The browning of white adipose tissue (WAT) enhances thermogenesis and represents a promising approach for combating obesity and metabolic disorders. MicroRNA-494 (miR-494) acts as a suppressor of browning in cultured adipocytes via regulation of peroxisome proliferator-activated receptor γ coactivator 1-α (PGC-1α), and its inhibition is expected to promote browning and thereby improve obesity and metabolic disorders. To assess its in vivo role and therapeutic potential, we generated miR-494 knockout (KO) mice using CRISPR/Cas9. KO mice showed increased browning of WAT and resistance to high-fat diet–induced obesity. Notably, they also exhibited improved glucose tolerance even under normal chow feeding without weight loss. Ex vivo analysis revealed enhanced β-adrenergic-stimulated oxidative phosphorylation directly induced by miR-494 deletion. Metabolomic and Seahorse analyses further suggested accelerated glucose metabolism independent of insulin secretion or sensitivity. Analysis of human adipose tissue transcriptomic data supported the association between low miR-494 expression and better glucose tolerance without weight differences. These findings suggest that suppression of miR-494 improves glucose metabolism through both insulin-dependent and insulin-independent mechanisms, independently of changes in body weight. Targeting miR-494 could represent a potential therapeutic strategy for obesity and various forms of diabetes.

Abstract Objective: Pancreatic ductal adenocarcinoma (PDAC) is a formidable challenge to cure and expected to be the second leading cause of cancer-related deaths in the US by 2030. Its aggressive nature stems from late diagnosis and early metastasis. PDAC cells interact with the host system and impair the immune response. This communication leads to the production of mediators by host cells, creating an environment that supports PDAC cells growth and the formation of premetastatic niches. Despite progress, the specific factors and mechanisms underlying immune modulation in PDAC progression are not fully understood. Methods: PDAC mice models (WT-KPCL and KOSAA-KPCL) were developed by administering luciferase-expressing KPC cells (KPCL) intraperitoneally into eight weeks old male C57BL/6 mice. The control groups (WT and KOSAA) received PBS. Plasma proteomics was analyzed by using Mass Spectrometry (MS). PDAC cells growth was visualized by bioluminescence using Lago X in-vivo imaging system. Results: During tumor progression, KPCL mice exhibited significant weight loss, with the loss of adipose tissue and skeletal muscle, indicating a hallmark of pancreatic cancer. To identify potential circulating molecules involved in tumor progression, we conducted proteomics analysis on plasma samples using MS. Comparative analysis revealed that out of 734 circulating proteins, 86 were upregulated and 35 were downregulated in tumor mice compared to controls. Serum Amyloid A 1 and 2 were found to be the most abundantly expressed proteins in the plasma of tumor mice, with a remarkable 1024-fold increase compared to controls. To determine the major source of SAA in tumor mice, we measured SAA1, 2, and 3 expressions in various tissues, including adipose tissue, liver, muscle, and tumor. Interestingly, adipose tissue displayed a substantial contribution to SAA levels in WT-KPCL. However, we were unable to detect SAA expression in the tumor tissue from KOSAA-KPCL indicating negligible source of SAA from KPCL cells. Intrigued by these findings, we studied the role of SAA in PDAC cell proliferation in-vivo. Luciferase-expressing KPC cells were injected into SAA-KO mice (KOSAA-KPCL) and controls (WT-KPCL). Unexpectedly, bioluminescence imaging revealed a dramatic growth of KPC cells in the pancreas of SAA-deficient animals. This was supported by a significantly higher rate of metastasis to the liver, increased by 62.5% in KOSAA-KPCL compared to WT-KPCL. SAA is retinol-binding protein and retinoic acid plays a vital role in the immune system. We found a decreased concentration of retinoic acid in the plasma of KOSAA-KPCL mice, with a significant decrease in CD8 expression in tumor tissue isolated from KOSAA-KPCL. These results suggest that SAA modulates immune response during PDAC cells growth. Conclusion: Our study demonstrates that adipose tissue is likely a major source of SAA, where the absence of SAA leads to enhanced PDAC cells growth and metastasis through immune modulation. These findings suggest the therapeutic potential of SAA for pancreatic cancer. Citation Format: Yuki Takamuku, Mengistu Lemecha, Keiichi Itakura. Differential growth of pancreatic ductal adenocarcinoma cells in serum amyloid A (SAA) deficient mouse model through immune modulation [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr C031.

