determine the overall trends. The dip in food intake that occurred in all groups after day 70 is likely the result of changing the housing location of all the mice due to institutional requirements. (F) Daily average food intake of pNAPE-EcN–treated mice normalized to the vehicle-treated mice.

Molecules absorbed through the colon enter the portal circulation, so that bacterially secreted NAPEs that had been subsequently converted to the biologically active NAEs by NAPE-PLD might exert effects in the liver in addition to their effects within the intestinal tract. When we examined liver NAE levels in mice that were euthanized 2 days after ending bacterial administration, we found that pNAPE-EcN treatment increased the levels of total NAE in liver by 44% (Figure 3). Treatment with pNAPE-EcN did not significantly alter either total plasma levels of NAE or NAPE or their N-acyl chain profiles (Supplemental Figure 12). Previous studies showed that orally administered NAEs induce a number of effects relevant to protection against obesity including the activation of fatty acid oxidation, inhibition of lipogenesis, and inhibition of lipid absorption (23, 25). We found no difference in lipid absorption between mice given standard drinking water and those treated with pNAPE-EcN (Supplemental Figure 13.) When we examined gene expression in livers collected from mice euthanized 4 weeks after ending bacterial administration, we found that mice treated with pNAPE-EcN had increased liver mRNA expression for genes linked to fatty acid oxidation such as peroxisome proliferator activator receptor α (Ppara) and delta (Ppard), carnitine palmitoyl transferase-1 (Cpt1), and acyl CoA oxidase (AOX), but not for genes linked to fatty acid synthesis such as fatty acid synthase (FAS) and sterol regulatory element–binding protein-1c (SREBP-1c) or to gluconeogenesis such as phosphoenolpyruvate carboxykinase (PEPCK) (Figure 4). They also had reduced expression of the stearoyl CoA desaturase-1 (Scd1) and lipid transporter Cd36 as well as reduced expression of inflammatory genes including Cd68 and monocyte chemotactic protein-1/chemokine C-C motif ligand 2 (Ccl2). To further examine the effect of pNAPE-EcN treatment on hepatic inflammation, we immunostained for infiltrating monocytes/macrophages using antibodies against F4/80 and CD11b. The livers of control mice had large numbers of immunoreactive cells (Figure 5). In contrast, pNAPE-EcN mice had markedly fewer immunoreactive cells. We also examined triglyceride levels in these livers and found significantly lower levels in pNAPE-EcN–treated mice than those in the other 3 groups (Table 1). Histological examination with both H&E and Oil Red O staining revealed markedly less hepatosteatosis in the livers of mice treated with pNAPE-EcN (Supplemental Figure 14).

Figure 3 Treatment with pNAPE-EcN increases hepatic NAE levels. NAEs were measured by LC/MS in liver collected from mice 2 days after ending a 9-week treatment with pNAPE-EcN or from untreated mice receiving standard drinking water (Water) during this same period (n = 10 mice per group; mean ± SEM). NAE levels were significantly different (2-way ANOVA, for treatment P < 0.0001, for NAE species P < 0.0001). Summed NAE levels were 1.70 ± 0.18 versus 2.45 ± 0.21 nmol/g liver for untreated versus pNAPE-EcN–treated mice, respectively.

Figure 4 Treatment with pNAPE-EcN induces expression of genes encoding for fatty acid oxidation, but not fatty acid synthesis, and reduces expression of inflammatory genes in the liver. Liver mRNA was measured by qRT-PCR using primers specific for each gene. β-actin (Actb) was used as a control, and all values were normalized to the vehicle group (mean ± SEM, n = 10 mice per group). *P < 0.05 by 1-way ANOVA for individual gene expression and by Dunnett’s multiple comparison test versus vehicle for pNAPE-EcN, but not for pEcN.

Figure 5 Treatment with pNAPE-EcN reduces infiltration of F4/80 and CD11b immunopositive leukocytes into liver. Slides were immunostained with either anti-F4/80 (A–D) or anti-CD11b (E–H) antibodies (DAB brown stain) and counterstained with hematoxylin. Representative photomicrographs are shown. Scale bars: 10 μm. (A and E) Standard drinking water only. (B and F) Vehicle-treated (0.125% gelatin) mice. (C and G) pEcN-treated mice. (D and H) pNAPE-EcN–treated mice.

