Mahmoud Mohammad

Transcriptome profiling of microRNA by Next-Gen deep sequencing reveals known and novel miRNA species in the lipid fraction of human breast milk.

While breast milk has unique health advantages for infants, the mechanisms by which it regulates the physiology of newborns are incompletely understood. miRNAs have been described as functioning transcellularly, and have been previously isolated in cell-free and exosomal form from bodily liquids (serum, saliva, urine) and tissues, including mammary tissue. We hypothesized that breast milk in general, and milk fat globules in particular, contain significant numbers of known and limited novel miRNA species detectable with massively parallel sequencing. Extracted RNA from lactating mothers before and following short-term treatment with recombinant human growth hormone (rhGH) was smRNA-enriched. smRNA-Seq was performed to generate 124,110,646 36-nt reads. Of these, 31,102,927 (25%) exactly matched known human miRNAs; with relaxing of stringency, 74,716,151 (60%) matched known miRNAs including 308 of the 1018 (29%) mature miRNAs (miRBase 16.0). These miRNAs are predicted to target 9074 genes; the 10 most abundant of these predicted to target 2691 genes with enrichment for transcriptional regulation of metabolic and immune responses. We identified 21 putative novel miRNAs, of which 12 were confirmed in a large validation set that included cohorts of lactating women consuming enriched diets. Of particular interest, we observed that expression of several novel miRNAs were altered by the perturbed maternal diet, notably following a high-fat intake (p<0.05). Our findings suggest that known and novel miRNAs are enriched in breast milk fat globules, and expression of several novel miRNA species is regulated by maternal diet. Based on robust pathway mapping, our data supports the notion that these maternally secreted miRNAs (stable in the milk fat globules) play a regulatory role in the infant and account in part for the health benefits of breast milk. We further speculate that regulation of these miRNA by a high fat maternal diet enables modulation of fetal metabolism to accommodate significant dietary challenges.

Effect of dietary macronutrient composition under moderate hypocaloric intake on maternal adaptation during lactation

BACKGROUND No evidence-based recommendations exist concerning what dietary macronutrient composition optimizes weight loss during lactation while maintaining milk production. OBJECTIVES The study was designed to test the following hypotheses: compared with a reduced-calorie, high-carbohydrate (H-CHO) diet, an isonitrogenous, isocaloric high-fat (H-F) diet will decrease milk production and carbohydrate oxidation, increase gluconeogenesis and hexoneogenesis, and not affect energy balance. DESIGN Seven healthy lactating mothers and their infants were studied on 2 occasions in random order for 8 d separated by 1-2 wk. On one occasion, the subjects received the H-F (30% of energy as carbohydrate and 55% as fat) diet and on the other occasion received the H-CHO (60% of energy as carbohydrate and 25% as fat) diet. Milk production, infant intakes, and substrate and hormone concentrations were measured. Glucose rates of appearance, production, gluconeogenesis, glycogenolysis, and hexoneogenesis were measured by using stable-isotope gas chromatography-mass spectrometric techniques, and energy expenditure and substrate oxidation were measured by using indirect calorimetry. RESULTS Milk volume, lactose, and protein concentrations were unaffected. Milk fat, energy, and infant intakes were higher (P < 0.05) during the H-F diet. Neither gluconeogenesis nor hexoneogenesis was different. During the H-F diet, energy expenditure and fat and protein oxidation rates were higher (P < 0.05), and the daily energy balance deficit was greater (P < 0.01). CONCLUSIONS Milk fat, energy output, and energy expenditure were higher during the H-F diet, which resulted in a greater negative energy balance. The lactating mothers adapted to a low carbohydrate intake by decreasing carbohydrate oxidation. Additional studies are warranted to determine whether a hypocaloric H-F diet might promote weight loss to a greater extent than the H-CHO diet while maintaining milk production.

