Elevated O-GlcNAcylation Enhances Metabolic Rate and Reduces the Excitability of Hypothalamic ARC Neurons in 10-month-old Male Mice

Abstract

Aging correlates with alterations in metabolism and neuronal function, which affect the overall regulation of energy homeostasis. Recent studies have highlighted that protein O-GlcNAcylation, a common post-translational modification regulating metabolic function, is linked to aging. In particular, elevated O-GlcNAcylation increases energy expenditure, potentially due to alterations in the neuronal function of the hypothalamic arcuate nucleus (ARC), a key brain region for energy balance and metabolic processes. However, its impact on metabolism and hypothalamic neuronal activity in aged mice remains unknown. This study investigates the effect of elevated O-GlcNAcylation on metabolic rate, motor behaviors, glucose tolerance, and neuronal excitability within the hypothalamic ARC in 10-month-old mice. We demonstrate that Oga^+/- mice with elevated O-GlcNAcylation levels show increased energy expenditure, but do not show significant alterations in motor function or glucose tolerance. Our ex vivo electrophysiology experiments revealed that Oga^+/- mice exhibited a reduced firing rate of hypothalamic ARC neurons, suggesting that the increased metabolism in these mice could be attributed to the reduced activity of ARC neurons. These findings indicate that O-GlcNAcylation plays a crucial role in maintaining metabolic balance and neuronal function in the aging brain.

INTRODUCTION

Aging is a multifaceted biological process that affects virtually every organ system in the body, leading to a decline in physiological functions and an increased susceptibility to various diseases [1]. Among the myriad changes that occur with aging, alterations in metabolic and neurological functions are particularly significant, as they can profoundly impact overall health and quality of life [2]. Increasing evidence suggests that these dysfunctions are closely linked to age-related impairments in the hypothalamus, a key brain region that integrates metabolic and neuroendocrine signals to maintain homeostasis [3]. Within the hypothalamus, the arcuate nucleus (ARC) plays a central role in regulating appetite, satiety, food intake, and energy expenditure, and is especially sensitive to age-related metabolic changes [3, 4]. At the molecular level, specific alterations within hypothalamic neurons, such as protein O-GlcNAcylation, are increasingly recognized as critical contributors to these age-associated metabolic disturbances [3].

O-GlcNAcylation is a post-translational modification that involves the attachment of N-acetylglucosamine (GlcNAc) to Ser/Thr residues. It occurs on both nuclear and cytoplasmic proteins and has a significant impact on various cellular processes such as transcription, ubiquitination, the cell cycle, and stress responses [4]. The O-GlcNAc cycling system is regulated by two enzymes, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), which add and remove O-GlcNAc, respectively. These enzymes are abundantly expressed in the brain and maintain cellular homeostasis [5]. In the context of aging, previous studies in mice have shown that OGA expression declines with age [2]. This decline is significant because reduced OGA activity leads to the accumulation of O-GlcNAcylated proteins, disrupting key cellular functions [4, 6, 7].

An imbalance in O-GlcNAcylation levels caused by reduced OGA activity has been linked to aging-related phenotypes such as cognitive decline, neurodegeneration, and metabolic disorders [5, 8]. The pathological role of OGA in aging involves a complex interplay of mechanisms that disrupt cellular homeostasis, ultimately contributing to neurodegeneration, metabolic dysfunction, and systemic inflammation [9-11]. A previous study demonstrated that increased O-GlcNAc levels potentiate energy expenditure, reduce body fat mass, and improve glucose tolerance in young adult (10-week-old) Oga^+/- mice [8]. However, despite the fact that the expression levels of OGA decrease along with age, the role of OGA in metabolic regulation and its potential neuronal contribution in middle-aged mice remains unclear.

This study aims to investigate the role of elevated O-GlcNAcylation in modulating energy expenditure and neuronal excitability within the hypothalamic ARC during aging, using 10-month-old Oga^+/- mice. In addition, we examined behavioral phenotypes and glucose tolerance in these mice. Our ex vivo electrophysiology experiments revealed that Oga^+/- mice showed a reduced firing rate of hypothalamic ARC neurons, suggesting that their increased metabolism may be attributed to the downregulated activity of ARC neurons. Collectively, our study highlights O-GlcNAcylation as a key regulatory mechanism for maintaining metabolic homeostasis in the aging brain.

MATERIALS AND METHODS

Animals

10-month-old male Oga^+/- mice on a C57BL/6J background were used, consistent with our previous study [7] . The experimental mice were maintained on a consistent 12-hour light/dark cycle and had unrestricted access to food and drink. The animal care and experimental procedures complied with the Guide for the Care and Use of Laboratory Animals and received approval from the Institutional Animal Care and Use Committee (IACUC) at the Korea Institute of Science and Technology (KIST) (Approval number KIST- IACUC-2021-129).

Metabolic rate analyses

The metabolic parameters including oxygen consumption (VO₂) and carbon dioxide production (VCO₂) of the experimental mice were assessed using indirect calorimetry at room temperature (22°C) with the Comprehensive Laboratory Animal Monitoring System (CLAMS) system (Columbus Instruments, Columbus, OH, USA) [12]. Each mouse was kept in isolation for a period of 2 to 3 days before the experiment to ensure proper acclimation. Following this, they were provided with chow diet and water in clear respiratory chambers measuring 20.5×10.5×12.5 cm. The activity, heat, energy expenditure, CO₂ generation, and O₂ consumption of the experimental animals were calculated for 5 days. The respiratory exchange ratio (RER) was calculated as the ratio of VCO₂ to VO₂ (RER=VCO₂/VO₂). Heat was calculated by multiplying the respiratory exchange ratio (RER) and VO₂. This value was then used, along with body weight, to determine the metabolic rate. Heat production (i.e., energy expenditure) was estimated using the Weir equation: Heat (kcal/day)=3.941×VO₂+1.106×VCO₂. The data were collected using Oxymax for Windows (version 5.40.14, Columbus Instruments), and analysis was performed using CLAX (version 2.2.15, Columbus Instruments). The respiratory quotient, exchange ratio (CO₂/O₂), and delta-heat values were calculated using the CLAX software based on the collected data.

Behavioral tests

Before beginning each behavioral task, all experimental mice (10 months old) were given at least 30 minutes to acclimate to the behavioral testing room. The behavioral tests were conducted on the same group of mice. WT mice were littermates of the Oga^+/- mice for all experiments. During these days, the mice were carefully handled for 10-minute sessions.

Open field test

A single experimental mouse was placed in a 40×40×40 cm open-field box and observed for 10 minutes to monitor its locomotor activities under 50 lux lighting conditions. The movement of the subjects was observed and recorded for 10 minutes using a camera positioned above. The data were then analyzed using the Any-maze software developed by Stoelting Co., USA.

Treadmill performance test

The comprehensive running duration was assessed using a treadmill from Jeung Do BIO & Plant CO., LTD (Seoul, Korea). During the experiment, the mice were given a 4-day adaptation period on the treadmill. They were required to run at a speed of 9 m/min with a 5-degree incline. On day 5, the practical running test took place. The speed started at 9 m/min on an inclined slope and gradually increased by 3 m/min every 3 minutes until it reached 33 m/min. The test concluded when a mouse stayed off the track for more than 20 seconds and demonstrated reduced responsiveness to stimuli. At that moment, the mouse was carefully taken out, and the duration of the intense running speed was noted.

Forelimb grip strength test

The assessment of forelimb grip strength was conducted using a Grip Strength Meter manufactured by Jeung Do BIO & Plant CO., LTD (Seoul, Korea). The digital force transducer measured the highest tensile force in grams when a mouse was held onto the bar. Three measurements were taken daily, with each measurement being only a minute apart. Each day, mice were selected at random for testing, and the inspector remained unaware of previous results. Tests were conducted in the vivarium during the afternoon light cycle (11 AM to 5 PM), and the device was consistently calibrated by the manufacturer.

Rotarod test

A rotarod test was used to evaluate the sensorimotor performance of mice. A mouse rotarod apparatus from BS Techno Lab, Inc. (Seoul, Korea) was used, with the rod initially set to rotate at 2 rpm. A set of four mice was placed on the rod, and over 5 minutes, the rod gradually increased its speed to 40 rpm. A mouse’s descent was detected by an interruption in the infrared beam, causing both the rod and the timer to stop. The duration of time that each mouse remained on the rod without falling was recorded, with a maximum limit of 5 minutes. Mice were given a 30-minute break between sessions, allowing for three trials each day for 3 days. Latency to descend and overall distance were evaluated before each fall, utilizing a padded pad underneath the apparatus to ensure safety.

Glucose tolerance test

As part of the experiment, the mice were administered an intraperitoneal injection of D-glucose (1 g/kg body weight) following a 15-hour fasting period for the glucose tolerance test (GTT). The blood glucose levels were measured from the tails of mice at 0, 15, 30, 60, and 120 minutes after the glucose injection using a glucometer.