To determine whether the effect of pNAPE-EcN was mediated solely by its ability to reduce food intake, we pair-fed a group of mice treated with pEcN with the same amount of food consumed each day by pNAPE-EcN–treated mice for a period of 4 weeks. The body weight of mice that were pair-fed for 4 weeks was significantly lower than that of mice treated with pEcN or standard water, but was still significantly higher than that of mice treated with pNAPE-EcN (Figure 6A). Pair-fed mice also had significantly less body fat than did pEcN- or water-only–treated mice, but they trended toward higher body fat levels than were seen in pNAPE-EcN–treated mice (Figure 6B). When we calculated feeding efficiency over the 4-week treatment period, both pEcN- and water-treated mice gained significantly more weight per kcal of food consumed than did pNAPE-EcN–treated mice, while the pair-fed mice showed intermediate feeding efficiency (Figure 6C). We then examined whether the changes in body composition induced by food restriction were sufficient to induce the expression of the fatty acid oxidation genes seen in pNAPE-EcN–treated mice. Despite having similarly low levels of triglyceride levels in their livers (Figure 7A), pair-fed mice failed to induce increased expression of hepatic fatty acid oxidation in a manner similar to that of pNAPE-EcN–treated mice (Figure 7B). We interpreted these results as indicating that this gene induction was the direct result of the delivery of NAEs to the liver afforded by pNAPE-EcN treatment rather than an indirect response to lower food intake or body fat.

Figure 6 Pair-feeding fails to fully recapitulate the effect of pNAPE-EcN treatment on body weight and body fat gain. A group of mice treated with pEcN (E) were were pair-fed (PF) by restricting them to the same number of calories of food as the pNAPE-EcN mice (N). An ad libitum–fed control group received standard drinking water (W). All values are the mean ± SEM; n = 9–10 mice per group. (A) Body weight gain during 4 weeks of treatment and pair-feeding. Two-way RM ANOVA, P < 0.0001 for time, P = 0.0003 for treatment. P < 0.05 by Bonferroni’s multiple comparison test for pair-fed versus pNAPE-EcN for days 16 to 25. P < 0.05 for pair-fed versus pEcN and versus standard water for day 28. (B) Body fat at week 0 (before beginning treatment) and week 4 (28 days after beginning treatment). No group differences were observed at week 0. Week 4 1-way ANOVA, P < 0.0001 by 1-way ANOVA; P < 0.05 by Dunnett’s multiple comparison test for pair-fed versus water or versus pEcN. Pair-fed versus pNAPE-EcN, not significant. (C) Calculated feeding efficiency, P < 0.0001 by 1-way ANOVA; P < 0.05 BY Dunnett’s multiple comparison test for pair-fed versus pEcN; pair-fed versus standard water or versus pNAPE-EcN, not significant.

Figure 7 Pair-feeding does not induce hepatic expression of fatty acid oxidation genes but does reduce lipid accumulation. All values are the mean ± SEM; n = 9–10 mice per group. (A) Liver triglyceride (TG) levels in mice given standard drinking water (W), or drinking water with pEcN (E), pNAPE-EcN (N), or pEcN with pair-feeding to pNAPE-EcN mice (PF). P = 0.0012 by 1-way ANOVA; *P < 0.05 versus standard water by Dunnett’s multiple comparison test. (B) Expression of mRNA encoding for proteins related to fatty acid oxidation. Ppara, P < 0.0001 by 1-way ANOVA; *P < 0.05 versus standard water by Dunnett’s multiple comparison test; Cpt1a, P < 0.0233 by 1-way ANOVA; *P < 0.05 versus standard water by Dunnett’s multiple comparison test; AOX, P = 0.1326 by 1-way ANOVA.

