The polyunsaturated n-3 fatty acid has been implicated as a health fatty acid . The present study showed that 8 weeks of intake of the high-fat fish oil diet, rich in EPA (eicosapentaenoic acid) and DHA (docosahexaenoic acid), caused only a small non-significant increase of body weight in comparison to the control diet, along with a light increase of caloric intake and feed efficiency. These results agree with previous findings of our laboratory [5–8] and indicate that the fish oil diet was protective against the potentially deleterious effects of high-fat intake. Accordingly, fish oil intake has been found to reduce the adverse effect of overfeeding in early life in rats .
The hypothalamus receives inputs from gastrointestinal-borne molecules, including the nutrients themselves and factors induced by nutrients, and this sensing affects the hypothalamic control of energy homeostasis . Hypothalamic neuroinflammation induced by high-fat intake of saturated or n-6 PUFAs has been shown to impair this homeostatic role of the hypothalamus . Contrarily, the chronic high-fat intake of fish oil diet decreased hypothalamic levels of IL-6 and TNF-α .
To better understand the effects of high-fat fish oil diet intake, we performed a proteomic analysis of the hypothalamus. We found that the 29 proteins differentially expressed in the Fish group belonged to 4 different main pathways: Metabolism of Carbohydrate, TCA cycle and Respiratory Electron Transport, Innate Immune System, and Cellular Response to Stress.
Included in the Metabolism of Carbohydrate pathway, the Glucose Metabolism pathway had 5 affected proteins. Phosphoglycerate mutase 1 was downregulated by 16% while the othe 4 proteins were upregulated: aspartate aminotransferase, mitochondrial (26%), Glucose-6-phosphate isomerase (15%), Malate dehydrogenase, mitochondrial (10%), and Alpha-enolase (13%).
Glucose-6-phosphate isomerase, Phosphoglycerate mutase 1, Alpha-enolase participate in the glycolytic pathway, but they do not play a regulatory role in the pathway. Glucose-6-phosphate isomerase catalyzes the isomerization of glucose-6-phosphate to fructose-6-phosphate. A 20% decrease of this protein in the cerebral cortex of diabetic rats after carotid occlusion has been shown to exacerbate the ischemia-induced damage . This enzyme is also known as neuroleukin and is involved in neuronal growth, differentiation, and motility, besides its function in glucose metabolism. Inhibition of this protein in PC12 cells increased the susceptibility to apoptosis . In the present work, its increased expression in the Fish group could represent a protective mechanism.
Phosphoglycerate mutase 1 acts on the conversion of 3-phosphoglycerate to 2-phosphoglycerate in glycolysis. Its levels have been found to increase in human glioma tissue, proportionally to tumor grade, as well as in C6 glioma cells, and its knockdown inhibited cell proliferation and promoted apoptosis in U87 glioma cells [22–23]. The increased expression of this protein in cancer cells is consistent with the increase of glycolysis as main source of energy supply to the tumor . On the other hand, its super-expression in mouse hippocampus facilitated phosphorylation of cAMP response element-binding protein (CREB) and increased cellular proliferation and neuroblast differentiation, responses indicative of a possible role of this protein in promoting tissue regeneration . In the HT22 hippocampal cell line, phosphoglycerate mutase 1 activation attenuated ischemia-induced cell damage and decreased reactive oxygen species levels. In rodents, super-expression of this protein decreased the damage of the CA1 hippocampal area, after 4 days of brain ischemia . The above data indicated that, although deleterious when expressed in cancer cells, phosphoglycerate mutase 1 had beneficial effects in the response of hippocampal cells to injury. We could not find papers on the hipothalamic status of this protein in response to challenges potentialy affecting the hypothalamus. Importantly, in rat liver, six weeks of a high-fat saturated diet induced steatosis and increased phosphoglycerate mutase 1 by 57%, while the combination of Fuzhuan brick tea restored enzyme levels to those of control animals and ameliorated the hepatic fat accumulation .
Alpha-enolase is one of the sub-units of the enzyme enolase, responsible for the interconversion of 2-phosphoglycerate and phosphoenolpyruvate in glycolysis. In human brain, alpha-enolase has been located mainly in the glia . A proteomic study has found increase of alpha-enolase in the hippocampus and cingulate gyrus of Alzheimer`s disease patients . On the other hand, analyzing oxidized proteins in the cortex Alzheimer`s disease patients another proteomic study found increased levels. The authors suggested that oxidation could lead to loss of protein function and impair glycolysis . This is in agreement with data showing glucose hypometabolism in this pathology . Interestingly, a prospective study found that the intake of n-3 fatty acids-rich seafoods by elderly subjects protected from the development of Alzheimer`s Disease, a finding that was associated with DHA intake .
