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Novel role of insulin in the regulation of glucose excretion by mourning doves (Zenaida macroura)
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Novel role of insulin in the regulation of glucose excretion by mourning doves (Zenaida macroura) Karen L. Sweazea a,b,∗ , Eldon J. Braun c , Richard Sparr a a b c
School of Nutrition and Health Promotion, Arizona State University, 550 North 3rd Street, Phoenix, AZ 85004, USA School of Life Sciences, Arizona State University, 427 E Tyler Mall, Tempe, AZ 85287, USA Department of Physiology, College of Medicine, Arizona Health Sciences Center, University of Arizona, 1501 N. Campbell, Tucson, AZ 85724, USA
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Article history: Received 15 August 2016 Received in revised form 18 January 2017 Accepted 17 February 2017 Available online xxx Keywords: Avian physiology Insulin metabolism Kidney Glomerular filtration rate Glucose excretion
a b s t r a c t In mammals, insulin primarily lowers plasma glucose (PGlu ) by increasing its uptake into tissues. Studies have also shown that insulin lowers PGlu in mammals by modulating glomerular filtration rate (GFR). Birds have naturally high PGlu and, although insulin administration significantly decreases glucose concentrations, birds are resistant to insulin-mediated glucose uptake into tissues. Since prior work has not examined the effects of insulin on GFR in birds, the purpose of the present study was to assess whether insulin can augment renal glucose excretion and thereby lower PGlu . Therefore, the hypothesis of the present study was that insulin lowers PGlu in birds by augmenting GFR, as estimated by inulin clearance (CIn ). Adult mourning doves (Zenaida macroura) were used as experimental animals. Doves were anesthetized and the brachial vein was cannulated for administration of [14 C]-inulin and insulin and the brachial artery was cannulated for blood collections. Ureteral urine was collected via a catheter inserted into the cloaca. Ten minutes following administration of exogenous insulin (400 g/kg body mass, i.v.) plasma glucose was significantly decreased (p = 0.0003). Twenty minutes following insulin administration, increases in GFR (p = 0.016) were observed along with decreases in urine glucose concentrations (p = 0.008), glucose excretion (p = 0.028), and the fractional excretion of glucose (p = 0.003). Urine flow rate (p = 0.051) also tended to increase after administration of insulin. These data demonstrate a significant role for insulin in modulating GFR in mourning doves, which may in part explain the lower PGlu measured following insulin administration. © 2017 Elsevier GmbH. All rights reserved.
1. Introduction In mammals, postprandial euglycemia is maintained through the glucose-lowering action of the pancreatic hormone insulin. The typical mechanism by which insulin maintains euglycemia is through activation of signaling pathways in skeletal and cardiac muscle fibers as well as in adipose cells that promote glucose uptake through the insulin responsive glucose transporter, GLUT4 (for review see Shepherd and Kahn, 1999). In addition, insulin has been shown to increase the glomerular filtration rate (GFR) in mammals, affecting glucose excretion (Tucker et al., 1992); a similar action of insulin has not been described for birds. Thus, the purpose of the current study was to examine the role of insulin in renal glucose excretion of birds.
∗ Corresponding author at: School of Life Sciences, Arizona State University, 427 E Tyler Mall, Tempe, AZ 85287-4501, USA. E-mail address: [email protected] (K.L. Sweazea).
