Role of Forkhead Transcriptional Factor O1 (FoxO1) in
Dopaminergic neuron system in the central nervous system
Dopaminergic (DA) neurons, though comprising less than 1% of the central nervous system (CNS), play a crucial role in regulating various physiological functions in mammals, including movement, mood, reward, cognition, attention, memory, sleep, and prolactin production These neurons are integral to the catecholaminergic system in the brain Recent advancements in immunohistochemistry have enhanced our ability to differentiate between various catecholamines, facilitating detailed mapping of the DA neuron system in rodents There are nine major DA cell groups, designated A8 to A16, which are extensively distributed from the mesencephalon to the olfactory bulb.
Figure 1 Dopaminergic cells and projections in the rodent brain Adapted from Bjorklund and Dunnett Trends Neurosci, 2007
Approximately 90% of dopamine (DA) neurons are located in the ventral mesencephalon of the midbrain, comprising three main cell groups: A8, A9, and A10 The A9 neurons in the substantia nigra project to the striatum, playing a crucial role in motor control through the nigrostriatal pathway Meanwhile, the A10 neurons in the ventral tegmental area connect to the limbic system and cortex, influencing emotional responses, reward behavior, and motivation via the mesocorticolimbic pathway Additionally, the A8 retrorubral DA neurons are believed to coordinate the functions of both the nigrostriatal and mesolimbic systems.
The five dopamine (DA) cell groups in the hypothalamus, ranging from A11 to A15, play a crucial role in the endocrine system While the function of A11 DA neurons projecting to the spinal cord remains unclear, A13 DA neurons are involved in regulating gonadotropin-releasing hormone within the hypothalamus Most DA neurons in the arcuate nucleus (A12) and the preoptic area/anterior hypothalamus (A14) are classified as endocrine neurons, releasing dopamine into the portal blood system for storage in the pituitary gland These neurons are essential for controlling the secretion of prolactin and growth hormone from the anterior pituitary and for regulating melanocyte-stimulating hormone release from the intermediate lobe of the pituitary gland.
Dopamine is synthesized from the amino acid tyrosine in presynaptic neurons by two sequential reactions which are catalyzed by enzyme tyrosine
In catecholaminergic neurons, dopamine is synthesized from L-tyrosine through the action of the rate-limiting enzyme, tyrosine hydroxylase (TH), and is subsequently converted into norepinephrine by aromatic amino acid decarboxylase (AADC) and dopamine-β-hydroxylase (DBH).
Dopamine, released from presynaptic neurons, binds to its receptors on postsynaptic neurons, activating key intracellular signaling pathways, including adenylate cyclase and protein kinase A There are five dopamine receptor types (D1 to D5), all part of the G-protein-coupled receptor family The D1 and D5 receptors enhance intracellular cAMP levels by coupling with Gαs, while the D2, D3, and D4 receptors reduce cAMP levels by coupling with Gαi and inhibiting adenylate cyclase Dopamine is removed from the synaptic cleft through reuptake by the dopamine transporter (DAT) or by enzymatic degradation via monoamine oxidase (MAO) and catechol-O-methyltransferase (COMT).
Role of dopaminergic system in energy balance and obesity
1.2.1 Dopamine, food reward and obesity
Extensive evidence highlights the crucial role of the dopamine (DA) system in integrating metabolic signals to regulate energy balance, often linked to its function in reward modulation The widespread distribution of DA neurons in the brain, particularly in the nigrostriatal, mesolimbic/mesocortical, and tuberoinfundibular (hypothalamic) pathways, indicates that each component uniquely influences various aspects of eating behavior Notably, DA neurons in the hypothalamus are essential for maintaining basal metabolic functions.
The regulation of food intake is influenced by the midbrain dopamine (DA) neurons originating in the ventral tegmental area (VTA) and projecting to the nucleus accumbens (NAc), which play a crucial role in food reward and motivation Additionally, emotional responses to feeding are driven by mesocortical DA neurons, while the sensory-motor aspects of eating are associated with DA neurons from the substantia nigra (SN) that project to the dorsal striatum, also known as the caudate putamen (CPu).
Dopamine (DA) in the hypothalamus plays a crucial role in regulating food intake, with distinct effects observed in the lateral hypothalamic area (LHA) and the ventromedial hypothalamus (VMH) Following a meal, dopamine levels in the LHA surge, correlating directly with food consumption, and high levels during eating signal meal termination, thus regulating meal size Pharmacological increases in LHA dopamine reduce food intake, while D2 antagonists can reverse the appetite-suppressing effects of amphetamines Conversely, dopamine levels in the VMH decrease after feeding, with the extent of this decrease linked to meal size, indicating that VMH dopamine concentration influences the duration between meals and overall meal frequency.
Dopamine (DA) signaling in the hypothalamus plays a crucial role in regulating food intake, while midbrain DA neurons are primarily involved in the reward and motivation aspects of feeding behavior The "dopamine hypothesis" suggests that DA neurons projecting from the ventral tegmental area (VTA) to regions like the nucleus accumbens (NAc), hippocampus, amygdala, and prefrontal cortex influence both "reward liking" and "reward wanting." Under normal conditions, these neurons exhibit tonic firing, resulting in a steady release of dopamine; however, significant environmental stimuli can trigger burst-firing, leading to phasic dopamine release This increased DA signaling directs an animal's focus toward reward cues and motivates goal-oriented behaviors aimed at obtaining rewards Additionally, heightened DA activity from VTA neurons is thought to enhance conditioned learning by associating rewards with salient environments Consequently, animals tend to engage in behaviors that elevate dopamine levels, such as drug self-administration or electrical stimulation of DA neuron-activating brain regions Conversely, reduced brain DA signaling can prompt excessive behaviors aimed at restoring dopamine levels.
A large body of literature has indicated the role of mesolimbic/mesocortical DA reward circuitries in motivational, reward aspects
Dopamine (DA) neurons in regions such as the amygdala, hippocampus, and orbitofrontal cortex play a crucial role in the hedonic value of food cues and the regulation of feeding behaviors Highly palatable foods, particularly those rich in sugars and fats, act as powerful natural rewards that activate the DA reward circuitry, enhancing eating behaviors even when energy requirements are met However, chronic consumption of these foods can lead to habituation in DA responses and disrupt neural circuits, resulting in altered brain reward systems and weakened control over eating Frequent intake of high-calorie foods can reset reward thresholds and contribute to overeating Additionally, similar to drug addiction, chronic intake of palatable foods reduces D2-receptor availability in the dorsal striatum, correlating with diminished reward responsiveness in obese individuals, which may further encourage overeating through compensatory mechanisms.
In addition to reduced D2 receptor level, other changes in striatal DA
Obese individuals, particularly those on a high-fat diet, exhibit significant changes in brain activity, characterized by lower basal and evoked dopamine levels in critical areas such as the nucleus accumbens, dorsal striatum, and medial prefrontal cortex compared to their lean counterparts This condition is linked to impairments in dopamine biosynthesis and release, leading to a deficiency in striatal dopamine signaling The reduced dopamine activity observed in obese individuals is likely associated with diminished expression of dopamine-related factors, including tyrosine hydroxylase, vesicular monoamine transporter, dopamine reuptake transporter (DAT), and D2 receptors.
Research indicates that the dopamine (DA) reward circuitry plays a significant role in obesity, as evidenced by neurogenetic, neuroimaging, and epidemiological studies Obese individuals show increased activation of brain reward circuits when exposed to palatable foods, in contrast to lean individuals.
Research indicates a significant link between reward sensitivity and body mass index (BMI), revealing that overweight individuals are more responsive to rewards than those of normal weight However, obese individuals (BMI > 30) experience higher levels of anhedonia, indicating a reduced sensitivity to rewards compared to their overweight counterparts Additionally, obese individuals exhibit diminished striatal dopamine (DA) signaling in response to palatable foods when compared to lean individuals Furthermore, those who gain weight show a decrease in striatal DA responses to the consumption of palatable foods, contrasting with individuals who maintain stable weight Collectively, these findings suggest that the reward-related DA pathways may become less responsive in the context of obesity.
