Main Research Findings

1) Research shows that reduced levels of α-MSH is related to hypothalamic stress-induced hepatic gluconeogenesis.

2) Administration of α-MSH was found to suppress food intake and increase psychomotor activity by affecting MC4 receptor-signaling pathways.

Selected Data
1) This study conducted by researchers Schneeberger et al involved experiments conducted on C57BL/6J mice as well as POMCMfn2KO mice. The animals were housed under controlled conditions, specifically a 12-hour light-dark cycle, with free access to water and either a normal chow diet (NCD) or a high-fat diet (HFD) containing 45% of calories from fat. Diet administration began at six weeks of age, and POMCMfn2KO male mice were studied between five and six weeks of age unless otherwise specified [1].
To assess physiological parameters, blood samples were collected from mice either through tail vein sampling or trunk bleeds using a capillary collection system. Blood glucose levels were determined using a glucometer. Several metabolic tests were carried out to evaluate glucose and substrate handling. These included glucose tolerance tests (GTT) with 2 g/kg of D-glucose, glucose-stimulated insulin secretion (GSIS) with 3 g/kg of D-glucose, pyruvate tolerance tests (PTT) with 1 g/kg of sodium pyruvate, and glycerol tolerance tests (GlyTT) with 1 g/kg of glycerol. All tolerance tests were performed on mice fasted overnight. Insulin sensitivity tests were also conducted, using 0.4 IU/kg of insulin administered to mice deprived of food for six hours. For all procedures, compounds were injected intraperitoneally, and blood glucose was measured at specified time points. In addition, plasma hormones, including insulin, leptin, epinephrine, norepinephrine, adrenocorticotropic hormone (ACTH), and corticosterone, were quantified using commercial ELISA kits. Plasma triglycerides were measured using a quantitative enzymatic assay [1].
The study further examined hypothalamic POMC neurons and α-MSH content through immunohistochemical techniques. Mice were perfused with paraformaldehyde, and their brains were processed into thin slices. The hypothalamic sections were washed, blocked, and incubated with antibodies targeting either α-MSH or POMC, followed by secondary antibodies tagged with fluorescent markers. Imaging was carried out using a Leica microscope, and α-MSH levels were quantified with ImageJ software. POMC neuron counts were obtained from defined sections of the arcuate nucleus. For biochemical analysis of α-MSH, hypothalamic tissue was homogenized in acid solution, centrifuged, and the supernatant analyzed by ELISA. Protein concentrations were determined by the Bradford method.
To assess gene expression, hypothalamic and liver tissues were harvested and frozen in liquid nitrogen. RNA was extracted using Trizol, and reverse transcription followed by quantitative real-time PCR was performed. Transcript levels were measured using an ABI Prism system and proprietary primer-probe sets targeting a panel of metabolic, stress response, and neuroendocrine genes, including Acaca, Atf4, Atf6, Cpe, Cpt1a, Creb1, Ddit3, Dgat2, Fasn, G6pc, Hprt, Hspa5, Pck1, Pcsk1, Pcsk2, Prcp, Scd1, and Xbp1s [1].
Western blot analysis was used to study protein expression and signaling pathways. Protein lysates were prepared from either pulverized liver tissue or dissected mediobasal hypothalamus samples. These lysates were resolved by SDS-PAGE, transferred to PVDF membranes, and probed with antibodies targeting key proteins involved in stress signaling, metabolism, and POMC processing. Targets included phosphorylated and total CREB, mitofusin-2, phosphorylated and total PERK, XBP1, phosphorylated eIF2α, ATF4, ATF6, CHOP, prohormone convertases PC1 and PC2, as well as structural proteins α-tubulin and β-actin. Protein detection was performed via chemiluminescence, and band intensities were quantified with ImageJ software [1].
The study also involved intracerebroventricular cannulation to directly administer compounds into the brain. Surgeries were performed on nine-week-old mice, and experiments took place at ten weeks of age. Control mice and POMCMfn2KO mice, as well as wild-type mice maintained on NCD or HFD for four days, were used. Following an overnight fast, animals received intracerebroventricular infusions of either artificial cerebrospinal fluid as a control, α-MSH, or tauroursodeoxycholic acid (TUDCA). Two hours after infusion, pyruvate tolerance tests were performed to assess metabolic effects. For TUDCA experiments, an additional i.c.v. injection was administered prior to the fasting period.
