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ENDOCRINE AND DIABETES
-Cell Function after 3-Week Daily Administration in Obese Diabetic (ob/ob) Mice
School of Biomedical Sciences, University of Ulster, Coleraine, Northern Ireland (K.S.L., N.I., F.P.M.O., P.R.F.); School of Biological Sciences, Queen's University of Belfast, Belfast, Northern Ireland (B.D.G., P.H., B.G.); and School of Life and Health Sciences, Aston University, Birmingham, United Kingdom (C.J.B.)
Received for publication
October 28, 2005
Accepted
April 26, 2006.
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Abstract |
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-cell function were examined in obese-diabetic (ob/ob) mice. Acute intraperitoneal administration of (Val8)GLP-1 (6.25-25 nmol/kg) with glucose increased the insulin response and reduced the glycemic excursion in a dose-dependent manner. The effects of (Val8)GLP-1 were greater and longer lasting than native GLP-1. Once-daily subcutaneous administration of (Val8)GLP-1 (25 nmol/kg) for 21 days reduced plasma glucose concentrations, increased plasma insulin, and reduced body weight more than native GLP-1 without a significant change in daily food intake. Furthermore, (Val8)GLP-1 improved glucose tolerance, reduced the glycemic excursion after feeding, increased the plasma insulin response to glucose and feeding, and improved insulin sensitivity. These effects were consistently greater with (Val8)GLP-1 than with native GLP-1, and both peptides retained or increased their acute efficacy compared with initial administration. (Val8)GLP-1 treatment increased average islet area 1.2-fold without changing the number of islets, resulting in an increased number of larger islets. These data demonstrate that (Val8)GLP-1 is more effective and longer acting than native GLP-1 in obese-diabetic ob/ob mice.
; Nauck et al., 2003; Deacon, 2004; Green et al., 2004b; Vilsboll and Holst, 2004). Glucose lowering induced by GLP-1 seems to be mediated by a potent insulin-releasing action as well as a range of other effects, including inhibition of glucagon secretion and gastric emptying, increased satiety, stimulation of glucose-uptake, and gluconeogenesis (Villanueva-Penacarrillo et al., 1994; Turton et al., 1996; Zander et al., 2002; Green et al., 2004b; Vilsboll and Holst, 2004). GLP-1 also seems to exert trophic effects on the cells, stimulating growth and differentiation and inhibiting cytokine- and free fatty acid- and streptozotocin-mediated apoptosis (Buteau et al., 1999; Zhou et al., 1999; Liu et al., 2004; Bregenholt et al., 2005).
In view of these attributes, GLP-1 is now the focus of pharmaceutical industry's attention. The duration of action of GLP-1 (t1/2 of 2-3 min) is limited by inactivation due to N-terminal degradation by the enzyme dipeptidyl peptidase IV (DPP IV) (Deacon et al., 1995). DPP IV is a ubiquitous cell surface and circulating enzyme found in large amounts at the brush border of kidney epithelium. Two main intervention strategies are under development to prevent degradation of GLP-1: specific inhibitors of DPP IV and subtle modifications of the GLP-1 molecule to generate analogs that are resistant to DPP IV. We have modified the N terminus of GLP-1 to generate a family of novel DPP IV-resistant analogs (Green et al., 2003, 2004b). (Val8)GLP-1 is a GLP-1 analog with profound resistance to DPP IV and greater biological activity than other N-terminally modified analogs and native GLP-1 (Green et al., 2003). Acute administration of (Val8)GLP-1, in combination with glucose, showed similar insulin-releasing activity to native GLP-1 and greater glucose lowering than GLP-1. The more potent antihyperglycaemic activity of (Val8)GLP-1 may therefore relate to other beneficial actions such as inhibition of glucagon secretion or extrapancreatic effects (Fehmann et al., 1995).
Although structural modification of GLP-1 may overcome degradation by DPP IV, this does not address the loss of GLP-1 by renal filtration (Meier et al., 2004). We and others have attempted to prevent renal filtration of GLP-1 by acylation (attaching long-chain fatty acid molecules) (Green et al., 2004a). Acylating peptides facilitate binding to plasma proteins, such as albumin, thereby minimizing their elimination by the kidney. LY315902 (Eli Lilly & Co., Indianapolis, IN), for example, is an acylated GLP-1 analog with an octanoyl fatty acid chain (Holz and Chepurny, 2003). NN2211 (Liraglutide; Novo Nordisk, Bagsværd, Denmark) contains a hexanoyl fatty acid group attached to the 
-amino group of Lys26 (Holz and Chepurny, 2003), and CJC-1131 (Conjuchem) contains a reactive chemical linker attached to the -amino group of Lys34 (Holz and Chepurny, 2003). NN2211 and CJC-1131 show sustained activities and half-lives greatly in excess of 8 h. Other attempts to acylate GLP-1 with palmitate (18-carbon fatty acid) produce analogs with moderately prolonged activities but with greatly reduced bioavailability (Green et al., 2004a). Albugon is a recombinant GLP-1-albumin protein that decreases the glycemic excursion in mice, but it has a reduced ability to activate the GLP-1 receptor (Baggio et al., 2004).