During obesity development, hyperactivity can increase the risk of overeating palatable foods However, as overeating progresses, the function of striatal dopamine (DA) signaling diminishes This reduction in DA signaling may lead to adaptive responses that drive individuals to overconsume palatable foods in an attempt to restore their natural dopamine reward circuitry.
Research indicates a significant connection between midbrain dopamine (DA) reward circuitry and obesity, particularly in the context of Parkinson's disease (PD), which involves the degeneration of dopamine-producing neurons in the midbrain A comprehensive longitudinal study has found a correlation between midlife obesity and the subsequent onset of PD Additionally, PD patients typically consume less food compared to control subjects, and treatment with dopamine receptor agonists increases the desire for palatable foods Conversely, long-term use of D2 receptor antagonists is associated with a heightened risk of weight gain and obesity.
Research indicates that the dopamine (DA) system significantly influences the development and progression of obesity by modulating reward responses to dietary content, especially in modern societies with easy access to highly palatable foods Dysfunction in the midbrain DA reward circuitry is likely a key factor exacerbating the obesity epidemic today Consequently, developing therapeutic strategies that enhance this reward system may offer effective solutions for managing obesity.
DA function may be beneficial for prevention and/or treatment of obesity and
Recent studies indicate that certain dopamine receptor agonists have been approved or are undergoing clinical trials to enhance glycemic control and insulin sensitivity in patients with type 2 diabetes However, further research at molecular, cellular, and circuit levels is essential to clarify the mechanisms involved and to better understand the interplay between homeostatic and reward circuits that influence food intake and energy balance.
Recent literature on feeding and obesity highlights dopamine's primary role in mediating reward, particularly through the midbrain dopamine circuitry, which regulates food consumption and energy intake However, the role of dopamine in energy expenditure, a crucial aspect of energy homeostasis, is often overlooked in discussions about obesity Despite the limited information on dopamine's influence in this area, evidence suggests that dopamine also plays a significant role in modulating physical activity, a key contributor to energy expenditure This gap in research emphasizes the need to consider dopamine's involvement in both reward and energy balance for a comprehensive understanding of obesity.
Total energy expenditure is comprised of three key components: basal metabolic rate (BMR), physical activity, and adaptive thermogenesis, all regulated by the central nervous system (CNS) The brain directs voluntary physical activities, spontaneous movements, and shivering for heat dissipation through somatic motor nerves BMR is primarily managed by the hypothalamus via neuroendocrine systems, while the CNS also oversees adaptive thermogenesis in brown adipose tissue (iBAT) through sympathetic projections Recent research indicates that dopamine (DA) activity plays a significant role in both voluntary physical activity and iBAT thermogenesis.
Physical activity contributes 8-15% of total daily energy expenditure, primarily through muscle activity While most energy is used during voluntary exercise, non-exercise activity thermogenesis (NEAT) also plays a significant role Key brain regions involved in regulating physical activity include the mesencephalic locomotor region, locus coeruleus, VTA, SN, NAc, and tuberomammillary nucleus Research using wheel-running rodent models suggests that the dopamine system partially regulates voluntary physical activity Beeler's work aims to integrate various perspectives on dopamine's function within the broader context of energy management and its impact on energy expenditure.
Modulation of dopaminergic system by metabolic hormones
Endocrine factors, alongside the nervous system, are crucial for maintaining body homeostasis, with hormones like insulin, leptin, and ghrelin indicating carbohydrate and lipid storage levels These hormones influence brain signals that adjust food intake and energy expenditure While their roles in the hypothalamus are well-documented, research is expanding to other brain areas, particularly the midbrain dopamine (DA) circuitry, which is vital for reward and motivation Notably, insulin, leptin, and ghrelin receptors are present in DA neurons in the ventral tegmental area (VTA) and substantia nigra (SN) Evidence shows that ghrelin activates, while insulin and leptin inhibit, the midbrain DA pathway, thereby regulating feeding behavior and energy balance.
Insulin receptor and downstream substrates such as IRS2 and PI3K are expressed in DA neurons 23,25,26 Direct administration of insulin into the VTA
Insulin plays a significant role in regulating food intake by influencing the dopamine (DA) reward system in the brain Research indicates that a high-fat, sweetened diet can lead to reduced food consumption, particularly in sated conditions Insulin enhances the firing rate of 50% of DA neurons in the ventral tegmental area (VTA), which is associated with decreased dopamine levels due to the up-regulation of dopamine transporters (DAT) through a PI3K-mTOR pathway Additionally, the absence of insulin receptors in tyrosine hydroxylase-positive neurons increases sensitivity to sucrose and contributes to weight gain and hyperphagia In humans, insulin treatment has been shown to lower food intake, even for highly palatable foods Overall, insulin appears to modulate food consumption by acting on the neural DA reward system, although the exact mechanisms remain to be fully understood.
Research indicates that leptin negatively influences the midbrain dopamine (DA) reward system, playing a crucial role in regulating feeding behavior and food intake Direct injections of leptin into the ventral tegmental area (VTA) reduce mesolimbic DA signaling by lowering the firing frequency of DA neurons, which in turn decreases dopamine levels in the nucleus accumbens (NAc) and leads to reduced food consumption Additionally, leptin inhibits excitatory synaptic transmission onto DA neurons in the VTA Genetic studies utilizing viral-mediated RNA to knock down leptin receptors in the VTA have demonstrated increased sensitivity to palatable foods and higher food intake A recent optogenetic study further supports these findings by activating DA neurons to assess the reward value of nutrients, confirming leptin's negative impact on food reward.
21 effects of leptin on the reward value via reduction of DA signaling 85
Leptin significantly influences food reward in humans, as evidenced by functional MRI studies that reveal a positive correlation between ventral striatum activation in response to food cues, leptin levels, and body mass index (BMI) In leptin-deficient adolescents, leptin supplementation has been shown to decrease ventral striatum activation when exposed to food cues Additionally, in obese patients without a genetic leptin deficiency, leptin injections after weight loss resulted in reduced activation of reward system regions in response to food cues compared to those who did not receive treatment.
Leptin's effects on the dopamine (DA) system, particularly within the mesolimbic pathway, remain complex and not fully understood, with multiple mechanisms at play Research indicates that leptin exerts a synaptic inhibitory effect on DA neurons in the ventral tegmental area (VTA) through JAK2/STAT3 and PI3K signaling pathways, while its role in reducing food intake in the VTA relies solely on JAK2/STAT3 activation Additionally, leptin treatment has been shown to enhance the activity of tyrosine hydroxylase (TH) in the VTA and nucleus accumbens (NAc), thereby influencing dopamine production Notably, ob/ob mice exhibit reduced TH levels and decreased DA content in both the VTA and NAc However, leptin also leads to a reduction in both basal and evoked dopamine release in the NAc, which may be attributed to increased dopamine transporter (DAT) activity Further research is necessary to clarify these interactions.
22 required to elucidate the molecular action of leptin in DA neurons.
Forkhead box transcriptional factor O1
Forkhead box transcriptional factor O1 (FoxO1), a key member of the FoxO family of transcription factors, is the predominant isoform among the four mammalian FoxOs: FoxO1, FoxO3, FoxO4, and FoxO6 FoxO1 is widely distributed throughout the body, with high expression levels in insulin-responsive organs such as the liver, skeletal muscle, adipose tissue, and brain, as well as in insulin-producing pancreatic β cells While FoxO3 is also ubiquitously expressed, it is primarily found in the ovary, spleen, heart, and brain FoxO4 shows the highest expression in skeletal and cardiac muscle, along with adipose tissue, whereas FoxO6 is predominantly found in the central nervous system (CNS) Within the CNS, FoxO1 is most abundant in the striatum and certain regions of the hippocampus, while FoxO3 is primarily expressed in the cortex, hippocampus, and cerebellum, and FoxO6 is prevalent in the hippocampus, amygdala, and cingulate cortex.