Electrophysiology

Male 10-month-old mice were anesthetized, and their brains were rapidly dissected. The techniques for slice preparation, cerebrospinal fluid buffer, and internal recording solution were identical to those described in our previously published work [12]. The brain slices containing the hypothalamic ARC region were utilized for electrophysiology experiments. The recording involved an artificial cerebrospinal fluid (CSF) solution and was maintained at a controlled temperature and oxygenation level throughout the experiment. The hypothalamic ARC neurons were subjected to whole-cell patching using a current-clamp configuration. The firing frequency of the neurons was then observed by using an internal solution.

Statistical analysis

Comparisons between two groups were conducted using unpaired two-tailed Student’s t-tests. To assess the effects of two independent variables, we adopted two-way ANOVA with Bonferroni’s multiple comparison test. The data analysis was conducted using GraphPad Prism 10 software (GraphPad Software Inc., La Jolla, San Jose, CA, USA) and the results were presented as the mean±SEM. The results were deemed statistically significant at *p<0.05 and ****p<0.0001.

RESULTS

Increased metabolism in middle-aged Oga^+/- mice

To evaluate the impact of heightened O-GlcNAcylation on energy expenditure and metabolic function in middle-aged mice, we used 10-month-old Oga^+/- mice. Haploinsufficiency of the Oga gene in these mice leads to an elevated level of O-GlcNAcylation [8]. To assess the energy expenditure of the mice, metabolic rate, food intake, and basal activity were assessed using metabolic cages and indirect calorimetry during both the light and dark periods for 7 days, comparing with wild type (WT) mice. All experimental mice were placed constantly in the metabolic cage chamber for 7 days (The data from the first 2 days were excluded for adaptation). Our 7-day metabolic rate analysis revealed a significant increase in metabolism in Oga^+/- mice compared to WT littermates during both light and dark phases, which was attributed to the elevated O-GlcNAcylation (Fig. 1A, B). VO₂ was significantly increased in Oga^+/- mice compared to WT controls across both light and dark phases accompanied by elevated VCO₂ and total energy expenditure (Fig. 1C, D). Interestingly, Oga^+/- mice had a higher RER than controls during the light phase, indicating greater carbohydrate use at rest, whereas wild-types showed a higher RER during the dark phase, suggesting genotype-dependent shifts in substrate use (Fig. 1E). This implies that OGA insufficiency alters diurnal fuel preference without changing activity. However, no significant differences were observed in body weight or daily food intake and physical activity between WT and Oga^+/- mice (Fig. 1F~I). These results indicate that OGA insufficiency promotes a sustained metabolic state without altering behavior, and may influence diurnal substrate preference in aged mice.

Fig. 1.

Increased metabolic rate analysis in 10-month-old Oga^+/- mice over 5 days. (A) Schematic representation of metabolic rate using metabolic cages from day 3~7. (B) Quantification of the averaged metabolic rate. Middle-aged Oga^+/- mice show significantly higher metabolic rate during both light and dark phases (interaction, ns; genotype, p<0.0001; phase, p<0.0001). (C) Quantification of carbon dioxide production (VCO₂) during light and dark phases (interaction, ns; genotype, p<0.0001; phase, p<0.0001). (D) Quantification of oxygen consumption (VO₂) during light and dark phases (interaction, ns; genotype, p<0.0001; phase, p<0.0001). (E) Calculated respiratory exchange ratio (RER) during light and dark phases (interaction, ns; genotype, p<0.0001; phase, p<0.0001). (F) Quantification of body weight measured on days 3 and 7 (interaction, ns; D3, p=0.5466; D7, p=0.7256). (G) Quantification of total food intake. (H, I) Quantification of total basal activities (Interaction, ns; light, p=0.9817; dark, p=0.7707). Data are expressed as mean±S.E.M. (n=3~4). The statistical differences were determined using two-way ANOVA with Bonferroni’s multiple comparison test (B~F and H~I) or unpaired two-tailed Student’s t-test. ****p<0.0001. ns, not significant.