Adipocyte browning is a potent therapeutic strategy for obesity and metabolic disorders. We previously reported that miR-494 served as a negative regulator of mitochondrial biogenesis during browning in cultured adipocytes. However, the in vivo evidence has not been provided. In this study, we examined the role of miR-494 in adipocyte browning and aging using miR-494 knockout (KO) mice. KO mice were generated using CRISPR/Cas9 system. KO mice had equivalent energy expenditure, physical activity, food intake and body weight compared to wild-type (WT) mice; had lower resting respiratory quotient (0.95±0.006 vs 0.91±0.004), less visceral fat (16.3±1.08 vs 13.4±0.96 g/kg body weight) and 3.7-fold increased expression of Ucp1 in subcutaneous fat. In addition, KO mice showed cold tolerance during 3 hours exposure to 4ºC and increased oxygen consumption than WT mice after administration of a β3 agonist in thermoneutral condition, indicating an increase in non-shivering thermogenesis. In adipose tissues of KO mice, increased expression of Pgc-1α, a target gene of miR-494, and tendency to increase in mitochondrial DNA were observed. Primary adipocytes derived from KO mice showed enhanced mitochondrial respiration driven by fatty acids as determined by extracellular flux analysis. Finally, KO mice showed an extended median lifespan 29% relative to WT mice (from 113.7 to 146.7 weeks). Aged KO mice (≥ 2 years old) showed reduced age-related changes, such as hair graying and low physical activity. In conclusion, deletion of miR-494 demonstrated increased adipocyte browning and survival advantage in mice. Although there are numerous studies that focused on promoting adipocyte browning, its long-term effect has not been well evaluated. Thus, our data showing the association among miR-494, adipocyte browning and longevity provide a novel insight into adipocyte biology and aging study. The gene sequence of miR-494 and its binding site to Pgc-1α are conserved from mice to humans; it is expected to be applied to human research. Disclosure L.Sugawara: None. K.Morino: Research Support; AstraZeneca, Astellas Pharma Inc., Sanwa Kagaku Kenkyusho, Ono Pharmaceutical Co., Ltd. H.Iwasaki: None. S.Ida: None. T.Yanagimachi: None. M.Lemecha: None. H.Maegawa: None. Y.Fujita: Research Support; Terumo Corporation, Boehringer Ingelheim Japan, Inc., Japan Society for the Promotion of Science, Mitsubishi Tanabe Pharma Corporation, Sumitomo Dainippon Pharma Co., Ltd. S.Kume: Research Support; Boehringer Ingelheim International GmbH, Japan Society for the Promotion of Science. Funding Grants-in-Aid for Scientific Research (22K08650)

During B cell development in bone marrow, large precursor B cells (large Pre-B cells) proliferate rapidly, exit the cell cycle, and differentiate into non-proliferative (quiescent) small Pre-B cells. Dysregulation of this process may result in the failure to produce functional B cells and pose a risk of leukemic transformation. Here, we report that AT rich interacting domain 5B (ARID5B), a B cell acute lymphoblastic leukemia (B-ALL) risk gene, regulates B cell development at the Pre-B stage. In both mice and humans, we observed a significant upregulation of ARID5B expression that initiates at the Pre-B stage and is maintained throughout later stages of B cell development. In mice, deletion of Arid5b in vivo and ex vivo exhibited a significant reduction in the proportion of immature B cells but an increase in large and small Pre-B cells. Arid5b inhibition ex vivo also led to an increase in proliferation of both Pre-B cell populations. Metabolic studies in mouse and human bone marrow revealed that fatty acid uptake peaked in proliferative B cells then decreased during non-proliferative stages. We showed that Arid5b ablation enhanced fatty acid uptake and oxidation in Pre-B cells. Furthermore, decreased ARID5B expression was observed in tumor cells from B-ALL patients when compared to B cells from non-leukemic individuals. In B-ALL patients, ARID5B expression below the median was associated with decreased survival particularly in subtypes originating from Pre-B cells. Collectively, our data indicated that Arid5b regulates fatty acid metabolism and proliferation of Pre-B cells in mice, and reduced expression of ARID5B in humans is a risk factor for B cell leukemia.