Since (Val8)GLP-1 differs by one amino acid from physiological form of mammalian GLP-1 and exhibits increased stability and acute biological activity, it offers particular promise for therapeutic use. However, whether this translates into improved duration of action and tangible metabolic long-term benefits in type 2 diabetes remains to be evaluated. In this study, we assessed the magnitude of the glucose-lowering and insulin-releasing actions of (Val8)GLP-1 compared with GLP-1. Furthermore, we describe the effects of 21-day long-term daily administration of (Val8)GLP-1 on feeding activity, body weight, basal glucose and insulin concentrations, glucose tolerance, pancreatic -cell function, insulin sensitivity, and islet morphology of obese diabetic (ob/ob) mice, a commonly used animal model of non-insulin-dependent diabetes.
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Materials and Methods |
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). In brief, (Val8)GLP-1 and GLP-1 were sequentially synthesized on a model 432A automated peptide synthesizer (Applied Biosystems, Foster City, CA) using standard solid-phase 9-fluorenylmethoxycarbonyl peptide chemistry. They were judged to be >99% pure by reversed-phase high-performance liquid chromatography using a Millenium 2010 chromatography system (Waters, Milford, MA). Peptide structures were confirmed using electrospray ionization-mass spectrometry, as described previously (Green et al., 2003).
Animals. The genetic background and characteristics of the ob/ob colony have been described previously (Bailey and Flatt, 1995). Males, aged 15 to 19 weeks, were housed individually in an air-conditioned room at 22 ± 2°C with a 12-h light (6:00 AM-6:00 PM):12-h dark cycle (6:00 PM-6:00 AM) cycle. Drinking water and a standard rodent maintenance diet (Trouw Nutrition Ltd., Cheshire, UK) were freely available. All animal experiments were carried out in accordance with the UK Animals (Scientific Procedures) Act 1986. No adverse effects were observed after administration of any of the peptides.
Evaluation of Acute and Long-Term Effects of (Val8)GLP-1 and Native GLP-1 in ob/ob Mice. Initial experiments were performed to evaluate the acute dose-dependent glucose-lowering and insulin-releasing effects of (Val8)GLP-1 and GLP-1 when administered by intraperitoneal injection to 18-h fasted ob/ob mice together with glucose (18 mmol/kg). On the basis of the results obtained, subsequent studies were conducted with a peptide dose of 25 nmol/kg. To demonstrate the longer term duration of action of (Val8)GLP-1, glucose tolerance (18 mmol/kg) and insulin release were assessed 4 h after administration of (Val8)GLP-1, GLP-1 (25 nmol/kg), or saline.
For long-term studies separate groups of ob/ob mice received once-daily subcutaneous injections (5:00 PM) of either (Val8)GLP-1, GLP-1 (25 nmol/kg in saline), or saline [0.9% (w/v) NaCl] over a 21-day period. Before the 21-day treatment period, animals were stratified so that all groups were of similar age, body weight, and diabetes status as judged by nonfasting plasma glucose concentration. Food intake and body weight were recorded daily. Blood samples were collected on days 0, 1, 3, 7, 11, 14, and 20 (9:00 AM) from the cut tail tip of conscious fed mice. Glucose tolerance (18 mmol/kg, intraperitoneally), meal tolerance (15 min refeeding after 18-h fast), peptide response (25 nmol/kg peptide with 18 mmol/kg glucose, intraperitoneally), and insulin sensitivity (50 U/kg insulin, intraperitoneally) tests were conducted on day 21. Procedures were commenced between 9:00 AM and 10:00 AM. For all experiments, blood samples were collected at the times indicated in the figures into chilled fluoride/heparin-coated microcentrifuge tubes (Sarstedt, Numbrecht, Germany) and centrifuged (30 s at 13,000g) using a Beckman microcentrifuge (Beckman Instruments, Buckinghamshire, UK). The resulting plasma was then aliquoted into fresh Eppendorf tubes and stored at -20°C before glucose and insulin analysis.
