High dietary protein intake, reducing or eliciting insulin resistance?

Abstract

Dietary proteins have an insulinotropic effect and thus promote insulin secretion, which indeed leads to enhanced glucose clearance from the blood. In the long term, however, a high dietary protein intake is associated with an increased risk of type 2 diabetes. Moreover, branched-chain amino acids (BCAA), a prominent group of amino acids, were recently identified to be associated with diabetes. Observational data and intervention studies do not point in the same direction regarding the effect of protein intake on insulin sensitivity and diabetes risk. Therefore, the first aim of this review will be to discuss human studies addressing high dietary protein intake and insulin action, with special attention for BCAA. In the second part, we will highlight the (patho) physiological consequences of high-protein diets regarding insulin action, in particular the role of the mechanistic target of the rapamycin pathway.

Introduction

Insulin resistance is defined as tissues no longer being sensitive to the physiological actions of insulin, in particular glucose uptake. The insulin-mediated uptake of glucose can decrease when tissues are chronically overexposed to high levels of insulin. Thus, prolonged hyperinsulinaemia can lead to insulin resistance and eventually type 2 diabetes mellitus.1 Lifestyle factors such as physical activity and diet have a key role in the development of insulin resistance. Many different diets for weight reduction and improvement of insulin sensitivity are advocated. Diets high in protein content and low in carbohydrates, such as the Atkins diet or Zone diet, have demonstrated a positive effect on body composition and body weight. However, the effects of those high-protein diets on insulin sensitivity are somewhat controversial. In the beginning of the 20th century, Jacobsen2 identified a high-protein diet as an insulin sensitivity improving strategy. Dietary proteins have an insulinotropic effect and thus promote insulin secretion, which indeed leads to enhanced glucose clearance from the blood.3 In the long term, however, a high dietary protein intake has been associated with an increased risk of type 2 diabetes.4, 5, 6, 7 Moreover, branched-chain amino acids (BCAA), a prominent group of amino acids, and a significant part of dietary protein, were recently strongly suspected to be associated with diabetes.8,9 Observational data and intervention studies do not point in the same direction regarding the effect of protein intake on insulin sensitivity and diabetes risk. Therefore, the aim of this review was first to discuss human studies addressing high dietary protein intake and insulin action, with special attention for BCAA. In the second part, we wanted to highlight the (patho)physiological consequences of high-protein diets regarding insulin action, and in particular the role of the mechanistic target of the rapamycin (mTOR) pathway.

Methods: Selection of relevant studies

There are many human studies available on high protein intake and insulin sensitivity. However, comparing these studies is difficult. This is mainly due to differences in the control diet used, duration of the study, energy balance, that is, with or without weight loss, the source and amount of protein used and differences in the type of subjects included: non-obese, overweight, non-diabetic and diabetic subjects. Comparing studies can be made easier when studies are organized by those factors. For the first part of this overview article, recent—year 2000 and onwards—human intervention studies were selected in which a dietary protein content of >20 energy percentage (En%) was used and which measured insulin sensitivity. The present review aims to give a broad overview on the subject, but it does not intend to be a complete systematic review. Furthermore, for a better comparison, studies were arranged by the duration of the intervention: short term (less than 6 months of intervention) versus long term (more than 6 months of intervention, and including observational studies). Within these topics, a distinction was made between the type of subjects prescribed the diets, followed by interventions with weight loss. For all studies mentioned, the amount of protein is expressed as En% considering that a high-protein diet refers to a protein content of >20 En%. For a summary of the studies cited in this overview article on the effect of high-protein diets on insulin action in humans, see Table 1.

Table 1 Overview of the effects of high-protein diets on insulin action in human studies

High dietary protein diets and insulin action

Short-term, energy balanced, high-protein diets

In healthy non-obese volunteers, data on the short-term consequences of manipulating protein intake on insulin action are limited and showing only minor effects. When healthy subjects were fed a high-fat diet (39.4 En%), high in protein content (25.7 En%), for 2 weeks, this did not have any effect on insulin and glucose homeostasis when compared with a normal protein (15.4 En%) high-fat diet (37.7 En%).10 Similarly, in a young and an old group of healthy subjects, a high-protein diet did not affect insulin sensitivity, and the acute insulin response to glucose also did not vary between age group or between diets. Here, 10 days of a high-protein diet (21 En% in the young group and 24 En% protein in the old group) was compared with a normal protein diet (11 En% in the young group and 12 En% protein in the old group). Furthermore, the fat content differed between diets, whereas the carbohydrate content was kept constant at 50 En%.11 In the same way, two months of a high-protein diet (29 En%) did not change insulin action and secretion compared with a normal protein diet (16 En%) with a similar fat content (30 En%) in healthy subjects.12 However, an acute high-protein diet (35 En%, 1 week), with very little carbohydrates (5 En%), caused an instant postprandial reduction in daily plasma insulin.13

