GHR1A mRNA Research Articles

PSI-7 Creation of gene-edited pigs for functional analysis of growth hormone receptor promoters in swine

Abstract Growth hormone (GH; somatotropin) has a central role in animal growth, reproduction, and lactation. The biological actions of GH are dependent on the GH receptor (GHR) that is found in important target tissues such as liver, muscle, adipose tissue, and bone. It is widely accepted that GH is the primary hormone controlling animal growth and that its actions are mediated by the GHR. In humans, laboratory species, and farm animals there are two promoter complexes that control GHR expression. The first promoter complex (P1) controls the liver-specific inducible expression of the GHR1A mRNA. The second promoter complex (P2) controls the non-tissue specific constitutive expression of GHR1B mRNA. Despite the central role that GH and the GHR have in animal growth, the independent function of each promoter complex has not yet been tested in any species. The objective was to perform a germ-line deletion of each GHR promoter complex to test their function in a porcine model. The CRISPR/Cas9 system was used to delete the P1 and P2 promoter complexes in porcine fetal fibroblasts and cloned to create two independent lines for each promoter deletion. Adult animals that were heterozygous (het) for the GHRP1 and GHRP2 deletions were mated to create litters of +/+ (wildtype, WT), +/- (het), and -/- (knockout, KO) pigs for the respective promoters. An RTPCR of liver and muscle confirmed that deletion of GHRP1 and GHRP2 effectively knocked out GHR1A and GHR1B mRNA. Piglets were weighed at birth and approximately monthly until 2 (GHRP1) or 5 (GHRP2) mo of age. Piglets from the GHRP1 line were +/+ (n = 3), +/- (n = 10), and -/- (n = 2) across two litters. Birthweights (1.5 ± 0.1, 1.4 ± 0.1, and 1.5 ± 0.2 kg, respectively) and body weight (BW) at 2 mo of age (32.7 ± 2.9, 27.0 ± 1.6, and 27.0 ± 3.5 kg, respectively) were similar (P > 0.10) for GHRP1 piglets. Piglets from the GHRP2 line were +/+ (n = 2), +/- (n = 6), and -/- (n = 9) across two litters. Birthweights were similar (1.4 ± 0.3, 1.3 ± 0.1, and 1.0 ± 0.1 kg, respectively) but GHRP2-/- pigs weighed less (P < 0.01) at weaning compared with GHRP2+/+ or GHRP2+/- piglets (6.4 ± 0.8, 6.5 ± 0.4, and 4.5 ± 0.4 kg, respectively). Monthly BW for the GHRP2-/- piglets were less (P < 0.001) when compared with GHRP2+/+ or GHRP2+/- piglets. At 5 mo of age, the GHRP2-/- piglets were approximately 50% of the BW of GHRP2+/+ or GHRP2+/- (89.6 ± 6.7, 89.6 ± 3.9, and 43.1 ± 3.2 kg, respectively). In conclusion, the deletion of GHRP2 (and GHR1B mRNA) had a profound effect on pig growth. The GHRP1 deletion (GHR1A mRNA) did not affect growth with the caveat that the GHRP1 litters had not reached maturity. This work was supported by USDA NIFA 2019-67015-29484.

Brief Research Report: Effect of Triiodothyronine on Hepatic Growth Hormone Receptor Expression in Primary Bovine Hepatocytes.

In previous studies, triiodothyronine (T3) was found to be lower in cows with ketosis and an effect of T3 on Growth Hormone Receptor (GHR) expression is described, e. g., in a human hepatoma cell line. Therefore, this study aimed to test whether T3 affects GHR messenger RNA (mRNA) expression in a well-established bovine hepatocyte model. Hepatocytes were kept in a sandwich culture and stimulated for 6 days with constant (10 μg/ml) or decreasing (from 10 to 5 μg/ml) T3 concentrations, and GHR, as well as IGF-1 mRNA expression, was measured using real-time polymerase chain reaction (RT-PCR). We could confirm in vitro that T3 has a stimulatory effect on GHR1A mRNA expression.

