Systemic and acute administration of parathyroid hormone-related peptide(1–36) stimulates endogenous beta cell proliferation while preserving function in adult mice
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- Williams, K., Abanquah, D., Joshi-Gokhale, S. et al. Diabetologia (2011) 54: 2867. doi:10.1007/s00125-011-2260-z
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A major focus in the treatment of diabetes is to identify factors that stimulate endogenous beta cell growth while preserving function. The first 36 amino acids of parathyroid hormone-related protein (PTHrP) are sufficient to enhance proliferation and function in rodent and human beta cells in vitro. This study examined whether acute and systemic administration of the amino-terminal PTHrP(1–36) peptide can achieve similar effects in rodent beta cells in vivo.
Adult male mice were injected with 40, 80 or 160 μg of PTHrP(1–36) per kg body weight or with vehicle for 25 days. Glucose and beta cell homeostasis, as well as expression of differentiation markers and cell cycle genes were analysed.
All three doses of PTHrP(1–36) significantly enhanced beta cell proliferation in vivo at day 25, with 160 μg/kg PTHrP(1–36) increasing proliferation as early as day 5. Importantly, the two higher doses of PTHrP(1–36) caused a significant 30% expansion of beta cell mass, with a short-term improvement in glucose tolerance. PTHrP(1–36) did not cause hypercalcaemia, or change islet number, beta cell size, beta cell death or expression of differentiation markers. Analysis of islet G1/S cell cycle proteins revealed that chronic overabundance of PTHrP(1–139) in the beta cell significantly increased the cell cycle activator cyclin D2 and decreased levels of cyclin-dependent kinase 4 inhibitor (p16Ink4a [Ink4a also known as Cdkn2a]), but acute treatment with PTHrP(1–36) did not.
Acute and systemic administration of PTHrP(1–36) increases rodent beta cell proliferation and mass without negatively affecting function or survival. These findings highlight the future potential therapeutic effectiveness of this peptide under diabetes-related pathophysiological conditions.
KeywordsAmino-terminal parathyroid hormone-related protein Beta cell proliferation Cyclin D2 In vivo p16INK4a
Inhibitor of kinase
Intraperitoneal glucose tolerance test
v-Maf musculoaponeurotic fibrosarcoma oncogene family
Cyclin-dependent kinase 4 inhibitor
Peptide at 40 μg/kg body weight
Peptide at 80 μg/kg body weight
Peptide at 160 μg/kg body weight
Proliferating cell nuclear antigen
Protein kinase C
Parathyroid hormone-related protein
Rat insulin promoter
Diabetes results from a loss of functional beta cells. Therefore a major goal in the treatment of diabetes is to find ways to induce endogenous beta cell regeneration without negatively affecting beta cell function. Proliferation is an important mechanism for normal postnatal growth and for regeneration after specific injury in rodent beta cells [1, 2, 3]. Importantly, beta cell proliferation has also been documented in humans, albeit less frequently [4, 5], providing a rational basis to search for molecules that can acutely enhance pancreatic beta cell replication and/or function in vivo. We examined the potential of parathyroid hormone (PTH)-related protein (PTHrP) in this context.
PTHrP is a 139 (rodent) or 173 (human) amino acid protein, which was first discovered as a tumour peptide that causes humoral hypercalcaemia of malignancy. It has since been shown to be produced in every tissue in the body, both during development and in adult life. Its receptor, the G-protein-coupled seven transmembrane PTH receptor-1 (PTHR1), which recognises the amino-terminal regions of PTHrP and its related member PTH, is also produced in the same or adjacent cells as PTHrP . PTHrP/PTHR1 signalling is critical for the development and differentiation of numerous tissues, and for the sustenance of life itself, as mutations in this pathway lead to embryonic/neonatal death in rodents and humans [6, 7, 8, 9].
PTHrP is a promising candidate for the promotion of endogenous beta cell proliferation for several reasons. PTHrP and its receptor, PTHR1, are both normally produced in rodent and human beta cells [10, 11, 12, 13, 14]. Indeed, several studies have now shown that PTHrP can enhance beta cell function and proliferation in vitro in human and rodent beta cells [13, 14, 15, 16, 17]. Of particular relevance is the finding that the first 36 amino acids of PTHrP are sufficient to manifest its salutary effects on beta cell proliferation and function in vitro [13, 14, 15, 16, 17]. Furthermore, we have found that full-length PTHrP also enhances beta cell growth and function in vivo, when produced under the rat insulin promoter (RIP) in transgenic mice [12, 15, 18, 19]. RIP-PTHrP (also known as Pthlh) transgenic mice are hypoglycaemic and hyperinsulinaemic, and have improved glucose tolerance, increased beta cell proliferation and enhanced beta cell mass. Finally, PTHrP(1–36) peptide is currently being used for its anabolic effects on bone in human clinical studies, demonstrating its probable safety as a potential therapeutic agent [20, 21].
