The dairy-derived peptide Miltin exerts anti-obesity effects by increasing adipocyte thermogenesis.

Growing research has highlighted that the consumption of dairy products improves the metabolic health in obese individuals by functioning as regulatory modulators. However, the molecular basis of this effect remains largely unknown. Herein, we report a dairy-derived peptide, which we named Miltin, that activates the thermogenesis of brown adipocytes and increases white adipocyte browning. Previously, Miltin was merely identified for its antioxidant capacity, although it is commonly present in different dairy products. In this study, we revealed the effect of Miltin in modulating adipose thermogenesis and further explored its potential in treating obesity through in vivo and in vitro strategies. The administration of Miltin in mice fed with a high-fat diet resulted in enhanced thermogenesis, improved glucose homeostasis, and reduced body mass and lipid accumulation, indicating the anti-obesity effect of Miltin. Genomic analysis revealed that Miltin modulates thermogenesis by inducing the activation of the MAPK signaling pathway by preferentially interacting with GADD45γ to promote its stability. Together, our findings indicate that Miltin's role in initiating the thermogenesis of adipocytes makes it a potential anti-obesity therapy for future development.

The secreted peptide BATSP1 promotes thermogenesis in adipocytes.

Although brown adipose tissue (BAT) has historically been viewed as a major site for energy dissipation through thermogenesis, its endocrine function has been increasingly recognized. However, the circulating factors in BAT that play a key role in controlling systemic energy homeostasis remain largely unexplored. Here, we performed a peptidomic analysis to profile the extracellular peptides released from human brown adipocytes upon exposure to thermogenic stimuli. Specifically, we identified a secreted peptide that modulates adipocyte thermogenesis in a cell-autonomous manner, and we named it BATSP1. BATSP1 promoted BAT thermogenesis and induced browning of white adipose tissue in vivo, leading to increased energy expenditure under cold stress. BATSP1 treatment in mice prevented high-fat diet-induced obesity and improved glucose tolerance and insulin resistance. Mechanistically, BATSP1 facilitated the nucleocytoplasmic shuttling of forkhead transcription factor 1 (FOXO1) and released its transcriptional inhibition of uncoupling protein 1 (UCP1). Overall, we provide a comprehensive analysis of the human brown adipocyte extracellular peptidome following acute forskolin (FSK) stimulation and identify BATSP1 as a novel regulator of thermogenesis that may offer a potential approach for obesity treatment.

Extracellular Vesicles Derived from Human Umbilical Cord MSC Improve Vascular Endothelial Function in In Vitro and In Vivo Models of Preeclampsia through Activating Arginine Metabolism.

Endothelial cell damage is an important feature of preeclampsia (PE). Human umbilical mesenchymal stem-cell-derived extracellular vesicles (HUMSCs-derived EVs) have been shown to have therapeutic effects on a variety of diseases and tissue damage. However, the therapeutic effect of HUMSCs-derived EVs on endothelial injury in PE remains unclear. This study explored the possible mechanism of HUMSCs-derived EVs in the treatment of endothelial cell injury. Tumor necrosis factor α- and lipopolysaccharide-induced endothelial dysfunction models were used to evaluate the therapeutic effect of HUMSCs-derived EVs on endothelial injury. We further constructed PE mouse models to explore the function of HUMSCs-derived EVs in vivo. The changes of metabolites in endothelial cells after HUMSCs-derived EVs treatment were analyzed by metabolomics analysis and further validated by cell experiments. HUMSCs-derived EVs treatment can alleviate endothelial cell injury in PE, involving cell proliferation, migration, angiogenesis, and anti-inflammatory. Importantly, administration of HUMSCs-derived EVs improves hypertension and proteinuria in PE mice, alleviates kidney damage, and promotes vascularization in the placenta. Furthermore, metabolomics analysis found that the arginine metabolic pathway is activated after HUMSCs-derived EVs treatment. We also observed increased arginine level, nitric oxide content, and nitric oxide synthase activity, and further experiments proved that activating the arginine metabolic pathway could alleviate endothelial dysfunction. Our results reveal that HUMSCs-derived EVs could ameliorate PE endothelial dysfunction by activating the arginine metabolic pathway and may serve as a therapeutic method for treating PE.