FoxOs proteins are highly conserved and structurally consist of four domains: (1) a highly conserved forkhead DNA-binding domain, (2) nuclear localization sequences, (3) nuclear export sequences, and (4) a C-terminal
FoxO proteins, including FoxO1, FoxO3, and FoxO6, are crucial for various cellular functions, with FoxO1, FoxO3, and FoxO6 sharing a similar length of approximately 650 amino acids, while FoxO4 is shorter at about 500 amino acids These three isoforms possess conserved Akt phosphorylation sites, which play a significant role in regulating their subcellular localization and transcriptional activity In contrast, FoxO6 is primarily located in the nucleus due to the absence of the C-terminal Akt-dependent site, although its transcriptional function is still affected by the phosphorylation of the remaining Akt-dependent sites.
The transcriptional activity of FoxO1, along with other FoxO proteins, is influenced by factors such as stability, subcellular localization, target gene specificity, and DNA binding activity A key regulatory mechanism for FoxO1 is its subcellular localization, which is modulated by post-translational modifications, including phosphorylation, methylation, acetylation, and ubiquitination Notably, serine/threonine phosphorylation by Akt, a downstream effector of PI3K and SGK, plays a crucial role by promoting the translocation of FoxO1 from the nucleus to the cytoplasm, thereby inhibiting its ability to activate target genes Additionally, the acetylation and deacetylation of FoxO1 are regulated by the activities of p300/CBP and sirtuins (SIRTs), respectively Furthermore, physical interactions between FoxO1 and various factors, including nuclear receptors such as estrogen and androgen receptors, also contribute to its regulatory mechanisms.
24 progesterone receptor 105 and PPAR 106,107 also modulate its transcriptional activity
FoxO1 is widely recognized as a crucial downstream target of the insulin/IGF1/PI3K signaling pathway, playing significant roles in cell cycle regulation, apoptosis, stress response, metabolism, and longevity.
FoxO1 plays a crucial role in regulating whole body metabolic homeostasis by binding to specific consensus forkhead recognition elements, such as (T/C)(G/A)AAACAA and its complement TTGTTT(T/C)(G/A), as well as similar sequences like the insulin response element T(G/A)TTT This interaction allows FoxO1 to stimulate or suppress the expression of various target genes.
Role of FoxO1 in peripheral regulation of metabolism
FoxO1 is a crucial downstream effector of insulin signaling, significantly influencing various metabolic functions In the liver, it is essential for the regulation of both glucose and lipid metabolism.
FoxO1 plays a crucial role in enhancing gluconeogenesis by directly up-regulating essential enzymes such as glucose-6-phosphatase (G6Pase) and phosphoenol-pyruvate carboxykinase (PEPCK), thereby promoting hepatic glucose production Additionally, FoxO1 is implicated in regulating de novo lipogenesis and the secretion of triglycerides from the liver.
FoxO1 plays a crucial role in regulating lipid metabolism by down-regulating glucokinase and other glycolytic enzymes, which are essential for directing carbons into the hexose monophosphate shunt to support lipogenesis It also inhibits SREBP1c, a key regulator of lipogenic gene transcription, and suppresses genes involved in fatty acid synthesis, including fatty acid synthase and ATP citrate lyase Additionally, FoxO1 enhances the expression of apolipoprotein C-III (apoC-III), which regulates lipoprotein lipase activity, and stimulates hepatic microsomal triglyceride transfer protein (MTP), the rate-limiting enzyme for assembling triglycerides and very low-density lipoproteins (VLDL), resulting in increased plasma triglyceride levels.
FoxO1 plays a crucial role in the pancreas by regulating β-cell formation and function, suppressing β-cell mass through reduced proliferation and differentiation during fetal development Despite this, FoxO1 protects β cells from oxidative stress-induced damage caused by excess glucose or lipid levels Additionally, it is vital for the development and function of adipocytes by inhibiting PPARγ activity Furthermore, FoxO1 acts as a negative regulator of skeletal differentiation, contributing to skeletal muscle loss and promoting muscle atrophy.
Role of FoxO1 in central regulation of metabolism
FoxO1 plays a crucial role in central metabolism regulation within the hypothalamus, acting as a downstream effector of the PI3K/Akt pathway that influences insulin and leptin actions It modulates the expression of hypothalamic neuropeptides by acting as a transcriptional suppressor of the POMC gene and an activator of the AgRP gene, competing with STAT3 for binding to their promoters Recent findings suggest that FoxO1 regulates energy expenditure and glucose homeostasis by targeting specific neuron subsets in the hypothalamus Additionally, its central action is believed to influence hepatic glucose production In summary, activated FoxO1 in the hypothalamus contributes to hyperphagia, increased body weight, decreased energy expenditure, and diminished leptin's anorexigenic effects.
Recent studies suggest that FoxO1 may have roles beyond the hypothalamus, as its broad neuronal deletion leads to a diminished re-feeding response, heightened locomotor activity, and increased resistance to diet-induced obesity Additionally, FoxO1 serves as a key downstream mediator of insulin signaling.
27 and leptin actions and the fact that both leptin/insulin receptors and the functional signaling pathways, IRS2/PI3K/Akt and JAK/STAT3, are present in
DA neurons 22-27 , the DA neuron system may be a potential target of FoxO1 action
Figure 3 FoxO1 mediates effect of insulin/leptin in the hypothalamus 122
Insulin and leptin binding to their respective receptors triggers the phosphorylation of IRS2, activating PI3K, which in turn phosphorylates Akt via PDK The activation of Akt leads to the phosphorylation and exclusion of FoxO1 from the nucleus, inactivating it FoxO1 represses POMC gene expression while activating AgRP gene expression The JAK/STAT3 pathway also regulates POMC and AgRP expression; upon leptin binding, JAK phosphorylates STAT3, resulting in its dimerization This active dimeric STAT3 translocates to the nucleus to inhibit AgRP expression and activate POMC gene expression, highlighting the intricate regulatory mechanisms involved in energy homeostasis.
HYPOTHESIS AND RESEARCH APPROACH
This study investigates the role of FoxO1 in dopamine (DA) neurons regarding the regulation of feeding and energy homeostasis It examines the hypothesis that FoxO1 acts as a positive regulator of food intake and a negative regulator of energy expenditure, based on its established metabolic functions in the central nervous system (CNS) To test this hypothesis, researchers utilize DA-specific FoxO1 knockout mice (KO, FoxO1).
KO DAT mice were created by crossing DAT IREScre mice with FoxO1 loxP/loxP (FoxO1 F/F) mice Control mice, known as wild-type (WT), were littermates that were homozygous for the floxed FoxO1 allele but lacked the DAT IREScre allele Following confirmation of the specific deletion of FoxO1 in dopaminergic (DA) neurons, a comparison was made between the metabolic parameters of FoxO1 KO DAT mice and those of WT littermate mice to explore the physiological role of FoxO1 in DA neurons.
In this study, the following specific aims were addressed:
1 Generate and validate the DA-specific FoxO1 knockout mice model
2 Characterize the metabolic phenotypes of DA-specific FoxO1 knockout mice a In normal metabolic condition with chow diet b In metabolic stress condition with high fat diet
3 Examine the effects of DA neuron FoxO1 ablation on metabolic determinants of energy balance
30 a Feeding behavior and food consumption b Energy expenditure
4 Figure out the molecular mechanism underlying the metabolic phenotypes of DA-specific FoxO1 knockout mice
MATERIALS AND METHODS
All animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of the Yonsei University Wonju College of Medicine (YWC-140428-1)
Mice were maintained in a controlled environment at a temperature of 22-24 °C, following a 12-hour light/dark cycle, with lights on at 06:00 a.m and off at 06:00 p.m They were fed either a normal chow diet (NC) containing 4.25% kcal from fat and 3.82 kcal/g, or a high-fat diet (HFD) comprising 60% kcal from fat and 5.24 kcal/g, sourced from Research Diets in the USA.
