The Potential Catalytic Effects of Fructose and its Epimers on Postprandial Carbohydrate

Metabolism in Subjects without Diabetes and a Systematic Review and Meta-analysis in all

Subjects

By

Catherine Rose Braunstein

A thesis submitted in conformity with the requirements

for the degree of Master of Science

Department of Nutritional Sciences

University of Toronto

© Catherine Rose Braunstein (2017)

ii

The Potential Catalytic Effects of Fructose and its Epimers on Postprandial Carbohydrate

Metabolism in Subjects without Diabetes and a Systematic Review and Meta-analysis in all

Subjects

Catherine Rose Braunstein

Master of Science

Department of Nutritional Sciences

University of Toronto

2017

ABSTRACT

The objective was to determine the ‘catalytic’ (doses

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ACKNOWLEDGMENTS

I could not have made it through this journey without the love and support of my family.

I am especially grateful for my parents Karl and Alison, and my sister, Caroline, who is my

closest and truest ally. My parents have put up with all of my worries and concerns, while

reminding me to lighten up and enjoy the journey. Their guidance helped me navigate the most

exciting and challenging 2 years of my life. They taught me what it means to work hard through

your weaknesses and face challenges head on, without their example I do not know where I

would be today. And as for Caroline, she is beyond her years in terms of her wisdom and

thoughtful advice that has kept me sane and grounded, especially in the last few years.

I would like to thank my lab members Jarvis Noronha, Vivian Choo, Effie Viguiliouk,

Vanessa Ha, Sarah Stewart, Rodney Au Yeung, Stephanie Nishi, Sonia Blanco Mejia, Tauseef

Kahn, Andrea Glenn, and Rebecca Noseworthy for all of them made it a joy to come to lab every

day. Each one of them continues to inspire me in my future endeavors and I feel truly grateful to

have known them. I am also forever indebted to Chris Ireland and Tauseef Kahn, without whom

I would not have learned some valuable skills in statistical analysis. And without Laura

Chiavaroli, I would never have gotten my start as a volunteer with this amazing group of people,

so I owe a lot of thanks to her.

The FACE trial could not have been efficiently and expertly executed without Andrea

Glenn, the research coordinator for the study and Bonnie Kennedy, the study nurse for the FACE

Trial. I would also like to thank Teruko Kishibe, Information Specialist, Scotiabank Health

Sciences Library at St. Michael’s Hospital, for her help in the development of search terms used

in the systematic review and meta-analysis presented in this thesis.

I am very grateful to my friends, new and old, in the Department of Nutritional Sciences.

Without their lighthearted fun, laughter, and support I wouldn’t have made so many great

memories and new friends. I am especially grateful for Olivia Moran, who has been a great

friend all these years throughout our undergraduate and graduate degrees at the University of

Toronto.

Further, I would thank my committee members Dr. Robert Josse and Dr. Thomas

Wolever, you have been instrumental guides during my time in the program. I am especially

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grateful to my external advisor, Dr. David Jenkins for his wisdom and encouragement over the

last few years. He has taught me what it means to be a passionate, dedicated scientist that stops at

nothing to get his research done. I can only hope that one day I will be at least half as

knowledgeable and passionate as he is about nutrition research.

Last but not least of all I owe the most gratitude to Dr. John Sievenpiper, who has been a

great supervisor to me over the last 3 years, starting from my undergraduate research to graduate

studies. John has always been a great mentor and it has truly been a pleasure to learn from, and

work with him.

Funding disclosure: The Tate and Lyle Nutritional Research Fund at the University of Toronto,

Banting & Best Diabetes Centre, Canadian Diabetes Association, and PSI foundation.

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TABLE OF CONTENTS

ABSTRACT……………………………………………………………………………………...ii

ACKNOWLEDGMENTS……………………………………………………………………...iii

TABLE OF CONTENTS…………………………………………………………………….….v

LIST OF ABBREVIATIONS…………………………………………………………………viii

LIST OF TABLES……………………………………………………………………………….x

LIST OF FIGURES……………………………………………………………………………..xi

CHAPTER I-INTRODUCTON…………………………………………………………………1

1.1 CATALYTIC FRUCTOSE AND ITS EPIMERS…………………………………………….2

CHAPTER II-LITERATURE REVIEW………………………………………………………4

2.1 FRUCTOSE AND ITS EPIMERS IN THE CONTEXT OF DIABETES AND

NUTRITION……………………………………………………………………………………..5

2.2 PATHOPHYSIOLOGY OF DIABETES……………………………………...…………...5

2.3 MEASURES OF INSULIN REGULATION……………………………..…………….......7

2.3.1 HOMA-IR…………………………………………………………………...………7

2.3.2 MATSUDA INSULIN SECRETION INDEX……………………………………...8

2.3.3 EARLY INSULIN SECRETION INDEX…………………………….………….....8

2.4 FRUCTOSE AND ITS EPIMERS: ALLULOSE, TAGATOSE, SORBOSE……….…...9

2.4.1 FRUCTOSE AND DIABETES…………………………………………………………......9

2.4.2 FRUCTOSE AND ITS EPIMERS………………………………………………………...12

2.4.2.1 ROLE OF FRUCTOSE AND ITS EPIMERS IN GLUCOSE

HOMEOSTASIS...........................................................................................................................12

2.4.3 ‘CATALYTIC’ DOSES OF FRUCTOSE…………………………………………………14

2.4.3.1 METABOLISM, ABSORPTION AND DIGESTION…………………………..15

2.4.3.2 ‘CATALYTIC’ FRUCTOSE AND GLYCEMIC CONTROL: ACUTE HUMAN

FEEDING TRIALS……………………………………………………………………...16

2.5 ALLULOSE…………………………………………………………………………………17

2.5.1 ABSORPTION AND DIGESTION: ANIMAL AND HUMAN STUDIES……………....18

2.5.2 ‘CATAYLTIC’ DOSES OF ALLULOSE IN ANIMALSTUDIES……………………….19

2.5.3 EFFECTS ON PLASMA GLUCOSE AND INSULIN: ANIMAL STUDIES…………....20

2.5.4 ACUTE FEEDING TRIALS IN HUMANS AT ‘CATALYTIC’ DOSES………………..21

2.6 TAGATOSE………………………………………………………………………………...22

2.6.1 ABSORPTION AND DIGESTION: ANIMAL AND HUMAN STUDIES………………23

2.6.2 EFFECTS ON PLASMA GLUCOSE AND INSULIN…………………………………....25

2.6.3 MECHANISM OF POSTPRANDIAL GLYCEMIC CONTROL……………………..….26

2.6.4 ‘CATALYTIC’ DOSES OF TAGATOSE IN ANIMAL STUDIES………………………26

2.6.5 ACUTE FEEDING TRIALS IN HUMANS AT ‘CATALYTIC’ DOSES………………..27

2.7 SORBOSE…………………………………………………………………………………...29

2.7.1 ABSOPRTION AND DIGESTION……………………………………………………….30

2.7.2 ‘CATALYTIC’ DOSES IN ANIMAL STUDIES…………………………………………30

2.8 GAPS IN THE LITERATURE……………………………………………………………31

CHAPTER III- RATIONALE AND OBJECTIVES…………………………………………32

3.1 RATIONALE……………………………………………………………………………...…33

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3.2 OBJECTIVES……………………………………………………………………………..…33

CHAPTER IV- AN ACUTE RANDOMIZED DOSE-FINDING EQUIVALENCE TRIAL

OF SMALL, CATALYTIC DOSES OF FRUCTOSE AND ALLULOSE ON

POSTPRANDIAL CARBOHYDRATE METABOLISM IN HEALTHY PARTICIPANTS:

THE FRUCTOSE AND ALLULOSE CATALYTIC EFFECTS (FACE) STUDY………35

CHAPTER V- THE ACUTE EFFECTS OF ‘CATALYTIC’ DOSES OF FRUCTOSE AND

ITS EPIMERS ON POSTPRANDIAL CARBOHYDRATE METABOLISM: A

SYSTEMATIC REVIEW AND META-ANALYSIS OF SINGLE-MEAL FEEDING

TRIALS…………………………………………………………………………………………59

CHAPTER VI-OVERALL DISCUSSION AND LIMITATIONS………………………...106

6.1 OVERALL DISCUSSION…………………………………………………………………107

6.2 OVERALL LIMITATIONS…………………………………….…………...……………..109

6.2.1 FACE TRIAL……………………………………………………………………………..109

6.2.2 SYSTEMATIC REVIEW AND META-ANALYSIS……………………………………109

6.3 MECHANISM OF ACTION…………………………………………………………….....109

6.4 CLINICAL IMPLICATIONS………………………………………………………………111

6.5 FUTURE DIRECTIONS…………………………………………………………………...111

CHAPTER VII-CONCLUSIONS…………………………………………………………....113

CHAPTER VII-REFERENCES…………………………………………………………..…115

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LIST OF ABBREVIATIONS

ALT- Alanine aminotransferase

A1P- Allulose-1-phosphate

AUC- Area under the curve

BMI- Body mass index

BW- body weight

CDA- Canadian Diabetes Association

CV- Coefficient of variation

d- Days

DDCT- Diabetes Control and Complications Trial

FACE- Fructose and allulose catalytic effects trial

FAS- Fatty acid synthase

FDA- Food and Drug Administration

FPG- Fasting plasma glucose

F1P- Fructose-1-phosphate

F6P- Fructose-6-phosphate

g- Grams

g/d- Grams per day

g/kg BW- Grams per kilogram of body weight

g/kg BW/d- Grams per kilogram of body weight per day

GDM- Gestational diabetes mellitus

GI- glycemic index

GLUT4- Glucose transporter 4

GRAS- Generally Recognized As Safe

GK- Glucokinase

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GKRP-Glucokinase regulatory protein

