Hypothalamic neurosphere progenitor cells in low birth-weight rat newborns: neurotrophic effects of leptin and insulin

Abstract

A low birth-weight (LBW) offspring exhibits reduced hypothalamic neural satiety pathways and dysregulated signaling leading to programmed hyperphagia and adult obesity. Hypothalamic appetite circuits develop during early life, under the influence of neurotrophic hormones (leptin and insulin). Notably, LBW newborns have reduced plasma leptin and insulin levels. As neurons and glia arise from neuronal progenitor cells (NPC), we postulated that a programmed impairment of NPCs may contribute to reduced hypothalamic neural pathway development in a LBW offspring. Control dams received ad libitum food, whereas study dams were 50% food-restricted from pregnancy day 10 to 21 (LBW). At day 1 of age, hypothalamic NPCs were cultured as neurospheres (NS) and treated with leptin/insulin. We analyzed in vitro NPC proliferation and differentiation into neurons/astrocytes, expression of signal molecules promoting proliferation (activated Notch1 and its downstream target, Hes1) and in vivo NPC proliferation and migration. LBW offspring had impaired in vivo evidence of NPC division and migration, and reduced in vitro evidence of proliferation and differentiation to neurons and astrocytes, under basal and stimulated conditions. The reduced Notch1 and Hes1 expression in LBW neurosphere, under both basal and stimulated conditions, suggests a reduced progenitor cell population or reduced cell density within the neurosphere.

Figure 1

A: Evidence of Hypothalamic NPCs and GFAP in 1 Day Newborn Brain from 1 day newborn was fixed, sectioned (20µm) and stained for markers of NPC (nestin), neuronal (NeuN), astrocyte (GFAP) and nuclei (DAPI). Upper images show evidence of hypothalamic NPCs around third venticular (V) region and lower images show hypothalamic surface (HS) GFAP (astrocyte). Images shown are at ×40 magnification. B: In Vivo NPC Proliferation and Migration: Hypothalamic Immunostaining of BrdU Incorporation Food-restricted (n=3) and control (n=3) pregnant dams were injected with BrdU (50 mg/kg/day, i.p.) from e17–e19. After birth, brains were collected from 1 day old Control () and LBW () newborn males. The images (×20) show hypothalamic BrdU (cell proliferation) and DAPI (nuclear marker) immunostaining around third venticular (V) region. C: In Vivo NPC Proliferation and Migration Food-restricted (n=3) and control (n=3) pregnant dams were injected with BrdU (50 mg/kg/day, i.p.) from e17–e19. After birth, brains were collected from 1 day old Control () and LBW () newborn males. Three brains per litter were frozen, and three sections per brain were immunostained. Cell proliferation was determined by counting BrdU positive cells in third ventricle and midline. Migration rate was determined by counting BrdU labeled cells in the area between 30 µm to 100 µm from midline. The average of BrdU-labeled cell numbers of three sections represented one brain and average of three brain cell numbers represented one litter. Values are mean±SE; *P<0.05 vs. Control.

Figure 1

A: Evidence of Hypothalamic NPCs and GFAP in 1 Day Newborn Brain from 1 day newborn was fixed, sectioned (20µm) and stained for markers of NPC (nestin), neuronal (NeuN), astrocyte (GFAP) and nuclei (DAPI). Upper images show evidence of hypothalamic NPCs around third venticular (V) region and lower images show hypothalamic surface (HS) GFAP (astrocyte). Images shown are at ×40 magnification. B: In Vivo NPC Proliferation and Migration: Hypothalamic Immunostaining of BrdU Incorporation Food-restricted (n=3) and control (n=3) pregnant dams were injected with BrdU (50 mg/kg/day, i.p.) from e17–e19. After birth, brains were collected from 1 day old Control () and LBW () newborn males. The images (×20) show hypothalamic BrdU (cell proliferation) and DAPI (nuclear marker) immunostaining around third venticular (V) region. C: In Vivo NPC Proliferation and Migration Food-restricted (n=3) and control (n=3) pregnant dams were injected with BrdU (50 mg/kg/day, i.p.) from e17–e19. After birth, brains were collected from 1 day old Control () and LBW () newborn males. Three brains per litter were frozen, and three sections per brain were immunostained. Cell proliferation was determined by counting BrdU positive cells in third ventricle and midline. Migration rate was determined by counting BrdU labeled cells in the area between 30 µm to 100 µm from midline. The average of BrdU-labeled cell numbers of three sections represented one brain and average of three brain cell numbers represented one litter. Values are mean±SE; *P<0.05 vs. Control.