In overweight and obese subjects, the short-term effect of increasing protein, without weight loss, is somewhat more diverse. Increasing dietary protein to 35 En% for 12 weeks with a whey supplement, compared with subjects supplemented with glucose (16 En% protein), resulted in improved insulin sensitivity in overweight subjects; carbohydrates were exchanged for protein, whereas fat intake was kept constant (29 En%).14 Yet, in a similar population, no effect on insulin sensitivity was seen after six weeks of a diet restricted in carbohydrates (13 En%), which were replaced by proteins (29 En%). However, although carbohydrates were replaced by protein, the fat (and consequently the energy) content of the test diets was also increased. Thus, the fact that insulin sensitivity did not change could be due to the increased fat content of the diet and could have less to do with the exchange of protein for carbohydrate.15

Similarly, after a period of initial weight loss, a low-fat diet (24 En%) supplemented with either casein or whey (35 En% protein) did not alter insulin sensitivity compared with a high-carbohydrate diet (16 En% protein, 63 En% carbohydrate).16 However, Weickert et al.17 demonstrated reduced insulin sensitivity after 6 weeks on a high-protein (25–30 En%), reduced-carbohydrate (40–45 En%) diet. This diet contained high amounts of legumes and dairy products, and was compared with a high-fiber diet (protein 15 En%, carbohydrate 55 En%). Yet, the observed effect weakened after 18 weeks.17 In type 2 diabetic patients, 5 weeks on a high-protein (30 En%), low-carbohydrate (20 En%) diet without weight loss improved insulin sensitivity compared with a normal protein (15 En%), normal carbohydrate (55 En%) diet. No differences were observed between the two diets in fasting insulin levels.18,19 The same high-protein diet did result in a decrease in both postprandial glucose and in the overnight fasting glucose concentration.20

Therefore, in short-term studies without weight loss in healthy, as well as in overweight and type 2 diabetic subjects, effects of high-protein diets on insulin sensitivity are inconclusive. In addition, when dietary protein is increased by decreasing the carbohydrate content of the diet, it is difficult to determine which is responsible for any effects on insulin sensitivity.

Short-term, energy-restricted, high-protein diets

In overweight, obese and type 2 diabetic subjects, the majority of studies using high-protein energy-restricted diets have focused on weight loss. Weight loss, however, is known to have a strong beneficial influence on insulin sensitivity.21 Improved insulin resistance was observed when a high-protein (27 En%) energy-restricted diet at the same time contained a low amount of carbohydrates (17 En%) compared with the baseline diet (protein 18 En%, carbohydrates 42 En%).22 Eight weeks of a high-protein (30 En%), reduced-carbohydrate (33 En%) energy-restricted weight-loss diet improved insulin sensitivity, compared with other weight loss diets (protein 19 En%, carbohydrate 51 En%), which were either high in fatty fish and legumes, or a balanced control diet.23 The same was observed after a 10-week energy-restricted diet relatively high in both fiber and protein (24 En%; carbohydrate 45 En%) compared with standard dietary advice (protein 19 En%; carbohydrate 46 En%).24 However, Rizkalla et al.25 showed that HOMA-IR improved more on a conventional energy-restricted weight-loss diet (21 En% protein, 44 En% carbohydrate), whereas B-cell function improved more after the high-protein weight-loss diet (33 En% protein, 40 En% carbohydrate). In overweight subjects, a high-protein (30 En%) very low carbohydrate (4 En%) energy-restricted diet did not offer any metabolic advantage on insulin sensitivity over a moderate-carbohydrate weight-loss diet.26,27 In addition, in studies considering obese type 2 diabetic subjects and aiming at weight loss, high-protein energy-restricted diets with carbohydrates exchanged for proteins showed no additional effect on insulin sensitivity above energy restriction.28, 29, 30 Sargrad et al.31 even found a high-carbohydrate (51 En%; protein 19 En%) energy-restricted diet to be superior to a high-protein diet (27 En%; carbohydrate 43 En%) in terms of improving glycaemic control and insulin resistance.

It might be concluded that the improved insulin sensitivity with high-protein energy-restricted diets in overweight, obese and type 2 diabetic subjects is, at least partly, dependent on weight loss. This was also confirmed by a recent meta-analysis considering high-protein energy-restricted diets, concluding that there is no effect of protein intake on glucose homeostasis; this effect was not adjusted for the extra weight loss of 0.79 kg in the high-protein diets compared with the standard protein diets.32

Long-term protein intake

In healthy subjects, fed for 6 month in energy balance, a diet high in protein (24 En%) compared with a normal protein diet (10 En%) induced a state of higher insulin resistance and glucose intolerance.33 Thus, in this study, a long-term consumption of a high-protein diet in healthy subjects seems to decrease insulin sensitivity. Moreover, in observational studies, long-term high dietary protein intake is associated with an increased risk of developing metabolic syndrome or diabetes type 2.4, 5, 6 However, the nurses’ health study observed that diets lower in carbohydrate and higher in protein and fat were not associated with an increased risk for diabetes type 2.34 Even a slight reduction of the risk was observed when vegetable sources of protein and fat were chosen. It was suggested that reducing the glycaemic load of a diet was the beneficial underlying factor of the reduced diabetes risk.34

Recently, attention was attracted by several studies pointing to an association between plasma BCAA levels and an increased risk of insulin resistance and/or type 2 diabetes.8,9,35,36 The three BCAA are valine, leucine and isoleucine, which are all considered essential amino acids. The BCAA content of mixed protein sources is about 20%.37 Higher levels of BCAA can be found in whey protein and to a lower extent in cod protein,14,37,38 with leucine being the most abundant. Of the dietary BCAA ingested, about 80% reaches the blood circulation.37,39,40 High protein intake or the intake of high levels of BCAA as supplements increases blood plasma concentrations of BCAA at least in shorter-term dietary interventions.11,41,42 Newgard et al.9 found a linear relationship between increased levels of plasma BCAA and decreased insulin sensitivity, as measured by an increase of the HOMA index. Levels of plasma BCAA were higher in obese as compared with lean Caucasian and African-American subjects.9 Similar results were found in an all Asian population, where increased levels of BCAA were observed in subjects diagnosed with insulin resistance by a high HOMA index.35 This inverse relationship between insulin sensitivity and BCAA levels, however, does not prove cause–effect relationships, as, BCAA levels may be high just because of insulin resistance. Since, tissues are no longer sensitive for the lowering effect of insulin on proteolysis leading to increased levels of circulating BCAA. Conversely, reducing body weight improves insulin sensitivity, thereby decreasing proteolysis resulting in decreased levels of circulating BCAA.

It was suggested that this relationship might be due to increased protein turnover and/or decreased rates of BCAA catabolism; increased BCAA levels may also be indicative of sarcopaenia.35 The consequence of weight loss on insulin resistance could even be predicted by decreased circulating BCAA levels.43 Thus, plasma BCAA, which also represents BCAA catabolism, was correlated with insulin resistance, with low BCAA concentrations predicting improved insulin sensitivity when subjects had moderately lost weight.35 Furthermore, in longitudinal studies, it has been shown that elevated plasma levels of BCAA and aromatic amino acids were good predictors of future development of type 2 diabetes.8,36 It has been suggested that hyperaminoacidaemia could promote diabetes via hyperinsulinaemia, leading to pancreatic beta cell exhaustion.8 On the other hand, another study demonstrated that peripheral insulin response was improved after weight loss in insulin-resistant offspring, yet this response was independent of plasma BCAA.44 Wurtz et al.45 also found no association between HOMA-IR and fasting amino-acid levels, indicating that amino acids do not have a role in the pathogenesis of insulin resistance.45 Besides, whether high blood concentrations of BCAA could be a cause or consequence of insulin resistance remains unclear.46 A recent meta analyses – which included 15 randomized controlled trials of more than 12 months – on the long-term effect of diets high in protein (>25 En% of protein) showed neither a positive nor a negative effect on glycaemic control compared with diets low in protein content (10–15 En%) in both healthy and insulin-resistant subjects.47

