The present invention relates to processes for treating milk and milk products obtainable by the treatment which will not be able to induce insulin resistance and/or exhibit a reduced insulinemic index and/or reduced mitogenic activity

Summary of the invention

Common chronic diseases of Western societies, such as coronary heart disease, diabetes mellitus, cancer, hypertension, obesity, dementia, allergic diseases, and acne are significantly influenced by dietary habits. Cow's milk and dairy products are nutritional staples in most Western societies. Milk and dairy product consumption is recommended by most nutritional societies because of their beneficial effects for calcium uptake and bone mineralization and as a source of valuable protein. However, the adverse long-term effects of milk and milk protein consumption on human health have been neglected. A hypothesis is presented, showing for the first time that milk protein consumption is an essential adverse environmental factor promoting most chronic diseases of Western societies. Milk protein consumption induces postprandial hyperinsulinaemia and shifts the growth hormone/insulin-like growth factor-1 (IGF-I) axis to permanently increased IGF-I serum levels. Insulin/IGF-1 signalling is involved in the regulation of fetal growth, T-cell maturation in the thymus, linear growth, pathogenesis of acne, atherosclerosis, diabetes mellitus, obesity, cancer and neurodegenerative diseases, thus affecting most chronic diseases of Western societies. Of special concern is the possibility that milk intake during pregnancy adversely affects the early fetal programming of the IGF-I axis which will influence health risks later in life. An accumulated body of evidence for the adverse effects of cow's milk consumption from fetal life to childhood, adolescence, adulthood and senescence will be provided which strengthens the presented hypothesis.

It is the main objective of this invention to treat cow milk or milk of other species like goat, horse, sheep and the like in a way that animal milk or dairy products will lose their ability to induce insulin resistance or be insulinotropic or to be mitogenic. As the somatotropic axis (GH-IGF-1-axis) is adjusted in the perinatal and early postnatal period for whole life, it is of greatest importance to avoid cow milk consumption in pregnancy or postnatal life in its usual form. If, however, breast-feeding is not possible, a modified cow milk or milk of another milk-giving species should be used which has by treatment not the ability to induce insulin resistance, i.e., this modified milk is not able to interfere with the GH-IGF-1-axis and will not increase human GH, insulin and IGF-I serum levels. Special targets for inactivation are bovine alpha- Lactalbumin and alpha-Lactalbumin of other species used for milk production for humans.

Insulin and the insulin-like growth factor system

The insulin-like growth factor (IGF) system is essential for normal embryonic and postnatal growth, and plays an important role in the function of a healthy immune system, lymphopoiesis, myogenesis and bone growth among other physiological functions. Growth hormone (GH) and IGFs play an important role in growth and tissue homeostasis. GH secreted by the anterior pituitary binds to GH-receptor, expressed on most peripheral cells of the body. In peripheral tissues and predominantly in the liver, GH induces the synthesis and secretion of the 7.65 kDa polypeptide hormone IGF-I, the mediator of the growth stimulating activity of GH. More than 90% of circulating IGFs are bound to IGF-binding protein-3 (IGFBP-3), the rest to IGFBP-I, -2, -4, -5, and -6, and less than 1% of IGFs circulate as free IGFs in the plasma. IGF-I signal transduction is mediated primarily by the IGF-1-receptor (IGFlR), a tyrosine kinase receptor, which is able to form heterodimers with insulin receptor (IR). IGF-2 binds to IGF-2-receptor (IGF2R), a scavenger receptor down-regulating IGF-2. IGF-2 is also able to bind to IGFlR. Insulin primarily binds to IR-A and IR-B, but also binds with lower affinity to IGFlR. IGF-I and IGF-2 bind to IR with lower affinity (Figure 1). IGFlR signal transduction is mediated primarily by the activation of the Ras-Raf-MAP kinase pathway and the phosphoinositide 3-kinase (PI3K)/Akt pathway. IGF-I acts a strong mitogen inducing cell growth and proliferation, but inhibits apoptosis [I]. The IR-B isoform is the form best known for the classic metabolic responses induced upon insulin binding and this isoform has low affinity for IGFs [I]. The IR-A isoform arises from alternative splicing of exon 11 encoded by the IR gene. Activation of the IR-A by insulin or IGF-2 leads to mitogenic responses similar to those described for IGFlR. Increased signaling via IR-A has been associated with the development of cancer [2]. In this regard, insulin and IGF-2 signal transduction via IR-A and IGF-I signaling via IGFlR induce and amplify mitogenic responses (Figure 1).

Milk and milk protein consumption increase IGF-I serum levels

Milk is a complex, bioactive substance honed by evolution to promote growth and development of the infant mammal. Cow's milk and dairy products derived from milk are widely consumed by children and adults of Western societies well after the age of weaning. It is important to note that cow's milk contains active IGF-I (4-50 ng/ml) and IGF-2 (40-50 ng/ml) [3, 4]. IGF-signaling belongs to the canonical pathways and networks regulated by estrogen and placental GH in the bovine mammary gland. Cows treated with recombinant bovine GH to improve milk yield showed increased levels of IGF-I in the milk [4]. High levels of IGF-I are still detectable after pasteurization and homogenization of milk [5]. Intriguingly, bovine and human IGF-I share the same amino acid sequences and therefore bovine IGF-I can bind to the human IGFlR [6].

Several lines of evidence indicate that IGFs in milk can survive digestion and remain bioactive in the serum of milk consumers. Studies have demonstrated intact oral absorption and plasma bioactivity of IGF-I in neonate and adult animals, especially when IGF-I was administered together with the protease inhibitor casein, the primary protein in milk. Intestinal absorption of growth factors in milk (IGF-I, betacellulin, prolactin, FGFs and others) may be increased by Western diet which is high in wheat products and potatoes. It has been recognized that wheat-derived gluten and potato-derived glycoalkaloids perturb and increase intestinal permeability resulting in increased intestinal protein absorption [6a, 6b, 6c]. High milk consumption in humans is associated with a 10-20% increase in circulating IGF-I levels among adults and a 20-30% increase among children [7-14]. Girls with a milk intake below 55 ml/day had significantly lower IGF-I serum concentration compared to girls consuming more than 260 ml/day [15]. In 2,109 European women, IGF-I serum levels positively correlated with the intake of milk [16]. A recent multicenter large cross-sectional analysis in 4731 men and women of the European Perspective Investigation into Cancer and Nutrition confirmed that the intake of dairy protein is an important dietary determinant of serum IGF-I levels [16a]. A recent meta-analysis further confirmed the association between cow milk consumption and serum IGF-I serum levels [16b]. It is important to notice that dairy products increase IGF-I levels more than any other dietary sources of protein like meat [9-16]. Moreover, milk-consumption raises the ratio of IGF-l/IGFBP-3 indicating an increased bioavailability of IGF-I [8-10, 12].

The insulinotropic effect of milk and milk products

Fermented and non-fermented milk products give rise to insulinaemic responses far exceeding what could be expected from their low glycaemic indexes (GI). Despite low GIs of 15 to 30, milk products produce three- to sixfold higher insulinaemic indexes (II) of 90-98 [17]. A large and similar dissociation of the GI and II exists for both whole milk (GI : 42 ± 5; II: 148 ±14) and skim milk (GI: 37 ± 9; II: 140 ± 13) [18]. It has been suggested that some factor within the protein fraction of milk is responsible for milk's insulinotropic effect [18]. Skim milk has been identified as a potent insulin secretagogue in type 2 diabetic patients [19]. Except for cheese with an insulin score of 45, milk and all dairy products including yoghurt, ice cream, cottage cheese, and fermented milk products have potent insulinotropic properties [20].

In a one-week intervention study of 24 prepubertal eight-year-old boys the effect of daily intake of 53 grams of either lean meat or skim milk (1.5 I per day) was studied with regard to insulin and IGF-I responses. In the skim milk group insulin significantly increased by 105% (from 22 to 45 pmol/l) and IGF- 1 significantly increased by 19% (from 209 to 249 ng/ml) [12]. There was no significant increase in either insulin or IGF-I in the meat group. This study clearly showed that milk protein consumption induces hyperinsulinaemia and increased IGF-I serum levels. The addition of an ordinary amount of 200 ml milk to a meal with a low GI increased the insulin response by 300% to a level typically seen from a meal with a very high GI like white bread [21]. The comparison of 43 breast-fed and 43 cow's milk formula fed one-week-old term infants showed higher insulin levels in the cow's milk formula-fed group at 90 and 150 minutes postprandial [22].

