[advances in food and nutrition research] volume 70 || role of proteins in insulin secretion and...

CHAPTER ONE

Role of Proteins in InsulinSecretion and Glycemic ControlViren Ranawana1, Bhupinder KaurClinical Nutrition Research Centre, Singapore Institute for Clinical Sciences, Singapore, Singapore1Corresponding author: e-mail address: [email protected]

Contents

1.

AdvISShttp

Introduction

ances in Food and Nutrition Research, Volume 70 # 2013 Elsevier Inc.N 1043-4526 All rights reserved.://dx.doi.org/10.1016/B978-0-12-416555-7.00001-1

2
2. Blood Glucose Homeostasis and the Role of Insulin 3 3. Classification of Proteins and Protein Quality 6 4. Impact of Proteins and Amino Acids on Insulin Secretion 9
4.1
Effect of proteins on insulin secretion 9 4.2 Effect of amino acids on insulin secretion 12 4.3 Impact of proteins and amino acids
on glucagon secretion
21 5. Impact of Proteins and Amino Acids on Glycemia 22
5.1
Effect of coingesting proteins and carbohydrateson blood glucose and insulin 27
5.2
Effect of amino acids on glycemia 31 6. Conclusions 36 Acknowledgment 38 References 38
Abstract

Dietary proteins are essential for the life of all animals and humans at all stages of the lifecycle. They serve many structural and biochemical functions and have significant effectson health and wellbeing. Dietary protein consumption has shown an upward trend indeveloped countries in the past two decades primarily due to greater supply and afford-ability. Consumption is also on the rise in developing countries as affluence is increasing.Research shows that proteins have a notable impact on glucose homeostasis mecha-nisms, predominantly through their effects on insulin, incretins, gluconeogenesis, andgastric emptying. Since higher protein consumption and impaired glucose tolerancecan be commonly seen in the same population demographics, a thorough understand-ing of the former’s role in glucose homeostasis is crucial both toward the preventionand management of the latter. This chapter reviews the current state of the art on pro-teins, amino acids, and their effects on blood glucose and insulin secretion.

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http://dx.doi.org/10.1016/B978-0-12-416555-7.00001-1

2 Viren Ranawana and Bhupinder Kaur

1. INTRODUCTION

Proteins are a major dietary component essential for the survival of

animals and humans. It is the principle macronutrient involved in the struc-

ture, function, and biochemistry of the human body. They function as hor-

mones, enzymes, and transport factors, and are the major component in all

structural units of the body (muscle, tissue, organs, nails, etc.). Proteome

research has shown that the human body has the genetic capacity to synthe-

size around 21,000 different proteins (Pearson, 2008). Proteins are contin-

ually synthesized and broken down in the body and its rate depends on

factors such as health, physiological status, and stage of life. The rate of pro-

tein turnover, in turn, affects body size, protein mass, and protein require-

ments ( Jeor et al., 2001). Consuming adequate proteins to maintain

optimum levels in circulating pools is therefore essential for proper growth,

reparation, and metabolism.

Both European and North American guidelines recommend that proteins

constitute 10–15% of total daily energy intake (DH, 1991; Klein et al., 2004).

A similar amount is recommended in diabetic dietary guidelines (Rodbard

et al., 2007). Higher amounts of proteins may be consumed depending on

age, body size, disease, pregnancy, lactation, fitness, and environment

(Henley, Taylor, Obukosia, & Steve, 2010; Jeor et al., 2001). Protein intake

in high-income countries is typically above the recommended levels of 0.8 g/

kg body weight (WHO, 2007). Data from Europe and North America show

that average protein intake in these countries are 109 g/person/day and 91 g/

person/day, respectively (de Boer, Helms, & Aiking, 2006; Fulgoni, 2008).

Rising affluence is also raising protein consumption in developing countries

(Popkin, 2001). Therefore, there is a change in the global dietary pattern

where a greater percentage of energy is obtained from proteins.

Proteins have a notable role in the prevention and management of

chronic noncommunicable diseases. High-protein diets have been shown

to beneficially affect blood glucose (Gannon, Nuttall, Saeed, Jordan, &

Hoover, 2003), blood pressure, heart health and cardiovascular disease

(Appel, 2003; Erdman & Committee, 2000), and cholesterol levels

(Forsythe, Green, & Anderson, 1986). High-protein meals have also been

indicated to have a greater thermic effect and increase energy expenditure

(Acheson et al., 2011). Therefore, there is greater focus on proteins also from

a therapeutic perspective. Increased protein consumption has also been

shown to be beneficial in weight loss (Halton & Hu, 2004) and has thus

3Proteins in Glycemic Control

become the basis of popular weight loss regimes (Malik & Hu, 2007) which

are followed by many (Blackburn, Phillips, & Morreale, 2001).

The global trend toward increased protein consumption combined with

its suggested positive impact on health and weight control implies that

higher protein diets are and will become commonplace in most societies.

Protein consumption is directly correlated with affluence, and incidentally

it is in the same affluent demographic that the greatest incidence of diabetes,

obesity, and related diseases can be typically seen (Fall, 2001). Therefore,

gauging the impact of proteins on glucose metabolism is of paramount

importance. Proteins affect glucose regulation primarily through their effects

on insulin. Likewise, a large volume of research has shown that consuming

proteins in combination with carbohydrates significantly alters glucose

homeostasis mechanisms. This chapter reviews studies that have looked at

the effects of proteins on glycemia and insulin secretion. This chapter col-

lates research so far and provides an overview of the current state of the art.

2. BLOOD GLUCOSE HOMEOSTASIS ANDTHE ROLE OF INSULIN

The blood glucose concentration is a dynamic but finely regulated

entity in the human body. In healthy individuals, the postabsorptive/fasting

concentration is precisely maintained between 4 and 6 mM/L (van den

Berghe et al., 2009). Postprandial blood glucose levels may go up to around

10 mM/L after consumption of a carbohydrate-rich meal. Glucose is the

principle fuel for the brain and red blood cells and a constant supply in

the blood is therefore critically important.

Both hypo- and hyperglycemia have been shown to adversely affect

health and well-being (Davidson, 2004). Hyperglycemia over time may

induce toxic effects on b-cells and produce reactive oxygen species. Abnor-mally high blood glucose concentrations desensitize b-cells to glucose whichlater results in apoptosis and cessation of insulin secretion. Prolonged expo-

sure to abnormally high blood glucose levels may also initiate cellular dam-

age through lipotoxic effects. Hyperglycemia has also been shown to

accelerate the onset of cardiovascular complications in type 2 diabetes due

to oxidative stress (Ceriello, 2006). Therefore, optimum glycemic control

is important toward avoiding a cascade of events leading to increased

morbidity and mortality (Ceriello et al., 2004; Ohkubo et al., 1995).

The blood glucose concentration is regulated by balancing the rate of

digestion and intestinal absorption of dietary carbohydrates, rate of removal


and release of glucose by the liver, rate of uptake of glucose by peripheral

tissue, and rate of loss and synthesis by the kidney (Nordlie, Foster, &

Lange, 1999). The apparent blood glucose concentration is therefore depen-

dent upon all these factors (Fig. 1.1).

In the fasted and postabsorptive states, blood glucose homeostasis is

maintained by the regulation of hepatic and renal glucose production and

the amount of glucose taken up predominantly by noninsulin-dependent

tissue (nervous system, red blood cells, skin, smooth muscles, etc.)

(Cherrington, 1999). In the postprandial state, glucose homeostasis is

maintained by controlling the rate of glucose appearance from the digestive

system and the uptake by noninsulin-dependent tissue, peripheral tissue

(skeletal muscle and adipocytes), kidney, and the liver. In the postprandial

hyperglycemic state, hepatic production ceases and tissues exclusively utilize

glucose derived from food (Moore, Cherrington, &Wasserman, 2003). The

appearance of glucose in the blood is from either exogenous (digestion of

food and absorption) or endogenous (liver and kidney) sources (Corssmit,

Romijn, & Sauerwein, 2001; Stumvoll et al., 1995). The principle

Plasma glucoseconcentration

4–6 mmol/L

Red blood cells

Brain and nervous system

Liver

Energy

Glucagon/adrenal hormones

Insulin/incretins-

+ -

+

Storageandenergy

Disposal andenergy

Gluconeogenesis

Glycolysis andgluconeogenesis

Adipose tissue

Skeletal muscle

Kidney

Food intake

Digestion and absorption

Glucosefrom food

Kidney

Liver

Figure 1.1 A graphical representation of glucose homeostasis.


mechanisms by which the liver regulates blood glucose homeostasis are

glycogenesis (storage and uptake of glucose), glycogenolysis (synthesis of

glucose from glycogen), and gluconeogenesis (synthesis of glucose from

nonglycogen sources; Nordlie et al., 1999). One of the principle substrates

for gluconeogenesis is amino acids (Mallette, Exton, & Park, 1969) and

alanine, in particular, has been showed to be a preferred substrate (Felig,

Pozefsky, Marliss, & Cahill, 1970). The kidney also produces glucose pri-

marily via gluconeogenesis and appears to be as important as the liver in this

respect. Glutamine, lactate, and glycerol have been suggested to be impor-

tant renal gluconeogenic precursors (Stumvoll, Meyer, Mitrakou,

Nadkarni, & Gerich, 1997).

Glucose uptake in peripheral skeletal muscle and adipose sugar is facili-

tated through the action of insulin. Insulin stimulates the translocation of the

GLUT-4 glucose transporter to the muscle plasma membrane and facilitates

the diffusion of glucose into the muscle. Insulin plays a very central role in

glucose homeostasis. It is a small globular protein that is secreted from the

b-cells in the pancreatic islets of Langerhans. A small quantity is also pro-

duced in the brain (Gerozissis, 2008). The daily output of insulin by the pan-

creas is approximately 40–50 units (15–20% of pancreatic insulin stores;

Keim, Levin, & Havel, 2006) and is secreted in response to macronutrient

metabolites (Woods, Lutz, Geary, & Langhans, 2006) and incretin hor-

mones (de Graaf, Blom, Smeets, Stafleu, & Hendriks, 2004; Drucker &

Nauck, 2006). While carbohydrate is the most potent insulin secretagogue,

proteins have also been shown to stimulate it, although not fat (Teff &

Kapadia, 2008). The majority of glucose absorption in the postabsorptive

state occurs in noninsulin-dependent tissue while the bulk of the glucose

in the postprandial state occurs in the insulin-dependent muscles (Kelley

et al., 1988). Therefore, insulin plays an important role in maintaining

postprandial glucose homeostasis. Insulin also suppresses endogenous

glucose production (Moore et al., 2003). Conversely, glucagon which is

produced in the a-cells of the pancreas stimulates the conversion of glycogen

to glucose during hypoglycemic conditions. It further safeguards against

hypoglycemia by regulating gluconeogenesis (Cherrington, Lacy, &

Chiasson, 1978).

Although dietary proteins and amino acids appear to have little direct

effect on blood glucose (Nuttall & Gannon, 1991), they influence glucose

homeostasis through their effects on insulin and glucagon secretion. Proteins

and some amino acids, in particular, have been shown to stimulate the secre-

tion of both insulin and glucagon. They have also been shown to


differentially affect incretin secretion (Hall, Millward, Long, & Morgan,

2003). A large volume of research has conclusively shown the importance

of proteins in glucose homeostasis (Gannon & Nuttall, 2010) and the need

to consider protein quality in its regulation (Millward, Layman, Tome, &

Schaafsma, 2008).