Figure 1

A: Evidence of Hypothalamic NPCs and GFAP in 1 Day Newborn Brain from 1 day newborn was fixed, sectioned (20µm) and stained for markers of NPC (nestin), neuronal (NeuN), astrocyte (GFAP) and nuclei (DAPI). Upper images show evidence of hypothalamic NPCs around third venticular (V) region and lower images show hypothalamic surface (HS) GFAP (astrocyte). Images shown are at ×40 magnification. B: In Vivo NPC Proliferation and Migration: Hypothalamic Immunostaining of BrdU Incorporation Food-restricted (n=3) and control (n=3) pregnant dams were injected with BrdU (50 mg/kg/day, i.p.) from e17–e19. After birth, brains were collected from 1 day old Control () and LBW () newborn males. The images (×20) show hypothalamic BrdU (cell proliferation) and DAPI (nuclear marker) immunostaining around third venticular (V) region. C: In Vivo NPC Proliferation and Migration Food-restricted (n=3) and control (n=3) pregnant dams were injected with BrdU (50 mg/kg/day, i.p.) from e17–e19. After birth, brains were collected from 1 day old Control () and LBW () newborn males. Three brains per litter were frozen, and three sections per brain were immunostained. Cell proliferation was determined by counting BrdU positive cells in third ventricle and midline. Migration rate was determined by counting BrdU labeled cells in the area between 30 µm to 100 µm from midline. The average of BrdU-labeled cell numbers of three sections represented one brain and average of three brain cell numbers represented one litter. Values are mean±SE; *P<0.05 vs. Control.

Figure 2

A: Nestin immunostaining of Neurosphere Sections Hypothalamic NPC from 1 day old Control () and LBW () newborn males were cultured in complete media. Neurospheres were sectioned (5 µm) and immunostained with nestin, Neu and GFAP. Images of LBW and Control are shown at 40× magnification. B: Basal Hypothalamic NPC Proliferation Hypothalamic NPC from 1 day old Control () and LBW () newborn males were cultured in complete media. Live images (magnification ×20), basal cell proliferation rate and nestin protein expression of LBW and Control NPCs. Values are fold change (mean ± SE); * P < 0.05 LBW vs. Control. C: Basal Hypothalamic NPC Differentiation Hypothalamic NPC from 1 Control () and LBW () newborn males were cultured in differentiating media. NPC were harvested and protein expression of neuronal marker (Tuj1)) and astrocyte marker (GFAP) were determined. Values are fold change (mean ± SE); * P < 0.05 LBW vs. Control.

Figure 2

A: Nestin immunostaining of Neurosphere Sections Hypothalamic NPC from 1 day old Control () and LBW () newborn males were cultured in complete media. Neurospheres were sectioned (5 µm) and immunostained with nestin, Neu and GFAP. Images of LBW and Control are shown at 40× magnification. B: Basal Hypothalamic NPC Proliferation Hypothalamic NPC from 1 day old Control () and LBW () newborn males were cultured in complete media. Live images (magnification ×20), basal cell proliferation rate and nestin protein expression of LBW and Control NPCs. Values are fold change (mean ± SE); * P < 0.05 LBW vs. Control. C: Basal Hypothalamic NPC Differentiation Hypothalamic NPC from 1 Control () and LBW () newborn males were cultured in differentiating media. NPC were harvested and protein expression of neuronal marker (Tuj1)) and astrocyte marker (GFAP) were determined. Values are fold change (mean ± SE); * P < 0.05 LBW vs. Control.

Figure 2

A: Nestin immunostaining of Neurosphere Sections Hypothalamic NPC from 1 day old Control () and LBW () newborn males were cultured in complete media. Neurospheres were sectioned (5 µm) and immunostained with nestin, Neu and GFAP. Images of LBW and Control are shown at 40× magnification. B: Basal Hypothalamic NPC Proliferation Hypothalamic NPC from 1 day old Control () and LBW () newborn males were cultured in complete media. Live images (magnification ×20), basal cell proliferation rate and nestin protein expression of LBW and Control NPCs. Values are fold change (mean ± SE); * P < 0.05 LBW vs. Control. C: Basal Hypothalamic NPC Differentiation Hypothalamic NPC from 1 Control () and LBW () newborn males were cultured in differentiating media. NPC were harvested and protein expression of neuronal marker (Tuj1)) and astrocyte marker (GFAP) were determined. Values are fold change (mean ± SE); * P < 0.05 LBW vs. Control.