Differential induction of insulin and IGF-I by milk protein fractions

The major protein fractions of cow's milk is casein (80%), the remaining 20% are whey proteins. Both, whey and casein contain specific proteins and peptides that may have growth stimulating effects. The effect of whey and casein fractions of milk on fasting concentrations of IGF-I and insulin has been examined in 57 eight-year-old boys who received over seven days either casein or whey protein fractions with protein amounts of casein or whey similar to the content of 1.5 I skim milk. In the casein group serum IGF-I increased by 15%, whereas there was no change in fasting insulin. In the whey group fasting insulin increased by 21%, with no change in IGF-I [23, 24, 24a]. The insulin response to a whey meal has been reported to be higher than that of a milk meal. This differential response suggests that the insulinotropic component of milk resides predominantly within the whey fraction of soluble milk proteins, whereas casein has a stronger IGF-I stimulating effect than does whey [23, 24, 24a] (Figure 2). It is conceivable that specific whey proteins or their enzymatically cleaved peptides function as secretagogues for the release of intestinal incretins like glucagon-like peptide- 1 or gastric inhibitory peptide (GIP, renamed to glucose-dependent insulinotropic polypeptide) which are known to stimulate pancreatic insulin secretion and release [25]. It has recently been demonstrated that whey proteins induce high GIP responses of enteroendocrine intestinal L-cells promoting insulin secretion of pancreatic beta-cells [25a, 25b]. GIP receptors are also expressed on adipocytes and stimulate adipogenesis [25c]. The whey protein-GIP signaling due to milk consumption will thus increase pancreatic insulin secretion and GIP-mediated adipocyte proliferation and differentiation promoting the development of obesity [25c]. Whey proteins further induce cholecystokinin (CCK) from intestinal I-cells [Hall WL et al. Casein and whey exert different effects on plasma amino acid profiles, gastrointestinal hormone secretion and appetite. Br J Nutr 2003; 89: 239]. A typical "Western" combination diet composed of milk proteins and food with high glycaemic index will have potentiating effects on serum insulin, IGF-I, GLP-I and GIP- levels thereby promoting anabolic signaling pathways involved in mitogenesis and antiapoptosis.

Insulin induces hepatic synthesis and secretion of IGF-I

The main source of circulating IGF-I is considered to be the liver. A study of seven insulin-dependent diabetic patients in whom insulin was withheld for 12 hours received insulin infusions (1.6 mil insulin/kg/min) after an overnight fasted state. Serum IGF-I, but not IGFBP-3, significantly increased during the insulin infusion, whereas hepatic IGFPB-I synthesis was reduced [26]. Mean serum baseline levels of IGF-I in arterial blood (166 μg/l) and hepatic vein (160 μg/l) blood increased during the 180 min of insulin infusion to 183 μg/l and 185 μg/l, respectively [26]. Thus, insulin infusion raised serum IGF-I levels by approximately 13%. It is conceivable that the milk protein-induced hyperinsulinaemia will stimulate hepatic IGF-I synthesis and secretion in normal individuals as well. Moreover, it has been shown in Mongolian children previously not used to milk consumption that a four week dietary daily intervention with 710 ml of UHT milk significantly raised serum growth hormone (GH) levels [41]. GH is the most important inducer of hepatic IGF-I synthesis and release.

The impact of milk consumption on fetal growth Both IGF-I and IGF-2 are expressed in fetal tissues from the earliest stage of pre-implantation to the final phase of tissue maturation before birth. IGF-2 is the primary growth factor supporting embryonic growth, with IGF-I increasing in importance later in gestation. Concentrations of IGF-I in the fetus are affected by nutrient supply to the fetus and nutrient-sensitive hormones [27]. Insulin positively regulates IGF-I levels [28]. In industrialized countries, one of 10 newborns is affected with fetal macrosomia which has been associated with an increased risk of developing diabetes type 2 later in life. Milk consumption during pregnancy has been associated with a higher birth weight of the offspring [29, 30]. The protein intake from dairy products but not cheese protein was associated with increase in birth weight [29]. Levels of IGF-I and IGFBP-3 appear to be regulated by several factors, such as insulin, GH and maternal factors [31]. Levels of IGF-I in cord sera of newborns small for gestational age (mean 48.7 ng/ml) were lower than those of newborns appropriate for gestational age (AGA) (56.4 ng/ml). Newborns large for gestational age (LGA) exhibited the highest IGF-I cord sera levels (96.1 ng/ml) [31]. A recent study showed significantly higher insulin, leptin, IGFBP- 3, and glucose concentrations in asymmetric LGA newborns than in symmetric LGA and AGA newborns [32]. Macrosomic neonates of diabetic mothers have significantly increased aortic intima-media thickness with higher serum IGF-I, IGFBP-3 and leptin concentrations than those of controls [33]. Umbilical cord serum IGF-I levels were correlated significantly with the IGF-I concentrations of the mothers [34]. Umbilical cord serum levels of free IGF-I, total IGF-I, IGFBP-2 and leptin have been demonstrated as predictors of birth weight [35]. Recently, normal variations in maternal glycaemia on birth size and birth outcomes has been investigated in nondiabetic mothers [36]. Each lmmol/l rise in mother ^' s 60-min glucose levels after oral glucose challenge in the 28 ^th week of gestation resulted in a 46 ± 8 g increase in offspring birth weight [36]. The mother ^' s higher fasting glycaemia, lower insulin sensitivity, and lower insulin secretion were independently related to greater offspring adiposity at birth [36]. As high milk and milk protein consumption induces hyperinsulinaemia which favors the development of insulin resistance, substantial milk and dairy product consumption during pregnancy may contribute to the development of higher birth weight [29, 30]. These data point to the important role of the insulin and the IGF-I axis in fetal growth and the impact of milk consumption during pregnancy for the development of fetal macrosomia.

Early programming of the GH-IGF-I axis

The GH-IGF-I axis is closely related to feeding in the newborn [37]. More recent data point to an early programming of the IGF-I axis within the first months of live. In early pregnancy maternal endocrine IGF-I programs the placenta for increased functional capacity throughout gestation [38]. IGFs play a critical role in fetal and placental growth throughout gestation [27, 39]. Increased maternal milk consumption during pregnancy enhances the nutrient supply for the fetus by an enlarged placenta. In the guinea pig administration of IGF-I during early pregnancy increased placental transport of glucose and amino acids and increased placental and fetal weights [38]. Intriguingly, an inverse association between IGF-I at 9 months and 17 years of age has been demonstrated in humans [40]. A 1 ng/ml higher IGF-I concentration at 9 months corresponded to 0.95 ng/ml lower IGF-I concentration at 17 years [40]. Breast-fed infants at two months exhibited an IGF-I serum level of 93.3 ±_ 23.6 ng/ml, whereas formula-fed two-months-old infants revealed increased IGF-I levels of 129.8 ± 39.8 ng/ml [40]. At the age of 17 years, the breastfed infants showed an increased IGF-I level of 328 ± 78.5 in comparison to decreased IGF-I levels of 292.9 ± 95.0 of not breast-fed adolescents of the same age [40]. These observations support the view that the IGF-I axis is programmed early in life [40]. As milk protein consumption during pregnancy is associated with increased serum levels of IGF-I and postprandial hyperinsulinaemia, a milk protein-mediated shift of the IGF-I axis to higher levels has to be expected early in pregnancy. One of the first effects of milk consumption during early pregnancy might increase maternal IGF-I levels which program the placenta for increased functional capacity throughout gestation thereby increase the risk of fetal macrosomia and other IGF-I- dependent diseases [38]. Recent epidemiological evidence points to an inverse correlation between the length of breast feeding and the degree and onset of early obesity in 2-year-old infants [38a]. A higher protein content of infant formula has been associated with higher weight in the first 2 y of life but has no effect on length. Lower protein intake in infancy might diminish the later risk of overweight and obesity [38b]. Cow milk formula-based early weight gain is explained by the early protein hypothesis. The early protein hypothesis suggests that a high protein intake with infant formula feeding, in excess of metabolic requirements, might induce increased circulating concentrations of insulin-releasing amino acids, which in turn might stimulate the secretion of insulin and IGFl, thereby inducing an increased weight gain during the first 2 y of life as well as increased adipogenic activity.