Gene regulation of UDP-galactose synthesis and transport: potential rate-limiting processes in initiation of milk production in humans

Lactose synthesis is believed to be rate limiting for milk production. However, understanding the molecular events controlling lactose synthesis in humans is still rudimentary. We have utilized our established model of the RNA isolated from breast milk fat globule from seven healthy, exclusively breastfeeding women from 6 h to 42 days following delivery to determine the temporal coordination of changes in gene expression in the carbohydrate metabolic processes emphasizing the lactose synthesis pathway in human mammary epithelial cell. We showed that milk lactose concentrations increased from 75 to 200 mM from 6 to 96 h. Milk progesterone concentrations fell by 65% at 24 h and were undetectable by day 3. Milk prolactin peaked at 36 h and then declined progressively afterward. In concordance with lactose synthesis, gene expression of galactose kinase 2, UDP-glucose pyrophosphorylase 2 (UGP2), and phosphoglucomutase 1 increased 18-, 10-, and threefold, respectively, between 6 and 72 h. Between 6 and 96 h, gene expression of UDP-galactose transporter 2 (SLC35A2) increased threefold, whereas glucose transporter 1 was unchanged. Gene expression of lactose synthase no. 3 increased 1.7-fold by 96 h, whereas α-lactalbumin did not change over the entire study duration. Gene expression of prolactin receptor (PRLR) and its downstream signal transducer and activator of transcription complex 5 (STAT5) were increased 10- and 2.5-fold, respectively, by 72 h. In summary, lactose synthesis paralleled the induction of gene expression of proteins involved in UDP-galactose synthesis and transport, suggesting that they are potentially rate limiting in lactose synthesis and thus milk production. Progesterone withdrawal may be the signal that triggers PRLR signaling via STAT5, which may in turn induce UGP2 and SLC35A2 expression.

Regulation of lipid synthesis genes and milk fat production in human mammary epithelial cells during secretory activation

Expression of genes for lipid biosynthetic enzymes during initiation of lactation in humans is unknown. Our goal was to study mRNA expression of lipid metabolic enzymes in human mammary epithelial cell (MEC) in conjunction with the measurement of milk fatty acid (FA) composition during secretory activation. Gene expression from mRNA isolated from milk fat globule (MFG) and milk FA composition were measured from 6 h to 42 days postpartum in seven normal women. Over the first 96 h postpartum, daily milk fat output increased severalfold and mirrored expression of genes for all aspects of lipid metabolism and milk FA production, including lipolysis at the MEC membrane, FA uptake from blood, intracellular FA transport, de novo FA synthesis, FA and glycerol activation, FA elongation, FA desaturation, triglyceride synthesis, cholesterol synthesis, and lipid droplet formation. Expression of the gene for a key lipid synthesis regulator, sterol regulatory element-binding transcription factor 1 (SREBF1), increased 2.0-fold by 36 h and remained elevated over the study duration. Expression of genes for estrogen receptor 1, thyroid hormone-responsive protein, and insulin-induced 2 increased progressively to plateau by 96 h. In contrast, mRNA of peroxisome proliferator-activated receptor-γ decreased severalfold. With onset of lactation, increased de novo synthesis of FA was the most prominent change in milk FA composition and mirrored the expression of FA synthesis genes. In conclusion, milk lipid synthesis and secretion in humans is a complex process requiring the orchestration of a wide variety of pathways of which SREBF1 may play a primary role.

Mechanisms to conserve glucose in lactating women during a 42-h fast

Little is known about how lactating women accommodate for their increased glucose demands during fasting to avoid maternal hypoglycemia. The objective of this study was to determine whether lactating women conserve plasma glucose by reducing maternal glucose utilization by increasing utilization of FFA and ketone bodies and/or increasing gluconeogenesis and mammary gland hexoneogenesis. Six healthy exclusively breastfeeding women and six nonlactating controls were studied during 42 h of fasting and 6 h of refeeding. Glucose and protein kinetic parameters were measured using stable isotopes and GCMS and energy expenditure and substrate oxidation using indirect calorimetry. After 42 h of fasting, milk production decreased by 16% but remained within normal range. Glucose, insulin, and C-peptide concentrations decreased with the duration of fasting in both groups but were lower (P < 0.05) in lactating women. Glucagon, FFA, and beta-hydroxybutyrate concentrations increased with fasting time (P < 0.001) and were higher (P < 0.0001) in lactating women during both fasting and refeeding. During 42 h of fasting, gluconeogenesis was higher in lactating women compared with nonlactating controls (7.7 +/- 0.4 vs. 6.5 +/- 0.2 micromol kg(-1) min(-1), P < 0.05), whereas glycogenolysis was suppressed to similar values (0.4 +/- 0.1 vs. 0.9 +/- 0.2 micromol kg(-1) min(-1), respectively). Mammary hexoneogenesis did not increase with the duration of fasting. Carbohydrate oxidation was lower and fat and protein oxidations higher (P < 0.05) in lactating women. In summary, lactating women are at risk for hypoglycemia if fasting is extended beyond 30 h. The extra glucose demands of extended fasting during lactation appear to be compensated by increasing gluconeogenesis associated with ketosis, decreasing carbohydrate oxidation, and increasing protein and FFA oxidations.

