The Role Of Hepatokine Manf In Glucose And Lipid Metabolism

Background: With the improvement of living standards,the number of obese people is increasing.Obesity has become a major health threat to human.Obesity is the result of a long-term imbalance of energy regulation(energy intake exceeds energy expenditure).Excess energy accumulates in adipose tissue in the form of triglycerides.There are two types of adipose tissue exist in mammals,white adipose tissue(WAT)and brown adipose tissue(BAT).WAT is known to be able to store excess energy,while BAT is responsible for dissipating chemical energy as heat through non-shivering thermogenesis.It was reported that a special type of adipose tissue called beige adipose tissue,which showing similar biochemical and morphological features as classical BAT,appeared in response to cold or hormonal stimulation within the WAT.This process is referred to as “browning”,which has been reported to be beneficial to obesity,glucose and lipid metabolism.Thus,activating beige adipocytes in WAT to increase energy expenditure might be a promising strategy to combat obesity and its complications.The browning process is regulated by various factors.Secreted factors are vital regulators to activate beige cells,such as Irisin,fibroblast growth factor 21(Fgf21),Bmp7/Bmp4 and so on.Dietary status can regulate energy metabolism.It has been found that feeding can increase thermogenesis in humans and rodents.Diet-induced thermogenesis is related to sympathetic nervous system and BAT.Β-adrenergic receptor,neuropeptide FF receptor-2(NPFFR2)and apolipoprotein A-IV have been proposed to play a role in feeding-induced thermogenesis.The expression and secretion of some secreted factors were reported to be upregulated by feeding,and these factors can promote browning to reduce body weight.Therefore,they may be potential regulators of diet-induced thermogenesis.For example,glucagon-like peptide-1(Glp-1)released by L cells locates in ileum and colon,creatine and Fgf21 secreted by adipose tissue.As one of the main organs responsible for metabolism,liver can synthesize and secrete a class of proteins(referred as hepatokine).They play an important role in glucose and lipid metabolism by acting on liver and other tissues via autocrine,paracrine or endocrine signaling.Feeding could induce release of hepatokine and regulate glucose and lipid metabolism.Feeding is known to induce the circulating level of proprotein convertase subtilisin/kexin type 9(Pcsk9)and angiopoietin like protein 8(Angptl8),which are responsible for regulating cholesterol and triglyceride metabolism.Serum Adropin levels were lower after fasting and protected mice from obesity,insulin resistance and dyslipidemia.However,whether the hepatokines induced by feeding could induce browning and regulate thermogenesis remains unclear.Mesencephalic astrocyte-derived neurotrophic factor(Manf)is a novel neurotrophic factor and a secreted protein induced by endoplasmic reticulum stress.Recent reports indicated that Manf may play an important role in diabetes,obesity,and other metabolic diseases.Manf was essential for proliferation and survival of pancreatic β cells in mice and protected human βcells from inflammatory-stress-induced death.Manf knockout resulted in hyperglycemia and hypoinsulinism.Circulating Manf was higher in children with type Ⅰ diabetes.Manf overexpression in the hypothalamus of mice resulted in obesity.Manf can also improve age-related metabolic dysfunction.This study aims to explore the role of Manf as a hepatokine in glucose and lipid metabolism and obesity.Methods: Wild type(WT)mice were fasted for 24 h or fasted for 20 h and then refed for 4 h.The change of secreted factors in livers from fasting and refeeding mice were measured by RNA-seq analysis.q PCR and Western Blot were then used to confirm the results of RNA-seq analysis.Liver-specific Manf transgenic(Tg)mice were created and fed a chow diet or high fat diet(HFD)for 12 weeks to study the role of Manf in glucose and lipid metabolism.Liver-specific Manf knockout(LKO)mice were generated to verify the role of Manf in energy metabolism.Fat-specific Manf knockout(FKO)mice were created to study whether adipocytes-secreted