Discordance between gut-derived appetite hormones and energy intake in humans

SUMMARY Gut-derived hormones affect appetite and are thought to play an important role in body weight regulation. Dietary macronutrient composition can influence gut-derived appetite hormone concentrations, thereby providing theoretical basis for why some diets might facilitate weight loss better than others. We investigated postprandial gut-derived appetite hormones in 20 inpatient adults after 2 weeks of eating either a low carbohydrate (LC) or a low fat (LF) diet followed by the alternate diet in random order. A LC meal resulted in significantly greater postprandial GLP-1, GIP, and PYY but lower ghrelin compared to an isocaloric LF meal (all p≤0.02). However, differences in gut-derived appetite hormones were incommensurate with subsequent ad libitum energy intake over the rest of the day, which was 551±103 kcal (p<0.0001) greater with the LC as compared to the LF diet. The effects of gut-derived appetite hormones on ad libitum energy intake can be dominated by other diet-related factors, at least in the short-term.


INTRODUCTION
Gut-derived hormones affect appetite.7][8] Circulating concentrations of gut-derived appetite hormones can be influenced by dietary macronutrient composition, [9][10][11][12][13] which provides a theoretical basis for why some diets may help facilitate weight loss better than others.We recently studied 20 inpatient adults who were exposed to two diets varying widely in the proportion of fat to carbohydrate for periods of 2 weeks each. 14In a randomized crossover design, after 2 weeks of eating a low carbohydrate (LC) diet (75% fat, 10% carbohydrate), the postprandial gut hormone responses to a representative LC liquid test meal were compared with an isocaloric low fat (LF) liquid test meal consumed after 2 weeks of eating a LF diet (10% fat, 75% carbohydrate).Subsequent ad libitum energy intake at lunch, dinner, and snacks for the rest of the day were measured to investigate whether postprandial responses were commensurate with subsequent intake during these dietary patterns.

Gut hormone responses and subsequent energy intake
At the end of each ad libitum feeding period, the LC diet resulted in greater fasting concentrations of GLP-1 and GIP, but similar concentrations of PYY and leptin, and lower concentrations of total ghrelin and active ghrelin, when compared to the LF diet (Table 1).
for use under a CC0 license.This article is a US Government work.It is not subject to copyright under 17 USC 105 and is also made available (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.After the breakfast test meal, ad libitum energy intake was greater during the LC diet at lunch (244±85 kcal; p=0.001) and dinner (193±86 kcal; p=0.04), but not snacks (114±63 kcal; p=0.12), such that the total ad libitum energy intake over the rest of the day was significantly greater (551±103 kcal; p<0.0001) as compared to when the same participants consumed the LF diet (Figure 2A).Within each diet pattern, there were no significant correlations between subsequent ad libitum energy intake and the mean postprandial active GLP-1 (LC diet: r=-0.The present cohort had a wide range of body mass indices, therefore diet by BMI interactions were explored to investigate if any of the differences in gut hormone responses were driven by BMI (Figure S1).The only significant diet by BMI interaction was observed for active ghrelin, which was greater in LF than LC in individuals with a BMI below 25 kg.m -2 (p<0.0001),but was not siginficiantly different in individuals with overweight (p=0.48) or obesity (p=0.25).
The observed differences in gut-derived appetite hormones during the LC test meal would be expected to result in reduced appetite and lower ad libitum energy intake as compared to the LF diet.However, the opposite result was observed, with the LC diet resulting in an additional ~500 kcal consumed over the remainder of the day following the test meal, as compared to the LF diet.This difference was similar to the overall ~700 kcal⋅d -1 difference between the diets averaged over two weeks. 14trations of the adipokine leptin were commensurate with the direction of ad libitum energy intake differences for the LC and LF diets.The lower leptin following the LC diet (vs LF) is in agreement with previous evidence comparing a ketogenic low carbohydrate diet with an isocaloric diet moderate in both carbohydrate and fat. 15The decrease in leptin following LC is likely explained by decreased insulin and glucose concentrations, which were lower in LC compared to LF. 14 Previous studies have shown that small increases in insulin induced by for use under a CC0 license.This article is a US Government work.It is not subject to copyright under 17 USC 105 and is also made available (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint this version posted October 19, 2023.; https://doi.org/10.1101/2023.05.10.23289718doi: medRxiv preprint glucose infusion of 2.5 mg•kg -1 •min were sufficient to offset the decrease in leptin observed with 16 hours of fasting 16 and 24 hours of ketogenic carbohydrate restriction reduced leptin concentrations independent from changes in ad libitum energy intake and preceding changes in adiposity. 17Therefore, the evidence suggests that changes in carbohydrate availability, rather than energy intake or energy balance, are key for altering leptin concentrations.Decreased leptin would theoretically increase appetite, as has been associated following weight loss. 18st it is possible that leptin, as a longer-term appetite signal, overrides the transient signals from gut-derived hormones, leptin concentrations did not correlate with ad libitum energy intake in the present study, so it is likely that other diet-related factors are more important in this context.

