Glucagon

ABSTRACT. Background: Glucagon-like peptide 2 (GLP-2) has a trophic effect on the intestine and enhances intestinal absorption in adult animals, but its effect in young rats is unknown. Glucocorticosteroids accelerate the ontogeny of the intestine, and in adult animals they increase the uptake of sugars and lipids. We hypothesized that GLP-2 and dexamethasone (DEX), when administrated to lactating rat dams, will enhance lipid uptake in the suckling and weanling offspring. Methods: Eight nursing rats were treated during lactation, 19 to 21 days, with GLP-2 (0.1 µg/g/d subcutaneously [sc]), DEX (0.128 µg/g/d sc), GLP-2 + DEX (GLP-2 0.1 µg/g/d sc plus DEX 0.128 µg/g/d sc), or placebo. Half of the offspring ("sucklings") were killed at 19 to 21 days of age, and half were killed 4 weeks later ("weanlings"). The rate of intestinal uptake of fatty acids (12:0, lauric; 16:0, palmitic; 18:0, stearic; 18:1, oleic; 18:2, linoleic; and 18:3, linolenic) and cholesterol were assessed using an in vitro ring technique. Results: GLP-2 and DEX resulted in loss of body weight in sucklings, which was prevented by giving the combination GLP-2 + DEX. The jejunal atrophy in sucklings given DEX was prevented by giving GLP-2 + DEX, but GLP-2 + DEX did not prevent the decline in jejunal and ileal villous height and crypt depth observed in weanlings given DEX. GLP-2 had little effect on lipid uptake in sucklings, whereas DEX or GLP-2 + DEX increased the uptake of lipids. In contrast, in weanlings there was malabsorption of several lipids with GLP-2 or GLP-2 + DEX, but not with DEX. Lipid uptake was lower in weanlings than in sucklings, and this age-associated decline was not altered by GLP-2 or DEX. Conclusions: The loss of body weight and the jejunal atrophy induced by DEX in sucklings is prevented by adding GLP-2. Giving DEX or GLP-2 + DEX to lactating mothers enhances lipid uptake in their suckling offspring. In marked contrast, a month after lactating dams were given GLP-2 or GLP-2 + DEX, there was reduced lipid absorption in the postweaning animals. Thus, giving GLP-2 + DEX during lactation may be useful to enhance lipid uptake in the suckling offspring, without adverse effects on body weight or intestinal characteristics. However, the late effects of this treatment on lipid absorption were of concern, and could be potentially deleterious to the nutritional well-being of the animal. (Journal of Parenteral and Enteral Nutrition 28:355-364, 2004)

Lipid absorption is the result of the sum of the processes of passive diffusion and protein-mediated transport.1,2 There is little information about the early development of lipid absorption in the intestine. Rats and mice have lingual lipases but no gastric lipases.3 The pancreas of the newborn has a low secretion of lipase and colipase, which is partially compensated for by the lingual and milk lipases.4 Also, this small capacity for digesting lipids is partially compensated for by a higher intestinal uptake of lipids in sucklings than in adults. This may be due in part to an increased fluidity of the brush border membrane (BBM),5,6 which allows for an enhanced passive diffusion of lipids. Moreover, there is greater metabolism of fat in sucklings compared with adult rats.7,8 During the ontogeny of the gut, there are age-dependent changes in the absorption of carbohydrates, lipids, and amino acids, which prepare the intestine for the diet changes that occur between the suckling and weanling periods. These alterations in absorption are possibly caused by changes in nutrient transporters, digestive enzymes, and BBM permeability.9-11 Interactions between diverse stimuli and genetic programming contribute to morphological and functional maturation of the intestine.4,10,12-14 These stimuli are induced by hormones, the enteric nervous system, the mucosa and mesenchyme, and by luminal factors such as diet and intestinal bacterial flora.15-17

