FIELD OF THE INVENTION:

The present invention relates to the field of infant nutrition and the effect that early-in-life nutrition has on later-in-life health.

BACKGROUND OF THE INVENTION

Breast-feeding is the preferred method of feeding infants. However, there are circumstances that make breast-feeding impossible or less desirable. In those cases infant formulae are a good alternative. The composition of modern infant formulae is adapted in such a way that it meets many of the special nutritional requirements of the fast growing and developing infant.

Early- in-life nutrition plays an important role in developmental programming. During a critical period of development, persistent effects on physiology throughout life may occur. Later in life it appears that subjects who were bottle fed early in life have an increased risk for disorders as obesity, cardiovascular diseases, hypertension and diabetes 2 during adulthood compared to subjects breast fed as infants.

Breast fed infants have higher leptin levels than bottle fed infants. This is explained by the presence of leptin in human milk. Furthermore, human milk fed infants have a lower leptin resistance and this may provide one potential mechanism for the long-term benefit of breastfeeding on adiposity.

Leptin is a hormone that is important for the regulation of food intake. Leptin informs the hypothalamus about the body's energy status. Leptin is derived from white adipose tissue and circulating leptin levels are therefore directly related to the total amount of white adipose tissue stores. Leptin production and secretion is also influenced by acute nutritional status; circulating leptin levels are low during fasting and high after a meal. Circulating leptin levels regulate the expression of neuropeptides in the hypothalamus that control feeding and energy expenditure. High circulating leptin promotes a negative energy balance by suppression of food intake and stimulation of energy expenditure. US2008/0242612 discoses the use of leptin containing milk fat globules. The goal was to increase leptin levels in a subject. The role of early-in-life diet on later-in-life fasting leptin levels is also disclosed in Singhal et al, 2002 Am J Clin Nutr 75:993-999. A nutrient enriched early-in-life diet for preterm infants increased fasting leptin concentrations later in life at the age of 16. It was postulated that the higher leptin concentrations associated with greater body fatness early in postnatal life, program the leptin-dependent feedback loop such that the regulation of body fat is less sensitive to leptin later in life.

WO 2010/027259 discloses nutritional compositions with coated lipid globules for infants and/or toddlers for the prevention of obesity later in life. SUMMARY OF THE INVENTION

The inventors employed an animal model which is representative of the physiological programming effects of the diet taken early in life. In this model animals are fed with different diets early in life and subsequently they are exposed to the same Western style, high fat, high cholesterol diet. This model allows to examine how the early-in-life diet, via body programming effects, have a health effect later in life and how the early-in-life diet influences the way the body handles a Western style, high fat and cholesterol rich diet later on.

The inventors unexpectedly found that an early-in-life diet similar in caloric density and macronutrient composition, with lipids having a similar fatty acid profile, but with lipid present in the form of lipid globules comprising a surface layer with phospholipids, increased leptin sensitivity later in life. Standard infant formula comprises lipid globules which are small and do not comprise a coating with phospholipids. This was used as a control early-in-life diet and was found to result in a lower leptin sensitivity later in life. This reflects the programming effect of the lipid globule component of the diet early in life on leptin sensitivity later in life. An increased leptin sensitivity later in life advantageously affects (self) regulation of food intake later in life and more in particular reduces the risk of the occurrence of hyperphagia later in life. This is of particular advantage for subjects growing up in an environment favoring a positive energy balance.

Therefore the present invention in particular relates to infant nutrition with lipid globules coated with phospholipids, to be administered early in life during infancy in order to increase leptin sensitivity, in particular later in life, improve self regulation of food intake, in particular later in life, improve regulation of satiety and/or prevent or reduce the risk of hyperphagia, in particular later in life. DETAILED DESCRIPTION OF THE INVENTION

The present invention concerns a method for

a) increasing leptin sensitivity,

b) improving self regulation of food intake,

c) improving regulation of satiety and/or

d) decreasing refeeding after fasting

in a human subject,

by feeding a nutritional composition comprising lipid, wherein the lipid is present, preferably the lipid essentially is present, in the form of lipid globules, said lipid globules comprising a core comprising triglycerides derived from vegetable origin and a surface layer comprising phospholipids

to the human subject early in life, preferably to the human subject having an age of 0 to 36 months.

In the context of the present invention, the method for increasing leptin sensitivity, improving self regulation of food intake, improving regulation of satiety and/or decreasing refeeding after fasting, is considered to be a non-medical method.

The invention can also be worded as the use of a nutritional composition comprising lipid, wherein the lipid is present, preferably the lipid essentially is present, in the form of lipid globules, said lipid globules comprising a core comprising triglycerides derived from vegetable origin and a surface layer comprising phospholipids for feeding a human subject early in life, preferably for feeding a human subject having an age of 0 to 36 months, for

a) increasing leptin sensitivity,

b) improving self regulation of food intake,

c) improving regulation of satiety and/or

d) decreasing refeeding after fasting.

Preferably the use is non-therapeutic. For some jurisdictions, the invention can also be worded as the use of lipid for the preparation of a nutritional composition wherein the lipid is present, preferably the lipid essentially is present, in the form of lipid globules, said lipid globules comprising a core comprising triglycerides derived from vegetable origin and a surface layer comprising phospholipids, for feeding a human subject early in life, preferably for feeding a human subject having an age of 0 to 36 months, for a) increasing leptin sensitivity,

b) improving self regulation of food intake,

c) improving regulation of satiety and/or

d) decreasing refeeding after fasting. The invention may even be worded as a nutritional composition comprising lipid, wherein the lipid is present, preferably the lipid essentially is present, in the form of lipid globules, said lipid globules comprising a core comprising triglycerides derived from vegetable origin and a surface layer comprising phospholipids, for use in feeding a human subject early in life, preferably for use in feeding a human subject having an age of 0 to 36 months, for use in

a) increasing leptin sensitivity,

b) improving self regulation of food intake,

c) improving regulation of satiety and/or

d) decreasing refeeding after fasting. The invention also concerns a method for preventing or reducing the risk of occurrence of hyperphagia in a human subject, by feeding a nutritional composition comprising lipid, wherein the lipid is present, preferably the lipid essentially is present, in the form of lipid globules, said lipid globules comprising a core comprising triglycerides derived from vegetable origin and a surface layer comprising phospholipids, to the human subject early in life, preferably to the human subject having an age of 0 to 36 months.

The invention can also be worded as the use of lipid for the preparation of a nutritional composition wherein the lipid is present, preferably the lipid essentially is present, in the form of lipid globules, said lipid globules comprising a core comprising triglycerides derived from vegetable origin and a surface layer comprising phospholipids, for feeding a human subject early in life, preferably for feeding a human subject having an age of 0 to 36 months, for preventing or reducing the risk of occurrence of hyperphagia.

