Dietary fat quality impacts genome-wide DNA methylation patterns in a cross-sectional study of Greek preadolescents

The type and the amount of dietary fat have a significant influence on the metabolic pathways involved in the development of obesity, metabolic syndrome, diabetes type 2 and cardiovascular diseases. However, it is unknown to what extent this modulation is achieved through DNA methylation. We assessed the effects of cholesterol intake, the proportion of energy intake derived from fat, the ratio of polyunsaturated fatty acids (PUFA) to saturated fatty acids (SFA), the ratio of monounsaturated fatty acids (MUFA) to SFA, and the ratio of MUFA+PUFA to SFA on genome-wide DNA methylation patterns in normal-weight and obese children. We determined the genome-wide methylation profile in the blood of 69 Greek preadolescents (∼10 years old) as well as their dietary intake for two consecutive weekdays and one weekend day. The methylation levels of one CpG island shore and four sites were significantly correlated with total fat intake. The methylation levels of 2 islands, 11 island shores and 16 sites were significantly correlated with PUFA/SFA; of 9 islands, 26 island shores and 158 sites with MUFA/SFA; and of 10 islands, 40 island shores and 130 sites with (MUFA+PUFA)/SFA. We found significant gene enrichment in 34 pathways for PUFA/SFA, including the leptin pathway, and a significant enrichment in 5 pathways for (MUFA+PUFA)/SFA. Our results suggest that specific changes in DNA methylation may have an important role in the mechanisms involved in the physiological responses to different types of dietary fat

Cephalic phase of insulin secretion and food stimulation in humans: a new perspective

Insulinemia and glycemia were measured at a 1-min interval at the hour of a lunch meal in human subjects. When no food was presented to naive subjects (n = 4), cyclic oscillations of insulinemia were found (period, 12-20 min; amplitude, 2.8-10.3 microU/ml). It is proposed that these spontaneous oscillations must be taken into consideration when evaluating the insulin response on cephalic contact with food stimuli; they might otherwise constitute a source of artifacts. Four subjects were then submitted to a series of four test meals scheduled at a 1-wk interval. Although their prandial glycemia remained comparable with preprandial values for the first 16 min of the meals, insulinemia often exhibited early peaks (within a few min after meal onset) whose amplitude appeared related to palatability conditions. Evidence suggests that the insulin peaks triggered by cephalic stimulation are Pavlovian reflexes that become conditioned to the test situation. A typical neuroendocrine response to alimentary frustration is also described. The results are discussed in perspective with animal works, in terms of the effects of neuroendocrine events on feeding behavior. </jats:p

Placental nutrient transporters adapt during persistent maternal hypoglycaemia in rats

Maternal malnutrition is associated with decreased nutrient transfer to the foetus, which may lead to foetal growth restriction, predisposing children to a variety of diseases. However, regulation of placental nutrient transfer during decreased nutrient availability is not fully understood. In the present study, the aim was to investigate changes in levels of placental nutrient transporters accompanying maternal hypoglycaemia following different durations and stages of gestation in rats. Maternal hypoglycaemia was induced by insulin-infusion throughout gestation until gestation day (GD)20 or until end of organogenesis (GD17), with sacrifice on GD17 or GD20. Protein levels of placental glucose transporters GLUT1 (45/55 kDa isotypes) and GLUT3, amino acid transporters SNAT1 and SNAT2, and insulin receptor (InsR) were assessed. On GD17, GLUT1-45, GLUT3, and SNAT1 levels were increased and InsR levels decreased versus controls. On GD20, following hypoglycaemia throughout gestation, GLUT3 levels were increased, GLUT1-55 showed the same trend. After cessation of hypoglycaemia at end of organogenesis, GLUT1-55, GLUT3, and InsR levels were increased versus controls, whereas SNAT1 levels were decreased. The increases in levels of placental nutrient transporters seen during maternal hypoglycaemia and hyperinsulinemia likely reflect an adaptive response to optimise foetal nutrient supply and development during limited availability of glucose.</jats:p

215-LB: Silencing of Fructose 1,6-Bisphosphatase (FBP1) in Liver Improves Glucose Homeostasis in Insulin-Resistant Rodent and Human Models