Based on these studies, we hypothesised that PTHrP(1–36) has the potential to enhance endogenous beta cell proliferation and/or function in vivo, when administered acutely in mice. Indeed, as demonstrated here, PTHrP(1–36) augmented beta cell proliferation as early as the 5th day of treatment, continuing to do so until day 25. This resulted in a significant 30% increase in beta cell mass with the two higher doses of PTHrP(1–36), without negatively affecting plasma glucose levels or beta cell differentiation, size and turnover. Although PTHrP(1–36) significantly improved glucose tolerance at the beginning, glucose tolerance returned completely to baseline levels by the end of the treatment, suggesting a potential desensitisation of the functional effect of PTHrP as the treatment progresses. Analysis of the cell cycle molecules regulated by PTHrP showed a significant increase in abundance of the activator cyclin D2 and a decrease in the cyclin-dependent kinase 4 inhibitor (p16Ink4a [Ink4a also known as Cdkn2a]), in transgenic mouse islets overproducing PTHrP(1–139) in the beta cell; this did not occur in the islets of mice treated acutely and systemically with PTHrP(1–36). Thus, systemic administration of PTHrP(1–36) peptide can acutely induce beta cell replication and mass with short-term improvement in glucose tolerance in normal adult mice, identifying this peptide as a potential agent for beta cell regeneration.
Balb/C male mice were injected s.c. with PTHrP(1–36) peptide at 40 (P-40 mice), 80 (P-80 mice) or 160 (P-160 mice) μg/kg body weight or vehicle (control). Injections were once daily for 5 days per week for 25 days, after which pancreases or islets were removed. PTHrP(1–36) was synthesised and assayed for activity as described previously [20, 21]. RIP-PTHrP transgenic mice bred on to a CD1 background were generated as described previously . All studies were performed with the approval of, and in accordance with, guidelines established by the University of Pittsburgh Institutional Animal Care and Use Committee. For further details on experimental design, see electronic supplementary material (ESM) Methods.
Glucose homeostasis and islet isolation
Blood glucose was measured 5 days per week for 25 days using a portable glucometer (Medisense, Bedford, MA, USA). An intraperitoneal glucose tolerance test (IPGTT) was performed on days 9 and 23 in mice that had been fasted for 16 to 18 h. These mice were injected i.p. with 2 g glucose per kg body weight [12, 15]. Islets that had been isolated for gene expression analysis from control and P-160 mice at day 5 or 25, and from 3- to 6-month-old RIP-PTHrP transgenic and normal mice were immediately handpicked in RPMI medium with 5.5 mmol/l glucose and 1% (vol./vol.) FBS .
Plasma was obtained from retro-orbital bleeds on days 8 and 25, and plasma calcium measured using a blood gas analyser (Rapid Lab 348; Bayer, Leverkusen, Germany).
Pancreatic histomorphometry and beta cell homeostasis
Histomorphometric analysis was performed on two to three insulin-stained pancreatic sections per animal to quantify the number of islets or ratio of beta cell area to pancreatic area using the Image J program (NIH) . Beta cell size, death and proliferation were quantified respectively as: (1) insulin-positive area per total number of insulin-positive cells (728 ± 36 beta cells/mouse); (2) percentage of condensed beta cell nuclei after co-staining for insulin and propidium iodide , or TUNEL (Promega, Madison, WI, USA) (1,043 ± 79 or 1,298 ± 174 beta cells/mouse); and (3) percentage of BrdU-positive (Amersham Pharmacia Biotech, Piscataway, NJ, USA) or proliferating cell nuclear antigen (PCNA)-positive (Santa Cruz Biotechnology, Santa Cruz, CA, USA) beta cells (1,538 ± 54 beta cells/mouse). Details on above, see also ESM Methods
RNA and protein analysis
RNA from islets of control and P-160 mice at day 25, or of 3- to 6-month-old RIP-PTHrP transgenic and normal mice was processed and analysed on a real-time PCR system (ABI 7300; Life Technologies, Carlsbad, CA, USA). Whole-islet extracts were analysed by immunoblot and quantitative densitometry was performed using the Image J program . Primers, primary antibodies and additional details are in ESM Table 1 and ESM Methods.