Although these findings were consistent with pNAPE-EcN treatment directly altering food intake and energy expenditure by the action of NAEs, we considered an alternative hypothesis for pNAPE-EcN action, which is that treatment with pNAPE-EcN indirectly leads to changes in adiposity by altering global gut microbiota. Changes in global gut microbiota, including an increased ratio of Firmicutes to Bacteroidetes, have been associated with obesity (9, 36–38). We anticipated that our treatment strategy would have very little long-term effect on the global microbial composition of the gut if our genetically modified EcN primarily colonized the gut by occupying niches previously held by native E. coli or other Proteobacteria. This is because Proteobacteria typically account for only a very small percentage of gut bacteria (~1% of adherent bacteria in the distal small intestine, ~3% of adherent bacteria in the descending colon, and <0.1% of bacteria in feces; R.L. Walzem, unpublished observations). To assess the effect of pNAPE-EcN treatment on global gut microbial composition, we performed 454 pyrosequencing of bacterial 16S rRNA obtained from the feces of treated mice. Fecal microbiota composition is a noninvasive, surrogate measure of intestinal microbiota composition with contributions from both bacteria shed by established intestinal colonies and ingested bacteria, which pass through the gut without adhering. Treatment with either pNAPE-EcN or pEcN markedly increased fecal levels of Proteobacteria compared with the levels found in vehicle-treated mice at the 8-week time point (Figure 8 and Supplemental Table 4). This increase in fecal Proteobacteria levels is consistent with large numbers of ingested EcN passing through the gut without adhering. Ingestion of either form of EcN also temporarily reduced the proportion of Firmicutes in the feces by week 8. Because pEcN reduced fecal Firmicutes levels but failed to alter body weight and body fat, this result does not support the notion that the acute change in fecal global microbial composition is responsible for the beneficial effects of pNAPE-EcN treatment. This interpretation is further supported by the microbial composition of feces collected at week 12, 4 weeks after EcN bacteria were no longer being ingested. At this time point, the global microbial composition of feces was very similar in the 3 groups, suggesting that treatment with pEcN or pNAPE-EcN only led to colonization of the relatively low-abundance niches available for E. coli in the gut and did not result in long-term alterations of the global composition of the gut microbiota. Because the mice colonized with pNAPE-EcN continued to show reduced food intake and body weight (Figure 2), these results support the notion that global changes in microbiota composition are not required for the beneficial effects of pNAPE-EcN treatment and that these effects are instead the result of the direct action of NAEs on target host tissues.

Figure 8 Relative abundance in feces of major bacterial phylla. Bacterial composition of feces collected during experimental week 8 (final week of treatment) and week 12 (fourth week of post-treatment follow-up) was determined by 16S rRNA sequencing (n = 9–10 mice per group). Treatment with either pNAPE-EcN (N) or pEcN (E) significantly decreased the abundance of Firmicutes compared with vehicle (V) and increased the abundance of Proteobacteria in excreted feces (week 8), but microbial composition reverted to that of the vehicle-treated animals by 4 weeks after ending bacterial administration (week 12).

To further characterize alterations in feeding behavior and energy expenditure induced by pNAPE-EcN, we performed metabolic monitoring after 4 weeks of treatment with pNAPE-EcN, pEcN, or standard drinking water. Mice were placed for 7 days in cages equipped with the Promethion system for monitoring indirect calorimetry, physical activity, and food and water intake. The mice were maintained on a high-fat diet and their designated treatment throughout these metabolic monitoring experiments. All groups had some body weight and fat loss during their time in the Promethion system, likely due to the novel environment (Supplemental Figure 15). Analysis was performed using data from the final 3 days of monitoring when mice had acclimated to the novel environment. Examination of the pattern of food intake over the 24 hours of the light-dark cycle showed that food intake primarily diverged during the dark phase, when mice typically are most active and consume the majority of their food (Figure 9A and Supplemental Figure 16). During the dark phase, we observed that meal duration was significantly shorter for mice treated with pNAPE-EcN (Figure 9B). We found no significant differences in meal duration between groups during the light phase. Intermeal intervals did not significantly differ between the groups in either the light or dark phase (Figure 9C). When meal size was categorized by food weight consumed during each 5-minute time block of monitoring, the pNAPE-EcN–treated mice consumed significantly fewer large meals (Figure 9D). These results suggest that treatment with pNAPE-EcN lowered the threshold of the high-fat diet consumption required to achieve satiation. The pattern of water intake was similar to that of food intake: pNAPE-EcN–treated mice consumed less water overall, and the greatest divergence in water intake occurred during the dark phase (Supplemental Figure 17).

