Ablation of AMPK-Related Kinase MPK38/MELK Leads to Male-Specific Obesity in Aged Mature Adult Mice

MPK38/MELK is an AMPK-related kinase that is highly phylogenetically conserved and has been implicated in many biological functions (1–3). Many studies are being conducted focusing on two functions of MPK38; one is a tumor-promoting function by its antiapoptotic action (1,4–7), and the other is a metabolic function by its proapoptotic action (8–18). In terms of the role of MPK38 in metabolism, recent studies have shown that MPK38 coordinately activates ASK1/TGF-β/p53 signaling, which is involved in metabolic homeostasis (8–11). MPK38 phosphorylates PDK1 at Thr³⁵⁴, thereby inhibiting its activity (12), and stimulates the activity of p53 by phosphorylating it at Ser¹⁵ (13). MPK38 also activates the apoptosis signal-regulating kinase 1 (ASK1) and transforming growth factor-β (TGF-β) signaling by directly phosphorylating ASK1 at Thr⁸³⁸ and SMADS (Ser²⁴⁵ of SMAD2, Ser²⁰⁴ of SMAD3, Ser³⁴³ of SMAD4, and Thr⁹⁶ of SMAD7), respectively (14,15). ASK1 ablation has been shown to be involved in metabolic disorders, such as obesity, nonalcoholic fatty liver disease, and fibrosis, in adipose and liver tissues (16–18). These findings suggest that MPK38 functions as a key regulator of the metabolism associated with ASK1/TGF-β/p53 signaling pathways. Indeed, obese mice display lower levels of activation of ASK1/TGF-β/p53 signaling and lower expression of MPK38 and its interacting partners (SMAD3, ASK1, and ZPR9) than control mice (19). The amelioration of the obesity-associated metabolic disturbance achieved by SMAD3 or ZPR9 replacement is mediated by an increase in MPK38 activity (11,20). These findings make MPK38 an attractive therapeutic target for obesity-associated metabolic disorders.

The current study shows that MPK38 plays a crucial role in the protection against late-onset obesity-induced metabolic disturbances and the age-related decline in testosterone concentrations in aged mature adult male, but not female, mice. We have also confirmed this sex-specific antiobesity effect of MPK38 by restoring its expression in diet-induced obese mice. These findings suggest that a reduction in MPK38 expression may be an important mediator of the obesity phenotype in aged mature adult male mice.

Detailed information for antibodies, oligonucleotides (Supplementary Table 1), and animals can be found in the Supplementary Material. MPK38 knockout mice (MPK38^−/−) were generated with C57BL/6N mouse embryonic stem cells harboring gene trap insertions (AR081; Mutant Mouse Regional Resource Center at UC Davis).

The tail blood glucose levels were measured at 0, 10, 20, 30, 45, 60, 90, and 120 min postinjection with an Accu-Check glucometer (6870228; Roche), according to the Mouse Metabolic Phenotyping Center recommendations for data presentation (21). The insulin level was measured by an ELISA kit (90080; Chrystal Chem), and the glucose level was quantified with an Accu-Check glucometer.

For the lipolysis assay, the glycerol content was determined by absorbance at a wavelength of 540 nm using the Free Glycerol Determination Kit (FG0100; Sigma-Aldrich). Isoproterenol-stimulated lipolysis was corrected for basal lipolysis by subtracting the amount of glycerol release in the absence of isoproterenol (19). The lipogenesis was assayed as previously described (19). The incorporated radiolabeled glucose was measured using liquid scintillation counting and normalized by total lipid content.

Serum levels of insulin and free fatty acids (FFA) were measured by ELISA kits (for insulin, from Millipore [EZRMI-13K], and for FFA, from Merck [MAK044]). Serum levels of triglyceride, total cholesterol, HDL (HDL-C) and LDL (LDL-C) cholesterol, and glucose were determined with a Hitachi 7080 Chemistry analyzer (Hitachi High-Tech Corporation, Tokyo, Japan). The serum triglyceride determination kit (TR0100; Sigma-Aldrich) was used for measurement of liver triglyceride levels. Total ketone levels were quantified with an enzyme colorimetric assay kit (415-73301 and 411-73401; FUJIFILM Wako Chemicals, Richmond, VA). Fatty acid oxidation was measured in fresh liver and white adipose tissue (WAT) homogenates using the previously established methods (19).

