High protein ingestion does not affect whole-body insulin sensitivity

High protein ingestion does not affect whole-body insulin sensitivity | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article High protein ingestion does not affect whole-body insulin sensitivity Oana Ancu, Astrid C. Hauge-Evans, Fulvia Draicchio, Diana-Elena Neculescu, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6555791/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Diet is critical in type 2 diabetes management, with high-protein diets (HPD), rich in branched-chain amino acids (BCAAs), proposed to enhance glycemic control, but overactivation of S6K1-related signaling pathway may contribute to IR pathogenesis by impairing insulin-stimulated glucose uptake in skeletal muscle. The study objective was to investigate the impact of varying dietary protein interventions on insulin sensitivity (IS) and molecular signaling in skeletal muscle in overweight or obese individuals, both acutely and over an up to 18-weeks period. The analysis performed in the 18-week data set used a subset of data with available adipose tissue biopsies randomised, controlled, isoenergetic dietary intervention, focusing on the here relevant HPD and control diets. IS was assessed using labelled intravenous glucose tolerance tests (IVGTT) in the acute study, while the euglycemic-hyperinsulinemic clamp (EHC) was used in the 18-week intervention. Key protein implicated in insulin signalling, such as IP6K1 and total AMPK protein content significantly decreased following the HP meal, alongside an increased p-AktThr 308 /Akt2 activity while S6K1 mRNA was lower after 6 weeks of HPD, compared to the control diet group, but not at 18 weeks. However, these differences at the molecular level did not translate into significant changes in whole-body IS. Health sciences/Endocrinology Health sciences/Molecular medicine Diabetes High protein Insulin resistance Obesity Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Article Highlights Excessive protein availability is known to cause overactivity of the S6K1 signaling nexus which may contribute to insulin resistance. Yet, the available research to date is limited to intravenous human protocols. Thus, our work aims to assess whole-body insulin sensitivity in response to a high protein meal, providing more ecologically relevant data. High protein intake does not affect insulin signalling but has impact on some signalling proteins in the skeletal muscle. Acute and moderate-term protein ingestion does not seem to cause whole-body insulin resistance Introduction Insulin resistance (IR) is the hallmark of metabolic syndrome and type 2 diabetes (T2Ds) and can be influenced by dietary interventions causing weight loss ( 1 ). Various diet models have been proposed to manage glycemic control, but high-protein diets (HPDs), rich in branched-chain amino acids (BCAAs; i.e., isoleucine, leucine, and valine) are commonly recommended ( 2 ). The relationship between high concentrations of circulating BCAAs and IR remains controversial. Elevated BCAAs concentrations may contribute to IR or could be a consequence of impaired insulin action and not a direct consequence of HPDs. Yet, elevated levels of plasma BCAAs have been linked to IR and could be a predictor of the development of T2Ds up to 12 years beforehand ( 3 – 6 ). BCAAs are potent activators of the protein synthesis pathway in peripheral tissue via the activation of mechanistic target of rapamycin complex 1 (mTORC1), in both resting and post-exercised skeletal muscle ( 7 ). mTORC1 is a serine-threonine protein kinase that acts as a crucial regulator of cell growth and metabolism, including lipid and protein synthesis, energy storage, and mitochondrial biogenesis, and dysregulation of this pathway has been implicated in numerous diseases, including cancer and metabolic syndrome ( 8 ). In the presence of persistent hyperaminoacidemia, overactivation of mTORC1 leads to the phosphorylation of ribosomal protein S6 kinase 1 (S6K1) and the subsequent deactivation of phosphatidylinositol 3-kinase (PI3K) ( 9 ). PI3K has an essential role in insulin-dependent glucose uptake via the activation of Akt, which promotes AS160 associated glucose transporter-4 (GLUT-4) translocation. This negative feedback loop on PI3K/Akt/AS160 can result in decreased GLUT-4 translocation and glucose uptake, contributing to the pathogenesis of IR and T2D ( 10 ). While acute hyperaminoacidemia is generally considered safe in healthy individuals, it can have various consequences in individuals with obesity, particularly when coupled with IR and other metabolic dysfunctions commonly observed in obesity. An investigation using obese Zucker rodents showed that hyperaminoacidemia impaired glucose tolerance, decreased insulin sensitivity, and increased hepatic glucose production (HGP) compared to control rats ( 11 ). In vivo human studies also show a decrease in insulin sensitivity during amino acid infusions, especially BCAA ( 12 , 13 ). However, considering that intravenous amino acid infusion bypasses the digestive system and the complex hormonal and neural signals involved in nutrient sensing and metabolic regulation that occur during normal dietary protein digestion, and additionally greatly exceed levels observed after oral protein intake, the metabolic responses observed with intravenous AA infusion may not fully reflect the dietary protein intake. We aimed to investigate the effects of moderate and high-protein meals, acutely, on both whole-body glucose metabolism and molecular markers implicated in insulin-stimulated glucose uptake in skeletal muscle in individuals with obesity. Comparisons were made between the trials to assess if the high protein meal induced intracellular dysregulation causing a downregulation in insulin signaling when compared with the moderate dose or a controlled meal. To provide additional metabolic insights on the impact of protein intake, we conducted further analysis on cellular signaling events in adipose tissue from a previously published 18-week controlled interventional study in overweight or obese participants ( 14 ), where protein intakes were manipulated. Materials and methods Study design The acute study employed a randomized order, double-blinded design with a 14-day washout period with 3 conditions differentiated by a single meal provided (Supplementary Fig. 1). Participants had either a chicken meal containing 50 grams of protein [moderate protein dose (MPD)], 100 grams of protein [high protein dose (HPD)] or 50 grams of protein with added fat [moderate protein dose and added fat (MPDAF)] to match the energy content of the HPD meal to investigate whether differences with the higher protein dose were dietary protein-specific or were attributable to the energy content of the meal. Participants were advised to spend approximately 15 minutes to consume the meal without being required to eat quickly. In the medium-term study, participants were randomly assigned to an 18-wk isoenergetic nutritional intervention in 111 overweight participants with features of the metabolic syndrome, divided into balanced groups (Supplementary Table 1). Details of that dietary intervention have been published ( 5 , 14 , 15 ). Diets were kept isoenergetic with identical dietary fat contents in all dietary groups and varying dietary protein and carbohydrate contents. Adipose tissue biopsies were performed in a subset (HP group, n = 7; control group, n = 6), sampled after 0, 6, and 18 weeks of dietary intervention. Gene expression data of that study have not been published previously. Ethical approval and participant recruitment Ethical approval for the acute experimental design and procedures was granted by the University of Roehampton Ethics Committee (reference number LSC 189/235) prior to the start of data collection. Participants were recruited through advertisements placed locally and online and through university-wide group emails. Participants for the medium-term study were recruited from the Metabolic-Syndrome-Berlin-Potsdam Study cohort (~ n = 2700) ( 16 ). The Ethics Committee of the University of Potsdam approved the study (BMBF FKZ 0313826). All experimental procedures were conducted in accordance with the World Medical Association’s revised declaration of Helsinki for Medical Research Involving Humans ( 17 ) and written informed consent was obtained from all participants. Inclusion/ exclusion criteria for participants To qualify for inclusion in the acute study, individuals needed to meet the following criteria: a non-diabetic state, as assessed by fasting blood glucose concentration below 7 mmol/L (< 126 mg/dL) or random plasma glucose below 11.1 mmol/L (< 200 mg/dL); a Body Mass Index (BMI) equal to or greater than 30 kg/m2; and an age range of 30–65 years. Exclusion criteria included: pregnant women or those taking hormone contra-conception therapy; individuals with secondary complications of metabolic syndrome (such as neuropathy, nephropathy, cardiovascular diseases, stroke, or hypertension); individuals with a known diagnosis of active cancer; current smokers; individuals requiring insulin or any other glycaemia-altering medication; individuals diagnosed with arthritis, rheumatism, or gout spondylitis; and individuals unable to mobilize independently. Details about inclusion and exclusion criteria in the medium-term study have been published ( 14 ) . Nine individuals with obesity ( mean age 46.1 ± 9.7 years, body mass 94.9 ± 9.3 kg, body mass index 33.2 ± 2.0 kg/m 2 , body fat 43.5 ± 10.2%, fasting blood glucose 4.5 ± 0.4 mmol/L, HbA1 c 36 ± 1 mmol/mol n = 5 males, n = 4 females) were recruited for participation in the acute study and attended the laboratories at University of Roehampton on 4 different occasions. Thirteen participants (control group = 6, HP group = 7) were included in the medium-term study analysis (mean age control group 57.1 ± 2.4 years and HP 54.8 ± 2.9, body mass control group 83.9 ± 4.2 kg and 86.4 ± 5.1 kg HP, control group BMI 30.6 ± 1.0 kg/m² and 32.5 ± 1.3 kg/m² for the HP group. Experimental protocol Participants attended the laboratory for the experimental protocol at ~ 9:00am on three occasions, having fasted for 12 hours prior, with each visit separated by 14–30 days. To allow for administration of glucose solution during the intravenous glucose tolerance test (IVGTT), a cannula was placed into the antecubital vein of one arm. A separate cannula was placed into a dorsal hand vein, in a retrograde direction, for frequent sampling of arterialised-venous blood, achieved through use of a thermoregulated hot box (~ 60°C) as previously described ( 18 , 19 ) . Muscle biopsies were collected under local anesthesia from the vastus lateralis with the conchotome method( 20 ) at baseline (0 minutes). Immediately following the baseline blood sample and muscle biopsy, participants were provided a chicken meal containing 50 grams of protein, 100 grams of protein or 50 grams of protein with added fat to match the energy content of the HPD meal. The fat that was added in the MPDAF consisted of 25g of