Human milk also contains proteins that serve the immune system either due to antimicrobial properties (e.g. immunoglobulins, lysosyme, haptocorrin, lactoperoxidase, α-lactalbumin) and proteins that provide essential components for the immune system such as various cytokines (e.g. interleukin (IL) 1β, IL 6, IL 8, IL 10, TNF-α, TGF-β).

Role of insulin in gut maturation

In this symposium, we elected to describe the story of one such bioactive peptide, the hormone insulin; a hormone that appears to be essential for gut development, posses anti-inflammatory properties and is present in human milk but is absent in infant formulas².

The insulin content of mature human milk is about 50 IU/ml and the level in colostrum is approximately 10 times higher. We have demonstrated that human milk insulin concentrations are similar in premature and full term infants regardless of gestational age³. Moreover, the insulin content of human milk remains unchanged even after more than one year of lactation⁴.

Radioligand binding experiments have documented the presence of insulin receptors in fetal, suckling, weaning and adult mammal enterocytes⁵. It is not surprising, therefore, that in vitro and in vivo studies in animal models have documented the ability of orally administered insulin to enhance gastrointestinal tract maturation and function². In preterm infants, enteral administration of insulin increased lactase activity, decreased gastric residuals and shortened the time to full feeds⁶.

Anti-inflammatory properties of insulin

In recent years insulin has been shown to have anti-inflammatory properties⁷. To begin with, glucose is a known pro-inflammatory agent and thus insulin anti-inflammatory properties are partially mediated by reducing glucose blood levels. In addition, insulin suppresses major proinflammatory transcription factors such as NF-kB, activator protein–1 (AP-1), and early growth response–1 (EGR-1).

Genes regulated by these transcription factors (such as monocyte chemoattractant protein 1, ICAM-1, and plasminogen activator inhibitor–1 (PAI-1) are also suppressed by insulin. Also, insulin suppresses reactive oxygen species generation and inhibits the generation of superoxide radicals, consistent with the potent antioxidant effect of insulin^7,8.

Recently, we demonstrated that oral insulin supplementation in the post weaning period reduces glucose and lipid blood levels in rats and mice (reviewed in ref. 2). These findings and the antioxidative effects of insulin prompted us to explore whether oral insulin supplementation in the post weaning period may attenuate the atherosclerotic process. In that study⁸, one-month-old male apo E knock out (E0 KO) mice were orally supplemented with human insulin (0.1, 0.5 and 1U/mL) or placebo, for 3 months.

At the end of the study, lipid peroxide serum levels were 18% lower (p<0.01), and serum paraoxonase activity was 30% higher (p<0.01) in mice supplemented with 1 U/mL insulin compared to controls. Insulin reduced mouse peritoneal macrophages lipid peroxide content and superoxide anion release by up to 44% and 62%, respectively (p<0.01). Most importantly, insulin reduced lesion size by 22%-37% (p<0.05) in all study groups (figure 1), suggesting that in E0-mice, oral insulin supplementation attenuates the atherosclerotic process⁸

We explored the effects of oral insulin supplementation in ischemia-reperfusion rat model⁹ and found that administration of oral insulin does not prevent ischemic damage but accelerated intestinal recovery, enhanced enterocyte proliferation and decreased cell death via apoptosis following the ischemia-reperfusion event. In that context, it is important to note that in humans suffering from myocardial infarction, the intravenous administration of insulin attenuated the increase in C-reactive protein blood levels as well as plasminogen activator inhibitor-1 (PAI-1) and of P47 phox (a key component of NADPH oxidase), but was unable to change the extent of the myocardial damage¹⁰.

Figure 1. Effects of oral insulin supplementation to E0 Mice on atherosclerotic lesion area and the number of lesions.


Insulin and intestinal mucosal integrity

Finally, we wanted to find out whether oral insulin supplementation may attenuate the intestinal damage following lipopolysaccharide (LPS) induced endotoxemia in a rat model. In that study, the addition of insulin to the drinking water (1U/ml) of rats treated with LPS (given I.P. at a dose of 10 mg/kg every 24 hours) did not prevent intestinal mucosal injury caused by LPS, but it enhanced intestinal mucosal recovery following LPS administration. Interestingly, oral insulin enhanced the up-regulating effect of LPS on Toll-like receptor (TLR) 4 expression in the proximal intestine, suggesting that TLR4 is not a major mediator of injury in this model (Sukhotnik I and Shamir R. Unpublished data).

References

1. Lonnerdal B. Nutritional and physiologic significance of human milk proteins. Am J Clin Nutr 2003;77(suppl): 1537S-43S.
2. Shehadeh N, Sukhotnik I, Shamir R. The gastrointestinal tract as a target organ for orally administered insulin. J Pediatr Gastroenterol Nutr 2006;43:276-81.
3. Shehadeh N, Khaesh-Goldberg E, Shamir R, et al. Insulin concentration in human milk: Postpartum changes and effect of gestational age. Arch Dis Child Fetal Neonatal Ed. 2003;88:F214-6.
4. Shehadeh N, Aslih N, Shihab S, et al. Human milk beyond one year post-partum: Lower content of protein, calcium and saturated very long chain fatty acids. J Pediatr 2006;148:122-4.
5. Fernandez-Moreno M, Fernandez-Gonzalez M, Diaz-Juarez J, et al. Interaction of insulin with small intestinal epithelial cells from developing rats. Biol Neonate 1988;54:289-93.
6. Shulman RJ. Effect of enteral administration of insulin on intestinal development and feeding tolerance in preterm infants: a pilot study. Arch. Dis. Child. Fetal Neonatal Ed. 2002;86:F131-3.
7. Dandona P, Mohanty P, Chaudhuri A, et al. Insulin infusion in acute illness. J Clin Invest. 2005;115:2069-72. 8. Shamir R, Shehadeh N, Rosenblat M, et al. Oral Insulin Supplementation Attenuates Atherosclerosis Progression in Apolipoprotein E-Deficient Mice. Arterioscler Thromb Vasc Biol 2003;23:104-10.
9. Sukhotnik I, Shehadeh N, Mogilner J, et al. Beneficial effects of oral insulin on intestinal recovery following ischemia-reperfusion injury in rat. J Surg Res. 2005;128:108-13.
10. Ghanim H, Aljada A, Hofmeyer D, et al. Circulating Mononuclear Cells in the Obese are in a Proinflammatory State. Circulation 2004;110:1564-71.
11. Shehadeh N, Shamir R, Berant M, et al. Insulin in human milk and the prevention of type 1 diabetes. Pediatric Diabetes 2001;2:175-7.
12. Meddings JB, Jarand J, Urbanski SJ, et al. Increased gastrointestinal permeability is an early lesion in the spontaneously diabetic BB rat. Am J Physiol Gastrointest Liver Physiol 1999; 39:G951-57.
13. Harada E, Syuto B. Precocious cessation of intestinal macromolecular transmission and sucrase development induced by insulin in adrenalectomized sucking rat. Comp Biochem Physiol A 1991; 99:327-31.
14. Wegmann DR, Eisenbarth GS. It’s insulin. J Autoimmun 2000; 15:286-91.