Immunohistochemistry. At the end of the experimental period, islet morphology was evaluated in four mice from each group. Tissue fixed in 4% paraformaldehyde/phosphate-buffered saline and embedded in paraffin was sectioned at 8 µm. After dewaxing, sections were incubated with blocking serum (Vector Laboratories, Burlingame, CA) before exposure to insulin antibody. Tissue samples were then incubated consecutively with secondary biotinylated universal, pan-specific antibody (Vector Laboratories) and streptavidin/peroxidase preformed complex (Vector Laboratories). After washing, the stained pancreatic tissue was counterstained with hematoxylin (BDH Chemicals, Dorset, UK) and then washed in acid methanol (500 ml of methanol, 500 ml of H2O, and 2.5 ml of concentrated HCl) before dehydration and mounting in Depex (BDH Chemicals). The stained slides were viewed under a microscope (Nikon Eclipse E2000; Diagnostic Instruments Inc., Sterling Heights, MI) attached to a model KY-F55B camera (JVC, London, UK) and analyzed using Kromoscan imaging software (Kinetic Imaging Limited, Faversham, Kent, UK). The average number and area of islets in each section were estimated in a blinded manner using ImageJ software (National Institutes of Health, Bethesda, MD) (Abramoff et al., 2004) calibrated with a stage micrometer (Graticules Limited, Tonbridge, Kent, UK). Approximately 60 to 70 random sections were examined from the pancreas of each mouse.
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). Results were expressed as means ± S.E.M. Plasma glucose, plasma insulin values, and islet area data were compared using the unpaired Student's t test. Incremental (
AUC0-60) values were compared using repeated measures one-way analysis of variation followed by the Student-Newman-Keuls post hoc test. AUC0-60 values were calculated using a computer-generated program using the trapezoidal rule (Burington, 1973) with baseline subtraction. Groups of data were considered to be significantly different if P < 0.05. It should be noted that in most cases these parameters did not regain baseline values by the end of the test period. Indeed, in separate glucose tolerance tests plasma glucose concentrations of untreated ob/ob mice only returned to baseline values at approximately 240 min (Fig. 1). Routine extension of the observation period in other tests was not possible, because there is a limit on the number of sequential blood samples that could be taken for glucose and insulin analyses in each mouse. |
Results |
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AUC0-60 confirmed this and revealed that a 6.25-nmol/kg (Val8)GLP-1 dose was more potent than 25 nmol/kg native GLP-1 (P < 0.001; Fig. 2). At doses of 12.5 and 25 nmol/kg, (Val8)GLP-1 was more effective than native GLP-1 in augmenting insulin release (P < 0.05-0.01; Fig. 2), despite lower glucose concentrations.
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Long-Term Effects of (Val8)GLP-1 in ob/ob Mice. Figure 4 shows the effects of daily administration of (Val8)GLP-1, GLP-1 or saline on body weight, food intake, and nonfasting plasma concentrations of glucose and insulin in ob/ob mice. GLP-1 had no significant effect on body weight or food intake. However, mice treated with (Val8)GLP-1 displayed significantly reduced body weights by day 16 (P < 0.05) without a significant change in food intake. Over the 21-day period, plasma glucose levels of ob/ob mice treated with saline ranged from 21 ± 3 to 25 ± 1 mM. Mice chronically treated with (Val8)GLP-1 had significantly lower plasma glucose levels after 18 days (P < 0.05) and elevated insulin levels after 9 days of treatment (P < 0.01). Glucose and insulin levels in GLP-1-treated mice did not differ from saline-treated mice on any of the days tested.
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Long-Term Effects of (Val8)GLP-1 on Glucose Tolerance in ob/ob Mice. Figure 5 shows the effects of intraperitoneal glucose (18 mmol/kg) on glucose and insulin concentrations of ob/ob mice treated for 21 days with either (Val8)GLP-1, GLP-1, or saline. Treatment with (Val8)GLP-1 significantly lowered plasma glucose levels from 15 min onward compared with saline-treated controls (P < 0.01); mice treated with GLP-1 showed lower plasma glucose levels from 30 min (P < 0.05). Treatment with (Val8)GLP-1 also significantly increased insulin responses to glucose at 15 and 30 min after injection (P < 0.01). Insulin responses in GLP-1-treated mice did not differ significantly from those of saline-treated mice.