Figure 9 Treatment with pNAPE-EcN reduces food intake and meal duration during the dark phase of the light cycle. Mice were given standard drinking water only, water with pEcN, or water with pNAPE-EcN for 4 weeks prior to metabolic monitoring and continued to receive treated water throughout the monitoring period (n = 9–10 mice per group). (A) Average cumulative food intake during the 24-hour light cycle. For each mouse, the cumulative food intake through each 5-minute block of a 24-hour light cycle (6 am–6 am) for each of the 3 days of monitoring was averaged. The mean cumulative food intake for each treatment group is shown. The 72-hour cumulative food traces for individual mice are shown in Supplemental Figure 16. (B) Effect of treatment on meal duration in light and dark phases (mean ± SEM). P = 0.8972 by 1-way ANOVA for light phase; P = 0.0554 by 1-way ANOVA for dark phase; *P < 0.05 versus standard water by Dunnett’s multiple comparison test. (C) Effect of treatment on intermeal interval (mean ± SEM). P = 0.0743 by 1-way ANOVA for light phase; P = 0.1085 by 1-way ANOVA for dark phase. (D) Effect of treatment on meal size (mean ± SEM). Each 5-minute block during which food hopper weight decreased by greater than 0.05 mg was scored as a meal and categorized from 1 (smallest) to 5 (largest), as described in Methods. Category 4, P = 0.0256 by 1-way ANOVA; all other meal categories, P = NS by 1-way ANOVA.

In addition to changes in energy intake, pNAPE-EcN treatment induced subtle changes in energy expenditure. While the lower body fat of pNAPE-EcN mice complicated direct comparisons of energy expenditure between groups, during the light phase of the 24-hour light-dark cycle (when mice are least active), the linear slope of the relationship between energy expenditure versus body weight increased more steeply for pNAPE-EcN–treated mice than for the other 2 groups (Figure 10A and Supplemental Figure 18A). The slopes during the dark phase were similar (Figure 10B and Supplemental Figure 18B). The increase in energy expenditure we observed for pNAPE-EcN–treated mice during the light phase was not due to increased physical activity, because pNAPE-EcN–treated mice did not show an increase in distance traveled within the cage, as calculated from beam breaks (Supplemental Figure 19). These results suggest that pNAPE-EcN might slightly increase resting metabolic rate.

Figure 10 Effect of pNAPE-EcN treatment on energy expenditure. (A) Energy expenditure during 12-hour light phase of the 24-hour light-dark cycle. Slopes for each group differed: pNAPE-EcN, y = 0.01016x + 0.02295; pEcN, y = 0.006475x + 0.1201; standard water, y = 0.002726 + 0.001779; differences in slope, P = 0.04038, F = 3.702 (n = 9–10 mice per group). (B) Energy expenditure during the dark phase of the 24-hour light cycle. Slopes for each group did not differ: pNAPE-EcN, y = 0.002704 + 0.3448; pEcN, y = 0.003438+0.3304; standard water, y = 0.004455 + 0.2857; differences in slope, P = 0.8734, F = 0.1362.

To examine whether the increased energy expenditure during the light phase resulted from increased fatty acid oxidation while resting, we analyzed the effect of pNAPE-EcN treatment on the respiratory quotient (RQ). Because all mice ate the same high-fat diet, no large-scale changes in the RQ would be expected, but animals with an increased reliance on their fat reserves might have a slightly lower RQ (i.e., favoring fatty acid oxidation over carbohydrate and protein oxidation) during periods of rest. Indeed, the pNAPE-EcN–treated mice consistently showed a slightly lower RQ than did the other 2 groups, although this apparent difference failed to reach statistical significance (Supplemental Figure 20).

To further characterize the effects of pNAPE-EcN treatment on glucose intolerance and insulin resistance, we performed several follow-up studies. To estimate the extent of protection from glucose intolerance afforded by pNAPE-EcN treatment, we treated mice with a high-fat diet, a high-fat diet and pNAPE-EcN, or a low-fat chow diet for 8 weeks. While administering pNAPE-EcN to mice fed a high-fat diet significantly improved homeostatic model assessment–estimated insulin resistance (HOMA-IR) index scores and the insulin response to glucose when compared with untreated mice receiving this diet, impairment in glucose tolerance was still evident in the pNAPE-EcN–treated mice when compared with mice fed a low-fat chow diet (Figure 11).