The invention can also be worded as a nutritional composition comprising lipid, wherein the lipid is present, preferably the lipid essentially is present, in the form of lipid globules, said lipid globules comprising a core comprising triglycerides derived from vegetable origin and a surface layer comprising phospholipids, for use in feeding a human subject early in life, preferably for feeding a human subject having an age of 0 to 36 months, for use in preventing or reducing the risk of occurrence of hyperphagia. The lipid in the nutritional compositions in the method or use according to the present invention is present in the form of lipid globules with a core comprising triglycerides derived from vegetable origin, preferably vegetable lipids, and a surface layer comprising cholesterol and phospholipids. Preferably essentially all the lipid in the nutritional compositions in the method or use according to the present invention is present in the form of lipid globules with a core comprising said triglycerides derived from vegetable origin and a coating comprising said phospholipids.

In the context of this invention, a human subject having an age of 0 to 36 months is also referred to as an infant.

Lipid component The present nutritional composition comprises lipid. The lipid provides preferably 30 to 60% of the total calories of the composition. More preferably the present composition comprises lipid providing 35 to 55% of the total calories, even more preferably the present composition comprises lipid providing 40 to 50% of the total calories. When in liquid form, e.g. as a ready-to- feed liquid, the composition preferably comprises 2.1 to 6.5 g lipid per 100 ml, more preferably

3.0 to 4.0 g per 100 ml. Based on dry weight the present composition preferably comprises 10 to 50 wt.%, more preferably 12.5 to 40 wt.% lipid, even more preferably 19 to 30 wt.% lipid.

Lipids include polar lipids (such as phospholipids, glycolipids, sphingomyelin, and cholesterol), monoglycerides, diglycerides, triglycerides and free fatty acids. Preferably the composition comprises at least 75 wt. %, more preferably at least 85 wt.% triglycerides based on total lipids.

The lipid of the present invention comprises vegetable lipids. The presence of vegetable lipids advantageously enables an optimal fatty acid profile, high in (poly)unsaturated fatty acids and/or more reminiscent to human milk fat. Using lipids from cow's milk alone, or other domestic mammals, does not provide an optimal fatty acid profile. This less optimal fatty acid profile, such as a large amount of saturated fatty acids, is known to result in decreased leptin sensitivity. Preferably the present composition comprises at least one, preferably at least two lipid sources selected from the group consisting of linseed oil (flaxseed oil), rape seed oil (such as colza oil, low erucic acid rape seed oil and canola oil), sunflower oil, high oleic sunflower oil, safflower oil, high oleic safflower oil, olive oil, coconut oil, palm oil and palm kernel oil. Preferably the present composition comprises at least one, preferably at least two lipid sources selected from the group consisting of linseed oil, canola oil, coconut oil, sunflower oil and high oleic sunflower oil. Commercially available vegetable lipids are typically offered in the form a continuous oil phase. When in liquid form, e.g. as a ready-to-feed liquid, the composition preferably comprises

2.1 to 6.5 g vegetable lipid per 100 ml, more preferably 3.0 to 4.0 g per 100 ml. Based on dry weight the present composition preferably comprises 10 to 50 wt.%, more preferably 12.5 to 40 wt.% vegetable lipid, even more preferably 19 to 30 wt.%. Preferably the composition comprises 45 to 100 wt.% vegetable lipids based on total lipids, more preferably 70 to 100 wt.%, even more preferably 75 to 97 wt.%. It is noted therefore that the present composition also may comprise non-vegetable lipids. Suitable non vegetable lipids include marine oils, microbial oils, egg fat and milk fat. Suitable and preferred non-vegetable lipids are further specified below.

Phospholipids and other polar lipids

The nutritional composition according to the present invention comprises phospholipids. Phospholipids are essential to form the surface layer, coating, of the lipid globules. Also in this coating dietary cholesterol can be located. Phosholipids are polar lipids. Polar lipids are amphipathic of nature and include glycerophospholipids, glycosphingolipids, sphingomyelin and/or cholesterol. Phospholipids refers to the sum of glycerophospholipids and sphingomyelin. According to the present invention the phospholipids are present as a coating of the lipid globule. By 'coating' is meant that the outer surface layer of the lipid globule comprises polar lipids, in particular phospholipids, whereas these polar lipids are virtually absent in the core of the lipid globule. The presence of phospholipids as a coating or outer layer of the lipid globule in the diet administered early in life was found to advantageously increase leptin sensitivity, in particular increase leptin sensitivity later in life. The presence of phospholipids as a coating or outer layer of the lipid globule in the diet administered early in life was found to advantageously increase satiety.

The present composition preferably comprises glycerophospholipids. Glycerophospholipids are a class of lipids formed from fatty acids esterified at the hydroxyl groups on carbon-1 and carbon-

2 of the backbone glycerol moiety and a negatively-charged phosphate group attached to carbon-

3 of the glycerol via an ester bond, and optionally a choline group (in case of phosphatidylcholine, PC), a serine group (in case of phosphatidylserine, PS), an ethanolamine group (in case of phosphatidylethanolamine, PE), an inositol group (in case of phosphatidylinositol, PI) or a glycerol group (in case of phosphatidylglycerol, PG) attached to the phosphate group. Lysophospholipids are a class of phospholipids with one fatty acyl chain. Preferably the present composition contains PC, PS, PI and/or PE, more preferably at least PC.

The present composition preferably comprises sphingomyelin. Sphingomyelins have a phosphorylcholine or phosphorylethanolamine molecule esterified to the 1 -hydroxy group of a ceramide. They are classified as phospholipid as well as sphingolipid, but are not classified as a glycerophospholipid nor as a glycosphingolipid. Sphingomyelin is especially preferred, preferably also since it interacts with cholesterol. Preferably the phospholipids are derived from milk lipids. A preferred source for phospholipids, particularly PC, is soy lecithin and/or sunflower lecithin.

The present composition preferably further comprises glycosphingolipids. The term glycosphingolipids as in the present invention particularly refers to glycolipids with an amino alcohol sphingosine. The sphingosine backbone is O-linked to a charged headgroup such as ethanolamine, serine or choline backbone. The backbone is also amide linked to a fatty acyl group. Glycosphingolipids are ceramides with one or more sugar residues joined in a β- glycosidic linkage at the 1 -hydro xyl position. Preferably the present composition contains gangliosides, more preferably at least one ganglioside selected from the group consisting of GM3 and GD3.

Preferably the weight ratio of phospholipids : glycosphingolipids is from 2:1 to 10:1 , more preferably 2:1 to 5: 1.

Preferably the present composition comprises 0.5 to 20 wt.% phospholipids based on total lipid, more preferably 0.5 to 10 wt.%, more preferably 1 to 10 wt.%, even more preferably 2 to 10 wt.% even more preferably 3 to 8 wt.% phospholipids based on total lipid.

Preferably the present composition comprises at least 5 wt.% sphingomyelin based on total phospholipids, more preferably at least 10 wt.%, even more preferably at least 15 wt.%, preferably no more than 60 wt.%.