G6P- Glucose-6-phosphate

G6PDH- Glucose-6-phosphate dehydrogenase

h- Hours

HAART- Highly active anti-retroviral therapy

HbA1c- Hemoglobin A1c

HDL- High density lipoprotein

HFCS- High fructose corn syrup

HOMA-IR- Homeostasis model assessment-insulin resistance

iAUC- Incremental area under the curve

IBS- Irritable bowel syndrome

IFG- Impaired fasting glucose

IGT- Impaired glucose tolerance

IHCL- Intrahepatocellular lipid

kcal- Kilo-calories

LDL-C- Low-density lipoprotein-cholesterol

Matsuda ISIOGTT- Matsuda insulin secretion index

MD- Mean difference

min- Minutes

mmol/L- millimoles per litre

mo- Months

mOsmol- milliosmol

MODY- Maturity on-set diabetes of the young

NIDDM- Non-insulin-dependent (type 2) diabetes mellitus

OGTT- Oral glucose tolerance test

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∆PI30-0/∆PG30-0- Early insulin secretion index

pmol/L- Picomoles per litre

SCFA’s- Short chain fatty acids

SRMA- Systematic review and meta-analysis

SMD-Standardized mean difference

S6P- Sorbitol-6-phosphate

TAG- Triacylgylcerol

TG-Triglycerides

TID- Latin: ter in die (english: three times a day)

TOST- Two one sided tests

T1DM- Type 1 diabetes mellitus

T2DM- Type 2 diabetes mellitus

US- United States

wk- Weeks

wt- Weight

y- Years

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LIST OF TABLES

CHAPTER II

Table 2.1. Risk factors for type 2 diabetes mellitus (adapted from 2013 CDA clinical practice

guidelines)).

CHAPTER IV

Table 4.1. Baseline characteristics of 25 participants in the FACE Trial.

Table 4.2. Primary and secondary outcomes for the effects of fructose on postprandial

carbohydrate metabolism.

Table 4.3. Primary and secondary outcomes for the effects of allulose on postprandial

carbohydrate metabolism.

Supplementary table 4.1. Self-reported adverse events showed no pattern associated with the

treatment type.

CHAPTER V

Table 5.1. Search strategy.

Table 5.2. Characteristics of studies investigating the effects of fructose, allulose, tagatose and

sorbose on postprandial carbohydrate metabolism.

Table 5.3. Continuous subgroup analyses for fructose and tagatose trials with the endpoint of

glucose iAUC.

Supplementary table 5.1. Dose response meta-regression analysis for the effect of tagatose on

glucose iAUC.

Supplementary table 5.2 A-B. Dose response meta-regression analysis for the effect of allulose

on glucose iAUC and insulin iAUC.

Supplementary table 5.3 A-B. Dose response meta-regression analysis for the effect of fructose

on glucose iAUC and insulin iAUC.

Supplementary table 5.4. Sensivity analyses with different correlation values in the

computation of the overall effect estimate for all outcomes.

Supplementary table 5.5. The GRADE assessment on effect of fructose and its epimers on

postprandial carbohydrate metabolism.

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LIST OF FIGURES

CHAPTER II

Figure 2.1. The proposed mechanism of the ‘catalytic’ effect of fructose and allulose in the

human hepatocyte.

CHAPTER IV

Figure 4.1. The CONSORT statement for participants in the FACE Trial.

Figure 4.2. No effect of fructose on incremental glucose and glucose iAUC in healthy

participants.

Figure 4.3. No effect of allulose on incremental glucose and glucose iAUC in healthy

participants.

Figure 4.4. Post-hoc subgroup analyses for the effect of fructose on the difference in glucose

iAUC between pooled doses and control.

Figure 4.5. Post-hoc subgroup analyses for the effect of allulose on the difference in glucose

iAUC between pooled doses and control.

Supplementary figure 4.1. Dose-response analysis for fructose dose (g) on the primary endpoint

of glucose iAUC.

Supplementary figure 4.2. Dose-response analysis for allulose dose (g) on the primary endpoint

of glucose iAUC.

Supplementary figure 4.3. Equivalence testing on the 95% CI for the effect of allulose and

fructose on the primary endpoint of glucose iAUC in healthy participants.

CHAPTER V

Figure 5.1. Summary of search and selection.

Figure 5.2 A-B. The Cochrane Risk of Bias Tool summary of findings for tagatose trials

investigating the effect of glucose iAUC.

Figure 5.3 A-B. The Cochrane Risk of Bias Tool summary of findings for fructose trials

investigating the effect of glucose iAUC.

Figure 5.4. Forest plot of acute, single-meal feeding trials investigating the effect of allulose on

glucose iAUC (mmol/L*min).

Supplementary figure 5.1. Summary of the risk of bias using The Cochrane Risk of Bias Tool

for the effect of tagatose on glucose iAUC.

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Supplementary figure 5.2. Summary of the risk of bias using The Cochrane Risk of Bias Tool

for the effect of fructose on glucose iAUC.

Supplementary figure 5.3. Forest plot of acute, single-meal feeding trials investigating the

effect of fructose on glucose iAUC (mmol/L*min).

Supplementary figure 5.4. Forest plot of acute, single-meal feeding trials investigating the

effect of tagatose on glucose iAUC (mmol/L*min).

Supplementary figure 5.5. Forest plot of acute, single-meal feeding trials investigating the

effect of allulose on insulin iAUC (pmol/L*min).

Supplementary figure 5.6. Forest plot of acute, single-meal feeding trials investigating the

effect of allulose on insulin iAUC (pmol/L*min).

Supplementary figure 5.7. Forest plot of categorical subgroup analyses for the effect of

tagatose on mean difference in glucose iAUC.

Supplementary figure 5.8. Forest plot of categorical subgroup analyses for the effect of fructose

on mean difference in glucose iAUC.

Supplementary figure 5.9 A-C. Linear dose response meta-regression for the effect of (A)

tagatose, (B) allulose, and (C) fructose dose on the mean difference (MD) in glucose iAUC.

Supplementary figure 5.10 A-B. Linear dose response meta-regression for the effect of (A)

allulose and (B) fructose dose on the mean difference (MD) in glucose iAUC.

Supplementary figure 5.11 A-B. Funnel plot for publication bias for mean difference in glucose

iAUC in (A) tagatose and (B) fructose.

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CHAPTER I. INTRODUCTION

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CHAPTER I. INTRODUCTION

1.1‘CATALYTIC’ FRUCTOSE AND ITS EPIMERS

Small, ‘catalytic’ doses (≤10g/meal or ≤36g/d) of fructose have been shown to acutely

increase hepatic concentrations of fructose-1-phosphate (F1P) and lead to increased glucokinase

(GK) activity in cell culture studies (1, 2). ‘Catalytic’ refers to the dose level of fructose and its

epimers that have been shown to initiate glycogen synthesis in hepatocytes and leading to

reductions in postprandial glucose levels. Numerous studies with: dogs (3, 4), rats (5-7), and

humans (8-17), have shown an increase in: hepatic glucose uptake (3), glycogen synthesis (4),

and improved glycemic control (10) due to ‘catalytic’ doses of fructose (17).

‘Catalytic’ doses of allulose have been shown to exert similar anti-hyperglycemic effects

(18-20). There have been numerous rat studies (5-7, 18, 21) and two human studies (19, 20) on

the effect of allulose (5% of the diet for rat studies and

3

Additionally, there is a lack of knowledge synthesis of acute randomized controlled trials

on the effect of ‘catalytic’ doses of fructose and its epimers (allulose, tagatose, and sorbose)

compared to other carbohydrates on postprandial carbohydrate metabolism. In order to pool all

the available evidence on the true effect of fructose and its epimers, we conducted a systematic

review and meta-analysis of acute, single-meal feeding trials. Our objective was to investigate

the effect of ‘catalytic’ doses of fructose and its epimers on postprandial carbohydrate

metabolism in single-meal feeding trials.

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CHAPTER II. LITERATURE REVIEW

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CHAPTER II. LITERATURE REVIEW

2.1 FRUCTOSE AND ITS EPIMERS IN THE CONTEXT OF DIABETES AND

NUTRITION

The ‘catalytic’ effects of fructose and its epimers can be summarized their ability

to stimulate hepatic glucose uptake and glycogen synthesis, leading to reduced postprandial

glycemia. These effects will be described in greater detail in the following review. The term

‘catalytic’ is not used here in the traditional sense of the word, which is to say that a catalyst is a

substance that facilitates a chemical reaction and does not itself get used up in the process.

Instead, in this body of literature ‘catalytic’ refers to the hepatic mechanism by which fructose

and its epimers stimulate glycogen synthesis to reduce postprandial glucose and insulin levels.

In the context of diabetes, fructose and its epimers may be useful as a novel dietary

treatment and prevention strategy for those with diabetes. Sugar intake is an especially regulated

aspect of the diet for those with diabetes. Sugars are under attack by the popular media and

international health organizations, creating a need for alternative, non-caloric sweeteners that are

safe for consumption for those with and without diabetes (25). Artificial sweeteners are currently

under scrutiny because of controversial research making claims that artificial sweeteners cause

unintended harm for consumers (26). This controversy strikes a need for other non-caloric

sweeteners that can be used in place of sugar, i.e. in baking formulations and food production,

while providing a similar taste and flavor profile as real sugar (27, 28). Such an opportunity can

possibly be filled by the fructose epimers including; allulose, tagatose, and sorbose, which are

non-caloric, naturally occurring, and may improve hepatic glucose handling (29, 30).

2.2 PATHOPHYSIOLOGY OF DIABETES

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Diabetes manifests slowly over time, such that impaired insulin action and insulin

production by pancreatic β-cells develops years before an individual has a diagnosis of diabetes

(31). Insulin resistance, or reduced insulin sensitivity, is defined as the impairment of insulin-

mediated glucose disposal in liver, muscle, and adipose tissues (31). Insulin resistance develops

due to a failure of insulin signaling wherein the tissues that would normally be responsive to

insulin are no longer able to take up glucose in an insulin-dependent manner (32). Declines in β-

cell function can be caused by genetics, age, diet, exercise, chronic hyperglycemia, and elevated

free fatty acids (FFAs) (31).