In summary, this study details a comprehensive experimental approach combining metabolic testing, hormone assays, histological analysis, gene expression studies, protein signaling evaluations, and targeted brain infusions. These methods were designed to investigate the role of POMC neurons, α-MSH signaling, mitochondrial function, and endoplasmic reticulum stress pathways in metabolic regulation under normal and high-fat dietary conditions. By integrating physiological, molecular, and neurobiological measurements, the study aimed to clarify the mechanisms underlying energy balance and metabolic adaptation in genetically modified and diet-challenged mice [1].
2) The study completed by the research team of Watanabe et al used young common goldfish weighing 3–5 g, obtained commercially and acclimated for two weeks before experimentation. To avoid any influence of sexual maturation on behavior, juvenile fish were selected. They were maintained in a temperature-controlled water tank at 20–24 °C under a 12-hour light/12-hour dark cycle to eliminate the effects of seasonal photoperiods. Fish were fed once daily at noon with a commercially available granular diet containing 32% protein, 5% fat, 2% fiber, 6% minerals, 8% water, and approximately 17% carbohydrates [2].
Several chemicals were used in the study including α-MSH, the melanocortin-4 receptor (MC4R), the antagonist HS024, and a neurotoxin, capsaicin. Stock solutions of α-MSH and HS024 were dissolved in saline containing 0.6% NaCl and 0.02% Na₂CO₃ at a concentration of 1 mM and stored at –80 °C. Capsaicin was dissolved in 20% dimethyl sulfoxide (DMSO) at 20 mM and diluted with saline before administration.
To assess the effects of α-MSH on food intake and psychomotor activity, fish were fasted for two days before experimentation. Intraperitoneal injections were performed under anesthesia using 0.05% MS-222, with fish receiving 20 μL/g body weight (BW) of either saline or α-MSH at doses of 10 or 100 pmol/g BW. Dosages were chosen based on earlier studies and plasma α-MSH levels. Following recovery from anesthesia, fish were placed in small experimental tanks supplied with food equivalent to 3% BW, and food intake was measured after 30 minutes by counting uneaten granules. Preference behavior was also examined in circular tanks divided into central and peripheral areas. After injections of α-MSH or saline, the time spent in each area and locomotor activity were recorded for 30 minutes using a video-tracking system [2].
Further experiments investigated how pretreatments influenced the anorexigenic and anxiogenic effects of α-MSH. Fish were injected with 100 pmol/g BW of α-MSH or saline two minutes after receiving intraperitoneal pretreatments with either 0.16 μmol/g BW of capsaicin or its vehicle, or intracerebroventricular pretreatments with 50 pmol/g BW of HS024 or saline. For intracerebroventricular injections, fish were anesthetized with 2 mM MS-222, placed in a stereotaxic apparatus, and a small part of the parietal bone was removed. A fine injection needle was inserted into the third ventricle, where 1 μL of saline, HS024, or Evans blue dye, to confirm accuracy, was injected. The injection site was sealed with adhesive, and accuracy was verified post-experiment. After these treatments, fish were tested for food intake and preference behavior in the same experimental setup. Prior work had shown that melanotan II, an MC4R agonist and MC5R antagonist, produced similar behavioral effects to α-MSH, justifying the use of HS024 to examine MC4R involvement [2].
To investigate the distribution of MC4R transcripts in the goldfish brain, fish were anesthetized, decapitated, and brains were dissected into several regions: olfactory bulb, telencephalon, optic tectum, diencephalon, cerebellum, vagal lobe, and medulla oblongata. Samples were flash-frozen in liquid nitrogen and stored at –80 °C. Total RNA was extracted from each region, and reverse transcription PCR was performed using specific primers for MC4R and elongation factor-1α (EF-1α), the latter serving as an internal control. Amplification conditions involved 40 cycles of denaturation, annealing, and extension, followed by a dissociation step. The primers were designed using known goldfish MC4R and EF-1α sequences. Expression levels were quantified relative to a standard curve of threshold cycle values.
In summary, this study was designed to investigate the role of α-MSH and MC4R signaling in regulating food intake and behavior in juvenile goldfish. The researchers used a combination of behavioral assays, pharmacological interventions with agonists, antagonists, and neurotoxins, and molecular techniques to examine receptor distribution in different brain regions. These approaches provided a comprehensive framework for understanding how melanocortin signaling influences feeding and anxiety-related behaviors in fish [2].