Human milk-derived extracellular vesicles alleviate high fat diet-induced non-alcoholic fatty liver disease in mice.

BACKGROUND: Nonalcoholic fatty liver disease (NAFLD), characterized by excessive hepatic lipid accumulation, imposes serious challenges on public health worldwide. Breastfeeding has been reported to reduce the risk of NAFLD. Extracellular vesicles (EVs) are bilayer membrane vesicles released from various cells into the extracellular space, participating in multiple life processes. Whether EVs from human milk exert metabolic benefits against NAFLD is worth investigating. METHODS AND RESULTS: In this study, the EVs were isolated from human milk collected from healthy mothers and quantified. Functional analyses were performed using the NAFLD mouse model and free fatty acid (FFA)-stimulated mouse primary hepatocytes. The results showed that human milk-derived EVs could effectively alleviate high fat diet-induced hepatic steatosis and insulin resistance in mice with NAFLD via inhibiting lipogenesis and increasing lipolysis. The FFA-induced lipid accumulation was also inhibited in hepatocytes after treatment with human milk-derived EVs. Mechanistically, the human milk derived-EVs cargo (proteins and miRNAs), which linked to lipid metabolism, may be responsible for these beneficial effects. CONCLUSION: The findings of this study highlighted the therapeutic benefits of human milk-derived EVs and provided a new strategy for NAFLD treatment.

Influence of gestational diabetes mellitus on lipid signatures in breast milk and association with fetal physical development.

Gestational diabetes mellitus (GDM) commonly leads to adverse pregnancy outcomes and long-term metabolic complications in offspring. Breastfeeding has been shown to rewrite the fetal "metabolic programming" resulting from maternal diabetes and finally lead to a lower risk of future metabolic disease. Lipids in breast milk act like hormones to promote infant growth and development, but there is minimal information invested thus far in constitution changes of lipids in breast milk, especially in the context of GDM. In the present study, we performed a lipidomics analysis to compare the lipid composition in breast milk collected from women with or without GDM. We further revealed the correlations of dysregulated lipids in breast milk with maternal glucose and infant physical development. A total of 833 lipid species from 15 classes were identified, 60 of which were found to be significantly altered in response to the high glucose, suggesting a remarkable lipid profiling change in breast milk induced by GDM. Our results showed significant associations between dysregulated lipids (e.g., neutral lipids, phospholipids, sphingolipids) and maternal glucose. Furthermore, correction analysis demonstrated that GDM related lipids were also associated with indicators of infant physical development, including body weight, length, and head circumference. These findings may help to understand the protective effects of breastfeeding especially during GDM pregnancy.

Vitamin B12 status and folic acid/vitamin B12 related to the risk of gestational diabetes mellitus in pregnancy: a systematic review and meta-analysis of observational studies.

BACKGROUND: This review was conducted to investigate the association between serum vitamin B12 levels as well as folic acid/vitamin B12 during pregnancy and the risk of gestational diabetes mellitus (GDM). METHODS: A comprehensive search of electronic databases (Embase, PubMed, and Web of Science) was performed. The odds ratios (ORs) with 95% confidence intervals (CIs) of GDM risk were summarized using a random effects model. We also performed subgroup analyses to explore the source of heterogeneity. RESULTS: A total of 10 studies, including 10,595 pregnant women were assessed. Women with vitamin B12 deficiency were at higher risk for developing GDM when compared with those who were vitamin B12 sufficient (OR, 1.46; 95% CI 1.21-1.79; I2: 59.0%). Subgroup analysis indicated that this association might differ based on sample size and geographical distribution. Elevated vitamin B12 levels may decrease the risk of GDM by 23%. The role of excess folic acid and low vitamin B12 levels in the occurrence of GDM is also controversial. CONCLUSION: In summary, vitamin B12 deficiency is associated with increased risk of GDM, it is necessary to pay more attention to the balance of vitamin B12 and folic acid. However, more in-depth studies across multiple populations are needed to verify these results.

A comparative lipidomic study of the human placenta from women with or without gestational diabetes mellitus.