Normal motor function in middle-aged Oga^+/- mice

Following metabolic analysis, we evaluated the basal motor function using a series of behavioral assays. In the open-field test, we found no significant differences in basal locomotor activity between Oga^+/- and WT mice (Fig. 2A). We next performed a treadmill test to assess the exercise tolerance and found no significant difference between the two groups (Fig. 2B). To evaluate the muscle strength, we conducted a grip strength test, which revealed a non-significant but notable trend toward decreased muscle strength in Oga^+/- mice (Fig. 2C). Finally, we conducted a rotarod test to assess the motor coordination and motor skills acquisition. No significant differences were observed in latency to fall or total distance moved between Oga^+/- and WT mice (Fig. 2D). Taken together, our behavioral analysis suggests that the elevated O-GlcNAcylation does not lead to significant alterations in motor function in middle-aged mice.

Fig. 2.

10-month-old Oga^+/- mice exhibit normal motor function. (A) Quantification of total distance traveled in an open field test. No significant difference was detected between WT and Oga^+/- mice. (B) Quantification of speed in a treadmill test. No significant difference was detected between WT and Oga^+/- mice. (C) Quantification of total hanging time in a grip strength test. Oga^+/- mice showed a non-significant trend toward reduced grip strength (p=0.0762). (D) Quantification of latency to fall off and total distance traveled in a rotarod test. There was no significant difference between WT and Oga^+/- mice. Illustration of the different tests were generated using BioRender.com. Data are expressed as mean±S.E.M. (n=3~4). Statistical differences between WT and Oga^+/- were determined using unpaired two-tailed Student’s t-test. ns, not significant.

Normal glucose tolerance in middle-aged Oga^+/- mice

Given that metabolic rate is highly associated with glucose tolerance [13], we assessed the glucose tolerance in the middle-aged Oga^+/- mice using an intraperitoneal glucose tolerance test (IPGTT) under fasting conditions (Fig. 3A). Following glucose injection, blood glucose levels in Oga^+/- mice were consistently higher than in WT mice; however, this difference was not statistically significant (Fig. 3B, C). This finding suggests that the increase in O-GlcNAcylation is not significantly associated with glucose tolerance in middle-aged mice.

Fig. 3.

Intraperitoneal glucose tolerance test (IPGTT) in 10-month-old Oga^+/- mice shows no significant alteration. (A) The schematic timeline of the IPGTT analysis. (B) IPGTT was performed using 10-month-old mice fed a chow diet, and no significant alteration was observed for Oga^+/- mice. (C) The area under the curve (AUC) was calculated from the IPGTT graph (B). Data are expressed as mean±S.E.M. (n=3~4). Statistical differences between WT and Oga^+/- were determined using unpaired two-tailed Student’s t-test. ns, not significant.

Reduced excitability of hypothalamic ARC neurons in middle-aged Oga^+/- mice

Systemic metabolism is regulated by hypothalamic neurons. Among various subpopulations of hypothalamic neurons, ARC neurons are responsible for the homeostatic control of food consumption and energy expenditure [14]. Given that increased metabolic rate was observed in the 10-month-old Oga^+/- mice, we next examined the activity of hypothalamic ARC neurons firing by performing whole-cell patch-clamp under a current-clamp mode (Fig. 4A). In the 10-month-old WT mice, ARC neurons exhibited a consistent pattern of action potential firings at a frequency of 2.3 ± 0.24 Hz (n=6) without any current injection. On the other hand, the rate of spontaneous firings was significantly reduced to 1.3 ± 0.29 Hz (n=7) in the 10-month-old Oga^+/- mice (Fig. 4B, C). This finding suggests that the intrinsic neuronal excitability of hypothalamic ARC neurons is decreased in middle-aged Oga^+/- mice. These results further implicate that elevated O-GlcNAcylation could reduce hypothalamic ARC neuronal activity in middle-aged mice, which could contribute to the increased metabolic rate.

Fig. 4.

10-month-old Oga^+/- mice show reduced excitability of hypothalamic ARC neurons. (A) Left, anatomical location of hypothalamic ARC. Right, schematic diagram of whole-cell patch clamp recording. Illustration was generated using BioRender.com. (B) Representative traces of spontaneous firing were recorded from hypothalamic ARC neurons under a current-clamp configuration. (C) Quantification of ARC neuronal firing frequency. Data are expressed as mean±S.E.M. (n=6~7). Statistical difference between WT and Oga^+/- was determined using unpaired two-tailed Student’s t-test. *p<0.05.