The brain was carefully removed from the skull, and the hypothalamus and cerebellum were extracted A coronal section of the midbrain was then selectively cut Using a low-power dissecting microscope, the cortex areas, bilateral substantia nigra (SN), and the midbrain regions containing the ventral tegmental area (VTA) were meticulously dissected and collected.
32 brain samples were snap-frozen on dry ice and stored at -80 °C for further analysis 124
3.4 Body weight and body composition
Body weight of WT and FoxO1 KO DAT mice, both male and female, was tracked weekly from weaning at 5 weeks old while on a normal chow diet For high-fat diet (HFD) experiments, male WT and FoxO1 KO DAT littermates were initially maintained on a regular chow diet until 8 weeks old, after which they were switched to HFD for an additional 6-12 weeks Body composition, including fat and lean mass, was assessed using nuclear magnetic resonance (LF90 Minispec, Bruker Corp., TX, USA).
3.5 Food intake and re-feeding experiments
In a study on food consumption, male mice aged 8-10 weeks with matched body weights were observed over a 7-day period on both chow diet and high-fat diet (HFD) The average food intake was measured in grams per kilogram of body weight For re-feeding experiments, male mice at 8 weeks and female mice at 26 weeks, also with comparable body weights, were individually housed and given normal chow and water ad libitum Following an overnight fast of 18 hours, with water available, rebound food intake, body weight, and blood glucose levels were recorded at specified time intervals.
The open field test was conducted in a chamber measuring 60 cm x 60 cm x 20 cm Following a 2-hour acclimation period, the activity of each mouse was recorded for 10 minutes using the SMART video tracking system version 3.0.04 (Panlab, Harvard Apparatus, MA, USA).
The sucrose preference test was conducted on 10-week-old male mice (n=6 per group), with body weights of 24.68 ± 0.77 g for WT and 23.73 ± 0.89 g for FoxO1 KO DAT (p > 0.4) Mice were acclimatized in cages with two drinking bottles—one with filtered tap water and the other with sucrose solutions of varying concentrations (1%, 2%, and 5%)—over a 3-day period Fluid consumption was recorded twice daily at 8:00 and 17:00 by weighing the bottles, and to prevent side bias, the positions of the bottles were alternated daily On the final day, mice were fasted overnight while having access to water, followed by the sucrose preference test.
34 monitored with 2% sucrose Sucrose preference was calculated as the percentage of the amount of sucrose solution consumed over total fluid consumption
After one week on a high-fat diet (HFD), the body weights of wild-type (WT) and knockout (KO) littermate male mice were measured at 32.07 ± 2.92 g and 29.38 ± 2.50 g, respectively, with no significant difference (p > 0.5) After six weeks on HFD, both groups showed body weights of 34.88 ± 2.58 g for WT and 29.65 ± 1.37 g for KO, again with no significant difference (p > 0.12) To assess metabolic rates, mice were individually housed in an eight-chamber Oxymax/CLAMS system for 72 hours in a fed state The respiratory exchange ratio (RER) and heat production (HP) were calculated using the formula (3.185 + 1.232 x RER) x V O2.
3.9 Glucose and insulin tolerance tests
Glucose tolerance tests (GTTs) were performed as described previously 128 For cohorts fed normal chow, male mice aged 24 weeks (n=5 each group; body weight, 30.93 ± 1.01 g and 29.74 ± 1.26 g for WT and KO,
35 respectively, p > 0.45) were used For cohorts fed HFD, 20-week-old male mice (n=5 each group; body weight, 37.16 ± 3.93 g and 31.38 ± 2.05 g for WT and
KO, respectively, p > 0.2) were used Glucose (1.5 g/kg body weight) was injected intra-peritoneally (i.p.)
In insulin tolerance tests (ITTs), 24-week-old male mice on a normal chow diet (n=5 per group; body weights of 31.22 ± 1.16 g for WT and 29.99 ± 1.26 g for KO, p > 0.49) and 20-week-old male mice on a high-fat diet (HFD) (n=4-6 per group; body weights of 40.11 ± 4.25 g for WT and 33.09 ± 2.36 g for KO, p > 0.15) were utilized Insulin was administered intraperitoneally at a dose of 0.9 U/kg (Eli Lilly, IN, USA).
At 32 weeks for male and 30 weeks for female mice, the rectal body temperature of FoxO1 knockout (KO) and wild-type (WT) littermates (n=5 per group) was measured using Thermalert TH-5 equipment (Physitemp Instruments Inc., NJ, USA) in a room temperature environment at 9:00 a.m Each mouse underwent three measurements, and the average temperature was calculated for accuracy.
Blood samples were collected from the tail-nicked blood drops for insulin and leptin measurements Insulin and leptin levels were measured by
ELISA kits (Morinaga Institute of Biological Science, Yokohama, Japan) in accordance with manufacturer’s instructions
Plasma or serum samples were obtained from mice maintained in a basal resting state during the daytime, with unrestricted access to food and water The norepinephrine levels in these samples were measured using an ELISA kit from LDN Labor Diagnostika Nord GmbH & Co KG, Nordhorn, Germany, following established protocols.
Dopamine levels from VTA and SN samples were analyzed at the Biomedical Research Center at the Asan Institute for Life Sciences in Seoul, Korea, with results expressed as pmole/mg of tissue The study utilized dopamine hydrochloride and dopamine-1,1,2,2-d4 hydrochloride sourced from Sigma-Aldrich, while Oasis wax cartridges were obtained from Waters All solvents, including water, were purchased from J T Baker.
Sample Preparation- Samples were weighed and then homogenized using Tissue Lyzer (QIAGEN, Hilden, Germany) with 400 μL of methanol The homogenate was incubated for 15 mins at 4 o C 200 μl of 1 μM
To prepare the sample for liquid chromatography-mass spectrometry (LC-MS/MS) analysis, 37 dopamine-1,1,2,2-d4 hydrochloride was added as an internal standard after incubation and mixed thoroughly The sample was then centrifuged at 14,000 rpm for 15 minutes, and the supernatant was collected, followed by the addition of an equal volume of 1% formic acid The mixture was conditioned using an Oasis wax 3cc cartridge with 1 mL of methanol and 0.5% formic acid, after which the sample solution was loaded and incubated for 10 minutes The cartridge was dried under vacuum, and 1 mL of methanol was used for elution, with the eluant subsequently dried under vacuum The final dried sample was stored at –20°C until analysis and reconstituted with 20 μL of 50% methanol before LC-MS/MS analysis.
Liquid Chromatography-Tandem Mass spectrometry (LC-MS/MS) -
Dopamine analysis was conducted using LC-MS/MS technology, specifically a 1290 HPLC from Agilent Technologies and a Qtrap 5500 from AB Sciex, utilizing a Pursuit 5 C18 reverse phase column (150 × 2 mm) A 3 μL sample was injected into the system and ionized via a turbo spray ionization source, with the mobile phases consisting of 0.1% formic acid in both water and methanol.
B, respectively The separation gradient was as follows: hold at 10% B for 5 mins, 10% to 70% B for 13 mins, 70% to 90% B for 0.1 min, hold at 90% B for 8.9 mins, 90% to 10% B for 0.1 min, then hold at 10% B for 2.9 mins LC flow was 200 μL/min, and column temperature was kept at 23 o C Multiple reaction
The monitoring of analytes was conducted in positive ion mode using 38 monitoring (MRM), with the extracted ion chromatogram (EIC) facilitating quantitation The specific transitions for dopamine and the internal standard were 154.1/137.1 and 158.1/141.1, respectively Data analysis was carried out using Analyst® software version 1.5.2 from AB Sciex, MA, USA.
RESULTS
4.1 Verification of DA-specific FoxO1 knockout mice model
To visualize DAT IREScre activity and evaluate FoxO1 deletion in dopamine (DA) neurons, tdTomato-FoxO1 knockout (KO) DAT mice were created by crossing tdTomato mice with FoxO1 KO DAT mice The results, illustrated in Figure 4A-C, indicate that Cre expression is confined to DA neurons, aligning with previous findings Additionally, double-staining with a TH antibody confirmed that DAT-cre expression is specific to DA neurons, with no expression observed in adrenergic neurons of the locus coeruleus or adrenal gland cells, as shown in Figures 4D and E.