The present composition preferably comprises cholesterol. The present composition preferably comprises at least 0.02 wt.% cholesterol based on total lipid, more preferably at least 0.03 wt.%, more preferably at least 0.04 wt.%, even more preferably at least 0.1 wt.% based on total lipid. Preferably the amount of cholesterol does not exceed 5 wt.% based on total lipid, more preferably does not exceed 2 wt.%, more preferably does not exceed 1 wt.% of total lipid, even more preferably does not exceed 0.3 wt% based on total lipid. Preferred sources for providing the phospholipids and optionally cholesterol are egg lipids, milk fat, buttermilk fat and butter serum fat (such as beta serum fat). Preferably the phospholipid and optionally cholesterol are derived from cow's milk. Preferably the phospholipid and cholesterol, are from the same source. This will enhance the presence of dietary cholesterol in a layer of phospholipid in the final nutritional product. Derived from milk includes milk lipid, cream lipid, butter serum lipid, whey lipid, cheese lipid and/or buttermilk lipid and isolated therefore. The buttermilk lipid is typically obtained during the manufacture of buttermilk. The butter serum lipid or beta serum lipid is typically obtained during the manufacture of anhydrous milk fat from butter. Preferably the phospholipids, glycosphingolipids and/or cholesterol are obtained from milk cream. The composition preferably comprises phospholipids, glycosphingolipids and/or cholesterol from milk of cows, mares, sheep, goats, buffalos, horses and camels. It is most preferred to use a lipid extract isolated from cow's milk. The use of phospholipids from milk fat advantageously comprises the milk fat globule membranes, which are more reminiscent to the situation in human milk. The concomitant use of phospholipids derived from domestic animals milk and trigycerides derived from vegetable lipids therefore enables to manufacture coated lipid globules with a coating more similar to human milk, while at the same time providing an optimal fatty acid profile. Suitable commercially available sources for milk phospholipids, including sphingomyelin, and/or cholesterol are BAEF, SM2, SM3 and SM4 powder of Corman, Salibra of Glanbia, and LacProdan MFGM-10 or PL20 from Aria. Preferably the source of milk phospholipids comprises at least 4 wt.% phospholipids based on total lipid, more preferably 7 to 75 wt.%, most preferably 20 to 70 wt.% phospholipids based on total lipid. Preferably the weight ratio phospholipids to protein is above 0.10, more preferably above 0.20, even more preferably above 0.3. Preferably at least 25 wt.%, more preferably at least 40 wt.%, most preferably at least 75 wt.% of the phospholipids is derived from milk phospholipids.

Fatty acid composition

Herein LA refers to linoleic acid and/or acyl chain (18:2 n6); ALA refers to a-linolenic acid and/or acyl chain (18:3 n3); LC-PUFA refers to long chain polyunsaturated fatty acids and/or acyl chains comprising at least 20 carbon atoms in the fatty acyl chain and with 2 or more unsaturated bonds; DHA refers to docosahexaenoic acid and/or acyl chain (22:6, n3); EPA refers to eicosapentaenoic acid and/or acyl chain (20:5 n3); ARA refers to arachidonic acid and/or acyl chain (20:4 n6); DPA refers to docosapentaenoic acid and/or acyl chain (22:5 n3). Medium chain fatty acids (MCFA) refer to fatty acids and/or acyl chains with a chain length of 6, 8 or 10 carbon atoms. LA preferably is present in a sufficient amount in order to promote a healthy growth and development, yet in an amount as low as possible to prevent occurrence of obesity later in life. The composition therefore preferably comprises less than 15 wt.% LA based on total fatty acids, preferably of 5 to 14.5 wt.%, more preferably of 6 to 10 wt.%. Preferably the composition comprises over 5 wt.% LA based on fatty acids. Preferably ALA is present in a sufficient amount to promote a healthy growth and development of the infant. The present composition therefore preferably comprises at least 1.0 wt.% ALA based on total fatty acids. Preferably the composition comprises at least 1.5 wt.% ALA based on total fatty acids, more preferably at least 2.0 wt.%. Preferably the composition comprises less than 10 wt.% ALA, more preferably less than 5.0 wt.% based on total fatty acids. The weight ratio LA/ ALA should be well balanced in order to reduce fasting leptin level and/or to prevent obesity and hypertriglyceridaemia later in life, while at the same time ensuring a normal growth and development. Therefore, the present composition preferably comprises a weight ratio of LA ALA of 2 to 15, more preferably of 2 to 7, more preferably of 4 to 7, more preferably of 3 to 6, even more preferably of 4 to 5.5, even more preferably of 4 to 5.

Since MCFA contribute to a healthy growth and development, the present composition preferably comprises at least 3 wt.% MCFA based on total fatty acids, more preferably at least 10 wt.%, even more preferably at least 15 wt.%. Preferably, the present composition advantageously comprises less than 50 wt.% MCFA based on total fatty acids, more preferably less than 40 wt.%, even more preferably less than 25 wt.%.

Preferably the present composition comprises n-3 LC-PUFA. More preferably, the present composition comprises EPA, DPA and/or DHA, even more preferably DHA. N-3 LC-PUFA reduces fasting leptin levels. Since a low concentration of DHA, DPA and/or EPA is already effective and normal growth and development are important, the content of n-3 LC-PUFA in the present composition, preferably does not exceed 15 wt.% of the total fatty acid content, preferably does not exceed 10 wt.%, even more preferably does not exceed 5 wt.%. Preferably the present composition comprises at least 0.2 wt.%, preferably at least 0.5 wt.%, more preferably at least 0.75 wt.% n-3 LC-PUFA based on total fatty acid content. Preferably the present composition comprises at least 0.2 wt.%, preferably at least 0.5 wt.%, more preferably at least 0.75 wt.% of the sum of DHA, DPA and EPA based on total fatty acid content.

As the group of n-6 fatty acids, especially arachidonic acid (AA) and LA as its precursor, counteracts the group of n-3 fatty acids, especially DHA and EPA and ALA as their precursor, the present composition comprises relatively low amounts of AA. The n-6 LC-PUFA content preferably does not exceed 5 wt.%, more preferably does not exceed 2.0 wt.%, more preferably does not exceed 0.75 wt.%, even more preferably does not exceed 0.5 wt.%, based on total fatty acids. Since AA is important in infants for optimal functional membranes, especially membranes of neurological tissues, the amount of n-6 LC-PUFA is preferably at least 0.02 wt.% more preferably at least 0.05 wt.%, more preferably at least 0.1 wt.% based on total fatty acids, more preferably at least 0.2 wt.%. The presence of AA is advantageous in a composition low in LA since it remedies LA deficiency. The presence of, preferably low amounts, of AA is beneficial in nutrition to be administered to infants below the age of 6 months, since for these infants the infant formulae is generally the only source of nutrition. Preferably in addition to the vegetable lipid, a lipid selected from fish oil (preferably tuna fish oil) and single cell oil (such as algal, microbial oil and fungal oil) is present. These sources of oil are suitable as LC-PUFA sources. Preferably as a source of n-3 LC-PUFA single cell oil, including algal oil and microbial oil, is used, since these oil sources have a low EPA/DHA ratio.. Thus in one embodiment the present composition further comprises at least one lipid selected from the group consisting of fish oil, marine oil, algal oil, fungal oil and microbial oil.

