The Function And Mechanism Of Skeletal Muscle Rev-erbs In Glucose And Lipid Metabolism

BackgroundMetabolic diseases arise from metabolic disorders associated with glucose,lipid,and protein.These include not only the common metabolic disorders（i.e.,diabetes,hypoglycemia,obesity,hyperuricemia,abnormal bone metabolism）,but also rare inherited metabolic diseases.In recent years,high-throughput profiling techniques are widely used in metabolic diseases with genomics,transcriptomics,proteomics,metabolomics,and other omics approaches.Multi-omics profiling,which has been shown to provide a new horizon for the study of the mechanism of metabolic diseases and to improve the diagnostic rate of rare inherited metabolic diseases,substantially furthers the development of the Precision Medicine.Chronotype describes a person’s behavioral preference in the biological rhythm Chronodisruption refers to the misalignment between the endogenous circadian clock and the external environmental cues.This includes shiftwork,jetlag,social jetlag,nighttime light,and nighttime eating.Such chronodisruption is believed to contribute to the pathogenesis of metabolic diseases,cancer,cardiovascular diseases,psychological and sleep disorders,and reduced life span.Chronotherapy involves a variety of strategies to maximize the benefit/risk ratio of any medical intervention through considering or harnessing the body’s endogenous biological rhythms.This includes chronopharmacology,chrononutrition,and chronoexercise.Chronotherapy has been reported to be able optimize the medical intervention for various diseases Therefore,the arrival of circadian medicine provides a new target to prevent and cure metabolic diseases.The mammalian master pacemaker is located in the suprachiasmatic nucleus（SCN）of the anterior hypothalamus.The SCN neurons receive photic information from the retina via synaptic transmission by axons of the retinohypothalamic tract（RHT）.SCN can autonomously control the neuronal firing and gene expression to general circadian rhythm,then transfer the signals to the whole body.The endogenous circadian rhythm arises from an autoregulatory feedback loop of the core clock genes,which include transcription factors CLOCK（Circadian locomotor output cycles kaput）,Bmal1（Bain and muscle ARNT-like 1）,Pers（Period）,Crys（Cryptochrome）,RORs（RAR-related orphan receptor）and Rev-erbs（nuclear receptor subfamily 1,group D）.It has been shown that most tissues in the mammal,such as skeletal muscle,heart,liver,and adipose tissue,possess cell-autonomous circadian oscillators.The peripheral clocks can work independently from the SCN to exert numerous documented effects on metabolism.Therefore,it has become a hot research topic in recent years.Skeletal muscle is the largest organ in the body,which comprises 40%of the total body mass Skeletal muscle is responsible for 85%of postprandial glucose uptake,and it serves as the most important reservoirs of amino acid.Dysregulation of skeletal muscle is a major contributing factor to the pathogenesis of type 2 diabetes,but the specific mechanism is not yet clearEctopic lipid accumulation in muscles can cause insulin resistance,a process referred to as lipotoxicity.However,this statement is not quite precise.Endurance athletes have higher intramuscular lipid contents associated with higher insulin sensitivity phenomenon known as the "Athlete paradox".In 1963,Philip Randle proposed a "glucose-fatty acid cycle" which describes the reciprocal relationship between fatty acid oxidation（FAO）and glucose oxidation.But Randle cycle may involve mechanisms beyond the original definition.Many studies have proposed that metabolic overload and increased fuel competition can induce accumulation of incompletely oxidized lipid intermediates and thereby cause insulin resistance,contributing to the development of insulin resistance and T2DM.Our team found that HDAC3（histone deacetylase 3）skeletal muscle knockout mice showed increased fatty acid oxidation and decreased glucose metabolism,which caused whole body glucose intolerance and insulin resistance.This study suggests that how the skeletal muscle handles lipid,rather than the lipid content,is responsible for the insulin sensitivity and glucose tolerance.Many studies showed HDAC3 has a tight relationship with circadian rhythm.The nuclear receptors Rev-erbα and Rev-erbβ repress gene transcription by recruiting nuclear receptor corepressors（NCORs）and HDAC3.Rev-erba and Rev-erbp are two unique orphan members of the nuclear hormone receptors（NHR）superfamily,which regulate a number of physiological functions including the circadian rhythm and metabolism.However,the functional roles of Rev-erbs in skeletal muscle is still controversial.In 2013,Woldt et.al found that whole-body Rev-erba null mice showed decreased endurance capacity and lower mitochondrial capacity.Since Rev-erba-/-mice displayed less voluntary locomotor activity,it is likely that reduced activity was the main cause of these phenotypes.On the basis of the above studies,we