Note: Descriptions are shown in the official language in which they were submitted.
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Composition
This invention relates to methods of reducing or preventing low-grade-
systemic inflammation, e.g. treating or preventing lifestyle-related diseases,
and to
compositions for use in such methods.
There is growing evidence that some diseases can be linked to modern
lifestyles. For example, the smoking of tobacco and consumption of alcohol or
drugs, as well as a lack of exercise, is thought to increase the risk of
developing
certain diseases, especially later in life. Diet is also a factor thought to
influence
susceptibility to many diseases. Diseases which are thought to be linked in
this way
to lifestyle include Alzheimer's disease, asthma, cancer, type 2 diabetes,
cardiovascular disease and obesity. Worldwide, chronic disease account
contribute
to about 60% of deaths (based on statistics from 2001 - World Health
Organisation)
and the incidence of such diseases is expected to rise to 75% by 2020, i.e. it
is a
significant and growing problem.
Recent studies have investigated some of the biological and physiological
mechanisms by which aspects of lifestyle, e.g. diet, can affect disease
states. Whilst
the effect of changing the type and amount of fats in the diet has received
considerable attention (e.g. the ratio of polyunsaturated (PUFAs) to saturated
fat
(SFA) in the diet), the role of carbohydrate has not received much attention.
Certain
epidemiological studies have suggested that dietary glycemic index (GI),
glycemic
load (GL) and fibre may be implicated in various lifestyle diseases, and in
particular
metabolic syndrome, but the studies are not all consistent and the mechanisms
behind this are not fully understood. Controversies regarding optimal dietary
carbohydrate or fat intake and quality still exist.
Low-grade systemic inflammation is a condition characterised by low level
inflammation throughout the body and has become recognised in recent years to
precede, and indeed possibly to predict, lifestyle diseases such as type 2
diabetes and
cardiovascular disease (CVD). Potential links between diet and low-grade
systemic
inflammation have been studied but these are still not well understood.
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In one recent study, Arbo et al. (Scandinavian Journal of Clinical and
Laboratory Investigations (2011) 71:330-339) show that the expression of genes
involved in immunological processes may be regulated by insulin levels, which
suggests inflammation as a possible consequence of postprandial
hyperinsulinemia.
However, whilst this study provides tantalising hints about how diet
composition
and the frequency of feeding might affect lifestyle diseases, it provides no
firm
guidance about how to go about formulating diet compositions or about treating
such diseases.
Given the prevalence of lifestyle diseases and their predicted increase as the
world's population ages, there is an acute need to find ways to combat them.
In
particular there is a need to find ways to reduce low-grade systemic
inflammation in
order to prevent or treat diseases associated therewith, e.g. cardiovascular
disease,
type 2 diabetes and obesity.
The present inventors have now discovered that providing specific
combinations of macronutrients in the diet can result in a marked reduction in
low-
grade systemic inflammation. In particular, providing a diet with a specific
ratio of
carbohydrate, protein and fat (on an energy basis) wherein the fat component
has a
defined ratio of omega 3 to omega 6 fatty acids and, more particularly, a
defined
monounsaturate content results in a marked reduction in inflammatory markers.
This finding provides a way to influence low-grade systemic inflammation by
modulation of the diet and therefore enables the prevention or treatment of a
wide
range of lifestyle-related diseases.
Accordingly, a first aspect the invention provides a method of treating or
preventing low-grade systemic inflammation, in a subject wherein the diet of
the
subject is modified so as to provide in the region of (or around) 27% of
energy (27
kcal%) from carbohydrate, in the region of (or around) 30% of energy (30
kcal%)
from protein and in the region of (or around) 43% of energy (43 kcal%) from
fat
and wherein the fat component of the diet has a ratio of omega 3 to omega 6
fatty
acids of about 1:3.
In a second, more particular, aspect of the invention, the method can be for
treating or preventing a lifestyle disease.
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The diet is typically provided by administration of a nutrient-containing
composition which comprises the said carbohydrate, protein and fat, as well as
other
nutritional components such as insoluble fibre, vitamins and minerals. The
nutrient
containing composition is preferably a meal replacement composition.
Accordingly, in another aspect the invention provides a method of reducing
or preventing low-level systemic inflammation in a subject, the method
comprising
administering to the subject a nutrient-containing composition comprising in
the
region of (or around) 27% carbohydrate (kcal%), in the region of (or around)
30%
protein (kcal%) and in the region of (or around) 43% fat (kcal%), wherein the
fat
component of the composition has a ratio of omega 3 to omega 6 fatty acids of
about
1:3.
The nutrient-containing composition is preferably designed to contain
substantially all of the daily energy requirement of the subject when
administered in
sufficient daily dosages.
In another aspect the invention provides a nutrient-containing composition
for use in reducing or preventing low-level systemic inflammation in a
subject, the
composition comprising in the region of (or around) 27% carbohydrate (kcal%),
in
the region of (or around) 30% protein (kcal%) and in the region of (or around)
43%
fat (kcal%), wherein the fat component of the composition has a ratio of omega
3 to
omega 6 fatty acids of about 1:3.
The invention also extends to the use of such a composition in the
manufacture of a medicament for reducing or preventing low-level systemic
inflammation in a subject.
The compositions of the invention may be used, or adapted for use, in the
general consumer market (e.g. as a meal replacement formula or nutrition bar),
in
medical nutrition (e.g. pre- or -post surgery), infant nutrition (e.g. as a
milk additive
or replacement formula) and sports nutrition (e.g. in a muscle-building and/or
bone-
strengthening formula).
As noted above, such methods, compositions and uses can more particularly,
additionally or alternatively be specified or defined to be for, or for use
in, treating
or preventing a lifestyle disease.
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In a further aspect the invention provides a nutrient-containing composition
(e.g. a meal-replacement composition) which comprises a carbohydrate component
providing in the region of (or around) 27% of the energy in the composition, a
protein component providing in the region of (or around) 30% of the energy in
the
composition and a fat component providing in the region of (or around) 43% of
the
energy in the composition, wherein the fat component of the composition has a
ratio
of omega 3 to omega 6 fatty acids of about 1:3. The compositions of the
invention
are suitable for, and typically adapted for, use in treating or preventing low-
grade
systemic inflammation, e.g. treating or preventing lifestyle diseases, as
described
herein.
In a preferred embodiment the nutrient-containing composition is designed
to provide substantially all of the dietary energy and nutritional needs of
the
recipient. The components of the composition, e.g. the carbohydrate component,
the
protein component and the fat component may be provided separately or in
admixture.
Further, it also preferred that the fat component of the composition
comprises at least about 28%, or more particularly, at least about 28.5%, by
weight
of monounsaturated fatty acids.
In a further preferred embodiment, the ratio of saturated fatty acids (SFA) to
monounsaturated fatty acids (MUFA) in the fat component of the diet or
nutritional
composition is about 1:3.
As discussed in more detail below, in further preferred embodiments of the
methods, compositions or uses of the invention, the diet or composition is
provided
or administered 4 to 8, preferably 4-7 or 4-6, times a day. The compositions
of the
invention may accordingly be provided in a form suitable for administration
multiple such times a day.
The present invention relates to the prevention or treatment of lifestyle-
related diseases, referred to herein as "lifestyle diseases" which term
includes in
particular any condition that is linked to low-grade systemic inflammation,
which is
also known as "metabolic inflammation". Low-grade systemic inflammation is a
condition of sub-clinical systemic inflammation (that is inflammation that is
not
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linked or confined to a particular tissue or organ of the body and which may
accordingly occur in multiple (i.e. at least 2, or at least 3) areas or
tissues of the
body). Low-grade systemic inflammation may thus occur in the endothelium and
other organ systems. It is typically characterised by upregulation or
elevation of
inflammatory processes in multiple locations in the body, e.g. in different
tissue
types, and may be distinguished from acute and/or local inflammation by the
absence of associated pain. Low-grade systemic inflammation does not typically
involve pain but it may result in uncomfortable symptoms such as light
swelling,
reddening of the skin, water retention and mild flu-like symptoms. It is
generally
regarded as a chronic condition and may more particularly be referred to as
chronic
low-grade systemic inflammation. Commonly, low-grade systemic inflammation
may be characterised by a two- to threefold increase in the systemic
concentrations
of cytokines such as TNF-cc, IL-6 and CRP.
Specific conditions linked to lifestyle and low-grade systemic inflammation
include obesity; metabolic syndrome; insulin resistance and conditions or
diseases
associated therewith or more generally with insulin regulation, including
notably
diabetes, particularly type 2 diabetes; cardiovascular disease, e.g.
atherosclerosis or
conditions or disorders of the heart; and kidney disease, e.g. nephritis or
diabetic or
hypertensive nephropathy.
Subjects to be treated according to the present invention will typically be
humans, but the results described herein are believed to be applicable to
other
animals, especially to mammals (e.g. cats, dogs, cows, horses or pigs). In one
embodiment, the subject to be treated is in need of general inflammatory
suppression, especially where the subject is awaiting or following surgical
treatment
and/or is a hospital inpatient. In another embodiment, the subject is a normal
or
healthy subject, e.g. a subject who does not present with chronic medical
conditions
or acute inflammatory conditions.
The present invention is based on the finding that controlling the diet can
lead to rapid changes in expression of genes involved in inflammatory
processes.
