Regulation Of Glucagon Receptor Gene Expression In Newborn Calf

As a result of a marked decline in dry matter intake (DMI) prior to parturition and a slow rate of increase in DMI relative to milk production after parturition, dairy cow experiences a negative energy balance. Changes in nutritional and metabolic status during the periparturient period predispose dairy cow to develop hepatic lipidosis and ketosis. Fat mobilization start in order to relieve this negative energy balance.In this course, glucagon(GLN) plays an important role in regulation of hepatic glyconeogenesis and dairy cow energy metabolism, especially fat metabolism by GLN binding to the glucagon receptor(GLNR). The objective of present study was to detect the distribution of GLNR mRNA in different tissues, and evaluate effects of neuroendocrine factors and metabolites on expression of GLNR mRNA in monolayer primary cultured neonatal calf hepatocytes in vitro to probe the regulation mechanism of neuroendocrine factors and metabolites on gluconeogenesis and lipid metabolism of ketosis and fatty liver in dairy cows.In the experiment of distribution of GLNR mRNA in tissues of neonatal calf,three male neonatal Holstein calves(40.5±5.28kg BW)were killed by carotid artery bloodletting.Within 30 minutes post-slaughter, 23 tissues were collected: cerebralcortex, cerebellar cortex, hypothalamus, aorta, jugular vein, heart,liver, spleen,lung, kidneycortex, pancreas, semitendinosus muscle,subcutaneous adipose,rumen,omasumreticulum,abomasum,duodenum,colon,ileum,cecum,rectum. Samples were snap-frozen in liquid nitrogen and subsequently stored at -80°C.The distribution of GLNR mRNA in central and peripheral tissues of newborn calves was detected by fluorescent quantitation PCR. The results shown: the GLNR mRNA was detectable in cerebral cortex, cerebellar cortex, hypothalamus, aorta, jugular vein, heart, liver, spleen, lung, kidney cortex, pancreas, semitendinosus muscle,subcutaneous adipose,rumen,omasum,reticulum,abomasum,duodenum,colon,ileum,cecum,rectum. High abundance of GLNR mRNA was found in hypophysis, hypothalamus, liver, aorta, subcutaneous adipose,cerebral cortex, cerebellar cortex, kidney cortex,lung tissue, indicating that these tissues may play an important role in mediating GLNR action. The wide spectrum of glucagon receptor gene expressing in tissues reveals that GLNR may have multiple physiological functions in calves.In the experiment of effects of neuroendocrine factors on expression of GLNR mRNA in monolayer primary cultured neonatal calf hepatocytes in vitro, healthy new born calves were killed by carotid artery bloodletting. Under the condition of asepsis, liver posterior lobe was collected, then sheared and digested for separating hepatocytes. After isolation, cells were seeded (1×106) onto custodite cell culture dishes with polylysine. After attachment, fluorescent quantitative RT-PCR were employed to determine the expressing level of GLNR mRNA in primary hepatocytes which were treated with different concentrations of insulin (0 nmol/L,1 nmol/L,10 nmol/L,100 nmol/L,1000 nmol/L), glucagon (0 nmol/L,1 nmol/L,10 nmol/L,100 nmol/L,1000 nmol/L) and leptin (0 ng/mL,2.5 ng/mL,5 ng/mL,10 ng/mL,50 ng/mL). The results showed that the expression level of GLNR mRNA in hepatic cells was gradually increased with increasing concentration of insulin in cell culture media, indicating that the insulin has a positive role on the expression of GLNR mRNA. With increasing concentration of glucagon in culture media, the expression level of GLNR mRNA in hepatic cells was gradually decreased, indicating that glucagon displays depressive effect on the expression of GLNR mRNA. Leptin displayed the dual effects on the expression of GLNR mRNA, the expression level of GLNR mRNA in hepatic cells was gradually increased and then decreased with increasing concentration of leptin in the media, indicating that leptin has the dual effects on the expression of GLNR mRNA.In the experiment of effects of metabolite on expression of GLNR mRNA in monolayer primary cultured neonatal calf hepatocytes in vitro.In order to determine the effects of different concentration of NEFA (0 mmol/L,0.5 mmol/L,1.0 mmol/L,1.5 mmol/L,2.0 mmol/L), BHBA (0 mmol/L,0.5 mmol/L,1.0 mmol/L,1.5 mmol/L,2.0 mmol/L) and GLU (0 mmol/L,1.5 mmol/L,2.8 mmol/L,5 mmol/L) in the cell culture media on the expression level of GLNR mRNA in primary cultured hepatocytes of new born calves, fluorescent quantitative RT-PCR were applied. The results showed that the expression level of GLNR mRNA in hepatic cells was gradually increased as increasing concentration of NEFA,BHBA,GLU in cell culture media, indicating that the metabolite NEFA,BHBA,GLU has a positive role in the expression of GLNR mRNA.As above-mentioned, the GLNR mRNA distributes in central and peripheral tissues of neonatal calf widely, which reveals GLNR has multiple physiologic functions. In, NEFA,BHBA,GLU can promote GLNR mRNA express,GLN is contrary to this result, but LEP has the dual effects on the expression of GLNR mRNA of hepatocytes in vitro.