Discussion
1) Researchers Schneeberger et al investigated how deletion of mitofusin 2 (Mfn2) in POMC neurons impacts glucose homeostasis. At twelve weeks of age, POMCMfn2KO mice developed marked obesity due to overeating and reduced thermogenesis, and they displayed major metabolic disturbances such as hyperglycemia, hyperinsulinemia, glucose intolerance, and insulin resistance. To determine whether these abnormalities arose from obesity itself or represented primary metabolic defects, the investigators also studied six-week-old POMCMfn2KO mice before differences in body weight or adiposity became apparent. Even at this early stage, weight-matched knockout mice showed fasting hyperglycemia, glucose intolerance, and insulin resistance. These findings established that glucose dysregulation in these animals occurs independently of obesity [1].
Next, the researchers examined possible mechanisms. Since pancreatic β-cell dysfunction often contributes to abnormal glucose control, they evaluated insulin secretion and islet morphology in young POMCMfn2KO mice. Plasma insulin levels, glucose-stimulated insulin secretion, islet architecture, and α- and β-cell mass were all normal. These results excluded impaired β-cell function as a cause of the glucose imbalance. They then tested glucose uptake across insulin-sensitive tissues using radiolabeled glucose. Uptake was similar between mutants and controls, with only a slight non-significant trend toward higher uptake in skeletal muscle, suggesting this could not explain the metabolic defects.
Attention turned to the liver, a key regulator of glucose metabolism through hepatic glucose production. Pyruvate and glycerol tolerance tests showed that POMCMfn2KO mice had significantly higher blood glucose levels, indicating enhanced gluconeogenesis. Transcriptomic analysis of liver tissue revealed broad dysregulation of genes, especially within pathways linked to glucose metabolism disorders. Genes critical for gluconeogenesis, including Creb1, glucose-6-phosphatase, and phosphoenolpyruvate carboxykinase, were upregulated, findings confirmed by quantitative PCR. Enzyme activity of PEPCK was also increased. Importantly, glycogen content in the liver was unchanged, highlighting increased gluconeogenesis rather than altered glycogen metabolism as the key disturbance. By contrast, hepatic lipid metabolism remained intact, with no changes in triglyceride levels, liver weight, hepatic triglyceride content, or expression of lipid-processing enzymes [1].
The researchers further explored hormonal contributions by assessing hypothalamic-pituitary-adrenal axis function. Levels of epinephrine, norepinephrine, adrenocorticotropic hormone, and corticosterone were similar between groups under both basal and stress conditions. Thus, counter-regulatory hormones did not account for the enhanced gluconeogenesis.
They then investigated the role of α-MSH, a neuropeptide released from POMC neurons known to regulate energy and metabolic control. Young knockout mice displayed reduced hypothalamic α-MSH content and fewer projections to the paraventricular nucleus. When α-MSH was administered directly into the brain, hepatic gluconeogenesis normalized: glucose levels during tolerance tests improved, expression of gluconeogenic genes decreased, and PEPCK activity returned to control levels. This confirmed that reduced α-MSH underlies the metabolic phenotype [1].