Manf has the same effect through paracrine/autocrine pathway.To study the therapeutical effect of Manf on glucose and lipid metabolism disorder.Diet-induced obesity(DIO)and ob/ob mice were s.c.injected with Manf-Fc weekly.Body weight was monitored daily.GTT and ITT were performed.Thermogenic genes were detected in subcutaneous adipose tissue.In vitro,primary adipocytes were isolated and treated with recombinant human Manf.m RNA and protein levels of thermogenic genes and related pathways were detected.Primary subcutaneous adipocytes were pretreated with SB203580 to confirm the mechanism of Manf-induced thermogenesis.To further study whether Manf plays a role in human obesity.Circulating MANF level of healthy participants and patients with overweight or obesity was quantified by enzyme-linked immunosorbent assay(Elisa).The correlation of serum MANF level with BMI was analyzed.Results: RNA-seq and western blot analysis showed that the expression and secretion of Manf in liver were regulated by food intake.Hepatic and circulating Manf levels were increased after refeeding.Manf protein level was downregulated in obese mice.No difference was observed among Tg,LKO and WT mice under chow diet.However,on an HFD,Manf overexpression protected mice from diet-induced obesity.Tg mice showed lower weight and smaller adipocyte size in epididymal and subcutaneous adipose tissue,increased energy expenditure and higher glucose uptake in subcutaneous adipose tissue.While locomotor activity was similar between Tg and WT mice.Further studies showed that Manf overexpression significantly increased the m RNA and protein levels of Ucp1 and other thermogenic genes in subcutaneous adipose tissue but did not affect activity of BAT.Manf overexpression in liver was also associated with increased lipolysis and decreased adipose inflammation in adipose tissue.Oil-red O staining and lipid quantitation of liver showed decreased lipid accumulation in livers of Tg mice.The ratio of liver weight to body weight was lower.GTT and ITT showed increased glucose tolerance and insulin sensitivity.Correspondingly,LKO mice showed increased body weight and adipocyte size,suppressed m RNA and protein levels of thermogenic genes in subcutaneous adipose tissue,decreased lipolysis and exacerbated inflammation in adipose tissue and lipid accumulation in liver.However,under both normal chow diet and HFD,FKO mice shared comparable body weight and tissue weights with control mice.Also,GTT and ITT results did not differ between control and FKO mice.Recombinant Manf-Fc reduced obesity and improved glucose tolerance and insulin resistance in both diet-induced and genetic obese mouse models.q PCR and Western Blot showed that Manf-Fc injection significantly upregulated thermogenesis in subcutaneous adipose tissue.In vitro,recombinant human Manf directly stimulated expression of Ucp1 and other thermogenic genes in primary adipocytes isolated from subcutaneous adipose tissue.However,no difference was observed in primary brown adipocytes incubated with recombinant human Manf.Consistently,adipocytes incubated with CM from Ad-Manf–infected hepatocytes showed higher m RNA levels of Ucp1,Cidea and Pgc-1α.In mechanism,Phosphorylated p38 MAPK(p-p38)was significantly increased at 15 min after the addition of Manf and decreased at 120 min.Moreover,the phosphorylation of p38 was further increased by a higher concentration of Manf.In contrast,the phosphorylation of Creb and Erk1/2 pathways was not changed.Manfinduced phosphorylation of p38 was suppressed by SB203580.Blockade of the p38 MAPK pathway greatly abolished the Manf-increased expression of thermogenic genes.In clinical study,serum Manf level was higher in patients with overweight and obesity than in controls.Manf appears to be positively associated with BMI.Conclusion: Manf is a feeding-induced hepatokine.Manf increases thermogenesis by promoting browning in subcutaneous white adipose tissue via p38 MAPK pathway,ameliorating diet-induced obesity and insulin resistance.The present study reveals the crucial role of Manf in regulating thermogenesis in adipose tissue,representing a potential therapeutic target for obesity and glucose and lipid metabolic disorders.