Two mitochondrial enzymes of the malate-aspartate shuttle (MAS), aspartate aminotransferase and malate dehydrogenase, showed increased levels in the Fish group. MAS transfers to mitochondria the electrons of the NADH generated in the citosol during glycolysis. Decreased levels of mitochondrial MAS proteins have been found in a proteomic analysis of postmortem brain tissue (putamen, thalamus and parietal lobe) of schemic stroke victims . In microglial Bv2 cells, the inhibition of aspartate aminotransferase decreased intracellular ATP levels and caused activation of apoptosis .
In the present study, aldo-keto reductase family 1 member B1, an enzyme important in detoxification processes, showed increased levels in the Fish group. In rats, increased levels of this enzyme were found in astrocytes after spinal cord injury, and associated with increased proliferation of astrocytes through stimulation of Akt pathways and glucose utilization . On the other hand, it has also been attributted a pro-inflammatory role through its involvement in prostaglandins metabolism . Increased levels have been reported in diabetes, asthma and sepsis and its inhibition decreased oxdative stress in these pathologies .
Additionaly, malate dehydrogenase is the last enzyme of the TCA cycle, convering malate to oxaloacetate with the production of 1 NADH. In synaptossomes, high levels of oxidative stress impaired NADH production by blocking aconitase and alpha-ketoglutarate dehydrogenase enzymes . In HT22 hippocampal cells, the induction of oxidative stress up regulated mitochondrial malate dehydrogenase mRNA levels by 22% . Since high-fat intake may induce oxidative stress , the increase of malate dehydrogenase seen in the present study could represent an attempt to maintain NADH levels in TCA.
The TCA cycle and respiratory electron transport pathway had other 4 affected proteins. Two proteins were upregulated: L-lactate dehydrogenase A chain (22%) and dihydrolipoyllysine-residue acetyltransferase component of pyruvate dehydrogenase complex, mitochondrial (20%). NADH dehydrogenase [ubiquinone] flavoprotein 2, mitochondrial, was downregulated by 16% and NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 9, mitochondrial, was downregulated by 24%
L-lactate dehydrogenase A chain is part of the lactate dehydrogenase enzyme, responsible for the conversion of glucose-derived pyruvate into lactate. In the brain, this enzyme is present mainly in astrocytes . Astrocytes form lactate and release it to neurons, where it is transformed in pyruvate . Lactate has been shown to have anorexigenic properties and L-lactate dehydrogenase is important to this function. The intracerebroventricular (i.c.v.) injection of pyruvate or lactate decreased food intake and body weight gain, effects abolished by the suppression of L-lactate dehydrogenase . Blocking L-lactate dehydrogenase centrally has been shown to also suppress the hypophagia induced by intraperitoneal lactate . The increased of L-lactate dehydrogenase found in the present study could explain, at least in part, the absence of excess caloric intake of the Fish group.
The protein dihydrolipoyllysine-residue acetyltransferase is a component of the E2 enzyme of the pyruvate dehydrogenase complex, responsible for formation of acetyl-CoA from glycolysis-derived pyruvate. This enzyme has been attributed a protective role in situations of metabolic impairment. After expose of rats to chronic mild stress, the up-regulation of its hippocampal levels protected against the development of depressive- like symptoms . Mild brain injury led to increased levels of the 3 enzymes of pyruvate dehydrogenase complex while severe injury decreased their levels. The authors indicated that the increased levels in mild brain injury was to protect the metabolism during a transiently malfunction of mitochondria .
The proteins NADH dehydrogenase [ubiquinone] flavoprotein 2 and NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 9, downregulated in the Fish group, are subunits of Complex I of the respiratory chain. Complex 1 has been found to generate more reactive oxygen species (ROS) than Complex III . Low levels of NADH dehydrogenase [ubiquinone] flavoprotein 2 has been associated lower ROS production and prolonged longevity . On the other hand, the knocking out of NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 9 in HEK293T cells impaired complex I assembly and the cells were unable to utilize galactose as a carbon source, indicating that oxidative phosphorylation was disrupted . Also, decressed levels of this enzyme have been found in the brain of Alzheimer’s Disease patients .