In adult animals, glucagon-like peptide 2 (GLP-2) enhances the absorption of sugars,18-20 amino acids such as leucine, and lipids such as triolein.21 It is not known if GLP-2 influences the absorption of lipids in young animals. Also, glucocorticosteroids such as dexamethasone (DEX) increase the intestinal uptake of sugars and lipids in adult rats,22,23 but it is not known if DEX alters lipid uptake in sucklings. Previous studies with rodents have shown that the lipid content of the maternal diet during pregnancy or lactation influences the absorption of nutrients in the offspring.24,25 Some lactating mothers might be given GLP-2 for treatment of the short bowel syndrome26 or be given glucocorticosteroids for medical conditions such as asthma or Crohn's disease. It is not known if the administration of GLP-2 or DEX to the lactating mother will affect the lipid uptake of the offspring. Accordingly, this study was undertaken to determine (1) the influence of GLP-2, DEX, and GLP-2 + DEX, when administered to lactating rat dams, on the in vitro intestinal uptake of lipids in the suckling and weanling offspring; (2) if these changes in lipid uptake are caused by variations in the intestinal morphology or mass, or by alterations in the abundance of selected cytosolic lipid binding proteins in the enterocytes; and (3) if GLP-2, DEX, or GLP-2 + DEX given to the lactating dams has a late effect on lipid uptake in the offspring after weaning.

MATERIALS AND METHODS

Animals

The principles for the care and use of laboratory animals, approved by the Canadian Council on Animal Care and by the Council of the American Physiologic Society, were observed in the conduct of this study. All experiments were approved by the Animal Ethics Board, University of Alberta. Eight 2-week-old pregnant Sprague-Dawley rats were obtained from Bio Science Animal Services, University of Alberta (Edmonton, Alberta, Canada).

The dams were randomized into 4 groups that received treatment with GLP-2, DEX, GLP-2 plus DEX, or placebo. The treatment was started after delivery and was continued until the offspring were 19 to 21 days old. GLP-2 was administrated in a dose of 0.1 µg/g body weight/d given subcutaneously [sc] twice per day at 7:00 AM and 9:00 PM. DEX was administrated in a dose of 0.128 µg/g body weight/d sc once per day at 21:00 hours. The regimen used for GLP-2 + DEX group was GLP-2 0.1 µg/g body weight/d sc twice per day at 7:00 AM and 9:00 PM plus DEX 0.128 µg/g body weight/d sc once per day at 9:00 PM. The placebo group received 0.9% saline in a volume equal with the volume of GLP-2 administrated daily per rat (depending on the weight of the nursing rat, the volume ranged from 0.46 mL to 0.50 mL) sc twice per day at 7:00 AM and 9:00 PM.

The number of offspring was decreased after delivery to 12 pups, which were housed with their nursing dams. At weaning, 8 offspring per group ("sucklings") were killed for the uptake studies, and 8 per group were killed for morphology and immunohistochemistry. The remaining postweaning animals ("weanlings") were killed for uptake studies at 7 weeks of age (Figure 1).

The animals were housed at a temperature of 21°C and each day were exposed to 12 hours of light and 12 hours of darkness. During the suckling period, the offspring received only the dam's milk. The weanlings were housed in pairs, and their water and food were supplied ad libitum. The dams and the weanlings were fed standard rat chow, PMI 5001 (Nutrition International LLC, Brentwood, MO). The diets were nutritionally adequate, providing for all known essential nutrient requirements. Body weights were recorded at the time of weaning and then weekly for the next 4 weeks.

Uptake Studies

Probe and marker compounds. The ^sup 14^C-labeled probes included cholesterol (0.05 mmol/L) and 6 fatty acids (0.1 mmol/L): lauric acid (12:0), palmitic acid (16:0), stearic acid (18:0), oleic acid (18:1), linoleic acid (18:2), and linolenic acid (18:3). The labeled and unlabeled probes were supplied by Amersham Biosciences Inc (Baie d'Urfe, PQ). The lipid probes were prepared by solubilizing them in 10 mmol/L taurodeoxycholic acid (Sigma Co., St Louis, MO) in Krebs-bicarbonate buffer, with the exception of 12:0 which was solubilized only in Krebs-bicarbonate buffer. ^sup 3^H-inulin was used as a non-absorbable marker to correct for the adherent mucosal fluid volume.27

Tissue preparation. Eight animals per treatment group were killed by an intraperitoneal injection of Euthanyl (sodium pentobarbital, 240 mg/100 g body weight). The whole length of the small intestine was rapidly removed and rinsed with 150 mL cold saline. The intestine was divided into 2 parts: the proximal half of the intestine beginning at the ligament of Treitz was termed the jejunum, and the distal half was termed the ileum. A 2-cm piece of each segment of jejunum and ileum was gently scraped with a glass slide. The mucosal scrapings and the remaining wall of the intestine were dried overnight in an oven at 55°C. The percentage of the intestinal wall composed of mucosa was calculated.