These changes were observed within a week of changing from a high carbohydrate
diet to the specific diet of the invention. The composition of the diet is
believed to
play a key role in the effects observed, as is the number of meals
administered per
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day on the diet. Thus, in one embodiment the nutrient-containing composition
of
the invention is administered at least 4 times daily, e.g. at least 5, 6, 7 or
8 times
daily, for example 4 to 8, 4 to 7 or 4 to 6 times daily. Preferably it is
administered 5
or 6 times and especially 6 times daily.
As is clear from the foregoing, the individual components of the
compositions need not be administered or provided in admixture and can
provided
singly or individually, or in any desired or convenient combination. The term
"composition" as used herein accordingly does not imply a single formulation.
A
composition according to the invention may accordingly contain, include or
comprise multiple separate component parts (e.g. two or more, three or more,
or four
or more separate component parts for separate administration). Accordingly a
composition of the invention may alternatively be defined as a kit of separate
nutritional component parts. Each component part may comprise e.g. a fat,
protein
or carbohydrate component, or a mixture of different components e.g. fats and
proteins, or a mixture of different fats or fatty acids etc. Other components
or
ingredients (e.g. vitamins or minerals as discussed further below) may be
included
as separate component parts or as part of a component part.
In a preferred embodiment the nutrient-containing composition of the
invention represents essentially all of the daily intake of food in the diet,
i.e. the
nutrient-containing composition replaces substantially all (or all) of the
food in the
diet of the recipient. The daily intake of the nutrient-containing composition
is
preferably chosen so as to be normocaloric, i.e. to provide the necessary
amount of
energy required by the recipient. However, in alternative embodiments the
daily
intake of the nutrient-containing composition may be chosen so as to be
hypercaloric
(i.e. to provide more energy that required by the recipient, especially if
weight gain
is desired, e.g. following illness or surgery), or to be hypocaloric (i.e. to
provide less
energy that required by the recipient, especially if weight loss is desired).
The daily
intake of energy required by a recipient may be determined by the skilled
person,
e.g. following general guidelines or by determining the amount required for a
given
subject based on projected energy expenditure (e.g. using methods known in the
art -
Harris et al., PNAS (1918) 4(12):370-373; and Brooks et al., Am J Clin Nutr
(2004)
79(suppl):921S-930S). Typical values for the daily energy requirement of a
human
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recipient are in the region of 2000-2500 kcal, e.g. around 2200 kcal, for
adult
women and 2500-3000 kcal, e.g. around 2700 kcal, for adult men (1 kcal is
equivalent to 1 Calorie (Cal) and to 4.184 kilojoules (kJ)). Values for active
individuals will typically be higher than this and for less active
individuals, e.g. the
elderly, and children it will typically be lower.
Administration of the nutrient-containing composition may be designed to
provide multiple daily dosages having an equal nutritional content, e.g. 6
daily
dosages each containing one sixth of the total daily requirement of nutrients.
In
other words the composition may be provided in isocaloric dosages.
Alternatively,
the administration profile may be designed to provide multiple daily dosages
having
an unequal nutritional content, e.g. 5 daily dosages wherein the first, third
and fifth
dosage each comprises 25% of the total daily requirement of nutrients and the
second and fourth dosage each comprises 12.5% of the total daily requirement
of
nutrients.
Administration is typically enteral, especially oral (e.g. by normal
ingestion),
although direct gastric administration via a feeding tube may also be
employed.
The form of the nutrient-containing composition will depend on the intended
use, e.g. clinical use in hospital or use at home, but it will typically be
administered
as a solid meal (e.g. as a meal replacement bar or other shaped food item) or
in a
liquid form (e.g. as a shake or a liquid meal replacement drink). The
composition
may come ready-prepared, e.g. separated into individual dosage forms, or may
be
provided in bulk for measuring into individual dosages.
In a preferred embodiment, especially when provided in bulk (i.e. in an
amount sufficient to provide multiple doses, e.g. more than 100 g, especially
more
than 250 or 500 g, or more that 1, 2, 5 or 10 kg), the composition is
preferably in the
form both of a powder, typically containing the dry ingredients (e.g. the
protein,
carbohydrate and any insoluble fibre, vitamins and minerals), and also an oil
(typically comprising the fatty components). The powder and the oil are
preferably
provided separately, e.g. for admixture by the end user, although they may
also be
provided ready mixed so that each dosage may simply be made up in the required
quantity with a liquid (e.g. water) before administration. In one embodiment,
the
fat-containing components may be provided in powder form (e.g. as encapsulated
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particles or as a lyophilised powder, optionally in the presence of one or
more
stabilisers), in which case they are preferably provided in admixture with the
other
dry ingredients.
As defined above, the compositions of the invention comprise a carbohydrate
component, a protein component and a fat component. The compositions also
preferably comprise the other nutritional components required in the diet, for
example insoluble fibre (i.e. essentially indigestible complex carbohydrates),
salt
(e.g. a source of chloride ions), vitamins and minerals. The fibre may be
provided
as part of the carbohydrate component of the diet. Where percentages by weight
are
mentioned hereinafter, they preferably relate to percentage by dry weight,
i.e.
relative to a powder or solid composition substantially in the absence of
water. The
term "dry weight" may, however, include non-solid components such as oils
which
are specifically defined as part of the composition.
The carbohydrate component of the composition is present at a level that
provides in the region of (or around) 27% of the total (calorific) energy of
the
composition, i.e. at a level of about 24-30 kcal%, preferably about 25-29 or
26-28
kcal%, most preferably about 27 kcal%. In a typical composition of the
invention,
this would equate to in the region of (or around) 30% by weight of
carbohydrate,
e.g. about 25-35% or 28-32% or 29-31% by weight, especially about 31% or about
30% by weight of carbohydrate. Preferred types of carbohydrate include one or
more of fructose, glucose, maltodextrin (e.g. corn maltodextrin) and
oligofructose
and preferably all of these types are included. Other carbohydrates that may
be
included in the composition include mixtures of carbohydrates such as glucose
syrup, e.g. corn syrup. In one embodiment, the carbohydrate component of the
composition is essentially free from lactose.
In a preferred embodiment the carbohydrate component comprises (e.g.
consists essentially of) one, more than one, or all of maltodextrin MGX18
(Maltodextrin powder - HFG, Shandong, China), preferably at a level of in the
region of (or around) 40% by weight (e.g. between 35 and 45%, and preferably
about 40%) of the carbohydrate component; Raspberry 225L (Obipektin AG,
CH-9200 Bischofszell, Switzerland) preferably at a level of in the region of
(or
around) 37% by weight (e.g. between 32 and 42% and preferably about 40%) of
the
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carbohydrate component; fructose, preferably at a level of in the region of
(or
around) 10% by weight (e.g. between 7 and 13% and preferably about 10%) of the
carbohydrate component; and oligofructose, preferably at a level of in the
region of
(or around) 13% by weight (e.g. between 10 and 16%, and preferably about 13%)
of
the carbohydrate component.
The protein component of the composition is present at a level that provides
in the region of (or around) 30% of the total (calorific) energy of the
composition,
i.e. at a level of about 27-33 kcal%, preferably about 28-32 or 29-31 kcal%,
most
preferably about 27 kcal%. In a typical composition of the invention, this
would
equate to in the region of (or around) 42% by weight of protein, e.g. about 38-
46%
or 40-44% by weight of protein, especially about 42% by weight of protein.
In one embodiment, the protein source is chosen so as to minimise specific
allergic reaction. In other words the protein component(s) is (are), or is
(are)
selected to be, non-allergenic, e.g. the protein is not derived from wheat
and/or is not
derived from milk and/or egg components which are known to be allergenic. In a
preferred embodiment, the protein component comprises proteins from multiple
sources in order to minimise general allergic reactions, e.g. the protein is
chosen
from a plurality of sources selected from meat (e.g. animal meat such as beef,
chicken and pork, and fish meat, such as cod, salmon, mackerel and tuna), egg,
milk
(e.g. whey and/or casein), vegetables (e.g. tubers such as potato and yam, and
legumes such as pea, soy bean and peanut), microorganisms (e.g. yeast) and
flour
(e.g. corn flour or wheat flour). Preferably, the protein component comprises
one,
two or all of whey protein, egg white and pea protein. In one embodiment, the
protein component of the composition is essentially free from gluten,
especially
wheat gluten.
In a preferred embodiment the protein component comprises (e.g. consists
essentially of) one, more than one, or all of whey protein 80 (Tine BA, 0051
Oslo,
Norway), preferably at a level of in the region of (or around) 55% by weight
(e.g.
between 50 and 60% and preferably about 55%) of the protein component; egg
white, preferably at a level of in the region of (or around) 13% by weight
(e.g.
between 10 and 16%, preferably about 13%) of the protein component; and pea
protein 90 (Pisane M9, Provital Industrie S.A., Belgium), preferably at a
level of in
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the region of (or around) 32% by weight (e.g. between 27 and 37% and
preferably
about 32%) of the protein component.