Establishing a model of primary bovine hepatocytes with responsive growth hormone receptor expression

The liver becomes resistant to growth hormone before parturition in dairy cows (uncoupling of the somatotropic axis). However, the mechanism of growth hormone insensitivity has not been fully described. The aim of the present study was to improve a previous model of adult bovine hepatocytes in a sandwich culture system to ensure growth hormone receptor (GHR) expression. First, we modified the protocol for hepatocyte retrieval and tested the effect of short (18 min) and long (up to 30 min) warm ischemia on hepatocyte viability. Second, we used medium additives that affect GHR expression in vivo-insulin (INS), dexamethasone (DEX), both (INS+DEX), or no hormone additives (CTRL)-to ensure the functionality of hepatocytes, as measured by lactate dehydrogenase activity and urea concentration in the medium. We also used reverse transcriptase PCR of hepatocytes to evaluate expression of albumin (ALB), hepatocyte nuclear factor 4α (HNF4A), nuclear factor-κ-B-inhibitor α (NFKBIA), cytosolic phosphoenolpyruvate carboxykinase (PCK1), and vimentin (VIM) mRNA. Moreover, we analyzed the expression of GHRtot (GHR), GHR1A, insulin-like growth factor-1 (IGF1), and IGF binding protein-2 (IGFBP2) in response to exposure to media with the different compositions. Modification of the protocol (changes in rinsing and perfusion times, buffer composition, and the volume and standardization of collagenase) led to increased cell counts and cell viability. Short warm ischemia with the modified protocol significantly increased cell count (4.7 × 107 ± 1.9 × 107 vs. 3.5 × 106 ± 1.5 × 106 vital cells/g of liver) and viability (79.1 ± 8.4 vs. 37.1 ± 8.9%). Therefore, we gathered hepatocytes from the liver after short warm ischemia with the modified protocol. For these hepatocytes, lactate dehydrogenase activity was lower in media with INS and with DEX than in media with INS+DEX or CTRL; urea concentrations were highest at d 4 for INS+DEX. As well, HNF4A and ALB were more highly expressed in hepatocytes cultured with INS and INS+DEX than in those cultured with DEX or CTRL, and the substitution of DEX suppressed VIM and NFKBIA expression but increased PCK1 expression. The expression of GHR, GHR1A, and IGF1 was suppressed by dexamethasone (DEX and INS+DEX), whereas INS distinctly increased GHR, GHR1A, and IGF1 mRNA expression. Hepatocytes in a sandwich culture showed influenceable GHR expression; this study provides a model that can be used in studies examining factors that influence the expression and signal transduction of GHR in dairy cows.

High concentrations of fatty acids and β-hydroxybutyrate impair the growth hormone-mediated hepatic JAK2-STAT5 pathway in clinically ketotic cows

The hepatic growth hormone (GH)-insulin-like growth factor (IGF)-I axis is essential for regulating intrahepatic lipid metabolism. Ketotic cows are characterized by high blood concentrations of fatty acids and β-hydroxybutyrate (BHB), which display lipotoxicity. The aim of this study was to investigate changes in the hepatic GH-IGF-I axis in ketotic cows and to determine the effects of fatty acids and BHB on the GH-IGF-I axis in calf hepatocytes. Liver and blood samples were collected from healthy (n = 15) and clinically ketotic (n = 15) cows. Hepatocytes were isolated from calves and treated with various concentrations of GH, fatty acids, and BHB. The results showed that clinically ketotic cows displayed a high blood concentration of GH, a low blood concentration of IGF-I, and decreased hepatic GHR1A expression as well as impaired hepatic Janus kinase 2 (JAK2)-signal transducer and activator of transcription 5 (STAT5) signaling. In vitro, GH treatment induced activation of the JAK2-STAT5 pathway to increase the mRNA expression and secretion of IGF-I in calf hepatocytes. More importantly, treatment with fatty acids or BHB significantly inhibited GHR1A mRNA and JAK2 protein expression, as well as the STAT5 phosphorylation level and phospho-STAT5 nuclear translocation; these effects markedly reduced IGF1 mRNA expression and secretion in calf hepatocytes. In summary, these results indicate that high blood concentrations of fatty acids or BHB can impair the intrahepatic GH-mediated JAK2-STAT5 pathway and downregulate IGF-I expression and secretion in ketotic cows.