Gestational diabetes mellitus (GDM) is always accompanied by lipid disorders. The placenta serves as a center for lipid synthesis and transport and plays a critical role in establishing GDM. Thus, the changes in the type and content of lipids in the placenta may contribute to the development of GDM. Here, we performed an untargeted lipidomic analysis to profile the alterations of lipids in the placenta induced by GDM. Principal component analysis (PCA) was used to reduce the dimensionality of lipid data, and orthogonal projections to latent structures-discriminate analysis (OPLS-DA) was launched to show the differences in the lipid profile between the GDM group and normal controls. Additional multivariate data processing was carried out, including classification, pathway analysis and correlation analysis between dysregulated lipids and maternal blood glucose levels. We finally identified 1202 lipids in positive mode and 924 lipids in negative mode, of which 63 lipids were strongly associated with GDM. Notably, most dysregulated lipids were clustered in two major subtypes: glycerophospholipids and glycerolipids. Consistently, a significant down-regulation of glycerophospholipid metabolism was observed from pathway analysis. In addition, we found that SHexCer(d50:1), TAG(15:0/20:6/20:6) and PE(18:1e/21:2) were positively correlated with blood glucose levels, while PC(12:0/22:3), PC(22:4e/18:5) and PE(18:1e/26:4) showed negative correlations. Combining these lipids with fasting blood glucose showed high accuracy in the discrimination of women with GDM. In general, we explored the placental lipidomic abnormalities induced by GDM, and these findings may help us understand the pathological mechanisms of GDM.

The mitochondrial-derived peptide MOTS-c relieves hyperglycemia and insulin resistance in gestational diabetes mellitus.

The most common complication during pregnancy, gestational diabetes mellitus (GDM), can cause adverse pregnancy outcomes and result in the mother and infant having a higher risk of developing type 2 diabetes after pregnancy. However, existing therapies for GDM remain scant, with the most common being lifestyle intervention and appropriate insulin treatment. MOTS-c, a mitochondrial-derived peptide, can target skeletal muscle and enhance glucose metabolism. Here, we demonstrate that MOTS-c can be an effective treatment for GDM. A GDM mouse model was established by short term high-fat diet combined with low-dose streptozotocin (STZ) treatment while MOTS-c was administrated daily during pregnancy. GDM symptoms such as blood glucose and insulin levels, glucose and insulin tolerance, as well as reproductive outcomes were investigated. MOTS-c significantly alleviated hyperglycemia, improved insulin sensitivity and glucose tolerance, and reduced birth weight and the death of offspring induced by GDM. Similar to a previous study, MOTS-c also could activate insulin sensitivity in the skeletal muscle of GDM mice and elevate glucose uptake in vitro. In addition, we found that MOTS-c protects pancreatic ß-cell from STZ-mediated injury. Taken together, our findings demonstrate that MOTS-c could be a promising strategy for the treatment of GDM.

A Novel Anti-Infective Peptide BCCY-1 With Immunomodulatory Activities.

Antibiotic resistance has been considered to be a global threat which underscores the need to develop novel anti-infective therapeutics. Modulation of innate immunity by synthetic peptides is an attractive strategy to overcome this circumstance. We recently reported that BCCY-1, a human ß-casein-derived peptide displays regulatory activities on monocytes, thereby enhancing their actions in innate immune responses. However, the function of peptide BCCY-1 in host defense against infection remains unknown. In this study, we investigated the in vivo characteristics and effects of peptide BCCY-1 in mouse models of bacterial infection. Following intraperitoneal injection, the peptide BCCY-1 exhibited high level of cellular uptake by monocytes without obvious toxicities. Results revealed that peptide BCCY-1, but not the scrambled version, stimulated the chemokine production and monocyte recruitment in vivo. Treatment with BCCY-1 enhanced the pathogen clearance and protected mice against lethal infections. Because the anti-infective effects of BCCY-1 was abolished by in vivo depletion of monocytes/macrophages rather than lymphocytes and granulocytes, we conclude that monocytes/macrophages are key effector cells in BCCY-1-mediated anti-infective protection. Additionally, BCCY-1 lacks direct antimicrobial activity. To our knowledge, a human ß-casein-derived peptide that counters infection by selective regulation of innate immunity has not been reported previously. These results suggest peptide BCCY-1 as a promising alternative approach and a valuable complement to current anti-infective strategy.