Galactose promotes fat mobilization in obese lactating and nonlactating women

BACKGROUND Galactose consumption as the only carbohydrate source results in little increase in plasma glucose and insulin concentrations when compared with fasting. Lower insulin might promote endogenous lipolysis during meal absorption, which may facilitate fat loss. OBJECTIVE The objective was to test the hypothesis that consumption of an isocaloric, isonitrogenous galactose drink will result in higher rates of lipolysis and fat oxidation than consumption of a glucose drink in obese lactating and nonlactating women. DESIGN Seven healthy, obese, exclusively breastfeeding women and 7 healthy, obese, nonlactating women were studied on 2 occasions according to a randomized, crossover, single-blinded design. Subjects received drinks providing ≈70% of the daily estimated energy requirement, of which 60% was either glucose or galactose. The primary outcomes were the rate of appearance (Ra) of glycerol and palmitate, and the secondary outcomes were glucose Ra, milk production, energy expenditure, and substrate oxidation. RESULTS Plasma glucose and insulin concentrations were lower (P < 0.05) and those of glycerol, palmitate, free fatty acids, and triglycerides were higher (P < 0.05) during galactose than during glucose feeding in both nonlactating and lactating women. During galactose feeding, glucose Ra was lower (P < 0.01) and glycerol, palmitate, and free fatty acid Ra were higher (P < 0.01) in both groups. During galactose feeding in all women combined, fat oxidation was higher (P = 0.01) and protein oxidation was lower (P < 0.01). Milk production, energy expenditure, and carbohydrate oxidation were similar between glucose and galactose feeding. CONCLUSIONS Galactose consumption is associated with higher endogenous fat mobilization and oxidation during meal absorption. Long-term studies are required to determine whether galactose as an exclusive carbohydrate source would promote body fat loss in obese subjects.

FABP4-Cre Mediated Expression of Constitutively Active ChREBP Protects Against Obesity, Fatty Liver, and Insulin Resistance

Carbohydrate response element binding protein (ChREBP) regulates cellular glucose and lipid homeostasis. Although ChREBP is highly expressed in many key metabolic tissues, the role of ChREBP in most of those tissues and the consequent effects on whole-body glucose and lipid metabolism are not well understood. Therefore, we generated a transgenic mouse that overexpresses a constitutively active ChREBP isoform under the control of the fatty acid binding protein 4-Cre-driven promoter (FaChOX). Weight gain was blunted in male, but not female, FaChOX mice when placed on either a normal chow diet or an obesogenic Western diet. Respiratory exchange ratios were increased in Western diet-fed FaChOX mice, indicating a shift in whole-body substrate use favoring carbohydrate metabolism. Western diet-fed FaChOX mice showed improved insulin sensitivity and glucose tolerance in comparison with controls. Hepatic triglyceride content was reduced in Western diet-fed FaChOX mice in comparison with controls, suggesting protection from fatty liver. Epididymal adipose tissue exhibited differential expression of genes involved in differentiation, browning, metabolism, lipid homeostasis, and inflammation between Western diet-fed FaChOX mice and controls. Our findings support a role for ChREBP in modulating adipocyte differentiation and adipose tissue metabolism and inflammation as well as consequent risks for obesity and insulin resistance.

Estradiol-17β Upregulates Pyruvate Kinase M2 Expression to Coactivate Estrogen Receptor-α and to Integrate Metabolic Reprogramming With the Mitogenic Response in Endometrial Cells

