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Endocrinology, doi:10.1210/en.2007-1761
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Endocrinology Vol. 149, No. 9 4382-4386
Copyright © 2008 by The Endocrine Society

Partial Deletion of Pten in the Hypothalamus Leads to Growth Defects that Cannot be Rescued by Exogenous Growth Hormone

Diana Choi, Kinh-Tung T. Nguyen, Linyuan Wang, Stephanie A. Schroer, Akira Suzuki, Tak W. Mak and Minna Woo

Institute of Medical Science (D.C., K.-T.T.N., M.W.) and Department of Medical Biophysics (L.W., S.A.S., T.W.M., M.W.), Ontario Cancer Institute, and The Advanced Medical Discovery Institute (T.W.M.), University of Toronto, Toronto, Ontario, Canada M5G 2M9; Department of Molecular Biology (A.S.), Akita University, School of Medicine, Akita 10-8543, Japan; and Department of Medicine and Keenan Research Centre in the Li Ka Shing Knowledge Institute (M.W.), St. Michael’s Hospital, Toronto, Ontario, Canada M5B 1W8

Address all correspondence and requests for reprints to: Minna Woo, Ontario Cancer Institute, 610 University Avenue, Room 8-113, Toronto, Ontario, Canada M5G 2M9. E-mail: mwoo{at}uhnres.utoronto.ca.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The GH/IGF-I axis plays a critical role in mammalian body growth. GH is secreted by the anterior pituitary, and its actions are primarily mediated by IGF-I that is secreted by the liver and other tissues. Local and circulating IGF-I action is largely mediated by the phosphoinositide 3-kinase signaling pathway, and phosphatase with tensin homology (PTEN) is a potent negative regulator of this pathway. Here we show that RIPcre+Ptenfl/fl mice, which exhibit PTEN deletion in insulin-transcribing neurons of the hypothalamus in addition to pancreatic β-cells, result in a small-body phenotype that is associated with an unexpected increase in serum IGF-I levels. We tested whether exogenous GH can override the growth defect in RIPcre+Ptenfl/fl mice. Our results showed no significant difference in their growth between the RIPcre+Ptenfl/fl mice injected with GH or vehicle. Together, PTEN in the hypothalamic insulin-transcribing neurons plays an essential role in body size determination, and systemic GH cannot overcome the growth defect in these mice.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
BODY GROWTH IS a complex phenomenon involving regulatory mechanisms largely influenced by GH (1). GH is a polypeptide hormone that stimulates cell replication in mammals, and it is synthesized, stored, and secreted by somatotrophs in the anterior pituitary gland. Most of the GH effects on growth are mediated through the actions of IGF-I, which binds to IGF-I receptor (IGF-IR) on target tissues to promote cellular growth. Historically, IGF-I was discovered as a growth factor produced only by the liver under the regulatory control of pituitary GH. However, more recently, it has been demonstrated that IGF-I is expressed in almost all tissues (2, 3, 4). IGF-I is secreted primarily from the liver as an endocrine hormone but also exerts its action through paracrine or autocrine mechanisms within a given tissue (4).

Liver IGF-I is the main constituent of circulating IGF-I (5). IGF-I production and secretion are regulated by circulating GH and IGF-I levels in a negative feedback system (6). IGF-I bioactivity is also regulated by a family of proteins known as the IGF-binding proteins (IGFBPs), six of which have been identified (7). These high-affinity proteins can modulate IGF-I action. Most of the circulating IGF-I is associated with a complex consisting of IGFBP-3 and the acid-labile subunit. Once the ternary complex dissociates, the IGF-I is able to reach the target tissues and interact with its receptors (8).

The cellular response to IGF-I is mediated primarily by the IGF-IR. The IGF-IR is a member of the family of tyrosine kinase growth factor receptors and is highly homologous to the insulin receptor (IR), especially in the tyrosine kinase domain (9). Upon ligand binding to the extracellular region, the intrinsic tyrosine kinase domain of the receptor is activated. Downstream of the receptor tyrosine kinases is the phosphoinositide 3-kinase (PI3K) signal transduction pathway, which is characterized by the activation of PI3K, which phosphorylates the phosphatidylinositol-4,5-bisphosphate (PIP2) to generate the second messenger phosphatidylinositol-3,4,5-trisphosphate (PIP3). PIP3 in turn interacts with downstream targets, including the protein kinase B (PKB)/Akt, which mediates many of the effects on cell growth (10, 11). Phosphatase with tensin homology (PTEN) is a dual-specificity phosphatase that dephosphorylates PIP3 to PIP2 and thus is a potent negative regulator of the PI3K pathway (12, 13).