3. CLASSIFICATION OF PROTEINS ANDPROTEIN QUALITY

Although high-quality dietary proteins are predominantly found in

animal foods, plant foods (legumes, cereals) too contain appreciable

amounts. Vegetable sources are the major protein source in most developing

countries where animal proteins are expensive and are of limited availability.

The quality of a protein depends on its characteristics, the food matrix they

are in, and how well they meet the metabolic needs of individuals (Millward

et al., 2008). Twenty one amino acids are involved in protein synthesis and

several more occur as metabolic intermediates that are not involved in pro-

tein synthesis. Anatomically, all amino acids are similar in that they contain

both an amino group and a carboxylic acid group attached to an (a) carbonatom.What sets them apart from each other are the characteristics of the side

chain attached to the a-carbon. Based on the chemical characteristics of the

side chain, amino acids can be grouped into distinct categories (Fig. 1.2).

In addition, the side chains are also characterized by the presence of groups

such as alcohol, thiol, indole, and imidazole (Reeds, 2000). These chemical

characteristics determine how each amino acid behaves in biochemistry.

Proteins are linear polypeptides made of amino acids and are naturally found

folded into secondary and tertiary structures. The secondary and tertiary

structures are formed through the formation of hydrogen bonds and due

to the interactions between side chains of amino acids. Therefore, the phys-

ical and functional characteristics of a protein depend on its amino acid

complement.

Early studies showed that all proteins were not nutritionally equal and

that some were more important toward nitrogen balance than others. Essen-

tial amino acids are those that cannot be synthesized by the human body

(Mercer, Dodds, & Smith, 1989) and must be met through the diet. Nine

amino acids are essential for humans (histidine, isoleucine, leucine, lysine,

methionine, phenylalanine, threonine, tryptophan, and valine), while the

remainder could be synthesized frommetabolic intermediates (alanine, argi-

nine, aspartate, asparagines, cysteine, glutamate, glutamine, glycine, proline,

Amino acids

Hydrophobic amino acids

1. Neutral amino acids

-Glycine -Cysteine

-Serine

-Threonine

-Aspartate

-Glutamate

-Arginine

-Histidine

-Asparagine

-Glutamine

-Lysine

-Proline

-Alanine

-Leucine

-Isoleucine

-Tryptophan

-Phenylalanine

-Tyrosine

-Valine

1. Neutral amino acids

2. Acidic amino acids

3. Basic amino acids

4. Amino acid amides

2. Aromatic amino acids

Hydrophilic amino acids

Figure 1.2 Classification of amino acids based on chemical nature.


serine, tyrosine, selenocysteine) and are therefore deemed nonessential.

Recently, some amino acids have been classified as conditionally essential

(arginine, proline, cysteine, glycine) as they can be synthesized from amino

acid precursors (Reeds, 2000). However, this depends on the availability of

the precursors, some of which are essential amino acids. Foods containing all

the essential amino acids in quantities adequate to meet growth and repair

are termed complete protein foods ( Jeor et al., 2001) and are exclusively

of animal origin. For the maintenance of proper nitrogen balance, it is

important to consume adequate amounts of complete proteins, or in their

absence, consume combinations of incomplete proteins with complemen-

tary amino acid compositions ( Jeor et al., 2001).

Table 1.1 Amino acid composition of some common protein sources

Amino acid

Composition (mg/100 g of food)

Cow’smilk

Cottagecheese

Humanmilk Casein Whey Beef Chicken Egg yolk

Eggwhite

Codfish Soy

Wheat,whole grain

Rice,white

Isoleucine 192 591 56 718 719 584 1069 820 571 679 1889 443 116

Leucine 324 1116 95 1262 1186 1199 1472 1370 922 1211 3232 898 222

Lysine 274 934 68 1077 1030 1161 1590 1202 739 1399 2653 359 97

Methionine 79 269 21 370 241 426 502 364 441 418 525 228 63

Cysteine 29 66 19 48 253 230 262 235 267 136 552 275 55

Phenylalanine 163 577 46 695 407 644 800 728 662 595 2055 682 144

Tyrosine 160 604 53 772 363 381 669 704 390 553 1303 275 90

Threonine 167 500 46 618 817 474 794 753 532 658 1603 367 96

Tryptophan 42 147 17 210 205 168 205 240 – 188 532 174 31

Valine 225 748 63 894 697 915 1018 998 536 731 1995 564 164

Arginine 121 497 43 497 375 1154 1114 1143 635 982 3006 648 224

Histidine 100 326 23 387 237 443 525 400 262 324 1051 357 63

Alanine 115 384 36 408 598 1005 682 759 676 877 1769 489 156

Aspartic acid 258 905 82 946 1269 1312 1834 1685 1223 1525 4861 722 253

Glutamic acid 690 2603 168 2924 2248 2121 3002 2208 1668 2297 7774 4328 524

Glycine 67 222 26 262 280 1238 1059 506 395 710 1736 569 122

Proline 330 1229 82 1531 786 922 829 617 406 512 2281 2075 127

Serine 210 639 43 801 622 570 781 1419 812 647 2128 620 141

Amino acid present based on mg/100 g of food (FAO, 1970; Schonfeldt, Hall, & Smit, 2012; USDA, 2012).


The amino acid composition of protein foods differ markedly

(Table 1.1). Animal proteins have a more balanced amino acid profile and

contain notable amounts of both essential and nonessential amino acids.

Plant proteins, on the other hand, often show deficiencies in certain essential

amino acids. For example, cereals and legumes are generally deficient in

lysine and methionine, respectively ( Jeor et al., 2001). Some nonessential

amino acids such as glutamic acid and aspartic acid are abundantly found

in all proteins.

4. IMPACT OF PROTEINS AND AMINO ACIDSON INSULIN SECRETION

4.1. Effect of proteins on insulin secretion
In early work, Floyd, Fajans, Conn, Knopf, and Rull (1966b) and Berger
and Vongaraya (1966) showed that proteins and amino acids significantly

increased insulin expression. Since then, many studies have confirmed that

both ingested proteins and intravenous administration of amino acids stim-

ulate insulin secretion (Fajans, Floyd, Knopf, & Conn, 1967; Gannon &

Nuttall, 2010).

Proteins are believed to induce insulin secretion both by the direct stim-

ulation of pancreatic b-cells by amino acids and via incretin hormones

expressed in response to meal composition (Nuttall & Gannon, 1991). Early

in vitro work using pieces of rabbit pancreas incubated in media containing

amino acids showed that insulin is stimulated in the presence of amino acids

(Milner, 1970). This was better demonstrated by Salehi et al. (2012) using

mouse pancreatic islets. The authors fed either whey protein or white bread

to human subjects and observed insulin expression by mouse islets when

they were exposed to the resulting serum. The results showed that amino

acids in serums increased insulin expression. The mechanisms by which

amino acids stimulate insulin appear to be different to that of glucose.

In an in vitro experiment utilizing bHC9 hyperplastic insulin secreting cells,

Ronner, Naumann, and Friel (2001) demonstrated that amino acids stimu-

late insulin also using different mechanisms to glucose. They proposed a

two-compartment model where insulin secretion occurs in response to an

amino acid sensor in the b-cells and when KATP channels have low activity.

This suggested that amino acid-mediated insulin secretion does not occur

when b-cells are poorly energized and when glucose concentrations are low.Circulating plasma amino acid levels also have effects on peripheral glu-

cose uptake mechanisms. Increased plasma amino acid levels have been


shown to induce skeletal muscle insulin resistance and reduce glycogen syn-

thesis by stalling glucose transport and phosphorylation (Krebs et al., 2002).

Elevated amino acid levels also activate intermediates important for protein

synthesis initiation such as p70 S6 Kinase and PHAS-I in the presence of

insulin (Patti, Brambilla, Luzi, Landaker, & Kahn, 1998). Increased amino

acid levels have further been shown to induce gluconeogenesis in the liver

(Nuttall, Ngo, & Gannon, 2008). This was most evident when amino acids

were fed in an impaired insulin secretion state as it showed the increased

plasma glucose levels in the absence of peripheral disposal (Krebs

et al., 2003).

Protein quality appears to notably affect the magnitude of the insulin

response. Early work carried out by Floyd and colleagues showed that con-

suming 500 g of either ground beef or chicken liver sharply increased plasma

insulin levels (Floyd et al., 1966b). Studies have shown that proteins from

various sources elicit different effects on insulin. A beef meal showed signif-

icantly higher plasma insulin levels than a cod fillet meal which was attrib-

uted to differences in their amino acid composition and also the differences

in protein digestibility (Soucy and Le Blanc, 1999). In an attempt to deter-

mine postprandial responses to different protein sources, Bowen, Noakes,

and Clifton (2006) fed 72 healthy lean and obese men 50 g of whey protein,

soy protein, and gluten and measured insulin, ghrelin, cholecystokinin

(CCK) and glucagon-like peptide-1 (GLP-1). They found that all the pro-

teins equally suppressed ghrelin, CCK, and GLP-1 but had different effects

on insulin secretions. This is suggestive of proteins’ directs effects on insulin

secretion. While all proteins failed to secrete insulin as much as glucose did,

the lowest expression was observed for gluten. Soy and whey proteins

showed higher and similar secretions. Nuttall and Gannon (1990) fed seven

healthy males 50 g of cottage cheese and egg white and measured insulin,

glucagon, and C-peptide and found that insulin, C-peptide, and glucagon

secretions were 50% less for egg white compared to cottage cheese. The

study also estimated the amount of metabolized protein in the test meals

and found that only 47% of the egg white had been metabolized compared

to 70% in cottage cheese. This data suggest that the lower hormonal response

to egg white was due to its lower digestibility. Previous studies on the bio-

logical value have also shown a low digestibility for egg protein and that it

depended on energy intake and maintenance (Inoue, Fujita, & Niiyama,

1973). Krezowski, Nuttall, Gannon, and Bartosh (1986) fed 50 g of lean

hamburger to healthy participants and showed that protein alone stimulated


only 28% of the insulin response seen when glucose was given. Although

they observed an effect of protein on glucagon, there was no impact on

C-peptide. Lang et al. (1998) fed 12 healthy participants egg albumin, casein,

gelatin, soy protein, pea protein, and wheat gluten (22.4% of total energy)

and found that all the proteins increased insulin secretion to the same degree.

Unlike the previously discussed studies, proteins were provided as part of a

mixed meal in this study and the lack of differences may have been due to

the influence of other macronutrients. Indeed, studies looking at the effects

of combined protein and carbohydrate meals have shown weaker effects

of protein quality on insulin (Claessens, Calame, Siemensma, van Baak, &

Saris, 2007).

A few studies have also looked at the effect of proteins in type 2 diabetics.