Mechanistically, the effects of α-MSH appeared to involve cAMP response element-binding protein (CREB), a transcription factor controlling genes with cAMP response elements. Transcriptome data showed Creb1 upregulation, and liver tissue from knockout mice contained elevated CREB protein and phosphorylated CREB (pCREB), the active form. Intracerebroventricular α-MSH administration normalized pCREB levels but not total CREB. Moreover, pharmacological inhibition of PKA-dependent CREB phosphorylation reduced gluconeogenesis, confirming CREB’s central role in this pathway [1].
The team further examined why α-MSH was reduced in these mice. Previous work had implicated hypothalamic endoplasmic reticulum (ER) stress in lowering α-MSH. Indeed, treatment of knockout mice with TUDCA, a chemical chaperone that alleviates ER stress, restored hypothalamic α-MSH levels and normalized pyruvate tolerance and gluconeogenic gene expression. These results demonstrated that ER-stress-driven α-MSH reduction is the cause of enhanced hepatic glucose production in the absence of obesity.
Finally, the researchers tested whether similar mechanisms occur under dietary stress. Normal mice were fed a HFD for only four days, a duration too short to cause weight gain. Despite unchanged body weight and leptin levels, these mice exhibited fasting hyperglycemia, hyperinsulinemia, impaired pyruvate tolerance, and increased hepatic gluconeogenic gene expression and PEPCK activity. Molecular analysis revealed enhanced hypothalamic ER stress, with increased expression of ER stress markers such as Atf4, Atf6, Xbp1s, and BiP, as well as greater PERK phosphorylation. Mfn2 expression was reduced in the hypothalamus, and α-MSH content and neuronal projections to the paraventricular nucleus were also diminished, despite unchanged Pomc mRNA, POMC protein content, and POMC neuron numbers. The decreased α-MSH-to-POMC ratio suggested defective POMC processing. Although proconvertase 1/3 was unchanged, PC2 expression increased, likely as a compensatory mechanism. Intracerebroventricular α-MSH administration rescued pyruvate tolerance in HFD-fed mice, further confirming that reduced hypothalamic α-MSH directly contributes to increased gluconeogenesis [1].
In summary, deletion of Mfn2 in POMC neurons disrupts glucose homeostasis not by altering pancreatic function or tissue glucose uptake, but by inducing hypothalamic ER stress, reducing α-MSH availability, and driving CREB-mediated hepatic gluconeogenesis. Short-term high-fat diet feeding recapitulates this mechanism, linking hypothalamic ER stress and α-MSH deficiency to glucose dysregulation even in the absence of obesity [1].
2) The study conducted by researchers Watanabe et al examined the effects of intraperitoneal administration of α-MSH on food intake, behavior, and locomotion in goldfish, as well as the influence of co-treatments with capsaicin and HS024, a selective MC4R antagonist. Additionally, the distribution of MC4R transcripts in various brain regions was investigated.
In the first set of experiments, the researchers administered α-MSH intraperitoneally at doses of 10 and 100 pmol per gram body weight. During the 30-minute observation period, α-MSH reduced food intake in a dose-dependent manner, with both doses causing a significant decrease compared to controls. Alongside its anorexigenic effect, α-MSH administration also influenced behavior. Specifically, it enhanced thigmotaxis, meaning the fish displayed an increased preference for the edges of the tank, which is interpreted as anxiogenic behavior. This effect was dose-dependent, with the higher dose of 100 pmol/g BW leading to a significant reduction in the time spent in the central area of the tank. However, α-MSH treatment had no effect on total swimming distance, indicating that locomotor capacity was unaffected. Thus, α-MSH exerted specific effects on feeding and anxiety-related behavior without altering general movement [2].