Under healthy conditions, insulin-dependent glucose uptake occurs via the glucose

transporter 4 (GLUT4) in skeletal muscle, cardiac muscle, and adipose tissue to result in glucose

uptake from the blood stream (31, 33). Insulin and exercise can acutely trigger the soluble

GLUT4 to skeletal and adipose cell surfaces for glucose intake (33). In T2DM, liver, skeletal,

and adipose tissues all lack functioning insulin signaling, thus preventing glucose uptake and

resulting in postprandial hyperglycemia (31). In adipocytes, insulin normally has anti-lipolytic

effects, but in a state of insulin resistance the result is an elevation of circulating FFAs (31). The

liver normally produces glucose in the presence of glucagon and suppresses glucose production

in the presence of insulin (31). However, insulin resistance causes the liver to overproduce

glucose via gluconeogenesis and glycogenolysis (32). Therefore, higher levels of insulin are

needed to induce glucose uptake via GLUT4 and this decreasing insulin sensitivity increases the

demand on pancreatic β-cells to produce more insulin (32). Once insulin levels are unable to

appropriately reduce blood glucose levels, the resulting chronic hyperglycemia develops into

T2DM (32). Hyperinsulinemia has been associated with blood vessel damage, increased blood

pressure, heart disease, obesity, osteoporosis, and cancer (34, 35).

7

2.3 MEASURES OF GLUCOSE AND INSULIN REGULATION

The gold standard method for the measurement of insulin resistance is the

hyperinsulinemic euglycemic clamp (HEC) method (36-38). The HEC method involves the

continuous infusion of insulin (at a pre-determined level) until a steady-state level is achieved

that fully suppresses endogenous glucose production, while at the same time a glucose solution is

infused at a variable rate to achieve a pre-determined blood glucose level (36). However, use of

this method is very costly and incurs a high burden on study participants (36, 37). This lead to

the development of several insulin sensitivity indices based on the 75g-OGTT, which is

relatively inexpensive and less burdensome on study participants compared to the HEC method

(36, 37).

The following clinical indices are commonly used to describe insulin regulation

including: homeostasis model assessment-insulin resistance (HOMA-IR), the Matsuda insulin

secretion index (Matsuda ISIOGTT), and the early insulin secretion index (∆PI30-0/∆PG30-0) (37).

All three indices can be calculated from the glucose and insulin levels from various time-points

of a standard 75g-OGTT (37). These measures are used as clinical indicators of diabetes onset in

healthy populations since insulin resistance is a major risk factor for diabetes development,

including other risk factors such as obesity, age, physical activity, and hyperinsulinemia (37, 39).

2.3.1 HOMA-IR

HOMA-IR uses fasting blood glucose and insulin (or c-peptide) levels to determine

insulin resistance from a single blood sample. The basis of the model relies on the assumption

that in the fasting state, hepatic glucose output is balanced by -cell output of insulin (40). The

simplified equation first described by Matthews et al.(41) for HOMA-IR is the product of fasting

8

glucose (mmol/L) and fasting insulin (µU/mL) divided by 22.5 (40). Then in 1996, the HOMA

model was updated (with a computer model) that accounts for variations in hepatic and

peripheral glucose resistance and renal glucose losses in hyperglycemic subjects (40, 42).

2.3.2 MATSUDA INSULIN SECRETION INDEX

The Matsuda ISIOGTT, as described by Matsuda and DeFronzo (43), uses values from a

75g-OGTT to determine whole body insulin sensitivity by accounting for hepatic and peripheral

insulin sensitivity. The Matsuda ISIOGTT produces values from 0-12, using fasting glucose

(mg/dL) and insulin (µU/mL) values during the OGTT in the following equation: 1000/ √(G0 *I0

*GMEAN *IMEAN) (37). The Matsuda ISIOGTT is highly correlated (r=0.73, P

9

2.4 FRUCTOSE AND ITS EPIMERS: ALLULOSE, TAGATOSE, SORBOSE

2.4.1 FRUCTOSE AND DIABETES

In the 1980’s, fructose was seen as a possibly beneficial sugar in the diabetic diet because

of its low glycemic index (GI) of 20 compared to glucose (GI=100) (45) and the ability of a 21%

fructose diet to reduce postprandial glycemia in those with T1DM and T2DM (46). More

recently, fructose has raised considerable concern in terms of its possible contribution to the

development of obesity (47). Since the 1980s in the US and the 1990s in Canada, high-fructose

corn syrup has been used in sugar-sweetened soft drinks instead of sugar (47, 48). In the US,

consumption of soft drinks and high fructose corn syrup (HFCS) containing products is

considerably higher than in Canada (48-50). Fructose has been implicated in the connection

between the rise in HFCS use and consumption that preceded the rise in obesity in the US (51).

Common concerns related to dietary fructose includes evidence that its consumption

contributes to development of metabolic and cardiovascular disorders (51-55). Fructose has been

implicated in de novo lipogenesis (56, 57), hyperuricemia (58, 59), insulin resistance (60), and

impaired glucose metabolism (61-63), all of which are features of the metabolic syndrome.

Fructose, unlike glucose, does not induce secretion of satiety hormones (insulin and leptin),

therefore it is thought that fructose that may reduce satiety and increase food intake leading to

weight gain and obesity development (51, 64). On the contrary, it is thought that the excess

calories associated with high fructose diets may be the driving factor that leads to development

of the metabolic syndrome (65). A review by Tappy et al.(53) concluded that there is no direct

evidence from human studies to warrant more serious metabolic consequences regarding

consumption of fructose over sucrose. However, a review by Basciano et al.(60) concluded that

fructose is a main contributing factor to the obesity epidemic and insulin resistance in T2DM. It

10

is evident that the debate continues to divide researchers, but the reviews described below can

offer some high-level evidence that fructose consumption at normal daily intake levels is safe to

consume and may even have some health benefits.

A systematic review and meta-analysis by Livesey et al.(66) found no significant effects

for fasting triacylglycerol (TAG) or body weight with fructose intakes of ≤100g/d. Livesey et

al.(66) also showed improvements in HbA1c for doses of fructose 100g/d, result in adverse health effects.

Ha et al.(17) found that isocaloric exchange of fructose for other carbohydrates

significantly decreased diastolic and mean arterial blood pressure, and there was no effect on

systolic blood pressure. Cozma et al.(67) found that isocaloric exchange of fructose for other

carbohydrates reduced HbA1c by 0.53%, which is similar to oral anti-hyperglycemic agents.

Sievenpiper et al.(17) found that ‘catalytic’ doses (≤10g/meal or ≤36g/d) reduce the glycemic

response to high-glycemic index meals, without adverse effects on body weight, TAG, insulin,

and uric acid. Chiu et al.(68) found that isocaloric exchange of fructose for other carbohydrates

does not induce NAFLD in healthy participants, however, hypercaloric trials with high doses

(104-220g/d) of fructose lead to significant increases in alanine aminotransferase (ALT) (mean

difference (MD): 4.94 U/I ((95% CI, 0.03-9.85)) and intrahepatocellular lipid (IHCL)

11

(standardized mean difference (SMD):0.45 (95% CI, 0.18 to 0.72)). Wang et al.(69) found that

isocaloric exchange of fructose for other carbohydrates, in hypercaloric diets (no effect for

isocaloric trials), showed a significant rise in postprandial triglycerides (SMD: 0.65 (95% CI: 0.3

to 1.01)). Sievenpiper et al.(70) found that isocaloric exchange of fructose for other

carbohydrates did not lead to an increase in body weight, however, high doses of fructose in

hypercaloric trials lead to modest, yet significant increases in body weight (MD: 0.53kg (95%

CI, 0.26 to 0.79kg)). Chiavaroli et al.(71) found that isocaloric exchange of fructose for other

carbohydrates did not affect any of the established lipid targets, however, fructose in

hypercaloric trials lead to significant increases in apolipoprotein B (MD:0.18mmol/L, 95% CI,

0.05 to 0.3)) and TG (MD:0.26 mmol/L(95% CI, 0.11 to 0.41)). Wang et al.(72) found that

isocaloric exchange of fructose for other carbohydrates did not affect uric acid in those with and

without diabetes, however, in hypercaloric trials extremely high doses (213-219g/d) of fructose

lead to increases in uric acid (MD:31.0 mmol/L (95% CI, 15.4-46.5)) in healthy individuals.

It is important to note that in all the above trials where diets were hypercaloric the

possible confounding effects from the excess calories cannot be excluded. Additionally, the

doses of fructose (>100g/d) that were associated with adverse health effects (in combination with

a hypercaloric diet) are doses that far exceed normal intake levels in Canada and the US.

Canadians consume 102g of total sugar per day (21% of daily energy intake), but the proportion

of fructose in the Canadian diet is unknown (48, 73). On average, Americans consume 130g of

total sugar per day (23% of daily energy intake) and of that amount only 48g of sugar consumed

is fructose (74). Therefore, average consumption of fructose does not reach the

supraphysiological doses described in the above trials and concern over results of such trials may

be exaggerated. A test of this idea was undertaken in a recent randomized, double-blind clinical

12

trial investigating the effect of diets with fructose containing sugars at average consumption

levels (either HFCS, fructose, glucose, or sucrose diets) on metabolic syndrome and CVD risk

factors found that effects of all diets were not clinically significant (75).

2.4.2 FRUCTOSE AND ITS EPIMERS

Fructose is a monosaccharide that is usually consumed as part of the disaccharide

sucrose, in fresh and dried fruits, vegetables, or added to processed foods in the form of HFCS.

Present in trace quantities alongside fructose are its various epimers: D-allulose (D-psicose), D-

tagatose, D-sorbose, and L-sorbose. L-sorbose has been found to induce hemolysis of dog

erythrocytes, a finding that ended investigation for use of L-sorbose as a food ingredient (76).