Detailed methods used in castration and ovariectomy (OVX) are described in the Supplementary Material. For the testosterone replacement, testosterone pellets (25 µg/day, SA-151; Innovative Research of America) or placebo pellets (25 µg/day, SC-111; Innovative Research of America) were subcutaneously implanted at the time of castration as previously described in detail (22). The serum levels of testosterone and estrogen were determined by ultra-high-performance liquid chromatography–tandem mass spectrometry coupled to an Agilent 6490 triple quadrupole with an Agilent 6490 triple quadrupole (QqQ) mass spectrometer using the detailed protocol described by McLeod et al. (23). The luteinizing hormone (LH) levels were detected with ELISA kits (E-EL-M3053; Elabscience). The quantification of LH levels was obtained by a microplate reader at 550 nm, with the wavelength correction set at 450 nm.

The stromal vascular fraction (SVF) cells were suspended in FACS buffer with propidium iodide (P4864; Sigma-Aldrich) and analyzed with BD LSRFortessa Cell Analyzer (BD Biosciences). Data analysis was carried out with FlowJo software version X.0.7 (Tree Star, Ashland, OR).

For generation of recombinant adenoviruses (11) expressing MPK38 (wild-type [WT] and kinase dead [K40R]), pEBG-MPK38 plasmids (WT and K40R) were used as templates for PCR with primers (Supplementary Table 1) with use of Advantage HD polymerase mix (cat. no. 639241; Takara Bio Inc.).

Values are shown as mean ± SEM unless otherwise indicated, and results are representative of at least three independent experiments. The statistical analyses were performed by one- or two-way ANOVA, followed by Tukey multiple comparison test, with use of GraphPad Prism 7.0 software (GraphPad Software).

The data sets generated or analyzed during the current study are available from the corresponding author upon reasonable request.

To determine the functions of MPK38 in vivo, we generated MPK38^−/− mice by gene trap insertion (Supplementary Fig. 1A). When fed a standard diet, 6- to 7-month-old MPK38^−/− male mice were visibly larger than their WT counterparts (Fig. 1A); however, these differences were not observed in MPK38^−/− female mice. MPK38^−/− male mice had higher liver, spleen, and epididymal fat masses than their age- and sex-matched WT counterparts (Fig. 1A and B). When fed a standard diet, MPK38^−/− and WT male mice had similar body masses for ∼4 months, but obesity became apparent in the MPK38^−/− male mice at ∼6 months of age, despite there being no statistically significant difference in food intake (Fig. 1C). Heterozygous MPK38^+/− male mice demonstrated intermediate levels of body mass gain (Fig. 1C, upper). These results suggest that the obesity induced by MPK38 deficiency is age, sex, and gene dose dependent.

Glucose (GTT) and insulin (ITT) tolerance tests revealed that MPK38^−/− male mice were glucose intolerant and less sensitive to insulin (Fig. 1D). In addition, they displayed significantly higher ratios of circulating glucose and insulin in fasting than in fed conditions (Fig. 1E). Consistent with this, insulin-stimulated in vitro 2-deoxy-glucose uptake was significantly lower in the WAT and muscle of MPK38^−/− male mice than in WT mice (Fig. 1F, left). Furthermore, MPK38 deficiency reduced the activation of the insulin receptor substrate (IRS)–phosphatidylinositol 3-kinase (PI3K) pathway and the expression of GLUT type 4 (GLUT4) and type 1 (GLUT1) in WAT and soleus muscle (Fig. 1F, right). In addition, the expression of mRNAs encoding gluconeogenic proteins, including the catalytic subunit of glucose 6-phosphatase (G6PC), PEPCK-1 (PCK1), and peroxisome proliferator–activated receptor γ coactivator 1-α (PGC1α), were much higher in the livers of MPK38^−/− mice than in WT mice (Fig. 1G, upper). When combined with the high blood glucose concentration in MPK38^−/− male mice (Fig. 1G, lower), these findings implicate MPK38 in the inhibition of hepatic gluconeogenesis.