DISCUSSION

Our study demonstrates that elevated O-GlcNAcylation plays a critical role in regulating metabolism and energy expenditure in middle-aged Oga^+/- mice without affecting total body weight or food intake. Notably, elevated O-GlcNAcylation does not impair insulin signaling or induce insulin resistance. Furthermore, it does not significantly impact age-related motor function. Additionally, our electrophysiological findings implicate that hypothalamic ARC neurons could be involved in the elevated O-GlcNAcylation-mediated increase in metabolic rate. Collectively, these findings provide valuable insights into the role of OGA in metabolic regulation and neuronal excitability in middle-aged mice, highlighting its potential impact on age-related metabolic adaptations.

O-GlcNAcylation and OGT expression are dynamically regulated during development and aging. Particularly, in the mammalian brain, both OGT expression and global O-GlcNAcylation levels have been reported to decrease with age after birth [15]. However, given that another report demonstrated that O-GlcNAcylation level is elevated in 2-year-old rats compared to 5-month-old rats [16], the aging-related reduction of O-GlcNAcylation level occurs mostly in the early aging. Therefore, it is important to examine the effects of Oga haploinsufficiency on metabolic regulation in young and middle-aged mice.

A previous study reported on the role of elevated O-GlcNAcylation in metabolism and energy expenditure in young adult (10-week-old) mice [8]. Young Oga^+/- mice exhibited increased energy expenditure and resistance to high-fat diet-induced obesity compared to WT mice, attributed to enhanced browning of white adipose tissue. This enhanced energy expenditure contributes to a leaner phenotype and improved glucose tolerance [8]. Consistently, our study demonstrated that middle-aged Oga^+/- mice exhibited increased energy expenditure, which was not accompanied by a significant increase in basal locomotor activity (Fig. 2A). Therefore, other factors, such as enhanced thermogenesis or respiration, may contribute to their elevated metabolic rate, a possibility that warrants further investigation. Meanwhile, it is worthwhile to note that unlike young Oga^+/- mice, middle-aged Oga^+/- mice showed no significant improvement in glucose tolerance and food intake. We also observed that Oga^+/- mice showed a trend toward reduced hanging time compared to wild-type controls (p=0.076) suggesting impaired muscular performance. While the limited sample size hindered drawing conclusions On the effect of Oga haploinsufficiency on muscle function, we should consider the possibility that OGA deletion and the associated increase in metabolic rate may not be universally beneficial in aging, particularly with respect to muscular function. Our findings suggest that O-GlcNAcylation may have a limited role in regulating systemic metabolism in middle-aged mice compared to younger mice. This hypothesis is also supported by a previous report demonstrating the reduction of O-GlcNAcylation level with aging [15].

The hypothalamic ARC plays a central role in regulating metabolism and energy expenditure by integrating peripheral energy signals and modulating neuronal activity [17]. Among several ARC neuronal subpopulations, pro-opiomelanocortin (POMC) and agouti-related peptide (AgRP) neurons are involved in regulating appetite, thermogenesis, and overall metabolic balance [18, 19]. Changes in ARC neuronal excitability, particularly reduced firing rates, have been linked to alterations in energy homeostasis, potentially contributing to metabolic disorders [19, 20]. Specifically, POMC neurons are implicated in increasing energy expenditure, whereas AgRP neurons primarily promote feeding behavior. In this study, we demonstrated that middle-aged Oga^+/- mice show a reduced firing rate of ARC neurons, suggesting a potential role of ARC neuronal activity in mediating the metabolic increase associated with elevated O-GlcNAcylation. Notably, this reduction in ARC neuronal activity was accompanied by increased metabolic rate without alterations in food intake, raising important questions about which ARC neuronal subpopulations are driving these effects. Because our electrophysiological recordings were not cell-type-specific, which is a limitation of this study, it remains unclear whether the reduced ARC activity we observed reflects suppression of AgRP neurons, POMC neurons, or both. Clarifying how reduced ARC activity contributes specifically to increased metabolic rate without affecting food intake will require future investigations.

This study faced certain limitations, including a limited number of mice available for behavioral experiments and the non-cell-type-specific recording of hypothalamic ARC firing. Additionally, although our findings reveal a correlation between reduced firing rates of ARC neurons and increased metabolic activity in Oga^+/- mice, the current study does not establish a causal relationship between these phenomena. Although this association is intriguing, we did not perform functional experiments such as chemogenetic or optogenetic manipulation of ARC neuronal activity that would directly test causality. We acknowledge this as a limitation and propose that future studies explore this mechanistic link to better understand the role of ARC neurons in regulating energy balance. Despite these limitations, our findings provide a foundation for further investigations into the developmental regulation of O-GlcNAcylation during mid- to late-stage aging, particularly in relation to metabolic processes and neuronal activity.