| In recent decades,the global overweight and obese population have soared,with the improvement of living standards and alterations of life style,especially decreased physical activity and increased high-calorie food intake.Obesity dramatically increased the risk of diabetes,cardiovascular diseases and many other diseases,seriously threatening human health.Therefore,it has become an important task to illuminate the mechanisms of body weight regulation in order to provide guidelines for the control of obesity epidemic.Nowadays,body weight is thought to be regulated by energy homeostatic mechanisms,in which the signal—leptin,secreted by adipocytes,acts in hypothalamic signaling pathways and regulates body weight through a bi-directional manner while obesity is considered to be a disorder of the homeostatic mechanisms.However,the hypothesis is still controversial.Epidemiologic studies reveal that body weight is collectively affected by genetic and environmental factors,and excess food-intake and high-energy diet are the major environmental factors leading to obesity.It was reported that Rcan2 regulates food intake through a leptin-independent pathway,and it could promote a rapid weight gain in mice when fed with a high-fat diet,which may provide evidence for the explanation of association between obesity and high-energy diet as well as the development of obesity epidemic.Rcan2 encodes two splicing variants with different transcription start sites,Rcan2-1 and Rcan2-3.Rcan2 knock-out mice(Rcan2-/-)were generated by replacement of exon 4 commonly used by Rcan2-1 and Rcan2-3 by a LacZ cassette mediated by homologous recombination.Thus,Rcan2-/-mice can be used to study the function and the expression pattern of Rcan2.However,the role of each isoform in body weight regulation and their expression patterns are unclear,which are the core questions to be answered in this study.In the present study,using CRISPR/Cas9 genome-editing technology,Rcan2-1-null mice(Rcan-1-/-)were generated by deleting 1 nucleotide(T)after the translational start site(ATG)in exon 2,and Rcan2-3-null mice(Rcan2-3-/-)were generated by deleting 2 nucleotides(AG)after ATG in exon 3.Homologous knock-out mice were produced by mating heterozygous mice.Starting from 4 weeks of age,a cohort of mice was placed on the high-fat diet(HFD)and their growth was monitored.There was no significant difference in growth rates between wild-type(WT)and Rcan2-3-/-mice during the 16-week monitoring period.However,the growth rate of Rcan2-1-/-mice was slower than that of WT mice.The weights of Rcan2-1-/-mice were significantly different from those of WT mice beginning at 6 weeks of age,the average weight difference reaching about 7g at the age of 20 weeks.Consistent with the body weight results,anatomical analyses demonstrated that white fat deposits(including epididymal and retroperitoneal white adipose tissue)and livers of Rcan2-1-/-mice were significantly lighter than those of WT mice,while those in Rcan2-3-/-mice were similar to the WT mice.The results indicate that Rcan2-1 may play a major role in weight regulation.To study the expression patterns of Rcan2-1(or Rcan2-3)in vivo,Rcan2-1LacZ(or Rcan2-3LaeZ)mice were generated by targeting the Rcan2-3-LacZ(or Rcan2-1-LacZ)fusion transcript in Rcan2+/-zygotes.As the X-Gal staining results showed,the expression loci of Rcan2-3 in Rcan2-3LacZ are quite similar to those of Rcan2 in Rcan2-/-mice:in the embryonic period,the expression was first detected at embryonic day 9.5(E9.5),and it was widely distributed in telencephalon,the mesencephalon-hindbrain junction,hindbrain,medulla and spinal cord at E11.5 and E12.5,and then at E13.5 its distribution was extended to mesencephalon and diencephalon,the expression of Rcan2-3 in the fetal brain gradually expanding along with the embryonic development;in adult mice,it was widely expressed in brain and the grey matter of spinal cord,particularly prominent in nervous nuclei such as PVN.Unlike Rcan2-3,lower expression level of Rcan2-1 was detected in mice,and it was weakly expressed at E15.5 and was merely expressed in certain areas of adult brain such as thalamus and cerebral cortex.Furthermore,to further determine the functions of Rcan2-1 and Rcan2-3 in weight regulation,FLAG-tagged transgenic mice were generated using Tol2 system.Unfortunately,Western blotting showed that no FLAG-tagged protein was detected in total protein extracted from tissues in the transgenic mice.It is speculated that the transgene expression may be affected by a few factors such as "position effects"mediated by the integration sites.In the present study,Rcan2-1-null and Rcan2-3-null mice models were generated using CRISPR/Cas9 system.It is revealed that the inactivation of Rcan2-1 rather than Rcan2-3 dramatically attenuated HFD-induced obesity,indicating the dominating function of Rcan2-1 in body weight regulation.In addition,the distributions of the two Rcan2 isoforms in mouse embryos and central nervous system(CNS)in adult were displayed visually through histochemical methods for the first time.The present study will provide new evidence for elucidating the weight regulation mechanisms of Rcan2. |
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