The fat component (i.e. the fat-containing component, typically including
fats and/or oils) of the composition is present at a level that provides in
the region of
(or around) 43% of the total (calorific) energy of the composition, i.e. at a
level of
about 40-46 kcal%, preferably about 41-45 or 42-44 kcal%, most preferably
about
43 kcal%. In a typical composition of the invention, because fat contains
roughly
double the calorific content by mass compared to protein and carbohydrate,
this
equates to in the region of (or around) 26% by weight of fats, e.g. about 22-
30% or
24-28% by weight of fats, preferably about 26%. The fat component is
preferably
derived from different sources to allow for an optimum combination of fat and
oil
types in the final composition. Examples of suitable sources of fats include
plant
sources (e.g. flax seed (linseed) oil, hemp oil, soya oil, canola (rapeseed)
oil,
vegetable oil, olive oil, coconut oil, borage oil and evening primrose oil,
and nut oils
from nuts such as walnuts) and animal sources (e.g. marine animals such as
seal and
squid, fish and shellfish, especially fish liver oils such as cod liver oil).
Preferably,
the fat component is derived from two or more, e.g. all, of the oils selected
from
rape seed oil, olive oil, coconut oil, flax seed oil and marine oils.
In a preferred embodiment, the fat component comprises (e.g. consists
essentially of) one, more than one, or all of rape seed oil, preferably at a
level of in
the region of (or around) 9% by weight (e.g. between 6 and 12%, preferably
about
9%) of the fat component; olive oil, preferably at a level of in the region of
(or
around) 79% by weight (e.g. between 70 and 88% and preferably about 79%) of
the
fat component; coconut oil, preferably at a level of in the region of (or
around) 9%
by weight (e.g. between 6 and 12% and preferably about 9%) of the fat
component;
flax seed oil, preferably at a level of in the region of (or around) 1.5% by
weight
(e.g. between 1 and 2%, preferably about 1.5%) of the fat component; and
marine
omega 3/6/7/9 oils, preferably at a level of in the region of (or around) 1.5%
by
weight (e.g. between 1 and 2%, preferably about 1.5%) of the fat component.
Marine omega 3/6/7/9 oils produced by Vitomega AS (Heimdal, Norway) are
especially preferred.
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The fat component of the composition must comprise both omega 3 and
omega 6 fatty acids. Typically these will be present in a sufficient amount to
provide the recommended daily allowance (RDA) of fatty acids when the
composition is administered, e.g. about 1-2 grams of omega 3 fatty acids per
day
and about 3-6 grams of omega 6 fatty acids per day. Thus, the fat component of
the
composition of the invention preferably comprises between about 0.5 and 2% by
weight of omega 3 fatty acids, especially between 0.75 and 1.5% by weight,
e.g.
about 1% by weight. The fat component of the composition of the invention
preferably comprises between about 1.5 and 6% by weight of omega 6 fatty
acids,
especially between 2.5 and 4.5% by weight, e.g. about 3% by weight.
The fat component of the composition preferably comprises at least about 28
%, or more particularly at least about 28.5% by weight of monounsaturated
fatty
acids, especially between 28 and 30% by weight. The ratio of monounsaturated
to
saturated fatty acids in the fat component of the composition is preferably
about 3:1.
Vitamins and/or minerals are preferably provided in the compositions of the
invention at levels at or around those recommended for daily intake, i.e.
around the
RDA levels.
Vitamins which may be included in the compositions of the invention
preferably include one or more of the following, especially all of the
following:
Vitamin C, Vitamin E, Thiamine, Riboflavin, Vitamin B6, Vitamin B12, Folic
acid,
Pantothenic acid, Niacin and Biotin. Other vitamins which may be included,
e.g. in
addition to those listed above, include one or more, and preferably all, of:
Vitamin
A, Vitamin D, Vitamin K, choline and beta-hydroxy-beta-methylbutyric acid
(HMB). The vitamins are preferably present in the necessary quantity to
provide the
RDA of each when a daily dosage of the composition is administered. Where the
composition is for administration in multiple daily dosages, the absolute
amount of
the vitamin will be typically reduced accordingly. For example, a single
dosage of
the composition of the invention which is intended for administration in six
equal
dosages per day may contain the following levels of each vitamin (one sixth of
the
total required daily amount): Vitamin C - between 10 and 100 mg, e.g. about 80
mg;
Vitamin E - between 2 and 5 mg, e.g. about 3.5 mg; Thiamine - between 0.2 and
0.6
mg, e.g. about 0.38 mg; Riboflavin - between 0.2 and 0.6 mg, e.g. about 0.42
mg;
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Vitamin B6 - between 0.2 and 0.8 mg, e.g. about 0.53 mg; Vitamin B12 - between
0.2 and 0.5 p,g, e.g. about 0.26 mg; Folic acid - about 0.051 i.ig;
Pantothenic acid -
between 0.5 and 5 mg, e.g. about 1.6 mg; Niacin - between 1 and 10 mg, e.g.
about
4.8 mg; and Biotin - about 0.042 vtg. Preferably the composition of the
invention
includes a vitamin C-containing extract of Acerola (e.g. Acerola C or Acerola
25-C
supplied by Obipektin AG, CH-3400 Burgdorf, Switzerland) which comprises in
the
region of (e.g. around) 17% by weight of vitamin C, e.g. between 12 and 30% or
between 15 and 20%, preferably about 17% by weight vitamin C. The extract of
Acerola preferably comprises about 0.5% by weight of the total composition.
In an especially preferred embodiment, the composition of the invention
contains a vitamin component comprising (e.g. consisting essentially of) about
97%
by weight of an Acerola C extract (which extract preferably comprises about
17%
by weight of vitamin C) and about 3% by weight of a multivitamin, the
multivitamin
comprising (e.g. consisting essentially of) about 25.5% by weight of Vitamin
C,
about 19.1% by weight of Vitamin E, about 2.7% by weight of Thiamine, about
3.0% by weight of Riboflavin, about 3.8% by weight of Vitamin B6, about
0.0019%
by weight (19 ppm) of Vitamin B12, about 0.0004% by weight (4 ppm) of Folic
acid, about 11.4% by weight of Pantothenic acid, about 34.4% by weight of
Niacin
and about 0.0003% by weight (3 ppm) of Biotin. The vitamin component is
preferably present in the composition at a level of about 0.5% by weight.
Minerals which may be included in the compositions of the invention
preferably include one or more of the following, especially all of the
following:
calcium, magnesium and potassium. Other minerals which may be included, e.g.
in
addition to those listed above, include one or more, and preferably all, of:
iron,
phosphorus, iodine, zinc, selenium, copper, manganese, chromium and
molybdenum. The term "mineral" as used herein is not intended to encompass
sodium chloride. The minerals are in a physiologically tolerable, e.g.
absorbable
ionic, form. Such forms of minerals for human nutrition are well known,
examples
of which include citrates (e.g. potassium citrate), phosphates (e.g. calcium
phosphate) and sulphates (e.g. zinc sulphate). Where the composition is for
administration in multiple daily dosages, the absolute amount of each mineral
will
be typically reduced accordingly. For example, a single dosage of the
composition
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of the invention which is intended for administration in six equal dosages per
day
may contain the following levels of each mineral (one sixth of the total
required
daily amount): calcium - between 50 and 200 mg, e.g. about 64 mg; magnesium -
between 20 and 80 mg, e.g. about 32 mg; and potassium - between 100 and 1000
mg, e.g. about 175 mg.
In an especially preferred embodiment, the composition of the invention
contains a mineral component comprising (e.g. consisting essentially of) about
23.5% by weight of calcium, about 11.6% by weight of magnesium and about
64.9% by weight of potassium. The mineral component is preferably present in
the
composition at a level of about 0.3% by weight. The values given herein for
the
amounts of minerals refer to the weight percent of the metal ion, e.g. Ca2+.
Salt (i.e. a source of chloride ions, preferable NaC1) is typically present in
the
composition at a level of around 0.5 to 1% by weight, especially around or
about
0.75% by weight, especially where the composition is a meal replacement
composition. For example, a single dosage of the composition of the invention
which is intended for administration in six equal dosages per day would
typically
contain about 0.68 g of salt (calculated as NaC1). The compositions of the
invention
are preferably designed to provide a daily dosage which comprises a total of
between 3 and 6 g of salt (calculated as NaCl) per day, e.g. around 4 g or
around 5 g
per day.
In addition to the components listed above, other ingredients may be added
to alter the properties of the composition. These include flavourings, which
may be
natural or synthetic, e.g. raspberry flavour, vanilla flavour, strawberry
flavour or
chocolate flavour; colourings, especially those which are designated with an
number between 100 and 199, e.g. riboflavin (E101) or cochineal (E120); and
preservatives, especially those which are designated with an "E" number
between
200 and 299, e.g. sodium benzoate (E211).
The compositions of the invention are preferably meal replacement
compositions. As such, they will typically not comprise additional nutrient
components. Preferably they are essentially free of other components besides
those
mentioned above. For example, the compositions of the invention may be free
from
corn flour, wheat flour, corn starch, potato starch, etc.
CA 02779883 2012-06-12
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In a further aspect the invention provides a nutrition-containing composition
as defined herein.