The intestine was everted and cut into small rings of approximately 2 to 4 mm each. These intestinal rings were immersed in preincubation beakers containing Krebs's buffer (pH 7.2) at 37°C, bubbled with oxygen plus bicarbonate (O2-CO2, 95:5 by volume), and were allowed to equilibrate for 5 minutes.28 Uptake was initiated by the timed transfer of the tissue rings from the preincubation buffer to a 5-mL plastic vial containing ^sup 3^H-inulin and ^sup 14^C-labeled lipids in Kreb's buffer bubbled with oxygen plus bicarbonate that had been equilibrated to 37°C in a shaking water bath. The intestinal rings were incubated in the lipid substrates for 5 minutes.

Determination of uptake rates. The rate of uptake of lipid was terminated by pouring the vial contents onto filters on an Amicon vacuum filtration manifold that was maintained under suction, followed by washing the intestinal rings 3 times with ice-cold saline. The tissue rings were placed on a glass slide and were dried overnight in an oven at a constant temperature of 55°C.

The dry weight of the tissue was determined, and the tissue was transferred to scintillation counting vials. The samples were saponified with 0.75-M NaOH, scintillation fluid was added, and radioactivity was determined by means of an external standardization technique to correct for variable quenching of the 2 isotopes.27 The rates of lipid uptake were determined as nanomoles of substrate absorbed per 100 mg dry weight of the whole intestinal wall per minute (Jd, nmol/100 mg tissue^sup -1^/min^sup -1^), or as nmol of substrate absorbed per 100 mg dry weight of the mucosa per minute (Jm, nmol/100 mg mucosal tissue^sup -1^/min^sup -1^).

Morphological Analysis

To determine the morphological characteristics of the intestine, a vertical section was prepared from the jejunum and from the ileum. Hematoxylin and eosin-stained slides were prepared from paraffin blocks. Crypt depth, villous height, villous width, vinous width at half height, and cell density were measured using the program MetaMorph 5.05r (Universal Imaging Corporation, Downingtown, PA). The group means were obtained according to 10 villi and 20 crypts per slide, with a minimum of four animals in each group.

Immunohistochemistry

Jejunal and ileal tissues were embedded in paraffin, and 4-µm to 5-µm sections were mounted on glass slides. The sections were heated and placed immediately in xylene (2 × for 5 minutes each), followed by absolute ethanol (2 × for 2 minutes each), and were then rinsed with tap water. The slides were incubated in a hydrogen peroxide/methanol solution, and rinsed with tap water. Then they were rehydrated, and the tissue was encircled on the slides with a hydrophobic slide marker (PAP pen; BioGenex, San Ramon, CA). The slides were incubated for 15 minutes in blocking reagent (20% normal goat serum), followed by primary antibody to the intestinal fatty acid binding protein (I-FABP) or to the liver fatty acid binding protein (L-FABP) for 30 minutes. Both antibodies were a generous gift from Dr L. B. Agellon, University of Alberta. The slides were incubated in LINK and LABEL, and with DAB solution (BioGenex). The slides were then washed, stained in hematoxylin, dehydrated in absolute ethanol, and cleared in xylene. The slides were photographed, and the area was labeled with antibody was determined using Metamorph 5.05r. The results were expressed as a ratio of the area which was antibody-positive vs the total area. Statistical analyses were based on a minimum of 4 villi per animal and 4 animals per group.

Statistical Analyses

The results were expressed as means ± SEM. The statistical significance of the differences between the 4 groups was determined by analysis of variance (ANOVA) for values of p

RESULTS

Body and Intestinal Weights and Villous Morphology

When the lactating dams were given GLP-2 or DEX, the mean body weight of their suckling offspring was less than in controls, and this decline was prevented with the combination of GLP-2 + DEX (data not shown). There was no difference in the body weights of weanling rats whose lactating dams had previously been given GLP-2, DEX, or GLP-2 + DEX.