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Long-Term Effects of (Val8)GLP-1 on Insulin Sensitivity in ob/ob Mice. Figure 6 shows the effect of intraperitoneal insulin (50 U/kg) on glucose concentrations in 21-day-treated mice. (Val8)GLP-1 treatment resulted in significantly lower plasma glucose levels 30 min after insulin injection (P < 0.01). The glucose AUC0-60 value was also significantly decreased (437 ± 111 versus 238 ± 51 mM x min; P < 0.001). GLP-1 treatment had no significant effect on insulin-induced glucose lowering.
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AUC0-60 value (Val8)GLP-1-treated mice was 36% greater (P < 0.05) than in saline-treated controls (765 ± 60 versus 564 ± 60 ng/ml x min). The glycemic and insulin responses were not changed by GLP-1 treatment. Food intake of (Val8)GLP-1-treated (0.8 ± 0.1 g/mouse/15 min), GLP-1-treated (0.8 ± 0.1 g/mouse/15 min), and saline-treated (0.7 ± 0.1 g/mouse/15 min) mice were not significantly different.
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AUC0-60 values were also significantly different (P < 0.05) between (Val8)GLP-1-(65 ± 15 mM x min, 1692 ± 104 ng/ml x min) and GLP-1 (286 ± 50 mM x min, 1306 ± 80 ng/ml x min)-treated groups.
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Discussion |
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; Green et al., 2004b). (Val8)GLP-1 is a novel N-terminally modified GLP-1 analog displaying profound resistance to DPP IV degradation (Green et al., 2003). No degradation fragments were detected when (Val8)GLP-1 was incubated for up to 12 h in purified DPP IV or pooled human plasma (Green et al., 2003). The replacement of alanine with the larger valine residue seems to cause sufficient steric hindrance to diminish the susceptibility of GLP-1 to DPP IV-mediated degradation, but this change is sufficiently subtle to retain the biological activities of GLP-1 at the cellular level (Green et al., 2003). In vitro, (Val8)GLP-1 has similar cAMP-stimulating and insulin-releasing activity to another position 8-substituted analog, (Abu8)GLP-1, but in vivo it demonstrated a significantly greater ability to lower glucose (Green et al., 2003). Analogs of GLP-1 developed by modification of His7 are stable to DPP IV but suffer losses in biological potency (Green et al., 2004b).
Acute in vivo effects of (Val8)GLP-1 were compared with native GLP-1 using obese diabetic (ob/ob) mice. This spontaneous model of obesity and diabetes is devoid of biologically active leptin and characterized by hyperphagia, obesity, hyperglycemia, defective -cell function, and severe insulin resistance (Bailey et al., 1982). Consistent with previous observations (Green et al., 2003), acute administration of native GLP-1 together with glucose augmented insulin release but had minimal effects on the glucose excursion of ob/ob mice. (Val8)GLP-1 produced a greater increase in glucose-mediated insulin release and lowered the glucose values up to 70% more than GLP-1. In contrast to a previous report (Green et al., 2004a), these more detailed observations also demonstrated a greater in vivo insulin-releasing activity of (Val8)GLP-1. Most notably in the present study, the glucose-lowering potency of 6.25 nmol/kg (Val8)GLP-1 given acutely was superior to a 4 times greater dose of native GLP-1. Furthermore, the effect persisted for more than 4 h after injection, indicating an extended duration of action of (Val8)GLP-1. Since (Val8)GLP-1 and GLP-1 can be expected to be equally susceptible to renal filtration, this suggests that it is degradation by DPP IV and not renal filtration, which immediately curtails hormone action. Interestingly, the glucose-lowering activity of (Val8)GLP-1 was accompanied by relatively modest increases in insulin concentrations. This could indicate that (Val8)GLP-1 may also possess prolonged/enhanced effects on glucose-lowering mechanisms, in particular inhibition of glucagon secretion and stimulation of glucose uptake and gluconeogenesis.
The major interest of the present study concerns evaluation of how the stability of (Val8)GLP-1 translates to improved diabetes control following long-term once-daily injection. Administration of (Val8)GLP-1 to adult ob/ob mice (25 nmol/kg/day) for 21 days resulted in progressive elevation of plasma insulin and a lowering of basal glucose concentrations. Body weight also declined, but this was not matched with any measurable change in food intake. Although there is evidence that GLP-1 can reduce feeding (Turton et al., 1996; Flint et al., 2000), this is not a consistent finding (Thiele et al., 1997). It is possible that small differences in meal pattern, physical activity, or enhanced metabolic efficiency may have been missed by the current study. Interestingly, studies in db/db mice showed that the GLP-1 analog exendin-4 (exenatide) also decreased body weight after 10 weeks and lowered both fasting glucose and insulin concentrations (Greig et al., 1999). In clinical studies, in type 2 diabetic patients, chronic treatment with exendin-4 improved glycemic control with a reduction in body weight (DeFronzo et al., 2005).