However D-tagatose, a stereoisomer of L-sorbose, was found to have no hemolytic effects on

dog erythrocytes (76). The fructose epimers have little to no caloric value (30, 77, 78) and have

sometimes been shown to elicit reductions in postprandial glycemia and insulinemia when

consumed at ‘catalytic’ doses (

13

diabetes mellitus (NIDDM) (85). GK is expressed in pancreatic β-cells and hepatocytes and

controls whole body glucose phosphorylation and metabolism via these two tissues (85).

Nonsense mutations in GK are the most common cause of decreases in GK activity leading to

NIDDM and the development of glucose intolerance by a gene dosing mechanism (85-88).

Vionnet et al. (86, 87) found that those with T2DM have 50% less GK than those without

T2DM. Heterozygous inactivating mutations in GK lead to mild hyperglycemia from birth,

which usually does not require pharmacologic intervention (89). Those with GK mutations tend

to have decreased beta-cell responsiveness to exogenous glucose and reduced ability to entrain

insulin oscillations to exogenous glucose (90).

GK allows pancreatic β-cells and hepatocytes to respond to extracellular glucose

concentrations and has a unique role in the relationship between islet glycolysis and glucose-

mediated insulin secretion (91). Glucose is the strongest dietary stimulus for insulin release,

while other sugars like mannose (92) and fructose (93) are weak stimulators of insulin release

(91). As a glucose sensor, GK sets the pace of glycolysis in pancreatic islets and has a primary

role in glycolytic flux during the first phase of insulin secretion. Insulin induces GK synthesis in

hepatocytes, which is also facilitated by glucose and glucocorticoids (91, 94-96).

Hepatic GK plays a vital role in maintaining glucose homeostasis by mediating hepatic

glucose uptake (glycogen synthesis) and glucose output through the conversion of glucose to

glucose-6-phosphate (G6P) (84, 97, 98). GK resides in the nucleus bound to Glucokinase

Regulatory Protein (GKRP) which inhibits GK from entering the cytosol and prevents the

conversion of glucose to glucose-6-phosphate (84, 99, 100). High glucose levels and F1P lead to

the dissociation of the GKRP-GK complex (84). Fructose-6-phospate (F6P) binds an allosteric

site on GKRP to form the inhibitory complex of GKRP and GK (2, 84, 101). F1P competes with

14

F6P for the allosteric site on GKRP, such that the displacement of F6P by F1P results in

dissociation of the F6P-GKRP-GK inhibitory complex (1, 2, 84, 101). As a result, GK is free to

translocate from the nucleus to the cytosol and induce glucose utilization via glycogen synthesis

(1, 2, 84, 97).

Analogues of F6P, such as sorbitol-6-phosphate (S6P), 2-deoxysorbitol-6-phosphate, and

mannitol-1-phosphate, have also been shown to inhibit GK in the presence of GKRP (2, 101,

102). S6P is the most potent inhibitor of GK and acts at concentrations 4 times lower than F6P

(101). Activation of GK occurs via binding of F1P, A1P, tagatose-1-phosphate, L-sorbose-1-

phosphate, and other analogues of F1P (101). Therefore, D-fructose and its epimers D-allulose,

D-tagatose, D-sorbose, and L-sorbose all act via the same pathway to disinhibit GK and increase

hepatic glycogen synthesis in the postprandial state (97, 101).

2.4.3 ‘CATALYTIC’ DOSES OF FRUCTOSE

The ‘catalytic’ dose effect refers to the mechanism by which F1P displaces F6P on

GKRP, leading to the translocation of GK from the nucleus to the cytosol, and induction of

hepatic glycogen synthesis (102). Small ‘catalytic’ (

15

of fructose per 100g of apple (108). Therefore, the benefits of ‘catalytic’ doses of fructose may

be achievable with a diet that includes regular fruit consumption.

2.4.3.1 METABOLISM, ABSORPTION AND DIGESTION

Fructose is absorbed by enterocytes of the small intestine by GLUT5 and SGLT4 on the

apical membrane, while GLUT2 may function in basal membrane transport (109-111). Fructose

absorption in the small intestine increases when it is simultaneously ingested with glucose (112)

and galactose (113), but the exact mechanism is under debate (114-117). It is important to note

that fructose naturally occurs in combination with glucose (as sucrose) or in association with

other sugars, so it is rarely, if ever consumed in isolation. For example, in the case of HFCS

where fructose is usually 42% or 55% of the mixture and the rest is glucose and water (118).

Figure 2.1 The proposed mechanism of the ‘catalytic’ effect of fructose and allulose in the human hepatocyte.

Adapted from Hossain et al. (97), Hawkins et al. (14), and Van Schaftingen et al. (2). In the

postprandial state, the hepatocyte is the main tissue target for absorbed fructose and allulose

(C-3 epimer of fructose). Fructose and allulose are phosphorylated (F1P and A1P) and enter

the nucleus to displace F6P from the GKRP. This frees GK to enter the cytosol and induce

glycogen synthesis.

16

Fructose is incompletely absorbed in the small intestine, such that absorption ranges from

37-80% in healthy adults when they consume 50g of fructose (117, 119). Fructose

malabsorption is due to an inability of transporters in the intestinal epithelium to absorb all the

fructose in the intestinal lumen (114). Malabsorption of fructose is common and is estimated to

affect up to 50% of the US population (120), whereas hereditary fructose intolerance has an

estimated incidence of 1:18,000 in the UK population and 1: 60,000 in the US population (114,

121-123). Fructose intolerance (not malabsorption) is a hereditary condition due to mutations in

the enzyme aldolase B, which is normally expressed in the liver and kidney and is responsible

for fructose catabolism (124). Doses of fructose that may cause malabsorption can range from 5-

50g in healthy adults, whereas those with irritable bowel syndrome (IBS) can only tolerate up to

25g of fructose (116, 125, 126). There is individual variation in colonic fermentation of

undigested fructose into short-chain fatty acids, which leads to variation in the symptoms of

malabsorption like flatulence and bloating experienced by individuals (117, 125, 127, 128).

2.4.3.2 ‘CATALYTIC’ FRUCTOSE AND GLYCEMIC CONTROL: ACUTE HUMAN

FEEDING TRIALS

The long-term benefits of ‘catalytic’ doses of fructose have already been described

above, but there have also been acute investigations into the beneficial effects of ‘catalytic’ doses

of fructose. Moore et al.(15) provided 11 healthy adults with 7.5g of fructose and a 75g-OGTT,

which resulted in a 19% reduction in glucose AUC compared to the 75g-OGTT control

(P

17

reduced 25% and 27% (P

18

intestine, is not metabolized into a significant source of energy (0.2kcal/g), and is excreted in the

urine (77). Recently, allulose has been showing promise as a low-calorie sugar substitute that has

anti-hyperglycemic and anti-lipidemic effects (29). ‘Catalytic’ doses (5g) and fructose (>7.5g) have been shown to improve

postprandial glycemic control in those with and without T2DM in a dose dependent manner (8,

15, 16, 77, 79, 80). Allulose does not produce a glycemic response, so its use by those with

T2DM would be considered a novel functional food therapy (79). In the interests of the general

population, the aforementioned benefits of allulose could serve as a mode of preventing T2DM

onset and weight gain.

2.5.1 ABSORPTION AND DIGESTION: ANIMAL AND HUMAN STUDIES

Absorption of allulose in animal (rat) studies have shown that allulose is absorbed in the

small intestine, excreted in the urine and feces, and fermented in the large intestine (132, 133).

One study in rats fed radioactive allulose (D-[U-14C] allulose) found that 97-98% of allulose

was excreted in the urine by paper chromatography (132). The same study found that 1.0% of

liver glycogen contained radioactivity and only 0.6% was exhaled, suggesting minimal

metabolism of allulose (132). Oral administration of 5g/kg body weight in Wistar rats showed

that allulose is absorbed in the digestive tract and excreted in the urine (11-15%) in the first 24h

(133). Allulose diets (0, 10, 20, and 30% for 3-d) fed to Wistar rats lead to short chain fatty acid

formation in the cecum and increased cecum weight and surface area in a linear trend, suggesting

that allulose can be fermented in the colon. Rats fed psicose at 30% of the diet experienced

diarrhea, but rats fed the lower doses experienced no adverse effects, suggesting minimal harm

due to allulose in the diet (133). Unfermented allulose was excreted in the feces ranging from 8-

19

13% in the first 24h (133). These results suggest that allulose is rapidly absorbed and excreted in

the urine and feces when rats are fed these doses.

In humans, similar evidence was found by Iida et al.(77) upon investigation of urinary

excretion, colonic fermentation, and small intestinal absorption of allulose in healthy volunteers.

Iida et al.(77) found that urinary excretion rates of allulose reached 70% over a 48-h period.

Allulose, like water, did not increase respiratory quotient or carbohydrate energy expenditure as

measured by indirect calorimetry. Finally, they found that allulose has low large intestinal

fermentability over the short term (12-h after ingestion) and after an 8-week adaptation period.

In terms of small intestinal absorption, Hishiike et al.(134) found that allulose absorption

was improved with a GLUT-5 gene inducer on human Caco-2 cell intestinal monolayer. Also,

allulose absorption was impaired when glucose and fructose were present, leading the authors to

suggest that GLUT-2 is responsible for transporting glucose, fructose, and allulose across the

basolateral membrane of enterocytes.

2.5.2 ‘CATAYLTIC’ DOSES OF ALLULOSE IN ANIMAL STUDIES

Allulose has been shown to prevent weight gain and fat mass gains due to its very low

caloric value. In a study of Wistar rats where they were fed diets of 5% fructose, 5% cellulose, or

5% allulose for 21 days, adipose tissue in the abdomen was significantly lower (P

20

summation, these studies in rats provide the evidence that allulose provides negligible caloric

energy, may impair hepatic lipogenesis, and lead to reduced weight gain.