Biological Properties of Milk-Derived Extracellular Vesicles and Their Physiological Functions in Infant.

Extracellular vesicles (EVs) are released by all cells under pathological and physiological conditions. EVs harbor various biomolecules, including protein, lipid, non-coding RNA, messenger RNA, and DNA. In 2007, mRNA and microRNA (miRNA) carried by EVs were found to have regulatory functions in recipient cells. The biological function of EVs has since then increasingly drawn interest. Breast milk, as the most important nutritional source for infants, contains EVs in large quantities. An increasing number of studies have provided the basis for the hypothesis associated with information transmission between mothers and infants via breast milk-derived EVs. Most studies on milk-derived EVs currently focus on miRNAs. Milk-derived EVs contain diverse miRNAs, which remain stable both in vivo and in vitro; as such, they can be absorbed across different species. Further studies have confirmed that miRNAs derived from milk-derived EVs can resist the acidic environment and enzymatic hydrolysis of the digestive tract; moreover, they can be absorbed by intestinal cells in infants to perform physiological functions. miRNAs derived from milk EVs have been reported in the maturation of immune cells, regulation of immune response, formation of neuronal synapses, and development of metabolic diseases such as obesity and diabetes. This article reviews current status and advances in milk-derived EVs, including their history, biogenesis, molecular contents, and biological functions. The effects of milk-derived EVs on growth and development in both infants and adults were emphasized. Finally, the potential application and future challenges of milk-derived EVs were discussed, providing comprehensive understanding and new insight into milk-derived EVs.

A human ß-casein-derived peptide BCCY-1 modulates the innate immune response.

In this study, we report a novel peptide corresponding to the sequence of human ß-casein (named BCCY-1), which was identified in our previous peptidome analysis of human milk and has great immunomodulatory activity. The results revealed that peptide BCCY-1, but not the scrambled version, enhanced monocyte migration without obvious toxicities. This selective effect was mediated via increased production of chemokines by peptide stimulated monocytes. Moreover, BCCY-1 exerted its modulatory effects by activating nuclear factor (NF)-κB and mitogen-activated protein kinase (MAPK) signaling. The abundances of peptide BCCY-1 and the peptides partially encompassing its fragment were found to be lower in preterm milk than in term milk. Our study may lead to new insights into the immunoregulatory effects of casein-derived peptides and facilitate the discovery of novel peptide-based food and pharmaceutical products.

Human milk derived peptide AOPDM1 attenuates obesity by restricting adipogenic differentiation through MAPK signalling.

BACKGROUND: Emerging evidence revealed peptides within breast milk may be an abundant source of potential candidates for metabolism regulation. Our previous work identified numerous peptides existed in breast milk, but its function has not been validated. Thus, our study aims to screen for novel peptides that have the potential to antagonize obesity and diabetes. METHODS: A function screen was designed to identify the candidate peptide and then the peptide effect was validated by assessing lipid storage. Afterwards, the in vivo study was performed in two obese models: high-fat diet (HFD)-induced obese mice and obese ob/ob mice. For mechanism study, a RNA-seq analysis was conducted to explore the pathway that account for the biological function of peptide. RESULTS: By performing a small scale screening, a peptide (AVPVQALLLNQ) termed AOPDM1 (anti-obesity peptide derived from breast milk 1) was identified to reduce lipid storage in adipocytes. Further study showed AOPDM1 suppressed adipocyte differentiation by sustaining ERK activity at later stage of differentiation which down-regulated PPARγ expression. In vivo, AOPDM1 effectively reduced fat mass and improved glucose metabolism in high-fat diet (HFD)-induced obese mice and obese ob/ob mice. CONCLUSIONS: We identified a novel peptide AOPDM1 derived from breast milk could restrict adipocyte differentiation and ameliorate obesity through regulating MAPK pathway. GENERAL SIGNIFICANCE: Our findings may provide a potential candidate for the discovery of therapeutic drugs for obesity and type 2 diabetes.

The Effect of FOXC2-AS1 on White Adipocyte Browning and the Possible Regulatory Mechanism.