Total body irradiation (TBI) is a commonly used conditioning regimen for hematopoietic stem cell transplant (HCT), but dose heterogeneity and long-term organ toxicity pose significant challenges. Total marrow irradiation (TMI), an evolving radiation conditioning regimen for HCT can overcome the limitations of TBI by delivering the prescribed dose targeted to the bone marrow (BM) while sparing organs at risk. Recently, our group demonstrated that TMI up to 20 Gy in relapsed/refractory AML patients was feasible and efficacious, significantly improving 2-year overall survival compared to the standard treatment. Whether such dose escalation is feasible in elderly patients, and how the organ toxicity profile changes when switching to TMI in patients of all ages are critical questions that need to be addressed. We used our recently developed 3D image-guided preclinical TMI model and evaluated the radiation damage and its repair in key dose-limiting organs in young (~8 weeks) and old (~90 weeks) mice undergoing congenic bone marrow transplant (BMT). Engraftment was similar in both TMI and TBI-treated young and old mice. Dose escalation using TMI (12 to 16 Gy in two fractions) was well tolerated in mice of both age groups (90% survival ~12 Weeks post-BMT). In contrast, TBI at the higher dose of 16 Gy was particularly lethal in younger mice (0% survival ~2 weeks post-BMT) while old mice showed much more tolerance (75% survival ~13 weeks post-BMT) suggesting higher radio-resistance in aged organs. Histopathology confirmed worse acute and chronic organ damage in mice treated with TBI than TMI. As the damage was alleviated, the repair processes were augmented in the TMI-treated mice over TBI as measured by average villus height and a reduced ratio of relative mRNA levels of amphiregulin/epidermal growth factor (areg/egf). These findings suggest that organ sparing using TMI does not limit donor engraftment but significantly reduces normal tissue damage and preserves repair capacity with the potential for dose escalation in elderly patients.