In conclusion, studies considering high-protein diets and insulin action are not univocal. A beneficial effect on insulin sensitivity with high-protein diets is mainly observed in overweight and insulin-resistant subjects, when weight loss is present. Next to weight loss, these effects are most frequently explained by the insulinotropic properties of amino acids or the decreased glycaemic load of the high-protein diets. In healthy subjects, short-term manipulation of protein intake did not affect insulin action, whereas a high-protein diet seems to be deleterious when the intake is prolonged. This is in line with the observed association between plasma BCAA levels, insulin resistance and diabetes risk. In the long term, increased insulin secretion and consequent hyperinsulinaemia might lead to reduced hepatic insulin sensitivity. Increased hepatic glucose output results in a decreased glucose control, although a direct effect in insulin action in insulin-sensitive tissue can also have a role.

Physiological pathways linking protein intake and insulin action

In the second part of this review, first the insulinotropic effect of dietary proteins will be discussed, thereafter the topic of BCAA and insulin resistance will be highlighted. Finally, the relationship of BCAA and insulin resistance will be linked via the mTOR pathway.

Insulinotropic effect of dietary proteins

It is well known that dietary proteins promote insulin secretion, which leads to enhanced glucose clearance from the blood by peripheral tissues.3 Many intervention studies have confirmed this effect and underscored that amino acids have an important role in mediating insulin and glucagon secretion.48,49 It has been shown that whey protein and its hydrolysates resulted in a direct postprandial increment of insulin levels, some to a larger extent than others.50, 51, 52 In lean healthy subjects, a single high-protein test meal (50 En% protein, 40 En% carbohydrate, 10 En% fat) with either whey, casein or soy protein lowered peak glycaemia significantly, as compared with a carbohydrate test meal (1.2 En% protein, 95.5 En% carbohydrate, 3.3 En% fat). Plasma insulin was significantly higher after a whey protein-rich meal compared with a high-glucose meal.50 Moreover, this higher insulin level was still present after 330 min when consuming whey, whereas this was not the case for glucose, casein and soy meals.50

Indeed, when conditions of elevated postprandial amino-acid levels were artificially created in healthy subjects, insulin and glucagon secretion were stimulated.48 However, elevation of plasma amino acids to postprandial levels also caused insulin resistance by direct inhibition of muscle glucose transport and/or glucose phosphorylation with subsequent reduction in rates of glycogen synthesis. Thus, on the other hand, amino acids might have a role in the modulation of peripheral insulin sensitivity and contribute to insulin resistance.53 The stimulating effect of protein on insulin might be a strategy in insulin-resistant subjects.54 However, it could be harmful in healthy subjects in the long run, owing to prolonged hyperinsulinaemia, which can lead to decreased insulin sensitivity.

BCAA and insulin resistance

In humans, high levels of plasma BCAA, which might partly be derived from dietary protein, are linked to insulin resistance and diabetes via insulin secretion and subsequent hyperinsulinaemia.8,9 Although high levels of BCAA can be found in whey protein,38 the relationship between diet, circulating BCAA and insulin resistance deserves further exploration. A cause–effect relationship is not established yet and it is still discussed whether plasma BCAA levels reflect long-term protein intake.35,45

When looking at the short-term effect of high protein intake, circulating BCAA increased by 25% after a 22 En% high-protein diet in young subjects without affecting insulin sensitivity.11 On the other hand, insulin secretion was increased when supplementing lean subjects with BCAA-rich whey protein, compared with supplementing with either egg, fish or turkey protein, but postprandial AA profiles were not measured in this study.55 In addition, a 4-week cod protein diet improved insulin sensitivity compared with an animal meat and milk protein diet with the same amount of protein; the lower concentration of BCAA in the cod, compared with the other animal protein source, was proposed to explain this observation, but this conclusion remains speculative as cod protein also has a relatively high BCAA content compared with usual mix protein diet (that is >20%).56 In the long term, 6 months of supplementation with leucine did not improve glycaemic control in elderly type 2 diabetic men.57 Similarly, no differences in glycaemic levels after increasing BCAA intake were observed in patients with chronic hepatitis C and insulin resistance.42 Here it was concluded that BCAA might even have a beneficial effect on glycated hemoglobin values in insulin-resistant subjects. Given the fact that plasma amino acids in the postprandial state also respond to insulin secretion with more sustained long-lasting changes than glucose levels, it was proposed that plasma BCAA might even serve as better indicators of impaired insulin sensitivity in prediabetic states than glucose levels.58