vegetable oil, which was the equivalent of 231 kcal. The software Dietplan 7 (Forestfield Software Ltd, U.K.) was used for food composition analysis (Table 1 ). Table 1 Meal composition analysis for the 3 trials completed in the acute study Trial Protein (g) Chicken (g) Added fat (g) Energy (kcal) MPD 50 156 0 231 HPD 100 312 0 462 MPDAF 50 156 25 462 Abbreviations: MPD, moderate protein dose; HPD, high protein dose; MPDAF, moderate protein dose and added fat Following the meal, a 4-hour labelled IVGTT was administered (28.4mg/kg [6,6- 2 H 2 ]Glucose and 250mg/kg unlabeled glucose), prepared under sterile conditions. Thereafter, frequent arterialized (~ 5mL) blood samples were drawn over the ensuing 240 minutes, as previously described ( 19 ). A second muscle biopsy was collected immediately post the IVGTT and ~ 240 minutes following the finish of trial meals. Muscle samples were washed in ice-cold saline immediately before being flash frozen in liquid nitrogen and transferred to − 80°C until analysis ( 20 ). A 5µL sample was used to determined glucose concentrations (Biosen C-Line, EKF Diagnostics, UK) before the remaining samples were centrifuged at 4°C, 6000 RPM, for 10 minutes. The resulting plasma was aliquoted (1.5 mL) and stored at -80°C until further analysis. Details related to EHC and adipose tissue sampling/preparation in the medium-term study have been published ( 14 ). Controlled meal preparation All meals for the acute study were prepared at University of Roehampton.The meals consisted of baked chicken which was prepared on the same day of the trial. The chicken was baked in the oven at 180°C for 30 min and blended before served. For the MDPAF trial (50 grams of protein plus fat added to match the energy content of the high-protein trial), vegetable oil was added to the blended chicken until the energy content of the HPD meal was matched. Details related to the dietary contents and supplements used in the medium-term study have been published ( 14 ) Blood analysis Insulin concentrations were determined in plasma using a commercially available ELISA (EIA-2935, DRG Instruments GmbH, Germany). Blood glucose concentrations were determined using Biosen C-Line (EKF Diagnostics, UK). Labelled glucose was measured in plasma by gas chromatography-mass spectrometry as previously described ( 21 ). Proton magnetic resonance spectroscopy (1H-MRS) for the measurement of hepatic lipid content was performed as described ( 22 ). For details, see Online Supporting Materials under ‘Supplemental data’ in the online version of the original paper ( 14 ). Data modelling Plasma insulin, glucose concentrations, and [6,6- 2 H 2 ]Glucose enrichments values were used to model the metabolic indices: insulin sensitivity (S I 2* ), glucose effectiveness (S G 2* ), and hepatic glucose production (HGP) using two-compartment modeling, as described previously( 21 , 23 , 24 ) (SAAMII Institute, Seattle, WA). The incremental area under the curve (iAUC) of IVGTT was calculated for insulin and glucose concentrations as described in ( 25 ). Additionally, the acute insulin response to glucose (AIRg) was calculated using the iAUC of insulin concentration in the first 10 minutes, while 2nd phase insulin secretion was determined using 11–240 minutes of the IVGTT using the trapezoidal rule. Disposition index (DI = S I 2* x AIRg) was calculated as described in ( 26 ). To assess whole-body IS, the medium-term duration study employed a EHC at baseline (week 0), and after 6 and 18 weeks for each of the dietary interventions ( 14 ) . Muscle analysis Muscle samples were lysed and analysed via western blot for protein analysis as described ( 21 ). The post-intervention muscle biopsy data were normalised to the average baseline expression across the three experimental conditions for each individual to account for individual variability in baseline levels. The primary antibodies used were: anti-total Akt2 (#3063, RRID:AB_2225186) ( 27 ), pAkt S473 (#9271, RRID:AB_329825), pAkt T308 (#9275, RRID:AB_329828), pS6K1 Thr389 (#9234, RRID:AB_2269803), pAMPKα1 Ser485 /AMPKα2 Ser491 (#4185, RRID:AB_2169402), anti AMPKα (#2532, RRID:AB_330331) (all Cell Signalling Technology), Anti-IP6K1 antibody (ab129595, RRID:AB_11157733) and recombinant anti-S6K1 (ab32359, RRID:AB_777802) (Abcam). Signals observed after the meal was normalised to the respective participant's average baseline signal. RNA extraction from subcutaneous adipose tissue Fat tissue biopsies were performed as previously described ( 14 ). Total RNA was extracted from subcutaneous fat tissue with Lipid Tissue RNA-Kit™ (Qiagen, Germany) according to the manufacturer’s protocol. RNA was reverse transcribed into cDNA using the High-Capacity cDNA Reverse Transcription Kit™ with random priming (Applied Biosystems, Germany). cDNA was labelled by Power SYBR Green™ master mix (Applied Biosystems, Germany) and analyzed in an ABI Prism 7900HT Sequence Detector (Applied Biosystems, Germany). For quantification the standard curve method was applied. Target genes were normalized to relative expression levels of ribosomal protein large protein 0 (RPLP0) which is an established housekeeping gene in adipose tissue. Fold changes were calculated from the ratio of means of the normalized quantities and their statistical significance was determined by unpaired Student’s t-test. Statistical analyses Statistical analyses were carried out using IBM SPSS Statistics (version 28.0.1.1.). In the acute study, differences between the conditions in muscle protein content were evaluated using ANOVA, with Bonferroni correction applied to account for multiple comparisons., or two-tailed Student’s T-test for paired sample analyses. Data from the medium-term study were analysed using a two-way repeated measures ANOVA (2×3, group by time) to assess differences between baseline, 6 weeks, and 18 weeks with Bonferroni correction applied for post-hoc pairwise comparisons. Data are expressed as mean (SEM), unless otherwise specified. Statistical significance was accepted at P < 0.05. Results Acute study Two compartment modelling of the IVGTTs showed no significant differences between meals (MPDAF, MPD and HPD) for insulin sensitivity (S I 2* ) (p = 0.14), glucose effectiveness (S G 2* ) (p = 0.11) or HGP (p = 0.88) (Fig. 1 ). From plasma analysis and further calculations, this study found no statistical differences between the 3 conditions (HPD, MPD and MPDAF) for iAUC glu (p = 0.72), iAUC ins (p = 0.01), AIRg (p = 0.82), 2nd Phase Insulin Secretion (p = 0.11), Disposition index (DI) (p = 0.24) and DI 2nd Phase Insulin Secretion (p = 0.08) (Fig. 2 ). Plasma amino acids data for the IVGTT study ( Supplementary Figs. 2 and 3) show a time-dependant increase in some BCAA. In the HPD trial, valine concentrations increased significantly from baseline at 60 minutes post-meal and remained elevated up to 4 hours. Similarly, in the MPD trial, valine concentrations increased from baseline between 60- and 120-minutes post meal ingestion, whereas no significant changes were observed in the MPDAF trial. In addition, at 180-minute post meal, valine’s concentration was significantly higher in the HPD trial compared to MPD and MPDAF (p = 0.032 and 0.012 respectively) with difference between HPD and MPDAF persisting at 240-minutes (p = 0.029). Leucine concentrations increased significantly from baseline in HPD between 60 and 180 minutes, while in MPD, increases were noted only at 60 and 80 minutes, with no significant changes in MPDAF. At 180 minutes, leucine was higher in HPD than MPDAF (p = 0.015) and MPD (p = 0.031). Isoleucine concentrations remained unchanged post-meal across all trials. However, total BCAA concentrations were significantly elevated from baseline between 80 and 180 minutes in HPD and from 60 to 80 minutes in MPD, with no changes in MPDAF. Total BCAA levels in HPD were significantly higher than MPDAF at 180 minutes (p = 0.035). From western blot analysis, protein content of IP6K1 (Fig. 3 A) decreased in the 4 hours following the HPD when compared with baseline (p = 0.048), with no significant differences in the MPD (p = 0.89), nor in the MPDAF (p = 0.33) for the same comparison (i.e. against averaged baselines within trial). pAkt Ser308 /Akt2 (Fig. 3 F) increased from baseline to the 4-hour sample in the HPD trial (p = 0.046). Finally, AMPK (Fig. 3 J) decreased from baseline in the HPD trial (p = 0.01), with no significant differences between the groups. No significant differences were observed between the conditions for Akt2 (Fig. 3 B), pAkt Ser473 (Fig. 3 C), pAkt Thr308 (Fig. 3 D), pAkt Ser473 /total Akt2 (Fig. 3 E), total S6K1 (Fig. 3 G), pS6K1 Thr389 (Fig. 3 H), pS6K1 Thr389 /total S6K1(Fig. 3 I), pAMPK α1 Ser485 /AMPK α2 Ser491 (Fig. 3 K), (pAMPK α1 Ser485 /AMPK α2 Ser491 )/ total AMPK (Fig. 3 L). Medium term study The medium-term study revealed a significant difference in the mRNA levels of S6KI between HP and control diet at 6 weeks (p = 0.046), which was not witnessed at 18 weeks for the same group comparison (p = 0.80). No significant differences were noted for mRNA expression for IRS-1, mTOR, 4E-BP, PPARγ, Akt, fatty acid synthase, hormone-sensitive lipase, or ATGL between baseline, 6 weeks and 18 weeks following both the control diet (CD) and high-protein diet (HPD) (Fig. 4 ). There was a significant difference observed for intrahepatic lipid content (IHL), which was significantly lower in the HP diet group at 6 weeks compared to CD (p = 0.03). The same comparison at 18 weeks showed a trend towards significance (p = 0.052) (Fig. 4 J). In addition, no changes in insulin sensitivity between groups at 6 or 18 weeks were seen, as measured using the EHC (Fig. 5 ). In the medium-term study, all diets were designed to be isoenergetic and there was no change in body weight in any of the dietary groups between baseline (week 0) and later time points including week 6 time point (p = 0.42). Dietary amino acid signature data for the medium-term study have been previously published by Hattersley and colleagues ( 5 ). Discussion Our acute study investigated the effects of different protein doses on the cellular mechanisms that are central in the regulation of insulin-stimulated glucose uptake and protein synthesis in skeletal muscle in individuals with obesity. For the first time in humans, this study employed the two-compartment IVGTT to assess if the different meals affected whole body insulin sensitivity. Despite variations in protein intake, the current data set showed no statistically significant differences for insulin sensitivity, glucose effectiveness, or HGP between conditions (HPD, MPD, and MPDAF). These findings are, of course, following a short-term dietary manipulation. We acknowledge that the use of the labelled IVGTT resulted in our data representing insulin secretion in response to both protein ingestion and iv glucose. However, as the IVGTT glucose load remained fixed for each volunteer and for each of their trials, we were able to isolate the insulin response to the protein meal. We have included additional analyses of a similar dietary intervention yet over an 18-week