Objective: Adipose tissue is the largest endocrine organ. When activated by cancer cells, adipocytes secrete adipocytokines and release fatty acids, which are then transferred to cancer cells and used for structural and biochemical support. How this metabolic symbiosis between cancer cells and adipocytes affects skeletal muscle and thermogenesis during cancer cachexia is unknown. Cancer cachexia is a multiorgan syndrome and how the communication between tissues is established has yet to be determined. We investigated adipose tissue secretory factors and explored their role in crosstalk of adipocytes, muscle, and tumor during pancreatic cancer cachexia. Methods: We used a pancreatic cancer cachexia mouse model generated by syngenic implantation of pancreatic ductal adenocarcinoma (PDAC) cells (KPC) intraperitoneally into C57BL/6 mice and Lcn2-knockout mice. For in vitro studies, adipocytes (3T3-L1 and primary adipocytes), cachectic cancer cells (Panc0203), non-cachectic cancer cells (Du145 cells), and skeletal muscle cells (C2C12 myoblasts) were used. Results: To identify molecules involved in the crosstalk of adipose tissue with muscle and tumors, we treated 3T3-L1 adipocytes with conditioned medium (CM) from cancer cells. Upon screening the secretomes from PDAC-induced adipocytes, several adipocytokines were identified, including lipocalin 2 (Lcn2). We investigated Lcn2 as a potential mediator of cachexia induced by adipocytes in response to PDAC. During tumor progression, mice exhibited a decline in body weight gain, which was accompanied by loss of adipose and muscle tissues. Tumorharboring mice developed drastic hypothermia because of a dramatic loss of fat in brown adipose tissue (BAT) and suppression of the thermogenesis pathway. We inhibited Lcn2 with an anti-Lcn2 antibody neutralization or genomic ablation in mice. Lcn2 deficiency significantly improved body temperature in tumor-bearing mice, which was supported by the increased expression of Ucp1 and b3-adrenergic receptor in BAT. In addition, Lcn2 inhibition abrogated the loss of fat and muscle in tumor-bearing mice. In contrast to tumor-bearing WT mice, the corresponding Lcn2-knockout mice showed reduced ATGL expression in iWAT and decreased the expression of muscle atrophy molecular markers MuRF-1 and Fbx32. Conclusions: This study showed that Lcn2 is causally involved in the dysregulation of adipose tissue-muscle-tumor crosstalk during pancreatic cancer cachexia. Therapeutic targets that suppress Lcn2 may minimize the progression of cachexia. Published by Elsevier GmbH. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Background: Adipose tissue considerably influence metabolic homeostasis and play a central role in regulating whole-body energy and glucose metabolism. Previously, we have reported that mice with the global deletion of Arid5b are lean and have less fat in white and brown adipose tissues. However, this role is unknown in adipocyte specific Arid5b knockout (5bFKO) mice. Aim: We investigated the effects of adipose-specific deficiency of Arid5b on systemic glucose homeostasis and adipose tissue metabolism. Result: Deletion of Arid5b in adipose tissue using adiponectin promoter-driven Cre recombinase significantly improved glucose tolerance with significant reduction of insulin level. Body weight of 5bFKO mice was significantly reduced at the later age. Core body temperature of 5bFKO mice was significantly higher compared to the control mice. Robust increase in the protein expression of uncoupling protein 1 (Ucp1) was observed in inguinal white adipose tissue (iWAT) of 5bFKO mice housed at ambient temperature. This phenotype was further confirmed by prolonged cold exposure where Ucp1 and mitochondrial proteins such as pyruvate dehydrogenase (PDH), mitochondrially encoded cytochrome c oxidase (MTCO1) and ATP5A were substantially increased in iWAT of adipocyte specific Arid5b deficient mice. Gene expression of beige adipogenesis markers such as cell death inducing DFFA like effector A (Cidea) and iodothyronine deiodinase 2 (Dio2) were also significantly increased indicating browning of iWAT. Furthermore, loss of Arid5B in fat led to significant reduction in macrophages and total immune cells in iWAT. All together these results show that the deletion of Arid5b could promote browning of iWAT. Conclusion: These findings demonstrate that Arid5b regulates thermogenic genes expression to promote beige adipocytes formation and controls adipose inflammation. Inhibition of Arid5b may provide a novel therapeutic approach for obesity and diabetes. Disclosure M. Shukare: None. R. Huang: None. A. Ehsani: None. G. Zhang: None. J. Chalise: None. R. H. Whitson: None. K. Itakura: None.

MYOD-induced microRNA-494-3p expression inhibits fast oxidative myotube formation by downregulating myosin heavy chain 2 (MYH2) in human induced pluripotent stem cells (hiPSCs) during skeletal myogenesis. However, the molecular mechanisms regulating MYH2 expression via miR-494-3p remain unknown. Here, using bioinformatic analyses, we show that miR-494-3p potentially targets the transcript of the E1A-binding protein p300 at its 3′-untranslated region (UTR). Myogenesis in hiPSCs with the Tet/ON-myogenic differentiation 1 ( MYOD1 ) gene (MyoD-hiPSCs) was induced by culturing them in doxycycline-supplemented differentiation medium for 7 days. p300 protein expression decreased after transient induction of miR-494-3p during myogenesis. miR-494-3p mimics decreased the levels of p300 and its downstream targets MYOD and MYH2 and myotube formation efficiency. p300 knockdown decreased myotube formation efficiency, MYH2 expression, and basal oxygen consumption rate. The binding of miR-494-3p to the wild type p300 3′-UTR, but not the mutated site, was confirmed using luciferase assay. Overexpression of p300 rescued the miR-494-3p mimic-induced phenotype in MyoD-hiPSCs. Moreover, miR-494-3p mimic reduced the levels of p300, MYOD, and MYH2 in skeletal muscles in mice. Thus, miR-494-3p might modulate MYH2 expression and fast oxidative myotube formation by directly regulating p300 levels during skeletal myogenesis in MyoD-hiPSCs and murine skeletal muscle tissues.