Chung et al.(24) fed obese Sprague-Dawley rats either a normal or high fat diet

supplemented with or without allulose, sucrose, or erythritol for 8 weeks. Allulose lead to weight

loss (P

21

Finally, in a study of 24 Wistar rats fed 5% allulose or 5% cellulose with a high-fat (25%

fat) or low-fat (5% fat) diet for 16 weeks (5). Allulose diets did not affect glucose tolerance, as

glucose tolerance decreased over time similarly between the 4 groups (5). Allulose diets did not

increase carcass weights compared to cellulose diets, suggesting no added energy due to 5%

alluose in the diet, which is consistent with previous findings that allulose is minimally

metabolized into energy (5, 7, 97, 132). Liver weight and liver glycogen content increased in the

rats on the high fat and low fat allulose diets compared to both cellulose diets, consistent with

other animal studies (5, 132).

2.5.3 EFFECTS ON PLASMA GLUCOSE AND INSULIN: ANIMAL STUDIES

Matsuo et al.(6) studied serum glucose and insulin responses by sacrificing rats before

and after meals. Rats on the allulose-only diet and the fructose-allulose (3:1) diet had reduced

plasma glucose and insulin levels before and after meals compared to rats on control and

fructose-only fed diets. Weight gain was lower on the allulose-only diet compared to those on

control and fructose-only diets. Similarly, liver weight was higher before and after meals in rats

on the allulose-only diet compared to those on control and fructose-only diets. This evidence is

consistent with the anti-hyperglycemic effects of allulose diets and the hepatic glycogen storage

mechanism responsible. Iida et al.(79) studied the single administration of 7.5g allulose in 8

healthy volunteers on blood glucose and insulin responses and found it had no effect on

concentrations of either component. These animal and human studies further demonstrate that

allulose given alone has no effect on glucose and insulin concentrations, and allulose provided in

the diet reduces glycemic and insulinemic responses to meals.

2.5.4 ACUTE FEEDING TRIALS IN HUMANS AT ‘CATALYTIC’ DOSES

22

Human studies have shown that allulose reduced glycemic responses to a carbohydrate

load in acute studies (19, 20). In an acute, randomized single-blinded trial by Iida et al. (19) 20

healthy volunteers ingested one of the 75g-oral maltodextrin test drinks with allulose added at 4

doses (0g, 2.5g, 5g, 7.5g). Significant, dose-dependent reductions in plasma glucose (5g,

p=0.017; 7.5g, p

23

bars, frozen desserts and similar low-calorie products (137, 139). Some health benefits of

tagatose include: the ability to reduce post-prandial glycemic responses (81-83, 140) without

causing hypoglycemia (83), prevention of dental caries(30), and low caloric value (137, 138).

Tagatose has been gaining popularity in the last decade as a low-calorie sweetener since it has a

similar bulking capacity and sweetness as sucrose, such that the only limit to its use is the

laxation effect (30). Tagatose has been shown to provide other benefits such as raising high

density lipoprotein (HDL) (141, 142), reducing body weight in those with T2DM (141), and

improving long term glycemic control by lowering hemoglobin A1C (HbA1c) (83, 143).

Tagatose has undergone a phase 3 clinical trial (clinical trials.gov identifier: NCT00955747) for

development as an ‘anti-diabetic’ drug under the brand name Naturlose, due to its ability to

reduce HbA1c, fasting glucose, LDL, and total cholesterol in patients with T2DM (143, 144).

2.6.1 ABSORPTION AND DIGESTION: ANIMAL AND HUMAN STUDIES

Laerke et al.(145) studied pigs and the intestinal digestibility of tagatose by investigating

the intestinal composition of sacrificed pigs who were fed a tagatose diet compared to a sucrose

diet for 18-d. The tagatose diet reduced small intestinal digestibility of sucrose, dry matter, and

gross energy. In the large intestine, tagatose was a substrate for bacteria in the cecum and the

proximal colon in the generation of short chain fatty acids (SCFAs) and gases. Pigs on the

tagatose diet also had lower digestibility of gross energy and sucrose, and none of the ingested

tagatose was recovered in the feces. Saunders et al.(138) used D-[U-14C] tagatose to measure

disposition of tagatose (100g/kg BW) in conventional, germ-free rats. Authors found a 20%

small intestinal absorption rate, and that microorganisms in the colon are primarily responsible

for digestion of oral tagatose in rats. Therefore, animal studies of tagatose ingestion show a

24

pattern of low small intestinal absorption (but no energy metabolism) and colonic fermentation

of tagatose into SCFAs.

Normén et al.(146) studied ileostomy bag effluents of 6 human subjects (ileostomy

patients) to investigate small intestinal absorption and excretion of tagatose (15g/d) and other

carbohydrates. Approximately 81% of ingested tagatose was absorbed by the small intestine. It

seems there is a large difference in small intestinal absorption rates of tagatose in human

ileostomy patients (146) compared to the 20% absorption rate found in pigs (145). Normén et

al.(146) suggested that some small intestinal fermentation may have played a role in the 60%

difference in tagatose absorption between human ileostomy patients and pigs. Therefore, the

ileostomy patients may not be representative of the small intestinal absorption rates for tagatose

in people with intact gastrointestinal tracts (82). In humans with an intact gastrointestinal tract,

the systemic absorption of tagatose is around 20% (primary tissue is the liver) and this is

metabolized into CO2, with little tagatose entering the circulation (147). Approximately 75% of

ingested tagatose is fermented by colonic microflora into SCFA’s and there is no trace of

tagatose in the feces. Rates for urinary tagatose excretion vary, but usually 5% of ingested

tagatose is excreted according to a study in rats (30).

Another factor to consider is the gastrointestinal side effects of tagatose consumption.

Consumption levels of tagatose above 15g per meal tend to cause laxation effects, nausea,

diarrhea, stool softening, increased flatulence and bloating (139). As stated, the highly variable

incomplete absorption of tagatose (like sugar alcohols and dietary fibre) in the small intestine can

lead to a variety of these symptoms in people, depending on how long it takes to adapt to the

presence of undigested carbohydrates. However, undigested carbohydrates (leading to SCFA

production in the colon) can lead to health benefits by acting as a prebiotic for intestinal

25

microflora, similar to inulin (148). Tagatose has been implicated as having prebiotic properties

as evidenced by its malabsorption in the small intestine and increased butyrate production by

colonic bacteria (149, 150). Venema et al.(150) found that daily tagatose doses between 7.5g and

12.5g promoted growth of lactobacilli with minimal gastrointestinal side effects. In the end,

tagatose acting as a pre-biotic is a beneficial effect that can outweigh the uncomfortable

gastrointestinal side-effects that come with higher consumption levels of tagatose.

Finally, energy values for tagatose (1.5kcal/g) are based on 100% absorption and

utilization of SCFA’s produced from tagatose in the rat colon, but Lu et al.(82) suggests this may

be an over estimation (151, 152). Taken together, not only does tagatose travel quickly through

the body without contributing to caloric intake, there is the added benefit of promoting the

growth of healthy microflora.

2.6.2 EFFECTS ON PLASMA GLUCOSE AND INSULIN

Tagatose, similar to the other fructose epimers, does not have any effect on postprandial

glucose level when taken alone in people with (n=8) and without T2DM (n=8) (83). In a phase 2

clinical trial, tagatose was tested at 2.5g TID, 5g TID, and 7.5g TID for 6-mo in 101 subjects

with T2DM (140). This multi-centre, international trial (11 sites in the US and 7 sites in India)

found that the most effective dose for significant reductions in HbA1c, fasting glucose, and body

weight was 7.5g TID. This is especially promising because subjects in this trial managed their

diabetes by diet and exercise alone. The phase 3 clinical trial was also a multicentre,

international, 6-mo randomized trial investigating the safety and anti-diabetic effects of tagatose

in 480 individuals with T2DM (143). This trial showed that a dose of 15g/d TID lead to a 20%

reduction in HbA1c in the tagatose fed arm compared to placebo. There were also significant

reductions in fasting glucose, LDL, TC, and the proportion of subjects with an HbA1c

26

These trials demonstrate that tagatose taken daily for 6-mo can have significant effects on

glycemic control, body weight, and blood lipids.

2.6.3 MECHANISM OF POSTPRANDIAL GLYCEMIC CONTROL

Bar et al.(153) found that rats fed diets with 5-20% tagatose for 29-31 days lead to

increased liver glycogen and liver weight. Diets with 10-20% tagatose lead to significant liver

enlargement, but livers would return to baseline size when tagatose was removed from the diet.

Bar et al.(153) noted that liver enlargement (in rats) in the absence of histopathology may be a

sign of increased work load in the liver. Surgeries of rats fed 20% tagatose diets (14.4g/kg

BW/d) revealed no adverse liver histopathology and no increase in lipid deposition. Therefore,

liver enlargement as a result of tagatose induced glycogen synthesis is not harmful to rats in the

short term.

In a double-blind crossover trial, Boesch et al.(154) fed 12 healthy men diets with 15g

tagatose TID compared to a control of sucrose 15g TID for 28 days. During the 28-d diets, liver

glycogen tended to increase more in the sucrose diet than in the tagatose diet, but the difference

was not significant. This short-term trial gives evidence that high in take (45g) of tagatose daily

for one month does not cause increased liver weight in healthy individuals.

2.6.4 ‘CATALYTIC’ DOSES OF TAGATOSE IN ANIMAL STUDIES

Similar to the study by Bar et al.(153) , Police et al.(142) conducted a study in genetically

hypercholesterolemic mice that were fed tagatose, sucrose, or the standard murine (control) diet

for 16-wks. Mice on the sucrose diet gained significantly more weight, and became

hyperglycemic compared to mice on the standard murine and tagatose diets. Mice on the sucrose

diet also had atherosclerosis and significantly higher cholesterol levels than control and tagatose

27

fed mice. Therefore, the tagatose and control diets were shown to be healthier long-term diets

than the sucrose rich diet for genetically hypercholesterolemic mice. Similar findings regarding

increased liver weights due to tagatose diets were found by Bar et al.(153), as described above. It

is evident that tagatose works via the same ‘catalytic’ mechanism as fructose and the other

epimers to reduce glycemic responses by producing the effects seen by Police et al.(142)

including increased liver weight and reduced body weight.