A prior study of 97 avian species found that plasma glucose (PGlu ) levels were 1.5–2 times those of mammals (n = 162) of comparable body mass (Braun and Sweazea, 2008). Data also show that many avian species are resistant to the glucose-lowering effects of insulin such that supraphysiological doses must be administered to achieve a significant reduction in PGlu (Sweazea and Braun, 2005; Sweazea et al., 2006; Braun and Sweazea, 2008). Plasma concentrations of insulin in doves are much lower (1.03 ± 0.1 ng/ml; Sweazea et al., 2006) than observed in mammals because of fewer insulin-producing -cells in the pancreas, a trend observed for several species of birds (Braun and Sweazea, 2008). In a study of Gallus gallus chicks, administration of porcine insulin significantly decreased PGlu levels and uptake was reportedly increased into tissues (Tokushima et al., 2005). However, studies of adult mourning doves (Zenaida macroura) showed that despite significantly decreasing PGlu levels, insulin failed to increase glucose uptake into skeletal muscle, heart, liver, kidney, adipose or brain cells (Sweazea et al., 2006). Moreover, extensor digitorum communis flight muscles of adult house sparrows (Passer domesticus) showed resistance
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Please cite this article in press as: Sweazea, K.L., et al., Novel role of insulin in the regulation of glucose excretion by mourning doves (Zenaida macroura). Zoology (2017), http://dx.doi.org/10.1016/j.zool.2017.02.006
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thesia induced by sodium pentobarbital (23.6 mg/kg body mass, i.p.), the left brachial artery was cannulated with heparinized PE-50 tubing to collect blood for analyses of inulin and glucose concentrations. The left brachial vein was cannulated with heparinized PE-50 tubing to facilitate the infusion of insulin and [14 C]-inulin. Ureteral urine samples were collected by a small cannula inserted into the cloaca with a funnel opening that was juxtaposed to the ureters to avoid fecal contamination. Animal protocols were approved by the Institutional Animal Care and Use Committees of the University of Arizona and Arizona State University. 2.2. Administration of [C14 ]-inulin and insulin
Fig. 1. Variations in (A) plasma and (B) urine glucose concentrations (mg/ml) as well as (C) urine flow rate (ml/min,) following insulin administration (400 g/kg body mass, i.v.). Data for individual birds are shown in gray symbols, with the same symbols representing the same bird in each graph. Black hexagons represent the mean ± SEM of the individual data. Time 0 (baseline) represents the last 10-min clearance period prior to the insulin dose. * p < 0.005 from baseline; # p < 0.05 from 10 min; n = 7.
to insulin-mediated glucose uptake (Sweazea and Braun, 2005). Similarly, a study of chickens found that the insulin signaling pathway is refractory in skeletal muscle (Dupont et al., 2004). These characteristics may be attributed to the absence of a GLUT4-like transport protein in adult birds (Carver et al., 2001; Duclos et al., 1993; Seki et al., 2003; Sweazea and Braun, 2006; Sweazea et al., 2006; Welch et al., 2013). Despite the lack of a GLUT4-like protein, exogenous insulin does significantly decrease plasma glucose concentrations in birds. The mechanism behind this glucose-lowering effect remains elusive. Since insulin has been shown to modulate renal excretion in mammals, the purpose of the present study was to examine whether insulin can similarly alter the glomerular filtration rate in birds. The hypothesis of the current study was that insulin lowers PGlu concentrations in doves through enhanced excretion. 2. Materials and methods 2.1. Animals Adult mourning doves (Zenaida macroura, n = 7) of both sexes (105–130 g body mass) were captured from the Tucson metropolitan area using walk-in style funnel traps and transported to the laboratory in ventilated plastic animal carriers. Following anes-