Lipid globule design

Coating with phospholipids and cholesterol

The present invention comprises phospholipids. Phospholipids are present as a coating on the surface of the lipid globule. By 'coating' is meant that the outer surface layer of the lipid globule comprises polar lipids, whereas these polar lipids are virtually absent in the core of the lipid globule. Not all phospholipids and/or glycolipids that are present in the composition need necessarily be comprised in the coating, but preferably a major part is. Preferably more than 50 wt.%, more preferably more than 70 wt. %, even more preferably more than 85 wt.%, most preferably more than 95 wt.% of the phospholipids and/or glycolipids that are present in the composition are comprised in the coating of lipid globules. The phospholipids are located on the surface of the lipid globule, i.e. as a coating or outer layer. A suitable way to determine whether the phospholipids are located on the surface of the lipid globules is laser scanning microscopy as given in WO 2010/0027259.

In standard infant formulae, due to the small size of the lipid globule (resulting in a high lipid surface) and the very low levels of phospholipids present (typically below 0.15 wt% based on total fat), the lipid globules are not coated with phospholipids, but are covered for the main part by protein, such as casein.

Lipid globule size

According to the present invention, lipid is present in the composition in the form of lipid globules. When in liquid form these lipid globules are emulsified in the aqueous phase. Alternatively the lipid globules are present in a powder and the powder is suitable for reconstitution with water or another food grade aqueous phase. The lipid globules comprise a core and a surface. The core preferably comprises vegetable fat and preferably comprises at least 90 wt.% triglycerides and more preferably essentially consists of triglycerides. Not all vegetable lipids that are present in the composition need necessarily be comprised in the core of lipid globules, but preferably a major part is, preferably more than 50% wt.%, more preferably more than 70 wt.%, even more preferably more than 85 wt.%, even more preferably more than 95 wt.%, most preferably more than 98 wt.% of the vegetable lipids that are present in the composition are comprised in the core of lipid globules. In one embodiment the core of the lipid globules comprises at least 70 wt.% triglycerides of vegetable origin, more preferably the core of the lipid globules comprises at least 85 wt.%, more preferably at least 95 wt.% triglycerides of vegetable origin.

In one embodiment the lipid globules of the present invention preferably have a volume- weighted mode diameter above 1.0 μπι, preferably above 3.0 μπι, more preferably 4.0 μπι or above, preferably between 1.0 and 10 μιη, more preferably between 2.0 and 8.0 μιη, even more preferably between 3.0 and 8.0 μπι, most preferably between 4.0 μπι and 8.0 μπι. Preferably in addition the size distribution is in such a way that at least 45 volume %, preferably at least 55 volume %, even more preferably at least 65 volume %, even more preferably at least 75 volume % has a diameter between 2 and 12 μηι. More preferably at least 45 volume %, preferably at least 55 volume %, even more preferably at least 65 volume %, even more preferably at least 75 volume % has a diameter between 2 and 10 μηι. Even more preferably at least 45 volume %, preferably at least 55 volume %, even more preferably at least 65 volume %, even more preferably at least 75 volume % has a diameter between 4 and 10 μηι. It was found that coated large lipid globules have an improved effect on increasing leptin sensitivity, in particular on increasing leptin sensitivity later in life.

The percentage of lipid globules is based on volume of total lipid. The mode diameter relates to the diameter which is the most present based on volume of total lipid, or the peak value in a graphic representation, having on the X-as the diameter and on the Y-as the volume (%).

Standard infant formulae or growing up milks have lipid globules with mode diameter below 0.5 um.

The volume of the lipid globule and its size distribution can suitably be determined using a particle size analyzer such as a Mastersizer (Malvern Instruments, Malvern, UK), for example by the method described in Michalski et al, 2001 , Lait 81 : 787-796.

Methods for obtaining lipid globules with a coating of phospholipids, and increased size are disclosed in WO 2010/0027258 and WO 2010/0027259. The lipid globule size can be manipulated by adjusting process steps by which the present composition is manufactured. A suitable and preferred way to obtain lipid globules coated with phospholipids is to increase the amount of phospholipids compared to amounts typically present in infant formula and to have these phospholipids present during the homogenization process, wherein the mixture of aqueous phase and oil phase is homogenized. In standard infant milk formula the lipid fraction (usually comprising vegetable fat, a small amount of polar lipids and fat soluble vitamins) is mixed into the aqueous fraction (usually comprising water, skimmed milk, whey, digestible carbohydrates such as lactose, water soluble vitamins and minerals and optionally non-digestible carbohydrates) by homogenization under high pressure resulting in small lipid globules. If no homogenization was to take place, the lipid part would cream very quickly, i.e. separate from the aqueous part and collect at the top. Digestible carbohydrate component

The present nutritional composition preferably comprises digestible carbohydrate. The digestible carbohydrate preferably provides 30 to 80% of the total calories of the composition. Preferably the digestible carbohydrate provides 40 to 60% of the total calories. When in liquid form, e.g. as a ready-to-feed liquid, the composition preferably comprises 3.0 to 30 g digestible carbohydrate per 100 ml, more preferably 6.0 to 20, even more preferably 7.0 to 10.0 g per 100 ml. Based on dry weight the present composition preferably comprises 20 to 80 wt.%, more preferably 40 to 65 wt.% digestible carbohydrates.

Preferred digestible carbohydrate sources are lactose, glucose, sucrose, fructose, galactose, maltose, starch and maltodextrin. Lactose is the main digestible carbohydrate present in human milk. The present composition preferably comprises lactose. The present composition preferably comprises digestible carbohydrate, wherein at least 35 wt.%, more preferably at least 50 wt.%, more preferably at least 75 wt.%, even more preferably at least 90 wt.%, most preferably at least 95 wt.% of the digestible carbohydrate is lactose. Based on dry weight the present composition preferably comprises at least 25 wt.% lactose, preferably at least 40 wt.%. Non digestible carbohydrates

Preferably the present nutritional composition comprises non-digestible oligosaccharides. Preferably the present composition comprises non-digestible oligosaccharides with a degree of polymerization (DP) between 2 and 250, more preferably 3 and 60. The non-digestible oligosaccharides as dietary fiber may have a beneficial effect on increasing leptin sensitivity and reducing the risk of occurrence of hyperphagia.

Preferably the present nutritional composition comprises fructo-oligosaccharides, galacto- oligosaccharides and/or galacturonic acid oligosaccharides, more preferably galacto- oligosaccharides, most preferably transgalacto-oligosaccharides. In a preferred embodiment the composition comprises a mixture of transgalacto-oligosaccharides and fructo-oligosaccharides. Suitable non-digestible oligosaccharides are for example Vivinal GOS (Friesland Campina DOMO), Raftilin HP or Raftilose (Orafti). Transgalacto-oligosaccharide is preferred since it may increase insulin sensitivity.

Preferably, the nutritional composition comprises of 80 mg to 2 g non-digestible oligosaccharides per 100 ml, more preferably 150 mg to 1.50 g, even more preferably 300 mg to 1 g per 100 ml. Based on dry weight, the composition preferably comprises 0.25 wt.% to 20 wt.%, more preferably 0.5 wt.% to 10 wt.%, even more preferably 1.5 wt.% to 7.5 wt.%. A lower amount of non-digestible oligosaccharides will be less effective in increasing leptin sensitivity, whereas a too high amount will result in side-effects of bloating and abdominal discomfort.