Abnormal lipid accumulation in the liver (Supplementary Fig. 1B) and substantial increases in the concentrations of circulating FFA, triglycerides, total cholesterol, HDL-C, and LDL-C were identified in the MPK38^−/− mice (Fig. 1H). MPK38^−/− mice showed a higher rate of lipogenesis (Fig. 1I, left) and higher expression of mRNAs encoding lipogenic proteins, including fatty acid synthase (FAS), sterol CoA desaturase 1 (SCD1), and sterol regulatory element–binding transcription factor 1c (SREBP1c), than WT mice (Fig. 1I, right). Furthermore, there were high serum activities of AST and ALT, which reflect the fatty liver phenotype (Supplementary Fig. 1C). Consistent with these findings, the mean adipocyte size was higher in the MPK38^−/− mice than in their heterozygous (MPK38^+/−) or WT counterparts (Fig. 1J). In addition, the expression of mRNAs encoding adipogenic regulators, including C/EBPα, peroxisome proliferator–activated receptor γ (PPARγ), and fatty acid binding protein 4 (FABP4), was much higher in MPK38^−/− mice (Supplementary Fig. 1D). MPK38^−/− mice also showed higher levels of inflammation, evidenced by high circulating concentrations of proinflammatory cytokines, including tumor necrosis factor-α (TNF-α), interleukin (IL)-6, IL-1β, and MCP1, and larger numbers of M1 macrophages, demonstrated with flow cytometry (Fig. 1K and L).

With regard to energy metabolism (Supplementary Fig. 2), MPK38^−/− mice showed lower energy expenditure, 24-h O₂ consumption, CO₂ production, and respiratory exchange ratio. Consistent with this, the expression of mRNAs encoding uncoupling protein 1 (UCP1), PGC1α, and muscle-type carnitine palmitoyltransferase 1 (mCPT1), which are important for thermogenesis (24,25), was considerably lower in MPK38^−/− than in WT mice. Overall, these results suggest that MPK38 has favorable effects on metabolism that prevent obesity in aged mature adult male mice.

We next determined whether MPK38 protects against high-fat diet (HFD)-induced metabolic disturbances specifically in male mice using 7-month-old WT, MPK38^+/−, and MPK38^−/− mice fed chow or an HFD. Compared with WT mice, MPK38^−/− male, but not female, mice became more obese and developed dysregulated glucose and lipid metabolism, as well as developing higher levels of inflammation, under HFD-fed conditions (Fig. 2).

HFD-fed MPK38^−/− male mice displayed much higher ratios of circulating glucose and insulin in fasting than in fed conditions (Fig. 2E), along with glucose intolerance and insulin resistance (Fig. 2F). Consistent with these observations, insulin-stimulated in vitro 2-deoxy-glucose uptake was significantly lower in the WAT and muscle of HFD-fed MPK38^−/− male mice (Fig. 2G). This contention was supported by the finding that HFD-fed MPK38^−/− male mice had higher expression levels of mRNAs encoding gluconeogenic proteins, including G6PC, PCK1, and PGC1α, alongside their higher blood glucose concentrations, than HFD-fed WT male mice (Fig. 2B and H).

HFD-fed MPK38^−/− males also demonstrated a higher rate of lipogenesis and higher expression levels of mRNAs encoding lipogenic proteins, including FAS, SCD1, and SREBP1c, in adipose tissue in comparison with HFD-fed WT males (Fig. 2I). In addition, HFD-fed MPK38^−/− males demonstrated higher levels of expression of key adipogenic regulators, including C/EBPα, PPARγ, and FABP4 (Fig. 2D), and greater inflammation (Fig. 2P and Q). However, these adverse effects of MPK38 deficiency in HFD-fed mice were not observed in females (Supplementary Fig. 3). These results indicate that MPK38 protects against diet-induced metabolic abnormalities in aged mature adult male, but not female, mice.