propose the following hypothesis:Rev-erbs affect insulin resistance and glucose tolerance through lipid-glucose cycle,while they do not directly affect mitochondrial function.To further validate the hypothesis,skeletal muscle-specific Rev-erbα/β double knockout mice（Rev-MKO）were constructed.Through multiple experimental approaches and multi-omics techniques,we conducted a comprehensive and systematic analysis of the function of Rev-erbs in skeletal muscle.In clinical research,we reported a rare metabolic derangement present in a 7-year-old boy who developed severe progressive abdominal fat deposition from the age of 7 months.He developed muscle atrophy and recurrent hypoglycemia that led to seizures at the age of 4 years.Imaging studies supported metabolic diseases,but the causes were unclear.Randle cycle could partially explain the pathophysiology and clinical manifestations:increased glucose utilization leads to hypoglycemia,while decreased fatty acid oxidation results in abnormal fat deposition.To make a definitive molecular diagnosis,we performed whole-genome sequencing（WGS）on the DNA isolated from peripheral blood cells of the child and his parents.In addition,we confirmed the suspicious variant by western blot and Sanger sequencing.Part Ⅰ Role of Rev-erbs in skeletal muscle functionObjectiveTo determine the role of Rev-erbs in skeletal muscle metabolism,strength,endurance capacity and mitochondrial function.Methods1.The muscle-specific Rev-erbs knockout mice（Rev-MKO or simply KO）were generated by crossbreeding mice bearing Rev-erbaloxP（Nr1d1m1.2Rev,MGI ID 5426700）and Rev-erbβloxP（Nr1d2tm1.1Rev,MGI ID 5426699）alleles with the MLC-Cre line（MGI ID 193 1135）.Littermate mice bearing Rev-erbαloxP and Rev-erbβlaxP alleles without Cre were used as a wild-type（WT）control.All data shown are from male mice.The knockdown efficiencies were determined by detecting Rev-erhα and Rev-erbβ mRNA levels via RT-qPCR.2.The weights of the mice were monitored weekly.Food intake,voluntary activity,energy expenditure,and oxygen consumption were monitored by Comprehensive Lab Animal Monitor System（CLAMS）.3.Mitochondrial copy number and oxidative phosphorylation（OXPHOS）were measured by RT-qPCR and Western blot,respectively.4.For measurement of muscle weights,mouse Tibialis anterior（TA）,Extensor Digitorum Longus（EDL）and Soleus（SOL）muscles were dissected,and the average of the left and right muscle weights was used.Immunofluorescence staining was performed to evaluate the muscle fiber size and muscle fiber type.5.Muscle strength was measured by grip strength and wire hanging test.Endurance capacity was tested under different exercise intensity during treadmill running.6.For measurement of fatty acid oxidation in vivo,mice were forced to run on the treadmill for 15 mins,then mice were i.p.injected with 2μCi[9,10-3H（N）]palmitate.After another 15mins running,serum was collected to measure[3H]H2O.Oxygen consumption and respiratory exchange ratio（RER）was measured indirect calorimetry.Lactate was measured after 30mins running on the treadmill.Triglyceride and glycogen contents in skeletal muscle were measured after 6h fasting.7.To further validate whether the metabolic change is muscle-autonomous,primary myoblasts were extracted and were differentiated into myotubes,2-[3H（N）]deoxy-d-glucose and[9,10-3H（N）]palmitate were used to determine glucose uptake and fatty acid oxidation,respectively.8.To determine whether Rev-erbs in skeletal muscle affect systemic insulin sensitivity and glucose tolerance,glucose and insulin were measured after 6h fasting,then,intraperitoneal glucose tolerance test（IPGTT）was performed.Results1.Rev-MKO mice showed efficient deletion of Rev-erbα and Rev-erbβ in TA,SOL,and EDL,but not in the heart,liver,or hypothalamus2.Rev-MKO mice displayed normal locomotor diurnal rhythm under the 12h light/12 h dark（LD）condition.Rev-MKO mice also had a normal volume of voluntary locomotor activity,food intake pattern,body weight,energy expenditure,and systemic fuel metabolism in the LD condition.These results are in contrast to the whole-body Rev-erbα or Rev-erbβ null mice that show reduced locomotor activity or altered food intake.3.Rev-MKO mice did not show changes in mitochondrial DNA copy number or OXPHOS protein levels.4.Muscle weight,fiber numbers and muscle fiber type composition of Rev-MKO mice were all comparable to WT muscles.5.Rev-MKO mice showed decreased strength during the grip strength and wire hanging test.Rev-MKO mice performed poor endurance at high-speed profile but showed enhanced endurance at middle-low-speed profile.Skeletal muscle mainly burns lipid during lower intensity exercise,but with increasing intensity there is greater reliance on glucose oxidation.Therefore,it is likely that Rev-erbs regulate the fuel preference in skeletal muscle.6.We used isotopes to determine the glucose and lipid oxidation capacity.Rev-MKO mice displayed increased fatty acid oxidation rate but producing less lactate during exercise.Rev-MKO showed lower respiratory exchange