Protein

The present nutritional composition preferably comprises proteins. The protein component preferably provides 5 to 15% of the total calories. Preferably the present composition comprises a protein component that provides 6 to 12% of the total calories. More preferably protein is present in the composition at most 9% based on calories, more preferably the composition comprises of 7.2 to 8.0% protein based on total calories, even more preferably of 7.3 to 7.7% based on total calories. Human milk comprises a lower amount of protein based on total calories than cow's milk. The protein concentration in a nutritional composition is determined by the sum of protein, peptides and free amino acids. Based on dry weight the composition preferably comprises at most 12 wt.% protein, more preferably of 9.6 to 12 wt.%, even more preferably 10 to 1 1 wt.%. Based on a ready-to-drink liquid product the composition preferably comprises at most 1.5 g protein per 100 ml, more preferably of 1.2 to 1.5 g, even more preferably of 1.25 to 1.35 g. The source of the protein should be selected in such a way that the minimum requirements for essential amino acid content are met and satisfactory growth is ensured. Hence protein sources based on cows' milk proteins such as whey, casein and mixtures thereof and proteins based on soy, potato or pea are preferred. In case whey proteins are used, the protein source is preferably based on acid whey or sweet whey, whey protein isolate or mixtures thereof and may include a- lactalbumin and β-lactoglobulin. More preferably, the protein source is based on acid whey or sweet whey from which caseino-glyco-macropeptide (CGMP) has been removed. Preferably the composition comprises at least 3 wt.% casein based on dry weight. Preferably the casein is intact and/or non-hydro lyzed. For the present invention protein includes peptides and free amino acids.

Other

The present nutritional composition is preferably particularly suitable for providing the daily nutritional requirements to a human with an age below 36 months, particularly an infant with the age below 24 months, even more preferably an infant with the age below 18 months, most preferably below 12 months of age. Hence, the nutritional composition is for feeding or is used for feeding a human subject. The present composition preferably comprises lipid, and protein and digestible carbohydrate wherein the lipid preferably provides 30 to 60 % of total calories, the protein preferably provides 5 to 20 %, more preferably 5 to 15 wt.%, of the total calories and the digestible carbohydrate preferably provides 25 to 75% of the total calories. Preferably the present nutritional composition comprises lipid providing 35 to 50% of the total calories, protein providing 6 to 12 % of the total calories and digestible carbohydrate providing 40 to 60 % of the total calories. In one embodiment, the protein provides 5 to 9 % of the total calories. The amount of total calories is determined by the sum of calories derived from protein, lipids and digestible carbohydrates.

The present composition is not human breast milk. The present composition is not (raw) cow's or other (raw) mammalian milk. The present composition comprises vegetable lipids. The composition of the invention preferably comprises other ingredients, such as vitamins, minerals according to international directives for infant formulae.

In one embodiment, the nutritional composition according to the invention or the nutritional composition for use according to the invention is a preterm formula, infant formula, follow on formula or growing up milk.

In order to meet the caloric requirements of the infant, the composition preferably comprises 50 to 200 kcal/100 ml liquid, more preferably 60 to 90 kcal/100 ml liquid, even more preferably 60 to 75 kcal/100 ml liquid. This caloric density ensures an optimal ratio between water and calorie consumption. The osmolarity of the present composition is preferably between 150 and 420 mOsmol/1, more preferably 260 to 320 mOsmol/1.

Preferably the composition is in a liquid form, with a viscosity below 35 mPa.s, more preferably below 6 mPa.s as measured in a Brookfield viscometer at 20°C at a shear rate of 100 s ¹ . In one embodiment, the present composition is a powder. Suitably, the composition is in a powdered form, which can be reconstituted with water or other food grade aqueous liquid, to form a liquid, or in a liquid concentrate form that should be diluted with water. It was found that lipid globules maintained their size and coating when reconstituted.

When the composition is in a liquid form, the preferred volume administered on a daily basis is in the range of about 80 to 2500 ml, more preferably about 450 to 1000 ml per day.

Application

The inventors surprisingly found that when in an animal model during infancy and childhood a food composition comprising lipid globules coated with phospholipids was fed, a different and significant effect on leptin sensitivity, in particular later in life, was observed compared to animals which during infancy and childhood had been fed a food composition having a similar fatty acid composition, but no phospholipids present in the form of a coating around the dietary lipid globule. The leptin sensitivity increasing effect thus was mainly due to the effects occurring during exposure to the Western style diet, i.e. the way the metabolism deals with the Western style, high fat, cholesterol rich diet. Circulating leptin levels were compared between diet groups at day 42 (direct diet effects) and at day 130 (programming effects). Circulating leptin was reduced by a diet containing phospholipid coated lipid globules compared to the control diet. This differs from the effect of human milk in feeding human infants, where the level of leptin is higher, probably due to the leptin in human milk, and is supposed to play a beneficial effect. This is also indicative that there is no significant amount of leptin present in diet 2 derived from cow's milk fat.

After the challenge with the Western style diet later in life the animals in the study group showed reduced plasma leptin compared to the animals in the control group. The caloric intake after fasting of animals in the study group was lower than that of control group, this was both directly after the programming diet and also after exposure to the western style diet during adulthood. This is indicative for regulation of food intake and satiety both directly and indirectly. During refeeding, study group animals reach a satiated state sooner than control group animals. Most likely the hypothalamic circuits may have been programmed towards less energy intake. Energy intake during adulthood after fasting, i.e. refeeding, was reduced by feeding a composition according to the invention during infancy. This is indicative for an improved regulation of satiety, or increased satiety, in particular the in-between meals satiety. Furthermore, it was observed that post prandial levels of triglycerides were prolonged with the diet of the present invention, comprising lipid globules coated with phospholipids, compared to diets with lipid globules not coated with phospholipids. This is indicative of an improved regulation of satiety, or increased satiety, in particular in between meals satiety, as a direct diet effect.

The inventors surprisingly found that leptin sensitivity of study group animals was higher than that of control group animals. Feeding after fasting increases circulating leptin and leptin induces satiety sooner when leptin sensitivity is higher. Therefore the diet of the present invention promotes leptin sensitivity, or alternatively prevents or reduces the risk on leptin resistance.

Leptin resistance is defined by high circulating leptin whereas the anorexic effect of leptin does not show. Animals that were raised on a composition according to the invention were more sensitive to the inhibiting effects of leptin, less leptin resistant than animals in the control group and showed reduced circulating leptin levels. Therefore, the composition according to the invention also prevents or reduces the risk of hyperphagia during adulthood, when exposed to a western style high fat high caloric dietary environment.

The present composition is administered to the human subject during the first 36 months of life. This period from birth up to childhood represents a critical timeframe during which nutrition programs the metabolism and body composition, still under development at this time, towards its function setting later in life. Infancy is also the critical period wherein diet can have an action on the development of the hypothalamic neurons, involved in feed intake and energy expenditure regulation. Without wishing to be bound by theory, the inventors assume that hypothalamic circuits may have been programmed towards less energy intake and that the expression of the long form of leptin receptor (ObRb) in the arcuate nucleus (ARC) of the hypothalamus is increased later in life in; the expression of the orexigenic neuropeptides Agouti-related peptide (AgRP), Neuropeptide Y (NPY), which reduce food intake and increase energy expenditure are affected and that the anorexigenic neuropeptides propo-opidmelanocortin (POMC), which is converted to alpha-melanocyte stimulating hormone (a-MSH) and cocaine- and amphetamine- regulated transcript (CART) are decreased later in life on diet 2 raised animals and that this explains the increased leptin sensitivity of the hypothalamus when during infancy the diet of the present invention was consumed.