Milk consumption shifts the GH-IGF-I axis in pre-pubertal children

After a month of drinking 710 ml of ultra heat treated whole milk daily, 10-11- year-old Mongolian children, previously not used to milk consumption, had a higher mean plasma level of IGF-I and higher ratio of IGF-l/IGFBP-3 [41]. The mean serum IGF-I levels were raised in the children after 4 weeks of milk consumption by 23.4% from mean pre-treatment values of 291 to 358 ng/ml [41]. Moreover, there was a significant increase in serum GH levels after 4 weeks of daily milk consumption. Thus, there is good evidence that milk consumption shifts the human intrinsic GH-IGF-I axis to unusual high levels.

Milk consumption and linear growth

Over the last centuries, body height significantly accelerated. Milk intake is the best source for utilization of calcium for bone growth and mineralization and is positively associated with IGF-I serum levels [15]. Milk consumption during pregnancy is associated with increased infant size at birth [29]. During a four- week intervention with daily milk intake of 710 ml, Mongolian children experienced a rapid linear growth (the equivalent of 12 cm/year). Girls grew a mean 1.1 + 0.2 cm and boys 1.0 + 0.2 cm [41]. The Growing Up Today Study showed that girls drinking less than 1 glass milk per week had a height of 150.6 cm, while girls drinking 2 or more glasses per day had a height of 151.9 cm (increase of 1.3 cm) [42]. Boys with a milk intake of less than 1 glass per week had a height of 150.1 cm, those with more than 2 glasses per day had height of 152.4 cm (increase of 2.3 cm) [43]. Further evidence for the growth- promoting effect of milk comes from studies in developing countries [23]. Milk and milk protein consumption is associated with an acceleration of linear growth and body height in industrialized countries.

Effect of IGFs and insulin on adreno-gonadal maturation and onset of puberty

The GH-IGF-I axis plays an important role for the ACTH-dependent production of dehydroepiandrosterone sulphate (DHEAS) of the human adrenal gland [44]. IGF-I is involved in ovarian androgen synthesis and has been implicated in the pathogenesis of ovarian hyperandrogenism and polycystic ovary syndrome (PCOS) [45]. IGF-I serum levels are increased in patients with PCOS who exhibit insulin resistance, anovulation, hyperandrogensism with acne and hirsutism. Proliferation and differentiation of adult testicular Leydig cells is the prerequisite for the increase of circulating plasma androgens during puberty. IGF-I is an essential local mediator of testicular steroidogenesis. In human testicular cell cultures, IGF-I stimulated testosterone secretion and cell proliferation, whereas apoptosis was inhibited [46]. Both insulin and leptin are thought to accelerate the timing of pubertal onset and to up-regulate the tempo of pubertal progression [47, 48]. The insulin sensitizing agent metformin decreases elevated serum IGF-I levels in patients with PCOS [49]. Girls with precocious pubarche and low birth weight reveal increased IGF-I serum levels and insulin resistance which leads to rapid progression of puberty. These girls are predisposed to develop PCOS [50]. Metformin treatment (425 mg/day) over two years of eight-year-old girls with precocious pubarche and low birth weight prevented the onset of early puberty by 0.4 years and significantly decreased serum levels of IGF-I, fasting insulin, DHEAS and testosterone [51]. In the untreated group (n = 19) of these girls IGF-I increased from 215 ± 10 to 289 ± 21 ng/ml (Δ 0-24 months: 74 ± 23), whereas IGF-I in the metformin-treated group (n = 19) only moderately increased from 197 ± 11 to 258 ± 22 ng/ml (Δ 0-24 months: 61 ± 24) after the 24 months intervention [51]. Fasting insulin increased in the untreated group, whereas only a moderate increase was observed in treated girls [51]. These data show that improvement of insulin resistance by metformin treatment is associated with a decrease in IGF-I levels in girls with precocious pubarche as well as adult patients with PCOS. A recent study indicated that the GH/IGF-1 axis and insulin resistance might be involved in the mechanism of adrenarche during prepuberty in normal girls [52]. Metformin shifts the IGF-I axis to lower levels thereby preventing an early onset of puberty. Thus, metformin treatment has the opposite effect compared to the effects of milk consumption, which shifts the IGF-I axis and insulin to higher levels [41]. From these data it can be concluded that milk and milk protein consumption has not only an impact on the acceleration of linear growth but also on the onset of puberty. Milk consumption, IGF-I serum levels and acne

Acne is regarded as an androgen-dependent disease of the pilosebaceous follicle. Its course, however, corresponds less closely to plasma androgen levels than it does to GH and IGF-I levels [53]. Significantly increased serum levels of IGF-I have been observed in women with post-adolescent acne as well as adult acne patients [54, 55]. In women, the total number of acne lesions correlated with serum IGF-I levels. In Western societies, acne is a nearly universal disease afflicting 79 to 95% of the adolescent population. In men and women older than 25 years, 40% to 54% have some degree of facial acne, and clinical facial acne persists into middle age in 12% of women and 3% of men [56]. Epidemiologic observations point to the role of Western diet in the development or aggravation of acne [56]. Cordain et al. [56] reported on 1200 Kitavan islanders of Papua New Guinea and 115 Ache hunter- gatherers of Paraguay who do not consume dairy products and have low glycaemic diets. No case of acne has been detected in these two non- westernized populations. Prospective cohort studies (Growing Up Today Study, based 1996) in 4,273 teenage boys and 6,094 teenage girls in the United States demonstrated a correlation between milk consumption and acne [42, 43]. In the study of boys, the strongest association has been found between intake of skim milk and acne [43]. It has been shown that high intakes of skim milk but not meat, increase serum IGF-I and IGFBP3-levels in eight year old boys [57]. Sebaceous glands express IGFlR [58]. IGF-I has been recognized as a mitogen and morphogen of sebaceous glands [58]. GH receptor has been found on the acini of sebaceous glands [59, 60]. Insulin as well as IGF-I stimulate sebocyte differentiation. However, when insulin or IGF-I were administered together with GH, the effect on sebocyte differentiation was potentiated compared to either hormone administered alone [60]. These data are in good agreement with the clinical association between increased IGF-I serum levels and increased facial sebum excretion in acne patients [61]. Thus, it is conceivable that a rise in insulin and IGF-I levels by milk consumption stimulates sebocyte proliferation and differentiation resulting in the development and progression of acne.

Endocrine disorders associated with increased IGF-I serum levels and acne

In prepubertal girls with premature adrenarche significantly higher ACTH- stimulated 17-hydroxy-pregnenolone and DHEA serum levels, high IGF-I, and low IGFBP-I have been reported [62]. It is remarkable, that premature pubarche shares many clinical characteristics with PCOS [62]. PCOS is associated with increased serum levels of IGF-I and DHEAS, hyperinsulinemia, insulin resistance, acne and hirsutism [63]. Twofold elevated serum levels of free IGF-I have been detected in women with PCOS [63]. Patients with acromegaly and GH hypersecretion have increased IGF-I serum levels, exhibit greasy skin with increased sebum excretion and often develop acne. Like in PCOS, patients with acromegaly often exhibit insulin resistance and hirsutism as well as increased susceptibility for cancer. An increased risk of prostate cancer has recently been observed in patients with severe acne [64]. It has recently been demonstrated that increased insulin/IGF-1 signaling reduces the nuclear concentration of the transcription factor FoxOl [64a] which has been identified as the key transcription factor of acne pathogenesis [64b]. A nuclear deficiency of FoxOl due to increased growth factor signaling results in increased androgen receptor transactivation, increased follicular keratinocyte proliferation (comedogenesis), increased sebaceous lipogenesis and deterioration of innate immunity with follicular hypercolonization with Propionibacterium acnes. On the other hand, isotretinoin treatment, the most effective anti-acne agent, decreased serum IGF-I levels and has been suggested to increase nuclear levels of FoxOl [64c].