period ( 14 ). Employing an euglycemic hyperinsulinemic clamp (EHC), this data performed in a subgroup of participants with available adipose tissue biopsies showed no differences between high protein and control diets on whole-body insulin sensitivity. We hypothesized that the higher protein ingestion would result in hyperaminoacidemia, leading to an increase in phosphorylation of S6K1( 28 ) and a subsequent decrease in insulin signaling at the molecular level. While we did observe an increase in amino acid concentration in the HPD trial in the acute study (Supplemental 2 & 3sh), there were no observed differences in muscle pS6K1 Thr389 for the same comparisons. In addition, S6K1 mRNA was significantly lower in the high protein compared to control, as measured in adipose tissue, at 6 weeks. Tremblay and colleagues used an EHC coupled with amino acid infusions leading to an approximately 2.5-fold increase in blood amino acid concentration and a 3.7-fold elevation in skeletal muscle S6K1 protein content ( 29 ). Building upon these insights, the present studies sought to examine the alterations in muscle S6K1 protein content four hours following the consumption of different sized protein meals, and over 18 weeks of high protein diet, hereby providing insights into the physiological dynamics of S6K1 postprandially. No significant changes were observed in either the total S6K1 protein content or its phosphorylated state at Thr 389 during the acute study for any of the conditions, indicating that the influence of plasma amino acid levels on S6K1 may be notably attenuated compared to infusion studies. However, the medium-term study showed a decrease in S6K1 mRNA expression in adipose tissue over the strictly controlled 6-week period with HPD when compared to an isoenergetic control diet (p = 0.046), whereas after 18 weeks, no significant differences were seen, which is in agreement with the findings observed in the entire cohort( 14 ) and likely related to an observed drop in the adherence to the HPD in the medium-term; as was supported by measurement of biomarkers of protein intake and entirely expected in medium -term dietary interventions in humans that does not include a fully controlled feeding intervention. These findings may suggest that medium-term high protein feeding is required to manipulate S6K1 and that this response may be tissue specific. Nevertheless, researchers noted an increased pS6K1 at Thr 389 in skeletal muscle induced by elevated insulin, as seen when performing EHC studies ( 29 ). The conflicting results regarding this signaling kinase may be attributed to variations in insulin response between studies. In our current dataset, no significant changes in insulin response were observed, which could explain the absence of differences in this signaling kinase between trials. It is worth noting that in the published work of Weickert (2011) there was a significant worsening in insulin sensitivity after 6 weeks of a HPD in a larger sample size [n = 23; 4.20 +/- 0.38 (0 week) to 3.71 +/- 0.36 mg · kg · min − 1 (6 week), p = 0.13]. The effect observed in the sample with available gene expression data used in this study (Control n = 6; HPD n = 7) agrees with observations in obese subjects with moderate type 2 diabetes ( 30 , 31 ). These authors showed a similar decrease of hepatic fat on high protein diets after 6 weeks by about 40%, and no changes of fasting amino acid levels or of the insulin signaling pathway ( 30 , 31 ). We also observed a decrease in muscle IP6K1 protein content in the acute study in the HPD trial when compared to baseline fasting samples. These results suggest that acutely, 4-hour post meal ingestion, IP6K1 content was downregulated by dietary protein intake only after a certain intake threshold, as no differences were seen in the MPD trial, or when energy intake was matched in the MPDAF. The regulation of IP6K1 in skeletal muscle is of interest due to its role in the modulation of glucose metabolism and insulin signaling( 21 , 32 – 35 ). An inhibition or genetic deletion of IP6K1 was shown to protect mice from hepatic steatosis by improving mitochondrial function and reducing gluconeogenic pathways while reducing lipolysis in adipose tissue ( 32 ). The inhibition of IP6K1 positively regulates numerous metabolic pathways and may therefore explain the positive effects of high protein diets observed in humans with metabolic dysfunction ( 36 ). Our acute data set also showed an increase in Akt2 activity, in agreement with previous publications ( 21 , 34 , 37 – 39 ). The heightened availability of BCAAs, particularly leucine, subsequent to high-protein meals, is acknowledged for its potential to activate mTOR and its downstream targets, including S6K1. In some in vitro studies, such activation has been linked to reduced insulin action on Akt, thereby impairing insulin signaling ( 40 , 41 ); and supported by previous findings where amino acid infusion caused a rise in IR and S6K1 activity ( 29 ). However, over the 18-week study, we did not observe significant changes in adipose tissue total Akt and pAkt, or Akt mRNA. Although we noted significantly elevated levels of BCAA acutely, over the 4 hours post meal, following the HPD trial, which outlasted those seen at lower doses of protein (MPDAF and MPD), substantial alterations in S6K1 were not seen acutely. Consequently, distinctly from the intravenous infusions studies where the digestion system is circumvented and unphysiologically high levels of AA are achieved ( 10 ), in the meal-induced context, the increased in pAkt Thr308 /Akt2 seen following the HPD meal could potentially be attributed to IP6K1 modulation rather than S6K1 activation, although the relatively small sample size in our studies is acknowledged. Additionally, our results may have missed important changes in insulin signaling owing to the time of the second muscle biopsy. The changes in IP6K1 muscle content are supported by a previous study that also noted Akt activation in response to high-protein intake correlated with change in IP6K1 content ( 42 ). Furthermore, we had previously published effects of isoenergetic HP vs control diet with comparable dietary fat contents on intrahepatic lipid (IHL) over an up to 18-week period ( 14 ). In the here analyzed subset of participants with available gene expression data, we observed a significant decrease in IHL in the HP group compared to the control diet after 6 weeks point, with a trend still observed after 18 weeks. These findings are consistent with the work of ( 43 ), who reported a significant 42.6% reduction in IHL among morbidly obese individuals following a calorie-restricted, isocaloric HPD, compared to a low-protein diet and upon intake of isocaloric high protein diets in patients with Type 2 Diabetes ( 31 ). Further, in the published data from the larger cohort ( 14 ), the drop in IHL from baseline following 6 weeks of isoenergetic HPD vs control diet happened despite unchanged body mass in all dietary groups, thereby excluding any weight-change related effects of the diets. Conclusion In conclusion, our findings contribute explaining the potential of high protein meals as a dietary intervention for enhancing metabolic health and glycemic control. While none of the meals tested (MPD, MPDAF, or HPD) induced significant alterations in insulin sensitivity or glucose effectiveness using a labelled IVGTT, the consumption of a high-protein meal (containing 100 grams of protein) resulted in a notable reduction in muscle IP6K1 levels and an increase in Akt activity, suggesting that protein consumption can improve insulin signaling, in an acute context. Declarations Data availability The datasets generated and/or analysed during the current study are not publicly available due to privacy restrictions and ongoing analyses but are available from the corresponding author on reasonable request. Grants RM and OA were supported by the Health Innovation Network, London. MOW and AFHP were supported by grants from the German Ministry of Education and Science (BMBF, 0313826A, and 0313826B), the German Institute of Human Nutrition (Potsdam-Rehbruecke), Charité University Medicine Berlin, scientific collaborators and regional companies (Rettenmayr Inc, Anona Inc, and Kathi Inc, Germany), which included the provision of raw materials for the dietary supplements Disclosures None of the authors reported any financial disclosures that were related to the study. None of the funding organizations or sponsors played any role in the design and conduct of the study; in the collection, analyses, and interpretation of the data; or in the preparation, review, or approval of the manuscript. Acknowledgements Conceived and designed research: OA, RM, AHE, NB, AP, MW. Performed experiments: OA, RM, FD, DN, NB, AP, MW. Analysed data: OA, RM, FD, DN, AP, MW. Interpreted results of experiments: OA, RM, FD, DN, NB, AP, MW. Prepared figures: OA, RM, AP, MW. Drafted manuscript, edited and revised manuscript, approved final version of manuscript: OA, RM, AHE, FD, DN, NB, RR, AP, MW. RM is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. References Ancu O, Mickute M, Guess ND, Hurren NM, Burd NA, Mackenzie RW. Does high dietary protein intake contribute to the increased risk of developing prediabetes and type 2 diabetes? 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Schulze MB, Hoffmann K, Boeing H, Linseisen J, Rohrmann S, Möhlig M, et al. An accurate risk score based on anthropometric, dietary, and lifestyle factors to predict the development of type 2 diabetes. Diabetes Care. 2007;30(3). World Medical Association Declaration of Helsinki: ethical principles for medical research involving human subjects. J Am Coll Dent. 2014;81(3). McGuire EAH, Helderman JH, Tobin JD, Andres R, Berman M. Effects of arterial versus venous sampling on analysis of glucose kinetics in man. J Appl Physiol. 1976;41(4). Mackenzie R, Maxwell N, Castle P, Brickley G, Watt P. Acute hypoxia and exercise improve insulin sensitivity (SI2*) in individuals with type 2 diabetes. Diabetes Metab Res Rev. 2011;27(1). Dietrichson P, Coakley J, Smith PEM, Griffiths RD, Helliwell TR, Tedwards RH. Conchotome and needle percutaneous biopsy of skeletal muscle. J Neurol Neurosurg Psychiatry. 1987;50(11). Naufahu J, Elliott B, Markiv A, Dunning-Foreman P, McGrady M, Howard D, et al. High-Intensity Exercise Decreases IP6K1 Muscle Content and Improves Insulin Sensitivity (S i 2 ∗) in Glucose-Intolerant Individuals. Journal of Clinical Endocrinology and Metabolism. 2018;103(4). Machann J, Thamer C, Schnoedt B, Stefan N, Haring HU, Claussen CD, et al. Hepatic lipid accumulation in healthy subjects: A comparative study using spectral fat-selective MRI and volume-localized 1H-MR spectroscopy. Magn Reson Med. 2006;55(4). Vicini P, Caumo A, Cobelli C. The hot IVGTT two-compartment minimal model: Indexes of glucose effectiveness and insulin sensitivity. Am J Physiol Endocrinol Metab. 