Amylin exerts osteogenic actions with different efficacy depending on the diabetic status

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Abstract

Amylin displays osteogenic features, but its role in diabetic osteopenia is unclear. We examined the possible osteogenic action of amylin infusion for 3 days into fructose-induced insulin-resistant (IR) and streptozotocin-induced type 2 diabetic (T2D) and normal (N) rats. Amylin failed to affect glycaemia or parathyroid hormone levels in any group, but reduced hyperinsulinemia in IR rats. In N rats, amylin increased bone formation rate and reduced osteoclast surface and erosive surface in the femoral metaphysis, and increased osteoprotegerin (OPG)/receptor activator of NFκB ligand (RANKL) mRNA ratio in the tibia. In T2D rats, amylin normalized trabecular structure parameters and increased osteoblast number and osteocalcin (OC) expression in long bones. In contrast, in IR rats, no apparent osteogenic effect of amylin in the femur was observed, although both OC and OPG/RANKL ratio were increased in the tibia. Our findings demonstrate a different osteogenic efficacy of amylin in two diabetic settings.

Highlights

Amylin can exert osteogenic effects in type 2 diabetic states. ► The efficacy of amylin to promote bone accrual differs in distinct diabetic settings. ► Insulin-resistance hampers the bone anabolic action of amylin.

Introduction

Amylin is a hormone synthesized by the pancreatic islet β-cell, from which it is released together with insulin in response to food intake and other stimuli (Kahn et al., 1990, Novials et al., 1993). Amylin regulates fuel metabolism in association with other metabolic, endocrine and neural influences (Cooper, 1994). This hormone shows the characteristics of a peripheral satiety signal (Barth et al., 2003, Lutz, 2005, Riediger et al., 2004), primarily acting at the hindbrain and the rostral central nervous system (Creutzfeldt, 2001, Young et al., 1996), after amylin binding to specific receptors in the area postrema (Christopoulos et al., 1999). Amylin decreases body weight gain and reduces adiposity (Roth et al., 2008). In addition, recent in vitro studies also indicate that amylin can restore the altered glucose homeostasis in target tissues such as adipose tissue and liver in states of insulin-resistance and type 2 diabetes mellitus (Moreno et al., 2011). On the other hand, an impaired amylin regulation appears to occur associated with hypertension in the setting of the metabolic syndrome (Kailasam et al., 2000). In fact, amylin analogs are now envisioned as putative therapeutic agents not only in obesity (Dunican et al., 2010) but also in obesity-related syndromes such as diabetes mellitus (Lebovitz, 2010, Osaka et al., 2008).

As mentioned above, amylin is co-secreted with insulin after a meal. It is well known that insulin as well as other hormones, including those of incretin character, which are postpandrially released, can act as anabolic stimulus to the skeleton (Clowes et al., 2005). In this scenario, the concerted action of these physiological factors together with diet-supplied substrates would positively contribute to bone formation. Therefore, it makes sense that amylin might act in concert with other postprandial factors in the physiological control of bone maintenance. Only a few studies have yet been performed to address this hypothesis. It was shown that s.c. injection of amylin at nM concentration into mouse calvariae for 5 days increased osteoblast numbers and osteoblastic activity; meanwhile, amylin inhibited osteoclast-mediated bone resorption (Cornish et al., 1995, Cornish et al., 1998b). Moreover, systemic injection of amylin into adult normal mice increased bone mass (Cornish et al., 1998a). Several in vitro reports have shown that amylin stimulates osteoblastic cell proliferation (Cornish et al., 1995, Cornish et al., 1998b, Villa et al., 1997), although some conflicting results have also been published in this regard (Ellegaard et al., 2010).

Amylin mimics many if not all the actions of glucagon-like peptide (GLP-1), an incretin with both insulinotropic and insulin-independent antidiabetic properties, affecting glucose and energy metabolism (Creutzfeldt, 2001, Sancho et al., 2005, Valverde et al., 1994). However, recent studies performed in normal and type 1 diabetic (T1D) patients (Asmar et al., 2010), and also in a nonhuman primate model (Bello et al., 2010), have suggested that the common effects of GLP-1 and amylin on gastric emptying, food intake and glucagon release are exerted in an independent manner from each other, and point to the potential advantage for the combined GLP-1 and amylin therapy (see Roth et al., 2012 for review).

Recently, continuous infusion for 3 days of either GLP-1 or its analog exendin-4 into normal rats was shown to have a dual effect on bone turnover: inhibiting bone resorption through an increased osteoprotegerin (OPG)/receptor activator of NFκB ligand (RANKL) ratio, and increasing bone formation as suggested by an augmented expression of osteocalcin (OC) in the tibia (Nuche-Berenguer et al., 2009, Nuche-Berenguer et al., 2010). Moreover, these peptides were able to improve the deleterious trabecular structure in the long bones using well characterized insulin resistant (IR) or type 2 diabetic (T2D) rat models (Nuche-Berenguer et al., 2009, Nuche-Berenguer et al., 2010). Thus, the question arises as to whether amylin might be as efficient as GLP-1 at correcting the altered bone quality in the setting of osteopenia as occurs in the latter diabetic states (Blakytny et al., 2011). In spite of the scanty experimental evidence in this respect, such assumption has recently been proposed for some chemically modified analogs of amylin (Kowalczyk et al., 2012). In view of these considerations, in the present study we aimed to unravel the putative osteogenic action of amylin in diabetic conditions.

Section snippets

Reagents

Rat amylin and insulin (Bachem AG, Bubendorf, Switzerland); streptozotocin (STZ), demeclocycline, and TRI Reagent™ for RNA isolation (Sigma–Aldrich, St. Louis, MO, USA); high-capacity cDNA reverse transcription kit and Taqman Universal PCR master mix as well as TaqMan probes for rat osteocalcin (OC, Rn00566386_g1), osteoprotegerin (OPG, Rn00563499_m1), receptor activator of NF-κB ligand (RANKL, Rn00569289_m1), and 18S (4319413E), from Applied Biosystems (Foster City, CA, USA);

Plasma measurements

In accordance with previous observations (Nuche-Berenguer et al., 2009, Moreno et al., 2011), basal plasma insulin was higher than normal in IR rats, while hyperglycemia together with hypoinsulinemia occurred in the T2D group; treatment with amylin for 3 days reduced insulinemia in IR rats, and did not modify glycemia in any group studied (not shown). Basal plasma amylin levels in the three groups were undistinguishable from each other (overall mean ± S.E.M.: 15.2 ± 1.8 pM, n = 20), and amylin

Discussion

While whether a decrease or an increase of bone mass occurs in T2D subjects is a matter of debate, the reported increase in fracture risk associated with this pathology has been attributed to poor bone quality and strength (Khazai et al., 2009). The mechanisms responsible for bone disease in this setting are yet ill-defined, but a reduction in bone formation with an increase or no changes in bone resorption have been reported in rats and humans with T2D (Fujii et al., 2008, Nuche-Berenguer et

Acknowledgments

This work was supported by CIBERDEM (CB/07/08/2007), Red Temática de Investigación Cooperativa en Envejecimiento y Fragilidad (RETICEF) (RD06/0013/1002) and other Grants (PI080922 and PI11/00449) from Instituto de Salud Carlos III. I.G.-R. and A.A. are recipients of a CIBERDEM contract; I. R.-A., B.N.-B. and P. M. are research fellows from Fundación Conchita Rábago de Jiménez Díaz; D.L. is the recipient of a research contract from Comunidad Autónoma de Madrid (S2009/MAT-1472). We thank Mark

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