Following cannulations, birds were administered a priming dose of [14 C]-inulin (1 Ci) followed by sustained infusion of 0.01 Ciinulin in 0.9% saline at a rate of 0.2 ml/min. Clearance of inulin (CIn ) can be used to estimate the glomerular filtration rate (GFR) as it is freely filtered, but not metabolized, reabsorbed or secreted by the renal tubules. Therefore, measurement of plasma and urine inulin concentrations is considered a gold standard for estimating filtration (Stevens and Levey, 2009; Hinojosa-Laborde et al., 2015). Baseline urine and blood samples were collected for three 10-min clearance periods. As these pre-insulin values were not different, the last 10-minute period was used to establish the baseline values (time 0) prior to administration of insulin. Following the baseline collections, porcine insulin (400 g/kg body mass in 0.9% saline) was administered intravenously and plasma and urine samples were collected at 10 and 20 min post-infusion. Porcine insulin was chosen as it has been used routinely in prior studies of birds (Julian and Abbott, 1998; Pal et al., 2002; Tokushima et al., 2005) as well as in veterinary medicine to treat rare cases of avian hyperglycemia, such as reported in macaws (Gancz et al., 2007). Moreover, the insulin receptor is highly conserved across species (Muggeo et al., 1979). Samples were weighed (assuming a specific gravity of 1) to measure urine flow rate (ml/min). Blood samples were centrifuged to separate the formed elements from plasma and urine samples were centrifuged to remove solid uric acid prior to freezing at −20 ◦ C until further analysis. 2.3. Quantification of plasma and urine glucose and inulin Urine and plasma glucose concentrations were quantified by the glucose oxidase method using a commercially available assay kit (Cat. 10009582; Cayman Chemical Company, Ann Arbor, MI, USA). An aliquot of 10 l urine and/or plasma was used for [14 C]-inulin determination. Samples were dark-adapted for 3 h prior to measuring radioactive inulin by liquid scintillation. GFR was calculated using the clearance formula: GFR = urine inulin concentration (counts/minute) x urine flow rate (ml/min)/plasma inulin concentration (counts/minute). 2.4. Statistical analyses Data in the figures represent changes within individual birds in gray as well as the mean ± SEM of the individual data shown in black. Data in the table are expressed as baseline mean ± SEM and average percent changes from baseline ± SEM. Raw data were analyzed by one-way repeated measures ANOVA and Holm–Sidak posthoc analyses. Percent changes in values were analyzed using paired t-tests. Statistical analyses were completed using SigmaStat 3.0 (Systat Software Inc., San Jose, CA, USA). A probability of ≤ 0.05 was considered statistically significant for all comparisons.
Please cite this article in press as: Sweazea, K.L., et al., Novel role of insulin in the regulation of glucose excretion by mourning doves (Zenaida macroura). Zoology (2017), http://dx.doi.org/10.1016/j.zool.2017.02.006
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Fig. 3. (A) Variations in glucose excretion (mg/min) by the kidney following insulin administration (400 g/kg body mass, i.v.). Data for individual birds are shown in gray symbols, with the same symbols representing the same bird in each graph. Black hexagons represent the mean ± SEM of the individual data. Time 0 (baseline) represents the last 10-min clearance period prior to the insulin dose. Glucose excretion calculated as urine flow rate (ml/min) × UGlu (mg/ml). (B) Variations in the fractional excretion of glucose from baseline following insulin administration (400 g/kg body mass, i.v.). Fractional excretion of glucose calculated as (UGlu /PGlu ) × (UInulin /PInulin )−1 . Data for individual birds are shown in gray symbols, with the same symbols representing the same bird in each graph. Black hexagons represent the mean ± SEM of the individual data. * p < 0.005 from baseline; # p < 0.05 from 10 min; n = 7.
Fig. 2. Variations in (A) glomerular filtration rate (ml/min), (B) filtered load of glucose (mg/min) and (C) glucose reabsorption (mg/min) following insulin administration (400 g/kg body mass, i.v.). Data for individual birds are shown in gray symbols, with the same symbols representing the same bird in each graph. Black hexagons represent the mean ± SEM of the individual data. Time 0 (baseline) represents the last 10-min clearance period prior to the insulin dose. Glomerular filtration rate was estimated by inulin clearance (UInulin (counts/minute) × urine flow rate (ml/min))/PInulin (counts/minute). Filtered load of glucose calculated as GFR (ml/min) × PGlu (mg/ml). Glucose reabsorption calculated as filtered load of glucose (mg/min) − glucose excretion (mg/min). * p < 0.02 from baseline; n = 7.