Attenuation of IGF-I/insulin signaling in the whole body leads to a small body size in primitive organisms, such as Caenorhabditis elegans or Drosophila melanogaster (14, 15) as well as in mammals (16, 17). Therefore, although it is clear that this signaling pathway plays a critical role in determining body size, which specific tissue signaling contributes to the body size outcome is less clear.

Here we show that enhancement of the PI3K signaling due to PTEN deletion in insulin-transcribing neurons in the hypothalamus is sufficient to control body size. We investigated whether systemic GH can override the control exhibited by insulin-transcribing neurons. Our results show that PI3K signaling in the hypothalamic insulin-transcribing neurons plays a critical role in body size determination that cannot be overcome by exogenous GH.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mouse protocol
RIPcre+Pten+/fl mice, expressing the Cre recombinase under the control of the rat insulin promoter (RIP), were generated as previously described (18). RIPcre+Pten+/fl mice were intercrossed to generate RIPcre+, Ptenfl/fl, Pten+/+, and Pten+/fl mice. Genotyping of the mice was performed by PCR amplification of ear clip DNA as previously described (19). Both male and female RIPcre+Ptenfl/fl mice were studied on a mixed 129J-C57BL/6 background with appropriate littermate controls. Both heterozygous (RIPcre+Pten+/fl) and wild-type (RIPcre+Pten+/+) mice were used as controls, and results from these mice were indistinguishable. Mice were maintained on a 12-h light, 12-h dark cycle with free access to water and standard irradiated rodent chow (5% fat; Harlan Teklad, Indianapolis, IN) and housed in pathogen-free barrier facilities at the central animal facility at the Ontario Cancer Institute (Toronto, Ontario, Canada). All animal experiments were approved by the Ontario Cancer Institute Animal Care Facility.

Growth measurement
Growth was determined by measuring body weight and body length (snout to anus) of mice.

Metabolic studies and hormone measurements
Two-week-old mice were not fasted because they were not weaned. All blood glucose levels were determined from tail venous blood with an automated glucose monitor (Precision Xtra; Abbott Diabetes Care, Inc., Alameda, CA). Blood was collected by cardiac puncture. Serum IGF-I and insulin levels were measured by RIA by the Mouse Metabolic Phenotyping Center, Hormone Analytical Subcore Unit (Vanderbilt University, Nashville, TN).

mRNA measurements
mRNA was extracted from the liver by TRIzol following the manufacturer’s protocol (Invitrogen, Toronto, Ontario, Canada) and treated with ribonuclease-free deoxyribonuclease (Invitrogen). Semiquantitative RT-PCR amplification was performed with a one-step RT-PCR kit (Invitrogen). IGFBP-1, IGFBP-3, and β-actin cDNA were amplified by PCR using specific primers. Densitometric analysis was performed using Image J software. To correct for differences in loading, we corrected densitometric values of IGFBP-1 and IGFBP-3 cDNAs with corresponding values of β-actin cDNA and calculated the IGFBP-1 to β-actin and IGFBP-3 to β-actin ratios.

Real-time PCR analysis
cDNA was synthesized using M-MLV reverse transcriptase kit (Invitrogen). IGF-I and GAPDH expressions were analyzed using SYBR Green PCR kit (Applied Biosystems Inc., Forster City, CA). Amplification reactions were performed in a final volume of 25 µl containing 12.5 µl of the SYBR Green PCR Master Mix, 0.25 µM of IGF-I-specific primers, and 2 µl 50-fold diluted cDNA solution. PCR was monitored in real-time using the ABI Prism 7900HT Real-time PCR System (Applied Biosystems). Experiments were performed in triplicate for each sample. IGF-I values were normalized with GAPDH values and expressed in arbitrary units relative to littermate control levels.