CONTEXT Proliferating cells reprogram their cellular glucose metabolism to meet the bioenergetic and biosynthetic demands and to maintain cellular redox homeostasis. Pyruvate kinase M (PKM) is a critical regulator of this metabolic reprogramming. However, whether estradiol-17β (E2) reprograms cellular metabolism to support proliferation of human primary endometrial stromal cells (hESCs) and the molecular basis of this reprogramming are not well understood. OBJECTIVES Our objectives were to study whether E2 induces reprogramming of glucose metabolism in hESCs and to investigate the potential roles of PKM2 in E2-induced metabolic reprogramming and proliferation of these cells. METHODS The oxygen consumption rate and extracellular acidification rate were assessed by a Seahorse XF24 analyzer. PKM2 expression was assessed by real-time RT-PCR and immunoblotting. RESULTS E2 induces a Warburg-like glucose metabolism in hESCs by inducing the expression of PKM. E2 also enhanced PKM splicing into the PKM2 isoform by upregulating the c-Myc-hnRNP axis. Furthermore, E2 induces PKM2 oxidation, phosphorylation, and nuclear translocation. In addition to its glycolytic function, PKM2 physically interacted with estrogen receptor-α (ERα) and functioned as an ERα coactivator. Small-molecule PKM2 activators ameliorated ERα transcriptional activity and abrogated the E2-induced hESC proliferation. CONCLUSIONS We show for the first time that E2-induced hESC proliferation is associated with a shift in glucose metabolism toward aerobic glycolysis, and the molecular basis for this metabolic shift is linked to the effects of E2 on PKM2. In addition, PKM2 acts as a transcriptional coactivator for ERα and small-molecule PKM2 activators inhibit ERα transcriptional activity and reduce E2-induced cell proliferation.

Comparative Analyses of Lung Transcriptomes in Patients with Alveolar Capillary Dysplasia with Misalignment of Pulmonary Veins and in Foxf1 Heterozygous Knockout Mice

Alveolar Capillary Dysplasia with Misalignment of Pulmonary Veins (ACDMPV) is a developmental disorder of the lungs, primarily affecting their vasculature. FOXF1 haploinsufficiency due to heterozygous genomic deletions and point mutations have been reported in most patients with ACDMPV. The majority of mice with heterozygous loss-of-function of Foxf1 exhibit neonatal lethality with evidence of pulmonary hemorrhage in some of them. By comparing transcriptomes of human ACDMPV lungs with control lungs using expression arrays, we found that several genes and pathways involved in lung development, angiogenesis, and in pulmonary hypertension development, were deregulated. Similar transcriptional changes were found in lungs of the postnatal day 0.5 Foxf1 +/− mice when compared to their wildtype littermate controls; 14 genes, COL15A1, COL18A1, COL6A2, ESM1, FSCN1, GRINA, IGFBP3, IL1B, MALL, NOS3, RASL11B, MATN2, PRKCDBP, and SIRPA, were found common to both ACDMPV and Foxf1 heterozygous lungs. Our results advance knowledge toward understanding of the molecular mechanism of ACDMPV, lung development, and its vasculature pathology. These data may also be useful for understanding etiologies of other lung disorders, e.g. pulmonary hypertension, bronchopulmonary dysplasia, or cancer.

De novo synthesis of milk triglycerides in humans

UNLABELLED Mammary gland (MG) de novo lipogenesis contributes significantly to milk fat in animals but little is known in humans. OBJECTIVE To test the hypothesis that the incorporation of (13)C carbons from [U-(13)C]glucose into fatty acids (FA) and glycerol in triglycerides (TG) will be greater: 1) in milk than plasma TG, 2) during a high-carbohydrate (H-CHO) diet than high-fat (H-FAT) diet, and 3) during feeding than fasting. Seven healthy, lactating women were studied on two isocaloric, isonitrogenous diets. On one occasion, subjects received diets containing H-FAT or H-CHO diet for 1 wk. Incorporation of (13)C from infused [U-(13)C]glucose into FA and glycerol was measured using GC-MS and gene expression in RNA isolated from milk fat globule using microarrays. Incorporation of (13)C2 into milk FA increased with increased FA chain length from C2:0 to C12:0 but progressively declined in C14:0 and C16:0 and was not detected in FA>C16. During feeding, regardless of diets, enrichment of (13)C2 in milk FA and (13)C3 in milk glycerol were ∼ 3- and ∼ 7-fold higher compared with plasma FA and glycerol, respectively. Following an overnight fast during H-CHO and H-FAT diets, 25 and 6%, respectively, of medium-chain FA (MCFA, C6-C12) in milk were derived from glucose but increased to 75 and 25% with feeding. Expression of genes involved in FA or glycerol synthesis was unchanged regardless of diet or fast/fed conditions. The human MG is capable of de novo lipogenesis of primarily MCFA and glycerol, which is influenced by the macronutrient composition of the maternal diet.