Milk consumption and obesity

IGF-I is required for terminal differentiation of pre-adipocytes into adipocytes [65, 66]. Milk consumption during pregnancy increased infant size and birth weight [29, 30]. Data from the Danish National Birth Cohort (n = 50,117) demonstrate a significant association between increase in birth weight and quantified intakes of protein from dairy products [29]. Umbilical cord serum IGF-I concentrations were higher in LGA newborns compared to AGA and SGA newborns [34]. Umbilical cord serum IGF-I levels were correlated significantly with fat mass of the newborn [34]. Milk intake associated with a rapid early growth rate may be a risk factor for obesity [29, 67]. The capacity of children ^' s serum to stimulate differentiation of pre-adipocytes into adipocytes is correlated with IGF-I and IGFBP3-levels [68, 69]. These observations are supported by clinical data demonstrating high IGF-I serum levels in obese children [70-72]. Obesity is a well known risk factor for the development of diabetes mellitus, arterial hypertension, cardiovascular disease and cancer. The compensatory hyperinsulinaemia that characterizes adolescent obesity chronically suppresses levels of IGFBP-I thereby increasing the bioavailability of free IGF-I [73]. The insulinotropic and IGF-I rising effect of milk and milk protein consumption will have further adverse effects on obesity. Moreover, milk consumption results in whey protein-mediated release of the incretin GIP [25a]. GIP receptors on adipocytes stimulate adipogenesis and anabolic reaction of adipocytes which promote obesity [25c].

Postnatal IGF-I axis, diabetes mellitus and hypertension

The IGF-I axis may be programmed by diet early in infancy [40]. An inverse relation between IGF-I levels during the first months of life and IGF-I levels in adulthood could be observed in 109 infants of the observational Copenhagen cohort study [40]. Low levels of IGF-I in the postnatal period are associated with high IGFl-levels in adolescence. Low levels of IGF-I are reported in SGA newborn infants [34]. Low birth weight is a recognized risk factor for the development of type 2 diabetes and hypertension in adulthood [74, 75]. Furthermore, longitudinal and cross-sectional studies have shown that low birth weight in girls with precocious pubarche are at risk for early onset of puberty and menses and further progress to anovulation, hyperinsulinaemic hyperandrogenism and PCOS [76]. It appears that the IGF-I axis in SGA-low birth weight newborns is drifted to higher IGF-I levels in adulthood promoting the development of diabetes type 2, hypertension and PCOS. Individuals who were SGA at birth may be at high risk in adulthood when consuming milk and dairy products which further increase the imbalanced IGF-I axis to even higher levels. The potent insulinotropic properties of milk and dairy protein induces hyperinsulinaemia and reactive hypoglycaemia similar to high glycaemic load carbohydrates which have been implicated as an underlying cause of certain diseases of insulin resistance [77].

Low birth weight is associated with hypertension in adulthood. The compensatory drift of the IGF-I axis to higher IGF-I levels in individuals with low birth weight and low IGF-I levels to higher IGF-I levels in adulthood may contribute to the development of hypertension. IGF-I receptors are up- regulated by angiotensin II [78]. In hypertensive animals there is an increased IGF-I mRNA and protein expression and IGF-I plasma levels in hypertensive patients have been related to pressure load [79, 80].

Studies in mice showed that underfeeding during the early postnatal period delayed growth, whereas overfeeding accelerated it. In both cases, final body size was permanently altered. Coordinated alterations in pituitary GH, plasma IGF-I and acid labile subunit, and gene expression of hypothalamic GHRH during postnatal development have been observed. These changes were consistent with the observed phenotypes. Alterations in the somatotropic axis persisted throughout adulthood. Although limited to the early postnatal period, both underfeeding and overfeeding led to reduced glucose tolerance later in life. These metabolic abnormalities were in line with defective insulin secretion in restricted mice and insulin resistance in overfed mice. Moreover, both restricted and overfed mice had increased arterial blood pressure, suggestive of vascular impairment. These findings in mice indicate a significant link between early postnatal diet, somatotropic development, and specific late onset diseases in mice. These data suggest that, together with other hormones like leptin, IGF-I may play a role in modulating hypothalamic stimulation of the developing somatotropic function [8Oa].

Milk, insulin, IGF-I and cancer

IGF-I is a known mitogenic hormone that stimulates growth, differentiation and metabolism in a variety of cell types [81]. IGF-I participates in the regulation of the cell cycle, inhibiting the processes of apoptosis and stimulating cell proliferation. IGF-I is a potential tumor promoter [82]. Several studies demonstrated a link between increased IGF-I serum levels with increased risk of breast, prostate, colorectal, and lung cancer [83]. High expression of IGFlRs has been detected in the majority of human cancers. Several studies have confirmed that IGF-I serum levels are related to premenopausal breast density, one of the strongest known breast cancer risk factors believed to represent epithelial and stromal proliferation [84-86]. A higher risk for cervical, ovarian and endometrial cancer is related to high IGF- 1 levels in post- and premenopausal women [87]. Plasma IGF-I levels and inherited variation in IGF-I have been implicated to be a risk factor in prostate carcinoma [88-90]. A high intake of dairy products and calcium has been associated with an increased risk of prostate cancer in a recent meta-analysis [91]. IGF-I and insulin act through the tyrosine kinase growth factor signaling cascade enhancing tumor cell proliferation [92]. Higher serum IGF-I in older men has been associated with increased risk of cancer death, independent of age, adiposity, lifestyle, and cancer history [92a].

Although, there is a large body of evidence that milk consumption increases IGF-I serum levels which are associated with increased risk of various cancers, two review articles came to the conclusion that there is no support for the association between dairy product consumption and risk of breast cancer [93, 94]. ParodP s [93] calculations are based on a IGF-I content in milk of 4 ng/ml, whereas 10-50 ng/ml were reported from other sources [3]. Moreover, the role of bioactive IGF-2 in milk (40-50 ng/ml), which increases IGF-signaling by cross-reaction with the IGFlR has not been considered [93]. Most important is the fact, that despite heat inactivation of growth factors, ultra-heat processed milk and fermented milk products retain the ability to raise human serum IGF-I and insulin levels more than other sources of dietary protein. High incidence rates of cancer are common in Scandinavia where milk protein consumption is very high. A prospective study of 25,892 Norwegian women clearly showed that consumers of 750 ml or more of full-fat milk daily had a relative risk of 2.91 for breast cancer compared with those women who consumed 150 ml or less [95]. The cancer promoting effect of low-fat milk in 7,12-dimethylbenz(A)anthracene-induced mammary tumors has been demonstrated in rats [96]. Commercial milk inhibited the regression of carcinogen-induced mammary tumors in ovariectomized rats [97]. Thus, substantial evidence supports the view that milk consumption raises IGF-I serum levels and promotes cancer.

Milk consumption in pregnancy, birth weight and risk of breast cancer

Milk consumption during pregnancy increases maternal IGF-I serum levels, birth weight and height of the newborn [29, 42, 43], all known risk factors of breast cancer [98, 99]. The intrauterine environment might contribute to the predisposition of women for breast cancer in adulthood [100]. The responsible /77-utero-mechanism has been linked to IGF-I [101]. Thus, the environmental breast cancer-promoting factor of Western societies could be associated with milk-induced IGF-signaling during early pregnancy. A recent study examined the association between IGF-I in infancy and later in life supporting the hypothesis that the IGF-I axis can be programmed early in life [40]. An imbalance in IGF-1-dependent T-cell maturation in the fetal thymus might affect the immune system's capacity to handle anti-tumor mechanisms later in life. Thus, fetal macrosomia could have a fatal outcome later in life. Individuals affected by genetic variations in the expression of IGF-I resulting in high IGF- 1 serum levels are at increased risk for cancer. Absence of the IGFl 19-CA repeat allele has been associated with high IGF-I levels during oral contraceptive use in nulliparous women in four different ethnic groups [102]. The IGFl 19-CA repeat allele modifies IGF-I levels, breast volume and possibly early-onset breast cancer risk after hormone exposure in young high- risk women [103, 104]. Intriguingly, absence of the common IGFl 19 CA- repeat allele is more common among BRCAl mutation carriers than among non-carriers from BRCAl families [105]. As the IGFl -19/-19 affects 6 to 13% of white women, this population subgroup may be especially vulnerable to further IGF-I elevations by milk protein consumption.

