Effects of peroxisome proliferator activated receptor gamma (PPARγ) agonist on fasting model applied neuron cultures

Introduction

In recent years, effects of calorie restriction (CR) and prolonged fasting on brain functions have been studied in experimental and clinical studies. In both cases, since there is a glucose restriction, brain uses ketone bodies as an alternative energy source. It is known that ketogenic diet is strongly effective in epileptic patients who are resistant to anti-epileptic drugs [1]. Ketogenic diet primarily consists of high-fats, moderate-proteins, and low-carbohydrates which leads the body to synthesize ketones as a result of low glucose in blood. Our group is working on the effects of the prolonged fasting model, an alternative to ketogenic diet, and ketone bodies in humans and neuronal cells.

The present study is inspired by the newly discovered positive effects of peroxisome proliferator-activated receptor (PPAR) gamma agonists (PPARγ), used for the treatment of Diabetes Mellitus (DM), on neuron and mitochondrial damage [2, 3]. Ketogenic diet is shown to activate PPARγ by many mechanisms [2, 4]. In prolonged fasting ketone bodies, secreted in high concentrations, can also activate PPARγ pathways.

Peroxisome proliferator-activated receptors (PPARs) are proteins belong to superfamily of nuclear hormone factors, that are nuclear receptors and ligand dependent transcription factors. Structurally, PPARs are similar to steroid or thyroid hormone receptors and are stimulated in response to small lipophilic ligands. It is shown that they have critical role on the regulation of mitochondrial biogenesis, inflammation, anti-oxidant defense and pathways related to carbohydrate and lipid metabolisms [5]. PPARs have three isotypes (PPARα, PPARδ and PPARγ) and play an essential role in the energy metabolism; however, they differ in the spectrum of their activity and tissue distribution; PPARγ regulates energy storage, whereas PPARα is expressed predominantly in the liver, muscle, heart and bone and PPARδ is ubiquitously expressed in whole body regulating the energy expenditure [3].

Thiazolidinediones (TZDs) are widely studied PPARγ ligands. Acting as sensors of hormones, vitamins, endogenous metabolites, and xenobiotic compounds, PPARγs are nuclear receptors which control the expression of a very large number of genes. PPARγ has been known to regulate adipocyte differentiation, fatty acid storage and glucose metabolism, which makes it a target of antidiabetic drugs [6], [7], [8]. PPARγ agonist improves insulin resistance by opposing the effect of TNF-α in adipocytes [9]. Moreover, PPARγ agonists are shown to have effects in Parkinson, Alzheimer disease and brain injury. They are thought to act on microglial cells inhibiting their activation. Recently, PPARs have been shown to modulate inflammation by inhibiting the production of proinflammatory molecules by glial cells. In vivo oral administration of the PPARγ agonist pioglitazone reduced glial activation and the accumulation of Aβ-positive plaques in the hippocampus and cortex [2, 3].

All these information about the effects of PPARγ on neurodegenerative damage made us think that, PPARγ agonist pioglitazone may also be effective on neuronal mitochondrial functions together with ketone bodies. Our aim was to investigate if pioglitazone can be an alternative in the treatment of neuron damage together with ketone bodies. In our study, SH-SY5Y (human neuroblastoma-ATCC/CRL 2266) cell cultures were performed and glucose deprived fasting model was applied to these neurons. In physiological conditions, fasting is accompanied by ketone bodies so neurons were treated by beta-hydroxybutyrate and further by pioglitazone to investigate the effects on the energy metabolites, mitochondrial functions and viability of the cells.

Materials and methods

Experiments

When cells were 80% confluent, normal media were withdrawn, flasks were washed with 2 mL of sterile phosphate buffered saline (PBS) and experiment media were added. 1 mL of the unused media were allotted for the measurements of energy metabolites (glucose, lactate, βOHB and Lactate dehydrogenase – LDH) before the experiments (initial values, i). Experiments were started the day before, and ended after 16-h incubation next morning. Experiment media were removed, 1 mL of them were allotted for the metabolic measurements again (end values, e). Cells were removed with Trypsin/EDTA and the viability rates were controlled with trypan blue. Then cells were homogenized for citrate synthase activity. In addition, microscopic pictures were taken (initial and after the experiment) for morphological examinations. Experiments were repeated 7 times under the same conditions.

Results

Fasting model optimization studies

To determine the incubation period for glucose deprivation model, 10⁵ cells were planted in 12-well flasks, each with an area of 4 cm², in the 6th passage and when the density was 80% normal growth media were removed and experiment media were added in three different groups: normal (N), fasting (F) and no glucose (G0). These media were added in a total volume of 2 mL according to the flask plan and cells were incubated in the aforementioned media for 4, 8, 12, 16, and 20 h. Media were collected from the wells at the beginning and at the end of each incubation, and the amount of glucose, lactate, LDH were analyzed (data not shown) together with cell viability (Figure 1). This experiment was repeated three times and 16 h of incubation period was decided by comparing all the results obtained. This decision was based on the LDH levels of the media, viability of the cells and the simulation of fasting for more than 12 h.