In a preferred embodiment the composition comprises (e.g. consists
essentially of):
- a carbohydrate component which provides about 27% of the energy in the
composition comprising maltodextrin MGX18 at a level of about 40% by
weight, Raspberry 225L at a level of about 37% by weight, fructose at a level
of about 10% by weight and oligofructose at a level of about 13% by weight;
- a protein component which provides about 30% of the energy in the
composition comprising whey protein 80 at a level of about 55% by weight;
egg white at a level of about 13% by weight and pea protein 90 at a level of
about 32% by weight;
- a fat component which provides about 43% of the energy in the
composition
comprising rape seed oil at a level of about 9% by weight, olive oil at a
level
of about 79% by weight, coconut oil at a level of about 9% by weight, flax
seed oil at a level of about 1.5% by weight and marine omega 3/6/7/9 oils at
a level of about 1.5% by weight;
- optionally and preferably salt (e.g. NaC1) at a level of about
0.75% by weight
of the composition; and
- optionally and preferably vitamins and minerals,
wherein the fat component of the composition has a ratio of omega 3 to
omega 6 fatty acids of about 1:3.
In a particularly preferred embodiment, the composition of the invention
comprises:
- a carbohydrate component which provides about 27% of the energy in the
composition comprising maltodextrin MGX18 at a level of about 40% by
weight, Raspberry 225L at a level of about 37% by weight, fructose at a level
of about 10% by weight and oligofructose at a level of about 13% by weight;
- a protein component which provides about 30% of the energy in the
composition comprising whey protein 80 at a level of about 55% by weight;
egg white at a level of about 13% by weight and pea protein 90 at a level of
about 32% by weight;
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- a fat component which provides about 43% of the energy in the
composition
comprising rape seed oil at a level of about 9% by weight, olive oil at a
level
of about 79% by weight, coconut oil at a level of about 9% by weight, flax
seed oil at a level of about 1.5% by weight and marine omega 3/6/7/9 oils at
a level of about 1.5% by weight;
- NaCl at a level of about 0.75% by weight of the composition;
- a vitamin component at a level of about 0.5% by weight of the
composition,
the vitamin component consisting essentially of about 97% by weight of an
Acerola C extract (which extract preferably comprises about 17% by weight
of vitamin C) and about 3% by weight of a multivitamin, the multivitamin
consisting essentially about 25.5% by weight of Vitamin C, about 19.1% by
weight of Vitamin E, about 2.7% by weight of Thiamine, about 3.0% by
weight of Riboflavin, about 3.8% by weight of Vitamin B6, about 0.0019%
by weight (19 ppm) of Vitamin B12, about 0.0004% by weight (4 ppm) of
Folic acid, about 11.4% by weight Pantothenic acid, about 34.4% by weight
Niacin and about 0.0003% by weight (3 ppm) Biotin; and
- a mineral component at a level of about 0.3% by weight of the
composition,
the mineral component consisting essentially of about 23.5% by weight of
calcium, about 11.6% by weight of magnesium and about 64.9% by weight
of potassium,
wherein the fat component of the composition has a ratio of omega 3 to
omega 6 fatty acids of about 1:3.
The invention will now be further described with reference to the following
non-limiting Examples and Figures in which:
Figure 1 shows - Mean glucose response to a single isocaloric meal of diet
HC and diet MC respectively, n = 7. Paired samples two-sided t-tests for: the
comparison of the area under the glucose response curve (AUC) 0 ¨ 120 min
after a
diet A meal (drawn as solid line) to after a diet B meal (drawn as dashed
line) (the
AUC is limited by a baseline drawn between the 0 and 120 min values for the
respective meal test) (P = 0.032), the glucose concentration at 30 min after
intake of
a diet AHC meal versus 30 min after a diet BMC meal (P --- 0.038), and the
glucose
CA 02779883 2012-06-12
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concentration at 120 min after intake of a diet AHC meal versus 120 min after
a diet
BMC meal (P = 0.002);
Figure 2 shows - Summary of the filtration of gene probes, selection of
differentially expressed genes in response to diet HC, diet MC, and
overlapping
genes between the two diets and the number of up- and down-regulated genes in
response to each diet;
Figure 3 shows - Pathways significantly regulated by the separate effect of
the ETC (red/light-grey upper bars) and/or the MC diet (blue/dark-grey lower
bars).
The pathways are grouped into Metabolic pathways, Apoptosis, Cell cycle,
proliferation and growth, Stress/immunity, and Other. Bars indicates -log(p-
values),
where the vertical line at 1.3 corresponds to a P 0.05 cut-off value for
pathway
significance level; and
Figure 4 shows - Regulation of differentially expressed genes downstream of
a selection of transcription factors in response to the FTC (AHC) and MC (BMC)
diets separately. Genes indicated by green or red labels, respectively, are
significantly down- or up-regulated on transcriptional level for the actual
before diet
to after diet comparison. Grey labels represent genes not significantly
changed in
the actual comparison. Genes outlined with blue are members of pathways
related to
apoptosis, proliferation/cell cycle regulation, or stress/immunity. All
relations are
curated from human studies and registered in the IPA library (Ingenuity
Pathway
Analysis version 8.0; Ingenuity Systems Inc, Redwood City, USA).
Example 1 - Anti-inflammatory meal replacement diet composition (referred
to as diet "MC")
A meal replacement diet was prepared with a defined quantity and quality of
each of the three main macronutrient components (carbohydrate, protein and
fat).
The diet was formulate to contain adequate amounts of fibre, minerals and
vitamins
as well as to provide specific levels of fats (e.g. a defined ratio of omega 3
to omega
6 fatty acids of 1:3) and carbohydrates (e.g. a distribution of
monosaccharides,
disaccharides and complex carbohydrates).
Table 1 below shows the amount of composition required (in grams) to
provide a 3000 kcal/day diet.
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Table 1 - Composition of MC diet.
Carbohydrates (27 kcal%)
Maltodextrin (MGX18)I 67.19 g
Raspberry 225L2 62.00 g
Fructose 18 g
Fibre (oligofructose) 21.00 g
Proteins (30 kcal%)
Whey protein 803 125.16 g
Egg white 29.11 g
Pea protein 904 72.77 g
Fats (43 kcal%)
Rape seed oil 12.07 g
Olive oil 110.10 g
Coconut oil 12.70 g
Flax seed oil 2.25 g
Marine omega 3/6/7/9 2.34 g
Vitamins/minerals
Acerola-C6 2.745 g
Vitamin C (mg) 17 1 %
Vitamin mix 0.0834g
Vitamin C (mg) 25.53598 %
Vitamin E (mg) 19.09647 %
Thiamine (mg) 2.70163 %
Riboflavin (mg) 3.03471 %
Vitamin B6 (mg) 3.84890 %
Vitamin B12 (jig) 0.00192%
Folic acid (lug) 0.00037 %
Pantothenic acid (mg) 11.36166 %
Niacin (mg) 34.41806 %
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Biotin (,ug) 0.00030 %
Salt (NaC1) 4.08 g
Mineral mix 1.635 g
Calcium (mg) 23.462 %
Magnesium (mg) 11.617%
Potassium (mg) 64.920 %
1 HFG, Shandong, China
2 Obipektin AG, CH 9200 Bischofszell, Switzerland
3 Tine BA, 0051 Oslo, Norway
4 Pisane M9 - Provital Industrie S.A., Belgium
5 capsules from Vitomega, Heimdal, Norway
6 Obipektin AG, CH-3400 Burgdorf, Switzerland
Example 2 - Control meal replacement diet composition (referred to as diet
"HC")
A control meal replacement diet was prepared to represent a typical western
diet, based on Norwegian and US official dietary recommendations. The diet
contained the same ingredients as the diet set out in Example 1, but the
amounts of
carbohydrate, protein and fat were altered to provide 65 kcal% from
carbohydrates,
15 kcal% from protein and 20 kcal% from fat. Diet HC contained half the amount
of saturated fat and half the amount of monounsaturated fat compared to diet
MC.
The amount and type of polyunsaturated fatty acid (PUFA) in diets MC and HC
was
exactly the same.
Example 3 - Feeding study
Background
The aim of present study was to investigate if and how the quantity of the
macronutrients in an iso- and normocaloric diet would affect chronic disease
risk in
CA 02779883 2012-06-12
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modestly overweight, otherwise healthy young subjects. In order to do so, two
meal replacement diets (MRDs) as defined in Examples 1 and 2 tested in a
randomized crossover fashion, meaning that all subjects consumed both diets
where
diet order was randomized. Both diets were similar in number of meals per day
(6),
quality and quantity of minerals, vitamins, fibre, PUFA (n-6/n-3), and ratio
SFA/monounsaturated fat (MUFA). Variable components were quantity of proteins
and SFA/MUFAs to compensate for difference in carbohydrate calories between
the
two diets. Transcriptomic, proteomic and metabolic data collected from blood
cells
and blood serum or plasma were analyzed and combined in a systems biological
fashion.
Subjects and methods
All subjects gave their written informed consent to participate in the study.
The study protocol and the ethical standards employed were approved by the
Regional Committee for Medical and Health Research Ethics, Central Norway
(REK ID 4.2007.515).