The activation of ASK1/TGF-β/p53 signaling by MPK38 was significantly less in the WAT of 7-month-old MPK38^−/− male mice than in their age- and sex-matched WT counterparts (Fig. 3A). Similarly, lower levels of ASK1/TGF-β/p53 signaling, and MPK38 expression and kinase activity, were also identified in ob/ob and HFD-fed males (19,20) (Supplementary Fig. 4).

To further investigate the sex-specific role of MPK38 in the amelioration of the metabolic disturbances associated with obesity, we restored WT MPK38 expression in 7-month-old HFD-fed male mice (Supplementary Fig. 5). This caused a significant reduction in adipocyte size (Fig. 3B) and in the expression of mRNAs encoding key adipogenic proteins (C/EBPα, PPARγ, and FABP4) (Fig. 3C). These findings indicate that MPK38 inhibits adipogenesis, leading to an increase in insulin sensitivity.

HFD-fed male mice infected with adenovirus expressing MPK38 (Ad-MPK38) displayed lower circulating glucose and insulin concentrations, and higher glucose tolerance and insulin sensitivity, than uninfected HFD-fed male mice (Fig. 3D and E). Ad-MPK38 infection increased 2-deoxyglucose uptake in both epididymal WAT and muscle (Fig. 3F, left), which may be explained by upregulation of the IRS-PI3K pathway and higher expression of GLUTs (Fig. 3F, right). In addition, the reduction in blood glucose may be explained by lower mRNA expression of hepatic gluconeogenic proteins (G6PC, PCK1, and PGC1α) (Fig. 3G).

Ad-MPK38 infection also reduced circulating FFA concentrations (Fig. 4A, left) and the expression of mRNAs encoding adipose lipogenic proteins (FAS, SCD1, and SREBP1c) (Fig. 4A, right) and increased isoproterenol-stimulated lipolysis (Fig. 4B, left) and the expression of mRNAs encoding adipose fatty acid oxidative proteins (PPARα, CPT1, and ACO) (Fig. 4B, right). Thus, Ad-MPK38 infection upregulated fatty acid utilization by increasing fatty acid oxidation via the mitochondrial and peroxisomal β-oxidation pathways (Fig. 4C). HFD-fed male mice infected with Ad-MPK38 also had lower circulating concentrations of triglycerides, total cholesterol, HDL-C, and LDL-C (Fig. 4D), as well as much lower hepatic lipid accumulation (Fig. 4E). These results suggest a positive role for MPK38 in lipid metabolism. Furthermore, Ad-MPK38 infection promoted ketone body production (Fig. 4F), increased the expression of mRNAs encoding ketogenic genes under fasting conditions (Fig. 4G), and reduced mTORC1 signaling (Fig. 4H). However, the beneficial effects of MPK38 restoration in 7-month-old HFD-fed male mice were not observed in age-matched female mice fed an HFD (Supplementary Fig. 6), indicating that the protective effect of MPK38 is sex specific.

To investigate the sexual dimorphism of late-onset obesity in MPK38^−/− mice, we analyzed the glucose and lipid metabolism of age- and sex-matched WT (MPK38^+/+) and MPK38^−/− mice 6 weeks after neutering (castration/OVX) or sham surgery because the neutering of mice affects glucose and lipid metabolism (26). Castration of 7-month-old MPK38^−/− male mice had little or no effect on the size distribution of adipocytes, according to histological analysis of WAT (Fig. 5A), or on the expression of mRNAs encoding key adipogenic regulators (C/EBPα, PPARγ, and FABP4) in WAT (Fig. 5B).

The castration of WT male mice increased glucose tolerance and insulin sensitivity versus sham controls (Fig. 5C and D) and reduced their circulating insulin and glucose concentrations under fasting conditions (Supplementary Fig. 7A and B). However, these effects were not observed in 7-month-old MPK38^−/− male mice when they were castrated. In addition, castration did not cause changes in insulin-stimulated in vitro 2-deoxyglucose uptake (Fig. 5E, left), IRS-PI3K pathway activation (Fig. 5E, right), or the expression of mRNAs encoding hepatic gluconeogenic genes (G6pc, Pck1, and Pgc1α) (Fig. 5F) in these mice.