Obesity has become a worldwide epidemic, and obesity-related problems are becoming more severe in public health. Increasing brown adipose tissue (BAT) mass or/and activity in mice and humans has been demonstrated to help lose weight and improve whole-body metabolism. Studies on the conversion of white adipose tissue (WAT) to BAT under certain conditions have provided new possibilities for treating obesity and the related disorders. It has been established that long non-coding RNAs (lncRNAs) play an important role in the regulation of mouse adipocyte differentiation and thermogenic programs; however, the function and potential mechanism of lncRNA in the process of human white adipocyte browning remains unclear. In the present study, we identified a lncRNA called Forkhead Box C2 antisense RNA 1 (FOXC2-AS1), which was first identified in osteosarcoma, and it was highly expressed in human adipocytes but decreased during the white adipocyte differentiation program. FOXC2-AS1 expression was also induced by the thermogenic agent forskolin. Lentivirus-mediated overexpression of FOXC2-AS1 in human white adipocytes did not affect lipid drop accumulation, but significantly promoted the browning phenotype, as revealed by the increased respiratory capacity and the enhanced protein expression levels of brown adipocyte-specific markers. In contrast, inhibiting FOXC2-AS1 with small interfering RNA led to attenuated thermogenic capacity in human white adipocytes. RNA-sequencing analysis and western blot were used to identify a possible regulatory role of the autophagy signaling pathway in FOXC2-AS1 to mediate white-to-brown adipocyte conversion. The autophagy inhibitor 3-methyladenine restored the reduced UCP1 protein level and thermogenic capacity caused by inhibiting FOXC2-AS1. Overall, the present study characterized the potential role of FOXC2-AS1 and further identified a lncRNA-mediated mechanism for inducing browning of human white adipocytes and maintaining thermogenesis, further providing a potential strategy for treating obesity and related disorder.

The role of microRNA-23b-5p in regulating brown adipogenesis and thermogenic program.

Enhanced brown adipose tissue (BAT) mass and activity have been demonstrated to promote the expenditure of excess stored energy and reduce prevalence of obesity. Cold is known as a potent stimulator of BAT and activates BAT primarily through the ß3-adrenergic-cAMP signaling. Here, we performed RNA-sequencing to identify differential miRNAs in mouse BAT upon cold exposure and a total of 20 miRNAs were validated. With the treatment of CL-316,243 (CL) and forskolin (Fsk) in mouse and human differentiated brown adipocyte cells in vitro, miR-23b-5p, miR-133a-3p, miR-135-5p, miR-491-5p, and miR-150-3p expression decreased and miR-455-5p expression increased. Among these deferentially expressed miRNAs, miR-23b-5p expression was differentially regulated in activated and aging mouse BAT and negatively correlated with Ucp1 expression. Overexpression of miR-23b-5p in the precursor cells from BAT revealed no significant effects on lipid accumulation, but diminished mitochondrial function and decreased expression of BAT specific markers. Though luciferase reporter assays did not confirm the positive association of miR-23b-5p with the 3'UTRs of the predicted target Ern1, miR-23b-5p overexpression may affect brown adipocyte thermogenic capacity mainly through regulating genes expression involving in lipolysis and fatty acid ß-oxidation pathways. Our results suggest that miRNAs are involved in cold-mediated BAT thermogenic activation and further acknowledged miR-23b-5p as a negative regulator in controlling thermogenic programs, further providing potential molecular therapeutic targets to increase surplus energy and treat obesity.

An Integrated Analysis of mRNA and lncRNA Expression Profiles Indicates Their Potential Contribution to Brown Fat Dysfunction With Aging.