There are few animal studies of the glycemic responses to tagatose because most pre-

clinical, non-human studies were focused on establishing the safety of tagatose as a food

ingredient for humans by ruling out genotoxicity (155), embryotoxicity (156), and teratogenicity

(157) in numerous rat studies. Once safety was established, human clinical trials began on the

effect of tagatose on glycemic responses, especially for those with T2DM, as described below.

2.6.5 ACUTE FEEDING TRIALS IN HUMANS AT ‘CATALYTIC’ DOSES

Like fructose and allulose, catalytic doses (

28

were not studied in relation to insulin response in this study, but it is important to note that

glucose AUC was reduced in the T2DM subjects.

A similar investigation was conducted by Lu et al.(82) as a pilot study for a phase 3

clinical trial with tagatose investigating the effect of dose and time of administration. In a

crossover study, 33 healthy participants were randomized to consume 10g of tagatose 0, 30, or

60 min before a 90g instant mashed potato meal as compared to the control who ingested 0g of

tagatose before the mashed potato meal. The second experiment involved consumption of 15g of

tagatose 0, 30, or 60 min prior to a 75g-OGTT or a potato meal. All tagatose treatments resulted

in non-significant decreases in postprandial glycemic responses to the potato and glucose loads.

There were no differences found due to tagatose dose (10g or 15g) and time of administration for

both the potato meal and the glucose load. The authors suggested that the lack of effect seen in

the healthy individuals in this study may be due to the strong glycemic control already present

with normal glucose homeostasis. This conclusion by the authors highlights that perhaps the

more appropriate population of study for the acute effects of tagatose on postprandial glycemic

control would be those with IGT and T2DM.

Kwak et al.(81) conducted a double-blinded crossover trial where 52 healthy and 33

hyperglycemic (IGT and T2DM) participants consumed a 5g tagatose containing drink or a

placebo drink with erythritol and 0.004g sucralose before consuming a standard breakfast meal.

In another crossover study by Kwak et al.(81), a subset of 17 volunteers from the healthy group

consumed a drink containing 10g of tagatose or a placebo drink. Treatment with the 5g tagatose

drink resulted in significant reduction in glucose AUC compared to placebo in the

hyperglycemic group (p=0.017). Of those participants in the hyperglycemic group, those with

T2DM had reduced glucose AUC compared to the placebo drink (p=0.032). Treatment with the

29

10g tagatose drink significantly reduced insulin AUC (p=0.009) and C-peptide AUC (p=0.004)

in healthy participants, but did not significantly reduce glucose AUC.

A trend towards a significant reduction in glucose AUC for healthy participants was

found by Kwak et al.(81) and Lu et al.(82) due to catalytic doses of tagatose. Although Lu et

al.(82) did not measure insulin response, Kwak et al.(81) found significant insulin AUC and C-

peptide reductions in healthy participants. Donner et al.(83) showed that a high (not catalytic)

dose of tagatose (75g) was also able to reduce insulin AUC in healthy participants, suggesting

that catalytic doses of tagatose are enough to induce similar effects on insulin levels as a high

dose of tagatose. These findings show that healthy and T2DM participants benefit from catalytic

tagatose supplementation in conjunction with high carbohydrate loads.

2.7 SORBOSE

The final rare sugar to be discussed is sorbose, a C-3 and C-4 epimer of fructose that is

non-caloric and has the potential to replace fructose in the diet, similar to allulose and tagatose

(158). Commercially, L-sorbose is a precursor in the production of Vitamin C (L-ascorbic acid)

and has been GRAS (GRN 49) by the FDA since 1974 for use in food packaging (159). Since

both D- and L-sorbose are rare in nature, few studies have looked at the effects of using either

form of sorbose as a low-calorie sweetener for those with T2DM (158, 160). L-sorbose was first

investigated for use as a non-caloric sweetener in the late 1970’s, but it was found that L-sorbose

causes hemolysis of canine erythrocytes (161-164). Although lysis of human erythrocytes did not

occur, there was still a risk of impaired cell functionality, which halted further testing into the

use of L-sorbose as a non-caloric sweetener (165). On the contrary, D-sorbose has been a source

of interest since more recently researchers discovered how to produce it from D-tagatose using a

30

bacterial enzyme called D-tagatose 3-epimerase (160). So far there have been no studies that link

D-sorbose to hemolysis, but the literature is limited.

2.7.1 ABSOPRTION AND DIGESTION

In a study of radioactive L-sorbose where un-adapted rats were fed diets of sorbose in

doses 0.04g-4.8g/kg BW, recovery in the urine, feces, and expired as CO2 was 5.3%, 46%, and

16%, respectively (166). In another group, rats were given L-sorbose at 3g/kg BW daily for 13-d

(adapted) and fed radioactive L-sorbose, these proportions changed in the urine, feces, and

expired CO2 to 8.9%, 6.6%, and 59%, respectively. L-sorbose was tested in vivo with colonic

micro-organisms from (un-adapted) human feces and it was found that L-sorbose was not

fermented after 24-h of incubation. However, when L-sorbose was added to adapted human feces

over a 36-h period the appearance of fermentation products like volatile fatty acids correlated

well with the disappearance of L-sorbose. Evidently, adaptation is required for intestinal

fermentation of L-sorbose to occur.

Wursch et al.(166) tested adaptation to sorbose in one human volunteer who consumed

20g L-sorbose twice a day for 10-d. Gastrointestinal discomfort and diarrhea subsided after the

first 3-d and sorbose disappeared from the feces after 5-d. Additionally, Wursch et al.(166) gave

3 volunteers 10-30g sorbose in a single dose and found that urinary recovery ranged from 8-13%

in the first 6-h. It seems that absorption tends to shift from mainly fecal excretion to colonic

fermentation (expired as CO2) in rats and humans as the host adapts to sorbose over a period of

sorbose consumption. Therefore, sorbose requires an adaptation period for colonic fermentation

to be maximized and for gastrointestinal side effects to subside.

2.7.2 ‘CATALYTIC’ DOSES OF SORBOSE IN ANIMAL STUDIES

31

Animal studies, particularly in rodents, have looked at the effects of D- and L-sorbose on

postprandial glycemic and insulinemic responses. However, since the literature is limited in this

area, studies of sorbose in animals will be discussed regardless of their dose. L-sorbose fed to

genetically diabetic mice in exchange for sucrose (200g/kg BW) lead to significant reductions in

body weight and blood glucose levels, but not insulin levels (167, 168). Similarly, Yamada et

al.(158) found that D-sorbose fed to Sprague-Dawley rats at 3% of the diet for 28-d lead to

significant reductions in blood glucose but not insulin levels compared to the control diet. Oku et

al.(169) tested various rare sugars in their ability to inhibit disaccharidases and found that D-

sorbose was significantly stronger than L-sorbose at inhibiting sucrase and maltase activity in rat

and human intestinal cells. So, both D- and L-sorbose have been shown to reduce blood glucose.

2.8 GAPS IN THE LITERATURE

There have been three trials (20, 79, 170) published on the short-term effects of

‘catalytic’ (doses

32

CHAPTER III: RATIONALE AND OBJECTIVES

33

CHAPTER III: RATIONALE AND OBJECTIVES

3.1 RATIONALE

Current literature on the acute effects of ‘catalytic’ doses (

34

3. To determine whether the effects of allulose and fructose are equivalent on the primary

endpoint of iAUC for plasma glucose across the 2 dose levels (5g and 10g) in healthy

participants.

4. To systematically review the existing evidence of acute, single-meal feeding trials to

determine the effect of fructose, allulose, tagatose, and sorbose on postprandial glucose

and insulin responses to other carbohydrates.

35

CHAPTER IV: AN ACUTE RANDOMIZED DOSE-FINDING EQUIVALENCE TRIAL

OF SMALL, CATALYTIC DOSES OF FRUCTOSE AND ALLULOSE ON

POSTPRANDIAL CARBOHYDRATE METABOLISM IN HEALTHY PARTICIPANTS:

THE FRUCTOSE AND ALLULOSE CATALYTIC EFFECTS (FACE) STUDY

36

4.1 ABSTRACT

Background: Recent literature suggests that catalytic doses (

37

4.2 INTRODUCTION

Recently, scientific research groups, the popular media, and international health

organizations have called for a reduction in free sugar intakes (25). Alternatives to sugar, such as

artificial sweeteners have also been targeted as ‘unhealthy’ due to some reports of adverse health

effects associated with their consumption (26). This has created an opportunity for ‘natural’ non-

caloric sweeteners to emerge in the food ingredient industry. One such group of non-caloric

sweeteners are the epimers of fructose including; allulose (psicose), tagatose, and sorbose.

Allulose is manufactured in the United States by Tate & Lyle under the brand name

Dolcia Prima®. There are many potential benefits of using allulose in place of other sweeteners

in food products. Allulose provides 0.2kcal/g compared to the usual 4kcal/g of other sugars

(fructose and glucose), so it is virtually non-caloric (172). Additionally, studies with allulose

have shown evidence of having antihyperglycemic effects similar to fructose when consumed in

small, ‘catalytic’ doses (

38

curve (iAUC); 2) to assess whether there was a dose response relationship over the proposed

range (0g, 5g, 10g) for fructose and allulose; 3) to assess whether the effects of fructose and

allulose were equivalent on the primary endpoint of glucose iAUC across the 2 doses (5g and

10g) compared with control (0g).

4.2.2 HYPOTHESES

Given the 3 objectives above, we had 3 corresponding hypotheses regarding the effects of

fructose and allulose on glucose and insulin responses to a 75g-OGTT in healthy participants: 1)

catalytic doses of fructose and allulose would significantly reduce glucose and insulin responses;

2) fructose and allulose would demonstrate a dose response on the primary endpoint of glucose

iAUC; 3) allulose and fructose will be equivalent in their effects on the primary endpoint of

iAUC for plasma glucose.