Immunohistochemistry using a specific FoxO1 antibody and a GFP-conjugated secondary antibody confirmed the targeted deletion of FoxO1 in dopamine (DA) neurons Immunofluorescence analysis revealed a strong FoxO1 signal in DA neurons of wild-type (WT) mice, while this signal was absent in the DA neurons of FoxO1 knockout (KO) DAT mice Additionally, allele-specific PCR genotyping indicated successful deletion of FoxO1 specifically in the substantia nigra (SN) and midbrain areas, including the ventral tegmental area (VTA), without affecting other brain regions such as the hypothalamus, cortex, and cerebellum, nor in peripheral tissues like adrenals, brown adipose tissue, muscles, and livers in WT mice.
The undetectable delta band in the hypothalamus may be attributed to a small population of dopamine neurons in the area Additionally, FoxO1 protein levels were significantly reduced in the midbrain, specifically in the ventral tegmental area (VTA) and substantia nigra (SN), of FoxO1 knockout dopamine transporter (DAT) mice, while remaining unchanged in other central nervous system regions and peripheral organs.
In the central nervous system (CNS), the levels of FoxO3a in dopaminergic (DA) regions of knockout (KO) mice remained unchanged compared to wild-type (WT) mice, as illustrated in Figure 8A and B In contrast, FoxO4 protein was not detectable in any brain regions, while FoxO1 levels were also examined.
KO DAT mice exhibited normal brain size and an unchanged number and size of dopamine (DA) neurons, indicating that the deletion of FoxO1 was successful and specific to DA neurons These findings suggest that the absence of FoxO1 in DA neurons does not impact the normal development of the brain or the DA neuron system Therefore, FoxO1 KO DAT mice serve as a valuable model for exploring the metabolic functions of FoxO1 in DA neurons in vivo.
Figure 4 Specific DAT-cre expression in DA neurons A, B, C, DAT-cre
58 expression in the midbrain (A), mediobasal hypothalamus (B) and premammillary nucleus (C) TdTomato (Td, red) was used to visualize DA neurons expressing DAT-cre DAPI stains nuclei (blue) Scale bar, 250 m D,
E, Co-localization between DAT-cre (red) and TH-immunoreactive cells (green) in the midbrain (D) and in the locus coeruleus (E, top panels) and adrenal gland (E, bottom panels) showing specific expression of DAT-cre in DA neurons, not in adrenergic neurons of the locus coeruleus or in the cells of adrenal gland DAPI stains nuclei (blue) Scale bar, 50 m (250 m for low magnification) Percentage of co-localization of DAT-cre expression and TH-positive cells in the midbrain is 92.4 ± 2.5% (recorded from 3 animals) VTA, ventral tegmental area SN, substantia nigra TH, tyrosine hydroxylase
In a study involving KO mice, the specific deletion of FoxO1 in dopamine (DA) neurons was visualized using green fluorescence The expression of DAT-cre in these neurons was highlighted with red fluorescence from TdTomato, while DAPI staining marked the nuclei in blue The scale bar provides a reference for measurement.
Figure 6 Allele-specific genotyping of WT and KO mice A, Allele-specific genotyping from different brain regions of FoxO1 F/+ , FoxO1 F/F and FoxO1
KO DAT mice B, Allele-specific genotyping of indicated peripheral organs SN, substantia nigra Hypo, hypothalamus Cb, cerebellum BAT, brown adipose tissue
Immunoblot analysis reveals the FoxO1 protein levels in various brain regions, including the substantia nigra, hypothalamus, and cerebellum, as well as in peripheral organs such as brown adipose tissue, comparing wild-type (WT) and knockout (KO) mice.
Figure 8 FoxO3a levels of WT and KO mice A, B, Immunoblots (A) and graphs (B) showing FoxO3a protein levels in the SN and midbrain samples of
WT and KO mice SN, substantia nigra
Figure 9 Brain size, DA neuron size and number of WT and KO mice A,
B, Representative figures (top) and graphs (bottom) showing the brain width (A) and brain weight (B) of WT and KO mice C, D, E, Representative figures (C) and graphs (D, E) showing the number and size of DA neurons of WT and
KO mice SN, substantia nigra VTA, ventral tegmental area
4.2 Metabolic phenotypes of DA-specific FoxO1 knockout mice on chow diet
The study on the FoxO1 KO DAT mice model revealed that, on a chow diet, the body weight of FoxO1 KO DAT mice was similar to that of WT littermates throughout the experimental period, indicating normal development alongside unchanged brain size and DA neuron characteristics Both groups exhibited comparable blood glucose levels in fed and fasted states, as well as similar insulin and leptin levels Notably, glucose tolerance tests (GTTs) and insulin tolerance tests (ITTs) demonstrated a significant improvement in glucose and insulin sensitivity in FoxO1 KO DAT mice at 22-24 weeks of age, despite no differences in insulin secretion These findings suggest that the deletion of FoxO1 in DA neurons enhances glucose and insulin sensitivity without impacting insulin secretion or normal development.
Figure 10 Body weight of WT and KO mice on chow diet A, B, Body weight measurement of male (A) and female (B) WT and KO mice on chow diet ♂, male ♀, female
The blood glucose levels of wild-type (WT) and knockout (KO) mice were analyzed while on a chow diet The study measured both fed and fasted blood glucose levels for male and female mice, denoted as ♂ for males and ♀ for females.
Figure 12 Insulin and leptin levels of WT and KO mice on chow diet
Plasma insulin and leptin levels of male (A and B) and female (C and D) of WT and KO mice on chow diet ♂, male ♀, female
Figure 13 Improved glucose homeostasis in KO mice on chow diet A, GTT
The study presents data on glucose tolerance tests (GTT) and insulin tolerance tests (ITT) conducted on wild-type (WT) and knockout (KO) male and female mice It highlights the insulin levels during the GTT for male mice and compares the AUC results for both WT and KO male mice in the GTT and ITT Additionally, it provides insights into the GTT and ITT outcomes for female mice, along with plasma insulin levels measured at 24 weeks of age.
4.3 Metabolic phenotypes of DA-specific FoxO1 knockout mice on high fat diet
To explore the impact of FoxO1 ablation in dopamine (DA) neurons under diet-induced metabolic stress, FoxO1 knockout (KO) DAT mice and wild-type (WT) mice were subjected to a high-fat diet (HFD) Results indicated that FoxO1 KO DAT mice experienced significantly less weight gain compared to WT mice, with a notably lower body weight observed after 7 weeks on the HFD Furthermore, the percentage of body weight gain in FoxO1 KO DAT mice was significantly reduced compared to their WT counterparts.
From the second week of high-fat diet (HFD), WT littermate mice showed significant differences that became more pronounced over time Notably, FoxO1 knockout (KO) DAT mice displayed a leaner phenotype, accompanied by a substantial reduction in total fat mass Additionally, these mice had lower serum insulin and leptin levels, reduced blood glucose levels, and demonstrated improved glucose and insulin sensitivity after 6-10 weeks on HFD.
In a study examining the effects of a high-fat diet (HFD) on male mice, KO mice demonstrated significantly less weight gain and reduced fat mass compared to their WT counterparts Weekly measurements indicated notable differences in body weight and percentage change, highlighting the impact of genetic factors on diet-induced obesity.
C, Body fat and lean mass of WT and KO male mice at 6 weeks on HFD HFD, high-fat diet
DISCUSSION
The dopamine (DA) system, crucial for motivation and reward, has garnered significant interest in metabolic research due to its role in hedonic feeding and the consumption of palatable foods Despite this attention, there is still a lack of comprehensive mechanistic understanding regarding how DA neurons regulate energy homeostasis, with existing information remaining limited and often contentious Recent advancements include the creation of DA neuron-specific FoxO1 models to further explore these dynamics.
