Orexin A attenuates palmitic acid-induced hypothalamic cell death

Abstract

Palmitic acid (PA), an abundant dietary saturated fatty acid, contributes to obesity and hypothalamic dysregulation in part through increase in oxidative stress, insulin resistance, and neuroinflammation. Increased production of reactive oxygen species (ROS) as a result of PA exposure contributes to the onset of neuronal apoptosis. Additionally, high fat diets lead to changes in hypothalamic gene expression profiles including suppression of the anti-apoptotic protein B cell lymphoma 2 (Bcl-2) and upregulation of the pro-apoptotic protein B cell lymphoma 2 associated X protein (Bax). Orexin A (OXA), a hypothalamic peptide important in obesity resistance, also contributes to neuroprotection. Prior studies have demonstrated that OXA attenuates oxidative stress induced cell death. We hypothesized that OXA would be neuroprotective against PA induced cell death. To test this, we treated an immortalized hypothalamic cell line (designated mHypoA-1/2) with OXA and PA. We demonstrate that OXA attenuates PA-induced hypothalamic cell death via reduced caspase-3/7 apoptosis, stabilization of Bcl-2 gene expression, and reduced Bax/Bcl-2 gene expression ratio. We also found that OXA inhibits ROS production after PA exposure. Finally, we show that PA exposure in mHypoA-1/2 cells significantly reduces basal respiration, maximum respiration, ATP production, and reserve capacity. However, OXA treatment reverses PA-induced changes in intracellular metabolism, increasing basal respiration, maximum respiration, ATP production, and reserve capacity. Collectively, these results support that OXA protects against PA-induced hypothalamic dysregulation, and may represent one mechanism through which OXA can ameliorate effects of obesogenic diet on brain health.

Keywords: Apoptosis; Hypocretin; Neurodegeneration; Neuroprotection; Palmitic acid; Reactive oxygen species.

Figure 1. Orexin A attenuates PA-induced hypothalamic cell death

A) mHypoA-1/2 cells were pretreated with OXA for 24 h and then incubated with PA in the presence or absence of OXA or DORA for an additional 24 h. PA significantly reduces cell viability (p<0.001 vs. vehicle control; C). OXA attenuates PA induced cell death (p<0.05 vs. OXA only and OXA+PA). DORA negates OXA induced cell survival (p<0.001 vs. OXA only and OXA plus PA). B) PA significantly increases caspase-3/7 activity (p<0.0001 vs. C). OXA reduces caspase 3/7 induced apoptosis (p<0.0001 vs. OXA only, p<0.001 vs. OXA+PA).

Figure 2. Orexin A reduces reactive oxygen species in mHypoA-1/2 cells

mHypoA-1/2 cells were pretreated with OXA for 24 h and then incubated with PA in the presence or absence of OXA for an additional 2 h. ROS is significantly increased following 2 h PA exposure (p<0.05 vs. C). OXA treatment attenuates PA-induced ROS production (p<0.001 vs. OXA only, p<0.05 vs. OXA+PA).

Figure 3. Orexin A increases Akt phosphorylation

mHypo-A1/2 cells were pretreated with OXA for 24 h and then exposed to PA in the presence or absence of OXA for an additional 1 h. Phosphorylated Akt is significantly increased following exposure to OXA in the presence or absence of PA (p<0.05 vs. C, p<0.001 vs. PA).

Figure 4. OXA stabilizes expression of anti-apoptotic gene Bcl-2

mHypoA-1/2 cells were pretreated with OXA for 24 h and then challenged with PA in the presence or absence of OXA for an additional 2 h. A) PA significantly reduces Bcl-2 expression (p<0.05 vs. C). OXA stabilizes Bcl-2 expression (p<0.05 vs. OXA only and OXA+PA). B) The Bax/Bcl-2 ratio is significantly increased following PA challenge (p<0.05 vs. C). However, OXA stabilizes Bax/Bcl-2 ratio (p<0.001 vs. OXA only, p<0.05 vs. OXA+PA).

Figure 5. OXA alters metabolic respiration in a dose dependent manner

Oxygen consumption rates (OCRs) were determined in mHypoA-1/2 cells treated with increasing concentrations of OXA (50, 150, 300 nM) for 2 h. OXA increases B) basal respiration (p<0.05 vs. C), C) ATP turnover (p<0.05 50, 300 nM OXA vs. C, p<0.001 150 nM OXA vs. C), D) maximum respiration (p<0.05 150, 300 nM OXA vs. C), and E) reserve capacity (p<0.05 150, 300 nM OXA vs. C).

Figure 6. Orexin induced metabolic respiration is directly mediated by orexin receptors

Oxygen consumption rates (OCRs) were determined in mHypoA-1/2 cells treated with OXA (300 nM) and/or DORA (1.16 nM) for 2 h. OXA increases B) basal respiration (p<0.0001 vs. C), C) ATP turnover (p>0.001 vs. C), D) maximum respiration (p<0.0001 vs. C), and E) reserve capacity (p<0.05 vs. C). Treatment with DORA attenuates OXA-increased B) basal respiration (p<0.0001 vs. OXA), C) ATP turnover (p<0.0001 vs. OXA), D) maximum respiration (p<0.0001 vs. OXA), and E) reserve capacity (p<0.0001 DORA vs. OXA, p<0.001 OXA+DORA vs. OXA).

Figure 7. Orexin stabilizes metabolic respiration

Oxygen consumption rates (OCRs) were determined in mHypoA-1/2 cells pretreated with OXA (300 nM) for 24 h and challenged with PA in the presence or absence of OXA for 6 h. PA significantly reduces B) basal respiration (p<0.0001 vs. C, OXA, OXA+PA), C)ATP turnover (p<0.0001 vs. C, OXA, p<0.001 vs. OXA+PA), D) maximum respiration (p<0.0001 vs. OXA, p<0.001 vs. C, OXA+PA), and E) reserve capacity (p<0.05 vs. C, OXA+PA, p<0.0001 vs. OXA). Treatment with OXA stabilizes PA-reduced B) basal respiration, C) ATP turnover, D) maximum respiration, and E) reserve capacity.