3. Results Results from the present study show that the infusion of exogenous insulin significantly decreases PGlu concentrations (p < 0.001, F-value = 25.619, df = 6; Fig. 1A). Holm–Sidak post hoc analyses show that PGlu was decreased within 10 min post insulin administration (p = 0.0003) and remained significantly decreased 20 min later (p = 0.00002). Urine glucose concentrations were significantly decreased with insulin administration (p = 0.008, F-value = 7.359, df = 6; Fig. 1B). Holm–Sidak post hoc analyses show that urine glucose concentrations were significantly decreased 20 min after administration as compared to baseline (p = 0.003). The observed trend for increased urine flow rate (p = 0.051, Chi-square = 6.0, df = 2; Fig. 1C) is supported by a significant increase in CIn (p = 0.040, F-value = 4.245, df = 6; Fig. 2A). The filtered load of glucose (PGlu x CIn ) did not significantly change although there was
considerable variability in the data (p = 0.335; Fig. 2B). The increase in CIn was not associated with an increase in systemic blood pressure as it did not change with insulin infusion (data not shown). The data show that renal glucose reabsorption was not significantly altered by insulin (p = 0.291, F-value 1.370, df = 6; Fig. 2C). In contrast, glucose excretion was significantly decreased (p = 0.028, F-value = 4.908, df = 6; Fig. 3A). Similarly, when the data for excretion of glucose are factored by GFR (fractional excretion of glucose), a significant decrease in fractional glucose excretion is observed by 20 min post-administration of insulin (p = 0.003, F-value = 9.706, df = 6; Fig. 3B). Examining the percent change in variables (Table 1) similarly shows that plasma and urine glucose as well as fractional excretion of glucose are significantly decreased following insulin administration whereas urine flow rate, glucose excretion and CIn are significantly increased. 4. Discussion The major findings of the present study are that exogenous insulin significantly lowers PGlu in mourning doves by increasing glomerular filtration leading to augmented renal flow rates. It is often assumed that insulin regulation of PGlu concentrations in birds results from enhanced insulin-mediated glucose uptake into tissues as observed in mammals. However, previous studies have shown that despite significantly lowering PGlu in mourning doves, insulin administration did not result in increased glucose uptake into any of the tissues examined (brain, skeletal muscle,
Please cite this article in press as: Sweazea, K.L., et al., Novel role of insulin in the regulation of glucose excretion by mourning doves (Zenaida macroura). Zoology (2017), http://dx.doi.org/10.1016/j.zool.2017.02.006
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Table 1 Insulin-mediated alterations of renal glucose handling in mourning doves (Zenaida macroura). Parameter
Baseline
% Change from baseline at 10 min (p-value, T-statistic)
% Change from baseline at 20 min (p-value, T-statistic)
318.0 ± 15.1 mg/dl
−30.55 ± 7.51 (0.007, 4.06)
−47.57 ± 13.3 (0.012, 3.56)
118.0 ± 35.4 mg/dl
−16.40 ± 7.32 (0.066, 2.24)
−156.3 ± 38.6 (0.007, 3.96)
0.041 ± 0.014 ml/min
38.17 ± 9.34 (0.006, −4.09)
41.50 ± 15.0 (0.033, −2.76)
0.224 ± 0.065 ml/min
46.10 ± 11.7 (0.008, −3.95)
45.66 ± 17.9 (0.043, −2.55)
6.96 ± 2.03 mg/min
31.57 ± 13.4 (0.057, −2.35)
21.65 ± 28.0 (0.478, −0.755)
6.45 ± 1.89 mg/min
31.45 ± 13.5 (0.059, −2.33)
24.41 ± 27.3 (0.414, −0.877)
0.508 ± 0.176 mg/min
29.27 ± 10.0 (0.027, −2.91)
−60.93 ± 48.2 (0.249, −1.28)
0.065 ± 0.012 AU
−38.29 ± 45.3 (0.427, 0.853)
−348.5 ± 257.19 (0.264, 1.23)
Plasma glucose
Urine glucose
Urine flow rate
Glomerular filtration rate
Filtered load of glucose
Glucose reabsorption
Glucose excretion
Fractional excretion
Bold font indicates significant difference from baseline (time 0). Data analyzed by paired t-tests. df = 6 for all comparisons. AU = arbitrary units.