Influence of macronutrient composition on gut hormone response
Although the LC and LF diets differed by more than just their macronutrient composition, it is likely that the differences in gut hormone responses were mainly due to macronutrient differences as previously reviewed. 19,20Early evidence in humans suggested that small increases in GLP-1 were observed after isocaloric carbohydrate (glucose), fat (double cream), or protein (lean turkey) ingestion, whereas GIP only responded to carbohydrate and fat. 21ver, regardless of nutrient, the food matrix also plays a large role in determining postprandial responses as demonstrated by isocaloric ingestion of glucose eliciting greater GLP-1 and GIP responses than whole food sources of carbohydrate, including brown rice or pearl barley. 21With regards to carbohydrate manipulation, the increase in PYY observed following LC in the present study resembles the results of a similar randomized crossover study in participants with obesity who consumed isocaloric low-carbohydrate or low-fat diets for one week before ingesting a representative breakfast meal. 22Similarly, high-fat drinks (38% carbohydrate, 50% fat) increase postprandial GLP-1 and PYY responses, without differences in postprandial ghrelin responses, compared to isocaloric (590 kcal) low-fat, high-carbohydrate drinks (84% carbohydrate, 3% fat), but these differences did not translate into differences in ad libitum energy intake in a subsequent lunch meal. 10Instead, ad libitum intake was associated with ghrelin responses, which contrasts with our results because total and active ghrelin were reduced with the LC diet in comparison to the LF diet and did not correlate with energy intake.
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Macronutrient manipulation, with food volume and energy density controlled, has been shown to alter postprandial GLP-1, GIP, PYY, active ghrelin, and total ghrelin responses, but did not alter subjective hunger or subsequent energy intake. 9,12,13Over the course of our study, the LF diet resulted in ~700 kcal⋅d -1 less ad libitum energy intake as compared to the LC diet without significant differences in self-reported appetite. 14Because postprandial responses of gutderived appetite hormones depend on the amount of food consumed, 23,24 the expected diet differences in postprandial ghrelin, GLP-1, GIP, and PYY during the ad libitum period would likely be even greater than we observed following the isocaloric meal tests and further emphasizes that the expected effects of these appetite hormone differences were dominated by other diet differences.

Diet-related factors affecting energy intake beyond gut hormones
Recent analysis of the meal characteristics that affect energy intake from our inpatient feeding studies suggests that energy density, eating rate, and proportion of hyper-palatable foods are positively associated with meal energy intake. 25Greater dietary energy density has consistently been shown to increase energy intake in short-term interventions. 26The LC diet of the present study had about double the energy density of the LF diet and mediated around 25% of the diet effect on meal energy intake. 25A quicker eating rate increases energy intake of presented meals without altering subsequent hunger. 27Eating rate could be related to sensory and physical properties of foods, like food texture; 28 for example, softer, less solid, less viscous foods are associated with increased eating rate. 29Eating rate in grams per minute was quicker in the LF meals at lunch (29±9 g⋅min -1 , p<0.0001) and dinner (14±9 g⋅min -1 , p=0.009) on the test meal day, compared to LC, so this factor is unlikely to explain our observations of increased energy intake on the LC test meal day.The volume and mass of food ingested is closely related to energy density, which may alter gastric distension and contribute to changes in gut hormone responses to meals. 30The mass of food eaten ad libitum was significantly greater following the LF diet at lunch and dinner, but not snacks (Figure 2B), compared to LC.This total difference across the day was consistent with the overall ~667 g⋅d -1 difference between the diets averaged over two weeks. 14Within each diet pattern, there were no significant correlations between postprandial gut hormone responses following the liquid test meals and subsequent mass of food eaten at lunch (first subsequent meal) or throughout the day of the test meal (Table S1), apart from a moderate negative correlation between food mass intake at lunch in the LF diet and leptin.Hyper-palatable foods have recently been defined using quantitative thresholds of for use under a CC0 license.This article is a US Government work.It is not subject to copyright under 17 USC 105 and is also made available (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint this version posted October 19, 2023.; https://doi.org/10.1101/2023.05.10.23289718doi: medRxiv preprint nutrient combinations that may drive excess intake; 1) fat and sugar (>20% energy, >20% energy), 2) fat and sodium (>20% of energy, >0.3% by weight), and 3) carbohydrates and sodium (>40% energy, >0.3% by weight). 31Across the entire 2 weeks, meals in the LC diet had a greater proportion of hyper-palatable foods than the LF diet, which may have mediated around 14% of the diet effect on meal energy intake. 25The availability of hyperpalatable foods in the US food system, by this definition, has increased from around 49% to around 69% in 30 years. 32ging cross-sectional evidence suggests that hyper-palatable foods may be more rewarding. 33More work is required to determine the utility of quantitative definitions of hyperpalatability and their influence on food intake.Whilst the alternative diet-related factors discussed may often be inter-related in real world settings, it is important for future work to isolate these diet-related factors and test their contribution to ad libitum energy intake in different dietary contexts (e.g.macronutrient manipulation or processing).