Western immunoblot analysis
Western immunoblot analysis was performed on protein lysates of hypothalamus, liver, and muscle samples as previously described (19). For the detection of IR and IGF-IR, 40 µg sample protein was loaded in each lane made up of 10% SDS-polyacrylamide gel and electroblotted into nitrocellulose membranes. Blots were blocked for 1 h in Tris-buffered saline with Tween 20 containing 5% nonfat dehydrated milk, followed by overnight incubation with rabbit polyclonal antibody against IR β-subunit and IGF-IR β-subunit (Santa Cruz Biotechnology, Santa Cruz, CA) diluted in Tris-buffered saline with Tween 20 containing 5% BSA. Western blot signal densities were analyzed using Image J software. IR and IGF-IR levels were normalized with {alpha}-tubulin levels and expressed in arbitrary units relative to littermate control levels.

GH treatment
RIPcre+Ptenfl/fl mice and their littermate controls were given sc injections of exogenous recombinant mouse GH (National Hormone and Pituitary Program, Torrance, CA) or vehicle using a regimen of increasing doses: 15 µg twice a day (first week) and 25 µg twice a day (second week). The choice of these dosages was made based on previously published data (20). The GH was diluted in 0.01 M NaHCO3 solution. Mice were injected starting at 1 wk of age everyday at 0900 and 2100 h for 2 wk. The weights and lengths of the mice were measured and recorded daily.

Statistical analysis
Data are presented as means ± SEM and were analyzed by independent-samples t test using the statistical software SPSS (version 11.0). P values ≤ 0.05 were accepted as statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Growth restriction in RIPcre+Ptenfl/fl mice
We and others have previously shown whole-body growth restriction in RIPcre+Ptenfl/fl mice compared with their controls (18, 21). In the present study, we focused on examining young mice (2 wk of age) with growth defects to better characterize and understand the role of PTEN in the hypothalamus that determines body size. The 2-wk-old RIPcre+Ptenfl/fl mice demonstrated proportionate whole-body growth restriction compared with their controls (Fig. 1Go, A and B). This was likely not due to a decrease in food intake because we observed milk-filled stomachs of similar sizes between mutant and control mice before weaning (Fig. 1CGo), and we also reported previously no difference in food intake in the immediate postweaning period (18).


Figure 1
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  		FIG. 1. RIPcre+Ptenfl/fl mice have postnatal growth retardation. Growth restriction was demonstrated by differences in weight (A) and length (B), measured from snout to anus, in mice at 2 wk of age (n ≥ 6 per genotype). *, P ≤ 0.05 for comparison between RIPcre+Ptenfl/fl and RIPcre+Pten+/+ or RIPcre+Pten+/fl. Error bars indicate SEM. C, No gross defect in food intake in mutant mice as shown by milk-filled stomachs in both genotypes. CON, RIPcre+Pten+/+ mice or RIPcre+Pten+/fl mice; –/–, RIPcre+Ptenfl/fl mice.

 
Serum levels of IGF-I and mRNA expression of liver IGF-I, IGFBP-1, and IGFBP-3
Given the importance of the GH/IGF-I axis in mammalian growth, we first examined systemic levels of IGF-I. Our previous study, which included RIPcre+Ptenfl/fl mice across a wide age range, showed an increasing trend of circulating IGF-I levels, which was statistically insignificant due to a small sample size (18). In the present study, we continue to observe higher serum IGF-I levels in the young RIPcre+Ptenfl/fl mice. The difference is statistically significant likely due to the more homogeneous age group (Fig. 2AGo).


Figure 2
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  		FIG. 2. RIPcre+Ptenfl/fl mice have increased serum IGF-I without altered liver IGF-I or IGFBP-1 or -3 transcript levels. A, Serum IGF-I was measured by ELISA (n ≥ 5 per genotype); B, real-time quantitative PCR quantification of IGF-I transcript levels in the liver (n ≥ 6 per genotype); C, semiquantitative RT-PCR for IGFBP-1 and IGFBP-3 transcript levels in the liver (n ≥ 6 per genotype). *, P ≤ 0.005. Error bars indicate SEM. CON, RIPcre+Pten+/+ mice or RIPcre+Pten+/fl mice; –/–, RIPcre+Ptenfl/fl mice.

 
Next, we tested whether the increased serum IGF-I levels in RIPcre+Ptenfl/fl mice corresponded to an increase in the production of IGF-I in the liver, because liver IGF-I is the major constituent of IGF-I in the systemic circulation. Surprisingly, we observed similar expression levels of liver IGF-I in RIPcre+Ptenfl/fl mice compared with control mice by real-time quantitative PCR analysis (Fig. 2BGo).