Milk, IGF-I and cardiovascular disease

The association between milk consumption and mortality from ischemic heart disease has been suggested in this journal 25 years ago [106]. A linear correlation between the consumption of unfermented milk proteins and male mortality of coronary heart disease has been demonstrated [107]. Animal models have shown that IGF-I is involved in stimulating atherosclerosis [108, 109]. IGFlRs are abundant in vascular smooth muscle cells and factors that stimulate atherosclerosis, such as angiotensin II up-regulate IGFlR expression [HO]. IGF-I secreted by activated monocytes can stimulate smooth muscle cell proliferation and extracellular matrix synthesis which lead to enlargement of the developing atheroma [111]. It is conceivable, that the relative small IGF-I polypeptide diffuses from the plasma into the early atheromatous lesions. In this regard, milk-derived IGF-I augments local IGF-1-dependent atherogenic effects.

IGF-I signaling and neurodegenerative diseases

The major risk factor for the development of neurodegenerative disease is aging [112]. Mechanistic links between the aging process and toxic protein aggregation, a common hallmark of neurodegenerative diseases, has been revealed. Lifespan is regulated by at least three different mechanisms, one of which is the insulin/IGF-1 signaling pathway. The insulin-IGF-1 pathway is the major candidate to link aging, proteotoxicity and late-onset neurodegenerative disease [113, 114]. It has been suggested that reducing insulin-IGF-1 signaling in the brain will enable cells to maintain the activity of protein quality-control mechanisms and clearance capabilities to a later age, thereby postponing the onset of neurodegenerative diseases [113]. Recent insights implicate the interconnection of IGFlR-signaling, regulation of lifespan, neurotrophin signaling and loss of neurogenic capacity and development of Alzheimer disease [115]. Prolonged milk-induced disturbance of the insulin- IGF-1 pathway has to be considered as a possible accelerator of neurodegenerative disorders. Intriguingly, circulating IGF-I is able to cross the blood-brain barrier and enter into the brain. Recent research points to the possibility that the brain is the site where reduced IGF-I signaling can consistently lead to an extended mammalian life span [114].

The IGF-axis and allergic and autoimmune disorders

The thymus is the only organ specialized in the establishment of immunological self-tolerance and stands at the crossroads between the immune and neuroendocrine systems [116]. The neuroendocrine system regulates the process of T-cell differentiation from the very early stages. T lymphocytes undergo in the thymus a complex educative process that establishes central T cell self tolerance of neuroendocrine principle. Neuroendocrine self-antigens correspond to peptide sequences that have been highly conserved throughout the evolution of one given family [116]. With regard to the insulin gene family, all members are expressed in the thymus network according to a precise hierarchy and topography of epithelial cells: IGF-2 (thymic cortex and thymic "nurse" cells) > IGF-I (thymic macrophages) > insulin (medulla) [116]. The blockade of thymic IGF-mediated signaling at the level of IGF ligands or IGFRs interferes with the early stages of T cell differentiation in fetal thymic organ cultures [117]. IGF-I stimulates thymus growth and T cell proliferation and development. Thymocytes (pre-T cells) express IGFRl and IGFR2. A number of data support the existence of a functional IGF-mediated signaling between stromal cells (thymic epithelial cells, macrophages) and immature T cells during their differentiation in the thymus [117]. The majority of T-cell precursors entering the thymus are eliminated by apoptosis, ensuring that only harmless T-cells without autoimmune and allergic potential survive apoptosis in the thymus. IGF-I activates the PI3K-pathway, involved in the activation of cell proliferation and inhibition of apoptosis [1, 82]. Consumption of boiled farm milk during pregnancy was positively associated with increased immunoglobulin E serum levels to cow ^' s milk and other food allergens [118]. The milk-induced maternal increase of the insulin-IGF-1 signaling might shift the insulin-IGF-1 axis in the fetal thymus, thereby damaging proper apoptosis of allergy- and autoimmune-prone T-cells explaining the co-appearance of atopic and autoimmune diseases later in life. Intriguingly, breast-fed humans have significantly lower serum IGF-I levels than those fed on a cow milk based formula [40]. Moreover, the consumption of milk and milk products in pregnancy would be a good explanation for the dominating maternal effect in the transmission of atopic diseases.

Growth Hormone

Growth hormone (somatropin, GH) is a peptide hormone that stimulates growth and cell reproduction. Growth Hormone induces the release of IGF-I and insulin. By its action, growth hormone induces insulin resistance. Therefore, it is suspected to be involved in the dysregulation of insulin and IGF-I during consumption of milk.

Ghrelin:

Ghrelin is a 28 amino acid peptide wherein the Ser-3 residue is n-octanoylated (Nature 402 (1999) 656-660). Ghrelin is predominantly produced in the human stomach. In the anterior pituitary somatotroph cells, ghrelin binds to GH secretagogue receptor, called GHS receptor type Ia. In this acylated form, ghrelin stimulates the secretion of GH. The deacylated peptide is inactive. Octanoyl is only one possible side chain. GH induces insulin resistance in humans and mammals for physiologic growth. Ghrelin has recently been detected in human breast milk, cow milk and goat milk (Aydin S et al. Ghrelin is present in human colostrums, transitional and mature milk. Peptides 27 2006; 878-882). The bovine GHRL gene has recently been characterized (Colinet F et al. Molecular characterization of bovine GHRL gene. Archiv Tierzucht 2009; 52:79-84). Bovine ghrelin is a peptide of 27 amino acids. During peak of lactation, plasma concentrations of bovine ghrelin and GHare greater in samples from high genetic merit Holstein-Friesian cows (Roche JR et al. Short communication : genetic selection for milk production increases plasma ghrelin in dairy cows. J Dairy Sci 2006; 89: 3471-3475). It is most likely that the plasma level of bovine ghrelin determines the level of ghrelin in cow milk. Bovine ghrelin has been detected recently in cow milk (Karatas F et al. Ghrelin and orotic acid increased in subclinical mastitis. Arch Physiol Biochem 2008; 114: 178-182). Formula fed infants had higher serum ghrelin levels (2654.86 versus 2132.96 pg/ml) and higher IGF-I levels (3.73 versus 3.15 ng/ml) in comparison to breast-fed infants (Savino F et al. Ghrelin, leptin and IGF-I levels in breast-fed and formula fed infants in the first years of life. Acta Paediatrica 2005; 94: 531-537). In cow milk consuming countries like the USA, serum ghrelin levels are higher in Caucasian men than Japanese men aged 40 to 49 years. Later are known to have lower daily milk intake (Matsunaga-Irie S et al. Serum ghrelin levels are higher in Caucasian men than Japanese men aged 40-49 years. Diabetes Obesity Metabol 2007; 9 : 591-593).

In this invention, bovine ghrelin is suspected to be the cause of increased human ghrelin levels which increase human pituitary GHsecretion and thus induce insulin resistance with hyperinsulinemia and increased IGF-I serum levels, unwanted adverse effects on human health.

Betacellulin:

Bovine betacellulin is found in substantial amounts in cow milk (1 930 ng/liter) and is quite stable and survives the pasteurization process and is even found in high concentrations in cheese. BTC passes the gut barrier and enters the systemic circulation to promote growth and most likely increases insulin secretion and beta-cell hyperplasia of pancreatic islet cells.

Betacellulin, a member of the epidermal growth factor (EGF) family, was purified from the conditioned medium of a cell line derived from mouse pancreatic beta cell tumors. Its primary translational product is composed of 178 amino acid residues, which contains a signal sequence, transmembrane and cytoplasmic domains in addition to the EGF-like domain.

Mature betacellulin is composed of 80 amino acid residues with extensive glycosylation and has a molecular weight of about 32 kDa. Betacellulin expresses in alpha, beta, and duct cells in normal adult pancreas, and converts rat pancreatic amylase-secreting cells (AR42J cells) to insulin expressing cells together with activin. In addition, PDX-I, one of the transcriptional factors for beta cells, induces insulin gene expression in a TCl cells in the presence of Betacellulin. Betacellulin also has the potential for the growth of a rat insulinoma cell line, INS-I cells, and the recombinant human betacellulin accelerates the improvement of glucose tolerance in mice with diabetes induced by selective alloxan perfusion. According to these observations, Betacellulin is thought to be necessary for differentiation and/or growth of the pancreatic beta cells.