Increased BCAA can also be a consequence of insulin resistance. An important player in this light is BCKD (branched-chain α-ketoacid dehydrogenase), the rate-limiting enzyme in BCAA oxidative catabolism. BCKD activity is inhibited by insulin, and as a result, BCAA catabolism is inhibited.46,59 Elevated plasma-free fatty acid levels, commonly observed in insulin-resistant subjects, reduce BCKD activity as well.46 Thus, it might be suggested that raised blood BCAA in insulin resistance actually reflects reduced BCKD activity and is a consequence rather than a cause of insulin resistance.46 However, although in vitro data support this concept, in vivo studies are nonconsistent, as no decrease of leucine oxidation was observed with hyperinsulinaemia–euleucinaemia.60

Yet, in rats fed a high-fat BCAA diet, insulin resistance was induced accompanied by activation of the mTOR pathway.61 The mTOR pathway is a nutrient-sensing pathway, which integrates nutrient sensing and insulin signaling to coordinate cell growth and metabolism (Figure 1),62,63 and it could have a crucial role in understanding the association between BCAA and insulin action. BCAA and other nutrients activate mTOR. Among other signals, this activation can lead, in a negative feedback loop, to phosphorylation of the insulin receptor substrate 1 (IRS1), leading to decreased insulin sensitivity. This provides a potential link between BCAA and insulin resistance or diabetes risk.

Figure 1
figure1

Nutrient-sensing pathways of mTORC1. BCAA and glucose activate mTORC1, resulting in the induction of protein synthesis, mitochondrial biogenesis, energy storage and lipid synthesis. Fatty acid oxidation and activity of the IRS1 are inhibited. Insulin, growth factors and TNFα activate mTORC1 via the phosphorylation (P) and inhibition of the mTOR inhibitor TSC1/2 (tuberous sclerosis complex heterodimer). Rapamycin and a drop in energy inhibit mTORC1 action. Raptor, regulatory-associated protein of mTOR; Rheb, Ras homolog enriched in brain; AMP-activated protein kinase (AMPK), AMP-activated protein kinase; White boxes and solid flashes represents activation; black boxes and dotted lines represents inhibition.

The mTOR pathway

mTOR is a serine–threonine protein kinase widely expressed in various tissues and involved in many important cellular functions. It was first identified in yeast as a target of rapamycin.64 In mammals, two multiprotein complexes containing mTOR were identified.63 mTORC1 is composed of a number of proteins, which include mTOR itself (the catalytic subunit of the complex), RAPTOR (regulatory-associated protein of mTOR), mLST8 (mammalian lethal with Sec13 protein 8), PRAS40 (proline-rich AKT substrate 40 kDa) and Deptor (DEP-domain-containing mTOR interacting protein). Similarly, the mTORC2 complex is composed of mTOR, mLST8 and Deptor, present in mTORC1, but also contains Rictor (rapamycin-insensitive companion of mTOR), mSIN1 (mammalian stress-activated protein kinase interacting protein) and Protor-1 (protein observed with Rictor-1). In contrast to mTORC1, mTORC2 is insensitive to the mTOR inhibitor rapamycin.65

The regulation of the mTOR complexes is intricate and yet not fully understood. It is activated in the fed state initiating anabolism and energy storage.66 mTORC1 integrates signals from nutrients such as amino acids and glucose but seems to be more particularly sensitive to amino-acid signaling and especially leucine (Figure 1). It is suggested that leucine activates mTOR by translocating Rag proteins and their binding to Raptor.67 This leads to a relocation of the complex to a perinuclear region containing the activator Rheb. An endogenous suppressor of mTORC1 is the tuberous sclerosis complex heterodimer (TSC1/2), which, when active, inhibits Rheb.66,68 Endogenous signals such as growth factors (Insulin, IGF), hormones and TNF lead to an activation of mTORC1 by phosphorylating and thereby deactivating TSC1/2. In turn, in a negative feedback loop activation of mTORC1 causes phosphorylation of IRS1. This inhibits the association with the insulin receptor and thereby lowers insulin sensitivity. In the fasted state, mTOR activity is supressed. During energy depletion, AMP-activated protein kinase is phosphorylated and inactivates mTOR either directly or via activation of TSC1/2. mTORC1 is considered to be an important regulator of many cellular processes, especially cell growth and metabolism. It positively regulates protein and lipid synthesis, as well as mitochondrial biogenesis and metabolism.

