Location: Children's Nutrition Research Center
2021 Annual Report
Objectives
Objective 1. In wild-type animals and transgenic animals lacking a functional Connexin43 gap junction within the adipocyte, characterize the effects of Connexin43 deletion on (1) the quantity of the milk a dam produces; (2) the composition of the milk, especially the lipid composition; and (3) other non-nutritional components in the milk.
Objective 2. Characterize the effects of a dam's adipocyte Connexin43 gap junction ablation on her offspring's body weight, adipose tissue mass, and late-life responses to metabolic challenges such as overnutrition.
Approach
Adipocytes, the primary cell type in the non-lactating breast, display a drastic morphological change during the lactation in rodent studies: lipid-filled cells undergo lipolysis to provide fatty acids for triglyceride synthesis and also as an energy source to support milk production; they come back as lipid-laden cells once the animal is done lactating. Our objective is to assess whether breast-milk composition can be regulated by maternal adipose tissue physiology – more specifically, adipose tissue gap junction. The results of this research can be leveraged to improve milk quantity and quality to benefit infant growth and future metabolic status. The central hypothesis is that adipose tissue responds to metabolic and hormonal cues to support milk production and that the Connexin43 gap junction is required to facilitate this process by coupling a group of adipocytes together. Genetic manipulation of adipose tissue Connexin43 will be used to determine the effects of Connexin43 deletion on milk production and milk composition as well as on the offspring's metabolic health.
Progress Report
We proposed to determine the quantity and quality of breast milk produced in wild-type animals and transgenic animals lacking a cellular structure that allows intercellular communications between two fat cells. Connexin43 (Cx43) protein constitutes gap junctions on cell membranes to allow two cells to exchange substances and information. Last year we successfully obtained the animal protocol for the proposed experiment and established breeders for experimental mice. During Fiscal Year 2021, using control and adipocyte Connexin43 knockout (Cx43 KO) female mice generated last year, we set up breeding to measure their capability in breast milk production, the composition of their milk, and the support of neonatal growth. Deletion of the Cx43 protein was achieved by adding 200 mg/kg of a specific antibiotic called doxycycline into the rodent diet. Control mice were treated with the antibiotic doxycycline as well to exclude any confounding effects doxycycline might have on breast milk production and breast milk composition. Those mice were impregnated by wild type male mice. Each of the study dams was given the same number of weight-normalized litters of one-day-old, C57BL/6J wild type pups, generated from separate C57BL/6J wild type mice breeding cages. Breast milk was extracted from those dams 9 days later after a 4-hour separation of the pups to allow milk to accumulate, and the weight of the pups was also measured. Macronutrients of the breastmilk were determined by measuring total protein levels, lactose concentration, and lipid abundance. In addition, other metabolic-relevant hormones and adipocytokines were determined using commercial assay kits. Our data showed no significant difference in the quantity of milk produced by control and adipocyte Cx43 KO mice dams through these experiments. However, lactose levels in breast milk were significantly lower from the Cx43 KO dams. And pups from Cx43 KO dams weighed 6.2% lower on Day 9, suggesting breast milk from Cx43 KO dams was less efficient in supporting neonatal growth. Other factors were not different in breast milk from the Cx43 KO mice dams compared to littermate control dams.
Accomplishments
1. Adipocyte Connexin43 gap junctions mediate breast milk nutritional composition. Nutritious breast milk is vital to support newborn growth and development. The mother's adipose tissue undergoes drastic morphological changes during lactation to support breast milk production. A specific structure in fat cells called Connexin43 gap junctions (Cx43 GJs) plays an important role in facilitating intercellular communication in adipose tissue however, whether Cx43 GJs are vital in adipose tissue remolding and breast milk production is unknown. Research scientists in Houston, Texas, conducted studies to measure breast milk production and quantify macronutrients in breast milk from mice lacking the Cx43 GJs. They found that mothers lacking the functional adipocyte Cx43 GJs produced breast milk with reduced levels of lactose, a vital sugar found in milk and important for supporting neonatal growth. Because many food components can modify adipose tissue Cx43 GJs levels, these data may support the idea of altering the lactating mother's diet to increase adipocyte Cx43 GJs.