Data are expressed as the mean±SE. Statistical significance, considered to be given at p ≤ 0.05, was determined by unpaired two-tailed Student’s t test or a one-way ANOVA with Dunnett’s post hoc test.
Effect of PTHrP(1–36) administration on body weight and plasma calcium
PTHrP(1–36) increased beta cell mass without affecting islet number, beta cell size or beta cell death
All three doses of PTHrP(1–36) enhanced beta cell proliferation
PTHrP(1–36) stimulates beta cell proliferation after only 5 days of treatment
Effect of PTHrP(1–36) on glucose homeostasis and beta cell differentiation
To determine whether PTHrP(1–36)-induced beta cell proliferation caused changes in expression of genes regulating the function or differentiation of the beta cell, we examined the transcriptional profile of these genes in islets isolated from control or P-160 mice at day 25. There was no significant change in expression of genes encoding islet hormones (insulin, somatostatin), glucose sensors (Glut2, glucokinase, Kir6.2) or transcription factors important for beta cell differentiation and function (pancreatic and duodenal homeobox 1 [Pdx1], v-maf musculoaponeurotic fibrosarcoma oncogene family [Maf], protein A [MafA], Maf protein B, neurogenic differentiation[Neurod], NK6 homeobox 1 [Nkx6.1]) (Fig. 6e). This suggests that the enhanced beta cell proliferation induced by PTHrP(1–36) treatment does not negatively affect beta cell function or differentiation.
PTHrP modulates expression of the G1/S cell cycle regulators, cyclin D2 and p16Ink4a, in islets
This study demonstrates that acute and systemic administration of amino-terminal PTHrP(1–36) peptide in rodents can stimulate beta cell proliferation and enhance beta cell mass in vivo, without negatively affecting beta cell function or survival. Previous studies have shown that transgenic production of the full-length PTHrP(1–139) in beta cells of RIP-PTHrP transgenic mice results in an increase in beta cell mass, proliferation and islet number, and in improved glucose homeostasis in these mice [12, 15, 18, 19]. In addition, in vitro studies have shown that the amino-terminal PTHrP(1–36) peptide is sufficient to enhance not only proliferation, in rodent insulinoma cell lines and primary human beta cells, but also function, increasing insulin mRNA and protein, and augmenting glucose-stimulated insulin secretion [13, 14, 15, 16, 17]. Based on these data, we examined whether PTHrP(1–36) peptide could induce similar effects in vivo, as a first step towards determining its therapeutic potential for the treatment of diabetes.
The major novel aspects of this study compared with previous in vivo studies using RIP-PTHrP transgenic mice are: (1) PTHrP is administered systemically rather than being expressed endogenously in the beta cell; (2) PTHrP treatment is acute, for a short time, rather than via continuous expression in the beta cell; (3) PTHrP is given to adult mice rather than being produced from conception of the beta cell in transgenic mice; and (4) only the amino-terminal PTHrP(1–36) peptide is being administered, rather than the entire PTHrP(1–139) protein being produced in transgenic mice.
Our choice of doses and duration of PTHrP(1–36) treatment was based on previous work showing that amino-terminal PTHrP and PTH have anabolic effects on bone using a similar treatment regimen [22, 23]. Indeed, we found that systemic administration of all three PTHrP(1–36) doses increased endogenous beta cell proliferation at 4 weeks, as well as in the early phase of treatment for the highest 160 μg/kg dose. Importantly, the two higher doses of PTHrP(1–36) caused a significant increase in beta cell mass. RIP-PTHrP transgenic mice exhibit a progressive increase in their beta cell mass, with no significant change at 1 week, and a significant twofold increase by 2 to 3 months, which further increases to threefold at 1 year . It is therefore likely that prolonged systemic administration of PTHrP(1–36) beyond 1 month could result in a similar progressive increase in beta cell mass, an issue that will be addressed in future studies. The enhanced beta cell mass in the PTHrP(1–36)-treated mice is likely to have been due to the increase in beta cell replication and not to changes in beta cell size, turnover or islet number, as none of these variables changed significantly in these mice. This was somewhat different from our observations in RIP-PTHrP transgenic mice, which showed a significant twofold increase in islet number at 2 months of age [18, 19]. This difference could stem from the fact that in the transgenic mice, PTHrP is probably produced early in embryonic life when the insulin promoter is first activated in the beta cell and, being a secretory protein, could potentially influence differentiation of neighbouring progenitor cells. In the current study, PTHrP(1–36) was injected once a day into adult mice and is therefore less likely to have had effects on islet development or number.