kidney, liver, adipose; Sweazea et al., 2006). This lack of insulinmediated glucose uptake has also been observed in isolated skeletal muscles from house sparrows (Sweazea and Braun, 2005) as well as chickens (Dupont et al., 2004), which may be attributed to the absence of an insulin-responsive GLUT4 transport protein in birds (Duclos et al., 1993; Carver et al., 2001; Seki et al., 2003; Dupont et al., 2004; Sweazea and Braun, 2006; Welch et al., 2013; Coudert et al., 2015). While a separate study reported GLUT4 protein expression in pigeon (Columba livia) leg muscle plasma membrane fractions, examination of the images from the western blots shows only a weak band and no attempt was made to sequence the protein (Diamond and Carruthers, 1993). In contrast to adult birds, insulin has been shown to increase glucose uptake into cardiac and skeletal muscles of Gallus gallus chicks (Tokushima et al., 2005). Similarly, insulin-mediated glucose uptake has been observed in isolated skeletal muscle cells and cultured myotubes from embryonic quail and Muscovy ducklings (Shanahan, 1984; Thomas-Delloye et al., 1999). It should be noted, however, that the antibody used to detect GLUT4 in ducklings reportedly crossreacts with GLUT1 (Thomas-Delloye et al., 1999). While adult avian muscle lacks GLUT4 expression, a recent study found that chicken skeletal muscle expresses a GLUT12 transport protein which may exhibit insulin responsiveness (Coudert et al., 2015). Thus, it is possible that age or species variations exist in, not only insulinmediated glucose transport into tissues, but perhaps regulation of glucose excretion as well. Similar to rodents, percent glucose excretion was significantly augmented in response to insulin in the present study. However, the fractional excretion of glucose (the amount excreted compared to the amount filtered) was not significantly altered at 10 min following the administration of insulin and was actually decreased 20 min post-administration. Therefore, one mechanism by which exogenous insulin may regulate glucose excretion by the avian kidney is through augmenting GFR resulting in an increase in urine flow rate leading to the observed enhanced percent change in glucose excretion at 10 min post-infusion. This may be attributed to reduced glucose contact time with proximal tubule transport proteins, thereby resulting in glucose loss in the urine. In fact, studies of laboratory rodents show that exogenous insulin acutely pro-
motes dilation of the afferent and efferent renal arterioles, with greater dilation of the afferent arteriole, thereby increasing GFR (Tucker et al., 1992). Moreover, others have shown that insulin increases permeability of the filtration barrier in mammals by acting directly on the podocytes through GLUT isoforms 1, 2, 3, 4, and 8 (Coward and Fornoni, 2015). Insulin has also been shown to regulate the contractility of podocytes by altering calcium transport and inducing actin remodeling, thereby increasing permeability of the filtration barrier (Coward and Fornoni, 2015). Thus, it is likely that the increase in CIn observed in the present study following insulin administration may have been due to altered vascular resistance of the glomerular afferent and/or efferent arterioles or changes in permeability of the filtration barrier. Neither the filtered load nor the reabsorption of glucose were significantly changed in response to insulin. The tendency for percent change in filtered glucose to increase 10 min post-infusion was likely compensated by partial recovery of glucose in the proximal renal tubules as evidenced by the tendency for percent change of glucose reabsorption to increase 10 min post-infusion. These findings suggest that under normal physiological conditions the tubular maximum for glucose reabsorption by the proximal renal tubules is not exceeded. However, additional studies are needed to elucidate the precise mechanism of the proposed insulin-mediated alterations of reabsorption in the avian kidney. The acute increase in GFR in doves administered insulin is similar to what has been reported for rats and humans. However, an increase in glucose excretion was not observed in rats and humans. In contrast to these acute effects of exogenous insulin, studies on dogs have shown that chronic intrarenal insulin infusion (7 days) modestly increases GFR but has no effect on renal plasma flow (Hall et al., 1991). Contrary to the observations in the present study where CIn and glucose excretion were increased, studies on isolated rabbit afferent and efferent arterioles suggest that insulin could decrease single nephron filtration rate (SNGFR) (Juncos and Ito, 1993). An increased resistance at the efferent, or a decreased resistance at the afferent, arteriole could mediate the increased CIn observed in the present study in addition to changes in the permeability of the filtration barrier.
