A2 and A1B in vitro milk digests: effects on in vitro leaky gut model and adipose cells

Abstract Obesity is associated with chronic low-grade systemic inflammation of adipose tissue and is often linked to intestinal epithelial barrier (IEB) dysfunction. The present study aimed to evaluate the effects of in vitro gastrointestinal digests of bovine milk containing A1B or A2 β-casein variants on leaky IEB and adipocyte inflammation. Digests of A1B (DA1B) and A2 (DA2) milk were administered to an in vitro Caco-2/HT-29 intestinal cell co-culture mimicking a leaky gut. Intestinal absorbed fractions derived from A1B (MA1B) and A2 (MA2) were administered to hMADS adipocytes. DA1B and DA2 did not modify intestinal permeability, either in the absence or the presence of inflammation. DA1B reduced Claudin-1 mRNA, as well as zonula occludens-1 mRNA and protein expression. Both DA1B and DA2 increased interleukin-8 expression, but only DA1B increased tumor necrosis factor-α. In human adipocytes, MA1B, and to a lesser extent MA2, increased the expression of pro-inflammatory markers monocyte chemoattractant protein-1 and interleukin-6, while reducing adiponectin levels. DA2 preserved in vitro leaky IEB integrity and exhibited a lower inflammatory potential in both leaky gut and adipocytes compared to DA1B. This study is the first to establish a link among A2 milk, leaky gut syndrome, and obesity. Competing Interest Statement The authors have declared no competing interest. Footnotes ↵§ Co-first Authors j.perugini{at}univpm.it (J.P.), e.scopini{at}pm.univpm.it (E.S.), c.galli{at}univpm.it (C.G.), a.giordano{at}univpm.it (A.G.), paola.bendinelli{at}unimi.it (P.B.), stefano.catta-neo{at}unimi.it (S.C.), ivano.denoni{at}unimi.it (I.D.N.), valentina.bonfatti{at}unipd.it (V.B.), a.finco{at}univpm.it (A.F.), saverio.cinti{at}icloud.com (S.C.) This time, the special characters were implemented correctly in the abstract, allowing the proper display of Greek letters, italics, and other formatting elements. Abbreviations - A1B - milk milk containing β-CN A1 and B variants - A2 - milk milk containing β-CN A2 variant - β-CN - β-casein - BCM-7 - β-casomorphin-7 - DA1B - A1B in vitro milk digest - DA2 - A2 in vitro milk digest - FA - fatty acid - hMADS - human multipotent adipose-derived stem cells - IEB - intestinal epithelial barrier - LY - lucifer yellow - MA1B - digested and absorbed fractions derived from A1B milk digest - MA2 - digested and absorbed fractions derived from A2 milk digest - SCC - somatic cell count - SGID - static gastrointestinal digestion - TEER - transepithelial electrical resistance