The intestinal microbiome produces short-chain fatty acids (SCFAs) from dietary fiber and has specific effects on other organs. During endurance exercise, fatty acids, glucose, and amino acids are major energy substrates. However, little is known about the role of SCFAs during exercise. To investigate this, mice were administered either multiple antibiotics or a low microbiome-accessible carbohydrate (LMC) diet, before endurance testing on a treadmill. Two-week antibiotic treatment significantly reduced endurance capacity versus the untreated group. In the cecum acetate, propionate, and butyrate became almost undetectable in the antibiotic-treated group, plasma SCFA concentrations were lower, and the microbiome was disrupted. Similarly, 6-wk LMC treatment significantly reduced exercise capacity, and fecal and plasma SCFA concentrations. Continuous acetate but not saline infusion in antibiotic-treated mice restored their exercise capacity (P < 0.05), suggesting that plasma acetate may be an important energy substrate during endurance exercise. In addition, running time was significantly improved in LMC-fed mice by fecal microbiome transplantation from others fed a high microbiome-accessible carbohydrate diet and administered a single portion of fermentable fiber (P < 0.05). In conclusion, the microbiome can contribute to endurance exercise by producing SCFAs. Our findings provide new insight into the effects of the microbiome on systemic metabolism.

BACKGROUND:Teff is a staple food in Ethiopia that is rich in dietary fiber. Although gaining popularity in Western countries because it is gluten-free, the effects of teff on glucose metabolism remain unknown. AIM:To evaluate the effects of teff on body weight and glucose metabolism compared with an isocaloric diet containing wheat. RESULTS:Mice fed teff weighed approximately 13% less than mice fed wheat (p < 0.05). The teff-based diet improved glucose tolerance compared with the wheat group with normal chow but not with a high-fat diet. Reduced adipose inflammation characterized by lower expression of TNFα, Mcp1, and CD11c, together with higher levels of cecal short chain fatty acids such as acetate, compared with the control diet containing wheat after 14 weeks of dietary treatment. In addition, beige adipocyte formation, characterized by increased expression of Ucp-1 (~7-fold) and Cidea (~3-fold), was observed in the teff groups compared with the wheat group. Moreover, a body-weight matched experiment revealed that teff improved glucose tolerance in a manner independent of body weight reduction after 6 weeks of dietary treatment. Enhanced beige adipocyte formation without improved adipose inflammation in a body-weight matched experiment suggests that the improved glucose metabolism was a consequence of beige adipocyte formation, but not solely through adipose inflammation. However, these differences between teff- and wheat-containing diets were not observed in the high-fat diet group. CONCLUSIONS:Teff improved glucose tolerance likely by promoting beige adipocyte formation and improved adipose inflammation.

Mitochondria are critical in heat generation in brown and beige adipocytes. Mitochondrial number and function are regulated in response to external stimuli, such as cold exposure and β3 adrenergic receptor agonist. However, the molecular mechanisms regulating mitochondrial biogenesis during browning, especially by microRNAs, remain unknown. We investigated the role of miR-494-3p in mitochondrial biogenesis during adipogenesis and browning. Intermittent mild cold exposure of mice induced PPARγ coactivator1-α (PGC1-α) and mitochondrial TFAM, PDH, and ANT1/2 expression along with uncoupling protein-1 (Ucp1) in inguinal white adipose tissue (iWAT). miR-494-3p levels were significantly downregulated in iWAT upon cold exposure (p < 0.05). miR-494-3p overexpression substantially reduced PGC1-α expression and its downstream targets TFAM, PDH and MTCO1 in 3T3-L1 white and beige adipocytes (p < 0.05). miR-494-3p inhibition in 3T3-L1 white adipocytes resulted in increased PDH (p < 0.05). PGC1-α, TFAM and Ucp1 mRNA levels were robustly downregulated by miR-494-3p overexpression in 3T3-L1 beige adipocytes, along with strongly decreased oxygen consumption rate. PGC1-α and Ucp1 proteins were downregulated by miR-494-3p in primary beige cells (p < 0.05). Luciferase assays confirmed PGC1-α as a direct gene target of miR-494-3p. Our findings demonstrate that decreased miR-494-3p expression during browning regulates mitochondrial biogenesis and thermogenesis through PGC1-α.

