Maternal High Fat Diet Programs Male Mice Offspring Hyperphagia and Obesity: Mechanism of Increased Appetite Neurons via Altered Neurogenic Factors and Nutrient Sensor AMPK

Abstract

Maternal high-fat (HF) is associated with offspring hyperphagia and obesity. We hypothesized that maternal HF alters fetal neuroprogenitor cell (NPC) and hypothalamic arcuate nucleus (ARC) development with preferential differentiation of neurons towards orexigenic (NPY/AgRP) versus anorexigenic (POMC) neurons, leading to offspring hyperphagia and obesity. Furthermore, these changes may involve hypothalamic bHLH neuroregulatory factors (Hes1, Mash1, Ngn3) and energy sensor AMPK. Female mice were fed either a control or a high fat (HF) diet prior to mating, and during pregnancy and lactation. HF male newborns were heavier at birth and exhibited decreased protein expression of hypothalamic bHLH factors, pAMPK/AMPK and POMC with increased AgRP. As adults, these changes persisted though with increased ARC pAMPK/AMPK. Importantly, the total NPY neurons were increased, which was consistent with the increased food intake and adult fat mass. Further, NPCs from HF newborn hypothalamic tissue showed similar changes with preferential NPC neuronal differentiation towards NPY. Lastly, the role of AMPK was further confirmed with in vitro treatment of Control NPCs with pharmacologic AMPK modulators. Thus, the altered ARC development of HF offspring results in excess appetite and reduced satiety leading to obesity. The underlying mechanism may involve AMPK/bHLH pathways.

Keywords: AMPK activator; developmental programming; hypothalamic arcuate nucleus; neuropeptides; neuroprogenitor cells.

Figure 1

Maternal HF (high fat) increases male offspring body weight, adiposity and food intake. (A) Male offspring body weights from birth to 48 weeks (wks) of age of Controls (●) and HF (●); inset of body weights from 1 day to 3 weeks of age. (B) Body fat and lean body mass and percentage of body weight of 12-month old male offspring. (C) Food intake from 4 to 48 weeks of age of male offspring from control (●) and HF (●) groups. Values are mean ± SE of n = 6 males from six litters. * p < 0.01 vs control.

Figure 2

Maternal HF diet alters offspring hypothalamic tissue expression of neuropeptides and neuronal phenotype. (A) POMC and AgRP protein expression in hypothalamus of 1-day-old males, and ARC of 12-month-old males from Control (■) and HF (□) groups. Values are fold change (mean ± SE) of n = 6 males from six litters per group (1 male/litter). * p < 0.001 vs Control. (B) ARC sections from representative control (■) and HF (■) 12-month-old males, immunostained for NPY (green) at × 20 and × 100; scale bar = 50 µm. Total NPY (mean ± SE) of n = 6 males from six litters per group (1 male/litter). * p < 0.01 vs control. Abbreviations: HF (high fat); POMC (proopiomelanocortin); AgRP (agouti-related protein); NPY (neuropeptide Y); ARC (arcuate nucleus); 3V (third ventricle).

Figure 3

Maternal HF diet alters offspring hypothalamic tissue expression of AMPK, pAMPK and bHLH factors. (A) Hes1, Mash1, Ngn3, and (B) AMPK and pAMPK protein expression in hypothalami of 1-day-old males, and ARC of 12-month-old males from control (■) and HF (□) groups. Fold change (mean ± SE) of n = 6 males from six litters per group (1 male/litter). * p < 0.01 vs control. Abbreviations: HF (high fat); AMPK (5’ AMP-activated protein kinase); pAMPK (phosphorylated 5’ AMP-activated protein kinase); Hes1 (Hairy and Enhancer of Split 1); Mash1 (Achaete-scute complex homolog-1); Ngn3 (neurogenin 3); GAPDH (Glyceraldehyde 3-phosphate dehydrogenase).

Figure 4

Maternal HF diet alters expression of AMPK, pAMPK and bHLH factors and promotes differentiation to NPY neurons in in vitro NPC culture. (A) NPC protein expression of AMPK, pAMPK and bHLH factors (Hes1, Mash1, Ngn3) in control (■) and HF (■) newborn males at 1 day of age. Fold change (mean ± SE) of n = 6 from six litters per group (1 male/litter). * p < 0.01 vs control. (B) NPCs from 1-day-old control (■) and HF (■) males were cultured in differentiating medium for 7 days, fixed and immunostained for NPY (green) and DAPI (blue). Scale bar = 50 µm values are percent of controls (mean ± SE). * p < 0.01 vs control. Abbreviations: HF (high fat); AMPK (5’ AMP-activated protein kinase); pAMPK (phosphorylated 5’ AMP-activated protein kinase); Hes1 (Hairy and Enhancer of Split 1); Mash1 (Achaete-scute complex homolog-1); Ngn3 (neurogenin 3); GAPDH (Glyceraldehyde 3-phosphate dehydrogenase); NPY (neuropeptide Y).

Figure 5

Activation of AMPK in vitro upregulates bHLH factors, suppresses appetite and increases POMC neuropeptide expression. (A) Control NPCs were treated in vitro with AMPK activator (0.5, 1, 2 mM; □) or vehicle (■). NPC protein expression with representative immunoblots of pAMPK, bHLH factors (Mash1, Ngn3) and (B) neuropeptides (POMC, AgRP). Values are fold change of vehicle treated (N = 5; mean ± SE). * p < 0.01 vs control. Abbreviations: AICAR (5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside); pAMPK (phosphorylated 5’ AMP-activated protein kinase); Mash1 (Achaete-scute complex homolog-1); Ngn3 (neurogenin 3); POMC (proopiomelanocortin); AgRP (agouti-related protein); GAPDH (Glyceraldehyde 3-phosphate dehydrogenase).

Figure 6

Suppression of AMPK in vitro downregulates bHLH factors and increases appetite and reduces satiety neuropeptide expression. (A) Control NPCs were treated in vitro with AMPK inhibitor (Compound C 1, 5, 25 µm; □) or vehicle (■). NPC protein expression with representative immunoblots of pAMPK, bHLH factors (Mash1, Ngn3) and (B) neuropeptides (POMC, AgRP). Values are fold change of vehicle treated (N = 5; mean ± SE). * p < 0.001 vs control. Abbreviations: Compound C (Dorsomorphin dihydrochloride); pAMPK (phosphorylated 5’ AMP-activated protein kinase); Mash1 (Achaete-scute complex homolog-1); Ngn3 (neurogenin 3); POMC (proopiomelanocortin); AgRP (agouti-related protein); GAPDH (Glyceraldehyde 3-phosphate dehydrogenase).