This study reveals that FoxO1 in dopamine (DA) neurons is essential for regulating feeding behavior, energy expenditure, and metabolic homeostasis in knockout (KO) mice Additionally, the transcriptional regulation of tyrosine hydroxylase (TH) expression by FoxO1 may serve as a molecular mechanism that underpins its metabolic functions within DA neurons.
Recent studies indicate that the role of FoxO1 in the central nervous system (CNS) may extend beyond its established metabolic functions in specific hypothalamic neurons, such as AgRP and POMC neurons in the arcuate nucleus (ARC) Research by Heinrich et al demonstrated that deleting FoxO1 in most hypothalamic neurons using transgenic mice with Nkx2.1-cre recombinase did not affect food intake or energy expenditure, nor did it prevent diet-induced obesity, highlighting the diverse functions of FoxO1 within the CNS.
Ren et al demonstrated that using synapsin-Cre to ablate FoxO1 in a broad range of neurons, while minimally affecting AgRP and POMC neurons, resulted in Syn-FoxO1 knockout (KO) mice displaying a catabolic energy metabolism phenotype This phenotype was characterized by increased locomotor activity and a diminished re-feeding response, attributed to heightened sensitivity to hormonal and nutritional signaling pathways in the central nervous system Additionally, Syn-FoxO1 KO mice exhibited increased energy expenditure and resistance to high-fat diet-induced obesity, highlighting the metabolic role of FoxO1 in neuronal populations beyond the hypothalamus.
The study investigates the metabolic role of FoxO1 in dopamine (DA) neurons, utilizing DAT-Cre recombinase activity to create DA neuron-specific FoxO1 knockout (KO) mice The findings reveal that, like Syn-FoxO1 KO mice, the FoxO1 DAT KO mice exhibit increased energy expenditure, enhanced glucose homeostasis, and resistance to weight gain induced by a high-fat diet (HFD) However, these KO mice did not show changes in re-feeding response or physical activity.
FoxO1 DAT knockout (KO) mice exhibited a greater preference for sucrose, linked to increased dopamine levels in the midbrain, which plays a crucial role in determining the reward value of nutrients Consequently, KO mice showed a tendency for higher food intake when fed a high-fat diet (HFD).
The increased reward value of palatable food or the compensatory feedback regulation of homeostasis due to elevated energy expenditure in KO mice could explain the observed differences in feeding behavior However, these variations in reward feeding are unlikely to account for the metabolic phenotypes seen in FoxO1 DAT.
In FoxO1 DAT knockout mice, the overall food intake remained similar, and the expression levels of hypothalamic neuropeptides related to feeding behavior, such as Agrp, Npy, Pomc, and Cart, showed no significant changes.
The FoxO1 DAT knockout (KO) mice exhibited significantly increased energy expenditure, primarily due to enhanced adaptive thermogenesis rather than increased physical activity This heightened thermogenesis in the iBAT of the FoxO1 DAT KO mice is likely linked to elevated sympathetic nervous system activity, as evidenced by increased norepinephrine levels Furthermore, the rise in dopamine levels may also play a role in boosting iBAT thermogenesis, supported by pharmacological studies indicating that dopamine reuptake inhibitors can activate this process The increase in adaptive thermogenesis, driven by enhanced central nervous system sympathetic activity, is further corroborated by the notable upregulation of Adrb3 expression and downstream signaling pathways, including p-Creb, p-p38 MAPK, and UCP1 in the iBAT of FoxO1 DAT KO mice.
BAT activation enhances overall glucose regulation and insulin sensitivity By converting metabolic substrates into heat rather than chemical energy, BAT significantly contributes to the clearance of free fatty acids.
102 fatty acids, triglycerides and glucose as well149,152,153
Triglycerides and fatty acids are the primary substrates for oxidative metabolism in brown adipose tissue (BAT), with glucose playing a minor role (5-15%) Initially, BAT utilizes its intracellular energy stores before relying on plasma substrates, meaning short-term activation of BAT has a minimal impact on overall plasma glucose utilization However, prolonged BAT activation can enhance plasma glucose oxidation, contributing up to 30% to increased resting energy expenditure In FoxO1 KO DAT mice, blood glucose levels remained stable under normal conditions but decreased after chronic high-fat diet (HFD) exposure, indicating improved glucose homeostasis compared to wild-type mice This suggests that mild BAT activation in FoxO1 KO DAT mice enhances lipid oxidative metabolism, leading to better glucose regulation Additionally, prolonged BAT activation during chronic HFD may further aid glucose clearance Enhanced expression of thermogenic genes and increased UCP1 induction in white adipose tissues (WATs) may also support adaptive thermogenesis, contributing to improved glucose homeostasis in these mice Overall, these findings highlight the significant role of increased thermogenesis and energy expenditure in glucose regulation.
103 expenditure probably due to enhanced sympathetic activity could be the major cause of the metabolic phenotypes observed in FoxO1 DAT KO mice
This study identifies dopamine (DA) neurons as a key target for the metabolic actions of FoxO1 in the central nervous system (CNS) The specific activity of DAT-cre confirms that the observed phenotypes in FoxO1 DAT knockout (KO) mice are directly linked to DA neurons Importantly, the findings demonstrate that FoxO1 in DA neurons is crucial not only for regulating feeding behavior but also for maintaining energy expenditure and metabolic homeostasis.
The dopamine (DA) neuron system plays a crucial role in the reward system, particularly in regulating feeding behavior linked to the reward value of food, which is associated with overeating and obesity Recent research indicates that midbrain DA neurons are not uniform; they receive inputs from various sources and project to multiple sites An intriguing question arises regarding whether a specific subset of DA neurons targeted by FoxO1 regulates energy expenditure, while other subsets modulate food reward Investigating the specific brain regions where FoxO1 influences these metabolic phenotypes is essential for future studies, such as the direct injection of adeno-associated virus (AAV)-mediated Cre expression into the midbrain of floxed FoxO1 alleles.
(FoxO1 F/F ) mice may help to determine if DA neurons in the midbrain or other brain regions govern these observed metabolic phenotypes of FoxO1 DAT KO mice
The current study reveals that FoxO1 directly regulates the expression of tyrosine hydroxylase (TH), the key enzyme in catecholamine synthesis This regulation may influence catecholamine production and provide insight into the phenotypes observed in FoxO1 DAT knockout mice Furthermore, this mechanism could serve as a crucial link between the dopamine neuron system and metabolic homeostasis By binding to the proximal region of the TH promoter, FoxO1 appears to inhibit TH transcription.
TH gene leading to decreased TH expression Consistent with in vitro results,
FoxO1 DAT KO mice exhibited a significant increase in TH mRNA and protein levels, potentially driving the elevated dopamine content in dopaminergic neurons This rise in midbrain dopamine levels may contribute to the heightened sucrose preference observed in these mice The close reciprocal interactions between dopaminergic and noradrenergic neurons suggest that increased dopamine could enhance norepinephrine levels, thereby boosting sympathetic nervous system activity Given that serum norepinephrine concentration reflects the overall norepinephrine levels in the body, it raises an intriguing question for future research: how does the central ablation of FoxO1 in dopaminergic neurons influence catecholamine synthesis in peripheral organs, such as sympathetic ganglions and the adrenal medulla?
CONCLUSION
Dopaminergic neurons play a vital role in integrating neuronal and hormonal signals to maintain energy balance, yet the mechanisms behind this regulation remain unclear FoxO1 is a crucial regulator of metabolism, food intake, and energy expenditure, essential for overall energy homeostasis While its role in the hypothalamus is well-documented, the function of FoxO1 in other regions of the central nervous system (CNS) is not well understood This study highlights the significance of FoxO1 in dopaminergic neurons, demonstrating its importance not only for feeding behavior but also for regulating energy expenditure and metabolic homeostasis.