Figure 11 Treatment with pNAPE-EcN reduces the insulin resistance index and increases insulin response in mice fed a high-fat diet. All values are the mean ± SEM; n = 10 mice per group. (A) Treatment with pNAPE-EcN (HF + N) improved basal glucose levels and glucose tolerance compared with mice treated with standard drinking water (HF + W), but not a low-fat chow diet (LF + W). Fasting glucose levels differed significantly between groups (P < 0.001 by 1-way ANOVA; P < 0.05 by Bonferroni’s post-hoc multiple comparison for HF + W versus HF + N or LF + W, and HF + N versus LF + W. Glucose AUC differed significantly between each treatment group (P < 0.0009 by 1-way ANOVA); P <0.05 by Bonferroni’s post-hoc multiple comparison for HF + W (50,841 ± 2,191 mg/dl/min) versus either HF + N (39,463 ± 1,800) or LF + W (24,573 ± 517), and for HF + N versus LF + W. (B) Treatment with pNAPE-EcN increased insulin responsiveness. Fasting levels of insulin were significantly higher in HF + W versus HF + N or LF + W mice (P < 0.0001 by 1-way ANOVA); P < 0.05 by Bonferroni’s post-hoc multiple comparison for HF + W versus HF + N or LF + W, but not for HF + N versus LF + W. The insulin AUC for each treatment differed significantly: P < 0.0001 by 1-way ANOVA; P < 0.05 by Bonferroni’s post-hoc multiple comparison for LF + W (69.5 ± 5.7 ng/ml/min, mean ± SEM) versus HF + W (193.9 ± 17.0) or HF + N (158.7 ± 8.4, P < 0.05). (C) Treatment with pNAPE-EcN improved the HOMA-IR score compared with a high-fat diet–only treatment. P < 0.0001 by 1-way ANOVA; P < 0.05 by Bonferroni’s post-hoc multiple comparison for HF + W versus HF + N or LF + W, but not for HF + N versus LF + W.

The improvement in glucose tolerance raised the possibility that pNAPE-EcN treatment increased insulin sensitivity. Insulin sensitivity could be improved if pNAPE-EcN treatment inhibited high-fat diet–induced phosphorylation of insulin receptor substrates that reduce activation of downstream signals in response to insulin. We therefore examined this signaling in response to both endogenous and exogenous insulin. As expected, the 4-hour fasting plasma insulin levels were lower in mice treated with pNAPE-EcN (1.14 ± 0.12 ng/ml) compared with levels in mice treated with pEcN (1.78 ± 0.17) or standard drinking water (1.96 ± 0.24; mean ± SEM, 1-way ANOVA, P = 0.0102), which is consistent with increased insulin sensitivity. Phosphorylation of the serine-threonine protein kinase AKT at serine 473 (p-S473-AKT) is a sensitive indicator of downstream activation of insulin targets. Consistent with pNAPE-EcN preserving sensitivity to insulin action during high-fat feeding, mice treated with pNAPE-EcN had higher levels of p-S473-AKT in their liver per unit of circulating insulin (p-AKT-INS) than did control mice (Figure 12A). When stimulated with a bolus of 0.75 IU/kg insulin, all groups of mice responded by increasing the level of p-AKT to a similar extent in liver and muscle (Supplemental Figure 21A), consistent with the notion that insulin resistance can be overcome with a sufficiently high concentration of insulin. Stimulation with this bolus of insulin also reduced glucose levels to a similar extent in all groups (Supplemental Figure 21B). Downstream activation of signaling cascades in response to insulin is reduced by inhibitory phosphorylation of the insulin receptor substrate-1 on serine 307 (p-IRS1) by inflammatory kinases like JNK. With saline-only stimulation, pNAPE-EcN–treated mice had lower liver p-IRS1 levels compared with those in control mice (Figure 12B). These mice also had lower levels of activated JNKs (determined by the extent of phosphorylation of the p46 and p54 isoforms) in liver tissue compared with levels in the control mice (Figure 12C). Insulin-stimulated mice did not have appreciable differences in activated JNKs compared with the saline-only–stimulated mice (not shown). Together, these data suggest that pNAPE-EcN treatment leads to lower levels of inhibitory phosphorylation of IRS1 by JNKs, thereby maintaining greater sensitivity to insulin.