A total of 32 healthy male (19) and female (13) volunteers aged 18-30 from
the Norwegian University of Science and Technology student population
participated in the study (Table 2). Study inclusion criteria were: body mass
index
(BMI; in kg/m2) >24.5 or <27.5; blood pressure less than 135 mmHg systolic and
80
mmHg diastolic; urine negative for protein or glucose dip-stix values; fasting
blood
glucose level inside normal range, high sensitivity (hs)CRP concentration <5
mg/L;
while exclusion criteria were regular use of prescription medication; receipt
of
inoculations within 2 month prior to the study or the intention to receive
such during
the study; diagnosis of chronic medical condition (e.g. diabetes,
cardiovascular
disease, anaemia, gastrointestinal disease); symptoms of allergy; planned or
current
pregnancy or lactation; acute inflammation as assessed on the basis of white
blood
cell count, platelet count or hsCRP; abnormal kidney, liver or metabolic
functions.
CA 02779883 2012-06-12
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Table 2 - Anthropometric and biochemical measures at baseline (n= 32,
fasting values).
Mean Range
Age (years) 24 19 ¨ 30
Height (m) 1.76 1.57 ¨ 1.90
Body mass (kg) 80.6 63.8 ¨ 97.5
BMI (kg/m2) 26.1 24.2 ¨ 28.0
Systolic blood pressure (mm Hg) 116 80 ¨ 138
Diastolic blood pressure (mmHg) 73 49 ¨ 89
Waist/hip ratio 0.78 0.68 ¨ 0.90
Glucose (mmol/L)I 4.77 4.10 ¨ 5.50
Insulin (pg/mL)2 2001 63 ¨ 4818
hsCRP (mg/L)I 0.76 0.15 ¨3.30
serum
2 EDTA-plasma.
We performed a randomized, controlled, cross-over diet intervention trial.
The study design consisted of two randomly assigned study arms stratified by
sex,
age (< 25 years or > 25 years), and waist circumference (females < 77 cm and
males
< 86 cm, or female > 77 cm and males > 86 cm at the most narrow part of the
waist)
using WebCRF (Unit for Applied Clinical Research, Norwegian University of
Science and Technology, Trondheim, Norway).
The method of Jorstad et al. (TRENDS in Plant Science (2007) 12(2):46-50)
was used to perform sample size estimation for detecting differences in gene
expression of diet (measurements 6 days apart) from pilot data of Brattbakk et
al.
(OMICS: A Journal of Integrative Biology, June 2011, ePub ahead of print). We
found that a sample size of around 20 subjects (calculation performed on men
only)
were needed to achieve an average power (expected proportion of correct
rejections)
of 0.8 and a positive false discovery rate (FDR) of 0.05. This means that
we would
expect to detect at least 80 % of the regulated genes with control of the
multiple type
I error, as defined by the FDR, at 5 %. Effect sizes for regulated genes were
CA 02779883 2012-06-12
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estimated from the pilot data, using a mixture model, that is, we assumed that
effect
sizes of interest in the current study are in the same order as in the pilot
study.
Two iso- and nonnocaloric liquid MRDs composed of 65:15:20 (diet HC,
high-carbohydrate, see Example 2) and 27:30:43 (diet MC, moderate-
carbohydrate,
see Example 1) energy percent (E %) of carbohydrates, proteins and fats,
respectively, were given as six isocaloric meals per day. This constituted all
nutrient
intakes during the study periods. The two study arms started with either study
diet
HC first or study diet MC first. We collected fasting blood samples at four
time
points, before and after each of the two diet periods. Diet periods were
separated by
eight days with normal eating habits. The study is registered in
ClinicalTrials.gov
with identifier NCT00733018. Microarray data are submitted to the ArrayExpress
(accession number E-TABM-1073)
Composition of meal replacement diets
Two dietary powder-mixes containing the carbohydrate and protein
components of the diets of Examples 1 and 2 were produced for the purpose of
the
study by Food Innovations AS, Lillestrom, Norway. Two different oil mixtures,
as
discussed above, were mixed by the study coordinator to obtain the desired
value of
SFAs, MUFAs and PUFAs. Other dietary factors such as fibre, PUFAs, vitamins,
and minerals were kept constant at the level of the recommended daily
allowances
(RDA) in both diets. The fibre content in both diets was 25 g per 3000 kcal.
The
glycemic load (GL) was calculated to be 2.71 times higher in diet HC than in
diet
MC. We adjusted the diets to be normocaloric to each individual based on
calculations of resting energy expenditure using Harris-Benedict equation
(Harris et
al., supra) multiplied by the self reported average daily physical activity
level - PAL
(Brooks et al., supra).
All food was provided; individually pre-packed with ID-code, diet date and
meal number, as six isocaloric meals per day to be mixed with water in a
provided
shaker. The diets were flavoured with raspberry flavour to optimize
compliance.
Nevertheless, the diets were not blinded because the taste and colour of the
diet
powders and the amount of oil in the two sets of meals were easy to
distinguish.
However, analyses of blood samples were performed blinded without knowledge of
CA 02779883 2012-06-12
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diet code. To the knowledge of the inventors, compliance was about 99.5%, as
registered in frequent, at least bi-weekly, personal dialogue between subjects
and the
study coordinator. Anyone who skipped more than 3 meals was excluded from the
study.
Body composition measurements
At each appearance, measures of body composition (body mass, body fat
percent, body water percent, visceral fat rating, muscle mass, physical
rating, basal
metabolic rate, metabolic age, and bone mass) were recorded by bioelectric
impedance analysis (BIA), using Tanita innerscan BC 545 segmental body
composition monitor (Tanita Corporation, Tokyo, Japan). We also measured body
weight at day 3 or 4 during each diet period, upon the subjects' collection of
the
second half of the pre-packed study meals. An eventual weight deviation within
the
diet period could thus be compensated half-way in the diet period by adjusting
the
size and caloric content of the remaining pre-packed meals.
Blood sampling
From all study participants venous blood samples were collected between
7.30 and 10.30 a.m. after an overnight fast. An initial sample was collected
as part
of the health check screening. Next, we collected samples at four time points;
before the start of each diet intervention period and the mornings after
completing
six days on the diets. We collected blood in appropriate blood collecting
tubes for
subsequent RNA isolation (K2EDTA-tubes, Greiner Bio-One, Kremsmiinster,
Austria) and biochemical analyses (clot separation tubes, Greiner Bio-One).
Tubes
for serum separation were left to coagulate at room temperature for 30 min
before
centrifugation at room temperature for 10 min at 2200 x g. Anti-coagulated
blood
for plasma separation was immediately placed on ice and centrifuged at 4 C
(1500 x
g, 10 min) within 30 min. Unless analyzed the same day, plasma and serum
samples
were kept on ice, aliquoted, and frozen at -80 C within 2-4 h, until further
analysis.
CA 02779883 2012-06-12
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Biochemical analyses
The clinical chemistry analyses were performed at The Department of
Medical Biochemistry at St. Olavs University Hospital, Trondheim, Norway. Of
these biomarkers, triglycerides, total- and HDL-cholesterol, glucose,
haemoglobin,
total leukocytes and differential count of leukocytes, platelets, hsCRP, and
uric acid
were analyzed before and after each diet period. LDL-cholesterol, the ratio of
triglycerides to HDL-cholesterol, the ratio of total- to HDL-cholesterol, and
the
atherogenic index (AIP, the logarithm of the ratio of triglycerides to HDL-
cholesterol) were calculated.
Protein analyses
Bio-Plex Diabetes Panel assay (Bio-Rad Laboratories Inc., Hercules, CA,
USA) was performed using Luminex xMAPTm technology on fasting EDTA-plasma
samples from before and after both diet periods from all subjects, with a Bio-
Plex
200 suspension array reader and analysis was done with the Bio-Plex Manager
5.0
software (Bio-Rad Laboratories Inc.). The analysis principle combines parallel
bead-based sandwich immunoassay with flow cytometry in a 96-well microplate
format. The diabetes panel is assembled to determine twelve diabetes related
biomarkers in the same assay using the same dilution of plasma samples. The
panel
biomarkers can be grouped into cytokines (interleukin-6 (IL-6), tumour
necrosis
factor-a (TNF-a), plasminogen activator inhibitor-1 (PAT-1)) adipokines
(leptin,
resistin, visfatin(nicotinamide phosphoribosyltransferase, Nampt)), gut
hormones
and incretins (ghrelin (appetite-regulating hormone), glucagon-like peptide-1
(GLP-
1), glucose-dependent insulinotropic polypeptide (GIP, Gastric inhibitory
polypeptide)), and glucose disposal hormones (insulin, glucagon, insulin
connecting
peptide (C-peptide)). We performed the analyses according to the
manufacturers'
protocol with exception of the following modification: a fifty percent
reduction in
the number of antibody labelled magnetic beads was used. We analyzed all
samples
from each subject in duplicate in the same assay to minimize inter-assay
variation.
Low- and high-level controls for all biomarkers were included in all assays.
Adiponectin (Mercodia, Uppsala, Sweden) and Serum Amyloid A (SAA, human)
(Invitrogen, Camarillo, CA, USA) were analyzed by ELISA technique in separate
CA 02779883 2012-06-12
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assays using EDTA-plasma, on a Dynex DS2 automatic ELISA instrument (Dynex
Technologies, Magellan Biosciences, Chantilly, VA, USA).
Postprandial meal test glucose responses
A subgroup of the subjects representing both sexes and intervention regimes
performed a postprandial meal challenge on the seventh day following each of
the
two six-day diet intervention periods. At sampling time point, fasting samples
for
determination of serum glucose were collected between 7.30 a.m. and 9.30 a.m.