We also examined IGFBP-1 and -3 mRNA levels in the liver by RT-PCR to assess whether the small-body phenotype in RIPcre+Ptenfl/fl mice was due to the attenuation of IGF-I activity by IGFBPs. Our results show similar IGFBP-1 and IGFBP-3 mRNA levels in RIPcre+Ptenfl/fl and control mice (Fig. 2CGo), suggesting that the differences in the total levels of serum IGF-I are not due to changes in liver transcript levels of IGFBP-1 or IGFBP-3.

Serum insulin and blood glucose levels
We have previously shown that adult RIPcre+Ptenfl/fl mice had increased peripheral insulin sensitivity as shown by their decreased fasting serum insulin levels and more pronounced glucose-lowering effects of insulin during insulin tolerance tests (18). Indeed, the 2-wk-old RIPcre+Ptenfl/fl mice had significantly decreased serum insulin levels despite lower blood glucose levels compared with control mice (Fig. 3Go, A and B) similar to our observation in older RIPcre+Ptenfl/fl mice. These results suggest that even the young mutant mice have increased insulin sensitivity (18).


Figure 3
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  		FIG. 3. RIPcre+Ptenfl/fl mice have decreased serum insulin levels and blood glucose levels vs. controls. A, Serum insulin levels of RIPcre+Ptenfl/fl mice and controls measured by RIA (n ≥ 6 per genotype); B, blood glucose levels in RIPcre+Ptenfl/fl mice vs. control mice (n ≥ 6 per genotype). *, P ≤ 0.005. Error bars indicate SEM. CON, RIPcre+Pten+/+ mice or RIPcre+Pten+/fl mice; –/–, RIPcre+Ptenfl/fl mice.

 
IGF-IR and IR expression in central and peripheral tissues
We examined IGF-IR and IR expression in the hypothalamus and peripheral insulin target organs such as liver and muscle in control and RIPcre+Ptenfl/fl mice to assess whether the differences in receptor expression can account for the IGF-I or insulin actions in the periphery. Our results showed similar IGF-IR or IR expression between the RIPcre+Ptenfl/fl and RIPcre+Pten+/+ mice in all tissues (Fig. 4Go, A and B), suggesting that the growth restriction in RIPcre+Ptenfl/fl mice is likely not due to changes in IGF-IR or IR expression in these central and peripheral tissues.


Figure 4
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  		FIG. 4. Similar IR and IGF-IR expression in hypothalamus, liver, and muscle tissues as shown by Western blot analysis of protein lysates of tissues from RIPcre+Ptenfl/fl and control mice at 2 wk of age (n ≥ 4 per genotype). Blots were probed with IR, IGF-IR, and {alpha}-tubulin as loading control. Error bars indicate SEM. CON, RIPcre+Pten+/+ mice or RIPcre+Pten+/fl mice; –/–, RIPcre+Ptenfl/fl mice.

 
Exogenous GH treatment
To assess whether providing exogenous GH may be able to rescue growth in the RIPcre+Ptenfl/fl mice, we sc injected recombinant mouse GH into RIPcre+Ptenfl/fl and control mice twice daily for 2 wk starting at 1 wk of age. We showed the biological efficacy of the recombinant GH because RIPcre+Pten+/+ or RIPcre+Pten+/fl mice injected with GH exhibited enhanced growth in both weight (Fig. 5AGo) and length (Fig. 5BGo) compared with the control mice injected with vehicle. In contrast, there was no significant difference between the RIPcre+Ptenfl/fl mice injected with vehicle and those injected with GH. These findings indicate that the GH was biologically active but was unable to rescue the small-body-size phenotype caused by a partial deletion of PTEN in the hypothalamus.