The direct transcriptional regulation of TH expression and catecholamine synthesis by FoxO1 highlights a crucial connection between metabolism and the dopamine (DA) system These insights could lead to the identification of new therapeutic targets within the DA system for addressing abnormal energy homeostasis and neurodegenerative diseases related to metabolism.
INTRODUCTION
Over the past thirty years, obesity has surged globally, becoming a widespread epidemic This condition arises from various factors, such as genetics, behavior, environment, physiology, and social influences, but fundamentally stems from an energy imbalance where calorie intake surpasses energy expenditure A significant contributor to this increased energy consumption is the excessive intake of highly palatable foods, particularly fast food.
The rise of obesity today can be primarily attributed to reduced energy expenditure linked to a sedentary lifestyle Gaining insight into the molecular mechanisms that regulate food intake and energy expenditure is crucial for developing effective therapeutic strategies for managing obesity and related metabolic disorders.
Recent advancements in genetic manipulation technologies have underscored the central nervous system's (CNS) vital role in regulating energy intake and body weight The hypothalamus, particularly its various nuclei such as the arcuate nucleus (ARC), ventromedial hypothalamus (VMH), and lateral hypothalamus area (LHA), has been a focal point in obesity and metabolism research These nuclei house crucial neurons like agouti-related peptide (AgRP) and pro-opiomelanocortin (POMC), which respond to the body's nutritional and hormonal signals Through intricate interactions and regulatory mechanisms, these neurons help balance energy expenditure with caloric intake, maintaining stable body weight However, food drive and consumption are influenced not only by the body's nutritional status (homeostatic feeding) but also by other external factors.
3 palatability (the rewarding aspect of food), environmental and social factors (non-homeostatic feeding) 7
The central dopaminergic (DA) system, crucial for motivation and reward, has gained significant attention in metabolic research due to its influence on hedonic feeding behaviors and palatable food consumption Evidence indicates that the DA system plays a vital role in energy homeostasis, primarily through its reward function that regulates feeding behavior and food intake However, some studies suggest that DA activity may also impact energy expenditure Further research is essential to clarify the role of DA neurons in regulating energy expenditure and to enhance understanding of how the central DA neuron system manages energy homeostasis.
Forkhead box protein O1 (FoxO1) is a vital transcription factor that regulates whole-body energy metabolism, significantly influencing the development and function of skeletal muscle, adipose tissue, and pancreatic β cells In the liver, FoxO1 is essential for gluconeogenesis and lipid metabolism Recent research has revealed that FoxO1 in the brain is instrumental in controlling food intake and body weight, particularly within the hypothalamus where it regulates energy balance FoxO1 mediates insulin and leptin's effects on AgRP and POMC neurons in the arcuate nucleus (ARC) by stimulating their transcription.
FoxO1 plays a crucial role in regulating energy expenditure and glucose homeostasis by acting on neurons that express SF-1 in the VMH, while also influencing behavioral manifestations The pan-neuronal deletion of FoxO1 in the CNS leads to a diminished re-feeding response, heightened locomotor activity, and resistance to diet-induced obesity, indicating its potential functions beyond the hypothalamus However, the metabolic roles of FoxO1 in brain regions outside the hypothalamus remain largely unexplored Notably, the dopamine (DA) neuron system, which expresses receptors for metabolic hormones like insulin and leptin, along with key signaling pathways such as IRS2/PI3K and JAK/STAT3, presents a promising area for investigating FoxO1's metabolic actions.
1.1 Dopaminergic neuron system in the central nervous system
Dopamine (DA) neurons, though comprising less than 1% of the central nervous system (CNS), play a crucial role in regulating various physiological functions in mammals, including movement, mood, reward, motivation, cognition, attention, memory, sleep, and prolactin production These neurons are integral to the catecholaminergic system in the brain Recent advancements in immunohistochemistry techniques have enhanced the ability to distinguish between different catecholamines, allowing for more detailed mapping of the DA neuron system in the rodent brain There are nine major DA cell groups, labeled A8 to A16, which are extensively distributed from the mesencephalon to the olfactory bulb.
Figure 1 Dopaminergic cells and projections in the rodent brain Adapted from Bjorklund and Dunnett Trends Neurosci, 2007
Approximately 90% of brain dopamine (DA) neurons are located in the ventral mesencephalon of the midbrain, encompassing three key cell groups: A8, A9, and A10 The A9 DA cells in the substantia nigra project to the striatum, playing a crucial role in motor control through the nigrostriatal pathway Meanwhile, A10 neurons in the ventral tegmental area connect to the limbic system and cortex, contributing to emotional regulation, reward behavior, and motivation via the mesocorticolimbic pathway Additionally, A8 retrorubral DA neurons are believed to coordinate the functions of both the nigrostriatal and mesolimbic systems.
The five dopamine (DA) cell groups in the hypothalamus, identified as A11 to A15, play a significant role in the endocrine system While the function of A11 DA neurons projecting to the spinal cord remains unclear, A13 DA neurons are involved in regulating gonadotropin-releasing hormone within the hypothalamus The majority of DA neurons found in the arcuate nucleus (A12) and the preoptic area/anterior hypothalamus (A14) function as endocrine neurons, releasing dopamine into the portal blood system for storage in the pituitary gland These neurons are crucial for controlling the secretion of prolactin and growth hormone from the anterior pituitary gland, as well as regulating melanocyte-stimulating hormone release from the intermediate lobe of the pituitary gland.
Dopamine is synthesized from the amino acid tyrosine in presynaptic neurons by two sequential reactions which are catalyzed by enzyme tyrosine
In catecholaminergic neurons, dopamine is synthesized from L-tyrosine through the action of the rate-limiting enzyme tyrosine hydroxylase (TH), followed by the conversion of dopamine into norepinephrine via aromatic amino acid decarboxylase (AADC) and dopamine-β-hydroxylase (DBH).
Dopamine, released from presynaptic neurons, binds to specific receptors on postsynaptic neurons, triggering intracellular signaling pathways, including adenylate cyclase and protein kinase A There are five types of dopamine receptors (D1 to D5), classified as G-protein-coupled receptors D1 and D5 receptors enhance intracellular cAMP levels by coupling with Gαs, while D2, D3, and D4 receptors lower cAMP levels by coupling with Gαi, thus inhibiting adenylate cyclase The clearance of dopamine from the synaptic cleft occurs through reuptake by the dopamine transporter (DAT) or via enzymatic degradation by monoamine oxidase (MAO) and catechol-O-methyltransferase (COMT).
1.2 Role of dopaminergic system in energy balance and obesity
1.2.1 Dopamine, food reward and obesity
Extensive evidence highlights the critical role of the dopamine (DA) system in integrating metabolic signals to regulate energy balance, often linked to its function in reward modulation The widespread distribution of DA neurons in the brain, particularly within the three major components—nigrostriatal, mesolimbic/mesocortical, and tuberoinfundibular (or hypothalamic) DA cell groups—indicates that each component uniquely influences various aspects of eating behavior Notably, DA neurons in the hypothalamus are essential for maintaining basal metabolic functions.
The regulation of food intake is influenced by midbrain dopamine (DA) neurons originating from the ventral tegmental area (VTA) and projecting to the nucleus accumbens (NAc), which play a crucial role in food reward and motivation Additionally, emotional responses to feeding are driven by mesocortical DA neurons, while the sensory-motor aspects of eating are associated with DA neurons from the substantia nigra (SN) that project to the dorsal striatum, also known as the caudate putamen (CPu).
Dopamine (DA) in the hypothalamus, particularly in the lateral hypothalamic area (LHA) and the ventromedial hypothalamus (VMH), plays opposing roles in regulating food intake Following a meal, dopamine levels in the LHA surge, correlating directly with food consumption, and this elevation helps signal meal termination Pharmacological increases in LHA dopamine reduce food intake, while D2 antagonists can reverse the appetite-suppressing effects of amphetamines, indicating that LHA dopamine acts to suppress eating Conversely, dopamine levels in the VMH decrease after feeding, with the extent of this decrease linked to meal size, suggesting that VMH dopamine influences the duration of the inter-meal interval and overall meal frequency.