Thereafter one study meal identical to all meals in the immediate previous
study
period was ingested within 5 min. After 30 and 120 min, venous blood samples
were
collected for subsequent serum glucose determination. We calculated the area
under
the glucose response curve (AUC) to each of the study meals by applying the
trapezoid rule for the intervals 0 to 30 min, and 30 to 120 min. We performed
a
baseline correction to adjust for the 120 min glucose concentration being
lower than
baseline, by subtracting the AUC for the 0 to 120 min interval from the sum of
AUCs 0 to 30 min and 30 to 120 min. Both the corrected AUCs and the 30 min and
120 min postprandial serum glucose concentrations were compared using paired
samples t-test.
Assessment of insulin sensitivity and resistance
The homeostasis model assessment (HOMA) determines insulin sensitivity
(% S), 13-cell function (% B) and insulin resistance (IR) based on fasting
blood
glucose (in mmol/L) and C-peptide (in nmol/L). We used the HOMA2 calculator
version 2.2.2 CD (Diabetes Trials Units, University of Oxford,
http://www.dtu.ox.ac.uk/homacalculator/index.php).
Isolation and quality determination of RNA from blood leukocytes
Leukocytes from EDTA-blood (K2EDTA-tubes, 9 mL) were captured
immediately after sampling on Leuk0LOCKTM filters using the Leuk0LOCKTM
Total RNA Isolation System (Applied Biosystems/Ambion, Austin, TX, USA)
according to the manufacturers' instructions
(http://www.ambion.com/techlib/prot/fm 1923.pdf). Plasma, erythrocytes and
CA 02779883 2012-06-12
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platelets were removed by filtering, while the leukocytes captured on the
LeukoLOCKTM filters were washed, and stabilized in RNAlater (Applied
Biosystems/Ambion) at -80 C until RNA extraction using the alternative
protocol
http://www.ambion.com/techlib/misc/leuko iso.pdf) (version 0602). The isolated
RNA was quantified and checked for protein and organic contaminations using
NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Delaware,
USA) at OD 260/280 and OD 260/230 respectively. RNA integrity was checked on
an Agilent 2100 Bioanalyzer (Agilent Technologies Inc., Santa Clara, CA, USA).
Microarray analyses
The gene expression analysis was performed by the Norwegian Microarray
Consortium in Trondheim, Norway. Briefly, 300 ng of total RNA was processed
into cRNA using Illuminaiv TotalPrepTm RNA Amplification Kit (Applied
Biosystems/Ambion, Austin, TX, USA). The cRNA concentration of each sample
was quantified using NanoDrop ND-1000 spectrophotometer, to ensure that equal
amounts of each sample were used for hybridization. Then, 750 ng cRNA was
hybridized onto HumanHT-12 Expression BeadChip v3.0 (Illumina, San Diego, CA,
USA) using Whole-Genome Gene Expression Direct Hybridization Assay
(Illumina). These BeadChips contain 48,804 probes derived from the National
Center for Biotechnology Information (NCBI) Reference Sequence (RefSeq) (Build
36.2, Rd l 22) and UniGene (Build 199) databases, of which 27,455 were
annotated
in Entrez Gene. The complete set of four samples from each subject was
hybridized
to the same 12-sample BeadChip to minimize intra-subject variation due to
inter-
array variation. A total of 128 samples from 32 subjects were hybridized on 11
BeadChips in randomized three-subject groups. After scanning, the results were
imported into Illumina BeadStudio where the quality of each array and scan was
tested.
Preprocessing of microan-ay data
Leukocyte gene expression profiling was done on the HumanHT-12
Expression BeadChip v3.0 (Illumina). Analyses of microarray data were
performed
in the R statistical analysis framework (R Development Core Team, 2011), using
the
CA 02779883 2012-06-12
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lumi (BeadArray Specific Methods for Illumina Microarrays) and the limma
library
(Smyth et al., Bioinformatics (2005) 21(9):2067-2075) add-on packages from
Bioconductor (http://www.bioconductor.org) (Gentleman et al., 2004), and the
Illumina add-on package (An R Library for pre-processing Illumina Whole Genome
Expression BeadChips) available from the Walter and Eliza Hall Institute for
Medical research www-pages at http://bioinfwehi.edu.au/software/index.htmi.
After removal of two outlier samples, background correction based on negative
controls, quantile-quantile normalization, signal log2-transformation, and
removal of
not detected or bad probes, 27 372 unique probes were left in the gene
expression
dataset. Data were analysed using moderated t- tests with individual as block
effect
in the limma package. Covariates used were time, diet and the time-diet
interaction,
sex and which diet that was administered first.
Three contrasts, i.e. the difference between the start-point and end-point of
both diet periods, were tested to determine the effect of the HC diet, the
effect of
diet MC diet, and the difference between the effect of the two diets. FDR
adjusted
p-values using the Benjamini-Hochberg step-up algorithm was produced for each
of
the three contrasts. Using a FDR cut-off of 5% this did not identify any
differentially expressed genes comparing the effect of the diets. Responding
to the
HC diet and the MC diet, a total of 3225 and 1370 differentially expressed
genes
were found, respectively, where 843 genes overlapped.
Pathway analysis was performed using Ingenuity Pathways Analysis 8.5
(IPA, Ingenuity Systems , Redwood City, CA, USA, www.ingenuity.com).
Statistical analyses
Analyses were performed using the R statistical analysis framework, and the
nlme library (Pinheiro etal., 2011). For each response a linear mixed effects
model
was fitted with a four levels fixed factor (before and after diet MC and HC).
A
random effect for each individual was used. Which sex and which diet that was
administered first were also included as covariates in the initial analyses,
but was not
found to be significant and not included in the final analyses. Analyses were
conducted either on the original scale (model 1), the log-scale (model 2) or
the
square root scale (model 3). The scale chosen was based on assessment of the
CA 02779883 2012-06-12
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normality of residuals using qq-plots and the Anderson-Darling test for
normality.
The same three contrasts as for the microarray analysis were investigated.
Estimated effects and 95% confidence intervals on the original scale for the
three
contrasts are presented in Table 3 below together with p-values and False
Discovery
Rate (FDR) adjusted p-values. P-values were calculated using t-tests, and the
FDR
adjusted p-values were calculated using the Benjamini-Hochberg step-up
algorithm
(Benjamini et al., J. Roy. Statist. Soc. Ser. B (1995) 57:289-300) for
controlling the
FDR for all the 32 responses and 3 contrasts (96 tests). Using a cut-off of 5%
for the
FDR 22 of the 96 tests are found to be significant. The linear mixed effects
degrees
of freedom (DF) are reported in Table 3.
Table 3 - Diet responses of markers regulating glucose, adipogenesis, insulin
resistance and HOMA indices (Mean for change day 6 ¨ day 0 with 95% CI; n= 32
for all analyses except glucagon and GLP-1 n= 31; paired samples t-test within
each
diet (AHC - Example 2; and BMC - Example 1) between day 0 and day 6; bold
indicates P < 0.05 between time points within diet).