Although some studies have shown that PTHrP stimulates beta cell proliferation [13, 15, 16], very little is known about the effect of PTHrP on regulation of the G1/S cell cycle molecules at the transcriptional and protein levels in rodent islets. We found a significant increase in cyclin D2 protein and a decrease in the inhibitor p16Ink4a protein, in islets from RIP-PTHrP transgenic mice with overproduction of PTHrP(1–139) in beta cells vs normal littermates. PTHrP has been shown to modulate a number of cell cycle molecules. Thus it inhibits production of the cyclin-dependent kinase-interacting protein/kinase-inhibitory protein and inhibitor of kinase (INK) family of cell cycle inhibitors that includes p15Ink4b (Ink4b also known as Cdkn2b), p27Kip1 and p57Kip2, as well as inducing transcription of cyclin D1 in different cell types in which it enhances proliferation, including chondrocytes, early osteoblastic cells and vascular smooth muscle cells [24, 25, 26, 27, 28]. Surprisingly, the increase in cyclin D2 and decrease in p16Ink4a abundance were not observed in islets isolated from P-160 mice after 5 days of PTHrP(1–36) treatment relative to control mice. This differential regulation of the cell cycle molecules in PTHrP(1–139) transgenic vs PTHrP(1–36)-treated islets could be the result of several factors including the following: (1) the effect of chronic PTHrP production vs acute treatment; (2) the effect of full-length PTHrP(1–139) and/or other PTHrP fragments vs only the amino-terminal PTHrP(1–36) peptide; (3) the effect of endogenous production vs exogenous treatment; and (4) possible local variations in effective concentrations of the protein. A recent report that is relevant to our findings found that mice lacking the nuclear localisation sequence and C-terminus of PTHrP (amino acids 85–139), but producing the amino-terminus PTHrP peptide showed decreased levels of cyclin D and increased levels of p16Ink4a in bone cells and embryonic fibroblasts , implying that the C-terminus region of PTHrP is required for the regulation of these cell cycle molecules. We observed the mirror effect of this in beta cells overproducing full-length PTHrP in vivo, but not in islets treated with only the amino-terminal PTHrP(1–36) peptide, again suggesting that the C-terminus region of PTHrP is important in the regulation of cyclin D2 and p16Ink4a. The question of whether the proliferative effects of PTHrP on rodent beta cells are mediated through the observed increase in cyclin D2 and/or decrease in p16Ink4a needs further verification.
RIP-PTHrP transgenic mice display hypoglycaemia and hyperinsulinaemia under fasting and non-fasting conditions, as well as improved glucose tolerance [15, 18, 19]. Systemic and acute administration of PTHrP(1–36) for 25 days did not alter plasma glucose in this study. However, there was a differential effect on glucose tolerance in the PTHrP-treated mice during the early vs the late phase of treatment. The significant improvement in glucose tolerance seen with the two higher doses of PTHrP(1–36) at day 9 was not observed at day 23, despite a 30% increase in beta cell mass in the P-80 and P-160 mice. The return of glucose tolerance to baseline values in these mice at day 23 is unlikely to be due to a negative impact of PTHrP(1–36) on beta cell function, as several studies have shown that PTHrP(1–36) increases insulin expression and enhances insulin secretion in rodent and human beta cells, similarly to full-length PTHrP [13, 15, 16, 17]. Thus, the short-term improvement in IPGTT vs the long-lasting effect on proliferation is more likely to be due to a signal-specific differential desensitisation/downregulation of the PTHR1. PTHrP is known to enhance beta cell function and proliferation through two distinct signalling pathways, namely activation of cyclic AMP with inhibition of the c-Jun N-terminal kinase pathway , and activation of the phosphatidylinositol 3-kinase and atypical protein kinase C (PKC) ζ pathway  respectively. In this context, there is evidence in osteoblasts that the desensitisation of PTHR1 is signalling pathway-specific, with a more pronounced effect on the cAMP/protein kinase-A pathway than on the phospholipase-C/PKC pathway . Similarly, it is possible that the beta cell also displays preferential attenuation of the cAMP, but not of the atypical PKCζ signalling pathway downstream of the PTHR1, leading to more short-lived effects of PTHrP(1–36) on glucose homeostasis vs beta cell proliferation. Importantly, there was no overall negative impact of PTHrP(1–36) treatment on glucose homeostasis in these mice.