1997;273(5 36-5). Utzschneider KM, Prigeon RL, Faulenbach M V., Tong J, Carr DB, Boyko EJ, et al. Oral Disposition index predicts the development of future diabetes above and beyond fasting and 2-h glucose levels. Diabetes Care. 2009;32(2). Bravata DM, Sanders L, Huang J, Krumholz HM, Olkin I, Gardner CD, et al. Efficacy and Safety of Low-Carbohydrate Diets: A Systematic Review. J Am Med Assoc. 2003;289(14):1837–50. Oana A, Jane N, Richie B, Peter W, Richard WA M. Disposition Index (DI) is not Improved with High-Intensity Intermittent Exercise in Adults with Hyperinsulinemia and Pre-Diabetes. Clinical Diabetes and Research. 2021;5(1). Raun SH, Ali M, Kjøbsted R, Møller LLV, Federspiel MA, Richter EA, et al. Rac1 muscle knockout exacerbates the detrimental effect of high-fat diet on insulin-stimulated muscle glucose uptake independently of Akt. Journal of Physiology. 2018;596(12):2283–99. Um SH, D’Alessio D, Thomas G. Nutrient overload, insulin resistance, and ribosomal protein S6 kinase 1, S6K1. Vol. 3, Cell Metabolism. 2006. Tremblay F, Krebs M, Dombrowski L, Brehm A, Bernroider E, Roth E, et al. Overactivation of S6 kinase 1 as a cause of human insulin resistance during increased amino acid availability. Diabetes. 2005;54(9):2674–84. Markova M, Hornemann S, Sucher S, Wegner K, Pivovarova O, Rudovich N, et al. Rate of appearance of amino acids after a meal regulates insulin and glucagon secretion in patients with type 2 diabetes: A randomized clinical trial. American Journal of Clinical Nutrition. 2018;108(2). Markova M, Pivovarova O, Hornemann S, Sucher S, Frahnow T, Wegner K, et al. Isocaloric Diets High in Animal or Plant Protein Reduce Liver Fat and Inflammation in Individuals With Type 2 Diabetes. Gastroenterology. 2017;152(3). Mukherjee S, Chakraborty M, Ulmasov B, McCommis K, Zhang J, Carpenter D, et al. Pleiotropic actions of IP6K1 mediate hepatic metabolic dysfunction to promote nonalcoholic fatty liver disease and steatohepatitis. Mol Metab. 2021;54. Mukherjee S, Haubner J, Chakraborty A. Targeting the inositol pyrophosphate biosynthetic enzymes in metabolic diseases. Vol. 25, Molecules. 2020. Barclay RD, Beals JW, Drnevich J, Imai BS, Yau PM, Ulanov A V., et al. Ingestion of lean meat elevates muscle inositol hexakisphosphate kinase 1 protein content independent of a distinct post-prandial circulating proteome in young adults with obesity. Metabolism. 2020;102. Ghoshal S, Mukherjee S, Chakraborty M, Msengi EN, Haubner J, Chakraborty A. Whole Body Ip6k1 Deletion Protects Mice from Age-Induced Weight Gain, Insulin Resistance and Metabolic Dysfunction. Int J Mol Sci. 2022;23(4). Chakkour M, Greenberg ML. Insights into the roles of inositol hexakisphosphate kinase 1 (IP6K1) in mammalian cellular processes. Vol. 300, Journal of Biological Chemistry. 2024. Chog ZZ, Li F, Maiese K. Activating Akt and the brain’s resources to drive cellular survival and prevent inflammatory injury. Vol. 20, Histology and Histopathology. 2005. Tremblay F, Marette A. Amino Acid and Insulin Signaling via the mTOR/p70 S6 Kinase Pathway. Journal of Biological Chemistry. 2001;276(41). Haydon CE, Watt PW, Morrice N, Knebel A, Gaestel M, Cohen P. Identification of a phosphorylation site on skeletal muscle myosin light chain kinase that becomes phosphorylated during muscle contraction. Arch Biochem Biophys. 2002;397(2). Patti ME, Brambilla E, Luzi L, Landaker EJ, Kahn CR. Bidirectional modulation of insulin action by amino acids. Journal of Clinical Investigation. 1998;101(7). Yip CK, Murata K, Walz T, Sabatini DM, Kang SA. Structure of the Human mTOR Complex I and Its Implications for Rapamycin Inhibition. Mol Cell. 2010;38(5). Kim J, Darè E, Rajasekaran SS, Ryu SH, Berggren PO, Barker CJ. Inositol pyrophosphates and Akt/PKB: Is the pancreatic β-cell the exception to the rule? Cell Signal. 2019;58. Xu C, Markova M, Seebeck N, Loft A, Hornemann S, Gantert T, et al. High-protein diet more effectively reduces hepatic fat than low-protein diet despite lower autophagy and FGF21 levels. Liver International. 2020;40(12). Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6555791","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":463332318,"identity":"e807e672-5ed4-4220-8358-c402792a7556","order_by":0,"name":"Oana Ancu","email":"","orcid":"","institution":"University of Roehampton","correspondingAuthor":false,"prefix":"","firstName":"Oana","middleName":"","lastName":"Ancu","suffix":""},{"id":463332319,"identity":"1fab66e0-f5cb-4eff-a49f-7b827bb7f987","order_by":1,"name":"Astrid C. Hauge-Evans","email":"","orcid":"","institution":"University of Roehampton","correspondingAuthor":false,"prefix":"","firstName":"Astrid","middleName":"C.","lastName":"Hauge-Evans","suffix":""},{"id":463332320,"identity":"118753c9-0136-466d-86e2-10cabd3dc7ea","order_by":2,"name":"Fulvia Draicchio","email":"","orcid":"","institution":"University of Roehampton","correspondingAuthor":false,"prefix":"","firstName":"Fulvia","middleName":"","lastName":"Draicchio","suffix":""},{"id":463332321,"identity":"36fb9bd8-0c10-4be1-922c-b68beb3a1283","order_by":3,"name":"Diana-Elena Neculescu","email":"","orcid":"","institution":"University of Roehampton","correspondingAuthor":false,"prefix":"","firstName":"Diana-Elena","middleName":"","lastName":"Neculescu","suffix":""},{"id":463332322,"identity":"774171f1-25c0-41e0-8651-ecb951839192","order_by":4,"name":"Ralph Rogers","email":"","orcid":"","institution":"Rogers Regenerative Medical Group","correspondingAuthor":false,"prefix":"","firstName":"Ralph","middleName":"","lastName":"Rogers","suffix":""},{"id":463332323,"identity":"aaa47545-9cd0-4a0b-a2a7-ba276dbf65d0","order_by":5,"name":"Nicholas A. Burd","email":"","orcid":"","institution":"University of Illinois at Urbana-Champaign","correspondingAuthor":false,"prefix":"","firstName":"Nicholas","middleName":"A.","lastName":"Burd","suffix":""},{"id":463332324,"identity":"c211ce07-c252-4de1-88c5-29fd37e536ba","order_by":6,"name":"Andreas F.H. Pfeiffer","email":"","orcid":"","institution":"Charité Universitätsmedizin Berlin","correspondingAuthor":false,"prefix":"","firstName":"Andreas","middleName":"F.H.","lastName":"Pfeiffer","suffix":""},{"id":463332325,"identity":"2991cca3-1d34-4c7b-92a7-bf99a184b0b9","order_by":7,"name":"Martin O. Weickert","email":"","orcid":"","institution":"University Hospitals Coventry and Warwickshire","correspondingAuthor":false,"prefix":"","firstName":"Martin","middleName":"O.","lastName":"Weickert","suffix":""},{"id":463332326,"identity":"0782a057-bcab-4b1e-9b18-635abbb25f03","order_by":8,"name":"Richard W.A. Mackenzie","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA6klEQVRIie2RMQrCMBSGUwKZCl3Tpb1CugiieJYnQr2BdJKK0C4eQG/hARyeFOwS7FrpoCK4u5SORp10iLoJ5pvCTz74fx4hBsNPQvMGou5zZsVahQE/yPDxxs8UW7jHJPtCcdJYiH5S+A4fbE6XVaYSZO5co3CJALCtgsU8HAo8ZyoB5i51vUpAhFEFQsoWR1QNS8Lcg8bw98dJDGyrlKK+K/47RZSUEkgQRD5jd0XcFF2xQIaMgBwEizRpqWFDO5D9aVs338uL2mqinu9QeuYRdjwvz9a7mW7+C1P77SFfGX/122AwGP6DKzg/VcAO5ZgTAAAAAElFTkSuQmCC","orcid":"","institution":"Coventry University","correspondingAuthor":true,"prefix":"","firstName":"Richard","middleName":"W.A.","lastName":"Mackenzie","suffix":""},{"id":463332327,"identity":"a64a0034-8e9d-4622-a275-274b44d42e59","order_by":9,"name":"Nicholas Hurren","email":"","orcid":"","institution":"University of Roehampton","correspondingAuthor":false,"prefix":"","firstName":"Nicholas","middleName":"","lastName":"Hurren","suffix":""}],"badges":[],"createdAt":"2025-04-29 11:08:25","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6555791/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6555791/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":83676382,"identity":"e541a609-277d-4aa0-a771-bbcb144f7574","added_by":"auto","created_at":"2025-05-30 14:55:39","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":283194,"visible":true,"origin":"","legend":"\u003cp\u003eInsulin Sensitivity (S\u003csub\u003eI\u003c/sub\u003e\u003csup\u003e2*\u003c/sup\u003e) (A), Glucose Effectiveness (S\u003csub\u003eG\u003c/sub\u003e\u003csup\u003e2*\u003c/sup\u003e) (B) and Hepatic Glucose Production (HGP) (C) calculated for each of the trials: MPDAF, MPD and HPD. n=9 for each group. Data expressed as mean ± SEM. Abbreviations: MPDAF, moderate protein dose and added fat; MPD, moderate protein dose; HPD, high protein dose. Data expressed as mean ± SEM. No significant differences between trials S\u003csub\u003eI\u003c/sub\u003e\u003csup\u003e2*\u003c/sup\u003e, p=0.14; S\u003csub\u003eG\u003c/sub\u003e\u003csup\u003e2*\u003c/sup\u003e, p=0.11; HGP, p=0.88.\u0026nbsp;\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6555791/v1/a04564945a63db9feb926270.png"},{"id":83675502,"identity":"c80dd801-96be-4364-a51f-060752746eda","added_by":"auto","created_at":"2025-05-30 14:47:39","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":392615,"visible":true,"origin":"","legend":"\u003cp\u003eIncremental area under the curve for glucose (iAUC\u003csub\u003eglu\u003c/sub\u003e) concentration during IVGTT (A);\u0026nbsp; iAUC\u003csub\u003eIns \u003c/sub\u003econcentrations during IVGTT (B); Acute insulin response to glucose (AIR\u003csub\u003eg\u003c/sub\u003e) (C); 2\u003csup\u003end\u003c/sup\u003e Phase Insulin Secretion (D); and Disposition index (DI) (E). n= 9 for each group. MPDAF = moderate protein dose added fat (50 g protein and 25 g fat), MPD = moderate protein dose (50 g), HPD = high protein dose (100 g). Data expressed as mean ± SEM.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6555791/v1/da557b37f5e837aeadecb797.png"},{"id":83675503,"identity":"f61fc192-0d31-41a0-9adc-6e1bdef6b6cf","added_by":"auto","created_at":"2025-05-30 14:47:39","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":464278,"visible":true,"origin":"","legend":"\u003cp\u003eSkeletal muscle protein content for IP6K1 (A), Akt2 (B), pAkt\u003csup\u003eSer473\u003c/sup\u003e(C), pAkt\u003csup\u003eThr308\u003c/sup\u003e (D), pAkt\u003csup\u003eSer473\u003c/sup\u003e/Akt2 (E), pAkt\u003csup\u003eThr308\u003c/sup\u003e/total Akt2 (F), total S6K1 (G), pS6K1\u003csup\u003eThr389\u003c/sup\u003e (H), pS6K1\u003csup\u003eThr389\u003c/sup\u003e/total S6K1 (I), Total AMPK (J), pAMPKα1\u003csup\u003eSer485\u003c/sup\u003e/AMPKα2\u003csup\u003eSer491\u003c/sup\u003e (K), pAMPK\u003csub\u003eα1\u003c/sub\u003e\u003csup\u003eSer485\u003c/sup\u003e/AMPK\u003csub\u003eα2\u003c/sub\u003e\u003csup\u003eSer491\u003c/sup\u003e/ total AMPK (L).\u0026nbsp; * denotes significant differences from baseline, P\u0026lt;0.05. n baseline=9, n MPDAF= 8, n MPD=7, n HPD=8. Data expressed as mean ± SEM. Abbreviations: MPDAF, moderate protein dose and added fat; MPD, moderate protein dose; HPD, high protein dose.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6555791/v1/16e15bb2fabb9ae9ffc97c0f.png"},{"id":83675505,"identity":"bd87b1a0-6148-4db2-8e44-96bd223063db","added_by":"auto","created_at":"2025-05-30 14:47:39","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":554944,"visible":true,"origin":"","legend":"\u003cp\u003eShow adipose tissue mRNA expression for S6K1 (A), IRS-1 (B), mTOR (C), 4EPB (D), PPARy (E), Akt (F), Fatty Acid Synthase (G), Hormone Sensitive Lipase (H), ATGL (I) and Intrahepatic Lipid Content (J) (fold change from baseline) at baseline, 6 and 18 weeks following a Control Diet (CD; n=6) and High Protein Diet (HPD; n=7). *Significant difference between groups at 6 weeks for S6K1 (p\u0026lt;0.05).