The present composition is advantageously administered to a human of 0 to 24 months, more preferably to a human of 0 to 18 months, even more preferably to a human of 0 to 12 months, most preferably to a human of 0 to 6 months of age. The present invention particularly aims to increase leptin sensitivity levels later in life and reduce the risk of occurrence of hyperphagia later in life. Preferably the composition is to be used in infants having a weight appropriate for gestational age.

The present composition is preferably administered orally to the infant. The present invention aims to increase leptin sensitivity, preferably later in life. Likewise, in an embodiment, the present invention reduces or prevents leptin resistance, preferably later in life. The present invention also aims to improve self regulation of food intake, improve regulation of satiety and/or decrease refeeding after fasting, preferably later in life. In one embodiment the present method is for increasing leptin sensitivity, reducing or preventing leptin resistance, improving self regulation of food intake, improving regulation of satiety and/or decreasing refeeding after fasting and/or for preventing or reducing the risk of occurrence of hyperphagia, in a human subject when said human subject has an age above 36 months, preferably when said human subject has an age above 5 years, particularly above 13 years, more particularly above 17 years.

Leptin sensitivity is expressed as the brain's sensitivity to the signals of leptin and; increased leptin sensitivity means a higher response to leptin (e.g. suppression of food intake) compared to the subjects who were bottle fed with standard infant formula during infancy. Preferably the increase in leptin sensitivity is at least 5 %, more preferably at least 10%. In one embodiment increasing leptin sensitivity means a higher capacity of leptin to suppress food intake compared to subjects who were bottle fed with standard infant formula during infancy. This increase in leptin sensitivity is preferably more reminiscent to the situation found in subjects who were breast fed for at least 3 months.

Human milk intake is associated with lower leptin concentrations relative to fat mass in adolescence (Singhal). Breast feeding has been shown to have a protective effect against obesity or overweight in later life. This might indicate that early nutrition influences the cross-talk between brain and adipocytes, ultimately resulting in a different leptin (sensitivity). Interestingly, in the current experiments animals exposed to diet 2 also showed a lower leptin to fat mass ratio later in life (d 130) compared to animals exposed to diet 1 (leptin (ng/ml): fat (g) ratio of 0.21 and 0.30).

Similarly, reducing leptin resistance means a lower resistance to leptin compared to the leptin resistance found in subjects who were bottle fed with standard infant formula during infancy. Also, improving self regulation of food intake and improving regulation of satiety means an improved self regulation of food intake respectively improved regulation of satiety compared to the self regulation of food intake and regulation of satiety found in subjects who were bottle fed with standard infant formula during infancy. And decreasing re feeding after fasting means a lower refeeding after fasting compared to the refeeding after fasting found in subjects who were bottle fed with standard infant formula during infancy. Further, reducing the risk of occurrence of hyperphagia means a lower risk of occurrence of hyperphagia compared to the risk of occurrence of hyperphagia found in subjects who were bottle fed with standard infant formula during infancy.

With later in life is meant an age exceeding the age at which the diet is taken, preferably exceeding said age with at least one year. Preterm and/or small for gestational age infants often encounter catch up growth early in life. This is generally seen as a risk factor for later in life obesity. So the composition of the present invention is advantageously used in preterm infants or small for gestational age (SGA) infants, in particular for feeding a preterm infant or an infant small for gestational age. A preterm or premature infant relates to an infant born before the standard period of pregnancy is completed before or on 37 weeks pregnancy of the mother, i.e. before or on 37 weeks from the beginning of the last menstrual period of the mother. SGA babies are those whose birth weight lies below the 10th percentile for that gestational age. Premature and/or SGA infants include low birth weight infants (LBW infants), very low birth weight infants (VLBW infants), and extremely low birth weight infants (ELBW infants). LBW infants are defined as infants with a weight less than 2500 g. VLBW infants as infants with a weight which is less than 1500 g, and ELBW infants as infants with a weight less than 1000 g.

In this document and in its claims, the verb "to comprise" and its conjugations is used in its non- limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. In addition, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the element is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article "a" or "an" thus usually means "at least one".

LEGENDS TO THE FIGURES

Figure 1 : Cumulative caloric intake in rats fed on diet 1 versus rats fed on diet 2 at day 42 after 22 h fasting when injected saline.

Figure 2 : Cumulative caloric intake in rats fed on diet 1 (A) or diet 2 (B) at day 42 after 22 h fasting, when injected with saline versus leptin.

Figure 3: Cumulative caloric intake later in life in rats raised on diet 1 or diet 2 (during day 16 to 42) at day 130 after 22 h fasting, when injected with saline.

Figure 4: Cumulative caloric intake later in life in rats raised on diet 1 (A) or diet 2 (B) (during day 16 to 42) at day 130 after 22 h fasting, when injected with saline versus leptin. EXAMPLES

Example 1:

Methods:

The following programming diets were used:

1) a control IMF: This diet comprises 255 g lipid per kg dry weight. Lipid globules were small. No polar lipids were added. Lipid comprised about 47 % digestible carbohydrates about 45 and protein about 8% of total calories. 2) an IMF of the present invention: This diet differed from diet 1 in that it comprised large lipid globules with a coating with phospholipids.

The amount of phospholipid in diet 2 was 1.6 wt.% based on total fat (and 0.11 wt% in diet 1). The amount of cholesterol in diet 2 was 0.03 wt% based on total fat. The amount of sphingomyelin was about 20 wt. % based on total phospholipids. Source of polar lipids was from milk fat.

Differences in lipid globule size were obtained by differences in homogenization pressure as described in example 1 of WO 2010/0027258. The pressure of homogenization was 10/5 for diet 2 and 550/50 for diet 1. The volume mode diameter of the lipid globules of diet 1 was 0.5 um. Less than 10 vol. % had a diameter between 2 and 12 μηι. The volume mode diameter of the lipid globules in diet 2 was 1.7 μηι. More than 45 vol.% had a diameter between 2 and 12 μηι.

The fatty acid composition of the experimental diets was very similar in respect of saturated, mono-unsaturated, poly unsaturated and long chain poly unsaturated fatty acids, with calculated linoleic acid (LA) of about 14 wt.% based on total fatty acids, with alpha-linoleic acid (ALA) of about 2.6 wt.% based on total fatty acids and with LA/ ALA weight ratio of about 5.4. The amount of DHA was 0.2 wt.% based on total fatty acids in all diets, and the amount of ARA was about 0.35 wt.% based on total fatty acids. EPA and DPA levels were about 0.04 and 0.01 wt.% based on total fatty acids.