Metabolic and endocrine profiles and hepatic gene expression in periparturient, grazing primiparous beef cows with different body reserves

The objective was to determine effects of prepartum BCS on metabolic/endocrine profiles and hepatic gene expression and their associations with cow and calf performance in grazing suckled-primiparous beef cows from −49 to 49 days postpartum (DPP). Twenty crossbred cows selected according to expected calving date, were classified at −35 DPP into thin (BCS<4.5) or moderate (BCS≥4.5) BCS groups and blocked by calving date. Blood samples were obtained weekly for metabolite and hormone analyses and liver biopsies were collected at −11, 7, 31, and 49 DPP. Cow BW and BCS were greater in moderate than thin cows throughout the period. Estimated energy intake was greater in moderate than thin cows Moderate BCS cows produced more milk than thin cows at 35 DPP and calves from moderate BCS cows had greater BW and average daily gain than calves from thin cows. Serum leptin tended to be greater while adiponectin was less in moderate than thin BCS cows. Overall serum insulin was less in moderate than thin cows while serum IGF-I during the prepartum was greater in moderate than thin BCS cows. Growth hormone receptor (GHR) mRNA was 2-fold greater at −11 DPP while GHR1A and IGF1 mRNA were 2.5-fold less at 49 DPP in moderate than thin BCS cows. The IGFBP2 mRNA decreased in moderate but increased in thin BCS cows from −11 to 49 DPP. These results were associated with changes in body reserves during prepartum and may indicate that prepartum differences in BCS lost can affect nutrient partitioning towards the mammary gland, and subsequent milk production and calf weight.

Effects of herbage allowance of native grasslands in purebred and crossbred beef cows: metabolic, endocrine and hepatic gene expression profiles through the gestation–lactation cycle

Our objective was to evaluate the metabolic, endocrine and hepatic mRNA profiles through the gestation–lactation cycle in purebred (PU: Angus and Hereford) and crossbred (CR: reciprocal F1 crosses) mutliparous beef cows (n=32), grazing on two herbage allowances of native pastures (2.5 v. 4 kg dry matter/kg BW; LO v. HI) and their associations with cow’s productive performance (calf birth weight, milk production and commencement of luteal activity). Cow BW, body condition score (BCS) and blood samples were collected monthly, starting at −165 days relative to calving (days), and every 2 weeks after calving until +60 days of lactation. Liver biopsies were collected at −165, −75, −45, −15±10, and +15 and +60±3 days. Metabolic, endocrine and hepatic gene expression profiles, and calf birth weight, milk yield and postpartum commencement of luteal activity were evaluated. Overall, the most pronounced changes in metabolic, endocrine and hepatic gene expression occurred during winter gestation (−165 to −45 days), when all cows experienced the onset of a negative energy balance (decreased BCS, glucose and insulin, and increased non-esterified fatty acid concentrations, P<0.008). Concentrations of insulin and IGF-I were greater (P<0.037) in HI than in LO cows. However, serum IGF-I concentrations and hepatic growth hormone receptor (GHR) and IGF1 mRNA decreased (P<0.05) during the winter gestation period only in HI cows. Although IGF-I concentrations decreased (P<0.05) during the early postpartum (−15 v.+15 days) for all cows, the typical molecular mechanism that control the uncoupling of the growth hormone-IGF1 axis during the transition period of the dairy cattle (reduced hepatic GHR1A and IGF-I mRNA) was not observed in this study. The hepatic mRNA expression of key transcripts involved in gluconeogenesis and fatty-acid oxidation were upregulated (P<0.05) during winter gestation (from −165 to −45, −15 or +15 days, depending on the cow groups). Particularly, acyl-CoA oxidase-1 mRNA was greater for CR than for PU cows during winter gestation (−75 and −45 days), and fibroblast growth factor-21 mRNA was downregulated (P<0.01) only for HI cows during the transition (−15 v. 15 days) and lactation period (+15 to +60 days, P<0.01). These results, together with the greater BCS, estimated energy intake, increased milk yield and shorter commencement of luteal activity in HI than in LO, and in CR than in PU cows (P<0.018), would indicate that HI and CR cows were able to adapt more efficiently to changes in nutrient and energy supply through the gestation–lactation cycle.

Antepartal insulin-like growth factor concentrations indicating differences in the metabolic adaptive capacity of dairy cows.