Background: Mitochondria play an essential role in the heat generation in beige adipocytes. Their number and function are regulated in response to external stimuli such as cold exposure and beta-3 adrenergic receptor (β3-AR) agonist. Previously, we have reported that miR-494 regulates mitochondrial biogenesis in the skeletal muscle. However, this remains unknown in beige adipocytes. Aim: We investigated the role of miR-494 on mitochondrial biogenesis during adipogenesis and browning. Result: C57BL/6J mice were subjected to intermittent mild cold exposure. The expression levels of peroxisome proliferator activated receptor gamma coactivator 1-alpha (PGC1-α) and mitochondrial proteins including mitochondrial transcription factor A (TFAM), pyruvate dehydrogenase (PDH), mitochondrially encoded cytochrome c oxidase (MTCO1) and uncoupling protein 1 (Ucp1) were strongly increased in inguinal white adipose tissue (iWAT). On the contrary, that of miR-494 resulted in 27% reduction (p < 0.05) in iWAT following 12°C cold exposure for 6 hours. Furthermore, β3-AR stimulation potently reduced miR-494 expression in 3T3-L1 beige cells. Overexpression of miR-494 substantially reduced the protein expression of PGC1-α and its downstream targets such asTFAM and MTCO1 (p < 0.05). In contrast, antisense of miR-494 significantly increased the expression of TFAM, MTCO1 and PDH (p < 0.05). Overexpression of miR-494 strongly decreased the oxygen consumption rate in 3T3-L1 beige cells and protein expression of PGC1-α and Ucp1 (p < 0.05) in primary beige adipocytes. Finally, we explored the direct target of miR-494 and found that 3`UTR region of PGC1-α is a direct target of miR-494 by luciferase assay. Conclusion: These findings demonstrate that miR-494 directly inhibits the expression of PGC1-α in adipose tissue. The decreased miR-494 expression during adipocyte differentiation removes its inhibitory effect, leading to stimulation of Ucp1 expression and mitochondrial biogenesis. Disclosure M. Lemecha: None. K. Morino: Research Support; Self; Astellas Pharma US, Inc., AstraZeneca, Sunstar Inc., CMIC Pharmascience, Kowa Pharmaceutical. T. Imamura: None. H. Iwasaki: None. N. Ohashi: None. H. Yamamoto: None. S. Ugi: Research Support; Self; Boehringer Ingelheim Pharmaceuticals, Inc., MSD K.K. H. Maegawa: Speaker's Bureau; Self; Astellas Pharma US, Inc.. Research Support; Self; Astellas Pharma US, Inc.. Speaker's Bureau; Self; Mitsubishi Tanabe Pharma Corporation. Research Support; Self; Mitsubishi Tanabe Pharma Corporation. Speaker's Bureau; Self; Sanofi. Research Support; Self; Sanofi. Speaker's Bureau; Self; Nippon Boehringer Ingelheim Co. Ltd.. Research Support; Self; Nippon Boehringer Ingelheim Co. Ltd.. Speaker's Bureau; Self; Daiichi Sankyo Company, Limited. Research Support; Self; Daiichi Sankyo Company, Limited, Takeda Pharmaceutical Company. Speaker's Bureau; Self; Takeda Pharmaceutical Company, Novo Nordisk A/S, Eli Lilly and Company.