rate during running Consistently,Rev-MKO mice showed higher triglyceride,but lower glycogen content in skeletal muscle after fasting7.Rev-MKO myocytes showed higher fatty acid oxidation and lower glucose uptake.So,the metabolic change is muscle-autonomous,Rev-erbs in skeletal muscle directly regulate glycolipid metabolism.8.Rev-MKO mice showed higher HOMA-IR（p=0.068）and glucose intolerance during IPGTT.Conclusions1.The muscle-specific Rev-erb knockout mice model is successfully constructed.Unlike the whole-body Rev-erba or Rev-erbp null mice,Rev-erbs in skeletal muscle do not affect food intake,body weight,energy expenditure,oxygen consumption and mitochondrial function.2.Rev-MKO show lower strength and different endurance capacity at different speed profile,this is because Rev-MKO drives fuel preference for lipid over glucose.3.Rev-erbs in skeletal muscle regulate systemic insulin sensitivity and glucose tolerance.Part Ⅱ Studies of the mechanism by which Rev-erbs regulate fuel preference in skeletal muscleObjectiveTo study the mechanism by which Rev-MKO drives fuel preference for lipid over glucose.Methods1.We explored the molecular changes underpinning the metabolic remodeling in Rev-MKO muscle through RNA-seq analysis.Volcano plots were made for the results from the differential expression analyses.In addition,we performed KEGG and Gene Set Enrichment Analysis（GSEA）analysis.Here,we focused on the metabolism-related gene sets.2.To assess the metabolic changes in skeletal muscle in Rev-MKO mice,metabolomics was performed.Metabolites were analyzed with MetaboAnalyst.3.ChIP-seq was performed to further explore the function of Rev-erbs in skeletal muscle.Functional enrichment analysis of ChIP-seq peaks were perfonned using GREAT software.Results1.Differentially expressed genes（DEGs）between KO and WT were highly enriched in metabolism and circadian rhythm.Many genes in amino acid catabolism and lipid metabolism were upregulated,while many genes in glycolysis and TCA cycle were downregulated in skeletal muscle of Rev-MKO mice2.Metabolomics study showed that amino acid,lipid,and TCA-related pathways were associated with Rev-erbs-induced metabolic changes3.ChIP-seq revealed that Rev-erba directly bind the promoter of metabolism-related genes,such as Pdk4,and Acsl1,which makes these genes presenting rhythmic expression.Great software analysis showed that ChIP-seq peaks were enriched in circadian rhythm,transcriptional regulation and metabolism-related pathwayConclusions1.Rev-erbs regulate the expression rhythms of important genes for circadian rhythm and metabolism.Amino acid and lipid metabolism related genes are upregulated,while glycolysis and TCA cycle related genes are downregulated in Rev-MKO mice2.Rev-erbs regulate the amino acid,lipid and TCA cycle metabolites in skeletal muscle3.Rev-erbs regulate metabolism-related genes expression via direct association with their promoter regionPart Ⅲ Genome sequencing analysis of a family with a child displaying severe abdominal lipid disposition and recurrent hypoglycemiaObjectiveTo determine the pathogenic variants in a child with inborn errors of metabolism.Methods1.Comprehensive physical examination,laboratory test and medical history collection were performed for the proband and his parents.2.Whole blood samples were collected from the proband and his parents.WGS was performed by using Illumina HiSeq X Ten3.Single nucleotide variants and insertion/deletions were functionally annotated with SnpEff.The Copy Number Variants（CNVs）were called using the CNVannotator4.Sanger sequencing was used to confirm these candidate pathogenic mutations Western blot was used to detect the mutant proteinsResults1.Abdominal distention was first noted in the proband at the age of 7 months and kept growing throughout childhood.At the age of 4 years,the proband suffered recurrent episodes of hypoglycemic seizures,with the glucose level at 0.78-1.78 mmol/1.The fasting insulin was<2.0μIU/ml.The proband was diagnosed with inborn errors of metabolism,but the etiology was not definitively established.2.According to ACMG guidelines,the PTEN（NM₀003 14.4:p.Pro283fs/c.849delA）was proposed as a pathogenic mutation.4.Sanger sequencing confirmed that the expected nucleotide deletion was present Western blot analysis further confirmed the significant reduction of the PTEN protein in the blood samples of the probandConclusions1.The proband was diagnosed with PTEN hamartoma tumor syndrome（PHTS）.This mutation causes a premature stop and gives rise to a partial loss of C2 domain and the total loss of C-terminal tail of the PTEN protein.Loss-of-function of PTEN results in the upregulation of PI3K-AKT pathway leading to increased insulin sensitivity and severe hypoglycemia during stress2.As a tumor suppressor gene,PTEN can affect normal cellular proliferation.Germline PTEN mutations are associated with increased lifetime risks for the formation of benign and/or malignant neoplasms.PTEN loss-of-function induced the adipose tumor growth in the proband.