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6555791/v1/bd62b56b41c6320a447e2856.png"},{"id":83675504,"identity":"71717eda-ca74-440f-bccc-a5c8bf389c04","added_by":"auto","created_at":"2025-05-30 14:47:39","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":203436,"visible":true,"origin":"","legend":"\u003cp\u003eShow insulin sensitivity at baseline, 6 and 18 weeks following a Control Diet (CD; n=6) and High Protein Diet (HPD; n=7). No significant differences were noted.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6555791/v1/db42a81a50dc854bca42d3e2.png"},{"id":86884233,"identity":"13a77985-9b80-4f6a-917e-96b53191a1b5","added_by":"auto","created_at":"2025-07-16 17:16:38","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2446693,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6555791/v1/4fab5de8-778e-4420-9cf5-b3915f1c9b2f.pdf"},{"id":83675496,"identity":"5b00c823-4e7d-4781-9569-e15bc36e4b32","added_by":"auto","created_at":"2025-05-30 14:47:39","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":41937,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigure1.docx","url":"https://assets-eu.researchsquare.com/files/rs-6555791/v1/02dddbe43d8db52a910e7b4e.docx"},{"id":83675500,"identity":"7a2f695e-3559-4aa1-8b10-667de512aab7","added_by":"auto","created_at":"2025-05-30 14:47:39","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":585863,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigure23.docx","url":"https://assets-eu.researchsquare.com/files/rs-6555791/v1/4c22adfb253dd652804e63e0.docx"},{"id":83675498,"identity":"12041e33-fdd8-4a0d-a91a-3d622ed5ec66","added_by":"auto","created_at":"2025-05-30 14:47:39","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":20440,"visible":true,"origin":"","legend":"","description":"","filename":"SupplTable1.docx","url":"https://assets-eu.researchsquare.com/files/rs-6555791/v1/ba4e45b764663ce00770ace6.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"High protein ingestion does not affect whole-body insulin sensitivity","fulltext":[{"header":"Article Highlights ","content":"\u003cp\u003eExcessive protein availability is known to cause overactivity of the S6K1 signaling nexus which may contribute to insulin resistance.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eYet, the available research to date is limited to intravenous human protocols. Thus, our work aims to assess whole-body insulin sensitivity in response to a high protein meal, providing more ecologically relevant data.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;High protein intake does not affect insulin signalling but has impact on some signalling proteins in the skeletal muscle.\u003c/p\u003e\n\u003cp\u003eAcute and moderate-term protein ingestion does not seem to cause whole-body insulin resistance\u003c/p\u003e"},{"header":"Introduction","content":"\u003cp\u003eInsulin resistance (IR) is the hallmark of metabolic syndrome and type 2 diabetes (T2Ds) and can be influenced by dietary interventions causing weight loss (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). Various diet models have been proposed to manage glycemic control, but high-protein diets (HPDs), rich in branched-chain amino acids (BCAAs; i.e., isoleucine, leucine, and valine) are commonly recommended (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). The relationship between high concentrations of circulating BCAAs and IR remains controversial. Elevated BCAAs concentrations may contribute to IR or could be a consequence of impaired insulin action and not a direct consequence of HPDs. Yet, elevated levels of plasma BCAAs have been linked to IR and could be a predictor of the development of T2Ds up to 12 years beforehand (\u003cspan additionalcitationids=\"CR4 CR5\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eBCAAs are potent activators of the protein synthesis pathway in peripheral tissue via the activation of mechanistic target of rapamycin complex 1 (mTORC1), in both resting and post-exercised skeletal muscle (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). mTORC1 is a serine-threonine protein kinase that acts as a crucial regulator of cell growth and metabolism, including lipid and protein synthesis, energy storage, and mitochondrial biogenesis, and dysregulation of this pathway has been implicated in numerous diseases, including cancer and metabolic syndrome (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). In the presence of persistent hyperaminoacidemia, overactivation of mTORC1 leads to the phosphorylation of ribosomal protein S6 kinase 1 (S6K1) and the subsequent deactivation of phosphatidylinositol 3-kinase (PI3K) (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). PI3K has an essential role in insulin-dependent glucose uptake via the activation of Akt, which promotes AS160 associated glucose transporter-4 (GLUT-4) translocation. This negative feedback loop on PI3K/Akt/AS160 can result in decreased GLUT-4 translocation and glucose uptake, contributing to the pathogenesis of IR and T2D (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWhile acute hyperaminoacidemia is generally considered safe in healthy individuals, it can have various consequences in individuals with obesity, particularly when coupled with IR and other metabolic dysfunctions commonly observed in obesity. An investigation using obese Zucker rodents showed that hyperaminoacidemia impaired glucose tolerance, decreased insulin sensitivity, and increased hepatic glucose production (HGP) compared to control rats (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). In vivo human studies also show a decrease in insulin sensitivity during amino acid infusions, especially BCAA (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). However, considering that intravenous amino acid infusion bypasses the digestive system and the complex hormonal and neural signals involved in nutrient sensing and metabolic regulation that occur during normal dietary protein digestion, and additionally greatly exceed levels observed after oral protein intake, the metabolic responses observed with intravenous AA infusion may not fully reflect the dietary protein intake.\u003c/p\u003e \u003cp\u003eWe aimed to investigate the effects of moderate and high-protein meals, acutely, on both whole-body glucose metabolism and molecular markers implicated in insulin-stimulated glucose uptake in skeletal muscle in individuals with obesity. Comparisons were made between the trials to assess if the high protein meal induced intracellular dysregulation causing a downregulation in insulin signaling when compared with the moderate dose or a controlled meal. To provide additional metabolic insights on the impact of protein intake, we conducted further analysis on cellular signaling events in adipose tissue from a previously published 18-week controlled interventional study in overweight or obese participants (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e), where protein intakes were manipulated.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003eStudy design\u003c/h2\u003e\n \u003cp\u003eThe acute study employed a randomized order, double-blinded design with a 14-day washout period with 3 conditions differentiated by a single meal provided (Supplementary Fig.\u0026nbsp;1). Participants had either a chicken meal containing 50 grams of protein [moderate protein dose (MPD)], 100 grams of protein [high protein dose (HPD)] or 50 grams of protein with added fat [moderate protein dose and added fat (MPDAF)] to match the energy content of the HPD meal to investigate whether differences with the higher protein dose were dietary protein-specific or were attributable to the energy content of the meal. Participants were advised to spend approximately 15 minutes to consume the meal without being required to eat quickly.\u003c/p\u003e\n \u003cp\u003eIn the medium-term study, participants were randomly assigned to an 18-wk isoenergetic nutritional intervention in 111 overweight participants with features of the metabolic syndrome, divided into balanced groups (Supplementary Table\u0026nbsp;1). Details of that dietary intervention have been published (\u003cspan class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e15\u003c/span\u003e). Diets were kept isoenergetic with identical dietary fat contents in all dietary groups and varying dietary protein and carbohydrate contents. Adipose tissue biopsies were performed in a subset (HP group, n\u0026thinsp;=\u0026thinsp;7; control group, n\u0026thinsp;=\u0026thinsp;6), sampled after 0, 6, and 18 weeks of dietary intervention. Gene expression data of that study have not been published previously.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eEthical approval\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eand participant recruitment\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eEthical approval for the acute experimental design and procedures was granted by the University of Roehampton Ethics Committee (reference number LSC 189/235) prior to the start of data collection. Participants were recruited through advertisements placed locally and online and through university-wide group emails. Participants for the medium-term study were recruited from the Metabolic-Syndrome-Berlin-Potsdam Study cohort (~\u0026thinsp;n\u0026thinsp;=\u0026thinsp;2700) (\u003cspan class=\"CitationRef\"\u003e16\u003c/span\u003e). The Ethics Committee of the University of Potsdam approved the study (BMBF FKZ 0313826). All experimental procedures were conducted in accordance with the World Medical Association\u0026rsquo;s revised declaration of Helsinki for Medical Research Involving Humans (\u003cspan class=\"CitationRef\"\u003e17\u003c/span\u003e) and written informed consent was obtained from all participants.\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eInclusion/ exclusion criteria for participants\u003c/h3\u003e\n\u003cp\u003eTo qualify for inclusion in the acute study, individuals needed to meet the following criteria: a non-diabetic state, as assessed by fasting blood glucose concentration below 7 mmol/L (\u0026lt;\u0026thinsp;126 mg/dL) or random plasma glucose below 11.1 mmol/L (\u0026lt;\u0026thinsp;200 mg/dL); a Body Mass Index (BMI) equal to or greater than 30 kg/m2; and an age range of 30\u0026ndash;65 years.\u003c/p\u003e\n\u003cp\u003eExclusion criteria included: pregnant women or those taking hormone contra-conception therapy; individuals with secondary complications of metabolic syndrome (such as neuropathy, nephropathy, cardiovascular diseases, stroke, or hypertension); individuals with a known diagnosis of active cancer; current smokers; individuals requiring insulin or any other glycaemia-altering medication; individuals diagnosed with arthritis, rheumatism, or gout spondylitis; and individuals unable to mobilize independently. Details about inclusion and exclusion criteria in the medium-term study have been published (\u003cspan class=\"CitationRef\"\u003e14\u003c/span\u003e) .