Figure 1:

Cell viabilities in the normal and glucose deprived model optimization studies (n=3). N, cells were incubated with DMEM/F12 + 10% FBS. F, cells were incubated with EBSS + 10% FBS. G0 (DMEM0 + 10% FBS, glucose-free medium), *p<0.01 when compared to the first 4 h of the same group.

Cell viability

Cellular viability was assessed by the MTT assay. Cell survival was expressed as percentage neuroprotection compared with normal group at 100% (Figure 2). Viability rates were not different in neurons incubated in the normal media with or without ketone and PPARγ agonist. However, viabilities of ketone and PPARγ agonist treated fasting group cells were higher (p<0.01, Figure 2).

Figure 2:

Cell viability is assessed with MTT assay. Data are expressed as mean ± SD (n=7). N, (normal) control group (DMEM F12 + 10% FBS-300 mg/dL glucose containing medium), NK, ketone added to normal medium (DMEM F12 + 10% FBS + 5 mM ßOHB), NKP, PPARγ agonist added to NK medium (DMEM F12 + 10% FBS + 5 mM ßOHB + 5 µM PPARγ agonist) F, glucose deprived media (EBSS + 10% FBS, 100 mg/dL glucose containing medium), FK, ketone added glucose deprived media (EBSS + 10% FBS, +5 mM ßOHB), FKP (PPARγ agonist added to FK medium (EBSS + 10% FBS, +5 mM ßOHB + 5 µM PPARγ agonist). *p<0.01, when compared to normal (N).

Citrate synthase activity

In our experiments, citrate synthase activity was measured in cells after 16 h of incubation with the experiment media. From the activity graphs, the time when the enzyme reaches the highest speed (Vmax) was calculated in minutes and is given in Figure 3. Cells incubated with normal medium reached the Vmax value fastest. It was observed that enzyme was working slowly as medium glucose content was decreased (Figure 3). In cells with the normal media, enzyme activity did not change significantly although mean velocity was slightly increased in cells containing both ketone and PPARγ agonist together (Figure 3). Enzyme velocity was found to be statistically increased in media containing ketone and further PPARγ agonist (Figure 3) in the fasting group. Additionally, enzyme activity observed to be faster in FK and FKP group than the F group which suggests in the deprivation of glucose, cells use ketone efficiently and may restore mitochondrial energy pathways in the presence of PPARγ.

Figure 3:

Citrate synthase activity was assessed after 16 h incubation with the experimental media. The time, when the enzyme reaches at the highest speed (Vmax) is calculated in minutes and given in the graphic (*p<0.01, when compared to normal). Data are expressed as mean ± SD (n=7). N, (normal) control group (DMEM F12 + 10% FBS-300 mg/dL glucose containing medium), NK, ketone added to normal medium (DMEM F12 + 10% FBS + 5 mM ßOHB), NKP, PPARγ agonist added to NK medium (DMEM F12 + 10% FBS + 5 mM ßOHB + 5 µM PPARγ agonist) F, glucose deprived media (EBSS + 10% FBS, 100 mg/dL glucose containing medium), FK, ketone added glucose deprived media (EBSS + 10% FBS, +5 mM ßOHB), FKP (PPARγ agonist added to FK medium (EBSS + 10% FBS, +5 mM ßOHB + 5 µM PPARγ agonist).

Discussion

In the present study, effects of peroxisome proliferator activated receptor gamma (PPARγ) agonist (pioglitazone) on glucose deprived, ketone added (fasting model) neuron cultures in terms of neuron viability, energy metabolism and mitochondrial functions were investigated in order to evaluate alternative treatment options in neurodegenerative diseases.

SH-SY5Y (human neuroblastoma-ATCC/CRL 2266) cell line was used in the study. SH-SY5Y is a human derived cell line used in vitro models of neuronal function and differentiation. The original cell line was isolated from a bone marrow biopsy taken from a four-year-old female with neuroblastoma. Normal nutrition and fasting models were applied to the 80% confluent cultured neurons by changing the glucose concentrations of the media [13]. Experiments were carried out by adding ketone and pioglitazone separately or together to the media.