The current studies raise numerous interesting questions. What would be the effect of higher doses of PTHrP(1–36) injected for varying durations on glucose and beta cell homeostasis? What would be the effect of treating mice with PTH instead of PTHrP, or with mutant peptides that activate specific signalling pathways ? It seems plausible that increasing the dose and/or duration of PTHrP(1–36) treatment would be likely to further enhance beta cell growth, based on the progressive increase in beta cell mass observed with age in RIP-PTHrP transgenic mice . However, it is possible that long-term treatment (for 3–6 months) with PTHrP(1–36) injected on a daily basis may not induce any further increase in beta cell mass, similarly to its effects in bone, where intermittent treatment is anabolic, but continuous or longer term treatment is catabolic or does not cause further increases in bone mass [22, 32]. Based on this, it may be more beneficial to treat mice less frequently (1–2 times per week) on a long-term basis to induce further increases in beta cell mass and perhaps improve glucose tolerance.
Another question is whether long-term systemic PTHrP(1–36) treatment could cause tumours or hypercalcaemia in mice? The doses of PTHrP(1–36) in this study did not cause hypercalcaemia over 25 days, nor did daily injections of PTHrP(1–36) in rats for 6 months  or overabundance of PTHrP in the beta cells of RIP-PTHrP transgenic mice . Furthermore, we have not detected insulinomas in RIP-PTHrP mice even after life-long (~2 years) production of PTHrP in their beta cells (R. C. Vasavada, unpublished observations). However, treatment duration and dose of the related PTH(1–34) peptide were found to be important determinants in the induction of bone tumours in rats . Therefore, it is critical to find the right dose and duration of PTHrP(1–36) treatment to balance the positive effects of PTHrP on the beta cell and its potential negative systemic effects.
While a number of growth factors, signalling and cell cycle molecules have the capacity to enhance rodent beta cell proliferation when expressed transgenically in the beta cell in vivo [35, 36, 37], only very few have this effect when administered systemically and acutely in vivo in non-diabetic rodents. In this regard, glucagon-like polypeptide-1 (GLP-1) and its analogues, as well as prolactin constitute two peptide families that induce rodent beta cell proliferation in vivo either when injected or infused continuously [38, 39, 40]. GLP-1 and its analogues are currently commercially available drugs for the treatment of diabetes [41, 42]. Like GLP-1, PTHrP(1–36) (1) systemically enhances endogenous rodent beta cell proliferation and mass, (2) acts through a seven transmembrane G-protein-coupled receptor activating many signalling pathways including the cyclic AMP pathway , (3) is safe to use in humans as demonstrated through clinical trials of its use in the treatment of osteoporosis [20, 21] and (4) therefore has the potential to be used in the future treatment of diabetes. Other growth factors such as gastrin, epidermal growth factor and islet neogenesis-associated protein have also been shown to increase beta cell mass when administered systemically in rodents [43, 44, 45, 46]. However, these growth factors increase beta cell mass through differentiation of new beta cells from non-beta cells, rather than by enhancing proliferation of pre-existing beta cells, as observed in our study. Therefore, it would be interesting to examine the effects of systemically administering PTHrP(1–36) in combination with one or more of these other growth factors to determine whether we can further augment beta cell mass through activation of differentiation and proliferation of beta cells.
In conclusion, this study clearly shows that the amino-terminal PTHrP(1–36) peptide is capable of enhancing rodent beta cell proliferation and mass in vivo, without negatively affecting beta cell function or survival, when administered acutely and systemically in normal adult mice. Future studies will determine whether this peptide can be used to induce endogenous beta cell regeneration in models of type 1 and type 2 diabetes or obesity. Given that clinical studies are currently ongoing to determine the ideal dose and treatment regimen of this peptide for the treatment of osteoporosis [20, 21], there is a good chance that PTHrP(1–36) could be a promising and safe therapeutic agent for diabetes.
This work was supported by grants from the National Institutes of Health (DK078060 and DK072264) and the Juvenile Diabetes Research Foundation (1-2008-46) to R.C. Vasavada. We are grateful to L. Alonso, N. Taesch, I. Cozar-Castellano, S. Velazquez-Garcia and S. Ernst for sharing their expertise and for their critical input. Thanks also to N. Vasavada for his assistance with the statistical analysis, and to C. Chin, D. Sipula, T. Rosa and S. Valle for superb technical assistance.
Each of the co-authors contributed to the conception and design, analysis and interpretation of data, and drafting and/or revision of the manuscript, as well as approving the final version to be published.
Duality of interest
A.F. Stewart is a member of Osteotrophin LLC. The other authors declare that there is no duality of interest associated with this manuscript.