Comparisons between diet and pharmacological or surgical interventions
Discordance between gut hormone responses and energy intake in the present study may appear to contradict the recent success of pharmacological gut hormone mimetics, including GLP-1 receptor agonists, but quantitative considerations of dose and exposure reconcile our findings.Specifically, the estimated active GLP-1 steady state average exposure concentration, C avg , for the present study had mean (95% CI) values of 0.034 (0.029, 0.043) nmol⋅L -1 for LF and 0.086 (0.071, 0.113) nmol⋅L -1 for LC, which are orders of magnitude lower than the C avg of both oral and subcutaneous semaglutide ranging from ~3 nmol⋅L -1 up to ~30 nmol⋅L -1 with oral and subcutaneous semaglutide, respectively (Figure 3A). 34Such high C avg with pharmacological treatment is due to the long half-life of semaglutide which has a similar binding affinity to the GLP-1 receptor as native GLP-1, 35 whereas the half-life of endogenous GLP-1 and GIP is minutes in humans. 36Unlike pharmacological intervention, diet-induced changes in gut hormone concentrations reflect conjoint changes of multiple hormones in a complex signalling system, so the quantitative exposure of GLP-1 from diet and pharmacological interventions cannot be compared directly, but this comparison highlights that the magnitude of change in GLP-1 from diet is not comparable to that of pharmacological interventions even with the concurrent changes of other hormones.
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The copyright holder for this preprint this version posted October 19, 2023.; https://doi.org/10.1101/2023.05.10.23289718doi: medRxiv preprint Pharmacological engagement of the GLP-1 receptor (e.g.exogenous GLP-1) may act differently from nutrient-stimulated gut hormones (e.g.endogenous GLP-1).For example, endogenous GLP-1 may affect the hypothalamus through neuronal signaling from the gut to brain via solitary tract neurons, whereas exogenous GLP-1 receptor agonists may directly engage hypothalamic and brainstem GLP-1 receptors. 6Further complicating this, GLP-1 can be produced centrally in brain regions which may directly alter appetite independent from diet-induced gut hormone secretion. 37With this context, changes in endogenous gut hormone concentrations induced by diet may be not be potent enough to affect energy intake.Indeed, mouse models that knockout the GLP-1 receptor, 38 or delete intestinal GLP-1 production throught GNG gene knockout, 39 do not result in a body weight or food intake phenotype, suggesting that endogenous GLP-1 has a limited effect on appetite in the normal physiological range.Some forms of bariatric surgery result in substantial increases in postprandial GLP-1 and PYY, [40][41][42] likely due to altered gastric emptying and intestinal nutrient delivery.The magnitude of post-surgical changes in postprandial gut hormone responses might be mechanistically linked to reduced appetite and energy intake.Infusion of GLP-1, PYY, and oxyntomodulin in healthy participants that mimics the concentrations observed following Roux-en-Y gastric bypass reduced energy intake at lunch and dinner by ~400 kcal. 3The active GLP-1 and PYY concentrations achieved were around 26 pmol⋅L -1 (85 pg⋅mL -1 ) and 80 pmol⋅L -1 (320 pg⋅mL -1 ) respectively.For active GLP-1, these concentrations are around 13-fold and 35-fold greater than mean postprandial concentrations following the LC and LF meals in the present study, and for PYY they are around 5-fold and 6-fold greater (Figure 3B).Infusing GLP-1 to achieve concentrations comparable with the LC condition of the present study (~15 pg⋅mL -1 ) delays gastric emptying without suppressing subjective appetite and ad libitum intake. 43aphysiological concentrations of GLP-1 achieved by infusion (~25 to 30 pg⋅mL -1 ) suppress subjective appetite, but effects on subsequent ad libitum energy intake are modest compared with higher concentrations (~100 to 240 kcal). 44,45Together, infusion studies indicate a doseresponse relationship between active GLP-1 and suppression of appetite, 46 and suggest that substantially greater increases in gut hormone concentrations are required to have meaningful effects on appetite and energy intake, likely greater than is achievable by diet interventions alone.
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Limitations and considerations
A limitation of the current study is that participants had no choice regarding the foods available for consumption.They could only choose the quantity of the foods eaten.While the gut-derived appetite hormones were not a dominant factor determining energy intake in this setting, it is possible that such differences in appetite hormones in a real-world setting might alter food choices at subsequent meals and thereby alter energy intake.Another limitation is that we used isocaloric liquid test meals that matched the macronutrient composition of the overall LC and LF diets.These test meals may have not been adequately representative of the effects of meals with solid foods.Furthermore, the results reported in this study were from analyses that were not pre-specified as primary or secondary outcomes of the main study and are hence exploratory in nature.Nevertheless, our study suggests that differences in dietary factors like energy density or proportion of hyper-palatable foods may play a greater role in appetite regulation than endogenous gut-derived appetite hormones, at least in the short term.Future research should aim to identify such diet differences that influence energy intake and evaluate whether their effects and their potential discordance with gut-dervied appetite hormones persist over time.
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Figures
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Figure 2 .
Figure 2. Total intake, and intake from lunch, dinner, and snacks throughout the day after isocaloric low carbohydrate (LC) or low fat (LF) meals following habituation to each diet in a randomized crossover design.Mean (range) of energy in the test meals was 777 (532 to 1043) kcal.Data are mean ± SEM and individual responses.n=20.p-values from paired t-test or Wilcoxon test.(A) Energy intake (EI) (kcal) (B) Mass intake (g)