\u003c/p\u003e\n\u003cp\u003eNine individuals with obesity ( mean age 46.1\u0026thinsp;\u0026plusmn;\u0026thinsp;9.7 years, body mass 94.9\u0026thinsp;\u0026plusmn;\u0026thinsp;9.3 kg, body mass index 33.2\u0026thinsp;\u0026plusmn;\u0026thinsp;2.0 kg/m\u003csup\u003e2\u003c/sup\u003e, body fat 43.5\u0026thinsp;\u0026plusmn;\u0026thinsp;10.2%, fasting blood glucose 4.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4 mmol/L, HbA1\u003csub\u003ec\u003c/sub\u003e 36\u0026thinsp;\u0026plusmn;\u0026thinsp;1 mmol/mol n\u0026thinsp;=\u0026thinsp;5 males, n\u0026thinsp;=\u0026thinsp;4 females) were recruited for participation in the acute study and attended the laboratories at University of Roehampton on 4 different occasions. Thirteen participants (control group\u0026thinsp;=\u0026thinsp;6, HP group\u0026thinsp;=\u0026thinsp;7) were included in the medium-term study analysis (mean age control group 57.1\u0026thinsp;\u0026plusmn;\u0026thinsp;2.4 years and HP 54.8\u0026thinsp;\u0026plusmn;\u0026thinsp;2.9, body mass control group 83.9\u0026thinsp;\u0026plusmn;\u0026thinsp;4.2 kg and 86.4\u0026thinsp;\u0026plusmn;\u0026thinsp;5.1 kg HP, control group BMI 30.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0 kg/m\u0026sup2; and 32.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3 kg/m\u0026sup2; for the HP group.\u003c/p\u003e\n\u003ch3\u003eExperimental protocol\u003c/h3\u003e\n\u003cdiv class=\"Heading\"\u003eParticipants attended the laboratory for the experimental protocol at ~\u0026thinsp;9:00am on three occasions, having fasted for 12 hours prior, with each visit separated by 14\u0026ndash;30 days. To allow for administration of glucose solution during the intravenous glucose tolerance test (IVGTT), a cannula was placed into the antecubital vein of one arm. A separate cannula was placed into a dorsal hand vein, in a retrograde direction, for frequent sampling of arterialised-venous blood, achieved through use of a thermoregulated hot box (~\u0026thinsp;60\u0026deg;C) as previously described (\u003cspan class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e19\u003c/span\u003e) .\u003c/div\u003e\n\u003cp\u003eMuscle biopsies were collected under local anesthesia from the vastus lateralis with the conchotome method(\u003cspan class=\"CitationRef\"\u003e20\u003c/span\u003e) at baseline (0 minutes). Immediately following the baseline blood sample and muscle biopsy, participants were provided a chicken meal containing 50 grams of protein, 100 grams of protein or 50 grams of protein with added fat to match the energy content of the HPD meal. The fat that was added in the MPDAF consisted of 25g of vegetable oil, which was the equivalent of 231 kcal. The software Dietplan 7 (Forestfield Software Ltd, U.K.) was used for food composition analysis (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e).\u0026nbsp;\u003c/p\u003e\u0026nbsp;\u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eMeal composition analysis for the 3 trials completed in the acute study\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eTrial\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eProtein (g)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eChicken (g)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eAdded fat (g)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eEnergy (kcal)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMPD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e156\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e231\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHPD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e312\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e462\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMPDAF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e156\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e462\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eAbbreviations:\u003c/strong\u003e MPD, moderate protein dose; HPD, high protein dose; MPDAF, moderate protein dose and added fat\u003c/p\u003e\n\u003cp\u003eFollowing the meal, a 4-hour labelled IVGTT was administered (28.4mg/kg [6,6-\u003csup\u003e2\u003c/sup\u003eH\u003csub\u003e2\u003c/sub\u003e]Glucose and 250mg/kg unlabeled glucose), prepared under sterile conditions. Thereafter, frequent arterialized (~\u0026thinsp;5mL) blood samples were drawn over the ensuing 240 minutes, as previously described (\u003cspan class=\"CitationRef\"\u003e19\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eA second muscle biopsy was collected immediately post the IVGTT and ~\u0026thinsp;240 minutes following the finish of trial meals. Muscle samples were washed in ice-cold saline immediately before being flash frozen in liquid nitrogen and transferred to \u0026minus;\u0026thinsp;80\u0026deg;C until analysis (\u003cspan class=\"CitationRef\"\u003e20\u003c/span\u003e). A 5\u0026micro;L sample was used to determined glucose concentrations (Biosen C-Line, EKF Diagnostics, UK) before the remaining samples were centrifuged at 4\u0026deg;C, 6000 RPM, for 10 minutes. The resulting plasma was aliquoted (1.5 mL) and stored at -80\u0026deg;C until further analysis. Details related to EHC and adipose tissue sampling/preparation in the medium-term study have been published (\u003cspan class=\"CitationRef\"\u003e14\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eControlled meal preparation\u003c/h3\u003e\n\u003cp\u003eAll meals for the acute study were prepared at University of Roehampton.The meals consisted of baked chicken which was prepared on the same day of the trial. The chicken was baked in the oven at 180\u0026deg;C for 30 min and blended before served. For the MDPAF trial (50 grams of protein plus fat added to match the energy content of the high-protein trial), vegetable oil was added to the blended chicken until the energy content of the HPD meal was matched. Details related to the dietary contents and supplements used in the medium-term study have been published (\u003cspan class=\"CitationRef\"\u003e14\u003c/span\u003e)\u003c/p\u003e\n\u003cp\u003eBlood analysis\u003c/p\u003e\n\u003cp\u003eInsulin concentrations were determined in plasma using a commercially available ELISA (EIA-2935, DRG Instruments GmbH, Germany). Blood glucose concentrations were determined using Biosen C-Line (EKF Diagnostics, UK). Labelled glucose was measured in plasma by gas chromatography-mass spectrometry as previously described (\u003cspan class=\"CitationRef\"\u003e21\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eProton magnetic resonance spectroscopy (1H-MRS) for the measurement of hepatic lipid content was performed as described (\u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e). For details, see Online Supporting Materials under \u0026lsquo;Supplemental data\u0026rsquo; in the online version of the original paper (\u003cspan class=\"CitationRef\"\u003e14\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eData modelling\u003c/h3\u003e\n\u003cp\u003ePlasma insulin, glucose concentrations, and [6,6-\u003csup\u003e2\u003c/sup\u003eH\u003csub\u003e2\u003c/sub\u003e]Glucose enrichments values were used to model the metabolic indices: insulin sensitivity (S\u003csub\u003eI\u003c/sub\u003e\u003csup\u003e2*\u003c/sup\u003e), glucose effectiveness (S\u003csub\u003eG\u003c/sub\u003e\u003csup\u003e2*\u003c/sup\u003e), and hepatic glucose production (HGP) using two-compartment modeling, as described previously(\u003cspan class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e) (SAAMII Institute, Seattle, WA).\u003c/p\u003e\n\u003cp\u003eThe incremental area under the curve (iAUC) of IVGTT was calculated for insulin and glucose concentrations as described in (\u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e). Additionally, the acute insulin response to glucose (AIRg) was calculated using the iAUC of insulin concentration in the first 10 minutes, while 2nd phase insulin secretion was determined using 11\u0026ndash;240 minutes of the IVGTT using the trapezoidal rule. Disposition index (DI\u0026thinsp;=\u0026thinsp;S\u003csub\u003eI\u003c/sub\u003e\u003csup\u003e2*\u003c/sup\u003e x AIRg) was calculated as described in (\u003cspan class=\"CitationRef\"\u003e26\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eTo assess whole-body IS, the medium-term duration study employed a EHC at baseline (week 0), and after 6 and 18 weeks for each of the dietary interventions (\u003cspan class=\"CitationRef\"\u003e14\u003c/span\u003e) .\u003c/p\u003e\n\u003cp\u003eMuscle analysis\u003c/p\u003e\n\u003cp\u003eMuscle samples were lysed and analysed via western blot for protein analysis as described (\u003cspan class=\"CitationRef\"\u003e21\u003c/span\u003e). The post-intervention muscle biopsy data were normalised to the average baseline expression across the three experimental conditions for each individual to account for individual variability in baseline levels. The primary antibodies used were: anti-total Akt2 (#3063, RRID:AB_2225186) (\u003cspan class=\"CitationRef\"\u003e27\u003c/span\u003e), pAkt\u003csup\u003eS473\u003c/sup\u003e (#9271, RRID:AB_329825), pAkt\u003csup\u003eT308\u003c/sup\u003e (#9275, RRID:AB_329828), pS6K1\u003csup\u003eThr389\u003c/sup\u003e (#9234, RRID:AB_2269803), pAMPK\u0026alpha;1\u003csup\u003eSer485\u003c/sup\u003e/AMPK\u0026alpha;2\u003csup\u003eSer491\u003c/sup\u003e (#4185, RRID:AB_2169402), anti AMPK\u0026alpha; (#2532, RRID:AB_330331) (all Cell Signalling Technology), Anti-IP6K1 antibody (ab129595, RRID:AB_11157733) and recombinant anti-S6K1 (ab32359, RRID:AB_777802) (Abcam). Signals observed after the meal was normalised to the respective participant\u0026apos;s average baseline signal.\u003c/p\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003ch2\u003eRNA extraction from subcutaneous adipose tissue\u003c/h2\u003e\n \u003cp\u003eFat tissue biopsies were performed as previously described (\u003cspan class=\"CitationRef\"\u003e14\u003c/span\u003e). Total RNA was extracted from subcutaneous fat tissue with Lipid Tissue RNA-Kit\u0026trade; (Qiagen, Germany) according to the manufacturer\u0026rsquo;s protocol. RNA was reverse transcribed into cDNA using the High-Capacity cDNA Reverse Transcription Kit\u0026trade; with random priming (Applied Biosystems, Germany). cDNA was labelled by Power SYBR Green\u0026trade; master mix (Applied Biosystems, Germany) and analyzed in an ABI Prism 7900HT Sequence Detector (Applied Biosystems, Germany). For quantification the standard curve method was applied. Target genes were normalized to relative expression levels of ribosomal protein large protein 0 (RPLP0) which is an established housekeeping gene in adipose tissue. Fold changes were calculated from the ratio of means of the normalized quantities and their statistical significance was determined by unpaired Student\u0026rsquo;s t-test.