Beyond ingredients: Supramolecular structure of lipid droplets in infant formula affects metabolic and brain function in mouse models

Abstract

Human milk beneficially affects infant growth and brain development. The supramolecular structure of lipid globules in human milk i.e., large lipid globules covered by the milk fat globule membrane, is believed to contribute to this effect, in addition to the supply of functional ingredients. Three preclinical (mouse) experiments were performed to study the effects of infant formula mimicking the supramolecular structure of human milk lipid globules on brain and metabolic health outcomes. From postnatal day 16 to 42, mouse offspring were exposed to a diet containing infant formula with large, phospholipid-coated lipid droplets (structure, STR) or infant formula with the same ingredients but lacking the unique structural properties as observed in human milk (ingredient, ING). Subsequently, in Study 1, the fatty acid composition in liver and brain membranes was measured, and expression of hippocampal molecular markers were analyzed. In Study 2 and 3 adult (Western-style diet-induced) body fat accumulation and cognitive function were evaluated. Animals exposed to STR compared to ING showed improved omega-3 fatty acid accumulation in liver and brain, and higher expression of brain myelin-associated glycoprotein. Early exposure to STR reduced fat mass accumulation in adulthood; the effect was more pronounced in animals exposed to a Western-style diet. Additionally, mice exposed to STR demonstrated better memory performance later in life. In conclusion, early life exposure to infant formula containing large, phospholipid-coated lipid droplets, that are closer to the supramolecular structure of lipid globules in human milk, positively affects adult brain and metabolic health outcomes in pre-clinical animal models.

Fig 1. Schematic overview of lipid globules.

Schematic overview of lipid globules with tri-layer of MFGM in human milk (A), lipid droplet with MFGM fragments at the droplet interface (B; Nuturis), lipid droplet in standard infant formula with MGFM dry blended (C) and standard infant formula without MGFM but with milk proteins at the droplet interface (D). Visual representation of the size and ratios of different MFGM and milk protein molecules does not fully reflect reality.

Fig 2

Transmission electron microscopy (TEM) images of experimental diets STR (A) and ING (B). A1-3: Large sized fat droplets displaying a thin interface mainly, indicating the presence of phospholipids. A2-3: a close-up on the interface of a lipid droplet with few associated casein micelles. B1: Small sized lipid droplets aggregated by proteins with MFGM fragments in the aqueous phase (B2). B3: A close-up on clustering of lipid droplets and their interfaces composed of protein. Solid arrows point to large casein/protein aggregates, clustering lipid droplets; dashed arrows point to interfacial proteins; dotted arrows point to MFGM fragments or vesicles in the aqueous phase. Sample preparation and TEM imaging was performed as previously described [45].

Fig 3. Relative mRNA expression of MAG, SYP and Iba 1 in hippocampus of mice exposed to experimental diets following previous maternal n-3 deficiency.

Relative mRNA expression in hippocampus of mice exposed to experimental diet (P16-42) with added phospholipid ingredient (ING, n = 4 litters [12 mice]) and experimental diet with adapted supramolecular lipid structure (STR1, n = 5 litters [15 mice]). MAG, myelin-associated glycoprotein, Syp, Synaptophysin; Iba 1, ionized calcium-binding adaptor molecule 1; ING1, group receiving diet with altered ingredient (Study 1, egg phospholipids); STR, group receiving diet with altered structure. Data are means ± SEM; * significant difference between ING and STR group (p < 0.05).

Fig 4. Body weight and body composition development during WSD challenge (week 6–14) of mice that had previously been exposed to experimental diets.

(A) body weight, (B) lean mass, (C) fat mass and (D) relative fat mass of adult mice on Western-style diet (weeks 6–14) that were previously exposed (postnatal day 16–42) to experimental diet with added phospholipid ingredient (ING2, n = 12) or experimental diet with adapted supramolecular lipid structure (STR2, n = 12). A group of mice that were raised on a neutral diet and exposed to low fat AIN-93-M were monitored and included as a reference for body weight and body composition development under non-challenged conditions (REF2, n = 12). ING2, group receiving diet with altered ingredient (Study 2, milk fat globule membrane); STR2, group receiving diet with altered structure; REF2, reference diet; P, postnatal day. Data are means ± SEM; *significant difference between ING and STR group (p < 0.05).

Fig 5. Body weight and body composition at day 98.

(A) body weight, (B) lean body mass (C) fat mass (D) relative fat mass of adult male mice that had previously been exposed (postnatal day P16-42) to experimental diet with added ingredient (ING3, n = 10) or experimental diet with adapted structure (STR3, n = 12). ING3, group receiving diet with altered ingredient (Study 3, milk fat globule membrane); STR3, group receiving diet with altered structure; REF3, reference diet; P, postnatal day. Data are means ± SEM; * = significant difference between ING3 and STR3 (p < 0.05).

Fig 6. Exploration behavior in the open field test and memory performance in novel object recognition test.

(A) total distance moved in open field, (B) relative time spent in center of the open field and (C) novel object recognition index of adult that had previously been exposed (postnatal day 16–42) to experimental diet with added ingredient (ING3, n = 10) or experimental diet with adapted structure (STR3, n = 12). ING3, group receiving diet with altered ingredient (Study 3, milk fat globule membrane); STR3, group receiving diet with altered structure. Data are means ± SEM; * = significant difference between ING3 and STR3 (p < 0.05).