Brown adipose tissue (BAT) can convert fatty acids and glucose into heat, exhibiting the potential to combat obesity and diabetes. The mass and activity of BAT gradually diminishes with aging. As a newly found regulator of gene expression, long non-coding RNAs (lncRNAs) exhibit a wide range of functions in life processes. However, whether long non-coding RNA (lncRNA) involves in BAT dysfunction with aging is still unclear. Here, using RNA-sequencing technology, we identified 3237 messenger RNAs (mRNAs) and 1312 lncRNAs as differentially expressed in BAT of 10-months-old mice compared with 6- to 8-week-old. The protein-protein interaction network and k-score analysis revealed that the core mRNAs were associated with two important aging-related pathways, including cell cycle and p53 signaling pathway. Gene set enrichment analysis indicated that these mRNAs might participate in lipid metabolism and brown fat dysfunction. Functional enrichment analyses demonstrated that dysregulated lncRNAs were associated with mitochondria, regulation of cellular senescence, cell cycle, metabolic and p53 signaling pathways. Moreover, we revealed that two lncRNAs (NONMMUT024512 and n281160) may involve in the regulation of their adjacent gene peroxisome proliferator-activated receptor alpha (Pparα), a thermogenesis regulator. Collectively, these results lay a foundation for extensive studies on the role of lncRNAs in age-related thermogenic degradation.

A novel endogenous antimicrobial peptide CAMP211-225 derived from casein in human milk.

A large number of bioactive peptides derived from breast milk have been identified to be multifunctional having anti-inflammatory, immunoregulatory and antimicrobial activities. Here, we report that an endogenous peptide located at ß-casein 211-225 amino acid from human breast milk (hereafter called CAMP211-225) presents specific antimicrobial activity against pathogenic E. coli and Y. enterocolitica. CAMP211-225 is a novel peptide that occurs at higher levels in preterm milk than in term milk. The minimal inhibitory concentrations (MIC) of CAMP211-225 against E. coli and Y. enterocolitica are 3.125 µg ml-1 and 6.25 µg ml-1, respectively, and the antimicrobial activity of CAMP211-225 was also confirmed by a disk diffusion assay. Further studies using fluorescence staining, scanning electron microscopy and a DNA-binding assay revealed that CAMP211-225 kills bacteria through a membrane-disrupting mechanism, but not by binding to intracellular nucleic acids. Neonatal necrotizing enterocolitis (NEC) is a devastating gastrointestinal disease in neonatal intensive care units. In our study, CAMP211-225 administration effectively reduced ileal mucosa damage in an experimental NEC mice model. These results suggest that the antimicrobial peptide CAMP211-225 may have potential value in the prevention and treatment of neonatal infections.

Fluorometric determination of the CCAAT/enhancer binding protein alpha by using gold nanoparticles and a labeled protein-binding DNA.

A method is described for the determination of the CCAAT/enhancer binding protein alpha (C/EBPα) which is a regulator in adipocyte differentiation. The method is based on quenching of the red fluorescence (with excitation/emission maxima at 548/562 nm) of Cy3-labeled DNA if it becomes adsorbed on positively charged gold nanoparticles (AuNPs). Fluorescently labeled dsDNA that can bind C/EBPα is introduced as a fluorescent probes. The dsDNA is electrostatically adsorbed on the positively charged AuNPs to quench their fluorescence. In the presence of C/EBPα, it will bind dsDNA which then diffuses away. The fluorescence of the AuNPs becomes restored. The fluorescent signal increases linearly in the 0.05 to 600 ng·mL-1 µM C/EBPα concentration range, and the detection limit is 29 pg·mL-1. The method is specific and was applied to analyze cell lysates and in-situ. Graphical abstractSchematic representation of a fluorometric method for determination of the CCAAT/enhancer binding protein alpha (C/EBPα). Fluorescently labeled dsDNA that can bind C/EBPα is introduced as a fluorescent probes. The dsDNA is electrostatically adsorbed on the positively charged AuNPs to quench their fluorescence. In the presence of C/EBPα, it will bind dsDNA which then diffuses away. The fluorescence of the AuNPs becomes restored.

Association of maternal folate status in the second trimester of pregnancy with the risk of gestational diabetes mellitus.