Evaluation of the spectrum of antidiabetic effects of 21-day treatment of ob/ob mice with (Val8)GLP-1 revealed substantial improvements of glucose tolerance and glycemic responses to feeding. This can be attributed in part to considerable improvements of -cell responsiveness. Islet number was not affected, arguing against stimulation of neogenesis over the time period studied. However, administration of (Val8)GLP-1 modestly enhanced islet area by increasing the proportion of larger islets in the pancreas, suggesting increased -cell numbers. This finding is in accordance with observations with exendin-4 in db/db mice (Greig et al., 1999; Young et al., 1999) and the reported ability of GLP-1 to stimulate -cell replication and inhibit apoptosis (Buteau et al., 1999; Zander et al., 2002). However, insulin sensitivity was also significantly improved by (Val8)GLP-1 administration. This may be due in part not only to reduced glucotoxicity indicated by the consistently lower glucose concentrations but also to the reduction in body weight imparted by (Val8)GLP-1 treatment. However, in contrast to (Val8)GLP-1, administration of native GLP-1 resulted in little change to insulin sensitivity, although improvement of glucose tolerance was significant.
The observation of significant improvements in glucose homeostasis and -cell function of ob/ob mice treated with (Val8)GLP-1 for 21 days suggests that the GLP-1 receptor is not down-regulated by prolonged exposure to the peptide. Consistent with this view, acute administration of (Val8)GLP-1 together with glucose retained ability to moderate the glycemic excursion and enhance insulin secretion. Indeed, these attributes of (Val8)GLP-1 given acutely were slightly enhanced after 21 days of treatment. This seems to reflect enhanced insulin sensitivity and improved -cell responsiveness. Likewise, long-term treatment with GLP-1 improved the glycemic excursion when the peptide was administered acutely with glucose, although there was no further improvement to the insulin response. The extent to which this reflects possible long-term exposure to the truncated metabolite GLP-1-(9-36) amide that acts as a weak antagonist at the GLP-1 receptor (Green et al., 2004c) is unknown. However, daily administration of the GLP-1 receptor antagonist, exendin(9-39) amide mildly impaired glucose homeostasis in normal mice due to changes of insulin secretion (Green et al., 2005). It remains to be seen whether DPP IV processing of GLP-1 to this degradation fragment results in any alterations of metabolism in obese-diabetic ob/ob mice.
It is possible that small differences in meal pattern, physical activity, or enhanced metabolic efficiency may have been missed by the current study. Long-acting GLP-1 analogs and exendin(1-39) can produce nausea and taste aversion (Thiele et al., 1997; Mark, 2003; Green et al., 2004b). It is not known whether (Val8)GLP-1 has such effects, but no adverse effects were noted during the 21-day treatment period.
In conclusion, this study shows that long-term treatment with (Val8)GLP-1 was associated with significant acute and long-term antidiabetic actions in ob/ob diabetic mice. Further development of stable long-acting GLP-1 analogs, such as (Val8)GLP-1, promises to provide new effective agents for diabetes therapy.
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Footnotes |
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: GLP-1, glucagon-like peptide-1; DPP IV, dipeptidyl peptidase IV; AUC, areas under plasma glucose and insulin curve; CJC-1131, HAEGTFTSDVSSYLEGQAA-{N--[
-Glu(N-
-hexadecanoyl)}-EFIAWLV-Lys34-GR-Lys-{2-[2-(2-maleimidopropionamido)ethoxy]ethoxy}acetamide; NN2211, HAEGTFTSDVSSYLEGQAA-Lys-{N--[-Glu(N--hexadecanoyl)}-EFIAWLV-Lys34-GRG; LY315902, Des-HAEGTFTSDVSSYLEGQAA-Arg26-EFIAWLV-Lys-(octanoyl)-GRG.
Address correspondence to: Dr. Brian Green, School of Biomedical Sciences, University of Ulster, Coleraine BT52 1SA, Northern Ireland, UK. E-mail: b.green{at}ulster.ac.uk
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, P < 0.05; 