Cows with different Insulin-like Growth Factor-I (IGF-I) concentrations showed comparable expression levels of hepatic growth hormone receptor (GHR). Suppressor of cytokine signaling 2 (SOCS2), could be responsible for additional inhibition of the GHR signal cascade. The aims were to monitor cows with high or low antepartal IGF-I concentrations (IGF-Ihigh or IGF-Ilow), evaluate the interrelationships of endocrine endpoints, and measure hepatic SOCS2 expression. Dairy cows (n = 20) were selected (240 to 254 days after artificial insemination (AI)). Blood samples were drawn daily (day -17 until calving) and IGF-I, GH, insulin, thyroid hormones, estradiol, and progesterone concentrations were measured. Liver biopsies were taken (day 264 ± 1 after AI and postpartum) to measure mRNA expression (IGF-I, IGFBP-2, IGFBP-3, IGFBP-4, acid labile subunit (ALS), SOCS2, deiodinase1, GHR1A). IGF-I concentrations in the two groups were different (p < 0.0001). However, GH concentrations and GHR1A mRNA expression were comparable (p > 0.05). Thyroxine levels and ALS expression were higher in the IGF-Ihigh cows compared to IGF-Ilow cows. Estradiol concentration tended to be greater in the IGF-Ilow group (p = 0.06). It was hypothesized that low IGF-I levels are associated with enhanced SOCS2 expression although this could not be decisively confirmed by the present study.

Insulin-like growth factor and growth hormone receptor in postpartum lactating beef cows

The objective of this study was to evaluate the plasma concentrations of insulin-like growth factor-I (IGF-I), and the mRNA hepatic expression of IGF-I and of the growth hormone receptors GHR and GHR 1A, in postpartum beef cows. Four Angus and four crossbred (Angus x Nelore) postpartum suckled beef cows were used. Liver and blood samples were collected every 10 days, from calving to 40 days postpartum, for gene expression and for β-hydroxybutyrate and IGF-I assays, respectively. Samples for progesterone assay were collected every other day, from day 10 to 40 postpartum. Three cows ovulated before 40 days postpartum. IGF-I concentration was higher in Angus x Nelore than in Angus cows. There was no difference in the expression of GHR, GHR 1A and IGF-I according to breed or ovulatory status. IGF-I concentrations were higher in crossbred cows, but have not changed according to postpartum ovulatory status. Moreover, changes in postpartum IGF-I concentrations are not associated with changes in liver GHR, GHR 1A and IGF-I mRNA expression in either breed.

Short communication: Growth hormone receptor expression in two dairy breeds during the periparturient period

The growth hormone receptor (GHR) 1A mRNA decreases after calving in the liver of Holstein dairy cows and may coordinate nutrient partitioning. The hypothesis that the decrease in GHR1A mRNA around the time of calving was characteristic of a second dairy breed was tested by examining Guernsey cows in addition to Holstein cows. Holstein and Guernsey cows were housed together and paired by parity and expected calving date. Liver biopsies and blood samples were collected prepartum (d −20±1) and postpartum on d 3, and d 14±1. The amounts of GHR1A, GHR1B, GHR1C, and insulin-like growth factor (IGF)1 mRNA were determined by quantitative reverse transcription polymerase chain reaction. Blood concentrations of growth hormone (GH) and IGF1 were measured by RIA. Both breeds underwent a decrease in GHR1A mRNA, a decrease in liver IGF1 mRNA, a decrease in blood IGF1, and an increase in blood GH after calving. The decrease in liver GHR1A and IGF1 mRNA after calving may be an inherent characteristic of dairy breeds that enables nutrient partitioning for greater milk production. Independent genetic selection in 2 dairy breeds seemingly exploited a similar mechanism, reduced GHR1A expression, to decrease blood IGF1 and increase blood GH, a key hormone involved in nutrient partitioning.

Growth Hormone Stimulates Hepatic Expression of Bovine Growth Hormone Receptor Messenger Ribonucleic Acid through Signal Transducer and Activator of Transcription 5 Activation of a Major Growth Hormone Receptor Gene Promoter

The objective of this study was to determine whether and how GH regulates hepatic expression of GH receptor (GHR) mRNA in cattle. Ribonuclease protection assays revealed that injection of GH in a slow-release formula increased both hepatic GHR and IGF-I mRNAs 1 wk after the injection. The increases in GHR and IGF-I mRNAs were highly correlated. Western blot analysis showed that the injection also increased liver GHR protein level. In cattle and other mammals, hepatic GHR mRNA is expressed as variants that differ in the 5'-untranslated region due to the use of different promoters in transcription and/or alternative splicing. We found that GH increased the expression of the liver-specific GHR mRNA variant GHR1A without affecting the other two major GHR mRNA variants in the bovine liver, GHR1B and GHR1C. In transient transfection analyses, GH could robustly activate reporter gene expression from a 2.7-kb GHR1A promoter, suggesting that GH augmentation of GHR1A mRNA expression in the liver is at least partially mediated at the transcriptional level. Additional transfection analyses of serially 5'-truncated fragments of this promoter narrowed the GH-responsive sequence element down to a 210-bp region that contained a putative signal transducer and activator of transcription 5 (STAT5) binding site. EMSAs demonstrated that this putative STAT5 binding site was able to bind to STAT5b protein. In cotransfection assays, deletion of this putative STAT5 binding site abolished most of the GH response of the GHR1A promoter. Like 1-wk GH action, 6-h (i.e. short-term) GH action also increased liver expression of GHR1A and total GHR mRNAs in cattle. These observations together suggest that GH directly stimulates the expression of one GHR mRNA variant, GHR1A, through binding STAT5 to its promoter, thereby increasing GHR mRNA and protein expression in the bovine liver.

Identification of zinc finger binding protein 89 (ZBP-89) as a transcriptional activator for a major bovine growth hormone receptor promoter

The objective of this study was to identify the transcription factors that regulate the expression of growth hormone receptor (GHR) 1A mRNA, a major GHR mRNA variant in the bovine liver. A deoxyribonuclease I footprint analysis revealed that the GHR1A promoter region −69 to −30 (relative to the transcription start site for GHR1A mRNA) contained binding sites for bovine liver nuclear proteins. Using a yeast one-hybrid analysis, zinc finger binding protein 89 (ZBP-89) was identified as a binding protein to this promoter region. Binding of ZBP-89 to the GHR1A promoter region −69 to −30 was further confirmed by an electrophoretic mobility shift assay. In cotransfection analyses, overexpression of ZBP-89 enhanced ( P < 0.01) the activity of the GHR1A promoter and this enhancement was dependent on the putative ZBP-89 binding site in the promoter. These results together indicate that ZBP-89 is a transcription factor that regulates the expression of GHR1A mRNA.

Growth hormone (GH) binding and expression of GH receptor 1A mRNA in hepatic tissue of periparturient dairy cows.

Growth hormone (GH) plays a role in metabolic adaptations that occur during lactogenesis. Liver GH receptor transcript (GHR 1A) is transiently decreased near parturition and may reduce GH-dependent signaling leading to low blood insulin-like growth factor I (IGF-I) concentrations in periparturient dairy cattle. We hypothesized that the decrease in GHR 1A mRNA at parturition was associated with decreased GH binding (i.e., GHR protein concentration) in liver. Blood and liver biopsy samples were collected from 12 Holstein cows on d -12 +/- 1, 3, and 17 relative to parturition. Total cellular RNA was isolated from a sub-sample of liver. Quantitative real-time polymerase chain reactions were used to measure GHR 1A, total GHR, IGF-I, and cyclophilin mRNA. Microsomal membranes were isolated from the remaining liver tissue and assayed for 125I-bGH binding. Plasma was assayed for GH and IGF-I concentrations. Liver GHR 1A mRNA, specific 125I-bGH binding to liver membranes, liver IGF-I mRNA, and plasma IGF-I concentrations were lower on d 3 relative to d -12. The GHR 1A mRNA, 125I-bGH binding, and plasma GH concentrations increased on d 17 but liver IGF-I mRNA and plasma IGF-I concentrations did not change between d 3 and 17. Total GHR mRNA and cyclophilin mRNA amounts were similar on d -12, 3, and 17. Across all days, 125I-bGH specific binding in liver was highly correlated with liver GHR 1A mRNA (R2 = 0.68) but not with total GHR mRNA. Saturation binding analysis showed that GHR concentration (Bmax) in liver on d 3 had decreased to only 5% of the amount on d -12. We conclude that decreased GHR 1A mRNA leads to decreased GHR protein concentration in liver. Reduced GHR in liver likely contributes to a decrease in liver IGF-I production and reduced concentrations of IGF-I in blood of periparturient cows.

Insulin restores GH responsiveness during lactation-induced negative energy balance in dairy cattle: effects on expression of IGF-I and GH receptor 1A.

Early lactation in dairy cattle is a period of severe negative energy balance (NEB) characterized by reduced blood glucose and insulin concentrations and elevated blood GH concentrations. The liver is refractory to GH during NEB and this uncoupling of the GH-IGF axis results in diminished plasma concentrations of IGF-I. Our objectives were to examine the effects of insulin administration during the immediate postpartum period on plasma IGF-I and GH concentrations and to examine the hepatic expression of total GH receptors (all GH receptor transcripts), GH receptor 1A (GHR 1A) and IGF-I. In addition, we examined adipose tissue for total GH receptor and IGF-I mRNA levels to establish the effects of chronic hyperinsulinemia on an insulin-responsive peripheral tissue. Holstein cows (n=14) were subjected to either a hyperinsulinemic-euglycemic clamp (insulin; INS) or saline infusion (control; CTL) for 96 h starting on day 10 postpartum. Insulin was infused i.v. (1 micro g/kg body weight per h), blood samples were collected hourly, and euglycemia was maintained by infusion of glucose. Insulin concentrations during the infusions were increased 8-fold in INS compared with CTL cows (2.33+/-0.14 vs 0.27+/-0.14 ng/ml (S.E.M.); P<0.001) while blood glucose concentrations were not different between treatments (45.3+/-2.2 vs 42.5+/-2.2 mg/dl; P>0.1). Plasma IGF-I increased continuously during the insulin infusion, and reached the highest concentrations at the end of the clamp, being almost 4-fold higher in INS compared with CTL cows (117+/-4 vs 30+/-4 ng/ml; P<0.001). Hepatic expression of GHR 1A and IGF-I mRNA was low in CTL cows, but was increased 3.6-fold (P<0.05) and 6.3-fold (P<0.001) respectively in INS cows. By contrast, in adipose tissue the changes in gene expression in response to insulin were reversed with decreases in both total GHR and IGF-I mRNA. The expressions of GHR 1A and IGF-I mRNA in liver tissue were correlated in INS (r=0.86; P<0.05), but not CTL cows (r=0.43; P>0.1). Insulin appears to be a key metabolic signal in coupling the GH-IGF axis, thus orchestrating a marked elevation in circulating IGF-I concentrations.

Involvement of hepatocyte nuclear factor-4 in the expression of the growth hormone receptor 1A messenger ribonucleic acid in bovine liver.

The GH receptor 1A mRNA (GHR 1A mRNA) is one of the major GHR mRNA variants that differ in the 5'-untranslated region. The GHR 1A mRNA is unique because it is exclusively expressed in liver. The objective of the present study was to understand the mechanism for the liver-specific expression of the GHR 1A mRNA in the bovine. Twenty-six kilobases of 5'-flanking region of the bovine GHR gene was cloned and sequenced. The first exon (exon 1A) that corresponded to the 5'-untranslated region of the GHR 1A mRNA was 15,250 bp upstream from exon 2 in the GHR gene. The major transcription start site for the GHR 1A mRNA was 19 bp downstream from a putative TATA box. Transient transfection analyses of the 5'-flanking region of exon 1A in liver cell lines vs. nonliver cell lines did not reveal a positively regulatory region responsible for the liver-specific expression of the GHR 1A mRNA perhaps because the liver cell lines do not recapitulate the in vivo hepatic environment. A putative regulatory region was then found by deoxyribonuclease I footprinting analyses of the proximal 5'-flanking region of exon 1A with nuclear extracts from bovine liver tissue. This regulatory region contained a putative binding site for the liver-enriched transcription factor hepatocyte nuclear factor-4 (HNF-4). Binding of HNF-4 in bovine liver to this putative HNF-4 binding site was confirmed by electrophoretic mobility shift assays. Overexpression of HNF-4 enhanced the transcriptional activity of the 5'-proximal region of exon 1A in various cell lines. Mutation of the HNF-4 binding site abolished the transactivation. In addition, the HNF-4 mRNA was found to be primarily expressed in liver and absent in most nonhepatic tissues in the bovine. Collectively, these observations suggest that the liver-enriched transcription factor HNF-4 plays a role in the expression of GHR 1A mRNA in bovine liver.