In suckling animals whose lactating dams were given GLP-2, there was no change in the characteristics of the jejunum (Table I) or the ileum (data not shown). When DEX was given, there was an approximately 50% fall in the weight of the jejunal wall, a 70% decline in the jejunal mucosal weight, and a 40% decline in the percentage of the intestinal wall comprised of mucosa. DEX had no effect on the characteristics of the ileum. When GLP-2 was added to DEX (GLP-2 + DEX), the marked loss in jejunal mass observed with DEX was not observed.

In weanling rats whose lactating dams were given GLP-2, the weight of the jejunal mucosa was greater than in those given placebo (Table II). DEX had no effect on the intestinal characteristics. GLP-2 + DEX markedly increased the weight of the jejunal submucosa and decreased the percentage of the jejunal wall composed of mucosa.

GLP-2 had no effect on the ileal morphology in sucklings (data not shown) or on the jejunal or ileal morphology of weanlings (Tables II and III). In contrast, in the jejunum of sucklings GLP-2 increased villous height, width, crypt depth, distance between villi, and distance between enterocytes in sucklings. DEX increased jejunal villous width, distance between villi, and distance between enterocytes (Table I). In contrast, in weanlings DEX was associated with a decline in all of these endpoints. GLP-2 + DEX had no effect on jejunal or ileal morphology in sucklings (Table I), but in weanlings GLP-2 + DEX was associated with a decline in all of the jejunal and ileal morphological parameters (Tables II and III).

Lipid Uptake

Because of the influence of the treatments on the intestinal characteristics and morphology (Tables I-III), the rate of lipid uptake was expressed according to the weight of the mucosa (Jm, nmol/100 mg mucosa^sup -1^/min^sup -1^; Tables IV-VII).

When the lactating mothers were treated with GLP-2, there was no change in the jejunal uptake of lipids in their suckling offspring, whereas the ileal uptake of 16:0 was increased and the ileal uptake of 18:2 was decreased (Table IV). DEX increased the jejunal uptake of 12:0, 18:0, 18:1, 18:3, and cholesterol. GLP-2 + DEX increased the jejunal uptake of 12:0, 18:0, 18:1, and cholesterol and increased the ileal uptake of 12:0, 16:0, and 18:1.

When uptake was examined in postweaning rats whose dams were exposed to GLP-2 1 month previously during lactation, there was reduced jejunal uptake of 12:0, 16:0, and 18:1, and reduced ileal uptake of 12:0 and 18:1. In contrast, the ileal uptake of 18:2 and cholesterol were increased with GLP-2 (Table V). DEX reduced the jejunal uptake of 18:1 and increased the uptake of 18:2. The combination of GLP-2 + DEX reduced the jejunal and ileal uptake of 18:1 and increased the ileal uptake of 18:2.

The jejunal uptake of 18:2 was lower in weanlings than in suckling rats given placebo (Table VI). Similarly, the ileal uptake of 18:0, 18:2, 18:3, and cholesterol was lower in weanlings than in suckling rats given placebo (Table VII). With GLP-2, the jejunal uptake of 12:0, 16:0, and 18:2 was lower in weanlings than in sucklings, whereas cholesterol uptake was increased in weanling rats vs sucklings. In the ileum of rats given GLP-2, the uptake of all fatty acids was lower in weanlings than in sucklings. The jejunal and ileal uptake of most fatty acids was lower in weanlings than in sucklings given DEX, and the same pattern was observed for rats given GLP-2 + DEX. Thus, lipid uptake falls with aging, and this process is not altered by GLP-2, DEX, or GLP-2 + DEX.

Immunohistochemistry

In sucklings, neither GLP-2 nor DEX had an effect on the jejunal or ileal abundance of I-FABP or L-FABP, whereas the ileal abundance of I- and L-FABP was increased in GLP-2 + DEX (data not shown).

DISCUSSION

Sucklings

GLP-2 given sc to lactating rats for 19 to 20 days in a dose of 0.1 µg/g body weight had no consistent effects on lipid uptake in either the jejunum or the ileum of the suckling offspring (Tables IV and V). These results were not expected, because it is known that in adult mice, the administration of GLP-2 at a dose of 5µg/d (approximately 0.16 µg/g body weight/d, greater than our dose of 0.1 µg/g body weigh t/d) for 10 days increased the absorption of ^sup 14^C triolein.21 Also surprisingly, the administration of GLP-2 to the lactating rat dams resulted in a decrease in body weight of the sucklings (data not shown). This is in contrast to the increase in the body weight of adults given GLP-2.26,29 GLP-2 is known as having a potent trophic effect on the intestine of adult animals.30,31 In this study, GLP-2 increased several morphological parameters in the jejunum of sucklings, including villous height, villous width, and crypt depth (Table I).