Figure 5
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  		FIG. 5. Growth of RIPcre+Ptenfl/fl is restricted compared with that of RIPcre+Pten+/+ littermates in weight (A) from 1 wk of age onward in both exogenous GH and vehicle treatment groups and in length (B) measured at d 14 after injections (n ≥ 5 in each group). Error bars indicate SEM. *, P ≤ 0.005 between GH-injected and vehicle-injected controls.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present study was prompted by our previous finding of growth restriction in the RIPcre+Ptenfl/fl mice (18). The small-body phenotype exhibited by these mice is attributed to the PTEN deficiency in the hypothalamic insulin-transcribing cells rather than the pancreatic β-cells because mice with postnatal PTEN deletion only in the β-cells by the Cre recombinase under the control of the inducible Pdx-1 promoter do not exhibit growth defects (22). We have previously shown that PTEN deletion in insulin-transcribing neuronal cells increases PI3K signaling in these cells (18). As a result, we would have expected an increase in body size because more primitive organisms demonstrate enhanced whole-body growth with increased PI3K signaling (14, 15). Instead, the enhanced PI3K signaling in the hypothalamic insulin-transcribing cells in the RIPcre+Ptenfl/fl mice led to an unexpected small-body phenotype. Given the essential role of the GH/IGF-I axis in mammalian growth, we examined whether there were any defects in serum IGF-I levels in the 2-wk-old RIPcre+Ptenfl/fl mice. We found that total circulating IGF-I levels were paradoxically increased with no apparent transcriptional changes in the liver in RIPcre+Ptenfl/fl mice compared with controls. Interestingly, previous studies have shown that serum IGF-I levels per se do not determine body growth (23, 24). For example, mice with liver-specific IGF-I deletion resulting in a 75% decrease in total serum IGF-I have normal body size (24). Our results further support that neither systemic IGF-I levels nor liver IGF-I production per se determines body size.

The hypothalamus is a complex region of the brain consisting of a multitude of different neuronal cell types involved in various functions. Much research has started to uncover the specific biological roles of each of the neuronal subtypes within the hypothalamus. However, the population of insulin-transcribing hypothalamic neurons remains as yet not fully characterized. What we have shown is that PTEN deletion in this particular neuronal population appears to have a negative impact on body growth, although the precise mechanisms are still unclear.

Here we showed using a genetic approach that IGF-I/insulin action in insulin-transcribing hypothalamic cells alone can determine whole-body size. We attempted to rescue the growth defect in RIPcre+Ptenfl/fl mice by administering exogenous GH. Because only insulin-transcribing cells were targeted in this model, peripheral GH and IGF-I tissue responses were expected to be normal. However, despite unmanipulated IGF-I signaling in the remainder of the body, exogenous GH was not able to rescue the small-body phenotype in RIPcre+Ptenfl/fl mice. This observation emphasizes the potency of this small population of hypothalamic neurons in its ability to regulate whole-body growth.

Although IGF-I is traditionally known as the downstream effector of GH, insulin has also been shown to play a role in body growth. This is exemplified in mice with IR deletion in the brain, liver, and pancreas, which exhibited severe growth restriction (16). Interestingly, our RIPcre+Ptenfl/fl mice exhibit low insulin levels, which may have a causal role in the growth defect observed in our mice.

The mechanisms behind the increased total IGF-I levels in the circulation in RIPcre+Ptenfl/fl mice are not entirely clear. Insulin sensitivity has been shown to positively correlate with systemic IGF-I levels (25). Thus, the serum IGF-I levels may simply be a reflection of the associated increase in insulin sensitivity in our PTEN mutant mice, as demonstrated by low fasting insulin levels and enhanced insulin effects during insulin tolerance tests (18).

In summary, we have identified that hypothalamic insulin-transcribing neurons play an essential role in determining body size. Despite PTEN deletion only in the insulin-transcribing cells, exogenous GH was unable to overcome the growth restriction exhibited by these RIPcre+Ptenfl/fl mice. Our findings highlight the critical importance of IGF-I/insulin signaling in insulin-transcribing hypothalamic cells in body size determination.


   Acknowledgments
 
We thank Sung Ah Jun for technical assistance.


   Footnotes
 
This work was supported by grants to M.W. from the Canadian Institutes of Health Research (CIHR) and the Canadian Diabetes Association. M.W. is a CIHR New Investigator. D.C. is supported by the Banting and Best Diabetes Centre-Novo Nordisk Studentship and the University of Toronto Open Fellowship.

Disclosure Statement: The authors have nothing to disclose.

First Published Online May 22, 2008

Abbreviations: IGFBP, IGF-binding protein; IGF-IR, IGF-I receptor; IR, insulin receptor; PI3K, phosphoinositide 3-kinase; PIP2, phosphatidylinositol-4,5-bisphosphate; PIP3, phosphatidylinositol-3,4,5-trisphosphate; PTEN, phosphatase with tensin homology; RIP, rat insulin promoter.

Received December 19, 2007.

Accepted for publication May 12, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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