Figure 12 Treatment with pNAPE-EcN preserves insulin sensitivity in liver by protecting against inhibitory phosphorylation of IRS1 by JNKs. Mice treated with standard water only (W), with pEcN (E), or with pNAPE-EcN (N) for 6 weeks were fasted for 4 hours and then injected intraperitoneally with either saline (to determine response to endogenous levels of insulin; n = 5 mice per group) or 0.75 IU/kg insulin (to determine response to exogenous, pharmacological levels of insulin; n = 4–5 mice per group). 15 minutes after injection, mice were euthanized and tissue collected. Data are the mean ± SEM. (A) Extent of activating phosphorylation of Ser473 of AKT (p-AKT) in liver of saline-injected mice. Values were normalized to plasma insulin (INS) and expressed relative to the average for water-only–treated mice. P = 0.0225 by 1-way ANOVA; *P < 0.05 versus standard water by Dunnett’s multiple comparison test. (B) Extent of inhibitory Ser307 phosphorylation of IRS1. Values were normalized to heat shock protein (HSP) and expressed relative to the average for standard water-only–treated mice. P = 0.0173 by 1-way ANOVA; *P < 0.05 versus standard water by Dunnett’s multiple comparison test. (C) Extent of activating phosphorylation of JNK isoforms p46 and p54. Values were normalized to HSP and expressed relative to the average for standard water-only–treated mice. For p46-JNK, P = 0.0084 by 1-way ANOVA; *P < 0.05 versus standard water by Dunnett’s multiple comparison test. For p54-JNK, P = 0.0134 by 1-way ANOVA; *P < 0.05 versus standard water by Dunnett’s multiple comparison test. There were no significant differences between groups stimulated with the exogenous insulin.

Although obesity in mice induced by a high-fat diet models many aspects of human obesity, consumption of a high-fat diet is not required for development of obesity in humans, particularly in individuals with a genetic predisposition toward overeating. Rodents carrying monogenic mutations in the gene for leptin or its receptor (e.g., ob/ob mice or Zucker rats) have been very useful models for understanding feeding and body weight regulation. However, mutations in these genes are rare in humans and cause extreme forms of obesity (39–41). The need to model the polygenic obesity more typical of human disease has led to the development of mice with polygenic obesity such as TallyHo/Jng (TallyHo) mice (42–45). Female TallyHo mice have been reported to rapidly gain excess weight and body fat (relative to C57BL6J mice) in the first 12 weeks of life, even while consuming a standard chow diet and without displaying the hyperglycemia seen in male TallyHo mice (45). We therefore tested whether pNAPE-EcN treatment could inhibit weight gain in female TallyHo mice fed a standard chow diet. TallyHo mice treated with pNAPE-EcN beginning at 5 weeks of age gained significantly less weight than did mice treated with vehicle or pEcN (Figure 13). This reduced body weight persisted for at least 4 weeks following the end of pNAPE-EcN treatment. The obesity induced by the low-fat chow diet in the vehicle-treated TallyHo mice was relatively modest in this study. For this reason, along with the difficulty of obtaining a large number of young TallyHo mice of similar age and body weight, our study was underpowered to detect statistically significant improvements in many of the metabolic parameters previously examined for C57BL6J mice fed a high-fat diet. Nevertheless, treatment of TallyHo mice with pNAPE-EcN tended to improve the same parameters such as lowering body fat, liver triglyceride levels, plasma leptin levels, expression of inflammatory genes in the liver, and infiltration of macrophages, while increasing expression of fatty acid oxidation genes in the liver (Supplemental Table 5 and Supplemental Figure 22). These data demonstrate that pNAPE-EcN can prevent obesity arising from a variety of causes and not simply obesity induced by high-fat diets.