It is unknown if GLP-2 crosses into mother's milk, although other hormones such as somatostatin do cross into milk.32,33 GLP-2 degradation in the intestinal lumen was not investigated, and it is possible that even if GLP-2 passed into the mother's milk, the GLP-2 might be sensitive to the intestinal proteases and could possibly be destroyed.34,35 Interestingly, the lack of effect of GLP-2 on lipid uptake is unlikely to be because of a lack of GLP-2 receptor (GLP-2R) expression, because the level of GLP-2R messenger ribonucleic acid (mRNA) is higher in fetal and neonatal rodent intestine than in adults.36,37 Furthermore, there was evidence for a biologic effect of GLP-2, such as its negative effect on body weight, its effect on preventing the loss of jejunal mass with DEX (Table I), its positive effects on jejunal morphology in sucklings (Table I), its effects on the jejunal mucosa and submucosa in weanlings given GLP-2 or GLP-2 + DEX (Table I), its enhancing effect on the ileal abundance of I- and L-FABP (data not shown), and its antiabsorptive effect on lipid uptake in weanlings (Table V). There are numerous peptides involved in the ontogeny of the intestine, such as hepatocyte growth factor, fibroblast growth factor, platelet-derived growth factor, and granulocyte colony-stimulating factor, and some of these peptides pass into milk.32,38-40 We speculate that GLP-2 either acts directly on the GLP-2R in the intestine or induces the release of other mediators that cross into mother's milk and are themselves responsible for these numerous effects of GLP-2 administered to the nursing mother. Alternatively, GLP-2 may have indirect effects on the offspring because of changes in milk production or consumption. Further research is required to determine the precise mechanism by which maternal GLP-2 influences lipid uptake in the offspring.

DEX treatment of lactating rats for 19 to 21 days in a dose of 0.128 µg/g body weight per day increased lipid uptake in the jejunum of sucklings (Table IV). This confirms the lipid uptake enhancing effect of DEX shown in previous experiments with adult animals treated with glucocorticosteroids for 4 weeks starting at 3 weeks of age.22,23 This enhanced uptake of lipids caused by DEX could be explained partially by a decline in the effective resistance of the unstirred water layer, as demonstrated by an increased uptake of 12:0.27 There was no correlation between the increase in lipid uptake and the intestinal characteristics or the animal's body weights. In the suckling offspring, DEX reduced the body weight and the weight of the jejunal wall and mucosa, although DEX resulted in an increase in villous height and width at the base, distance between villi, and enterocyte size (Table I). The loss in body weight was anticipated because DEX has catabolic effects on the whole body.41,42 Also, DEX induces atrophy of the intestine43,44 by decreasing both enterocyte turnover45 and cell proliferation by blocking growth hormone signaling.46,47

The enhancement in the jejunal uptake of lipids with DEX occurred despite the lack of change in the enterocyte cytosolic I- and L-FABP (data not shown). GLP-2 + DEX increased lipid uptake in the jejunum and ileum, but the abundance of I- and L-FABP in the jejunum did not change. Previous studies in adult rats also failed to show a correlation between changes in the intestinal lipid binding proteins and lipid uptake.48 In addition, other workers have shown that a change in the abundance or a deficiency in I-FABP did not result in parallel alterations of intestinal lipid uptake.49,50 Also, taking into account the complementary roles of L-FABP and ileal binding protein, it has been suggested that L-FABP is not necessary for lipid uptake into enterocytes.51

Glucocorticosteroids such as DEX act on gene transcription by way of their effects on nuclear receptors.52,53 DEX could potentially influence other lipid binding proteins not measured in this studies (such as caveolin-1, the SR-BI scavenger receptor, the plasma membrane fatty acid binding protein [FABP^sub pm^], the fatty acid transporter [FAT/CD36], the fatty acid transporter-4 [FATP4], the cholesterol transport protein, and the ileal binding protein), and might thereby modify lipid uptake. The fluidity of the BBM increases in fetal rats whose mothers received DEX54 or in adult rats given DEX,55 and this increase in BBM fluidity could possibly contribute to increased lipid uptake. We did not measure the BBM fluidity in this study.