Subsequently rodent diets were prepared comprising 282 g IMF 1 or 2 per kg. The rest of the diet was AIN-93G protein, carbohydrates and fibre. All lipid (about 72 g per kg rodent diet) present in the diet was derived from the IMF. 16 male Wistar rat pups were randomly assigned to one of 2 experimental groups at postnatal (PN) day 16 weaned at day 21.The experimental programming diets were continued until day 42 (n=8 per diet). From day 42 to day 133 all pups were fed the same diet based on AIN-93G diet with an adjusted lipid fraction (containing 20 wt.% lipid of which 50 wt.% lard and 1 % cholesterol, based on total lipid), comprising 4520 kJ per 100 g, 52 wt% digestible carbohydrates, 4.75 wt.% fibers, and 17.9 wt.% protein, which is representative for a Western style, high fat, high cholesterol diet. In the Western style diet the weight ratio LA/ALA was 9.15. No LC-PUFA were present. Food and water were available ad libitum, unless stated otherwise

At the end of the period for programming diet, (at PN day 39 and 42),and at the end of the experiment (at PN day 127 and 130) animals were subjected to a leptin challenge test to assess leptin sensitivity.

For the leptin challenge test animals were fasted for 22 hours and 1 hour before start of the dark phase animals received an intraperitoneal (i.p.) injection of leptin solution 2 mg/kg (Recombinant Rat Leptin. Peprotech, London, UK) or saline. For the next 24 hours animals were housed individually in food registration cages with free access to food and water. Food intake was automatically recorded every 5 minutes (Food Intake Monitor for Rat, MED Associates Inc., Georgia, Vermont, USA). Three days later the procedure was repeated with the other injection so that all animals received both leptin and saline. The succession of i.p. injections with leptin and saline was equally distributed over the sessions. Animals were fed their respective diets in the food registration cages. At PN day 39 and 42 animals in the diet 1 and diet 2 groups received their diet in a pelleted form instead of dough.

A basal blood sample was drawn at PN day 37 and day 125. Animals were dissected on PN day 133 and blood, fat depots and organs were collected, weighed and snap frozen for further analysis.

Plasma leptin levels were measured in triplicate in a 96-wells plate (Leptin Rat ELISA, DRG Diagnostics, Marburg, Germany).

All data are expressed as means ± SEM. Statistical analyses were performed using SPSS 15.0 (SPSS Benelux, Gorinchem, The Netherlands).

Results:

Direct diet effects:

No statistically significant effect on body weight on day 42 or growth between day 21 and day 42 was observed. Energy intake by animals fed diet 1 and 2 during day 21 to day 42 could not be measured accurately because these diets consisted of dough.

Fasting plasma leptin at day 37 was lower in animals fed diet 2 than in animals fed control diet 1. (0.90 ± 0.25 and 1.55 ± 0.33 ng/ml). The plasma leptin levels were not correlated to bodyweight at day 42 (data not shown).

At the end of the programming diet intervention (PN day 39 and 42), differences in caloric intake between dietary groups was investigated. Figure 1 shows the average cumulative caloric intake at PN day 42 after 22 hours of fasting (Saline control). The caloric intake of animals fed diet 2 appears to be reduced over time compared to that of diet 1 animals (p=0.076). This was especially the case in the after the 12 h refeeding period. This is indicative for a reduced risk on compensatory food intake or overcompensatory food intake such as hyperphagia. Caloric intake after a leptin injection was not different between diet 1 and diet 2 group. However, when the effect of leptin on food intake regulation was compared, i.e. leptin sensitivity, the food intake of diet 2-fed animals was inhibited by leptin at various time points within the first 12 hours whereas in the diet 1 fed-animals there was only inhibition for 5 hours (Figure 2, leptin) Programming effects:

Bodyweight and growth between day 42 and day 133 was not different between diet 1 and 2. From day 42 to day 130, the average daily energy intake was not statistically different between the two diet groups. At day 125 the diet 2-fed animals had lower plasma leptin than the diet 1-fed animals (5.3 ± 0.8 ng/ml and 10.3 ±1.9 ng/ml, respectively). In diet 2 the leptin levels were correlated to body weight, fat mass, relative fat mass (% of total body weight) at day 133 and also correlated with the fasting leptin levels observed at day 37, as can be seen in table 1. In contrast, in diet 1 fed animals, the leptin levels at PN 125 were only correlated to dissection body weight. This is indicative for a more balanced growth. This is indicative for a better regulation between leptin levels and adipose tissue. Interestingly, in the current experiments animals exposed to diet 2 also showed a lower leptin to fat mass ratio later in life (compared to animals exposed to diet 1 (leptin (ng/ml): fat (g) ratio of 0.21 and 0.30).

Table 1. Correlations between leptin levels at PN 125 and dissection BW, FM fat% and leptin levels at PN day 37 per diet group

Differences in caloric intake between dietary groups was investigated. Figure 3 shows the average cumulative caloric intake at PN day 127 or 130 after 22 hours of fasting (Saline control during later in life leptin challenge test). The cumulative caloric intake of animals raised on diet 2 is reduced over time compared to that of diet 1 animals (p<0.001).

Caloric intake after a leptin injection was not different between animlas raised on diet 1 or 2, although it was numerically reduced in diet 2 raised animals. However, the reaction to a leptin challenge with respect to cumulative food intake was different for both diet groups. Animals raised on diet 1 did not show a reduction in cumulative food intake after a leptin injection compared to that observed after the saline control injection (Figure 4).

In contrast, in the diet 2 raised animals, food intake during the leptin challenge was reduced by the leptin challenge compared to the saline control food intake in those animals at 2 and 3 hours post-injection (P=0.04 and p=0.08, respectively) and from 1 1 hours post-injection until the end of the challenge (P<0.03; Figure 4).

Although there was no difference in weight, the diet 2 programming diet resulted in statistically lower fat mass, including visceral fat mass, than the diet 1. However, the muscular tibialis, representative for lean body mass was not decreased in diet 2, but slightly increased. So the body composition was improved. The differences in fat mass at PN day 130 were not caused by differences in energy intake during the WSD challenge. Conclusions:

Circulating leptin levels were compared between diet groups at day 37 (direct diet effects) and at day 125 (programming effects). Circulating leptin was reduced by diet 2 compared to diet 1. This differs from the effect of human milk feeding in with human infants, where the level of leptin is higher, probably due to the leptin in human milk, and is supposed to play a beneficial effect. This is also indicative that there is no significant amount of leptin present in diet 2 derived from cow's milk fat.

After the challenge with the Western style diet later in life the animals in the diet 2 group showed reduced plasma leptin compared to the animals in the diet 1 group. This was also in line with reduced fat deposition observed in diet 2 raised animals. Irrespective of fat mass, an altered production of leptin in adipocytes might also have contributed to the observed differences. In similar studies with mice, mR A levels of leptin were reduced by diet 2 in isolated RP WAT adipocytes (data not shown). This is indicative for a better regulation of the adipocyte functionality.

The caloric intake after fasting of animals raised on diet 2 of the present invention was lower than that of diet 1 raised animals, this was both directly after the programming diet (day 39-42) and also after the western style diet during adulthood (day 127-130). This is indicative for regulation of food intake and satiety both directly and indirectly. During refeeding, diet 2 animals reach a satiated state sooner than diet 1 fed animals. Likely the hypothalamic circuits may have been programmed towards less energy intake. Energy intake during adulthood was reduced by former diet 2 feeding.