\u003c/p\u003e\n \u003cp\u003eStatistical analyses\u003c/p\u003e\n \u003cp\u003eStatistical analyses were carried out using IBM SPSS Statistics (version 28.0.1.1.). In the acute study, differences between the conditions in muscle protein content were evaluated using ANOVA, with Bonferroni correction applied to account for multiple comparisons., or two-tailed Student\u0026rsquo;s T-test for paired sample analyses. Data from the medium-term study were analysed using a two-way repeated measures ANOVA (2\u0026times;3, group by time) to assess differences between baseline, 6 weeks, and 18 weeks with Bonferroni correction applied for post-hoc pairwise comparisons. Data are expressed as mean (SEM), unless otherwise specified. Statistical significance was accepted at P\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eAcute study\u003c/h2\u003e \u003cp\u003eTwo compartment modelling of the IVGTTs showed no significant differences between meals (MPDAF, MPD and HPD) for insulin sensitivity (S\u003csub\u003eI\u003c/sub\u003e\u003csup\u003e2*\u003c/sup\u003e) (p\u0026thinsp;=\u0026thinsp;0.14), glucose effectiveness (S\u003csub\u003eG\u003c/sub\u003e\u003csup\u003e2*\u003c/sup\u003e) (p\u0026thinsp;=\u0026thinsp;0.11) or HGP (p\u0026thinsp;=\u0026thinsp;0.88) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFrom plasma analysis and further calculations, this study found no statistical differences between the 3 conditions (HPD, MPD and MPDAF) for iAUC\u003csub\u003eglu\u003c/sub\u003e (p\u0026thinsp;=\u0026thinsp;0.72), iAUC\u003csub\u003eins\u003c/sub\u003e (p\u0026thinsp;=\u0026thinsp;0.01), AIRg (p\u0026thinsp;=\u0026thinsp;0.82), 2nd Phase Insulin Secretion (p\u0026thinsp;=\u0026thinsp;0.11), Disposition index (DI) (p\u0026thinsp;=\u0026thinsp;0.24) and DI 2nd Phase Insulin Secretion (p\u0026thinsp;=\u0026thinsp;0.08) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Plasma amino acids data for the IVGTT study (\u003cb\u003eSupplementary Figs.\u0026nbsp;2 and 3)\u003c/b\u003e show a time-dependant increase in some BCAA. In the HPD trial, valine concentrations increased significantly from baseline at 60 minutes post-meal and remained elevated up to 4 hours. Similarly, in the MPD trial, valine concentrations increased from baseline between 60- and 120-minutes post meal ingestion, whereas no significant changes were observed in the MPDAF trial. In addition, at 180-minute post meal, valine\u0026rsquo;s concentration was significantly higher in the HPD trial compared to MPD and MPDAF (p\u0026thinsp;=\u0026thinsp;0.032 and 0.012 respectively) with difference between HPD and MPDAF persisting at 240-minutes (p\u0026thinsp;=\u0026thinsp;0.029).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eLeucine concentrations increased significantly from baseline in HPD between 60 and 180 minutes, while in MPD, increases were noted only at 60 and 80 minutes, with no significant changes in MPDAF. At 180 minutes, leucine was higher in HPD than MPDAF (p\u0026thinsp;=\u0026thinsp;0.015) and MPD (p\u0026thinsp;=\u0026thinsp;0.031).\u003c/p\u003e \u003cp\u003eIsoleucine concentrations remained unchanged post-meal across all trials. However, total BCAA concentrations were significantly elevated from baseline between 80 and 180 minutes in HPD and from 60 to 80 minutes in MPD, with no changes in MPDAF. Total BCAA levels in HPD were significantly higher than MPDAF at 180 minutes (p\u0026thinsp;=\u0026thinsp;0.035).\u003c/p\u003e \u003cp\u003eFrom western blot analysis, protein content of IP6K1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA) decreased in the 4 hours following the HPD when compared with baseline (p\u0026thinsp;=\u0026thinsp;0.048), with no significant differences in the MPD (p\u0026thinsp;=\u0026thinsp;0.89), nor in the MPDAF (p\u0026thinsp;=\u0026thinsp;0.33) for the same comparison (i.e. against averaged baselines within trial). pAkt\u003csup\u003eSer308\u003c/sup\u003e/Akt2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF) increased from baseline to the 4-hour sample in the HPD trial (p\u0026thinsp;=\u0026thinsp;0.046). Finally, AMPK (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eJ) decreased from baseline in the HPD trial (p\u0026thinsp;=\u0026thinsp;0.01), with no significant differences between the groups. No significant differences were observed between the conditions for Akt2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB), pAkt\u003csup\u003eSer473\u003c/sup\u003e(Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC), pAkt\u003csup\u003eThr308\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD), pAkt\u003csup\u003eSer473\u003c/sup\u003e/total Akt2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE), total S6K1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG), pS6K1\u003csup\u003eThr389\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH), pS6K1\u003csup\u003eThr389\u003c/sup\u003e/total S6K1(Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eI), pAMPK\u003csub\u003eα1\u003c/sub\u003e\u003csup\u003eSer485\u003c/sup\u003e/AMPK\u003csub\u003eα2\u003c/sub\u003e\u003csup\u003eSer491\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eK), (pAMPK\u003csub\u003eα1\u003c/sub\u003e\u003csup\u003eSer485\u003c/sup\u003e/AMPK\u003csub\u003eα2\u003c/sub\u003e\u003csup\u003eSer491\u003c/sup\u003e)/ total AMPK (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eL).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eMedium term study\u003c/h2\u003e \u003cp\u003eThe medium-term study revealed a significant difference in the mRNA levels of S6KI between HP and control diet at 6 weeks (p\u0026thinsp;=\u0026thinsp;0.046), which was not witnessed at 18 weeks for the same group comparison (p\u0026thinsp;=\u0026thinsp;0.80). No significant differences were noted for mRNA expression for IRS-1, mTOR, 4E-BP, PPARγ, Akt, fatty acid synthase, hormone-sensitive lipase, or ATGL between baseline, 6 weeks and 18 weeks following both the control diet (CD) and high-protein diet (HPD) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e\u003cb\u003e).\u003c/b\u003e There was a significant difference observed for intrahepatic lipid content (IHL), which was significantly lower in the HP diet group at 6 weeks compared to CD (p\u0026thinsp;=\u0026thinsp;0.03). The same comparison at 18 weeks showed a trend towards significance (p\u0026thinsp;=\u0026thinsp;0.052) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eJ). In addition, no changes in insulin sensitivity between groups at 6 or 18 weeks were seen, as measured using the EHC (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). In the medium-term study, all diets were designed to be isoenergetic and there was no change in body weight in any of the dietary groups between baseline (week 0) and later time points including week 6 time point (p\u0026thinsp;=\u0026thinsp;0.42). Dietary amino acid signature data for the medium-term study have been previously published by Hattersley and colleagues (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eOur acute study investigated the effects of different protein doses on the cellular mechanisms that are central in the regulation of insulin-stimulated glucose uptake and protein synthesis in skeletal muscle in individuals with obesity. For the first time in humans, this study employed the two-compartment IVGTT to assess if the different meals affected whole body insulin sensitivity. Despite variations in protein intake, the current data set showed no statistically significant differences for insulin sensitivity, glucose effectiveness, or HGP between conditions (HPD, MPD, and MPDAF). These findings are, of course, following a short-term dietary manipulation. We acknowledge that the use of the labelled IVGTT resulted in our data representing insulin secretion in response to both protein ingestion and iv glucose. However, as the IVGTT glucose load remained fixed for each volunteer and for each of their trials, we were able to isolate the insulin response to the protein meal.\u003c/p\u003e \u003cp\u003eWe have included additional analyses of a similar dietary intervention yet over an 18-week period (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). Employing an euglycemic hyperinsulinemic clamp (EHC), this data performed in a subgroup of participants with available adipose tissue biopsies showed no differences between high protein and control diets on whole-body insulin sensitivity. We hypothesized that the higher protein ingestion would result in hyperaminoacidemia, leading to an increase in phosphorylation of S6K1(\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e) and a subsequent decrease in insulin signaling at the molecular level. While we did observe an increase in amino acid concentration in the HPD trial in the acute study (Supplemental 2 \u0026amp; 3sh), there were no observed differences in muscle pS6K1\u003csup\u003eThr389\u003c/sup\u003e for the same comparisons. In addition, S6K1 mRNA was significantly lower in the high protein compared to control, as measured in adipose tissue, at 6 weeks.\u003c/p\u003e \u003cp\u003eTremblay and colleagues used an EHC coupled with amino acid infusions leading to an approximately 2.5-fold increase in blood amino acid concentration and a 3.7-fold elevation in skeletal muscle S6K1 protein content (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). Building upon these insights, the present studies sought to examine the alterations in muscle S6K1 protein content four hours following the consumption of different sized protein meals, and over 18 weeks of high protein diet, hereby providing insights into the physiological dynamics of S6K1 postprandially. No significant changes were observed in either the total S6K1 protein content or its phosphorylated state at Thr\u003csup\u003e389\u003c/sup\u003e during the acute study for any of the conditions, indicating that the influence of plasma amino acid levels on S6K1 may be notably attenuated compared to infusion studies. However, the medium-term study showed a decrease in S6K1 mRNA expression in adipose tissue over the strictly controlled 6-week period with HPD when compared to an isoenergetic control diet (p\u0026thinsp;=\u0026thinsp;0.046), whereas after 18 weeks, no significant differences were seen, which is in agreement with the findings observed in the entire cohort(\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e) and likely related to an observed drop in the adherence to the HPD in the medium-term; as was supported by measurement of biomarkers of protein intake and entirely expected in medium -term dietary interventions in humans that does not include a fully controlled feeding intervention. These findings may suggest that medium-term high protein feeding is required to manipulate S6K1 and that this response may be tissue specific. Nevertheless, researchers noted an increased pS6K1 at Thr\u003csup\u003e389\u003c/sup\u003e in skeletal muscle induced by elevated insulin, as seen when performing EHC studies (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). The conflicting results regarding this signaling kinase may be attributed to variations in insulin response between studies. In our current dataset, no significant changes in insulin response were observed, which could explain the absence of differences in this signaling kinase between trials.