Interest in the high folate status of pregnant women has increased due to its role in the prevention of neural tube defects (NTDs). The effect of increased red blood cell (RBC) folate status during the second trimester of pregnancy on gestational diabetes mellitus (GDM) remains unclear. We measured RBC folate concentrations by competitive protein-binding assay and obtained clinical information from electronic medical records. Logistic regression analysis was used to explore the associations of RBC folate concentrations with risks of gestational diabetes mellitus (GDM). We further assessed the potential nonlinear relations between continuous log-transformed RBC folate concentrations and GDM risk by using the restricted cubic splines. We observed high RBC folate concentrations in GDM patients compared to control group [median (interquartile range, IQR), GDM vs. controls: 1,554.03 (1,240.54-1,949.99) vs. 1,478.83 (1,124.60-1,865.71) nmol/L, p = .001]. Notably, high folate concentrations were significantly associated with an increased risk of GDM [RR per 1-SD increase: 1.16 (1.03, 1.30), p = .012] after adjustment for maternal age, parity, and body mass index (BMI) at enrollment. In the restricted cubic spline model, a test of the null hypothesis of the linear relationship was rejected (p = .001). Our study firstly showed that maternal RBC folate concentrations during the second trimester of pregnancy increase the risk of GDM in a Chinese population. Further randomized clinical trials (RCTs) are warranted to confirm the adverse effect.

A Comparative Peptidomic Characterization of Cultured Skeletal Muscle Tissues Derived From db/db Mice.

As an important secretory organ, skeletal muscle has drawn attention as a potential target tissue for type 2 diabetic mellitus (T2DM). Recent peptidomics approaches have been applied to identify secreted peptides with potential bioactive. However, comprehensive analysis of the secreted peptides from skeletal muscle tissues of db/db mice and elucidation of their possible roles in insulin resistance remains poorly characterized. Here, we adopted a label-free discovery using liquid chromatography tandem mass spectrometry (LC-MS/MS) technology and identified 63 peptides (42 up-regulated peptides and 21 down-regulated peptides) differentially secreted from cultured skeletal muscle tissues of db/db mice. Analysis of relative molecular mass (Mr), isoelectric point (pI) and distribution of Mr vs pI of differentially secreted peptides presented the general feature. Furthermore, Gene ontology (GO) and pathway analyses for the parent proteins made a comprehensive functional assessment of these differential peptides, indicating the enrichment in glycolysis/gluconeogenesis and striated muscle contraction processes. Intercellular location analysis pointed out most precursor proteins of peptides were cytoplasmic or cytoskeletal. Additionally, cleavage site analysis revealed that Lysine (N-terminal)-Alanine (C-terminal) and Lysine (N-terminal)-Leucine (C-terminal) represents the preferred cleavage sites for identified peptides and proceeding peptides respectively. Mapped to the precursors' sequences, most identified peptides were observed cleaved from creatine kinase m-type (KCRM) and fructose-bisphosphate aldolase A (Aldo A). Based on UniProt and Pfam database for specific domain structure or motif, 44 peptides out of total were positioned in the functional motif or domain from their parent proteins. Using C2C12 myotubes as cell model in vitro, we found several candidate peptides displayed promotive or inhibitory effects on insulin and mitochondrial-related pathways by an autocrine manner. Taken together, this study will encourage us to investigate the biologic functions and the potential regulatory mechanism of these secreted peptides from skeletal muscle tissues, thus representing a promising strategy to treat insulin resistance as well as the associated metabolic disorders.

Long non-coding RNA Bhmt-AS attenuates hepatic gluconeogenesis via modulation of Bhmt expression.

Dysregulation of gluconeogenesis contributes to the pathogenesis of metabolic disease, such as type-2 diabetes. The role of long non-coding RNAs (lncRNAs) in the pathogenesis of diabetes has recently received increased attention. In the present study, we identified a novel lncRNA, betaine-homocysteine methyltransferase-antisense (Bhmt-AS), and examined its expression patterns under pathophysiological conditions. Our results revealed that the expression of Bhmt-AS was significantly increased in the livers of fasted and db/db mice and was induced by gluconeogenic hormonal stimuli. The Bhmt-AS was also shown to be a concordant regulator of Bhmt expression. Functionally, depletion of Bhmt-AS suppressed hepatic glucose production both in vivo and in vitro. Adenovirus-mediated hepatic knockdown of Bhmt-AS improved pyruvate tolerance, glucose tolerance, and insulin sensitivity. Furthermore, overexpression of Bhmt restored the decreased glucose production caused by knockdown of Bhmt-AS in primary hepatocytes. Taken together, we uncovered a novel antisense lncRNA (Bhmt-AS) that is co-expressed with Bhmt and concordantly and specifically regulates Bhmt expression both in vitro and in vivo to regulate hepatic gluconeogenesis.