The castrated WT male mice demonstrated more rapid lipogenesis than sham controls, which was accompanied by higher mRNA expression of hepatic and adipose lipogenic genes, including Fas, Scd1, and Srebp1c, but lower concentrations of circulating FFA (Fig. 6A and Supplementary Fig. 7C). However, castration of 7-month-old MPK38^−/− male mice did not induce these effects; it had no effect on liver triglyceride storage or circulating total cholesterol, HDL-C, or LDL-C concentrations (Supplementary Fig. 7C and D).

Furthermore, the effects of castration on the mRNA expression of lipolytic genes, such as hormone-sensitive lipase (HSL), adipose triglyceride lipase (ATGL), and β-3 adrenergic receptor (ADRB3) (Fig. 6B); fatty acid utilization (Fig. 6C); the mRNA expression of key genes involved in fatty acid oxidation, such as PPARα, CPT1, and ACO (Fig. 6D, left); isoproterenol-stimulated lipolysis (Fig. 6D, right); liver lipid and triglyceride content (Supplementary Fig. 7C and E); and circulating triglyceride concentrations (Fig. 6D, middle) were not found in 7-month-old MPK38^−/− male mice. In addition, the castration of WT male mice markedly increased ketone body production and ketogenic gene expression under fasting conditions versus sham controls, but these effects were not induced by the castration of 7-month-old MPK38^−/− male mice (Fig. 6E and F). As expected, castrated MPK38^−/− male mice did not show changes in the phosphorylation of S6 at Ser^240/244 in response to fasting or in mTORC1 signaling pathway activation (Supplementary Fig. 7F). All these effects were further confirmed by testosterone replacement exerting opposite effects (Supplementary Fig. 8). Moreover, the phenotype of castrated 7-month-old MPK38^−/− male mice was not shared by age-matched OVX MPK38^−/− female mice (Supplementary Fig. 9). Taken together, these findings suggest an age-specific role for MPK38 in castration, but not OVX-induced alterations, in the glucose and lipid metabolism of mice.

Because AMPK regulates steroid hormone synthesis (27,28), we performed liquid chromatography–tandem mass spectrometry to quantify serum testosterone and estrogen concentrations in sham-operated and neutered WT and MPK38^−/− mice. Seven-month-old uncastrated MPK38^−/− males exhibited serum testosterone concentrations similar to those of age-matched castrated WT males (Fig. 7A, left), but there was no difference between the serum estrogen concentrations of WT and MPK38^−/− males of this age (Fig. 7A, right). By contrast, no equivalent effect was identified in non-OVX MPK38^−/− females of the same age with regard to serum estrogen concentrations (Fig. 7B, left). As expected, OVX also had no effect on the serum testosterone concentrations of the female mice (Fig. 7B, right). All of these findings indicate that MPK38 may play a key role in testosterone production in 7-month-old male mice.

We next determined whether alterations in steroidogenic gene expression are involved in the regulation of testosterone production by MPK38. The expression of 10 genes (29) was measured by quantitative PCR with use of RNA extracted from the testes of 7-month-old WT and MPK38^−/− male mice that had been fed a standard diet: steroidogenic acute regulatory protein (Star) and scavenger receptor b1 (Scarb1), which are implicated in cholesterol transport, and transcriptional activators (Nr4a1, Nr4a3, Crebl2, Cited4, and cJun), a repressor of steroidogenesis (c-Fos), and protein phosphatase methylesterase 1 (Ppme1) and cyclin-dependent kinase 12 (Cdk12), which were used as reference genes. As expected, the expression of Star, Scarb1, and the five transcriptional activators was lower, but that of c-Fos was higher, in 7-month-old MPK38^−/− male mice than in age-matched WT controls, whereas the expression of Ppme1 and Cdk12 was not affected by genotype (Fig. 7C). However, these differences in expression were not identified between 4.5-month-old MPK38^−/− and WT males (Fig. 7D). Furthermore, equivalent differences were not found between the ovaries of WT and MPK38^−/− females, regardless of age, suggesting that MPK38 plays no role in estrogen production in female mice (Fig. 7E and F). Similar results were also obtained in the analysis of other steroidogenic genes (30,31) including cytochrome P450 family 11 subfamily A member 1 (Cyp11a1), cytochrome P450 family 17 subfamily A member 1 (Cyp17a1), and 17β-hydroxysteroid dehydrogenase (17β-hsd), that were important for steroid hormone synthesis (Supplementary Fig. 10). When taken together, these findings suggest that MPK38 contributes to testosterone production in aged mature adult males but not estrogen production in females of the same age.