Change Diet Mean 95% CI
Glucose (mmol/L)1 AHC 0.03 -0.11, 0.17
0.645
BMC 0.02 -0.15, 0.18 0.818
Insulin (pg/mL)2 AHC -313 -681, 55
0.0814
BMC -449 -780, -119 0.009
C-peptide (pg/mL)2 AHC -215 -491, 62
0.124
BMC -397 -612, -181 0.001
hsCRP (mg/L)1 AHC 0.14 0.01, 0.27
0.043
BMC 0.27 -0.07, 0.60 0.113
Adiponectin (mg/L)2 AHC -0.57 -
1.01, -0.13 0.013
BMC -0.21 -0.68, 0.26 0.364
PAT-1 (pg/mL)2 AHC 632 -
561, 1824 0.3044
BMC -265 -490, -40 0.022
Glucagon (pg/mL)2 AHC -45 -98, 8 0.091
BMC -86 -144, -29 0.005
GLP-1 (pg/mL)2 AHC -191 -404, 23
0.078
BMC -236 -384, -88 0.003
HOMA2 IR AHC -0.16 -
0.35, 0.03 0.100
BMC -0.29 -0.45,-0.13 0.001
CA 02779883 2012-06-12
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HOMA2 B (%) AHC -10.2 -28.3, 8.0 0.261
BMC -21.2 -33.8, -8.6 0.002
HOMA2 S (%) AHC 11.3 -5.6, 28.1 0.182
BMC 23.4 9.0, 37.9 0.002
Triglycerides (mmol/L)I AHC -0.13 -0.24, -0.03
0.015
BMC -0.26 -0.36, -
0.15 <0.001
Total cholesterol (mmol/L) I AHC -0.31 -0.43, -
0.18 <0.001
BMC -0.39 -0.55, -
0.24 <0.001
LDL-cholesterol (mmol/L) I AHC -0.08 -0.20, 0.05 0.215
BMC -0.22 -0.35, -0.08
0.002
HDL-cholesterol (mmol/L) I AHC -0.15 -0.22,
-0.09 <0.001
BMC -0.07 -0.16, 0.02
0.109
Triglycerides/HDL-cholesterol ratio' AHC -0.05 -0.13, 0.04 0.305
BMC -0.17 -0.25, -
0.08 <0.001
Atherogenic index' AHC -0.02 -0.07, 0.03
0.373
BMC -0.10 -0.15, -
0.05 <0.001
TNF-ct (pg/mL)2 AHC -5.1 -10.5, 0.3 0.062
BMC -7.6 -13.0, -2.3 0.007
IL-6 (pg/mL)2 AT-IC -2.8 -5.7, 0.1 0.0594
BMC -4.5 -8.0, -1.0
0.0064
Serum amyloid A (mg/L)2 AHC 0.84 -2.5, 4.2 0.612
BMC 0.00 -4.2, 4.2 0.999
GIP (pg/mL)2 AHC -11.8 -25.6, 2.0 0.091
BMC -11.8 -24.3, 0.6 0.062
Ghrelin (pg/mL)2 AHC -14.8 -127.7, 98.1
0.791
BMC -3.6 -96.5, 89.2
0.937
Leptin (pg/mL)2 AHC -435 -950, 80 0.095
BMC -376 -754, 2 0.051
Visfatin (pg/mL)2 AHC -1638 -3424,
148 0.0354
BMC -2105 -3946, -
263 0.0344
Resistin (pg/mL)2 AHC 203 100, 306 <0.001
BMC 140 62,217 0.001
Uric acid (mon) AHC -23 -41, -4 0.017
BMC -31 -50, -13 0.002
Leukocytes (x 109/L)3 AHC -0.78 -1.11, -
0.46 <0.001
BMC -0.45 -0.85, -0.05
0.028
Monoeytes (%)3 AHC 1.09 0.56, 1.62
<0.001
BMC 0.48 -0.15, 1.12 0.130
Eosinophiles (%)3 AHC 0.19 -0.22, 0.59 0.351
BMC -0.32 -0.64, 0.00
0.048
Neutrophiles (%)3 AHC -1.41 -3.64, 0.83 0.209
BMC -0.03 -2.95, 2.88 0.982
Lymphocytes (%)3 AHC 0.22 -1.90, 2.33
0.834
BMC -0.35 -3.04, 2.33 0.789
Basophiles (%)3 AHC 0.09 -0.12, 0.31 0.374
BMC -0.16 -0.40, 0.07
0.169
CA 02779883 2012-06-12
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Platelets (x 109/L)3 AHC -2.4 -19.8, 15.0
0.779
BMC -6.4 -18.9, 6.1
0.301
sample matrix is serum
2
sample matrix is EDTA-plasma
3
sample matrix is EDTA-blood
4
p-values are calculated using logio transformed data because data are not
normally-distributed.
Results
Anthropometric and biochemical measurements
Thirty-two subjects (13 women and 19 men) completed the randomized
cross-over diet intervention of two six-day intervention periods separated by
eight
days normal dieting. Baseline biochemical and anthropometric characteristics
for all
subjects who completed the study are found in Table 2 above. Observed weight
changes of more than 1 kg halfway during any of the diet periods was reason to
adjust the quantities of the remaining meals. Even though the mean body mass
was
reduced (P <0.001) by 1.02 kg after six days on diet HC (80.3 8.8 to 79.3
8.6
kg) and 0.95 kg (80.3 8.5 to 79.4 8.8 kg) after six days on diet MC, or
0.91 kg
during the first diet period, and 1.06 kg during the second diet period, the
weight
changes were not significantly different between the two diets or intervention
regimes. The total weight loss during the whole diet intervention (20 days)
was in
average 1.56 kg (1.96 %).
Microarray analysis
Microarray hybridization was performed on leukocyte RNA collected from
all 32 subjects before and after each of the interventions, six days each on
diet HC
and MC. Changes in gene expression were determined by comparison of the
microarray results of the samples from before the diet intervention (day 0)
with
those after day 6 of the intervention. Dieting on the HC diet resulted in
changes in
expression of 3225 genes, whereas the MC dieting resulted in changes in
expression
CA 02779883 2012-06-12
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of 1370 genes; of these, 843 genes overlapped between the groups. Among the
latter, all except 10 genes changed in the same direction (see Figure 2).
Gene expression changes
In order to determine the role of the various genes that changed expression in
response to each diet, pathway analysis was performed. Significant enrichment
(P <
0.05) of differentially expressed genes was observed in several pathways
related to
metabolism, apoptosis, cell cycle/proliferation/growth, stress/immunity and a
variety
of other pathways (see Figure 3). In general, the HC diet induced changes in
gene
expression to a much larger extent (number of genes and higher log2 ratio)
than
consumption of the MC diet, including both up- and down-regulation of genes
within the same pathway. HC diet consumption led to stimulation of a number of
genes including NF-x13 signaling (RELA), STAT3, STAT5A, STAT5B, SRC, AHR,
and TRAIL family member (TNFSF10) regulating both apoptosis,
proliferation/cancer and stress/immunity, but also more specific regulators of
apoptosis such as CASP2, BAX, clAP (BIRC3), ICAD (DFFA), JAK2, MAP3K14,
and DAXX. Upregulated genes relevant to proliferation and cancer are genes
involved in(3-catenin signaling (CTNNB1), LEF/TCF (TCF7L2, TCF4), frizzled
(FZD1), CEBPA, and adenylate cyclase/protein kinase A signaling (PRKACA,
PRKACB, ADCY7). Apoptosis-relevant genes downregulated after HC dieting
included CASP8, BID, MDM2, FASLG, and CEBPB; while the proliferation and
cancer relevant genes in this category were NOTCHI, c-myc (MYC), raf (ARAF,
RAF1), BRCA1, and SHIP (INPP5D). SHIP and raf are also mediators in the
insulin
signaling pathway. Together with the P13-kinase (PIK3R3, PIK3R2, PIK3R4),
atypical protein kinase C (PRKCZ), Akt (RAC2), PPP1CC, ERK1/2 (MAPK1,
MAPK3), syntaxin (STX4), GSK3 (GSK3B), and RAPGEF1 they are mediating
glucose uptake and glycogen synthesis in insulin target cells. These genes are
all
down-regulated during HC dieting. MC dieting showed upregulation of genes
including gene coding for G protein subunit Py (GNG2), CDC42, FOX03, VCAMI,
of relevance to apoptosis, proliferation/cancer; while down-regulated genes in
this
category included NF-xB signaling (NFKB1), ICAM1, TNFSF12,CCR5, CD4OLG,
and MMP3, all belonging to the atherosclerosis signalling pathway, and gene
coding
CA 02779883 2012-06-12
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for G protein subunit a (GNA 1 5), IL] 5 , and PAIP2 with relevance to
apoptosis,
proliferation, cancer, stress, and immunity. Very few genes showed
differential
regulation by both diets. However, among them were genes for two growth
factors;
thymidine phosphorylase (TYMP) where a high TYMP expression at tumour sites is
correlated with tumour growth, induction of angiogenesis, and metastasis
(Bijnsdorp
et al., 2008); and granulin (GR1V) which contributes to the regulation of
early
embryogenesis, to adult tissue repair, inflammation, dementia and elevated
levels
are observed in cancers and dementia (Bateman et al., Bioessays (2009)
31(11):1245-1254). Both genes were up-regulated in response to the HC diet,
and
down-regulated in response to the MC diet. Moreover, RNF4, a regulator of DNA
demethylation whose expression is regulated down following the HC diet and up
following the MC diet. Several transcription factor members of the pathways in
Figure 3 were identified showing differential expression exclusively in
response to
either HC or MC diet. The interrelationship among these and their respective
downstream effects on apoptosis, proliferation, cancer and stress/immunity is
suggested in Figure 4. Approved HUGO (http://www.genenames.org/) gene
nomenclature of all gene symbols is described here in italics, and is
presented in
Figure 4.
Glucose, insulin and lipid metabolism
A significant decrease in fasting blood (serum) C-peptide levels during the
MC diet was observed, compared to baseline, along with glucagon levels. No
significant changes in serum glucose or insulin levels were observed upon
completion of each of the two six-day diet periods, although a trend for
reduction of
serum insulin after the MC diet could be observed, see Table 3. A trend in
hsCRP
concentration increase in response to the HC diet was evident, while the anti-
inflammatory adiponectin concentration showed a decreasing trend during the
same
diet. PAI-1, and GLP-1 concentrations showed a decreasing trend in response to
the
MC diet, see Table 3.
Based on changes in fasting blood glucose and C-peptide concentrations in
response to the two diet interventions, we applied the homeostasis model
assessment
(HOMA2 computer model), allowing for an insight into the underlying
CA 02779883 2012-06-12
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pathophysiological disorders regulating insulin resistance, pi-cell
dysfunction and
adipogenesis. The HOMA2 computer model for calculation of insulin resistance
(HOMA2 IR), 13-cell function (HOMA2 % B) as well as the insulin sensitivity
(HOMA2 % S) is calibrated to a reference population, where the HOMA2 IR is
normally 1, and the HOMA2 % B as well as the HOMA2 % S are normally 100 %.
In fasting samples, responses to six days on diet MC assessed as the mean
HOMA2-
IR and HOMA2 % B was decreased, while the HOMA2 % S was increased
compared to the respective fasting baseline samples (Table 3). The % S and % B
must be seen in relation to each other, as an increased insulin sensitivity
may be
compensated by decreased 13-cell function.