4.3 METHODS

4.3.1 STUDY PARTICIPANTS

Table 4.1 represents the baseline characteristics of the 25 healthy participants of the

FACE trial. 25 of 27 participants completed the study. The inclusion criteria were as follows:

male and (non-pregnant) female healthy volunteers between 18 and 75 years of age, no regular

medication use (except birth control and PRN medications), no complementary or alternative

medicine use, a body mass index (BMI) between 18.5 and 30 kg/m2, no prediabetes or diabetes

(HbA1c ≥6%, FBG ≥6.1mmol/L (173)), free of any major disease, no psychiatric illness, non-

smoking, and no heavy alcohol use. Participants were asked to keep their regular diet and

physical exercise the same as usual, the only instruction was to consume at least 150g of

carbohydrate in the 3-d leading up to a visit and refrain from excess alcohol consumption (> 3

39

drinks/d). Participants who did not fast the evening before a clinic visit or follow study protocol

were asked to repeat the study visit. Any changes to diet or exercise would have been reported in

the clinical health and diet questionnaire that study staff administered at the beginning of each

visit.

4.3.2 POWER CALCULATION

The power calculation was based on the sample size needed for equivalence testing for

the effect of 5g and 10g allulose compared to 5g and 10g fructose on the primary endpoint of

plasma glucose iAUC. Assuming equivalence margins (+δ, -δ) set at ±20% (174), with

significance level of α=0.05, power to detect a significant difference set at 1-β=80% and an

estimated intra-subject standard deviation of 16.25% (175) , a total recruitment of 24 participants

were needed.

4.3.3 STUDY DESIGN

The trial was designed as an acute, randomized crossover, dose-finding equivalence trial

of small, catalytic doses of fructose and allulose on plasma glucose and insulin responses to a

75g-OGTT with either fructose or allulose added (0g, 5g, or 10g). There was a total of 6 drinks

consumed by each subject, including 2 control 75g-OGTT drinks (0g allulose and 0g fructose)

and 4 treatment 75g-OGTT drinks (5g and 10g of allulose and fructose). The study was designed

Table 4.1. Baseline characteristics of 25 healthy participants in the FACE trial.

Characteristics Total, n=25 Males, n=13 Females, n=12

Age (y) 37.3 ± 16.1 40 ± 15.4 35 ± 17.0

Weight (kg) 69.3 ± 13.9 78.3 ± 11.7 59.5 ± 8.4

BMI (kg/m²) 24.7 ± 3.4 25.7 ± 3.9 23.6 ± 2.6

SBP (mmHg) 116 ± 8.4 120 ± 8.7 112 ± 6.1

DBP (mmHg) 70 ± 8.4 68.2 ± 8.7 71.2 ± 8.2

WC (cm) 81.2 ± 11.4 87.7 ± 11.0 74.3 ± 7.1

Mean ± SD, SBP=systolic blood pressure, DBP=diastolic blood pressure.

40

as a randomized crossover trial such that participants got the 75g-OGTT drink (control) twice in

order to increase precision of the control measurements. Neither the study staff nor the study

participants knew the identity of the drinks. After a 10 to 12-h overnight fast, study staff

administered a clinical diet and health questionnaire and took anthropometric measurements

(body weight, waist circumference, blood pressure) before blood draws were taken from

participants. Participants were instructed to maintain their regular exercise and dietary patterns,

and to ensure they were consuming at least 150g of carbohydrate per day in the 3-d leading up to

a clinic visit. Participants were instructed about what constitutes 150g of carbohydrate to

enhance compliance. Once an intravenous (IV) catheter was affixed to the participant’s arm by a

registered nurse, blood draws (15mL) were taken at -30-min, 0-min (point of consumption), 30-

min, 60-min, 90-min, and 120-min. The participant’s consumption of the study drink was timed

to be within 5 minutes. Clinic visits were scheduled at least one week apart to ensure that all test

substances were cleared from the body.

4.3.4 STUDY DRINKS

The 500mL study drinks (Tate & Lyle, London, UK), consisted of a 75g-OGTT with 0g,

5g, or 10g of fructose or allulose added (175). All drinks were designed to appear similar in taste,

texture, and appearance so that neither the participants nor the study staff could detect the

identity of the drink. Flavour and colour enhancements were used to mask the differences

between the treatment drinks. The drinks were coded by the manufacturer (Tate & Lyle) and

only the study staff member (not responsible for study administration) doing the randomization

of subjects to treatment drinks knew the identity of the coding. Study administrators and

supervising doctors did not have any knowledge of the identity of the study drink coding.

4.3.5 PROTOCOL FOR BLOOD SAMPLE COLLECTION

41

The World Health Organization (WHO) guidelines were used to determine the protocol

for the OGTT (176). Participants came to the Clinical Nutrition and Risk Factor Modification

Centre, St. Michael’s Hospital, Toronto, Canada on 6 separate mornings after a 10 to 12-h

overnight fast. A registered nurse inserted an IV catheter into the forearm of participants and

wrapped a heating blanket around their forearm for the duration of the test for the ease of

multiple venous blood draws. Blood samples were collected in 7mL tubes containing fluoride

oxalate. Two fasting blood samples were taken, one at -30-min and 0-min, at which point

participants ingested one of the 6 drinks within a period of 5-min. Additional samples were taken

at 30-min, 60-min, 90-min and 120-min. Participants received breakfast after the oral treatment

and a small monetary compensation of $50 at the end of each visit and transit fare less than $10.

4.3.6 OUTCOMES

The primary outcome was the difference in incremental area under the curve (iAUC) for

plasma glucose for the 5g and 10g doses of fructose and allulose to the 75g-OGTT compared to

control (75g-OGTT). The secondary outcomes for all doses include: iAUC for plasma insulin;

the early insulin secretion index (∆PI30-0/∆PG30-0); the Matsuda whole body insulin sensitivity

index (Matsuda ISIOGTT); and time of maximum concentrations (Tmax), total maximum

concentrations (Cmax), and mean incremental changes in plasma glucose and insulin responses.

The following endpoints were exploratory: the pooled dose (5g and 10g) analyses; the Insulin

Secretion and Sensitivity Index (ISSI-2) (177); total AUC for glucose and insulin; mean glucose

and insulin responses; and incremental Cmax for glucose and insulin concentrations.

4.3.7 PLASMA ANALYSES

All OGTT were performed in the morning after an overnight fast of 10-12h. Blood

samples were drawn at -30-and 0-min (point of consumption), then at 30, 60, 90, and 120-min

42

for measurement of plasma glucose and insulin. All screening blood samples were analyzed by

the St. Michael’s Hospital Core Laboratory, Toronto, Canada. Study samples were separated by

centrifuge for glucose and insulin analyses and immediately frozen at -72 oC for later analyses.

All study samples were stored at the University of Toronto, Toronto, Canada, until study

completion. Mount Sinai Hospital, Pathology and Laboratory Medicine, Toronto, Canada,

performed plasma glucose and insulin analyses. Plasma glucose was measured with

Roche/Hitachi MODULAR P (Roche Diagnostics, Indianapolis, IN, USA) analyzer using the

hexokinase method (178, 179). Plasma insulin was measured with MODULAR ANALYTICS

E170 (Roche Diagnostics, Indianapolis, IN, USA) immunoassay analyzer and

electrochemiluminescence immunoassay kit (180).

4.3.8 DATA ANALYSES

Plasma glucose and insulin curves were plotted as incremental change over time and

iAUC was calculated for each participant geometrically, ignoring areas below the fasting value

(181). The -30-min and 0-min glucose and insulin samples were averaged to provide a single

fasting measurement (0-min). Separate analyses were conducted for fructose and allulose but

data for the 2 controls (75g-OGTT) were pooled to increase precision for comparison to the 2

dose levels. The Matsuda ISIOGTT was calculated using the 75g-OGTT plasma glucose (PG) and

plasma insulin (PI) outcome: 10 000/ √([FPGFPI] ([PGMEAN PIMEAN]), where PG is

expressed in mg/dl (0.0555mmol/L) and PI in U/ml (6pmol/L) (182). The early insulin

secretion index (∆PI30-0/∆PG30-0) was calculated as the change in PI from 0-min to 30-min

divided by the change in PG over the same period (183).

Statistical analyses were performed using STATA (V.13.1, College Station, USA) and

SAS (V 9.2, SAS Institute Inc., Cary, NC, USA). Significance level for the primary endpoint of

43

plasma glucose iAUC at 5g and 10g doses (for fructose and allulose) was held at p

44

Subgroup analyses for fructose and allulose comparing the effect of treatments (5g and

10g allulose or fructose) compared to control (0g) were performed in STATA using the xtmixed

procedure.

Equivalence testing was used to determine whether the 90% CI for the effect of allulose

(using fructose as a reference) on the primary endpoint (iAUC for plasma glucose) fell within the

equivalence margins, which were set at ±20% (184). If the 90% CI fell completely within the

margins, then allulose was considered equivalent to fructose. If the upper or the lower bound of

the 90% CI fell outside the margin, then the assessment was inconclusive. If the 90% CI fell

completely above or below the margin, then allulose was considered superior or inferior to

fructose on the primary endpoint. The 90% CI was used because the equivalence test is made up

of two one sided tests (TOST), where the CI= (1-2α)*100%, with a significance level of α=0.05.

The clinical health questionnaires were administered each visit by study staff and

included; body weight, blood pressure, waist circumference, and detailed questions about the last

meal before the overnight fast, bowel movements, medications, activity the night before and the

morning of the clinic visit. Additionally, study staff collected ethnicity data after the study was

completed via telephone. Participants were called a maximum of 3 times and after which they

were considered a non-responder. For the 8 participants who were called 3 times or their

voicemail service was not initiated, study staff filled in the category from memory.

4.4 RESULTS

Recruitment took place from November 2015 to July 2016. The CONSORT statement for

the healthy participants of the FACE study can be seen in Figure 4.1 (185). Of the 53 individuals

screened over the telephone, 22 were excluded and 31 moved on to the screening blood

45

assessment. Four subjects were excluded based on screening blood work and 27 subjects were

randomized to a treatment sequence. There were two drop outs after randomization, one subject

refused to participate and one subject could not participate due to a conflict with work. Twenty-

five subjects completed the trial.