MTOR pathway in the liver

The liver acts as a buffer for the peripheral availability of nutrients, most importantly in maintaining blood glucose levels. The mTOR pathway is suggested to have an important role in this regulatory metabolism. In the past years, several genetic engineered mice elucidated mTOR function in the liver. During fasting, liver mTORC1 is responsible for providing peripheral organs with ketone bodies as an energy source.69 Adaptation to a high-protein diet leads to increased mTOR phosphorylation and, consequently, its activation in the liver of rats.70 Overactivation of the mTOR pathway in turn was reported to cause higher sensitivity toward hepatic steatosis when fed a high-fat diet.71 Furthermore, it was associated with decreased hepatic insulin sensitivity.62 Leucine deprivation on the other hand lowered mTOR phosphorylation and improved insulin sensitivity.72 Loss of mTORC1 function in the liver enhanced glucose tolerance and increased insulin sensitivity.73 As a consequence of deregulated hepatic insulin signaling, mTORC1-dependent effects on lipid metabolism were reported.74 In primary hepatocytes, mTORC1 activation lead to the initiation of lipogenesis via SREBP1c,75 whereas inhibition by rapamycin increased fatty acid oxidation.76 Data in humans are scarce. However, it is suggested that post-transplantational side effects after treatment with rapamycin impairs lipid and glucose metabolism via mTORC1-dependent mechanisms.77

MTOR pathway in the muscle

In the muscle, mTORC1 mainly has a role in protein synthesis,78 and muscle-specific loss of function of mTORC1 by depletion of Raptor causes musclar dystrophy. This is not the case with loss of mTORC2 function.79 In addition, mTORC1 is lately also suspected to be involved in muscular insulin resistance78 as in glucose homeostasis. It was suggested that amino acids activate the mTOR pathway and thereby inhibit insulin-stimulated glucose uptake into skeletal muscle via phosphorylation of IRS1.78 This was demonstrated in vitro and could be reversed by treatment with the mTOR inhibitor rapamycin.80,81 Also in vivo, rats fed a high-fat BCAA diet developed insulin resistance accompanied by increased phosphorylation of mTOR in IRS1 in skeletal muscle.9 In humans, amino acid-induced insulin resistance in skeletal muscle was assessed during a hyperinsulinaemic clamp.80 In muscle biopsies, a combination of amino acid and insulin infusion strongly increased IRS1 phosphorylation, indicating inhibited insulin-dependent glucose uptake.80

Concluding remarks

It is possible that healthy individuals consuming a high-protein non-restricted diet, containing more than 20 En% of protein (that is, consuming more than the Population Reference Intake of 0.83 g protein/kg/day), can lead to hyperinsulinaemia and in the long term can cause insulin resistance. However, as a dietary strategy, high-protein non-energy-restricted diets, containing more than 20 En% of protein, might be helpful for obese people in reducing body weight and subsequently increasing insulin sensitivity also owing to the insulinotropic effect of dietary protein. Yet, the results on circulating BCAA levels in relation to insulin resistance should be further explored on the effect of diet on these metabolic biomarkers. It is still discussed whether there is a relationship between long-term protein intake and BCAA plasma levels and whether high BCAA levels are a cause or consequence of insulin resistance. One possible mechanism can be the activation of mTOR by nutrients (for example, BCAA) leading to a phosphorylation of the IRS1. This supresses IRS1 function and might lead to a decrease in insulin sensitivity. The role of mTOR activation by BCAA in insulin resistance is not clear yet. However, activation of this pathway should be more explored when looking at the effect of high-protein diets on insulin resistance.

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Acknowledgements

JS was supported by a Marie Curie European Reintegration Grant within the 7th European Community Framework Programme.

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Rietman, A., Schwarz, J., Tomé, D. et al. High dietary protein intake, reducing or eliciting insulin resistance?. Eur J Clin Nutr 68, 973–979 (2014). https://doi.org/10.1038/ejcn.2014.123

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