Figure 3 . 34 (
Figure 3. Comparisons between dietary macronutrient induced changes in gut hormone responses and pharmacological or bariatric surgery induced changes.(A) estimated mean active GLP-1 steady state average exposure concentrations, C avg , achieved by low carbohydrate (LC) or low fat (LF) diet were orders of magnitude lower than the both oral and subcutaneous semaglutide using values median (90% exposure ranges) from Overgaard et al. 34 (B) peak active GLP-1 and PYY concentrations following a LC or LF test meal were orders of magnitude lower than peak concentrations observed during a mixed-meal test following Roux-en-Y Gastric Bypass surgery (RYGB) using data from Tan et al. 3 Data are mean ± SEM.

Table 1 . Fasting concentrations of gut-derived appetite hormones and leptin in the second week of low carbohydrate (LC) or low fat (LF) diet. Data are mean ± SEM, n=20.
This article is a US Government work.It is not subject to copyright under 17 USC 105 and is also made available (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.; body mass index ≥20 kg⋅m -2 ; body weight ≥53 kg; able to complete daily bouts of stationary cycling at a moderate rate and intensity with a heart rate (HR) equal to or greater than This article is a US Government work.It is not subject to copyright under 17 USC 105 and is also made available (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint test meal was 75% carbohydrate, 10% fat, and 15% protein.Blood samples were obtained at 0, 10, 20, 30, 60, 90, 120, 180, 240, 300, and 360 minutes after the meals in tubes containing a protease inhibitor cocktail (including DPPIV inhibitor and aprotinin) to measure GLP-1, GIP, PYY, total ghrelin, active ghrelin, and leptin using multiplex immunoassays (Meso Scale Diagnostics).After the breakfast mixed meal tests, ad libitum food intake was measured over the rest of the day including lunch, dinner, and snacks by weighing the remaining food and beverages to calculate the amount of each food consumed and energy intake was calculated using ProNutra software (v.3.4,Viocare) with nutrient values derived from the USDA National Nutrient Database for Standard Reference, Release 26 and the USDA Food and Nutrient Database for Dietary Studies, 4.0.Statistical analyses were performed using SAS (v.9.4; SAS Institute) and Prism (v.9.5.0;GraphPad).Mean plasma concentrations were calculated by dividing total area under the curve (tAUC) by 360 minutes.Active GLP-1 C avg was estimated by multiplying the 6-hour postprandial tAUC by 3 (18 h) and multiplying the postabsorptive (fasting) concentration by 360 minutes (6 h), to get 24-hour exposure, and dividing by 24.The conversion factor used for GLP-1 was 1 pmol⋅L -1 = 3.297 pg⋅mL -1 .Data were checked for normality using Shapiro-Wilk test, differences between conditions were assessed using paired t-tests for normally distributed and Wilcoxon tests for non-normally distributed data.Simple linear correlation was used to explore associations between gut hormone responses and ad libitum energy intake.Diet by BMI interactions were checked using 2-way ANOVA, with post-hoc Bonferroni tests used to identify differences.Significance was accepted as p≤0.05.for use under a CC0 license.This article is a US Government work.It is not subject to copyright under 17 USC 105 and is also made available (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint this version posted October 19, 2023.; https://doi.org/10.1101/2023.05.10.23289718doi: medRxiv preprint