Participants tolerated the treatment drinks well and there was no pattern to the instances

of lightheadedness, which we attributed to the process of taking the blood draws and drinking the

OGTT, but not the treatment type itself (Supplementary table 4.1).

4.4.1 PRIMARY OUTCOME: GLUCOSE iAUC

There were no significant effects on glucose iAUC after intake of fructose (5g, p=0.93;

10g, p=0.25) or allulose (5g, p=21;10g, p=0.50). For fructose (Figure 4.2), there appeared to be

an additive effect of fructose dose instead of the predicted catalytic effect, which according to

Telephone Screening Total: 53

Randomized: 27

Screening lood Assessment: 31

Excluded:

Completed Study: 25

Dropouts after randomization: 2

Refused to participate: 1

Study conflicted with work schedule: 1

Excluded: 22

Figure 4.1. The CONSORT statement for the participants in the FACE trial.

46

our hypotheses would have produced reductions in glucose iAUC. For the 5g and 10g allulose

(Figure 4.3) treatments, the magnitude of the reduction in glucose iAUC (-16% and -10%,

respectively) was comparable to what was reported in previous trials with catalytic doses of

allulose (20, 79). The high within-subject %CV in the participants’ glycemic responses to the

control (75g-OGTT) is one possible explanation for why we found a similar (yet non-significant)

magnitude of effect with allulose to previous studies. Pooling the doses (5g and 10g) for fructose

and allulose also did not result in significant changes in glucose iAUC.

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

0 30 60 90 120

Incr

emen

tal

Glu

cose

(m

mo

l/L

)

Time (min)

0g + 75g-OGTT

Fructose 5g + 75g-OGTT

Fructose 10g + 75g-

OGTT

Figure 4.2. No effect of fructose on incremental glucose and glucose iAUC in healthy participants.

Data are least squares means (SEM). N=25.

0

50

100

150

200

250

300

0g 5g 10g

Glu

cose

iA

UC

(mm

ol/

L*

min

)

Fructose dose

47

4.4.2 SECONDARY OUTCOMES

For fructose (Table 4.2) and allulose (Table 4.3) there were no significant effects found

for any secondary outcomes.

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

0 30 60 90 120

Incr

emen

tal

Glu

cose

(m

mo

l/L

)

Time (min)

0g + 75g-OGTT

Allulose 5g + 75g-OGTT

Allulose 10g + 75g-OGTT

Figure 4.3. No effect of allulose on incremental glucose and glucose iAUC in healthy participants.

Data are least squares means (SEM). N=25

0

50

100

150

200

250

300

0g 5g 10g

Glu

cose

iA

UC

(mm

ol/

L*

min

)

Allulose dose

48

Table 4.2. Primary and secondary outcomes for the effects of fructose on postprandial carbohydrate metabolism.

Outcome 0g 5g 10gPooled Doses vs.

0g

0g vs. 5g

P-value*

0g vs. 10g

P-value*

Pooled Doses

vs. 0g

P-value*

Primary Outcome

iAUC Glucose (mmol/L*min) 224 (24) 231 (24) 257 (24) 20 (19) 0.93 0.25 0.30

Secondary Outcomes

Mean glucose (mmol/L) 6.8 (0.2) 6.9 (0.2) 7.0 (0.2) 0.1 (0.1) 0.84 0.37 0.33

Total AUC glucose (mmol/L) 854 (25) 864 (25) 880 (25) 17.9 (31) 0.94 0.68 0.56

Total Cmax glucose (mmol/L) 8.6 (0.3) 8.8 (0.3) 8.9 (0.3) 0.2 (0.2) 0.74 0.58 0.38

Mean incremental glucose

(mmol/L)1.5 (0.2) 1.8 (0.2) 1.6 (0.2) 0.2 (0.1) 0.15 0.89 0.22

Incremental Cmax glucose

(mmol/L)3.3 (0.3) 3.7 (0.3) 3.5 (0.3) 0.2 (0.3) 0.38 0.77 0.30

Tmax glucose (min) 41.4 (3.6) 40.8 (3.6) 39.6 (3.6) 1.2 (3.8) 0.99 0.89 0.76

iAUC insulin (pmol/L*min) 59498 (7404) 67795 (7404) 62097 (7404) 5448 (3761) 0.17 0.76 0.19

Mean insulin (pmol/L) 504.0 (59.8) 561.9 (59.8) 526.8 (59.8) 40.4 (29.6) 0.31 0.63 0.22

Total AUC insulin (pmol/L) 68192 (7313) 75494 (7313) 70241 (7313) 4675 (7497) 0.80 0.32 0.29

Total Cmax insulin (pmol/L) 894 (98) 976 (98) 886 (98) 37 (60) 0.68 0.70 0.99

Mean incremental insulin

(pmol/L)431.4 (56.3) 458.3 (56.3) 497.8 (56.3) 46.6 (48) 0.61 0.14 0.14

Incremental Cmax insulin

(pmol/L)821.1 (94.4) 818.0 (94.4) 911.4 (94.4) 43.6 (58.9) 0.71 0.54 0.89

Tmax insulin (min) 45.0 (4.3) 43.2 (4.3) 54.0 (4.3) -3.6 (5.0) 0.93 0.22 0.48

Matsuda ISIOGTT 3.9 (0.5) 3.6 (0.5) 4.0 (0.5) 0.1 (0.2) 0.55 0.68 0.34

∆PI30-0/∆PG30-0 348 (64) 357 (64) 214 (64) 63 (51) 0.96 0.02 0.10

ISSI-2 290 (23) 276 (23) 306 (23) 0.8 (16) 0.86 0.39 0.67

Data are least squares mean (SEM). N=25. P-values

49

Post-hoc subgroup analyses for fructose (Figure 4.4) did not show any significant effect

modification by the subgroups investigated including: age (

50

African-American, Caribbean, and Other).

Post-hoc subgroup analyses for allulose (Figure 4.5) included the following subgroups:

age (

51

difference between the two HOMA-IR categories (p=0.07).

For fructose, two-way repeated measures ANOVA demonstrated that there was a

significant effect of time (P < 0.0001) but no effect of treatment (P=0.32) on mean incremental

changes in plasma glucose, with no interaction for either parameter (P=0.83) (Figure 4.2). For

allulose, two-way repeated measures ANOVA demonstrated that there was a significant effect of

time (P < 0.0001) but no effect of treatment (P=0.41) on mean incremental changes in plasma

glucose, with no interaction for either parameter (P=0.83) (Figure 4.3).

-200 -150 -100 -50 0 50 100 150 200

Subgroup Level N Difference in plasma glucose iAUC (Pooled doses-Control), mmol/L*min MD [95% CI]

P-value for

interaction

term*

Total 25 -28.8 [-59.2, 1.7]

Age, years < 30 12 -31.6 [-75.5, 12.2] 0.86

> 30 13 -26.1 [-68.3, 16.1]

Sex Male 13 -28.8 [-72.8, 15.1] 1.00

Female 12 -28.7 [-70.9, 13.5]

BMI, kg/m2 < 25 14 -22.4 [-66.1, 21.4] 0.69

≥ 25 11 -34.6 [-76.7, 7.4]

Baseline HbA1c, % < 5.1 12 -15.2 [-58.5, 28.1] 0.39

≥ 5.1 13 -41.3 [-82.9, 0.3]

Baseline fasting plasma glucose < 5.5 12 -25.7 [-69.6, 18.2] 0.85

mmol/L > 5.5 13 -31.6 [-73.7, 10.6]

Baseline fasting plasma insulin < 61.5 12 -37.5 [-81.1, 6.2] 0.59

pmol/L > 61.5 13 -20.7 [-62.7, 21.3]

Baseline HOMA-IR < 2.55 12 -1.4 [-42.7, 39.9] 0.07

≥ 2.55 13 -54.0 [-93.7, -14.3]

Ethnicity**

Caucasian 12 -35.1 [-74.6, 4.4] 0.31

South Asian 5 -10.2 [-71.4, 50.9]

East Asian 2 -32.8 [-129.5, 64.0]

African American 2 -97.8 [-194.5, -1.1]

Other 3 36.6 [-42.4, 115.5]

Carribbean 1 -92.3 [-232.0, 41.5]

Figure 4.5. Post-hoc subgroup analyses for the effect of allulose on the difference in glucose iAUC between pooled doses and control.

Beneficial effect Adverse effect

P

52

There was no significant dose response effect between dose (g) and glucose iAUC for

fructose (p=0.47) (Supplementary figure 4.1) or allulose (p=0.61) (Supplementary figure 4.2).

Supplementary figure 4.3 presents the results of the equivalence test between the effects

of fructose and allulose on the primary endpoint of glucose iAUC. The 90% CI for the effect of

allulose compared to fructose (with fructose as the reference) on glucose iAUC was inconclusive

with the 5g dose, 10g dose, and when the doses were pooled since all 90% CI’s crossed the a

priori equivalence margins of ± 20% (174). The 90% CI at all dose levels tended to favour

allulose more than fructose, meaning that allulose showed a stronger trend for reduction in

glucose iAUC as compared to fructose, although allulose was not superior to fructose.

4.5 DISCUSSION

This is the first study in a Canadian population of the catalytic effects of fructose and

allulose on postprandial carbohydrate metabolism. The results of the FACE trial involving 25

healthy adults showed that catalytic doses of fructose and allulose did not significantly affect

postprandial glucose and insulin responses to a 75g-OGTT. Although the magnitudes of the

effects seen in this trial are comparable to previous studies, we were unable to detect significance

due to a high degree of within-subject variability in the glucose data. There was a 50-unit

difference between the glucose iAUC for the randomly assigned ‘allulose’ control and the

‘fructose’ control, which translates to a 20% difference in participants’ responses to the same

cont