Figure 3

A: Evidence for NPC and Neuronal Leptin Receptor NPC grown in complete or differentiating media were immunostained for leptin receptor (ObRb), neuronal marker (Tuj1) and nuclear stain (DAPI). B: Leptin and Insulin Stimulated Hypothalamic NPC Proliferation Hypothalamic NPC from 1 day old Control () and LBW () males were cultured in complete media. On second day of seeding, NS were treated with leptin/insulin every 48h for 8 days and cell proliferation rate was determined. Values are percentage of untreated Control (mean ± SE); * P < 0.05 vs. untreated NS; “^C” P < 0.05 LBW vs. Control. C: Leptin and Insulin Stimulated Hypothalamic Nestin Expression Hypothalamic NS from 1 day old Control () and LBW () males were cultured in complete media. On second day of seeding, NS were treated with leptin/insulin every 48h for 8 days. Nestin protein expression (marker of NPC) was determined. Values are mean ± SE; * P < 0.05 vs. untreated NS; “^C” P < 0.05 LBW vs. Control.

Figure 3

A: Evidence for NPC and Neuronal Leptin Receptor NPC grown in complete or differentiating media were immunostained for leptin receptor (ObRb), neuronal marker (Tuj1) and nuclear stain (DAPI). B: Leptin and Insulin Stimulated Hypothalamic NPC Proliferation Hypothalamic NPC from 1 day old Control () and LBW () males were cultured in complete media. On second day of seeding, NS were treated with leptin/insulin every 48h for 8 days and cell proliferation rate was determined. Values are percentage of untreated Control (mean ± SE); * P < 0.05 vs. untreated NS; “^C” P < 0.05 LBW vs. Control. C: Leptin and Insulin Stimulated Hypothalamic Nestin Expression Hypothalamic NS from 1 day old Control () and LBW () males were cultured in complete media. On second day of seeding, NS were treated with leptin/insulin every 48h for 8 days. Nestin protein expression (marker of NPC) was determined. Values are mean ± SE; * P < 0.05 vs. untreated NS; “^C” P < 0.05 LBW vs. Control.

Figure 3

A: Evidence for NPC and Neuronal Leptin Receptor NPC grown in complete or differentiating media were immunostained for leptin receptor (ObRb), neuronal marker (Tuj1) and nuclear stain (DAPI). B: Leptin and Insulin Stimulated Hypothalamic NPC Proliferation Hypothalamic NPC from 1 day old Control () and LBW () males were cultured in complete media. On second day of seeding, NS were treated with leptin/insulin every 48h for 8 days and cell proliferation rate was determined. Values are percentage of untreated Control (mean ± SE); * P < 0.05 vs. untreated NS; “^C” P < 0.05 LBW vs. Control. C: Leptin and Insulin Stimulated Hypothalamic Nestin Expression Hypothalamic NS from 1 day old Control () and LBW () males were cultured in complete media. On second day of seeding, NS were treated with leptin/insulin every 48h for 8 days. Nestin protein expression (marker of NPC) was determined. Values are mean ± SE; * P < 0.05 vs. untreated NS; “^C” P < 0.05 LBW vs. Control.

Figure 4

A: Leptin Stimulated Hypothalamic Neuronal Differentiation from NPC Hypothalamic NPC from 1 day old Control () and LBW () males were cultured in differentiating media. On second day of seeding, NPC were treated with leptin every 48h for 8 days. NPC were harvested and protein expression of neuronal markers, DCX (immature neurons), and Tuj1 and NeuN (mature neurons) were determined. Values are mean ± SE; * P < 0.05 vs. untreated NPC; “^C” P < 0.05 LBW vs. Control. B: Insulin Stimulated Hypothalamic Neuronal Differentiation from NPC Hypothalamic NPC from 1 day old Control () and LBW () males were cultured in differentiating media. On second day of seeding, NPC were treated with insulin every 48h for 8 days. NPC were harvested and protein expression of neuronal markers, DCX (immature neurons), and Tuj1 and NeuN (mature neurons) were determined. Values are mean ± SE; * P < 0.05 vs. untreated NPC; “^C” P < 0.05 LBW vs. Control. C: Leptin and Insulin Stimualted Hypothalamic Astrocyte Differentiation from NPC Hypothalamic NPC from 1 day Control () and LBW () males were cultured in differentiating media. On second day of seeding, NPC were treated with leptin or insulin every 48h for 8 days. NPC were harvested and protein expression of astrocyte marker, GFAP, were determined. Values are mean ± SE; * P < 0.05 vs. untreated NPC; “^C” P < 0.05 LBW vs. Control.