In order to determine the glycemic response to a single meal from each of the
two diets, a meal challenge test was performed using a random subgroup of the
participants at the day following each of the two six-day isocaloric diet
intervention
periods. As shown in Figure 1, the postprandial serum glucose at 30 min was
significantly higher in response to an HC diet meal than after a MC diet meal.
At
120 min the serum glucose was significantly lower following both meals than in
fasting samples, but the hypoglycaemia was significantly greater in response
to diet
HC. The corrected AUC for the glucose response was larger for a diet HC meal
compared to a diet MC meal.
Blood lipid profiles were changed in response to both diet periods, where
triglyceride concentrations were reduced during HC and further reduced during
MC.
Total cholesterol was decreased during both HC and MC diet periods. LDL-
cholesterol was decreased during the MC diet, while HDL-cholesterol was
decreased in response to the HC diet. The triglyceride/HDL-cholesterol-ratio
was
decreased in response to the MC diet, and the atherogenic index (AIP,
log10(triglycerides/HDL-cholesterol-ratio)) was reduced by 53 % in response to
the
MC diet (Table 3). AIP has been shown to correlate with LDL-particle diameter,
and a more negative value is suggested to be a predictor of a marker of a
lipoprotein
phenotype with decreased cardiovascular risk.
CA 02779883 2012-06-12
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Blood cytokines and subsets of leukocytes
The pro-inflammatory cytokines TNF-cc and IL-6 showed trends of reduced
concentrations in response to the MC diet, while resistin was significantly
increased
in response to both MC and HC diets. The blood concentration of uric acid,
which
at elevated levels forms crystals accumulating in synovial fluid triggering a
cytosolic
sensor, the inflammasome leading to inflammation via activation of NF-KB, is
significantly decreased in response to both diets. The total number of
leukocytes
decreased during both diets, while the monocyte subfraction of leukocytes
increased
during the HC diet. The eosinophile subfraction diminished during diet MC
(Table
3).
Discussion
We performed a controlled, randomized crossover trial and show that six
days consumption of a meal replacement diet with reduced carbohydrate, higher
protein and fat (BMC) significantly improves the atherogenic index, main blood
cytokines/adipokines (TNF-a (TNF), IL6, PAT-I, adiponectin), and also improves
HOMA indices (reduces insulin resistance and improves insulin sensitivity,
improves 3-cell function) in slightly overweight subjects, compared to a high
carbohydrate (AHC) diet. The diet interventions resulted in diet-specific
changes in
leukocyte gene expression profiles characterized by differential effects on
metabolic
pathways, apoptosis, proliferation/cell cycle regulation, and stress/immunity.
This
includes activation of genes for transcription factors NF-KB, STAT3/5, CTNNp-
LEF/TCF, CEBPA in response to AHC and inhibition of gene for NF-KB while
activation of FOX03 during BMC dieting.
The strength of our study resides in its randomized crossover design, defined
diet macronutrient quality and quantity, the inclusion of both sexes, and a
frequent
eating pattern (six meals per day) on a normocaloric diet. The latter is
important,
since overeating, which refers to the overconsumption of energy, both per meal
and
day, that is inappropriately large for a given energy expenditure, thus, leads
itself to
obesity. Moreover, the combination of data on blood lipids, cytokines,
adipokines
and leukocyte transcriptomics allow the identification and a more
comprehensive
understanding of mechanisms underlying disease risk associated with diet
CA 02779883 2012-06-12
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carbohydrate quantity. To the knowledge of the inventors, the current study is
unique in that respect.
The meal replacement diet used in this study allowed for a highly defined
meal composition, in contrast to the use of conventional diets, and enabled
the
definition of the mechanisms involved in progression of lifestyle diseases,
which is
essential to study specific aspects like macronutrient quantity and quality
independently, and in a controlled manner, hence the choice of replacement
diets
and a cross-over design. The number of subjects was chosen based on power
calculations from transcriptome expression data in a pilot study.
A health promoting trend was to some extent observed in response to the
control (HC) diet, possibly partly due to common beneficial features between
the
diets (e.g. high fibre, high meal frequency with even distribution of caloric
intake,
adequate micronutrient and essential fatty acid intake, and weight reduction).
However, this common health promoting trend was enforced and reached
significance in response to the experimental (MC) diet. Weight reduction,
increase
in PUFA, and optimal n-3 to n-6 balance, share the common property of reducing
inflammation. Although the intervention was intended to be normocaloric, a
small
but significant weight reduction was observed on both diets. However, there
was no
significant difference in weight reduction between the diets, suggesting that
the
weight reduction itself does not influence the health promoting trend.
Reduction in
triglycerides is highly correlated with increase in PUFA, especially the n-3
variety.
The daily intake of fish oil capsules and other PUFAs were common in both
diets.
Also, there was a significant reduction in triglycerides in response to both
diets,
suggesting a common beneficial effect of PUFAs. Reduction of the pro-
inflammatory adipokine, visfatin and uric acid, was observed in response to
both
diets. The mean number of meals before entering the intervention was 3.8;
during
intervention it was 6, so an increase in meal number can be expected to have a
beneficial effect on postprandial hyperglycaemia independent of meal
composition.
In summary, the two diets share several common inflammation lowering
properties which may explain the common trend. However, the difference between
the diets in ability to reach significance in improved HOMA indices and
atherogenic
index, and improvement in main cytokine/adipokine concentrations, may be
CA 02779883 2012-06-12
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attributed to a shift in relative macronutrient composition. Inflammation is
an
important early variable in the metabolic response to diet via postprandial
hyperglycaemia. Compared to the control diet, the experimental diet
significantly
decreased the 30 min postprandial hyperglycaemia and postprandial
hypoglycaemia
at 2 hours. This correlated with improvements in HOMA indices, demonstrating
increased insulin sensitivity, reduced insulin resistance and improved n-cell
function. Leukocytes have active metabolism and insulin receptors, and we have
shown that monocytes are responsive to insulin stimulation (Arbo et al.,
Scandinavian Journal of Clinical and Laboratory Investigations (2011)
71(4):330-
339). However, knowledge of insulin action in leukocytes is sparse, although
it has
been speculated whether impaired presence of GLUT4 presence on the monocyte
cell surface could be an indicator of systemic insulin resistance. During HC
dieting,
the gene expression of several insulin signaling mediators was down-regulated,
suggesting a change in the insulin sensitivity homeostasis in leukocytes.
Accompanying these diet differences, we observed a significant improvement in
the
most important cytokine/adipokines regulating insulin resistance (TNF-a, IL-6,
PAI-
1, and adiponectin) in response to MC dieting. Metabolic inflammation in
adipose
tissue is regulated by subsets of leukocytes, most importantly monocytic
cells, but
also by T lymphocytes. Among the few changes in leukocyte subsets in response
to
dieting, we note a significant increase in monocytes in response to the HC
diet, a
pro-inflammatory property. All these changes suggest that compared to the HC
diet,
the MC diet elicits a more anti-inflammatory response. At the transcriptional
level
this was reflected in that pathways belonging to the processes apoptosis,
proliferation/cell cycle regulation, and stress/immunity were among the most
notable
pathways significantly exhibiting gene expression changes due to the two
diets.
Also, several transcription factors showed differentially regulation including
NF-x13,
STAT3 and 5, CEBPA/B. Among these, the most prominent regulator involved in
all processes is NF-KB. It was activated in response to the HC diet and
inhibited in
response to the MC diet, suggesting an important role in regulating the early
diet
specific changes in the current study. FOX03 has important, but cell type
specific
regulatory roles in leukocytes. In mice, FOX03 inhibits NF-KB and thereby T
cell
activity. However, in neutrophilic inflammation FOX03 is needed to sustain the
CA 02779883 2012-06-12
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pro-inflammatory environment, through suppression of FASLG and neutrophil
apoptosis. FOX03 was activated in response to the MC diet.
Inflammatory conditions in selected organs increase the risk of cancer.
Inflammatory components in the microenvironment of tumours include leukocytes,
cytokines, and complement components, orchestrated by transcription factors,
such
as NF-KB and STAT3. As cancer may be seen as the consequence of deregulation
of the balance between proliferation and apoptosis, there may be important
roles for
diet composition in cancer development in certain types of cancer. The current
study showed that genes for the transcriptions factors NF-KB, STAT3, and other
tumour promoting factors (SRC, TP, GRN) are activated and BRCA/ (a tumour
suppressor gene regulating breast cancer development) is inhibited in response
to the
HC diet. The transcription factor CEBPA has the capacity to induce monocytic
maturation and was induced in response to the HC diet suggesting that it may
contribute to the monocyte driven inflammation in circulation and remote
tissue, e.g.
adipose tissue. Thus, there are reasons to believe that the metabolic changes
taking
place in response to dieting include transcription factors, proteins, lipids
and other
metabolites and that these may be used to identify the link between
postprandial
hyperglycemia, inflammation and insulin resistance.
The results presented above show that a reduction in diet carbohydrate
quantity towards a caloric macronutrient balance seems to be an important
single
factor in reducing postprandial hyperglycemia-induced systemic low-grade
inflammation, associated with lifestyle disease development and supports the
use of
the compositions of the invention in the treatment of lifestyle diseases.