Insulin resistant individuals display elevated fasting and post-prandial glucose levels which are mainly driven by inadequate inhibition of gluconeogenesis (GNG) and are rarely normalized. The aim of this study was to investigate whether direct inhibition of GNG could be a new therapeutical approach to improve glucose homeostasis using in vitro and in vivo models of insulin resistance. Fructose 1,6-bisphosphatase (FBP1) is key in controlling GNG in liver and kidney and loss of function patients display hypoglycaemia episodes and lactate acidosis. Systemic FBP1 inhibition using small molecules has shown to improve glucose homeostasis. To assess the clinical efficacy and safety potential of silencing FBP1 selectively in the liver we dosed insulin resistant DIO rats with a hepatocyte specific GalXC-FBP1 siRNA entity. GalXC-FBP1 siRNA markedly reduced FBP1 mRNA level in the liver by over 90%. This was associated with a complete lack of glucose excursion following a pyruvate challenge indicating inhibition of GNG. Blood glucose level decreased similarly in rats treated with GalXC-FBP1 siRNA or vehicle during an insulin challenge or a prolonged fast suggesting that hepatic FBP1 silencing is not inducing hypoglycaemia. Insulin sensitivity and hyperinsulinemia were improved. Plasma lactate and liver enzymes were not elevated, but a significant 2-fold increase in liver triglycerides was observed in the GalXC-FBP1 siRNA group. Using human hepatocytes in a Liver-on-Chip in vitro model FBP1 silencing reduced glucose production by 30% supporting the relevance of this approach in humans. Collectively these data suggest that liver specific FBP1 silencing has the potential to improve insulin sensitivity in DIO rats without inducing hypoglycaemia but may be associated with a risk of liver steatosis overtime. This concept highlights the difficulty of blocking liver GNG without re-directing the metabolic intermediates fluxes toward triglycerides accumulation in liver. Disclosure C. Fledelius: Employee; Novo Nordisk A/S. D. Demozay: Employee; Novo Nordisk A/S. H. Iversen: None. R. S. Ingvorsen: None. L. B. Eriksen: None. A. Blois: Employee; Novo Nordisk. Y. Montauban: None. R. Rijnbrand: Employee; Novo Nordisk. W. Han: None. J. F. Jeppesen: Employee; Novo Nordisk A/S. </jats:sec

Immunohistochemistry.

(DOCX)</p

Western blotting.

(DOCX)</p

Fig 2 -

Result summary of nutrient transporter and InsR distribution (A) and protein levels (B) in placenta. A: Schematic illustration of the blood-placenta barrier and cellular distribution of the glucose transporters GLUT1 and GLUT3, amino acid transporters SNAT1 and SNAT2, as well as the insulin receptor (InsR) as shown by immunohistochemistry in controls in the present study. As it is not possible to differentiate between the GLUT1-45 and GLUT1-55 isotypes by the immunohistochemistry, these are not specified. For each transporter and the InsR, bold text indicates higher signal as compared to the same transporter not in bold in the same cell type (evaluated qualitatively, not quantitatively). Besides the localisation to the plasma membrane, as illustrated, all transporters and the InsR were also detected intra-cellularly in trophoblasts and foetal endothelial cells (not shown). The cell layers separating the maternal from the foetal circulation in rats are one loosely connected trophoblast layer (not shown), two trophoblast cell layers, and the foetal microvascular endothelial cells, where the trophoblast cell layers and basal membrane of the foetal vascular endothelial cell layer constitute the blood-placenta barrier [13, 14]. The main difference between the rat and human blood-placenta barrier is that the latter only contains one layer of trophoblast cells [13, 14]. B: Differences in placental protein levels in insulin-infused groups compared to controls as assessed by western blotting. ↑, increased levels; ↓, decreased levels; ↔, no change to levels, (↑), trend for increased levels.</p

Placental glucose transporter protein levels, fold changes and SD.

A: Representative examples of the visualised bands cropped from the original image. Full length blot pictures are included in S1 Fig. Band sizes: HPRT (housekeeping gene), 25 kDa; GLUT1, approximately 40 and 60 kDa, respectively; GLUT3, 40 kDa. *pB: GD17. Group CTRL-INT, n = 7; HI-INT, n = 8. C: GD20. GLUT1-45: Group CTRL, n = 12; HI-GD20, n = 9 (1 extreme outlier identified by ROUT test); HI-GD17, n = 10. GLUT1-55: Group CTRL, n = 12; HI-GD20, n = 10; HI-GD17, n = 10. GLUT3: Group CTRL, n = 12; HI-GD20, n = 9 (1 extreme outlier identified by ROUT test); HI-GD17, n = 10. Statistical analysis: GD17: two-tailed Mann Whitney test. GD20: Kruskal-Wallis test, post hoc Dunn’s multiple comparisons test. NS, not statistically different.</p

Maternal and foetal liver glycogen and lipid concentrations on GD20, individual (symbols) and means and SD.

Left panel: Maternal levels. Right panel: Foetal levels. A+B: Glycogen, C+D: Triglycerides, E+F: Cholesterol, G+H: FFA. Group CTRL, n = 21; group HI-GD20, n = 16; group HI-GD17, n = 20 for all maternal and foetal measurements except for maternal triglyceride levels, where n = 20 in the CTRL group and foetal triglyceride levels, where n = 15 in the HI-GD20 group, as one extreme outlier was identified in each of these groups (29.4 and 10.1 μmol/g, ROUT test) and excluded. FFAs, free fatty acids. *ppost hoc Tukey’s multiple comparisons test.</p