To determine how sex hormones, such as testosterone and estrogen, regulate the kinase activity and expression of MPK38, we first performed immunoblot analysis of dihydrotestosterone (DHT)- and estradiol (E2)-treated adipocytes from 4.5-month-old WT male and female mice. DHT increased the stability of MPK38, whereas MPK38 stability was reduced by E2 treatment (Fig. 8A). However, exposure of the cells to MG132, a proteasomal inhibitor, increased the stability of MPK38 versus untreated controls (Fig. 8A), which implies that the proteasome pathway plays a role in the degradation of MPK38 (20). Consistent with this, DHT reduced, but E2 increased, the endogenous ubiquitination of MPK38, regardless of sex (Fig. 8B). We also determined whether DHT and E2 have effects on MPK38 degradation through MDM2 because MPK38 physically interacts with MDM2 (32). Compared with control cells that were not treated with DHT or E2, cells treated with DHT showed lower levels of endogenous MPK38-MDM2 complex formation, whereas those treated with E2 showed higher levels of complex formation, whether the cells were derived from male or female mice (Fig. 8C). To further dissect the mechanism whereby testosterone increases the stability of MPK38, we determined the effects of DHT on complex formation by MPK38 and ZPR9 or TRX because ZPR9 and Trx act as a stabilizer and destabilizer of MPK38, respectively (20,32). As expected, treatment with DHT significantly increased endogenous MPK38-ZPR9 complex formation but reduced MPK38-TRX complex formation in both male and female mice (Fig. 8D). However, E2 treatment had the opposite effect on complex formation. We also determined whether DHT and E2 influence the ASK1/TGF-β/p53 signaling induced by MPK38. Compared with untreated control cells, DHT treatment resulted in a significant increase in ASK1/TGF-β/p53 signaling, whereas E2 treatment had the opposite effect, in both male and female mice (Fig. 8E). Similar effects of DHT and E2 on lipid metabolism and MPK38 kinase activity were also observed in hepatocytes, suggesting that MPK38 deficiency equivalently affects WAT and liver in mice (Supplementary Fig. 11). These data indicate that testosterone positively regulates, and estrogen negatively regulates, MPK38-dependent ASK1, TGF-β, and p53 signaling by differentially regulating the stability of MPK38 in both sexes.

In the current study, we found that functional inactivation of MPK38 led to late-onset obesity in males, even when fed a standard chow diet, which was associated with abnormal glucose, lipid, and energy metabolism, as well as inflammation, and that heterozygous MPK38^+/− male mice displayed moderate levels of obesity-induced metabolic disturbances in comparison with MPK38^−/− male mice (Fig. 1), proving haploinsufficiency of MPK38. In addition, Comparative Toxicogenomics Database (CTD) suggests a possibility that MPK38 may be associated with metabolic disorders even though genome-wide association studies are not available at the moment.

Testosterone is a male sex hormone that has important effects on carbohydrate, fat, and protein metabolism (33). In rodents, the insulin responsiveness of male WAT was increased by castration, whereas OVX reduced the insulin responsiveness of female WAT (26). However, in nonhuman primates, testosterone stimulates insulin-stimulated Akt phosphorylation and lipogenesis in the WAT of castrated males (34). In middle-aged men, the age-related decline in testosterone increases the obesity-associated risks of diabetes, cardiovascular diseases, and mortality by increasing body fat mass, in particular that of visceral fat (35,36). However, testosterone replacement therapy in hypogonadal males reverses these metabolic changes (37). The current study shows that the 7-month-old MPK38^−/− male mice have circulating testosterone concentrations similar to those of castrated WT males (Fig. 7A), suggesting that MPK38 has a role in testosterone production in aged mature adult male mice. Furthermore, serum LH levels are significantly decreased in proportion to the testosterone levels in 7-month-old MPK38^−/− males (Supplementary Fig. 12). These findings raise a strong possibility that MPK38 deficiency may affect the crucial components of the hypothalamic-pituitary-gonadal axis, leading to central hypogonadism associated with obesity. Nevertheless, the reverse effects of testosterone replacement are not found in 7-month-old MPK38^−/− males in comparison with age-matched WT controls (Supplementary Fig. 8). Therefore, we cannot rule out the possibility that alterations in other potential targets including metabolic enzymes by MPK38 deficiency may contribute to the sexual dimorphism of late-onset obesity in MPK38^−/− mice.