O-GlcNAcylation is a post-translational modification that is characterized by the addition of N-acetylglucosamine (GlcNAc) to proteins by O-GlcNAc transferase (Ogt). The degree of O-GlcNAcylation is thought to be associated with glucotoxicity and diabetic complications, because GlcNAc is produced by a branch of the glycolytic pathway. However, its role in skeletal muscle has not been fully elucidated. In this study, we created skeletal muscle-specific Ogt knockout (Ogt-MKO) mice and analyzed their glucose metabolism. During an intraperitoneal glucose tolerance test, blood glucose was slightly lower in Ogt-MKO mice than in control Ogt-flox mice. High fat diet-induced obesity and insulin resistance were reversed in Ogt-MKO mice. In addition, 12-month-old Ogt-MKO mice had lower adipose and body mass. A single bout of exercise significantly reduced blood glucose in Ogt-MKO mice, probably because of higher AMP-activated protein kinase α (AMPKα) protein expression. Furthermore, intraperitoneal injection of 5-aminoimidazole-4-carboxamide ribonucleotide, an AMPK activator, resulted in a more marked decrease in blood glucose levels in Ogt-MKO mice than in controls. Finally, Ogt knockdown by siRNA in C2C12 myotubes significantly increased protein expression of AMPKα, glucose uptake and oxidation. In conclusion, loss of O-GlcNAcylation facilitates glucose utilization in skeletal muscle, potentially through AMPK activation. The inhibition of O-GlcNAcylation in skeletal muscle may have an anti-diabetic effect, through an enhancement of glucose utilization during exercise.

Adipose tissues considerably influence metabolic homeostasis, and both white (WAT) and brown (BAT) adipose tissue play significant roles in lipid and glucose metabolism. O-linked N-acetylglucosamine (O-GlcNAc) modification is characterized by the addition of N-acetylglucosamine to various proteins by O-GlcNAc transferase (Ogt), subsequently modulating various cellular processes. However, little is known about the role of O-GlcNAc modification in adipose tissues. Here, we report the critical role of O-GlcNAc modification in cold-induced thermogenesis. Deletion of Ogt in WAT and BAT using adiponectin promoter-driven Cre recombinase resulted in severe cold intolerance with decreased uncoupling protein 1 (Ucp1) expression. Furthermore, Ogt deletion led to decreased mitochondrial protein expression in conjunction with decreased peroxisome proliferator-activated receptor γ coactivator 1-α protein expression. This phenotype was further confirmed by deletion of Ogt in BAT using Ucp1 promoter-driven Cre recombinase, suggesting that O-GlcNAc modification in BAT is responsible for cold-induced thermogenesis. Hypothermia was significant under fasting conditions. This effect was mitigated after normal diet consumption but not after consumption of a fatty acid-rich ketogenic diet lacking carbohydrates, suggesting impaired diet-induced thermogenesis, particularly by fat. In conclusion, O-GlcNAc modification is essential for cold-induced thermogenesis and mitochondrial biogenesis in BAT. Glucose flux into BAT may be a signal to maintain BAT physiological responses.

Amla is one of the most important plants in Indian traditional medicine and has been shown to improve various age-related disorders while decreasing oxidative stress. Mitochondrial dysfunction is a proposed cause of aging through elevated oxidative stress. In this study, we investigated the effects of Amla on mitochondrial function in C2C12 myotubes, a murine skeletal muscle cell model with abundant mitochondria. Based on cell flux analysis, treatment with an extract of Amla fruit enhanced mitochondrial spare respiratory capacity, which enables cells to overcome various stresses. To further explore the mechanisms underlying these effects on mitochondrial function, we analyzed mitochondrial biogenesis and antioxidant systems, both proposed regulators of mitochondrial spare respiratory capacity. We found that Amla treatment stimulated both systems accompanied by AMPK and Nrf2 activation. Furthermore, we found that Amla treatment exhibited cytoprotective effects and lowered reactive oxygen species (ROS) levels in cells subjected to t-BHP-induced oxidative stress. These effects were accompanied by increased oxygen consumption, suggesting that Amla protected cells against oxidative stress by using enhanced spare respiratory capacity to produce more energy. Thus we identified protective effects of Amla, involving activation of mitochondrial function, which potentially explain its various effects on age-related disorders.