A meal with glucose and cottage cheese resulted in a higher insulin response

than a meal with other proteins such as fish or soy (Gannon, Nuttall, Neil, &

Westphal, 1988). Themajor stimulus for insulin secretionwas the increase in

incretins that were stimulated in response to proteins (or its digestion prod-

ucts) in the intestine. This implies that the insulin response was related to the

protein digestion rate. Gannon, Nuttall, Damberg, Gupta, and Nuttall

(2001) fed 10 untreated type 2 diabetics either 50 g of lean beef or water

and measured insulin for the subsequent 8 h. They found that beef showed

a threefold increase in insulin compared to water and that these levels

remained for up to 7 h. An increase in C-peptide and glucagon was also

observed. Another study investigated the effect of 50 g of protein from lean

hamburger on insulin in type 2 diabetics and reported similar effects (Nuttall,

Mooradian, Gannon, Billington, & Krezowski, 1984). The same group

compared the insulin response to 25 g of protein from cottage cheese and

egg white in type 2 diabetics in another study (Gannon, Nuttall, Lane, &

Burmeister, 1992). While both proteins stimulated insulin secretion, the

response for cottage cheese was 3.6 times greater compared to that of egg

white. This was also the same for C-peptide concentrations. Again, cottage

cheese was metabolized more (81%) than egg white (52%) and elicited a

serum amino acid N content twice that of egg white and this may explain

the greater hormonal responses. The data therefore suggest that insulin

secretion in response to proteins is hyperstimulated in type 2 diabetics.

However, more data are required before firm conclusions can be made.

A very limited number of studies have looked at the effect of proteins on

insulin in diabetics and there are no studies looking at effects of combined

proteins on insulinemia. These are areas worthy of further study.


4.2. Effect of amino acids on insulin secretionA greater volume of research has looked at the effects of amino acids on insu-

lin secretion. This is reasonable as the impact of proteins on glucose homeo-

stasis have been shown to be mediated through the effects of their

constituting amino acids (Bos et al., 2003). The amino acid composition

of dietary proteins differ markedly (Table 1.1). Therefore, the protein type

ingested will determine the composition of postprandial circulating amino

acids and thereby the metabolic response to the protein meal.

Amino acids appear to affect insulin secretion and glucose homeostasis in

unique ways. Early work carried out in the 1960s showed that both single

and combined boluses of amino acids significantly increased insulin secretion

(Fajans, Floyd, Knopf, &Conn, 1967; Floyd et al., 1966b).While themajor-

ity of amino acids influence insulin secretion, some have been suggested

to be notably insulinogenic (phenylalanine, arginine, lysine, alanine,

leucine, and isoleucine; Newsholme, Brennan, Rubi, & Maechlen, 2005;

Nuttall & Gannon, 1991). Amino acids are postulated to stimulate insulin

through different mechanisms (Henquin & Meissner, 1981). Cationically

charged amino acids stimulate insulin by polarizing the plasma membrane,

and amino acids cotransported withNaþ has been shown to stimulate insulin

secretion by depolarizing the plasma membrane via Naþ transport and acti-

vating voltage-dependent Caþ channels (Newsholme et al., 2005). Amino

acid oxidation can also increase ATP stores and thus activate Caþ channels

leading to insulin release. In the following section, the insulinogenic poten-

tial of individual amino acids is discussed.

Although several studies have shown that alanine plays a notable role in

gluconeogenesis (Felig et al., 1970), a limited number of studies have inves-

tigated its effect on insulin secretion in humans. Studies have shown that oral

administration of alanine increases insulin secretion in normal, diabetic, and

obese subjects (Genuth, 1973; Genuth & Castro, 1974). These effects were

seen when obese individuals were given 50 g/day, and when normal and

diabetic subjects were given 0.1 and 0.5 g/kg body weight of alanine per

day. In vitro studies show that alanine is consumed by islet cells (Dixon,

Nolan, McClenaghan, Flatt, & Newsholme, 2003; Hellman, Sehlin, &

Taljedal, 1971) and that it induces insulin secretion from them (Dunne,

Yule, Gallacher, & Petersen, 1990; McClenaghan, Barnett, Ah-sing,

et al., 1996). These studies showed that alanine induces insulin by depolar-

ization of the membrane through cotransportation with Naþ. Other studies

have shown that alanine does not induce insulin secretion from rat b-cells


(Sener & Malaisse, 2002). However, these differences may be due to the use

of different cell lines all of rat origin. Although the literature more heavily

suggests that alanine is proinsulinogenic, this is based on a very limited num-

ber of studies. Data from more studies are required.

Arginine has been shown to stimulate insulin secretion from b-cellsthrough derived nitrogen oxides (Schmidt, Warner, Ishii, Sheng, &

Murad, 1992). Dupre, Curtis,Waddell, andBeck (1968) infused healthy par-

ticipantswith 15 gof arginine intraduodenally and intravenously over 40 min

and found that insulin increased in both instances. Similarly, Floyd et al.

(1966b) infused 172 mMof arginine to healthy subjects and observed an insu-

lin increase (maximal increase of 81 mU/ml). Interestingly, in both studies,

intravenous infusion also resulted in increased blood glucose, suggesting that

argininemay be glucogenic.However, intraduodenal infusion had no impact

on glucose possibly due to plasma arginine concentrations being less. More

recent work looking at the effects of oral arginine on insulin secretion found

no effects. Gannon, Nuttall, and Nuttall (2002b) fed nine healthy subjects

1 mM/kg bodyweight of arginine andmeasured insulin, plasma amino acids,

and glucagon for the subsequent 2 h. They found that arginine did not

increase insulin secretion although it did increase glucagon.These results sug-

gest that arginine at levels usually found inmeals is not insulinogenic although

it may be at higher levels (as observed by Dupre and colleagues). In vitro

work carried out using rat pancreatic cells show that arginine stimulates insu-

lin secretion through cationic amino acid transportation into the islet cells

resulting in membrane depolarization (Herchuelz, Lebrun, Boschero, &

Malaisse, 1984). These effects were seen when the cells were exposed to

an arginine concentration of 10 mM. Gerich, Charles, and Grodsky (1974)

observed a nonphasic insulin release in perfused rat pancreas when it was

exposed to 6 mM of arginine. Therefore, the literature on arginine’s effect

on insulin secretion is equivocal. In vitro studies show that arginine stimulates

insulin secretion from pancreatic cells which suggests that exposure of b-cellsto higher arginine concentrations stimulates insulin. More data from whole

body studies are required to ascertain if arginine levels typically found in

dietary proteins have a significant effect on insulin secretion.

Glycine is an amino acid found in large quantities in gelatin (approxi-

mately 30% of total amino acids; Gannon &Nuttall, 2010). Beef protein also

contains a relatively higher amount compared to other proteins (Table 1.1).

Previous work showed that gelatin induced a significantly large insulin

response (Gannon et al., 1988) and this was attributed to the presence of


large amounts of glycine. Gannon, Nuttall, and Nuttall (2002a) fed nine

healthy subjects 1 mM/kg lean body mass glycine and measured insulin

for the subsequent 2 h. They found that glycine only modestly increased

insulin secretions. However, it significantly increased plasma glucagon con-

centrations. Gonzalez-Ortiz, Medina-Santillan, Martinez-Abundis, and

Von Drateln (2001) gave six healthy nonobese first-degree relatives of type

2 diabetics a morning dose of 5 g of glycine and then measured insulin secre-

tion and action using a hyperglycemic–hyperinsulinaemic clamp. The study

showed that glycine increased insulin secretion although it had no effect on

insulin action. These studies suggest that glycine stimulates insulin secretion

to a modest level.

Lysine is one of three basic amino acids and is essential to human beings.

It is found in larger quantities in red meat. A limited number of studies have

investigated the effect of lysine on insulin secretion and these show a notable

effect. Floyd et al. (1966b) gave 30 g of lysine intravenously to nine healthy

male subjects and observed a moderate insulin secretion (maximal increase

52 mU/ml). In an attempt to determine the effect of lysine on insulin release,

Kalogeropoulou, Lafave, Schweim, Gannon, and Nuttall (2009) fed

1 mM/kg lean body mass of lysine to 13 healthy subjects and measured insu-

lin excursions over the subsequent 2.5 h. The study showed an insignificant

increase in insulin following lysine ingestion (compared to a water control).

However, there was a significant rise in glucagon. Similarly, Isidori, Lo

Monaco, and Cappa (1981) fed 1200 mg of lysine to 15 males and found

no effect on insulin but a rise in glucagon. These results suggest that lysine

has little effect on insulin secretion when given in doses representative of

dietary levels but stimulates glucagon. In vitro work also showed no effect

of lysine on insulin release. Milner (1970) incubated pieces of rabbit

pancreas in 5 mM solutions of lysine and found that it did not significantly

stimulate insulin release.

Isoleucine, an isomer of leucine is a branched chain amino acid (BCAA)

found in most proteins. Floyd et al. (1966b) infused three healthy subjects

with either 7.5 or 15 g of isoleucine and found a weak effect on insulin secre-

tion. Nuttall, Schweim, and Gannon (2008) fed nine healthy subjects

1 mM/kg lean body mass of isoleucine and measured insulin for the subse-

quent 2.5 h. They too observed no effect of isoleucine on insulin expression.

They also saw no effect on glucagon although plasma glucose decreased.

This may suggest a possible role of isoleucine in increasing glucose uptake.

Another study using dogs similarly saw no effect of isoleucine on glucagon

(Rocha, Faloona, & Unger, 1972). In vitro studies using rat and rabbit cells


show conflicting results. Bolea, Pertusa, Martın, Sanchez-Andres, and Soria

(1997) found that isoleucine stimulated insulin secretion in fresh mouse islet

cells as did one and 10 mM/L concentrations in BRIN-BD-11 cells

(McClenaghan, Barnett, O’Harte, & Flatt, 1996). However, Milner (1970)

found no significant effects when rabbit pancreas was incubated in medium

containing 5 mM of isoleucine. These studies predominantly suggest that

isoleucine alone has little effect on human insulin secretion. However, this

conclusion is based on a small number of studies.

Leucine is one of the most extensively studied BCAAs. It has been shown

to play a notable role in insulin-induced mRNA translation and skeletal

muscle synthesis (Kimball, Farrell, & Jefferson, 2002). Studies have also con-

clusively shown that leucine in combination with glucose significantly

upregulates insulin secretion. However, a fewer number of studies have

looked at the effects of leucine alone on insulin. Milner (1970) reported that

leucine was the only essential amino acid that stimulated insulin in the

absence of glucose. A study published by Fajans, Knopf, Floyd, Power,

and Conn (1963) studied the effect of 0.2 g/kg of body weight of leucine

on insulin secretion in healthy subjects. They found that both intravenous

and oral doses were insulinogenic. These results were confirmed in another

study by the same group using 30-g intravenous boluses on 10 healthy

humans (Floyd et al., 1966b). Kalogeropoulou, Lafave, Schweim,

Gannon, and Nuttall (2008) gave 13 healthy subjects 1 mM/kg lean body

mass leucine and measured insulin for the subsequent 2.5 h. The study

reported a small but significant increase (approximately 200 mU/min/ml)

in insulin compared to a water control. It also resulted in a significant

increment in glucagon. McArthur, Kirtley, and Waife (1963) gave two

healthy males oral doses of leucine at various levels and found that higher

levels (750 mg/kg body weight) induced hypoglycemia while lower levels

(150 mg/kg body weight) did not. While the results suggest that high levels

of leucine may be insulinogenic, this cannot be confirmed as the study did not

measure insulin. Interestingly, Cochrane, Payne, Simpkiss, andWoolf (1956)

showed that those with idiopathic hypoglycemia were more sensitive to

leucine and showed marked reductions in blood glucose when given

150 mg/g body weight of leucine, compared to normal subjects. Since leu-

cine’s influence on blood sugar has been indicated to be through its effects

on insulin (Fajans et al., 1963) this suggests that those with idiopathic hypo-

glycemiamay be producingmore insulin in response to leucine. Indeed, other

studies have shown that leucine upregulates insulin secretion in those

with disordered metabolism. Loridan, Sadeghi-Nejad, and Senior (1971)


found that leucine hypersecreted insulin in obese hyperinsulinaemic

children compared to normal children. Similar findings were reported

by Kelly et al. (2001) and the authors attributed this to impaired physiology

of glutamate dehydrogenase (GDH), an enzyme linked with leucine-

induced insulin secretion. Indeed, Li et al. (2003) demonstrated that b-cellsensitivity to leucine depends upon GDH activation. The method of insu-

lin secretion by leucine has been suggested to be different to that of other

amino acids (Fajans, Floyd, Knopf, Guntsche, et al., 1967). Leucine has

been shown to stimulate b-cells by increasing mitochondrial metabolism

through GDH activation which increases ATP through transamination

of leucine into a-ketoisocaproate (Newsholme et al., 2005). Leucine

metabolism has also been suggested to activate the mTOR signaling path-

way in b-cells and stimulate insulin.

In summary, the literature suggests that leucine stimulates insulin secre-

tion by b-cells even in the absence of glucose. There appears to be a dose–

response relationship between plasma leucine and insulin secretion in

normal subjects. However, this may be impaired in those with physiological

disorders. Leucine at levels usually found in food appears to have only a

modest effect on insulin secretion.

Phenylalanine is a nonpolar essential amino acid found in most animal

and plant proteins. Early work carried out by Floyd et al. (1966b) showed

that a 30 g intravenous infusion of phenylalanine modestly increased insulin

secretion (maximal increase 28 mU/ml). Guttler, Kuhl, Pedersen, and Paby

(1978) demonstrated that similar effects can be observed also by far smaller

phenylalanine doses. The authors gave six healthy adult males oral doses of

0.6 mM/kg body weight of phenylalanine. Insulin and glucagon levels

began rising within 10 min of amino acid ingestion and peaked at twice

the baseline level at 30 min. The study also observed a concomitant increase

in serum tyrosine which maximized at 2 h. Nuttall, Schweim, and Gannon

(2006) gave six healthy subjects 1 mM/kg body weight of phenylalanine and

measured insulin and glucagon for the following 2.5 h. The authors

observed a significant increment in insulin secretion following phenylala-

nine compared to the water control. However, the temporal insulin

response pattern differed from that seen by Guttler et al. in that phenylala-

nine induced a 10-min initial lag followed by a high and sustained response

during the remaining period. Phenylalanine also stimulated a significant rise

in glucagon (approximately 2000 pg/min/ml) compared to the control.

In vitro studies show less conclusive effects. In one study, 5–80 mM of phe-

nylalanine did not stimulate a significant insulin secretion from perfused


pancreatic cells (Landgraf, Landgraf-Leurs, & Horl, 1974). Similarly, Milner

(1970) observed no insulin secretion when pieces of rabbit pancreas were

incubated in 5-mMphenylalanine. Interestingly, the study showed that phe-

nylalanine inhibited insulin secretion in the presence of glucose. Although

in vitro studies show poor effects, the majority of human studies show an

effect of phenylalanine on insulin secretion also at physiological doses found

in food. One review highlighted that phenylalanine stimulated the greatest

insulin response of all amino acids (Gannon & Nuttall, 2010).

Proline is a readily absorbed amino acid that is found in higher quantities

in dairy, meats, and collagen. Nuttall, Gannon, and Jordan (2004) investi-

gated the effect of physiological levels of proline on insulin secretion. They

fed eight young healthy participants 1 mM/kg lean body mass of proline and

measured serum insulin over the following 150 min. The authors found that

proline only very slightly increased insulin levels and had no impact on glu-

cagon. While no studies have been conducted to investigate higher doses of

proline on insulin, the data so far suggest that proline alone at levels com-

monly found in food has little impact on insulin secretion.

Floyd et al. (1966b) evaluated the insulinogenic potential of valine (30 g),

methionine (30 g), histidine (30 g), threonine (7.5, 8.5, 15, and 22.5 g), and

tryptophan (2.5, 5, and 7.5 g) when they were infused intravenously to 2–10

healthy subjects. The authors found that all these amino acids had little effect

on insulin secretion. In an effort to determine the effect of ornithine on insu-

lin secretion, Bucci, Hickson, Wolinsky, and Pivarnik (1992) administered

oral boluses of 40, 100, and 170 mg/kg body weight of L-ornithine to

12 body builders. Serum insulin was not significantly affected by ornithine

at all three levels. While this suggests that ornithine has no effects on insulin

secretion, further studies are needed to confirm it. For instance, citrulline

(metabolizable from ornithine) has been shown to potentiate insulin secre-

tion from b-cells through nitric oxide production (Nakata & Yada, 2003).

In vitro and animal studies also provide indicative data on the effect of

amino acids on insulin secretion. Kuhara, Ikeda, Ohneda, and Sasaki

(1991) gave intravenous infusions of 17 amino acids each at doses of

3 mM/kg body weight to sheep and measured insulin, glucagon, and

growth hormone secretions. While leucine was the most insulinogenic, ala-

nine, glycine, and serine showed the next highest stimulation. The authors

concluded that straight chain amino acids were more insulin and glucagon

secretory than short-chain amino acids, and that BCAAs were insulinogenic

but had no effect on glucagon. In agreement, Hutton, Sener, and Malaisse

(1980) showed that rat islet cells produced insulin when they were exposed


to 10 mM of valine, leucine, or isoleucine. Studies using rodent-derived

BRIN-BD11 cells showed that 1 and 10 mM of alanine, arginine, gluta-

mine, glycine, leucine, lysine, proline, and serine all increased insulin

expression by up to fivefold in the presence of nonstimulatory glucose levels.

Glutamine was also shown to be insulinogenic by Li et al. (2004) using

mouse islet cells. The authors suggest that glutamine may be playing a central

role (as a signaling molecule) in amino acid- and glucose-stimulated insulin

secretion. Glutamic acid is one of few amino acids present in relatively

higher amounts in the plasma irrespective of time of day and meal compo-

sition, and is omnipresent in considerable amounts in food proteins

(Wurtman, Rose, Chou, & Larin, 1968). Therefore, it is possible that glu-

tamine plays a central role in amino acid-mediated insulin secretion. There

appear to be no human studies evaluating the effect of glutamate on insulin

secretion possibly due to gastrointestinal distress caused by it at physiological

doses (Gannon & Nuttall, 2010). Interestingly, cysteine has been shown to

inhibit insulin expression. Kaneko, Kimura, Kimura, and Niki (2006)

exposed mouse islet cells to L-cysteine and found that it reduced insulin

expression presumably due to the production of H2S by the amino acid.

However, these findings remain to be confirmed in human trials.

Human studies investigating amino acid effects on insulinemia provide

the most reliable data however, and these show different results to rodent

studies. A review by Gannon and Nuttall (2010) included a hierarchical list

of insulinogenic amino acids They reported that the most insulinogenic

amino acids are phenylalanine and glycine and the least insulinogenic, argi-

nine, tyrosine, and histidine. There is limited information from human stud-

ies on aspartic acid, asparagine, tryptophan, ornithine, threonine, serine,

tyrosine, and histidine on human insulin secretion, and no data on the

impact of cysteine and selenocysteine. Cysteine analogues have been shown

to stimulate insulin from rat pancreatic islets (Ammon, Hehl, Enz, Setiadi-

Ranti, & Verspohl, 1986) and this requires confirmation in human studies.

Interestingly, these findings are opposite to those reported by Kaneko et al.

(2006). Although cysteine is found in very small quantities in food proteins

investigating higher than physiological doses on insulin, secretion may pro-

duce therapeutically useful data. It may also be interesting to investigate

selenocysteine’s effects on insulin secretion as Selenium has been shown

to stimulate insulin (Iizuka, Sakurai, & Hikichi, 1992) and hold insulin

mimetic properties (Stapleton, 2000).

A considerable number of studies have shown that two or more amino

acids increase insulin secretion through synergy. Early work carried out by


Floyd et al. (1966b) showed that amino acid combinations induced greater

insulin secretion than single amino acids. A combination of 10 essential

amino acids produced a maximal insulin increase of 120 mU/ml compared

to when the same amino acids were given separately (which produced a

maximal ranging between 3 and 80 mU/ml). Similarly, 30 g portions of

six to eight amino acid containing mixes produced significantly greater

plasma insulin responses compared to when given alone. To test the syner-

gistic effects of amino acids, Floyd et al. (1970) conducted an experiment

using 11 healthy young males. The subjects were given 15 g intravenous

infusions of one or two amino acids and insulin was measured. Infusing

two amino acids produced a greater insulin response than the sum of the

separate effects. Arginine and leucine, and arginine and phenylalanine were

particularly good synergistic combinations. Arginine given with casein

increased the total insulin response in rats in another study (Sugano,

Ishiwaki, Nagata, & Imaizumi, 1982). Arginine combined with lysine or his-

tidine, and leucine and histidine did not produce synergistic effects. However,

Isidori et al. (1981) measured the insulin response in 15 healthy males follow-

ing an oral dose of lysine and arginine (1200 mg of each) and found that their

combination resulted in a greater insulin response than when each was given

separately. Sugano et al. (1982) showed that soya bean protein given in com-

bination with lysine increased total insulin secretions. Floyd, Fajans, Conn,

Knopf, and Rull (1966a) fed healthy participants 500 g of either beef or

chicken liver and found that plasma insulin and leucine increased markedly.

The magnitude of the insulin rise was far greater than that seen with leucine

alone in previous studies suggesting a possible synergistic effect between

amino acids found in the proteins. For example, these proteins are also good

sources of arginine, lysine, and glutamine. Other studies have shown that

glutamine in combination with leucine significantly augments insulin secre-

tion (Sener, Somers, Devis, & Malaisse, 1981). Although glutamine has been

shown to be poorly insulinogenic in some work (Gannon & Nuttall, 2010),

others show that it can induce insulin in the presence of allosteric GDH

activators (Newsholme et al., 2005). It is possible therefore that leucine and

glutamine synergistically increase insulin secretion by increasing catabolic

fluxes and the former acting as an allosteric activator of GDH (Malaisse

et al., 1982). In vitro work using mouse islet cells showed that a leucine ramp

(0–25 mM) in the presence of 2-mM glutamine increased insulin secretion by

10-fold although a glutamine ramp (0–25 mM) alone had no effect on insulin

(Li et al., 2004). Other in vitro cell studies showed that both arginine

and phenylalanine strongly stimulate insulin in the presence of glutamine


(van Loon, Saris, Verhagen, & Wagenmakers, 2000) suggesting a central

role for glutamine in amino acid-mediated insulin secretion.

Amino acid combinations also appear to increase insulin response in the

presence of glucose. van Loon et al. (2000) fed eight healthy males mixtures

of free amino acids (arginine, glutamine, leucine, phenylalanine) and protein

hydrolysates (whey, pea, wheat, casein) along with a standard amount of glu-

cose (114.2 g/L) and measured insulin. The study showed that the ingestion

of protein with carbohydrate increased insulin expression by nearly 100%

compared to carbohydrate alone. Leucine, phenylalanine, and arginine were

particularly insulinogenic when consumed with carbohydrates. The study

also showed that the insulin response was significantly positively correlated

with plasma leucine, phenylalanine, and tyrosine concentrations. In an

in vitro study, insulin secretion was augmented when a perfused rat pancreas

was exposed to 2, 5 and 10 mM of leucine and isoleucine in the presence

5.6 mM of glucose (Pek, Santiago, & Tai, 1978).

The greater insulinotropicity of certain food proteins may also be due to

synergistic associations between amino acids comprised in each. Gannon

et al. (1988) observed that gelatin induced a insulin release that was approx-

imately 270%more than that seen for glucose. Approximately 55–65% of the

amino acids in gelatin are made up of glycine, proline, and hydroxyproline

and it is therefore reasonable to postulate that the insulinotropicity of gelatin

is largely due to these amino acids. However, individual administration of

these amino acids (with or without glucose) had little effect on insulin secre-

tion (Gannon et al., 2002a; Nuttall et al., 2004). This suggests that the

enhanced insulinotropicity of gelatin may be due to synergistic effects

between these amino acids. Gannon et al. (1988) also showed that cottage

cheese potentiated an insulin response that was 360% greater than that of

glucose. The predominant protein in cottage cheese is casein and this is

relatively rich in proline and phenylalanine compared to other proteins

(Lavigne, Marette, & Jacques, 2000). Phenylalanine and proline alone or

in combination with glucose had relatively smaller effects on insulin secre-

tion. Therefore, the greater effects seen with casein may be again due to

synergism. Milk proteins have been shown to be more insulinogenic

(Elmstahl & Bjorck, 2001), particularly the whey fraction (Nilsson,

Stenberg, Frid, Holst, & Bjorck, 2004). While this may be due to synergistic

associations between the amino acids in dairy proteins, whey protein, in par-

ticular, has shown to also induce a greater incretin response (Nilsson et al.,

2004). Whey proteins independently stimulate GLP-1 and gastric inhibitory

peptide (GIP) secretion which can, in turn, influence insulin expression


(Luhovyy, Akhavan, & Anderson, 2007). There is very little data on the

effect of individual amino acids on incretins and this is an area that requires

focus in future research. Salehi et al. (2012) found that insulin secretion in

response to amino acids was augmented when GIP was present. Another

study showed that GIP and also GLP-1 increased insulin secretion in the

presence of amino acids (Fieseler et al., 1995).

4.3. Impact of proteins and amino acidson glucagon secretion

It is evident from the studies discussed above that proteins and amino acids

induce glucagon secretion. Postprandial plasma amino acid excursions have

been shown to stimulate glucagon secretion independent of glycemic status

(Krebs et al., 2003). Kuhara et al. (1991) assessed the metabolic response to

17 individual amino acids and found that all the amino acids stimulating glu-

cagon also stimulated insulin, suggesting that all amino acids potentiating

glucagon release also induce insulin. However, other studies disprove this.

Rocha et al. (1972) evaluated the effects of 20 individual amino acids at a

dosage of 1 mM/kg body weight in dogs. Of the 20 amino acids, 17 caused

a notable increase in plasma glucagon. Asparagine, glycine, and phenylala-

nine were the most glucagonogenic while valine, leucine, and isoleucine

failed to stimulate the hormone. Unlike Kuhara et al. (1991), this study

did not observe a relationship between glucagon and insulin stimulations.

The study suggested that amino acids entering the gluconeogenic pathway

as pyruvate seemed to be more glucagonogenic than those entering as

a-ketoglutarates and succinyl CoA. Human studies also show that glycine,

lysine, and phenylalanine are glucagonogenic (Gannon & Nuttall, 2010).

However, they showed asparagine to only moderately stimulate glucagon.

Similar to animal work, human studies also showed that leucine and isoleu-

cine did not induce glucagon although valine stimulated a modest release.

Histidine, proline, glutamine, and tyrosine did not stimulate glucagon in

humans either (Gannon & Nuttall, 2010). Ingestion of whole proteins

has also been shown to induce glucagon release (Gannon et al., 1992,

2001; Krezowski et al., 1986; Nuttall & Gannon, 1990). Gannon et al.

(1992) observed that the glucagon response area under the curve (AUC)

closely correlated with the amount of protein metabolized (egg white and

cottage cheese). This may suggest that glucagon plays a key role in protein

metabolism and uptake. Indeed, one study showed that elevated plasma

glucagon levels stimulate gluconeogenesis from amino acids in the liver


(Boden, Rezvani, & Owen, 1984). However, these effects need be con-

firmed in more studies and using different protein types.

5. IMPACT OF PROTEINS AND AMINO ACIDSON GLYCEMIA

Research looking at the effects of protein on blood glucose dates back at

least a century with early work carried out on animals. Reilly and colleagues

(1898) treated rabbits and dogs with phlorizin (a competitive inhibitor of

sodium/glucose cotransporter and lowers glucose amounts in blood) and

found that the amount of glucose appearing in the urine was comparable

to the increase in nitrogen appearing in the urine (termed dextrose to nitrogen

ratio or D/N; Reilly, Nolan, & Lusk, 1898). The urinary nitrogen increase

represented the dietary protein-derived amino acids that were deaminated

and subsequently converted into glucose and other products. Only urinary

glucose was measured in these studies and it was still unknown then if protein

had an effect on blood glucose concentrations. Janney (1915) reported that for

various proteins given orally to phlorizin-treated dogs, the D/N ratio was

unique for each protein, varying from 50% to 80% of the protein given, pre-

sumably due to differences in amino acid composition. In a subsequent study,

Janney showed that the results obtained from phlorizin-treated dogs were use-

ful for studying the impact of dietary proteins on blood glucose in humans

with diabetes ( Janney, 1916). Different amino acids and their ability to con-

vert into glucose was first documented by Dakin (1913). The amino acids

were administered subcutaneously to phlorizin-treated dogs. With the

exception of valine, leucine, isoleucine, lysine, histidine, phenylalanine,

and tryptophan, all other amino acids yielded large amounts of urinary

glucose. This was the first study to suggest that the glycemic potency of

different amino acids were not equal. However, all these studies only

measured urinary glucose.

In 1913, Jacobsen reported that ingestion of egg white protein did not

result in an increase in blood glucose in normal subjects (Jacobsen, 1913).

Type 2 diabetic subjects ingesting 250 g meat (containing 50 g protein)

showed stable glucose concentrations over the subsequent 5 h (McLean,

1924). But when the subject was given 25 g glucose on another occasion

(the amount of glucose that theoretically could have been produced from

the 50 g protein in the 250 g meat), blood glucose concentrations increased

nearly threefold. Another study reported that ingesting 3 pounds of beef

(136 g protein, about 68 g glucose) did not increase blood glucose


concentrations over 8 h of the study in both normal and diabetic subjects

(Conn & Newburgh, 1936). Other studies also showed similar findings.

Glucose concentrations did not increase with ingestion of protein in both

normal and type 2 diabetic subjects, but there was increased insulin stimu-

lation and production (Berger & Vongaraya, 1966; Fajans, Floyd, Knopf, &

Conn, 1967; Fajans, Floyd, Knopf, Guntsche, et al., 1967).

Subjects, with and without diabetes, did not seem to show an increase in

blood glucose levels after ingestion of protein but the latter showed a stron-

ger insulin response compared to nondiabetics (Berger & Vongaraya, 1966).

Blood glucose concentrations were not exhibiting significant changes even

though there seemed to be large amounts of glucose being produced in sub-

jects and consistent rises in blood urea nitrogen (indicating protein utiliza-

tion). According to Gannon and Nuttall (2010), although the D/N ratio

estimated the maximum amount of glucose that could be produced from

an ingested protein, these amounts were not apparent under normal phys-

iological conditions. Several other studies later also showed no change in

plasma glucose concentration from protein ingestion.When normal subjects

(Krezowski et al., 1986) ingested 50 g beef protein, the plasma glucose

concentration remained unchanged during the 4 h of the study, consistent

with earlier findings (Berger & Vongaraya, 1966; Rabinowitz, Merimee,

Maffezzoli, & Burgess, 1966). In type 2 diabetic subjects (Nuttall et al.,

1984), glucose concentrationwas stable with relatively no change when pro-

tein only was given. Again, the ingestion of protein had very little effect on

glucose production in normal subjects and diabetics.

The glucose appearance rate following the ingestion of proteins in nor-

mal subjects and type 2 diabetics was investigated in two studies. Normal

subjects ingested 50 g of protein in the form of cottage cheese and the glu-

cose appearance rate in plasma was compared against a water control using a

constant infusion of 3H-glucose (Khan, Gannon, &Nuttall, 1992). The total

amount of protein deaminated and converted to urea was 29.3 g with the

glucose appearing in the circulation as a result of amino acid metabolism

was approximately 10 g (Khan et al., 1992). Based on the gluconeogenic

potential of cottage cheese (42.3 g of glucose from 50 g of cottage cheese

protein), this accounted for about 43% protein metabolized (or 23% of

the total amount of protein ingested) with the fate of the rest remaining

unknown (Khan et al., 1992). In type 2 diabetics, insulin and glucagon con-

centration also increased following protein ingestion (Gannon et al., 2001).

The net amount of glucose estimated to be produced in type 2 diabetic sub-

jects (based on the quantity of amino acids deaminated) was approximately


12 g from ingesting 50 g of lean beef meat protein. However, the amount of

glucose appearing in the circulation of this group of people was only 2 g.

The peripheral plasma glucose concentration decreased by about 1 mM after

ingestion of either protein or water, confirming that ingested protein does

not result in a net increase in glucose concentration, and results in only a

modest increase in the rate of glucose disappearance in type 2 diabetics

(Gannon et al., 2001). A much lower amount of glucose was entering the

circulation of normal and type 2 diabetic subjects from ingesting protein

and the fate of the remaining metabolized amino acids was unknown.

The metabolic response of two different protein sources was compared

by Nuttall and Gannon (1990), who looked at the ingestion of egg white

and cottage cheese alone on glycemia. Egg white stimulated a slight increase,

whereas cottage cheese stimulated a slight decrease in serum glucose concen-

tration in healthy subjects. Both stimulated an increase in serum insulin,

C-peptide, and glucagon concentrations. Although the role of individual

amino acids on these parameters was not determined, the increase in circu-

lating amino acid concentrations appeared to have stimulated these effects.

Nuttall and Gannon (1991) critically reviewed studies investigating

plasma glucose and insulin response to macronutrients in nondiabetics

and type 2 diabetics. Protein generally was identified as not affecting glucose

concentrations in both groups but protein stimulated insulin secretion, with

it being more pronounced in type 2 diabetics. The null effect of proteins on

blood glucose was ascribed to an early hypothesis stating that increased pro-

duction and release of glucose from the liver led to a concomitant increased

uptake and utilization of glucose by peripheral tissues (Unger &Orci, 1976).

Ingestion of protein results in an increased glucagon concentration that

would stimulate gluconeogenesis from amino acids in the liver. The

increased insulin concentration would also stimulate peripheral tissues to

remove glucose produced and to store it as glycogen (Unger & Orci,

1976). Studies carried out afterward, however, have shown the picture to

be more complex. For example, protein quality has been demonstrated to

affect glucagon and insulin expression.

In summary, controlled feeding studies of known amounts of proteins

did not result in the predicted increase in peripheral glucose concentration

in normal and type 2 diabetic subjects. In fact, there were decreases in blood

glucose even though amino acids can be potentially used for gluconeogen-

esis. These studies showed lower glucose concentrations in diabetic subjects

than in nondiabetics. Therefore, protein ingestion collectively appears to

have a limited effect on glycemia. Reasons for this remain unclear and


require investigation in future work. The protein source also appeared to

determine the nature of the metabolic response presumably due to differ-

ences in amino acid composition.

Protein source and composition appears to affect blood glucose concen-

trations and this was evident in a study where different beverages were con-

sumed in isovolumetric amounts before a meal. Milk (2% fat) resulted in the

lowest postmeal blood glucose compared to other beverages such as soy,

chocolate milk (1% fat), orange juice, and cow’s milk-based infant formula

(Panahi et al., 2013). Milk (2% fat) had higher carbohydrate content than soy

beverage, but the higher protein content in the former (18 g vs. 14 g)

suggested that the amount and composition of protein played a role in post-

prandial blood glucose. The chocolate milk (1% fat) had similar sugar con-

tent as orange juice but had a greater blood glucose lowering effect. This

study showed the significant glucose lowering effects of milk protein. Earlier

studies have shown that milk protein stimulates an increase in postprandial

insulin response with corresponding reduction in postprandial blood glucose

levels (Ostman, Liljeberg, & Bjorck, 2001). The glycemic and insulinemic

effects of human and bovine milk was compared with white wheat bread in

healthy subjects (Gunnerud, Heinzle, Holst, Ostman, & Bjorck, 2012).

In addition to human and bovine milk, subjects were also served test meals

consisting of reconstituted bovine whey or casein protein. All test meals

were standardized in terms of lactose content (25 g). Human milk showed

the lowest insulin response in comparison to other meals and the bovine

whey meal was the highest. Human milk was more insulinotropic per unit

protein compared to bovine milk, possibly due to the higher proportion of

whey protein. Positive correlations were seen between individual plasma

amino acids and serum insulin and plasma incretion secretion in the post-

prandial phase, as well as negative correlations with the glycemic response.

This suggests that amino acids play an important role in the insulinotropic

properties of milk proteins and contributes to the observed lower postpran-

dial glycemia. Although human milk has a lower protein content than

other milk types it is rich in whey protein. Human whey protein is a potent

GLP-1 secretagogue rendering it stronger insulinogenic properties than

bovine whey.

Glucose and insulin responses were observed in healthy women follow-

ing consumption of isocaloric meals containing protein from cod, bovine

milk, or soy (Von Post-Skagegard, Vessby, & Karlstrom, 2006). The cod

protein meal had a larger insulin response AUC than the soy protein meal.

The serum insulin response showed a larger AUC for the milk protein meal


than the cod protein meal. The insulin/C-peptide ratio was higher after the

milk protein meal compared to the cod and soy protein meals at 120 min.

The insulin/glucose ratio was lower after the cod protein meal compared to

milk and soy protein meals at 120 min. This study showed that three differ-

ent protein meals with similar protein content had different metabolic

responses. The authors attributed these differences to the effects of different

kinds of proteins on insulin secretion and/or to the insulin extraction rates in

the liver (Von Post-Skagegard et al., 2006). Also, the specific amino acid

composition in the meal may have played an important role in these differ-

ences. An earlier study by Soucy and Leblanc (1998) showed that healthy

subjects had plasma insulin levels increased significantly, with a higher

insulin/glucagon ratio, when fed with a beef rather than cod meal. The rea-

son for different insulin levels was speculated to be due to the predominant

amino acids present. Beef increased the plasma histidine levels while cod fish

gave higher levels of arginine and lysine (Soucy & Leblanc, 1998) and this is

consistent with the amino acid profiles of these proteins (Table 1.1). How-

ever, histidine alone has been shown to have little impact on insulin

(Gannon & Nuttall, 2010) and the effects seen probably may have been

due to synergistic effects. Differences in absorption, digestion, and gut fac-

tors were also proposed to be factors contributing to different insulin levels

(Soucy & Leblanc, 1998). The plasma concentrations of histidine after a fish

meal was lower compared with a beef meal in another study on healthy

males although these differences were not significant (Uhe, Collier, &

O’Dea, 1992). Plasma glucose response also declined significantly and insu-

lin secretion increased significantly for both types of proteins. However, this

study used a different type of fish (Gummy shark) compared to the study by

Soucy and Leblanc (1998) (cod) and this may explain the different outcomes.

The ratio of animal-to-plant (A/P) protein has also shown to be a poten-

tial factor affecting insulin secretion. A population-based study collected die-

tary data using validated semiquantitative food frequency questionnaires,

and associations were made between intakes of total protein as well as the

A/P protein ratio and with cardiometabolic risk factors (Mirmiran,

Hajifaraji, Bahadoran, Sarvghadi, & Azizi, 2012). The findings showed that

a higher ratio of A/P protein ratio was related with lower serum fasting

glucose.

In summary, protein source and content seem to significantly affect

their capacity to decrease postprandial glycemia. The different glucose

and insulin responses in healthy individuals after ingesting different protein

sources could be caused by their unique effects on insulin secretion,


particularly due to their specific amino acid composition. Proteins exhibit an

insulinotropic effect but differ in their capacity to stimulate insulin release.

This is possibly due to differences in incretin effects and the insulinotropicity

of amino acids. Besides specific amino acids, the amount of protein, ratio of

A/P protein, and/or differences in absorption and digestibility of the protein

source are important factors to consider.

5.1. Effect of coingesting proteins and carbohydrateson blood glucose and insulin

There are studies showing a reduction in postprandial glucose response

when proteins and carbohydrates are coingested compared to the uptake

of carbohydrates alone. Krezowski et al. (1986) reported a rapid decline

in glucose concentration and lower mean glucose in normal individuals

when protein was ingested with glucose. The synergistic effect on insulin

secretion when protein was ingested with glucose could not be confirmed

similar to an earlier finding byRabinowitz et al. (1966). The addition of glu-

cose to the protein meal resulted in a delayed rise in glucagon but subse-

quently increased to levels greater than or equal to that observed after

ingestion of protein alone.

But synergistic effects were observed in an earlier study when protein

was ingested with glucose in type 2 diabetic subjects. Nuttall et al. (1984)

gave 0, 10, 30, and 50 g protein with 50 g glucose to type 2 diabetic subjects.

With 50 g protein (from lean hamburger), a significant lowering of the glu-

cose AUC was observed as compared with the glucose treatment. The

plasma glucose area above the baseline following a glucose meal was reduced

34% when protein was given with glucose in contrast to the glucose con-

centration remaining unchanged and then declining when only protein

was given. The mean insulin area was considerably greater when glucose

was givenwith protein than when glucose or protein was given alone.When

various amounts of protein were given with 50 g glucose, the insulin area

response was essentially first order. Following a second glucose þ protein

meal, the plasma glucose area was markedly reduced, being only 7% as large

as after the first meal, suggesting an additive beneficial effect when proteins

and carbohydrates are repeatedly consumed. The glucagon response was not

measured in this study.

As a follow up, Gannon et al. (1988) looked at the metabolic response in

type 2 diabetic subjects given single breakfast meals consisting of 50 g glu-

cose, or 50 g glucose plus 25 g protein in the form of lean beef, turkey, gel-

atin, egg white, cottage cheese, fish, or soy. Plasma insulin concentrations


increased further and remained elevated compared to glucose ingested alone.

The relative area under the insulin response curve was greatest for cottage

cheese ingested with glucose (360%) and least with egg white ingested with

glucose (190%), compared to glucose alone. Overall, each of the seven dif-

ferent protein sources ingested with glucose resulted in a reduced glucose

response with the exception of the egg white meal. The glucagon concen-

tration increased following protein þ glucose meals, in contrast to glucose

alone which had decreased glucagon concentrations. The 5-h insulin

response to the ingested protein was similar from these different sources

of proteins except the magnitude of the response varied greatly between cot-

tage cheeseþglucose and egg whiteþglucose, which was speculated to be

due to the difference in amino acid composition of the proteins. The simul-

taneous ingestion of glucose with cottage cheese or egg white protein

decreased the glucose area response by 11% and 20%, respectively in another

study by Gannon et al. (1992). Similar to Gannon et al. (1988), the insulin

area response was also greater after ingesting cottage cheese compared to egg

white suggesting that the glucose lowering effect was insulin mediated. The

insulin area response was twofold from ingesting cottage cheese in normal

subjects (Nuttall & Gannon, 1990) but in this study it was up to fourfold

higher for type 2 diabetics. Thus in subjects with diabetes, cottage cheese

was a more potent insulin secretagogue relative to egg white protein than

in normal individuals. The glucagon area response to both protein sources

were similar (Gannon et al., 1992); but in normal subjects, cottage cheese

protein showed a twofold greater glucagon response than egg white protein

(Nuttall & Gannon, 1990). The metabolic response by type 2 diabetics to

different proteins coingested with glucose is therefore markedly different

to that of normal individuals.

In vitro, whey had an insulinogenic effect by preferential elevation of

plasma concentrations of plasma amino acids, GIP and GLP-1 responses

(Salehi et al., 2012). Whey protein showed insulinotropic and glucose

lowering effects in several in vivo studies. The addition of a whey protein

supplement to a 50-g glucose drink reduced postprandial glycemia in a

dose-dependent manner (Petersen et al., 2009). Another study showed that

when whey protein (10–40 g) was consumed before a high carbohydrate

meal, blood glucose and insulin levels reduced in a dose-dependent manner

(Akhavan, Luhovyy, Brown, Cho, & Anderson, 2010). Whey protein con-

sumed in relatively small amounts just before a meal reduced postmeal blood

glucose while reducing insulin response. However, in another study, when

whey protein was consumed before a carbohydrate meal, insulin and


incretin hormone secretion was stimulated and slowed gastric emptying

which led to a marked reduction in postprandial glycemia in type 2 diabetics

(Ma et al., 2009). This is in agreement with other studies showing that

whey is insulinotropic (Nilsson et al., 2004). In the study by Akhavan

et al. (2010), a whey hydrolysate which was used as a comparator did not

reduce blood glucose response compared to whey protein suggesting that

whole proteins also possibly stimulate noninsulinotropic hypoglycemic

pathways. Premeal protein ingestion may be a useful strategy for blood glu-

cose control in healthy and diabetic individuals. This is an area worthy of

further exploration.

The blood glucose lowering and insulinotropic effect of whey protein

was also seen in type 2 diabetic subjects. The addition of whey to high gly-

cemic index meals was found to improve blood glucose control in type 2

diabetics (Frid, Nilsson, Holst, & Bjorck, 2005). A test meal consisting of

rapidly digested and absorbed carbohydrates supplemented with whey stim-

ulated insulin release and reduced postprandial blood glucose excursions

compared to the same meal which was supplemented with lean ham and

lactose in place of whey (Frid et al., 2005). Whey protein used in the test

meal also produced postprandial GIP responses which were higher than

when lean ham was used (Frid et al., 2005). A study by Ang, Muller,

Wagenlehner, Pilatz, and Linn (2012) on type 2 diabetics found no signif-

icant differences in glucose responses after ingestion of drinks containing

slowly digested isomaltulose with different protein types (i.e., fast-absorbing

whey/soy vs. slow-absorbing casein). But the results suggested that proteins

increased postprandial insulin and that insulin action was lower for the fast-

absorbing whey/soy than for slow-absorbing casein. A fast-absorbing pro-

tein mixture therefore does not appear to be beneficial for glycemic control

in type 2 diabetic patients as it reduces insulin action to a greater extent than

slow-absorbing proteins. Fast and slow absorbed dietary proteins have also

shown to have dissimilar digestion kinetics (Bos et al., 2003) and differen-

tially affect protein anabolism and oxidation (Boirie et al., 1997). Therefore,

the absorption rate of proteins appears to have significant implications in

insulin secretion and glucose metabolism (Calbet & Maclean, 2002).

Using healthy individuals Karamanlis et al. (2007) compared the

glycaemic response to drinks containing either glucose, gelatin or glucose+

gelatin Karamanlis et al. (2007). The blood glucose response was less after

glucoseþgelatin than after glucose alone. The study also showed that gastric

emptying rate was lowest when proteins and carbohydrates were coingested.

The rate of gastric emptying and incretin, hormone (GLP-1 and GIP)


responses are known to be major determinants of postprandial blood glucose

excursions (Rayner, Samsom, Jones, & Horowitz, 2001). Therefore, it

appears that protein ingested with carbohydrates can reduce glycemic

response partly by slowing the rate of gastric emptying.

Moghaddam, Vogt, andWolever (2006) observed a dose–response effect

on glycemic response and insulin sensitivity in nondiabetic individuals when

fed soy protein (0, 5, 10, or 30 g) after intake of 50 g glucose. A dose of 30 g

soy protein caused a significant reduction in glycemic response. Other stud-

ies have investigated the impact of coingesting more than one protein

type with glucose on glycemia. Spiller et al. (1987) evaluated increasing

levels of two combined proteins (milk protein: soy protein 1: 2 ratio,

amount ranging between 0 and 50 g) along with 58 g carbohydrate

(maltodextrinþ fructoseþ lactose) on serum glucose and insulin. Mean

AUC above fasting for glucose decreased with increasing protein dose. Pro-

tein appeared to exert a dose-dependant effect on glucose response in normal

fasted subjects fed test meals consisting primarily of carbohydrate.

In contrast, the serum insulin curves did not show a protein dose-dependent

effect (Spiller et al., 1987). But overall, protein increased the insulin response

compared to the meal with no protein. Increasing levels of protein and its

impact on postprandial blood glucose has also been studied in type 2 dia-

betics. A diet consisting of 30% protein was able to reduce postprandial

blood glucose and improve overall glucose control compared to the nation-

ally recommended 15% protein diet (control) in a sample of 12 adults with

type 2 diabetes (Gannon et al., 2003). Similar results were observed by

Nuttall et al. (1984).

Other studies have observed no protein dose–response effects on blood

glucose. Various amounts of protein (10, 30, 50 g of lean hamburger) plus

glucose (50 g) ingested by healthy subjects did not alter serum glucose

response compared to that observed with 50 g glucose alone (Westphal,

Gannon, &Nuttall, 1990). Interestingly, there was also no increase in insulin

concentration when proteins were ingested with glucose, except in the 50 g

protein treatment where a modest but prolonged increment was seen. The

sum of the insulin areas for the meals containing either 50 g protein or 50 g

glucose was 100% that of the combined protein and glucose treatment, indi-

cating that these insulin responses were additive (Westphal et al., 1990).

However, Nuttall et al. (1984) observed that subjects with type 2 diabetes

showed a linear relationship between the quantity of protein ingested and

the insulin response, suggesting that the insulin response is much more sen-

sitive to protein quantity in persons with noninsulin-dependent diabetes.


With glucose present in the protein meal, there was no significant rise

in glucagon concentration for �60 min, regardless of protein dose

(Westphal et al., 1990). This data support previous studies showing that

the ingestion of either protein or glucose results in glucagon stimulation

and suppression, respectively (Muller, Faloona, Aguilarparada, & Unger,

1970). Circulating glucagon concentrations also appear to depend on the

ratio of protein to carbohydrate in the meal (Ahmed, Nuttall, Gannon, &

Lamusga, 1980).

Some studies have shown that protein hydrolysates enhance postprandial

insulin response and reduce postprandial serum glucose levels in type 2

diabetics ( Jonker et al., 2011; Manders, Koopman, et al., 2006; Manders,

Praet, et al., 2006; Manders et al., 2005; van Loon et al., 2003).These studies

showed that although the b-cell response to glucose is impaired in this

group, the insulin secretory response to amino acids remains functional.

In addition, hydrolysates are advantageous in that they have faster digestion

and quicker availability of amino acids than whole proteins (Koopman

et al., 2009).

Overall, the metabolic response to ingested protein alone either results in

little or no increase in blood glucose concentrations. Protein coingested with

glucose increases insulin secretion and reduces plasma glucose rise and this is

more apparent in diabetics than healthy individuals. Protein ingestion stim-

ulates a rise in glucagon secretion and is suppressed in the presence of glucose

ingestion. Proteins could stimulate a rise in insulin and glucagon, but these

effects depend on protein type and quantity. The glucose and insulin

responses also appear to have a dose-dependent relationship with the quan-

tity of protein (and carbohydrate) ingested. However, there is inadequate

data regarding the extent and limits of this relationship.

5.2. Effect of amino acids on glycemiaStudies on dietary proteins have made it clear that they have different effects

on circulating glucose, insulin, and glucagon concentrations both when

consumed alone or with glucose. Similarly, amino acid type has also been

shown to significantly affect insulin secretion (section 4). This section will

focus on the effects of amino acids on glycemia.

Amino acids released from proteins can be gluconeogenic, insulinogenic,

or both.Gluconeogenic amino acids directly contribute to de novo synthesis of

glucose and participate in recycling of glucose carbon via the glucose-amine

cycle. These amino acids give rise to a net production of pyruvate or TCA


cycle intermediates such as a-ketoglutarate and oxaloacetate, all of which aregluconeogenic precursors of glucose. The literature contains reports of both

positive and negative impact of amino acids, ingested with or without glu-

cose, on glycemic regulation.

Studies have reported that the intravenous administration of amino acids

decrease glucose disposal, induce hyperinsulinemia and hyperglycemia, and

potentially lead to insulin resistance (Ferrannini & Mari, 1998; Krebs et al.,

2002). Intravenous administration of individual amino acids and mixtures of

amino acids were accompanied by large increases in blood glucose with

acute increases in plasma amino acids (Floyd et al., 1966a). A decreased glu-

cose uptake and increased secretion of insulin and glucagon were also

observed. Gannon and Nuttall (2010) pointed out the importance of study-

ing the effect of amino acids on the glucose concentration when adminis-

tered orally rather than intravenously, since some amino acids are

metabolized by the intestinal cells and liver, and others pass through the liver

into the peripheral circulation.

Early studies showed that alanine given to obese subjects prior to ther-

apeutic starvation resulted in an increased insulin concentration and

decreased glucose concentration (Genuth, 1973). When oral doses of gly-

cine were given to normal and diabetic adults, a moderate reduction in

blood sugar concentrations was observed in both groups (Cochrane et al.,

1956). Leucine administered orally resulted in a decrease in blood glucose

in most of the subjects and an increase in plasma insulin (Fajans et al.,

1963). Phenylalanine (7 g) when ingested by normal-weight men increased

glucagon and insulin concentrations but showed no change in plasma glu-

cose concentrations (Guttler et al., 1978). These studies conclusively show

that amino acids have different effects on glucose and insulin. They also

suggest that amino acids have a greater effect on insulin secretion than on

glucose modulation.

In an earlier study, gelatin, when ingested with glucose, strongly

potentiated a glucose-stimulated increase in insulin in type 2 diabetics

(Gannon et al., 1988). A study was later carried out to determine if gly-

cine (the major amino acid in gelatin) stimulated insulin secretion or

reduced glucose response when ingested with glucose (Gannon et al.,

2002b). The results showed that glycine when ingested with glucose

did not significantly affect the plasma glucose concentration, similar to

earlier studies (quoted by Cochrane et al., 1956). When glycine was

ingested with glucose, the insulin peak occurred later and was slightly less

than when glucose was ingested alone (with glucose, there was a rapid rise


in insulin concentration that corresponded with a rise in glucose con-

centration). This suggests that glycine alone cannot be credited for the

insulinotropic effect of gelatin.

Intravenous studies showed that out of a mix of amino acids, arginine was

the most potent in increasing circulating insulin concentrations and decreas-

ing blood glucose in healthy subjects (Floyd et al., 1966a). Arginine admin-

istered with glucose attenuated blood glucose concentrations. In another

study, Floyd et al. (1970) showed that arginine brought about the greatest

insulin release in healthy subjects compared to histidine and leucine

(all given as 30 g doses). Arginine given as an intraduodenal infusion to

healthy volunteers resulted in an increase in serum insulin while blood

glucose concentrations did not change (Dupre et al., 1968). The effect of

L-arginine administered orally on blood glucose concentrations in healthy

subjects was investigated in another study (Gannon et al., 2002b). L-arginine

was given with and without glucose on two separate occasions. When

L-arginine was ingested with glucose, it attenuated and prolonged the blood

glucose rise. The study showed that arginine did not stimulate an increase in

insulin when ingested alone and neither did it synergize when ingested with

glucose to stimulate insulin secretion. Arginine was shown to induce large

increases in plasma citrulline along with reduced glucose production

(Apostol & Tayek, 2003). The production of citrulline and nitric oxide from

arginine seemed to play important roles in blood glucose regulation.

An in vitro study with isolated rat islets in 1980 showed that in the presence

of glucose, arginine potentiated the effect of glucose which increased with

increasing amino acid concentrations (Khalid & Rahman, 1980). Other

in vitro studies showed that the stimulation of insulin by arginine was depen-

dent on the ambient glucose concentration (Blachier et al., 1989; Levin,

Grodsky, Hagura, Smith, & Forsham, 1972). However, human studies

showed that arginine ingested with glucose did not stimulate insulin secre-

tion and attenuated the blood glucose rise (Gannon et al., 2002a,2002b).

This attenuation was not caused by delayed gastric emptying and the mech-

anism remains to be determined. Arginine also stimulated a modest increase

in glucagon concentration when ingested alone, and a decrease when

ingested with glucose (Gannon et al., 2002b). Arginine’s effects on insulin

secretion remains equivocal. However, it appears to play a notable role in

blood glucose control through its effects on metabolic pathways.

Leucine has been long known as a potent insulin secretagogue (Fajans

et al., 1963; Floyd et al., 1966a; McArthur et al., 1963). Intravenous

administration of 30 g leucine with 30 g glucose was reported to


synergistically increase plasma insulin concentrations but not affect blood

glucose (Floyd et al., 1970). Later studies also showed similar findings.

Kalogeropoulou et al. (2008) showed that leucine when ingested with glu-

cose modified glycemia, insulin, and glucagon response in healthy subjects.

On its own, leucine did not affect serum glucose concentrations but mod-

estly increased insulin. When leucine was ingested with glucose, a strong

attenuation in glucose response (by 50%) was seen. A synergistic effect of

ingested glucose with leucine on insulin secretion was observed whereby

the insulin area response increased by an additional 66%. Leucine alone

increased glucagon concentration while coingestion with glucose attenuated

the decrease in expected glucagon when glucose alone was ingested

(Kalogeropoulou et al., 2008). Leucine stimulated pancreatic insulin release

and therefore decreased plasma glucose concentrations when ingested with

glucose. This synergistic effect could either be a direct effect of leucine or

through a leucine-stimulated release of gut incretins. In vitro studies have also

shown that leucine is an insulin secretagogue (Newsholme et al., 2005; Xu,

Kwon, Cruz, Marshall, & McDaniel, 2001) and improves blood glucose

clearance (Nishitani et al., 2002).

Isoleucine when ingested by healthy subjects also showed similar results

as leucine (Nuttall, Schweim, et al., 2008). When isoleucine was ingested

alone, it decreased glucose but did not affect insulin. When isoleucine

was ingested with glucose, a lower plasma concentration of the amino acid

was observed. However, insulin expression was increased by 43%more than

that following ingestion of glucose alone. The glucose increase was less and it

decreasedmore rapidly following isoleucineþ glucose, compared to glucose

alone. Consequently, the glucose area response was markedly attenuated and

isoleucine had little effect on the glucagon concentration. Isoleucine alone

does not appear to stimulate insulin and glucagon but seems to decrease glu-

cose concentrations. But when ingested with glucose, insulin secretion was

stimulated and glucose response decreased. This suggests that isoleucine

modulates blood glucose both through insulin-dependent and -independent

mechanisms.

Lysine ingestion showed no increases in blood glucose in healthy subjects

in one study (Floyd et al., 1966a). Another recent study also showed only a

slight decrease in serum glucose in healthy subjects (Kalogeropoulou et al.,

2009). But the authors observed an increase in glucagon and insulin secretions.

Floyd and colleagues observed that lysine was also potent in stimulating

the release of insulin (Floyd et al., 1966a). When lysine was ingested with

glucose, there was a striking attenuation of the glucose response without an


accompanying increase in insulin response, which was a different response to

that seen with leucine (Kalogeropoulou et al., 2008) and isoleucine (Nuttall,

Schweim and Gannon, 2008). Lysine ingestion with glucose also decreased

glucagon concentrations (Kalogeropoulou et al., 2009). It appears that lysine

attenuates blood glucose through noninsulin-mediated pathways.

The metabolic response to proline with and without glucose in

nondiabetic subjects was reported by Nuttall et al. (2004). Proline ingestion

without glucose increased plasma proline concentrations 13-fold with no

change or a slight decrease in circulating glucose. However, plasma proline

concentrations are decreased by 50% when proline was coingested with glu-

cose. Ingestion of proline with glucose also attenuated the glucose response

and did not affect the insulin response compared with glucose alone. Proline

also facilitated a glucose-stimulated decrease in glucagon concentration.

Therefore, proline appears to be having a noninsulin-mediated hypoglyce-

mic effect.

Ingestion of phenylalanine alone increased glucagon in healthy subjects

but had only a modest effect on insulin (Nuttall et al., 2006). It did not affect

blood glucose concentrations compared to water. Ingestion of phenylala-

nine with glucose showed that the plasma glucose area response was

decreased by 66% and insulin area responses were the sum of the responses

to phenylalanine alone and glucose alone. Phenylalanine seems to notably

attenuate the glucose-induced rise in plasma glucose when ingested with

glucose.

In an attempt to determine the metabolic effects of glutamine, Greenfield

et al. (2009) fed glutamineþwater, glucoseþwater and water to normal,

obese, and obese subjects with impaired glucose tolerance over three sepa-

rate days in random order. The glucoseþwater as expected showed a mar-

ked increase in plasma insulin concentrations for all three groups.

Glutamineþwater increased insulin and glucagon significantly in normal,

obese, and obese subjects with impaired glucose tolerance but did not affect

blood glucose concentrations in all three groups (Greenfield et al., 2009).

However, the insulin and glucagon response to glutamineþwater was

greatest in obese subjects with impaired glucose tolerance, followed by obese

and then normal subjects. This concurs with other studies showing that

amino acid-mediated insulin and glucagon expression is hyperstimulated

in those with impaired glucose tolerance.

The effect of coingesting amino acids and/or protein with carbohydrate

mixtures on blood glucose was studied by a few investigators. A mixture

of amino acids (leucine, isoleucine, valine, lysine, and threonine) was


compared to whey to study if the former would have similar or better

insulinotropic properties than the latter in healthy subjects (Nilsson,

Holst, & Bjorck, 2007). The amino acid mixture resulted in similar glycemic

and insulinemic responses as whey with no additional effects of GIP and

GLP-1. An increased insulin-stimulated glucose disposal and reduced post-

prandial glucose concentration was observed in type 2 diabetics when they

coingested a carbohydrate with a mixture of casein hydrolysate and amino

acids (leucine and phenylalanine;Manders, Koopman, et al., 2006;Manders,

Praet, et al., 2006; Manders et al., 2005). In another study, a beverage con-

sisting of an amino acid mixture and carbohydrate (isoleucine, leucine, cys-

teine, methionine, valineþ100 g dextrose) was compared to a control

(100 g dextrose only) to observe if there was an improvement in the glucose

response of healthy overweight subjects to an oral glucose tolerance test

(OGTT;Wang et al., 2012). Interestingly, plasma glucagon was significantly

higher than the control treatment, but a reduction in plasma glucose

response was also observed. There were no differences in the plasma insulin

responses between treatments. The amino acid mixture appeared to lower

the glucose response to an OGTT in subjects in an insulin-independent

manner which was consistent with Nuttall, Schweim, et al. (2008) and

in vitro studies (Doi, Yamaoka, Fukunaga, & Nakayama, 2003; Nishitani

et al., 2002). The insulinotropic properties of protein and/or amino acid

mixtures (with a carbohydrate) could have clinical significance in the treat-

ment of type 2 diabetes by accelerating blood glucose disposal through both

insulin-dependent and -independent mechanisms.

In summary, the literature shows that amino acids alone have little effects

on blood glucose and insulin. However, some such as BCAAs, glutamine,

and phenylalanine have glucose and insulin modulating effects when given

alone. Amino acids when consumed in combination with carbohydrates

show greater effects on blood glucose and insulin. Specific amino acids

showing notable effects are arginine, proline, phenylalanine, glutamine,

and BCAAs.

6. CONCLUSIONS

The review reaffirms the notable effects proteins and amino acids exert

on insulin and glycemia. Proteins and amino acids influence insulin secretion

and glycemic control by directly stimulating b-cells and activating insulin

secretary mechanisms, by stimulating incretin responses, modulating


gluconeogenesis in liver and kidney, and by influencing gastric emptying.

Dietary proteins when consumed alone appear to increase insulin expression

although not as much as glucose. They have no impact on blood glucose.

The metabolic effects of proteins are conditional upon their quality. Dairy

proteins and whey protein, in particular, appears to be significantly

insulinotropic. While animal and fish proteins have also shown to be mod-

erately insulinotropic, data on vegetable proteins are equivocal. The com-

bined ingestion of proteins and carbohydrates appear to markedly attenuate

blood glucose and hyperstimulate insulin secretion. These effects also

depend upon protein quality. The reduction in glycemia and increase in

insulinemia are more marked in type 2 diabetics compared to normal indi-

viduals. Proteins may also be attenuating the glycemic response by slowing

gastric emptying. There is evidence to suggest that the ratio of animal and

plant protein in a meal can affect insulin secretions.

Individual amino acids also appear to play an important role in glucose

metabolism. The majority of amino acids when given alone at least modestly

affected insulin secretion. A few such as BCAAs and phenylalanine appear to

have a greater effect on insulin secretion. Only BCAAs seem to be able to

attenuate the blood glucose response when ingested alone. The glucose low-

ering and insulin secretion effects of amino acids are enhanced when they are

coingested with glucose. Amino acids such as arginine, proline, phenylala-

nine, glutamine, and BCAAs have greater glucose attenuating effects in this

regard. Arginine, BCAAs, and glutamine appear to be better insulin secre-

tagogues when taken in combinationwith glucose. The effect of amino acids

on glucose and insulin depends on amino acid type, mode of administration

(intravenous or oral), dosage, and combination. Oral administration of

amino acids stimulates glucose homeostasis metabolic pathways (involving

incretins and the liver) that intravenous dosages may bypass. While physio-

logically greater doses of amino acids often show better effects, amounts rep-

resentative of quantities typically found in food sometimes may show a lesser

impact. Amino acid combinations have been shown to have a significantly

greater effect on glycemia and insulin secretion than any given alone. This

highlights the significance of synergistic effects and the importance and

practical usefulness of considering amino acid combinations rather than

singular types.

These studies collectively demonstrate the complexity of protein’s effects

on glucose homeostasis and the equivocal nature of the current state of the

art. The literature so far produces as many questions as it does answers.

A large amount of the basic knowledge in this area comes from studies


carried out over 50 years ago. These studies are often poor in design in terms

of subject numbers, their characteristics, and experimental control. Some of

them need repeating taking advantage of modern experimental methods and

knowledge, and with greater rigor. Although some groups have attempted

to carry out systematic series of studies to investigate effects of proteins and

amino acids on glycemia and insulin, there is still much that is unknown. For

example, systematic studies on lesser investigated amino acids are required.

Very little is also known how novel dietary protein types (isolates, hydroly-

sates, concentrates, novel plant protein extracts, etc.) and processing

methods affect proteins’ impact on blood glucose and insulin. A systematic

line of studies targeted at investigating the synergistic effects of both amino

acids and proteins is crucial. There is also very little data on the metabolic

effects of dietary protein combinations both in diabetics and nondiabetics.

Similarly, dose–response studies must be initiated to discern the effects of

protein quantity, quality, and their combination on blood glucose and insu-

lin. To our knowledge, no studies have so far looked at the long-term effects

of protein quality on glycemia and insulin secretion. This aspect of research

must receive attention as it will also firmly establish the long-term therapeu-

tic potential of proteins in glycemic control and disease management.

A point of concern is the contrasting data often seen between in vitro and

in vivo studies. While human studies may be considered the gold standard

of evidence, in vitro work is useful in understanding the mechanistic aspects.

It is imperative that future research focuses on narrowing the divide between

in vitro and in vivo findings so that a more clarified picture can be elucidated.

Specific dietary guidelines for protein quality are yet to be developed for dia-

betics and nondiabetics. This review highlights the importance of more

research before firm guidelines and recommendations can be established.

ACKNOWLEDGMENTThe authors are grateful to the Singapore Institute for Clinical Sciences for facilitating this

chapter.

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