Figure 4

A: Leptin Stimulated Hypothalamic Neuronal Differentiation from NPC Hypothalamic NPC from 1 day old Control () and LBW () males were cultured in differentiating media. On second day of seeding, NPC were treated with leptin every 48h for 8 days. NPC were harvested and protein expression of neuronal markers, DCX (immature neurons), and Tuj1 and NeuN (mature neurons) were determined. Values are mean ± SE; * P < 0.05 vs. untreated NPC; “^C” P < 0.05 LBW vs. Control. B: Insulin Stimulated Hypothalamic Neuronal Differentiation from NPC Hypothalamic NPC from 1 day old Control () and LBW () males were cultured in differentiating media. On second day of seeding, NPC were treated with insulin every 48h for 8 days. NPC were harvested and protein expression of neuronal markers, DCX (immature neurons), and Tuj1 and NeuN (mature neurons) were determined. Values are mean ± SE; * P < 0.05 vs. untreated NPC; “^C” P < 0.05 LBW vs. Control. C: Leptin and Insulin Stimualted Hypothalamic Astrocyte Differentiation from NPC Hypothalamic NPC from 1 day Control () and LBW () males were cultured in differentiating media. On second day of seeding, NPC were treated with leptin or insulin every 48h for 8 days. NPC were harvested and protein expression of astrocyte marker, GFAP, were determined. Values are mean ± SE; * P < 0.05 vs. untreated NPC; “^C” P < 0.05 LBW vs. Control.

Figure 4

A: Leptin Stimulated Hypothalamic Neuronal Differentiation from NPC Hypothalamic NPC from 1 day old Control () and LBW () males were cultured in differentiating media. On second day of seeding, NPC were treated with leptin every 48h for 8 days. NPC were harvested and protein expression of neuronal markers, DCX (immature neurons), and Tuj1 and NeuN (mature neurons) were determined. Values are mean ± SE; * P < 0.05 vs. untreated NPC; “^C” P < 0.05 LBW vs. Control. B: Insulin Stimulated Hypothalamic Neuronal Differentiation from NPC Hypothalamic NPC from 1 day old Control () and LBW () males were cultured in differentiating media. On second day of seeding, NPC were treated with insulin every 48h for 8 days. NPC were harvested and protein expression of neuronal markers, DCX (immature neurons), and Tuj1 and NeuN (mature neurons) were determined. Values are mean ± SE; * P < 0.05 vs. untreated NPC; “^C” P < 0.05 LBW vs. Control. C: Leptin and Insulin Stimualted Hypothalamic Astrocyte Differentiation from NPC Hypothalamic NPC from 1 day Control () and LBW () males were cultured in differentiating media. On second day of seeding, NPC were treated with leptin or insulin every 48h for 8 days. NPC were harvested and protein expression of astrocyte marker, GFAP, were determined. Values are mean ± SE; * P < 0.05 vs. untreated NPC; “^C” P < 0.05 LBW vs. Control.

Figure 5

A: Leptin Induced Notch1 Signaling during NPC Proliferation Hypothalamic NPC from 1 day old Control () and LBW () males were cultured in complete media. On second day of seeding, NPC were treated with leptin every 48h for 8 days. NPC were harvested and protein expression of Notch1 activation (cleaved Notch1, ICD) and Hes1 were determined. Values are mean ± SE; * P < 0.05 vs. untreated NPC; “^C” P < 0.05 LBW vs. Control. B: Insulin Induced Notch1 Signaling during NPC Proliferation Hypothalamic NPC from 1 day old Control () and LBW () males were cultured in complete media. On second day of seeding, NPC were treated with insulin every 48h for 8 days. NPC were harvested and protein expression of Notch1 activation (cleaved Notch1, ICD) and Hes1 were determined. Values are mean ± SE; * P < 0.05 vs. untreated NPC; “^C” P < 0.05 LBW vs. Control.

Figure 5

A: Leptin Induced Notch1 Signaling during NPC Proliferation Hypothalamic NPC from 1 day old Control () and LBW () males were cultured in complete media. On second day of seeding, NPC were treated with leptin every 48h for 8 days. NPC were harvested and protein expression of Notch1 activation (cleaved Notch1, ICD) and Hes1 were determined. Values are mean ± SE; * P < 0.05 vs. untreated NPC; “^C” P < 0.05 LBW vs. Control. B: Insulin Induced Notch1 Signaling during NPC Proliferation Hypothalamic NPC from 1 day old Control () and LBW () males were cultured in complete media. On second day of seeding, NPC were treated with insulin every 48h for 8 days. NPC were harvested and protein expression of Notch1 activation (cleaved Notch1, ICD) and Hes1 were determined. Values are mean ± SE; * P < 0.05 vs. untreated NPC; “^C” P < 0.05 LBW vs. Control.