To reduce the capacity of cow ^' s milk and milk products including cheese to promote human beta-cell hyperstimulation and hyperplasia with concomitant increased insulin serum levels and increased IGF-I serum levels, raw milk has to processed in a way that bovine betacellulin is extracted, eliminated or functionally disrupted. This betacellulin-free milk will decrease the risk of human breast cancer and other EGFR-dependent human cancers as well as diabetes type II. Increased permanent stimulation of the pancreatic beta-cells will early lead to impaired function. Betacellulin binds to Erbl and Erb4-receptor homodimers and all Erb receptor heterodimers and thus is an important nutritional stimulus of Erb- dependent carcinoma cells.

Alpha-lactalbumin: The key element

Alpha-lactalbumin is an important whey protein in cow's milk (~1 g/l), and is also present in the milk of many other mammalian species. In primates, alpha-lactalbumin expression is upregulated in response to the hormone prolactin and increases the production of lactose. Alpha-lactalbumin forms the regulatory subunit of the lactose synthase (LS) heterodimer and β-1,4- galactosyltransferase (beta4Gal-Tl) forms the catalytic component. Together, these proteins enable LS to produce lactose by transferring galactose moieties to glucose. As a monomer, alpha-lactalbumin strongly binds calcium and zinc ions and may possess bactericidal or antitumor activity. A folding variant of alpha-lactalbumin, called HAMLET, likely induces apoptosis in tumor and immature cells. When formed into a complex with GaI-Tl, a galactosyltransferase, alpha-lactalbumin enhances the enzyme's affinity for glucose by about 1000 times, and inhibits the ability to polymerize multiple galactose units. This gives rise to a pathway for forming lactose by converting GaI-TI to lactose synthase. The molecular weight is 14178 Da, and the isoelectric point is between 4.2 and 4.5. One of the main structural differences with beta-lactoglobulin is that it does not have any free thiol group that can serve as the starting point for a covalent aggregation reaction. As a result, pure alpha-lactalbumin will not form gels upon denaturation and acidification.

Alpha-lactalbumin is hydrolyzed in the intestine and induces the synthesis of glucose-dependent insulinotropic polypeptide (GIP) (also known as gastric inhibitory polypeptide) in K-cells. This incretrin upregulates insulin synthesis in the pancreatic beta-cells.

Progesterone in milk fat: co-inductor of insulin resistance

Steroid hormones are known to cross the blood-milk barrier. This effect has been recurred to for the diagnosis of the pregnancy of cows by analyzing the progesterone content of their milk (Fritsche S, Steinhart H. Occurrence of hormonally active compounds in food : a review. Eur. Food Res. Technol. 1999; 209: 153-179). Since the steroid hormone progesterone is distributed in the lipid phase, the concentration of progesterone in dairy products depends on their fat content. Thus, progesterone concentrations are found to be 1.4 μg/l in low-fat milk, 10 μg/l in whole milk, 44.2 mg/kg in Gouda, 41.8-72.7 μg/kg in cream and up to 300 μg/kg in butter (Fritsche S, Steinhart H. Occurrence of hormonally active compounds in food : a review. Eur. Food Res. Technol. 1999; 209: 153-179). In a young cow that has not yet had a calf (heifer), from 16.7 to 37.9 μg/kg of progesterone is detected in the fat tissue, whereas from 239 to 336 μg/kg of progesterone was determined in the fat of pregnant cows (Fritsche S, Steinhart H. Occurrence of hormonally active compounds in food : a review. Eur. Food Res. Technol. 1999; 209: 153-179). Thus, pregnant cows have a tenfold progesterone content in their fat.

Since it has to be considered that progesterone continues to be accumulated in the tissue in perpetually pregnant dairy cows of Western dairy farming and passes into the milk, an unphysiologically increased progesterone content of milk as compared to former times is to be assumed from the "industrialized technology of milk recuperation". This could be another unrecognized risk factor of Western life style.

When milk and fat-containing dairy products are ingested in high amounts and preferentially, the high progesterone content of these products allows for a possible daily progesterone intake of from 40 to 50 μg, which reaches the dose range of the progestogen fraction of an ethical oral contraceptive.

In addition to the described insulinotropic load on the human body from the intake of whey proteins, the extremely resistant progesterone of the lipid phase of dairy products adds to the load from steroid hormones. The daily uptake of bovine progesterone must be seen in an overall consideration with the uptake of progestogen-containing oral contraceptives, since the progestogenic effects of nutritive and iatrogenic progestogens are additive.

The expression of insulin receptors and insulin sensitivity changes physiologically during the menstrual cycle of females due to fluctuations of the gonadal steroids (Diamond MP, Simonson DC, De Fronzo RA. Menstrual cyclicity has a profound effect on glucose homeostasis. Fertil Steril 1989; 52: 204-208; Valdes C, Helkind-Hirsch K. Intravenous glucose tolerance test- derived insulin sensitivity changes during the menstrual cycle. J Clin Endocrinol Metab 1991; 72: 642-645). The intake of oral contraceptives and especially of progestogens (desogestrel and etonogestrel, Implanon ^® ) increases insulin resistance (Godsland IF, Walton C, Felton C, proudler A, Patel A, Wynn V. Insulin resistance, secretion, and metabolism in users of oral contraceptives. J Clin Endocrinol Metab 1991; 74: 64-70; Cagnacci A, Ferrari S, Tirelli A, Zanin R, Volpe A. Insulin sensitivity and lipid metabolism with oral contraceptives containing chlormadinone acetate or desogestrel : a randomized trial. Contraception 2009; 79: 111-116; Meyer C, Talbot M, Teede H. Effect of Implanon on insulin resistance in women with polycystic ovary syndrome. Au N Z J Obst Gyn 2005; 45. 155-158). Therefore, it is extremely alarming that no balancing considerations about the potentiating effect of iatrogenically administered and nutritively ingestible progestogens from the consumption of dairy products and eggs have been made to date.

The perpetual pregnancy of dairy cows and oral contraception are biologically similar mechanisms that flood us humans with unphysiological doses of pregnancy hormones and shift our hormone axes towards pregnancy. This could also be a good explanation for the decline of natural fertility in Western industrial nations. Progesterone stimulation from milk, dairy products and oral contraceptives has negative impacts on insulin resistance. In patients suffering from PCOS, it could be shown that the existing insulin resistance deteriorates significantly when the contraceptive etonogestrel (Implanon ^® ) is implanted (Meyer C, Talbot M, Teede H. Effect of Implanon on insulin resistance in women with polycystic ovary syndrome. Au N Z J Obst Gyn 2005; 45. 155- 158). Thus, PCOS patients represent a special risk group, being disposed to developing diabetes mellitus, cardiovascular diseases and cancer (Fϋrstenberger G, Senn H-J. Insulin-like growth factors and cancer. Lancet 2002; 3: 298-302; Druckmann R, Rohr UD. IGF-I in gynaecology and obstetrics: update 2002. Maturitas 2002; 41 (Suppl 1) : S65-S83). Not only increased insulin and IGF-I serum levels are to be expected therefrom, but also a progesterone-induced increase of the autocrine production of GH in peripheral tissues. Thus, it could be shown recently in mammary carcinomas of bitches that progesterone increases the peripheral concentration of GH , which stimulates the local and systemic IGF-I secretion (Cagnacci A, Ferrari S, Tirelli A, Zanin R, Volpe A. Insulin sensitivity and lipid metabolism with oral contraceptives containing chlormadinone acetate or desogestrel : a randomized trial. Contraception 2009; 79: 111-116). This observation illustrates that a molecular cross-talk between the GH/IGF-1 axis and steroid hormones, such as progesterone, exists in mammals (Cagnacci A, Ferrari S, Tirelli A, Zanin R, Volpe A. Insulin sensitivity and lipid metabolism with oral contraceptives containing chlormadinone acetate or desogestrel : a randomized trial. Contraception 2009; 79: 111-116).

Prolactin

Prolactin is a 23-kDa protein hormone that binds to a single-span membrane receptor, a member of the cytokine receptor superfamily, and exerts its action via several interacting signaling pathways. Prolactin is a multifunctional hormone that affects multiple reproductive and metabolic functions and is also involved in tumorigenicity. In addition to being a classical pituitary hormone, prolactin in humans is produced by many tissues throughout the body where it acts as a cytokine [116a]. To meet the increased demand for insulin during pregnancy, the pancreatic islets undergo adaptive changes including enhanced insulin secretion and beta-cell proliferation. These changes peak in mid- pregnancy and return to control levels by parturition.

Lactogens (placental lactogen and/or prolactin) induce this up-regulation and remain elevated throughout gestation [116b]. There is no doubt that prolactin is a most important inducer of postnatal beta-cell proliferation (postnatal beta- cell burst) and insulin secretion.

Intriguingly, colostrum of various species including human and bovine colostrum (500-800 ng/ml) contains very high amounts of prolactin. Cow milk contains high levels of bovine prolactin (6-8 ng/ml) [3]. Human prolactin compared to bovine prolactin exhibits 73.1% protein identity and 80.3% DNA identity. Prolactin is known to be absorbed in the intestine during the postnatal period. However, continuous cow milk consumption in infancy and adulthood may result in increased intestinal prolactin absorption, especially in conditions with disturbed intestinal permeability barrier. High intake of wheat gluten and potato glycoalkaloids which are common in Western diet [6a, 6b, 6c] may increase intestinal absorption of bovine prolactin and other milk-derived growth factors affecting pancreatic beta-cell stimulation, thus promoting the insulinotropic effect of milk consumption. Most importantly, plasma prolactin and growth hormone levels are modified by the type of diet (Ishizuka B et al. Pituitary hormone release in response to food ingestion : evidence for neuroendocrine signals from gut to brain. J Clin Endocrinol Metab 1983;57: 1111). Alpha-lactalbumin is a unique protein of milk which contains high amounts of tryptophane, the precursor of serotonin (5- hydroxytryptamine) which stimulates the secretion of pituitary GH and prolactin (Markus CR. Dietary amino acids and brain serotonin function; implications for stress-related affective changes. Neuromol Med 2008; 10: 247). In this regard, milk and especially whey proteins containing alpha-lactalbumin will increase postprandial prolactin and GH levels which exert adverse mitogenic effects on pancreatic beta-cells (promotion of type 2 diabetes mellitus), on adipocytes (promotion of obesity) and other somatic cells like cancer cells (promotion of cancer).

Discussion and hypothesis

Our "inborn belief" of the beneficial effect of cow's milk in human nutrition is challenged. Humans are the only species on earth allowed to consume milk, an evolutionary designed sophisticated growth-signaling system, lifelong after weaning. Cow's milk consumption and most likely other dairy products have an enormous impact on the human GH/insulin/IGF-1 axis, disturbing most sensitive hormonal regulatory signaling networks, interfering with IGFlR- signalling from fetal life to senescence (Figure 3). The presented hypothesis elucidates that man-made manipulation of the human insulin/IGF-1 axis is involved in the development of most chronic diseases of Western societies (Table 1).

Table 1

Potential hazards of milk protein consumption due to enhanced insulin/IGF- l/IGF-2 signaling

For the first time, apparently unrelated etiopathological processes in disease development could be related to disturbed insulin/IGF-1 signaling which is most critical for the early programming of various systems in the human organism. The prenatal and postnatal period may be the most sensitive periods for milk-induced disease development. This hypothesis explains most chronic diseases of Western societies on the basis of over-stimulated, proliferative responses and reduced inhibition of apoptotic mechanisms by IGF-I. Cow milk protein consumption has been identified as the basic environmental factor promoting a permanent shift of the insulin/IGF-1 axis to higher levels which are inadequate for humans. The ease of access of milk protein products and their extended distribution in Western nutrition leads to a permanent manipulation of an intrinsic and most sensitive hormonal signaling system in man. Cow milk and cow milk protein consumption with its high insulin and IGF-I stimulatory effects has to be regarded as a violation of a physiological principal in mammalian nutrition developed during the eons of mammalian evolution. The short-sighted view on the beneficial effects of milk consumption on bone formation and bone mineralization ignores already well documented facts of harmful disease- and cancer promoting effects of milk protein consumption. It is a principle of science to propose a hypothesis to understand a phenomenon. The simplicity of a hypothesis and the possibility to deduce explanations for various facts increases its degree of probability. This scientific principle can be applied to insulin/IGF-1 signaling in various cell systems. According to the presented hypothesis cow milk consumption has to be regarded as a health hazard for humans which afford immediate intervention. It is of special concern, that pregnancy and the postnatal period are most sensitive programming periods in human life which should not be manipulated by an evolutionary developed growth-stimulating system of another mammalian species imposed on the human IGF-I axis. Consequently, gynecologists should not advise pregnant women to consume milk during pregnancy. The hormonal changes during the first months of intrauterine and postnatal life may affect adult health outcomes predisposing to cancer, allergies and other diseases. Dermatologists and pediatricians should recommend to reduce milk consumption in patients with acne. In this context, persistent acne in adulthood with increased IGF-I serum levels should be considered as an indicator disease of increased cancer risk like in PCOS and acromegaly. Pediatricians and general practitioners should consider milk restriction in patients with obesity to reduce the synergistic insulinotropic effects of milk- and hyperglycemic carbohydrate enriched diets. Oncologists should advise their cancer patient families to refrain from milk and milk protein consumption. Especially, persons with already increased IGF-I serum levels and those with genetic variations with elevated IGF-1/IGFlR signaling should refrain from milk consumption. Caution is also necessary in patients with a familial risk of atherosclerosis as well as neurodegenerative diseases which might be postponed in onset by a reduction of milk- and milk protein- intake. It is most important to evaluate safe limits for the daily consumption of milk proteins. A better understanding of the mechanism of the insulinotropic and IGF-I raising effects of milk proteins might lead to targeted enzymatic or biophysical destruction of these adverse mitogenic and antiapoptotic effects.

Currently, Asian populations increase their milk consumption and the Dietary Guidelines for Americans 2005 recommend that Americans should increase their intake of dairy products. Industrial and marketing efforts are undertaken to increase the amount of whey protein intake. Furthermore, it is proposed to increase the content of alpha-lactalbumin in infant formula. An urgent global multidisciplinary approach is necessary to study the adverse effects of chronically over-stimulated insulin-IGF-1-signaling induced by milk and dairy product consumption in humans on a basis of controlled and randomized open studies. The presented epidemiological, biochemical, clinical and circumstantial evidence supports the presented hypothesis that cow's milk consumption is a major health hazard and should be recognized as a promoter of most common chronic diseases of industrialized countries (Figure 3). Long-term reduction in milk and dairy product consumption could have an enormous impact on disease programming, mortality, onset of chronic diseases and costs for our health care systems. As milk consumption is a major component of Western countries, a restriction of milk and milk product consumption will be difficult to achieve.

Objective of the Invention

It is the object of the present invention to overcome the drawbacks of prior art, especially by reduction of the insulinotropic effects of milk. The solution to the health hazards of milk is the modification/attenuation of milk ^' s insulinotropic and mitogenic properties.

A measure of the insulinotropic effect of milk is the insulinemic index of milk. The insulinemic index of milk ranges between 90-148 [Ostman EM, LiIj berg Elmstahl HGM et al. : Inconsistency between glycemic and insulinemic reponses to regular and fermented milk products. Am J Clin Nutr 2001; 74:96- 100; Hoyt G, Hickey MS, Cordain L. Dissociation of the glycaemic and insulinaemic responses to whole and skimmed milk. Br J Nutr 2005; 93: 175- 177; Holt S, Brand Miller J, Petocz P. An insulin index of foods: the insulin demand generated by 1000-kJ portions of common foods. Am J Clin Nutr 1997; 66: 1264-1276]. Major contributors to milk ^' s high insulinemic index are the GIP secretion induced by whey proteins, especially alpha-lactalbumin, the growth factors of the whey protein fraction, especially bovine prolactin and betacellulin and the IGF-I which is transported in the casein fraction of milk.

One embodiment of the invention is a process for treating milk comprising the steps of

- treating casein to reduce the ability of casein to induce IGF-I in a human being upon consumption together with

- treating whey proteins to reduce the ability of whey proteins to induce insulin in a human being upon consumption or

- treating milk to reduce the ability of milk to induce GH in a human being upon consumption or

- treating milk to reduce the content or activity of alpha-lactalbumin

- treating milk to reduce the content or activity of ghrelin or

- treating milk to reduce the content or activity of betacellulin

- treating milk to reduce the content or activity of prolactin.

In other words, the method of the invention comprises the reduction of the content or activity of at least one of alpha-lactalbumin, ghrelin, betacellulin or prolactin. The major focus of the invention is the reduction of alpha- lactalbumin as a most important precursor for pituitary GH and prolactin secretion as well as intestinal incretin formation (GIP, GLP-I and cholecystokinin)

The method of the invention is also a method of treating milk to reduce the ability of milk to induce GH and prolactin in a human being upon consumption. A further embodiment is a method of treating casein to reduce the ability of casein to induce IGF-I in a human being upon consumption together with treating whey proteins to reduce the ability of whey proteins to induce insulin in a human being upon consumption.

In one embodiment milk is adjusted to have an insulinemic index between 40 and 80. The insulinemic index is defined according to Holt S, Brand Miller J, Petocz P. An insulin index of foods: the insulin demand generated by 1000-kJ portions of common foods. Am J Clin Nutr 1997; 66: 1264-1276]

In a preferred embodiment, the insulinemic index is in the range of 40 to 50.

Preferably the process comprises the step of a hydrolytic treatment. Preferably the hydrolytic treatment is conducted until the ability of casein to induce IGF-I and/or the ability of whey proteins to induce insulin and/or the ability of milk to induce growth hormone and/or the content or activity of ghrelin or betacellulin or alpha-lactalbumin is reduced.

This can be tested by administering to persons an amount of untreated milk and an amount of treated milk according to the process of the invention and measure the amount of IGF-I, insulin, growth hormone, ghrelin or betacellulin in the person.

The hydrolytic treatment can be conducted using one or more proteases, preferably from microorganisms, bacteria or fungi.

Other methods useful in this process comprise chromatographic treatments, treatments with absorptive materials, filtration and/or heat treatment steps.

One very preferred treatment is directed against content or activity of ghrelin and/or betacellulin. The treatment either comprises a hydrolytic treatment to hydrolyze the peptide structure of ghrelin and/or betacellulin.

A further possibility to reduce the activity of bovine ghrelin would be the addition of an anti-ghrelin Spiegelmer (Kobelt P et al. Anti-ghrelin Spiegelmer NOX-BI l inhibits neurostimulatory and orexigenic effects of peripheral ghrelin in rats. Gut 2006; 55:788-792; Shearman LP et al. Ghrelin neutralization by a ribonucleic acid-SPM ameliorates obesity in diet-induced obese mice. Endocrinology 2006; 147: 1517-1526). This would also work with betacellulin Spiegelmer.

A further possibility is the specific inactivation of ghrelin by hydrolyses of the acyl group on Ser-3.

A further possibility is the removal of ghrelin and/or betacellulin by absorption, especially immunoabsorption.

Filtration can for example be used to separate soluble whey proteins from casein proteins which have a higher molecular weight.

Absorptive material may be used to bind materials from the milk, especially when used in form of an affinity treatment. For removal of alpha-lactalbumin, affinity adsorption is suitable. As a coating material, galactose or galactosyltransferase are useful. They could be coated on beads, surfaces or the like.

In one embodiment, alpha-lactalbumin is denatured by high-pressure treatment as described in Huppertz T, Fox PF, de Kruif KG, Kelly AL. High- pressure induce changes in bovine milk proteins: a review. Biochim Biophys Acta 2006; 1764: 593-598.

A method to increase the content of alpha-lactalbumin is described in Kiesner C et al. (2000) Manufacturing of α-lactalbumin-enriched whey systems by selective thermal treatment in combination with membrane processes. Lait 80: 99-111. In a modified way, this method can also be used to reduce the content of alpha-lactalbumin.

For the removal of prolactin affinity binding procedures are applied. Biotechnologically produced prolactin binding proteins are fixed to adsorber systems or beads to remove free prolactin and bovine lactogen of milk.

Also a prolonged heat treatment of the milk assists in reducing the insulinotropic effects.

A further embodiment of the invention additionally comprises a step to reduce bovine IGF-I in milk.

Based on the understanding that there are at least eight major signalling pathways for the insulinotropic effect of milk: (the induction of insulin secretion by whey proteins, especially alpha-lactalbumin and prolactin, induction of growth hormone secretion by ghrelin and GIP, the insulinotropic EGFR-mediated effect of betacellulin, upregulation of serum IGF-I by the casein fraction of milk, and milk progesterone and androgen precursors in the fatty fraction of milk). A person skilled in the art is able to modify the milk to adjust the respective levels of insulinotropic factors in a way that milk or milk- derived products, especially the alpha-lactalbumin amount of the whey protein fraction for the production of infant formula will exhibit an insulinemic index in the comparable range of the insulinemic index of human breast milk. The produced future whey fraction will be adjusted in a way that infant formula feeding to a human newborn will exert postprandial insulin, glucose-dependent insulinotropic polypeptide and IGF-I responses comparable to the postprandial and long-term responses of physiologic breast-feeding.

A further embodiment of the invention is milk or a product from milk wherein the milk has been treated according to the process of the invention.

A further embodiment is a milk or a product from milk, wherein

- casein is modified to reduce the ability of casein to induce IGF-I in a human being upon consumption and

- whey proteins are modified to reduce the ability of whey proteins to induce insulin in a human being upon consumption or

- milk is modified to reduce the ability of milk to induce growth hormone in a human being upon consumption or - milk is modified to reduce the content or activity of alpha-lactalbumin

- milk is modified to reduce the content or activity of ghrelin or

- milk is modified to reduce the content or activity of betacellulin

- milk is modified to reduce the content or activity of prolactin.

In the process for the modification of milk of the present invention the whey protein fraction is preferentially modified especially alpha-lactalbumin.

A further embodiment of the invention is an transgenic animal having genetic modifications to produce milk with

- modified casein genes to reduce the ability of casein to induce IGF-I in a human being upon consumption of the milk and/or

- modified whey protein genes to reduce the ability of whey to induce insulin in a human being upon consumption of the milk or

- reduced ability to induce growth hormone in a human being upon consumption or

- reduced content or activity of alpha-lactalbumin

- reduced content or activity of ghrelin or

- reduced content or activity of betacellulin

- reduced content or activity of prolactin.

In a further embodiment the whole growth hormone containing basic whey fraction (with IGF-I, IGF-I, PDGF, FGF, TGFbeta, betacellulin, prolactin and alpha-lactalbumin) is removed from pasteurized raw milk. One possible method follows the technique of LACTERMIN production by ultrafiltration and column chromatography described in Dyer et al. Food and Chemical Toxicology 46 (2008), 1659-1665. The combination of ion exchange chromatography and micro- and ultrafiltration of milk is appropriate.

The use of the treated milk of the invention is especially useful for the preparation of milk products, especially in the field of baby food and infant formula.

In this case it may be useful to keep part of the insulinotropic effect; i.e. reduce to a level comparable to human milk.

The milk of the invention is further useful for the preparation of a product for the treatment or prevention of disease selected from diabetes type II and obesity.

Less insulinotropic and/or less mitogenic milk will be of advantage for cancer patients and patients with increased cancer risk, especially IGF-R dependent cancers, especially Erb expressing cancers like breast cancer and prostate cancer and prolactin-driven cancers.

The milk of the invention with reduced insulinemic index is useful for adolescents to prevent and treat acne and other hyperproliferative diseases like psoriasis.

Figure legends

Figure 1 shows IGF-I, IGF-2 and insulin signal transduction and receptor cross-reactivity Figure 2 shows insulinotropic and IGF-I inducing effect of milk protein and milk protein fractions

Figure 3 shows synopsis of milk and milk protein induced disturbances of insulin/IGF-1 signaling from fetal life to senescence and associated chronic diseases of Westernized societies.

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Abbreviations

ACTH : adrenocorticotropic hormone

AGA: appropriate for gestational age

DHEAS: dehydroepiandrosterone sulfate

DHT: dihydrotestosterone

GH : growth hormone

GHR: growth hormone receptor

IGF: insulin-like growth factor

IGFBP: IGF binding protein

IGFlR: IGF-I receptor

IGF2R: IGF- 2 receptor

IR: insulin receptor

LH : luteinizing hormone

LGA: large for gestational age

MAPK: mitogen activated protein kinase

PCOS: polycystic ovary syndrome

PI3K: phosphoinositide-3-kinase

SGA: small for gestational age

SREBP: sterol response element binding protein