\u003c/p\u003e \u003cp\u003eIt is worth noting that in the published work of Weickert (2011) there was a significant worsening in insulin sensitivity after 6 weeks of a HPD in a larger sample size [n\u0026thinsp;=\u0026thinsp;23; 4.20 +/- 0.38 (0 week) to 3.71 +/- 0.36 mg \u0026middot; kg \u0026middot; min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (6 week), p\u0026thinsp;=\u0026thinsp;0.13]. The effect observed in the sample with available gene expression data used in this study (Control n\u0026thinsp;=\u0026thinsp;6; HPD n\u0026thinsp;=\u0026thinsp;7) agrees with observations in obese subjects with moderate type 2 diabetes (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). These authors showed a similar decrease of hepatic fat on high protein diets after 6 weeks by about 40%, and no changes of fasting amino acid levels or of the insulin signaling pathway (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWe also observed a decrease in muscle IP6K1 protein content in the acute study in the HPD trial when compared to baseline fasting samples. These results suggest that acutely, 4-hour post meal ingestion, IP6K1 content was downregulated by dietary protein intake only after a certain intake threshold, as no differences were seen in the MPD trial, or when energy intake was matched in the MPDAF. The regulation of IP6K1 in skeletal muscle is of interest due to its role in the modulation of glucose metabolism and insulin signaling(\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan additionalcitationids=\"CR33 CR34\" citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e). An inhibition or genetic deletion of IP6K1 was shown to protect mice from hepatic steatosis by improving mitochondrial function and reducing gluconeogenic pathways while reducing lipolysis in adipose tissue (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). The inhibition of IP6K1 positively regulates numerous metabolic pathways and may therefore explain the positive effects of high protein diets observed in humans with metabolic dysfunction (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eOur acute data set also showed an increase in Akt2 activity, in agreement with previous publications (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan additionalcitationids=\"CR38\" citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e). The heightened availability of BCAAs, particularly leucine, subsequent to high-protein meals, is acknowledged for its potential to activate mTOR and its downstream targets, including S6K1. In some \u003cem\u003ein vitro\u003c/em\u003e studies, such activation has been linked to reduced insulin action on Akt, thereby impairing insulin signaling (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e); and supported by previous findings where amino acid infusion caused a rise in IR and S6K1 activity (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). However, over the 18-week study, we did not observe significant changes in adipose tissue total Akt and pAkt, or Akt mRNA. Although we noted significantly elevated levels of BCAA acutely, over the 4 hours post meal, following the HPD trial, which outlasted those seen at lower doses of protein (MPDAF and MPD), substantial alterations in S6K1 were not seen acutely. Consequently, distinctly from the intravenous infusions studies where the digestion system is circumvented and unphysiologically high levels of AA are achieved (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e), in the meal-induced context, the increased in pAkt\u003csup\u003eThr308\u003c/sup\u003e/Akt2 seen following the HPD meal could potentially be attributed to IP6K1 modulation rather than S6K1 activation, although the relatively small sample size in our studies is acknowledged. Additionally, our results may have missed important changes in insulin signaling owing to the time of the second muscle biopsy. The changes in IP6K1 muscle content are supported by a previous study that also noted Akt activation in response to high-protein intake correlated with change in IP6K1 content (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFurthermore, we had previously published effects of isoenergetic HP vs control diet with comparable dietary fat contents on intrahepatic lipid (IHL) over an up to 18-week period (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). In the here analyzed subset of participants with available gene expression data, we observed a significant decrease in IHL in the HP group compared to the control diet after 6 weeks point, with a trend still observed after 18 weeks. These findings are consistent with the work of (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e), who reported a significant 42.6% reduction in IHL among morbidly obese individuals following a calorie-restricted, isocaloric HPD, compared to a low-protein diet and upon intake of isocaloric high protein diets in patients with Type 2 Diabetes (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). Further, in the published data from the larger cohort (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e), the drop in IHL from baseline following 6 weeks of isoenergetic HPD vs control diet happened despite unchanged body mass in all dietary groups, thereby excluding any weight-change related effects of the diets.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn conclusion, our findings contribute explaining the potential of high protein meals as a dietary intervention for enhancing metabolic health and glycemic control. While none of the meals tested (MPD, MPDAF, or HPD) induced significant alterations in insulin sensitivity or glucose effectiveness using a labelled IVGTT, the consumption of a high-protein meal (containing 100 grams of protein) resulted in a notable reduction in muscle IP6K1 levels and an increase in Akt activity, suggesting that protein consumption can improve insulin signaling, in an acute context.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eData availability\u003c/p\u003e\n\u003cp\u003eThe datasets generated and/or analysed during the current study are not publicly available due to privacy restrictions and ongoing analyses but are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Grants\u003c/p\u003e\n\u003cp\u003eRM and OA were supported by the Health Innovation Network, London. MOW and AFHP were supported by grants from the German Ministry of Education and Science (BMBF, 0313826A, and 0313826B), the German Institute of Human Nutrition (Potsdam-Rehbruecke), Charit\u0026eacute; University Medicine Berlin, scientific collaborators and regional companies (Rettenmayr Inc, Anona Inc, and Kathi Inc, Germany), which included the provision of raw materials for the dietary supplements\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eDisclosures\u003c/p\u003e\n\u003cp\u003eNone of the authors reported any financial disclosures that were related to the study. None of the funding organizations or sponsors played any role in the design and conduct of the study; in the collection, analyses, and interpretation of the data; or in the preparation, review, or approval of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Acknowledgements\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eConceived and designed research: OA, RM, AHE, NB, AP, MW. Performed experiments: OA, RM, FD, DN, NB, AP, MW. Analysed data: OA, RM, FD, DN, AP, MW. Interpreted results of experiments: OA, RM, FD, DN, NB, AP, MW. Prepared figures: OA, RM, AP, MW. Drafted manuscript, edited and revised manuscript, approved final version of manuscript: OA, RM, AHE, FD, DN, NB, RR, AP, MW. RM is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAncu O, Mickute M, Guess ND, Hurren NM, Burd NA, Mackenzie RW. Does high dietary protein intake contribute to the increased risk of developing prediabetes and type 2 diabetes? Vol. 46, Applied Physiology, Nutrition and Metabolism. 2021. \u003c/li\u003e\n\u003cli\u003eLynch CJ, Adams SH. Branched-chain amino acids in metabolic signalling and insulin resistance. 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Mol Cell. 2010;38(5). \u003c/li\u003e\n\u003cli\u003eKim J, Dar\u0026egrave; E, Rajasekaran SS, Ryu SH, Berggren PO, Barker CJ. Inositol pyrophosphates and Akt/PKB: Is the pancreatic \u0026beta;-cell the exception to the rule? Cell Signal. 2019;58. \u003c/li\u003e\n\u003cli\u003eXu C, Markova M, Seebeck N, Loft A, Hornemann S, Gantert T, et al. High-protein diet more effectively reduces hepatic fat than low-protein diet despite lower autophagy and FGF21 levels. Liver International. 2020;40(12). \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Diabetes, High protein, Insulin resistance, Obesity","lastPublishedDoi":"10.21203/rs.3.rs-6555791/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6555791/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eDiet is critical in type 2 diabetes management, with high-protein diets (HPD), rich in branched-chain amino acids (BCAAs), proposed to enhance glycemic control, but overactivation of S6K1-related signaling pathway may contribute to IR pathogenesis by impairing insulin-stimulated glucose uptake in skeletal muscle.\u003c/p\u003e \u003cp\u003eThe study objective was to investigate the impact of varying dietary protein interventions on insulin sensitivity (IS) and molecular signaling in skeletal muscle in overweight or obese individuals, both acutely and over an up to 18-weeks period.\u003c/p\u003e \u003cp\u003eThe analysis performed in the 18-week data set used a subset of data with available adipose tissue biopsies randomised, controlled, isoenergetic dietary intervention, focusing on the here relevant HPD and control diets. IS was assessed using labelled intravenous glucose tolerance tests (IVGTT) in the acute study, while the euglycemic-hyperinsulinemic clamp (EHC) was used in the 18-week intervention.\u003c/p\u003e \u003cp\u003eKey protein implicated in insulin signalling, such as IP6K1 and total AMPK protein content significantly decreased following the HP meal, alongside an increased p-AktThr\u003csup\u003e308\u003c/sup\u003e/Akt2 activity while S6K1 mRNA was lower after 6 weeks of HPD, compared to the control diet group, but not at 18 weeks. However, these differences at the molecular level did not translate into significant changes in whole-body IS.\u003c/p\u003e","manuscriptTitle":"High protein ingestion does not affect whole-body insulin sensitivity","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-30 14:47:34","doi":"10.21203/rs.3.rs-6555791/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"eafb6efa-44e6-45e8-848a-4a057ab76569","owner":[],"postedDate":"May 30th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":49198283,"name":"Health sciences/Endocrinology"},{"id":49198284,"name":"Health sciences/Molecular medicine"}],"tags":[],"updatedAt":"2025-07-16T17:08:29+00:00","versionOfRecord":[],"versionCreatedAt":"2025-05-30 14:47:34","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6555791","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6555791","identity":"rs-6555791","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}