DEX crosses into mother's milk and directly influences the intestine of the suckling offspring.15,32,56 The decrease in body weight of the offspring and the atrophy of the intestinal mucosa are likely the result of DEX down-regulating the insulin-like growth factor I (IGF-1) hepatic endocrine axis and inducing catabolism in the whole body and in the intestine.57 Interestingly, after DEX treatment, the IGF-1 decline is greater in the wall of the intestine than in the villi,42 and this may explain the decrease in intestinal characteristics (Table I).

GLP-2 + DEX also increased lipid uptake in sucklings in both the jejunum and the ileum (Table IV). Interestingly, this enhancement was similar to the effects of DEX in the jejunum. This enhancing effect of DEX or of GLP-2 + DEX could be partially caused by a decrease in the effective resistance of the intestinal unstirred water layer (Table IV). The mucosal mass was decreased in DEX and was restored to normal in GLP-2 + DEX (Table I). Thus, alterations in the mucosal mass or morphology are not the cause of the increased uptake of lipid in GLP-2 + DEX.

Weanlings

The jejunal and ileal uptake of lipids was lower in weanlings than in sucklings (Tables VI and VII). A similar age-dependent decline in lipid absorption was demonstrated by Frost et al8 and by Flores et al,7 measuring the absorption of ^sup 14^C-labeled substrates. This age-associated fall in lipid uptake may be due a decline in the fluidity of the BBM.5,6 The content of lipids in the intestinal lumen influences lipid uptake, so the age-associated decline in lipid uptake may be the result of the switch from the high-lipid milk diet consumed by sucklings to the high-carbohydrate diet that is eaten at weaning.15,58

This decline in lipid uptake in early life appears to be a process that is not affected by GLP-2, DEX or GLP-2 + DEX (Tables VI and VII). In contrast to the relative lack of effect of GLP-2 on lipid uptake in sucklings (Table IV), 1 month after the administration of GLP-2 to the lactating dams, there was now reduced jejunal and ileal uptake of lipids (Table V). Furthermore, the stimulating effect of DEX on lipid uptake in suckling was lost in weanlings, but GLP-2 + DEX resulted in a mixed picture in weanlings, with the increase in the uptake of some lipids and a decrease in others (Table V). We did not establish the mechanisms of these late effects of GLP-2, DEX, or GLP-2 + DEX on lipid uptake. It is known that there are late effects of early nutrition on the absorptive function of the small intestine.59 Also, variations of the type of lipids in the diet of pregnant or lactating mothers modify the normal ontogeny of intestinal nutrient absorption.25,60,61 This late appearance of an effect of GLP-2 in weanlings raises the possibility that the GLP-2 given to lactating dams resulted in some alterations that led to a change in the ontogeny of lipid absorption in the intestine. Some human mothers might require glucocorticosteroids to be given for health reasons during lactation. The catabolic effects of DEX on body weight and on the intestine of the offspring could be reversed by given GLP-2 with DEX. However, this potential benefit needs to be weighed against the adverse effect of an increase in 2 of the lipid-binding proteins in the intestine and the mixed effect of GLP-2 + DEX on lipid uptake. The late enhancement in the abundance of I-and L-FABP, and in the uptake of some lipids a month after the lactating mothers were given GLP-2 + DEX, raises the possibility that this abnormal intestinal absorption of lipids may continue in later life and might thereby contribute to abnormalities in lipid metabolism, which could be detrimental to the welfare of the animal.

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Claudiu Iordache, MD, MSc*; Laurie Drozdowski, MSc*; Tom Clandinin, PhD*; Gary Wild, MD, PhD[dagger]; Zoe Todd*; and Alan B. R. Thomson, MD, PhD*

From the * Nutrition and Metabolism Group, University of Alberta, Edmonton, Alberta, Canada; and the [dagger] Division of Gastroenterology, Department of Medicine, McGill University, Edmonton, Alberta, Canada

Received for publication January 15, 2004.

Accepted for publication June 8, 2004.

Correspondence: Dr. A. B. R. Thomson, Gastroenterology, Department of Medicine, University of Alberta, 205 College Plaza, 8215 112 Street, Edmonton AB T6G 2C8, Canada. Electronic mail may be sent to alan.thomson@ualberta.ca.

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