Estrogen, a female sex hormone, has also been shown to have a significant effect on metabolism. In rats, OVX leads to a marked increase in energy storage, and this is prevented by estrogen replacement (38). However, the results of the current study show that there is no difference in the effects of neutering on metabolism between WT and MPK38^−/− female mice (Supplementary Fig. 9), suggesting that MPK38 does not have a role in the effects of estrogen on metabolism in female mice. Our data also show that E2, the principal circulating estrogen, inhibits the activity of MPK38, regardless of sex (Fig. 8E), but activates AMPK by increasing the phosphorylation of its α-catalytic subunit (39).

We also show that MPK38 stimulates steroidogenesis by increasing the expression of genes encoding proteins involved in cholesterol transport (Scarb1 and Star) (Fig. 7). In contrast to the effects of AMPK (29), MPK38 increases the expression of two activators (c-Jun and Nr4a1) of steroidogenesis. NR4A1 prevents AMPKα activation in hepatocytes by sequestering liver kinase B1 (LKB1), an AMPK upstream kinase, in the nucleus (40). The current study shows that NR4A1 is also involved in the inactivation of MPK38 because it stimulates the nuclear localization of MPK38 and its positive regulator ZPR9, and the cytoplasmic localization of its negative regulator TRX1, which leads to the inactivation of AMPK in the cytoplasm (Supplementary Fig. 13 and Supplementary Fig. 14A). However, the nuclear localization of LKB1 induced by NR4A1 does not affect the activity of MPK38 because LKB1 has no effect on MPK38 (Supplementary Fig. 13 and Supplementary Fig. 14B). These findings suggest that MPK38, unlike AMPK, modulates the expression of transcriptional regulators that play crucial roles in steroidogenesis in a sex- and age-dependent fashion (Fig. 7C–F).

In summary, the data presented here provide evidence that MPK38 acts as a metabolic regulator in aged mature adult male, but not female, mice, in which it prevents obesity-induced metabolic disturbances, and suggest that MPK38 may be an attractive therapeutic target for obesity in middle-aged men.

Acknowledgments. The authors thank E.-Y. Lee (National Institute of Horticultural and Herbal Science, Eumsung), J.-H. Mang (Clinical Research Associates Korea Incorporated, Cheongju), S.-H. Yeon (NEXEL Co., Ltd., Seoul), and J.-H. Kwak (Immunology Laboratory, Department of Biochemistry, Chungbuk National University) for excellent technical assistance during animal experiments. The authors also thank Dr. C.-S. Choi (Department of Internal Medicine, Gil Medical Center, Gachon University Graduate School of Medicine, Incheon, Korea) and Dr. K.-T. Kim (Department of Life Sciences, Pohang University of Science and Technology, Pohang, Korea) for warm and kind help in initial characterization of knockout mice and animal studies and Dr. K. Lee (Hormone Research Center, School of Biological Sciences and Technology, Chonnam National University, Gwangju, Korea) for excellent technical assistance in castration and OVX studies.

Funding. This work was supported by grants from the National Research Foundation of Korea (2018R1A2A2A05018692) and the Research Year of Chungbuk National University in 2019.

Duality of Interest. No potential conflicts of interest relevant to this article were reported.

Author Contributions. H.-A.S. performed all experiments and data analysis and wrote all experimental protocols. H.H. designed, supervised, and interpreted all studies and wrote the manuscript. H.-A.S. and H.H. are the guarantors of this work and, as such, had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis.