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Endocrinology Vol. 138, No. 5 1988-1994
Copyright © 1997 by The Endocrine Society

Articles

Liver and Kidney Growth Hormone (GH) Receptors Are Regulated Differently in Diabetic GH and GH Antagonist Transgenic Mice1

Nian-yi Chen, Wen Y. Chen and John J. Kopchick

Edison Biotechnology Institute, Molecular and Cellular Biology Program, and Department of Clinical Research, Ohio University College of Osteopathic Medicine, Ohio University, Athens, Ohio 45701

Address all correspondence and requests for reprints to: Dr. John J. Kopchick, Edison Biotechnology Institute, Konneker Research Laboratories, The Ridges, Ohio University, Athens, Ohio 45701.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Elevated GH levels are frequently seen in poorly controlled type I diabetics and have been implicated in diabetic complications. Studies of GH and GH antagonist (GHA) transgenic mice with streptozotocin (STZ)-induced diabetes have revealed that GH has a permissive effect for diabetic nephropathy, and that expression of a GHA gene protected mice against diabetic kidney lesions. To investigate whether kidney GH receptor (GHR) and/or GH-binding protein may play a role in diabetic nephropathy, we evaluated GH-specific binding and messenger RNA levels for GHR/GH-binding protein in mouse livers and kidneys from bovine (b) GH or bGHA transgenic (Tg) mice and their nontransgenic (NTg) littermates with or without STZ-induced diabetes. We found that liver-specific GH binding is significantly higher in both bGH- and bGHA-Tg mice compared to that in their NTg controls. In contrast, kidney GH binding is significantly lower in bGH-Tg mice compared to that in NTg littermates. These results indicate that regulation of mouse GHR expression is tissue specific. STZ-induced diabetes decreased GH-specific binding in both liver and kidney of NTg and GHA-Tg mice, but not in bGH-Tg mice. The lowered GHR binding in diabetic NTg and GHA-Tg mice suggests the involvement of insulin in the regulation of GHR expression. The down-regulation of kidney GHR in GHA-Tg mice in combination with the presence of GHA may partially explain the protective mechanism of GHA against diabetic kidney lesions.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ALTHOUGH controversial, augmented GH secretion in diabetic patients is believed to be associated with diabetic nephropathy (1, 2, 3, 4). To investigate the role of GH in diabetes, we had previously examined the effect of streptozotocin (STZ) on transgenic (Tg) mice expressing GH and GH antagonist (GHA) genes. We found that GH transgenic (GH-Tg) mice without hyperglycemia exhibited kidney glomerulosclerosis resembling human diabetic nephropathy, whereas GHA-Tg mice did not possess detectable kidney lesions (3, 4). Yet, liver GH receptors (GHRs) were up-regulated in both mouse lines (4, 5). STZ-induced diabetes (STZ-diabetes) in these mice resulted in decreased liver GH binding in GHA-Tg and nontransgenic (NTg) mice, but not in GH-Tg mice. Based on these data, we proposed that 1) GHR levels may be related to diabetic end-organ complications; and 2) hepatic GHR expression is regulated by both GH and insulin.

It is believed that up to 50% of circulating serum GH is bound to GH-binding protein (GHBP) (6), the structure of which corresponds to the extracellular domain of GHR (7, 8, 9). Although the physiological role of GHBP is uncertain, measurements of GHBP have been used as an estimate of hepatic GHR density (10, 11, 12). A decrease in GHBP has been shown in young patients with insulin-dependent diabetes mellitus (IDDM) (12). Therefore, it is believed that hepatic GH binding is reduced by the onset of diabetes, resulting in GH resistance in some IDDM patients (12).

Although high GH levels have been frequently observed in IDDM patients (13, 14, 15, 16), decreased insulin-like growth factor I and retarded growth have been reported in diabetic rats and humans (17, 18, 19, 20). These findings indicate that diabetes may be associated with a defect in GHR or postreceptor signaling systems in GH target tissues.

GHAs are GH analogs with amino acid substitutions of Gly (G) at position 119 (bGH) or 120 (hGH) (5, 21, 22). We have previously shown that when bovine (b) GH or human (h) GH was mutated with an amino acid with a bulky side-chain, such as Lys (K) or Arg (R) at position G119 or G120, respectively, the resulting molecules bound to GHRs, but blocked the biological activities of native GH (5, 21, 22). In vivo studies in our laboratory have demonstrated that Tg mice expressing bGH-G119K (R) or hGH-G120R (K) genes exhibited dwarf phenotypes (5, 21, 22). In vitro studies have shown that these GH analogs inhibited GH-induced differentiation and proliferation of preadipocytes and GH-induced tyrosine phosphorylation of a GH-signaling molecule, Stat5 (23, 24).

Since GH-Tg mice developed kidney lesions resembling those found in human diabetic patients, whereas GHA-Tg mice did not, we attempted to determine whether these mice may be used as animal models to study the pathophysiology of human diabetic nephropathy. In this regard, Striker et al. found that glomerulosclerosis develops in a progressive manner in GH-Tg mice (1).

To further investigate GHR/GHBP’s role in diabetic nephropathy, we examined the expression and regulation of liver and kidney GHR/GHBP in GH-Tg and GHA-Tg mice in the absence or presence of STZ diabetes.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Female Tg and NTg mice, 4–6 weeks of age, were treated with or without STZ (Sigma Chemical Co., St. Louis, MO) as described previously (4, 12). Two Tg mouse lines were used in this study. One expressed the bGH transgene and exhibited an enhanced growth phenotype. It will be referred to as GH-Tg. The other expressed a GHA, bGH-G119K, and possessed a dwarf phenotype. It will be referred to as GHA-Tg (5, 6). Experimental parameters were compared among diabetic GH-Tg mice, GHA-Tg mice, and their NTg littermates and also compared with corresponding nondiabetic GH-Tg, GHA-Tg, and NTg controls. All mice were fed ad libitum with Purina rodent chow 5004 (Ralston-Purina, St. Louis, MO) and were killed after 12 weeks of STZ-diabetes.

STZ treatment
Mice were injected ip with STZ (Sigma) at 80 µg/g·day or a vehicle solution at 80 µl/g·day for 5–7 days as previously described (4). Blood glucose levels were elevated to 250–300 mg/dl in STZ-treated mice.

Serum insulin and blood glucose assays
Fasting serum insulin levels and fasting blood glucose levels were assayed before the animals were killed using a RIA kit (Linco’s Research, St. Charles, MO) and a One Touch glucometer (Lifescan, Milpitas, CA).

RRA for GHR
Liver or kidney GHR binding assays were performed as previously described (6). Livers and kidneys were homogenized in 4–5 vol ice-cold homogenization buffer (0.3 M sucrose, 10 mM EDTA, 50 mM HEPES, 0.1 mM N-tosyl-L-phenylalanine chloromethyl ketone, and 0.5 mM phenylmethylsulfonylfluoride, pH 8.0) using a Brinkmann homogenizer (Westbury, NY). The homogenates were centrifuged at 20,000 x g for 15 min. The resulting supernatants were then centrifuged at 100,000 x g for 1 h. The final pellets, containing microsomal membranes, were suspended in ice-cold 10 mM HEPES buffer, and total protein concentrations were determined using the Bio-Rad assay system (Richmond, CA). The suspensions were stored at -20 C.

For competitive receptor binding assays, 0.2 mg total protein was incubated with 125I-labeled hGH (2 ng/ml; SA, 100 µCi/µg) in the presence or absence of 5 µg/ml unlabeled hGH in reaction buffer (20 mM HEPES, 10 mM CaCl2, 0.1% BSA, and 0.05% NaN3, pH 8.0) at room temperature for 3 h. Reactions were terminated by adding 1 ml ice-cold reaction buffer followed by centrifugation (16,000 x g). Radioactivity associated with the membrane pellets was determined using a {gamma}-counter (Gamma 5500, Beckman, Fullerton, CA). All assays were performed in triplicate and repeated four to eight times. ANOVAs were used to determine the statistical differences in GH binding levels between the treatment groups.

Semiquantification of mouse (m) GHR and mGHBP messenger RNA (mRNA)
RNA extraction.
Liver or kidney total RNA was extracted using RNAzol (Cinna Biotecx, Friendswood, TX). Total RNA concentrations were determined by OD260 readings. The integrity of the RNA was assessed by visual inspection of the ethidium bromide-stained gels after agarose-formamide gel electrophoresis.

Reverse transcription-PCR (RT-PCR) studies.
As mRNA of GHR and GHBP in rats and mice are generated by alternative splicing of a single gene (Fig. 1AGo) (10, 25, 26), one common upstream sense primer ("a") encompassing bp 326–351 (5'-TGC CCT GAT TAT GTC TCT GCT GGA AAA-3'), which is located in exon 5, was used for PCR amplification of both GHR and GHBP complementary DNA (cDNA; Fig. 1BGo). Two different antisense primers were designed for specific RT of GHR and GHBP mRNAs. The GHR-specific antisense primer ("b") encompassing bp 816–837 (5'-ATC CAG TTT CCA TGG TTC TTA-3') was designed in exon 8 of mGHR cDNA, and the GHBP-specific antisense primer ("c") encompassing bp 887–907 (5'-ATC CCT AGC CTT GTG GGC ACC-3') was designed from exon 8A of the same gene (Fig. 1BGo) (26). RT and PCR amplification of cDNA fragments of GHR and GHBP were performed using a GeneAmp RT/PCR kit with rTth DNA polymerase (Perkin-Elmer/Cetus, Norwalk, CT). The sizes of the corresponding amplified products were 511 bp for GHR and 581 bp for GHBP.



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  		Figure 1. RT-PCR primer design based on the alternative precursor mRNA splicing schemes of GHR and GHBP. A, Two different precursor mRNA splicing reactions give rise to GHR mRNA (with exon 8A removed) and GHBP mRNA. B, Primers were designed according to cDNA sequences of GHR and GHBP. a, Upstream primer for GHR and GHBP. b, Downstream primer specific for GHR. c, Downstream primer specific for GHBP.

 
Competitive PCR.
Competitive PCR was used for quantification of mGHR or mGHBP mRNAs. Two plasmids with altered GHR genes were generated for use as internal controls for competitive PCR (Fig. 2Go). Plasmid pPCRII-mGHRI (27), which encodes mGHR cDNA exons 2, 3, 4, 5, 6, 7, and part of exon 8, was digested with HincII and religated (Fig. 2Go). The resulting plasmid, p{Delta}mGHR, is 206 bp shorter than the parental plasmid with a deletion of a fragment between +451 (exon5) and +657 (exon 7). Amplification of p{Delta}mGHR with primers "a" and "b" would produce a PCR product of 287 bp.



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  		Figure 2. Construction of p{Delta}mGHR plasmid. The parental plasmid pPCRII-mGHR-I, which contains exons 2–8 of mGHR cDNA, was cleaved with HincII, and the longer fragment (4563 bp) was religated. The resulting plasmid is 206 bp shorter than the parental plasmid.

 
p{Delta}mGHBP was generated by insertion of DNA sequence complimentary to the antisense GHBP primer into the HpaI site located in exon 7 (+657) of plasmid pPCRII-mGHRI using oligo-directed mutagenesis (Fig. 3Go). This construction allows amplification with the common primer "a" and GHBP-specific primer "c," producing a PCR product of 360 bp. This PCR product from p{Delta}mGHBP is 220 bp shorter than that of wild-type mGHBP cDNA with same pair of primers. The concentration of the purified altered DNAs was determined by comparison with {lambda}/HindIII DNA standards (Life Technologies, Grand Island, NY) and densitometric scanning of the agarose gel.



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  		Figure 3. Construction of p{Delta}mGHBP plasmid. A 21-bp sequence complimentary to the GHBP cDNA antisense primer was inserted into the HpaI site of the parental plasmid, pPCRII-mGHR-I, by oligo-directed mutagenesis.

 
Competitive PCR was performed following a previously described procedure (28) with modifications. An initial reaction was performed to determine the range and relative amounts of mutated and test cDNAs to be added to each tube and the optimal PCR conditions. Then, 2 master RT reactions were generated, each one with a gene-specific downstream primer (i.e. specific for either GHR or GHBP). Each master RT reaction contained 1 µg total RNA multiplied by the tube numbers needed for a series of competitive PCRs. After an incubation at 70 C for 10 min, each RT reaction mixture was divided into 5 PCR tubes. To each tube, a 75-µl aliquot of master mixture containing 10 x chelating buffer, MgCl2, sense primer, and H2O was added, followed by the addition of 5 µl altered cDNAs at five concentrations ranging between 0.5–4 pg/µl. PCR was performed with an initial cycle of 94 C for 2 min, followed by 35 3-step cycles of 94 C for 1 min, 56 C for 1 min, and 72 C for 1 min, and a final cycle of 72 C for 5 min.

Analysis of PCR products.
Ten to 15% of the reaction mixtures were separated by electrophoresis on a 2% agarose gel and visualized with ethidium bromide staining followed by UV transillumination. Photographs taken with Polaroid 667 film (Polaroid Co., Cambridge, MA) were then scanned by densitometry (model GS-670, Bio-Rad), and the intensity of the bands was quantified using a Molecular Analysis program (Bio-Rad). The densitometric values of the test and mutated DNA bands were calculated, and the ratio of each set of bands was plotted as a function of the amount of mutated template DNA added. A straight line was drawn by linear regression analysis. The quantity of GHR (or GHBP) cDNA (pg/µg total RNA) in the test samples was calculated to be the amount of mutated DNA at which the mutant/test band density ratio was equal to 1 (28). Competitive PCR was repeated twice for each sample.

ANOVA was used to determine the statistical differences in the amount of mRNA between treatment groups.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Hyperglycemia was successfully induced by STZ treatment in GH-Tg, GHA-Tg, and NTg mice. As shown in Table 1Go, blood glucose levels in the three mouse lines were significantly elevated (P < 0.001) compared to those in their nondiabetic controls, and serum insulin levels were significantly decreased (P < 0.05 or P < 0.001). Without STZ-diabetes, serum insulin concentrations were considerably higher in GH-Tg mice than in NTg mice (P < 0.01).


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  		Table 1. Fasting blood glucose and serum insulin levels from GH, GHA-Tg, and NTg mice with or without STZ treatment (+ or - STZ)

 
GH specific-binding in liver and kidney
As previously reported (13), liver GH-specific binding was up-regulated in nondiabetic GH-Tg (P < 0.01) and GHA-Tg (P < 0.05) mice compared to that in their NTg littermates (Fig. 4AGo). STZ treatment resulted in a significant decrease in GH specific binding in GHA-Tg (P < 0.001) and NTg mice (P < 0.05). In contrast, GH-specific binding remained high in GH-Tg mice even after diabetes was induced (Fig. 4AGo).



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  		Figure 4. GH-specific binding levels in mouse liver and kidney. GH-Tg, GHA-Tg, and NTg mice were treated with or without streptozotocin (- STZ or + STZ). Ligand-receptor binding assays were performed using 125I-labeled hGH. A, Changes in liver GH-specific binding in diabetic animals. a, Different from - STZ control (P < 0.05). b, Different from - STZ control (P < 0.001). c, Different from NTg control (P < 0.01). d, Different from NTg control (P < 0.05). B, Changes in kidney GHR binding in diabetic animals. d, Different from NTg control (P < 0.05). e, Different from - STZ control (P < 0.05).

 
Kidney GH-specific binding showed a different profile from that of liver in nondiabetic mice. GH-specific binding was significantly lower (P < 0.05) in GH-Tg and GHA-Tg mice compared to that in the NTg littermates (Fig. 4BGo). As in liver, STZ treatment significantly decreased GH-specific binding in GHA-Tg and NTg mice (P < 0.05; Fig. 4BGo). Kidney GH-specific binding in diabetic GHA mice also showed a significant difference (lower) compared to that in NTg mice (P < 0.05; Fig. 4BGo). GH-specific binding in GH-Tg mice was not significantly changed by STZ-diabetes.

GHR and GHBP mRNA levels in liver and kidney
Liver and kidney GHR and GHBP mRNA levels were semiquantified. DNA fragments of 511 and 580 bp corresponding to GHR and GHBP mRNA, respectively, were successfully amplified from mouse livers and kidneys by RT-PCR using a common upstream primer "a" and a gene-specific downstream primer "b" or "c" (Fig. 1Go). Mutated DNA plasmids, p{Delta}GHR or p{Delta}GHBP, that possess the same mGHR or mGHBP sequences at upstream and downstream primer sites were coamplified and competed with wild-type mGHR or mGHBP cDNA at or near an equimolar basis. The signal densities of PCR products from a known amount of mutated DNA were used as internal standards for judging the quantity of test cDNA.

Addition of competitor cDNA, p{Delta}GHR, or p{Delta}GHBP in increasing amounts (0.5–4 pg) to identical PCR reaction tubes containing liver or kidney RNAs resulted in a series of cDNA bands with decreased intensities. Figure 5Go represents a typical experiment showing kidney GHR cDNA from a NTg mouse treated with (lanes 1–5) and without (lanes 7–11) STZ competed with mutated plasmid DNA, p{Delta}mGHR. mRNA concentrations of kidney GHR with and without diabetes were 1.9 and 3.3 pg, respectively.



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  		Figure 5. Quantification of mouse kidney GHR mRNA by competitive PCR. The upper panel represents the general scheme of the reaction. A constant amount (1 µg) of total tissue RNA and increasing amounts (0.5–4 pg) of mutated plasmid DNA, p{Delta}mGHR, were added to a series of five reaction tubes containing the same PCR reagents. The middle panel corresponds to ethidium bromide-stained gel after PCR amplification and gel electrophoresis. Lanes 1–5 represent PCR reactions for GHR cDNA from a nondiabetic (- STZ) mouse kidney. Lanes 7–11 represent PCR reactions for GHR cDNA from a diabetic (+ STZ) mouse kidney. The bottom panel represents regression curves of the density ratios of wild-type (wt) GHR/p{Delta}mGHR vs. input of p{Delta}mGHR. Curve 1, - STZ; curve 2, + STZ. Concentrations of wild-type GHR cDNA (picograms per µg total RNA) were calculated from the equivalence points, y = 1.

 
As shown in Table 2Go, RNA semiquantification by competitive PCR demonstrated elevated levels of liver GHR mRNA (P < 0.05) in GH-Tg mice, but not in GHA-Tg mice, compared to those in NTg mice. After STZ treatment, liver GHR mRNA levels were significantly decreased (P < 0.05) in the three mouse lines, with GH-Tg mice still having the highest levels. Liver GHBP mRNA levels were also elevated (P < 0.01) in GH-Tg mice, but not in GHA-Tg mice compared to those in NTg mice. Liver GHBP mRNA levels in GH-Tg mice increased (P < 0.05) after STZ treatment, whereas those in GHA-Tg and NTg mice decreased (P < 0.05).


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  		Table 2. Liver GHR/GHBP mRNA quantification by competitive PCR

 
Kidney GHR mRNA levels were lower (P < 0.05) in GH-Tg mice than those in NTg controls (Table 3Go). Comparable kidney GHR mRNA levels were seen in NTg and GHA-Tg mice. STZ treatment resulted in a significant decrease in kidney GHR mRNA in NTg and GHA-Tg mice (P < 0.05; Table 3Go), but had no significant effect on GH-Tg mice. Kidney GHBP mRNA levels were significantly higher in GH-Tg mice than in NTg mice (P < 0.05; Table 3Go). Comparable GHBP mRNA levels were found in NTg and GHA-Tg mice. STZ treatment caused a significant decrease in kidney GHBP mRNA in GH-Tg (P < 0.01), GHA-Tg (P < 0.05), and NTg mice (P < 0.05; Table 3Go).


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  		Table 3. Kidney GHR/GHBP mRNA quantification by competitive PCR

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Elevated GH levels have been reported in IDDM patients (17, 18, 19, 20, 21). Also, we previously demonstrated that Tg mice that express bGH or hGH developed kidney glomerulosclerosis resembling that seen in human diabetic patients, whereas GHA-Tg mice did not (4). Moreover, GHA-Tg mice were protected from STZ-induced kidney lesions (4). These findings raised the possibility of GH involvement in diabetic nephropathy. However, the role of GHR/GHBP in diabetic nephropathy remained unclear.

GHR expression is known to be regulated by a number of variables, including GH and insulin (29, 30, 31, 32, 33). It has been reported that liver GHRs decrease in diabetic rats, and this decrease could be reversed by insulin therapy (29). Although the mechanism is unknown, GH is also known to up-regulate liver GH receptors (5, 30, 31, 32, 33). The high liver GH-specific binding levels in GH-Tg mice seen in this study are consistent with the earlier reports. As shown by competitive PCR, GHR mRNA levels in GH-Tg mouse livers were elevated, indicating that the up-regulation of liver GHR by GH may occur at pretranslational levels due to enhanced transcription and/or increased stability of mRNA in these mice. Interestingly, liver GH-specific binding levels were also higher in GHA-Tg mice. As liver GHR mRNA levels in these dwarf mice are not elevated, the mechanisms for up-regulation of GH binding in GH-Tg and GHA-Tg mice are apparently different.

In addition to GH, insulin has been shown to influence liver GHR levels (29, 30). Therefore, the high insulin levels found in nondiabetic GH-Tg animals may also contribute to the elevated GHR levels in these mice.

STZ-diabetes greatly decreased liver GH-specific binding in GHA-Tg mice, but not in GH-Tg mice, which further suggests that GHR levels in the liver are regulated differently in GH-Tg and GHA-Tg mice. The decrease in GHR binding in diabetic NTg and GHA-Tg mice adds support to the concept that insulin has a permissive effect on GHR binding (29, 30). The lower GHR mRNA levels in these mice, which had low serum insulin levels due to STZ treatment, suggest that regulation of GHR expression by insulin is also at the pretranslational level. Although liver GHR binding levels remained high in diabetic GH-Tg mice, GHR mRNA was significantly lower after STZ treatment. This may suggest that decreased insulin levels also affected GHR gene transcription or GHR mRNA stability in these mice. However, constantly high circulating GH levels in these mice may maintain the high GHR binding levels through other mechanisms, such as stimulation of GHR recycling and/or cooperativity.

The high liver GHBP mRNA levels in nondiabetic GH-Tg mice seen in this study suggest that mouse GHBP is also up-regulated by GH. As GHBP mRNA levels decreased in NTg and GHA-Tg mice after STZ treatment, insulin may also play a role in GHBP expression. However, the slight elevation of liver GHBP mRNA in diabetic GH-Tg mice may indicate that liver GHBP levels are more sensitive to the change in the GH concentration than to that in the insulin level.

The kidney GH binding profile in the various mouse lines is different from that seen in the liver. Down-regulation of GH-specific binding as well as GHR mRNA levels was observed in GH-Tg mice compared to those in their NTg controls. This again suggests that GHR expression may be altered at transcriptional levels via expression of the GH transgene, although this change is opposite that found in the liver. The observation of low GH binding levels in GH-Tg mice that possess high GH levels and severe glomerulosclerosis indicates that the nephropathic effect of GH is ligand (GH) specific. In other words, mice that overexpress GH may develop kidney lesion independent of their low kidney GHR levels. Also, there may be a low threshold value at which kidney GHRs are sufficient to mediate the nephropathic effect of GH.

Our findings of down-regulation of kidney GH binding as well as kidney GHR mRNA by overexpression of GH is opposite earlier reports of up-regulation of hepatic GHR by GH (4, 5, 31). This difference suggests that GHR regulation is tissue specific. One possible mechanism for this observation could be at the transcriptional level. To date, five different 5'-untranslated regions have been reported for the GHR/GHBP gene in different rat tissues (34), and eight have been identified for the hGHR gene (11, 35). It is believed that different 5'-untranslated variants are the results of different promoters acting in a tissue-specific manner (34). It is possible that different tissue-specific transcriptional regulatory sequences are contained in these 5'-untranslated regions, which may help to explain the different GHR expression levels seen in the liver and kidney in response to the same hormone.

Similar to what was found in the liver, STZ treatment resulted in a significant decrease in both GH-specific binding and GHR mRNA levels in the kidneys of GHA-Tg and NTg mice, but not in bGH-Tg mice. The low GHR levels in combination with the blockage of endogenous GH action in GHA-Tg mice may explain the protection of the kidneys from diabetic lesions in these mice.

In contrast to GHR mRNA levels, significantly elevated kidney GHBP mRNA levels were found in GH-Tg mice. The role of kidney GHBP is unknown. The coexistence of the elevated kidney GHBP with glomerulosclerosis raises a possibility that GHBP may mediate the nephropathic effect of GH in the kidney. Reduction of kidney GHBP mRNA after STZ treatment in these mice suggests a possible influence of insulin on kidney GHBP expression.

In this study, liver GHR binding levels were high in GH-Tg mice, in which insulin and GH levels are relatively high. This suggests that high insulin levels may contribute to the up-regulation of hepatic GH binding. However, fasting blood glucose and kidney GH binding levels in these mice did not significantly change. This may suggest a tissue-specific manner for insulin’s effects.

In summary, mouse GHR expression in the liver and kidney appears to be regulated by GH and insulin. Regulation of GHR expression and function by either GH or insulin was tissue specific. The consistently decreased levels of GH binding in diabetic GHA-Tg mice suggest that a GHA may protect the kidneys from diabetic nephropathy via two routes, i.e. 1) by blockage of the action of endogenous GH, and 2) through down-regulation of GHR.


   Acknowledgments
 
The authors thank Dr. Yihua Zhou for supplying the plasmid pPCRII-mGHRI, and Dr. Karen Coschigano for reviewing this manuscript.


   Footnotes
 
1 This work and J.J.K. were supported in part by the Ohio State’s Eminent Scholar Program, which includes a grant by Milton and Lawrence Goll, and by grants from Sensus Corporation and the Juvenile Diabetes International Foundation (no. 195061). Back

Received August 15, 1996.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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