When pioglitazone is added to the normal (N) and fasting (F) media alone it does not cause any statistically significant difference in metabolic parameters and cell viability in neurons (Table 2). However, when we simulate physiologic fasting conditions with ketone addition to the media (F and FK vs. FKP), metabolic parameters and cell survival rates were all changed in a positive manner (Table 3 and Figure 2). In the fasting group when pioglitazone was added to ketone containing medium (FKP), lactate and LDH levels were significantly lower than the F and FK group. Additionally neurons used ketones more efficiently in the presence of PPARγ agonist (Table 3). These findings made us think that, when pioglitazone added to the media, neurons might have used their mitochondria more efficiently (Figure 3). So we have conducted an other study with citrate synthase activity of the cells. Since citrate synthase is the first enzyme of Krebs cycle which takes place only in the mitochondrial matrix, activity measurements of citrate synthase in a cell homogenate, is accepted to show the mitochondrial capacity of a cellular community [14]. Growing evidence suggests the involvement of mitochondrial dysfunction in the pathogenesis of many major neurodegenerative and neuroinflammatory disorders. This dysfunction is associated with defects in mitophagy, bioenergetics, mitochondrial functions such as electron transport chain activity reductions and increased mitochondrial-generated oxidative stress [2]. Therefore, pharmacological approaches to enhance mitochondrial function, reduce reactive oxygen species (ROS) or enhance antioxidant defence are essential for the new therapeutic approaches to neurodegenerative and neuroinflammatory diseases.

It is shown that, knockout of PPARγ is embryonically lethal [15, 16]. Moreover, PPARγ activation is associated with increased lipid and carbohydrate metabolism together with adipocyte differentiation and reduction of plasma glucose concentration [17]. PPARγ agonists are currently in clinical use for the management of type 2 diabetes and pioglitazone, one of the most frequently used PPARγ agonists, was also been proven to have anti-inflammatory effects by PPARγ activation and regulating myelin gene expression in brain cells [18]. It is known that, PPARγ is expressed in different parts of brain. High levels of expression of PPARγ have been found in discrete areas of the central nervous system (CNS), including the dopaminergic cells in the basal ganglia [19, 20]. PPARγ, is also thought to regulate inflammatory response by suppressing the expression of more than one proinflammatory genes [21]. Moreover it is shown that inhibiting mammalian target of rapamycin (mTOR) pathway with PPARγ agonists had an anti-inflammatory effect on status epilepticus models in rats [22]. mTOR pathway inhibition is one of the major mechanisms of fasting in humans leading to ketogenesis [23].

In the present study, we have showed positive effects of PPARγ agonist on neuron survival, energy metabolism and mitochondrial functions which were enhanced with ketone body (βOHB) presence in the media. Changes in mitochondrial functions and survival rates had been studied with PPARγ agonists, pioglitazone and rosiglitazone on differentiated SH-SY5Y neuroblastoma cells before [24]. Miglio et al. [24] showed PPARγ agonists promoted biogenesis of mitochondria in glucose deprived neurons while did not significantly change mitochondrial membrane potential. However, to our knowledge this is the first study showing positive effects of pioglitazone on neurons can be synergistically increased by ketones.

Ketone body supply and metabolism is essential in the brain. Ketolysis accounts for 70% of the energy supply during prolonged fasting (the majority is from βOHB), and the rest, comes from glucose derived from different sources: glycerol, amino acids, lactate and pyruvate catabolism [23]. Apart from their roles in the fasting metabolism, more recently, ketone bodies have been shown to have direct signaling capabilities [1]. There are multiple mechanisms through which ketone bodies have been thought to affect cellular signaling by modulating the activity of histone deacetylases (HDACs) [2, 23]. Ketone bodies, particularly βOHB, also shown to have anti-inflammatory effects by reducing inflammatory cytokine production, which may further limit neuronal cell loss and facilitate tissue regeneration following injury. With these aforementioned findings and in the light of our results we can suggest that PPARγ agonists and ketones may act synergistically in neuron protection. It has been shown that PPARγ agonist (pioglitazone) treated rats had increased ß-oxidation [25] which can enhance ketogenesis explaining the synergistic effects of these two on neuron protection.

There are certain limitations of this study. The present study is performed in secondary cell cultures with neuroblastoma cells which can have some differences in the regulation of metabolic pathways hence our results must be confirmed by primary neuron culture studies. Second limitation is, we have used βOHB as key ketone body in the treatments and did not test acetoacetate which is another ketone body synthesized by the liver. Another limitation is although it is shown that pioglitazone as the main molecule affects the metabolism, its metabolite hydroxypioglitazone is shown to be effective in vivo [25]. We have not tested its effects in the experiments. Finally mitochondrial functions were measured with metabolic parameters and citrate synthase activity in the present study that need to be confirmed with other investigations such as flowcytometric membrane potential analysis.

However, PPARγ agonist addition to glucose deprived and ketone added (fasting model applied) neurons caused positive improvements with metabolic parameters, mitochondrial functions, and survival rates. Changes in mitochondrial citrate synthase activity, lactate and LDH levels of neuronal cells treated with PPARγ and βOHB have not been previously pointed out in the literature. Based on all these findings; we can conclude that improvements in neuron damage were recorded after pioglitazone (PPARγ agonist) treatment especially in the presence of ketones. We suggest that the PPARγ agonists in fasting states show significant promise as therapeutic agents in neurological diseases and repositioning of pioglitazone may be a new approach for the treatment of neurodegenerative diseases. However, the role of pioglitazone in the treatment of neurodegenerative diseases and the integration with ketones should be further investigated by functional genetic pathway analysis and clinical studies.