Hypothalamic dopamine (DA) signaling plays a crucial role in regulating food intake, while midbrain DA neurons are primarily involved in the reward and motivation aspects of feeding behavior The "dopamine hypothesis" suggests that DA neurons projecting from the ventral tegmental area (VTA) to regions like the nucleus accumbens (NAc), hippocampus, amygdala, and prefrontal cortex influence both "reward liking" and "reward wanting." Under normal conditions, DA neurons exhibit tonic firing, but salient environmental stimuli can trigger burst-firing and phasic DA release This heightened DA signaling directs an animal's attention to reward cues and motivates goal-directed behaviors Additionally, increased DA signaling from VTA neurons is believed to enhance conditioned learning by associating rewards with specific environments Consequently, animals are driven to engage in behaviors that elevate dopamine levels, such as drug self-administration or electrical stimulation of DA-activating brain regions Conversely, reduced DA signaling can lead to excessive behaviors aimed at restoring dopamine levels.
A large body of literature has indicated the role of mesolimbic/mesocortical DA reward circuitries in motivational, reward aspects
SCREENING FOR AMPK ACTIVATORS AND RESEARCH HYPOTHESIS
In a study screening various phytochemicals for their ability to activate AMPK signaling in the HepG2 cell line, several small structure polyphenols demonstrated potential effects, including caffeic acid, vanillic acid, p-coumaric acid, vanillin, and gallic acid Among these, gallic acid (3,4,5-trihydroxybenzoic acid; GA) stands out as a promising candidate due to its natural occurrence in a variety of plants, microalgae, and some bacteria, and its demonstrated ability to enhance glucose homeostasis through anti-obesity and hypo-lipidemic effects observed in in vivo studies However, the specific molecular mechanisms by which gallic acid exerts these effects remain to be fully understood.
This study investigates the hypothesis that GA activates AMPK signaling, resulting in positive metabolic effects on glucose homeostasis Using a HepG2 cell culture system, the research examines the in vitro effects of GA on AMPK signaling and its metabolic consequences, including autophagy induction, lipid accumulation, and mitochondrial biogenesis Additionally, a diet-induced obese mouse model is utilized to explore the in vivo effects of GA treatment on AMPK signaling and glucose homeostasis.
Figure 35 Screening for AMPK activators from natural compounds Veh, vehicle Res, resveratrol EGCG, epigallocatechin gallate -neo En, -neo endorphin met-Enke, met-enkephalin
All animal experiments in this study were approved by the Institutional Animal Care and Use Committee (IACUC) of the Yonsei University Wonju College of Medicine (YWC-131023-1)
Dulbecco’s modified Eagle’s medium (DMEM) was obtained from HyClone (Thermo Scientific, UT, USA) Fetal bovine serum (FBS) and penicillin-streptomycin (P/S) were purchased from Gibco (Life Technologies,
NY, USA) Gallic acid was purchased from Alfa Aesar Company (Ward Hill,
Metformin and compound C were sourced from Sigma Chemical Co in Missouri, USA, while Bafilomycin A1 was obtained from Invitrogen in California, USA All other reagents were also purchased from Sigma-Aldrich, unless specified otherwise Stock solutions of gallic acid and metformin were prepared using Invitrogen® UltraPure water and stored at -20°C.
HepG2 cells were maintained in DMEM media containing 10% FBS
153 and 1% P/S and cultured in a humidified atmosphere of 5% CO2 at 37 o C
The constructs utilized for Sirt1 silencing are detailed in previous studies Specifically, the shRNAs include a Scramble sequence and a shRNA-Sirt1 sequence, which were subcloned into the RNAi-Ready pSIREN-DNR-DsRed-Express Donor Vector from Clontech.
CA, USA) via BamHI and EcoRI sites The vectors were transiently transfected into the cells using Lipofectamin® 2000 Reagent (Life Technologies, NY, USA)
HepG2 cells stably transfected with mCherry-LC3-GFP were cultured on coverslips and subjected to either a 50 μM GA treatment or serum starvation for 12 hours Following this, the cells were fixed with 3.7% paraformaldehyde for 15 minutes and stained with DAPI for 5 minutes at room temperature The coverslips were then mounted using VECTASHIELD Mounting Medium from Vector Laboratories and analyzed using a laser scanning confocal microscope (TCS, SPE, Leica Microsystems) An independent researcher quantified the number of puncta per cell.
3.6 Oil Red O staining and triglyceride content
Cells were incubated with oleate (Sigma-Aldrich, MO, USA, Cat No O7501) in a 10% BSA solution for 12 hours to promote lipid accumulation Following this, the cells received treatment with 20 µM or 50 µM of GA for an additional 12 hours After treatment, the cells were washed with 1X PBS and fixed using 3.7% formaldehyde in 1X PBS.
Cells were fixed and incubated at room temperature for one hour before being washed three times with water and stained using Oil Red O solution for 40 minutes After staining, excess dye was removed with three additional washes of water and one wash with 60% isopropanol, followed by complete drying of the stained cells The lipid droplets were then dissolved in 4% Nonidet P-40 in isopropanol, and their concentration was quantified by measuring absorbance at 540 nm.
Cells were harvested and washed with 1X PBS before being lysed in ice-cold buffer A, which contained HEPES, KCl, EDTA, EGTA, DTT, and NaF, along with protease and phosphatase inhibitors After centrifugation, the cytoplasmic supernatant was collected, while the nuclear pellet was lysed in ice-cold buffer C, composed of HEPES, NaCl, EDTA, EGTA, DTT, and NaF.
The supernatant nuclear fraction was collected by centrifugation of a solution containing protease and phosphatase inhibitors Subsequently, 50 µg of protein from the total nuclear extract was immunoprecipitated using a PGC1α antibody from Santa Cruz Biotechnology Inc.
In this study, the protein-antibody complex was isolated using protein A/G-coupled agarose beads from Santa Cruz Biotechnology (Cat No sc-2003), following the manufacturer's protocol Subsequently, the isolated protein was analyzed through Western blotting to assess its characteristics.
C57BL/6 mice, sourced from RaonBio in Korea, were kept in a controlled environment with temperatures between 22-24 °C and a 12-hour light/dark cycle Male littermate mice, aged ten to twelve weeks, were divided into two groups, with 5-7 mice in each group, and housed individually throughout a 9-week experimental period During this time, the mice received intraperitoneal injections of either normal saline or a dosage of 10 mg/kg body weight.
GA dissolved in saline Body weight and food intake were monitored daily before injection After one week of normal chow (Research Diets, IN, USA, Cat
In a study involving mice (No 7001), the subjects were placed on a high-fat diet (Research Diets, NJ, USA, Cat No D12492), which consisted of 60% of calories derived from fat, providing 5.24 kcal/g Blood glucose levels were monitored throughout the experiment Upon completion of the study, the mice were sacrificed, and their organs were collected for subsequent analysis.
3.9 Glucose and insulin tolerance tests
The glucose tolerance test (GTT) involved fasting mice for 18 hours while allowing free access to water Following the measurement of their fasted glucose levels, glucose was administered via intraperitoneal injection at a dosage of 1.2 g/kg body weight, with blood samples subsequently collected from a tail nick for analysis.
In the insulin tolerance test (ITT), mice were fasted for two hours and given water ad libitum After measuring basal glucose levels, 0.9 U/kg body weight of regular insulin (Eli Lilly, IN, USA) was administered intraperitoneally Blood glucose levels were monitored at 15, 30, 60, 90, 120, and 150 minutes post-injection using the glucose oxidase method with a commercial glucometer (Ascensia Contour, Bayer HealthCare, IN, USA).
White adipose tissues (WATs) were fixed in 4% neutral-buffered formalin and paraffin-embedded, followed by sectioning into 4-μm slices These slices were stained with hematoxylin and eosin (H&E) for imaging Representative images were captured using a Nikon Digital Camera DXM1200 microscope system from three independent animals per group, showcasing the results from each mouse.