Differential Responses of the Growth Hormone Axis in Two Rat Models of Streptozotocin-Induced Insulinopenic Diabetes
Total Page：16
File Type：pdf, Size：1020Kb
263 Differential responses of the growth hormone axis in two rat models of streptozotocin-induced insulinopenic diabetes E Kim, S Sohn, M Lee, J Jung, R D Kineman1 and S Park Department of Pharmacology, Kyunghee University School of Medicine and Institute for Basic Medical Sciences, Seoul 130-701, Korea 1Department of Medicine, University of Illinois at Chicago and Jesse Brown VA Medical Center, Research and Development, Chicago, Illinois 60612, USA (Requests for offprints should be addressed to S Park; Email: [email protected]) Abstract The impact of streptozotocin (STZ)-induced, insulino- and hypothalamic GHRH, pituitary GH mRNA levels of penic diabetes on the GH axis of rats and mice differs HI STZ-treated rats were 58% of controls. However, from study to study, where this variation may be related to pituitary receptor mRNA levels for GHRH and ghrelin the induction scheme, severity of the diabetes and/or increased and those for somatostatin (sst2, sst3 and sst5) the genetic background of the animal model used. In decreased following HI STZ treatment. The impact of order to begin differentiate between these possibilities, we LO STZ treatment on the GH axis differed from that compared the effects of two different STZ induction observed following HI STZ treatment, despite com- schemes on the GH axis of male Sprague–Dawley rats: (1) parable changes in circulating glucose and insulin. a single high-dose injection of STZ (HI STZ, 80 mg/kg, Specifically, LO STZ treatment did suppress circulating i.p.), which results in rapid chemical destruction of the IGF-I levels to the same extent as HI STZ treatment; pancreatic -cells, and (2) multiple low-dose injections of however, the impact on hypothalamic NPY mRNA STZ (LO STZ, 20 mg/kg for 5 consecutive days, i.p.), levels was less dramatic (158% of vehicle-treated controls) which results in a gradual, autoimmune destruction of where NPY immunoreactivity was increased only within -cells. STZ-treated animals were killed after 3 weeks of the paraventricular nucleus. Also, there were no changes hyperglycemia (>400 mg/dl), and in both paradigms in circulating GH, hypothalamic GHRH or pituitary circulating insulin levels were reduced to <40% of receptor expression following LO STZ treatment, with vehicle-treated controls. HI STZ-treated rats lost weight, the exception that pituitary sst3 mRNA levels were while body weights of LO STZ-treated animals gradually suppressed compared with vehicle-treated controls. Taken increased over time, similar to vehicle-treated controls. As together these results clearly demonstrate that insulino- previously reported, HI STZ resulted in a decrease in penia, hyperglycemia and reduced circulating IGF-I levels circulating GH and IGF-I levels which was associated are not the primary mediators of hypothalamic and with a rise in hypothalamic neuropeptide Y (NPY) pituitary changes in the GH axis of rats following HI STZ mRNA (355% of vehicle-treated controls) and a fall in treatment. Changes in the GH axis of HI STZ-treated rats GH-releasing hormone (GHRH) mRNA (45% of were accompanied by weight loss, and these changes are vehicle-treated controls) levels. Changes in hypothalamic strikingly similar to those observed in the fasted rat, which neuropeptide expression were reflected by an increase in suggests that factors associated with the catabolic state are immunoreactive NPY within the arcuate and paraven- critical in modifying the GH axis following STZ-induced tricular nuclei and a decrease in GHRH immunoreactivity diabetes. in the arcuate nucleus, as assessed by immunohisto- Journal of Endocrinology (2006) 188, 263–270 chemistry. Consistent with the decline in circulating GH Introduction if the appropriate dose is given (Candela et al. 1979, Junod et al. 1969, Rossini et al. 1977). HI STZ-induced A commonly used method to induce insulin-dependent insulinopenia is typically associated with rapid weight loss, diabetes in rodents is a single high-dose injection (i.p. or due to a decrease in fat stores, leading to a fall in i.v.) of streptozotocin (HI STZ). In this paradigm, STZ circulating leptin (Havel et al. 1998, Sindelar et al. 1999) induces pancreatic -cell necrosis, which is evident 4 h and a compensatory rise in hypothalamic neuropeptide Y following STZ injection. Blood glucose levels peak (NPY), an orexigenic signal (Ishii et al. 2002, Marks et al. 1–3 days following STZ injection and remain elevated 1993, White et al. 1990). In rats (Sprague–Dawley and Journal of Endocrinology (2006) 188, 263–270 DOI: 10.1677/joe.1.06501 0022–0795/06/0188–263 2006 Society for Endocrinology Printed in Great Britain Online version via http://www.endocrinology-journals.org Downloaded from Bioscientifica.com at 10/01/2021 12:53:43PM via free access 264 E KIM and others · Changes in GH axis in two models of STZ-induced diabetes Wistar), these metabolic changes have been associated principles and procedures outlined in the NIH Guide for with suppression of the growth hormone (GH) axis, the Care and Use of Laboratory Animals. which includes a decrease in hypothalamic GH-releasing hormone (GHRH) and pituitary GH mRNA levels and a reduction in pulsatile GH release, and mean circulating STZ-induced diabetes insulin-like growth factor (IGF)-I levels (Busiguina et al. HI STZ In order to induce rapid-onset diabetes, rats were 2000b, Olchovsky et al. 1990). Despite these changes, the treated with vehicle (citrate buffer, pH 4·5; n=5)orSTZ pituitary of the HI STZ-treated rat is more sensitive to the (80 mg/kg, i.p.; n=7) between 14·00 and 16·00 h. Body stimulatory actions of GHRH (Sheppard et al. 1989a, weights and blood glucose levels were determined on day 1989b) and GH secretagogue (GHS) receptor (GHS-R) 0 (time of STZ treatment) and on days 2, 4, 7, 14 and 21. agonists (ipamorelin; Johansen et al. 2003) and less sensi- HI STZ-treated animals lost weight (Fig. 1A) and all tive to the inhibitory actions of somatostatin (SRIH; treated animals displayed hyperglycemia (>22·2 nmol/l or Bruno et al. 1994, Sheppard et al. 1989b). 400 mg/dl) 2 days after STZ injection and remained Mice (ICR background) treated with HI STZ hyperglycemic until their death (Fig. 1B). (200 mg/kg, i.p. injection) also lose weight and exhibit a dramatic reduction in circulating GH and IGF-I and a LO STZ To generate a model resembling a more gradual decrease in pituitary GH and hypothalamic GHRH onset of diabetes, rats received vehicle (n=5) or STZ expression, which is associated with pituitary GHRH (20 mg/kg, i.p.; n=7) for 5 consecutive days as previously hypersensitivity, in vitro (Murao et al. 1995). It should be described (Li et al. 2000a, Like & Rossini 1976). Body noted that these changes in the GH axis are strikingly weight and blood glucose levels were measured at day –4 similar to those observed in the fasted rat (Park et al. 2004, (time of first STZ treatment), day 0 (time of last STZ Tannenbaum et al. 1979) and therefore might be related treatment) and on days 2, 4, 7, 14, 21 and 28 following the to the catabolic condition and not to the absolute circu- last day of STZ injection. Body weights were not sup- lating levels of insulin and glucose. This hypothesis is pressed with treatment (Fig. 1A) and blood glucose levels consistent with a report showing BALB/c mice, when began to rise to 262 mg/dl at 2 days after the last injection. treated with a single i.v. injection of HI STZ (250– All LO STZ-treated animals showed hyperglycemia 300 mg/kg), have elevated circulating GH levels, which (>22·2 nmol/l or 400 mg/dl) 7 days after last injection was associated with hypoinsulinemia and hyperglycemia and remained hyperglycemic until their death (Fig. 1B). without dramatic weight loss or ketosis (Flyvbjerg et al. HI STZ-induced diabetic animals were killed 21 days 1999), a response similar to that reported in poorly following bolus STZ treatment, and LO STZ animals controlled type I diabetic humans (Cohen & Abplanalp were killed 4 weeks after the last STZ injection. Therefore 1991, Ismail et al. 1993, Krassowski et al. 1988). However, both groups of animals were exposed to hyperglycemia from these studies it is difficult to say with certainty if the ff for 3 weeks. Blood, pituitaries and hypothalami were variable e ects of STZ on circulating GH levels are due to collected and stored at 70 C until further analysis. the severity of catabolic condition or if the differences are more related to species or genetic background of the animal model used. To help clarify this issue we have Measurement of glucose, GH, IGF-I and insulin compared the impact of diabetes on the GH axis of male concentrations Sprague–Dawley rats, where diabetes was induced by either HI STZ treatment or by multiple low-dose injec- Glucose levels were measured using blood from the tail tions of STZ (LO STZ), where LO STZ results in vein by GlucoDr Blood Glucose Meter (Allmedicus, hypoinsulinemia and hyperglycemia >7 days following Korea; maximal reading 600 mg/dl). Serum GH concen- the last STZ injection, with the severity of the disease trations were measured by rat GH RIA kit (Amersham increasing over time due to the gradual autoimmune Biosciences Co.). Total serum IGF-I levels were assayed destruction of pancreatic -cells (Li et al. 2000a, 2000b, using a rat IGF-I RIA kit (Amersham) after acid/ethanol Like & Rossini 1976). extraction according to the manufacturer’s instructions. Serum insulin concentrations were assessed using the rat insulin RIA kit (Amersham). Materials and Methods RNA isolation Animals Total hypothalamic and pituitary RNA were recovered Male Sprague–Dawley rats (7–8 weeks; 220–250 g) were using standard procedure reported previously (Kamegai housed under controlled environmental conditions et al.