Figure 1: Changes in A) food intake, B) thigmotaxis, and C) locomotor activity, following intraperitoneal administration of α-MSH.
The second part of the study tested whether these α-MSH-induced effects could be influenced by capsaicin or HS024. Capsaicin, a neurotoxin that affects sensory neurons, was administered intraperitoneally at 160 nmol g⁻¹ BW, while HS024 was injected intracerebroventricularly at 50 pmol/g BW. The results showed that α-MSH at 100 pmol/g BW significantly decreased food intake compared to controls. Administration of capsaicin or HS024 alone had no effect on feeding. Interestingly, co-injection of capsaicin with α-MSH also failed to influence the anorexigenic response, suggesting that the α-MSH effect on feeding is not mediated by pathways sensitive to capsaicin. In contrast, HS024 markedly inhibited the anorexigenic action of α-MSH. Statistical analysis using two-way ANOVA with Bonferroni correction confirmed a significant antagonistic interaction between α-MSH and HS024. These findings strongly support the conclusion that α-MSH reduces food intake in goldfish through mechanisms dependent on the activation of MC4R [2].

Figure 2: Changes in α-MSH-induced anorexigenic action following A) intraperitoneal administration of capsaicin or B) intracerebroventricular administration of HS024.
Behavioral responses were also tested under the same treatment conditions. Consistent with earlier observations, α-MSH at 100 pmol/g BW enhanced thigmotaxis, reducing the time spent in the central tank area. Capsaicin, whether administered alone or with α-MSH, had no effect on this behavioral pattern. However, HS024 significantly attenuated the α-MSH-induced anxiogenic behavior. Statistical analysis again confirmed the significance of this interaction. These results further reinforced the role of MC4R signaling in mediating both the anorexigenic and anxiogenic actions of α-MSH in goldfish [2].
The study also measured total swimming distance during the 30-minute post-injection period to assess potential motor effects of the treatments. Across groups, goldfish swam approximately 20–50 meters during this period. Neither capsaicin nor HS024, when administered alone or in combination with α-MSH, altered locomotor activity. Statistical analyses indicated no significant differences in swimming distance between groups, suggesting that the effects on feeding and anxiety-like behavior were not confounded by changes in physical activity.
In the third section, the researchers focused on the localization of MC4R expression in the goldfish brain. The brain was divided into seven regions: the olfactory bulb, telencephalon, optic tectum, diencephalon, cerebellum, vagal lobe, and medulla oblongata. Real-time PCR analysis revealed significant regional differences in MC4R mRNA expression. In contrast, expression of elongation factor-1α, used as a control gene, was consistently high across all brain regions without significant variation. Among the brain regions examined, the diencephalon exhibited the highest levels of MC4R transcripts, while the cerebellum showed only very low expression. These findings suggest that MC4R-mediated signaling in the diencephalon likely plays a central role in the regulation of feeding and anxiety-related behaviors in goldfish [2].
Taken together, the results of this study demonstrate that α-MSH exerts potent anorexigenic and anxiogenic effects in goldfish through mechanisms dependent on MC4R activation. While capsaicin-sensitive pathways do not appear to be involved, HS024 effectively blocked both the feeding suppression and anxiety-like behaviors induced by α-MSH. Importantly, these behavioral and metabolic effects occurred without alterations in locomotor activity, highlighting the specificity of α-MSH’s actions. Furthermore, the strong expression of MC4R transcripts in the diencephalon underscores the central role of this brain region in mediating α-MSH’s influence on feeding and behavior [2].

Disclaimer

**LAB USE ONLY**
*This information is for educational purposes only and does not constitute medical advice. THE PRODUCTS DESCRIBED HEREIN ARE FOR RESEARCH USE ONLY. All clinical research must be conducted with oversight from the appropriate Institutional Review Board (IRB). All preclinical research must be conducted with oversight from the appropriate Institutional Animal Care and Use Committee (IACUC) following the guidelines of the Animal Welfare Act (AWA).

Citations

[1] Schneeberger M, Gómez-Valadés AG, Altirriba J, et al. Reduced α-MSH Underlies Hypothalamic ER-Stress-Induced Hepatic Gluconeogenesis. Cell Rep. 2015;12(3):361-370. doi:10.1016/j.celrep.2015.06.041

[2] Watanabe K, Konno N, Nakamachi T, Matsuda K. Intraperitoneal administration of α-melanocyte stimulating hormone (α-MSH) suppresses food intake and induces anxiety-like behavior via the brain MC4 receptor-signaling pathway in goldfish. J Neuroendocrinol. 2024;36(11):e13435. doi:10.1111/jne.13435

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