Most importantly, leptin sensitivity of diet 2 raised animals was higher than that of diet 1 raised animals. Again this was observed at day 42, but also surprisingly [to an even pronounced effects] on day 130. Feeding after fasting increases circulating leptin and leptin induces satiety sooner when leptin sensitivity is higher. Therefore the diet of the present invention promotes leptin sensitivity directly and indirectly. Or alternatively prevents or reduces the risk on leptin resistance. Directly and later in life.

Leptin resistance is defined by high circulating leptin whereas the anorexic effect of leptin does not show. Animals that were raised on diet 2 were more sensitive to the inhibiting effects of leptin, less leptin resistant than animals in the diet 1 group and showed reduced circulating leptin levels. Directly and later in life. Diet2 therefore also prevents or reduces the risk of hyperphagia during adulthood, when exposed to a western style high fat high caloric dietary environment.

Likely the hypothalamic circuits may have been programmed towards less energy intake. Without wishing to be bound by theory it is hypothesized that the expression of the long form of leptin receptor (ObRb) in the arcuate nucleus (ARC) of the hypothalamus is increased later in life in diet 2 raised animals. The expression of the orexigenic neuropeptides Agouti-related peptide (AgRP), Neuropeptide Y (NPY), which reduce food intake and increase energy expenditure is affected later in life in diet 2 raised animals. The anorexigenic neuropeptides propo-opidmelanocortin (POMC), which is converted to alpha-melanocyte stimulating hormone (a-MSH) and cocaine- and amphetamine -regulated transcript (CART) is decreased later in life on diet 2 raised animals. This will explain the increased leptin sensitivity of the hypothalamus is increased in diet 2 raised animals. In short, the results of the present study demonstrate that diet 2 of the present invention administered early in life improves leptin sensitivity directly and programs for reduced leptin resistance during adulthood when challenged with a Western Style Diet (WSD)20 %. Also, the results of the present study demonstrate that diet 2 of the present invention administered early in life improve self regulation of food intake, improve regulation of satiety and/or decrease refeeding after fasting during adulthood when challenged with WSD. Furthermore, the results of the present study are indicative that diet 2 of the present invention administered early in life reduces or prevents the risk of the occurrence of hyperphagia during adulthood.

Example 2:

Methods:

The following programming diets were used:

1) a control IMF: similar to IMF 1 of example 1.

2) an IMF with added pospholipids: This diet differed from diet 1 in that it comprised large lipid globules and comprised phospholipids not present in the form of a coating around the lipid globule. 3) an IMF of the present invention: This diet differed from diet 1 in that it comprised lipid globules with a coating with phospholipids.

For IMF 1 no added phospholipids were present. The amount of vegetable (soy-derived) glycerophospholipids was 0.12 wt.% based on total fat for IMF 1. IMF 2 and 3 comprised 5.7 wt.% phospholipids based on total fat, of which about 98 % derived from the butter milk powder and about 2 % already present in the standard IMF, derived from vegetable oils. As a source SM2 powder of Corman was used comprising about 76 % glycerophospholipids, based on total phospholipids and further comprises about 24 wt.% sphingomyelin based on total phospholipids. The mode diameter of the lipid globules was 0.96 um.

For IMF 2 the SM2 powder comprising the phospholipids were dry blended after the homogenization, sterilization and spray dry step in order to prevent coating of the lipid globules with phospholipids. In IMF 1 and IMF 2 the lipid globules are coated mainly with protein (especially casein). For IMF 3 the SM2 powder was present in the aqueous phase during the homogenization step resulting in a coating of the lipid globules with the phospholipids.

Macronutrient and fatty acid composition was similar to example 1 and similar in all diets, except that no LC-PUFA were present. The size of the lipid globule in IMF2 was 0.83 um and of IMF 3 1.55 um

Subsequently rodent diets were prepared comprising 282 g IMF 1 , 2 or 3 per kg. The rest of the diet was AIN-93G protein, carbohydrates and fibre. All lipid (about 72 g per kg rodent diet) present in the diet was derived from the IMF. Offspring of C57 BL6 mice nests were culled to 4 male 2 female pups. On day 15 one group of 42 pups were separated from the dams and fasted for 2 h. One group of background-pups (n=7) was sacrificed and blood and liver specimen were obtained: the t=0 group.

After 2 h of fasting, at t=0 male pups (n=35) were given a gavage of warm soy oil (0.25 ml/pup). Pups were sacrificed at t=l , 2,3,5 and 8 h after gavage (n=7 per time point). From each pup body weight, and a blood sample, liver specimens, and fresh (wet) fecal droppings were obtained. The remaining pups were assigned to 3 different experimental diets until day 28. The pups remained in the nests with their dams until day 21 when the dam was removed.

On day 28 all pups were fasted for 2 h. One group of background-pups (n=7) was sacrificed and blood and liver specimen were obtained: the t=0 group.

At t=0 the other pups (n=35) were given a gavage of warm soy oil (0.25 ml/pup): the fat challenge. Pups were sacrificed at t=l ,2,3,5 and 8 h after gavage (n=7 per time point). From each pup body weight, and a blood sample, liver specimens, and fresh (wet) fecal droppings were obtained.

From the blood samples obtained at day 15 and 28, triglyceride levels were determined colorimetrically (Sigma kit).

Results:

A good growth of the mice was observed with all 3 diets.

The plasma triglyceride levels in time are disclosed in table 2. It can be deduced from the t=0 levels on day 28 that fasting plasma triglyceride levels are beneficially lower in pups having been exposed to the experimental diet with IMF 3 in the previous 2 weeks. Also the lower levels of plasma triglycerides to which the values return after 8 h after the fat challenge are indicative of a lower basal levels of plasma triglycerides with diet 3. These values are comparable to the values observed in breast milk fat pups (day 15, t=0) and t=8.

Interestingly, the postprandial time course of plasma triglycerides after having consumed a standard fat load (the fat challenge) differs in mice having been exposed to a diet with IMF 3 compared to mice having been exposed to diets with IMF 1 and IMF 2. This difference can mainly be explained by the difference in location of the phospholipids (i.e. coated around the lipid globule), but not by the fatty acid composition or the composition or presence of the phospholipids as such. Postprandial levels of plasma triglycerides in mice having been exposed to a diet with IMF 3 were lower in maximal concentration, the maximal concentration was achieved later in time and plasma triglycerides were elevated for a more prolonged time. The relative response, setting the t=0 value on 0 %) is however higher with IMF 3, which is indicative of a difference use of the triglycerides, since the resulting plasma profile is the sum of appearance and disappearance. This finding is indicative for no peak levels but a more gradual entry of the fat in the body: This is beneficial since there is no need to store the excess plasma triglycerides in adipose tissue, but the triglycerides can be use immediately for heat and energy production (β -oxidation). Assumably the sustained and more stable level of plasma triglycerides of mice exposed to IMF 3 will be translated into different signalling messages in the body with regard to e.g. satiety, energy status and adipose metabolism.

Table 2: Postprandial triglyceride level (n=4-7): means (s.e.m.). n.d.=not determined.

The results of the present study are indicative that a diet according to the present invention administered early in life increases satiety later in life. It is noted that the postprandial effects on triglyceride level are determined with the same fat load and are not a direct effect of structural differences.