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A limitation of the present study is the use of porcine insulin as opposed to an avian isoform. This may be one reason why such high doses of insulin are required before measuring a glucose-lowering effect in birds as chicken insulin binds to receptors (both avian and mammalian) with high affinity (Muggeo et al., 1979). However, it should be noted that studies using chicken insulin at high doses have shown the absence of changes in glucose uptake into tissues of several species of birds, including mourning doves (Sweazea et al., 2006; Sweazea and Braun, 2005). Porcine insulin also binds with relatively high affinity and the insulin receptor is highly conserved across species (Muggeo et al., 1979). Several prior studies have reported using porcine insulin in avian research (Julian and Abbott, 1998; Pal et al., 2002; Tokushima et al., 2005). Moreover, porcine insulin antibodies have been used to block the effects of insulin in prior studies of birds (Dupont et al., 2008; Patwardhan et al., 2004) as well as in veterinary medicine to treat rare cases of hyperglycemia in macaws (Gancz et al., 2007). Therefore, it is unlikely that the use of chicken insulin would have produced a different effect than the porcine insulin used in the current study. In conclusion, the data from the present study demonstrate that the glucose-lowering effect of exogenous insulin may be more complex than previously thought in birds and involves an increase in GFR with the subsequent disposal of glucose in the urine. To our knowledge, a mechanism of glucose regulation through augmented renal excretion by insulin has not been previously demonstrated in birds. However, it should be emphasized that these effects were observed with exogenous administration of insulin. Thus, when administering insulin at doses sufficient to reduce blood glucose in birds, it is important to consider the potential effects of insulin on renal excretion as well as changes in tissue uptake of glucose, which is the primary mechanism of insulin-mediated glucose regulation in mammals. Acknowledgements The authors thank Donna Fergusen and Jennifer Vranish for their assistance with the experiments. This work was supported by new faculty start-up funds provided by Arizona State University (to KLS). References Braun, E.J., Sweazea, K.L., 2008. Glucose regulation in birds. Comp. Biochem. Physiol. B 151, 1–9. Carver, F.M., Shibley Jr., I.A., Pennington, J.S., Pennington, S.N., 2001. Differential expression of glucose transporters during chick embryogenesis. Cell. Mol. Life Sci. 58, 645–652. Coudert, E., Pascal, G., Dupont, J., Simon, J., Cailleau-Audouin, E., Crochet, S., Duclos, M.J., Tesseraud, S., Coustard, S.M., 2015. Phylogenesis and biological characterization of a new glucose transporter in the chicken (Gallus gallus), GLUT12. PLoS ONE 10, e0139517. Coward, R., Fornoni, A., 2015. Insulin signaling: implications for podocyte biology in diabetic kidney disease. Curr. Opin. Nephrol. Hypertens. 24, 104–110. Diamond, D.L., Carruthers, A., 1993. Metabolic control of sugar transport by derepression of cell surface glucose transporters: an insulin-independent recruitment-independent mechanism of regulation. J. Biol. Chem. 25, 6437–6444.
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Duclos, M.J., Chevalier, B., Le Marchand-Brustel, Y., Tanti, J.F., Goddard, C., Simon, J., 1993. Insulin-like growth factor-I-stimulated glucose transport in myotubes derived from chicken muscle satellite cells. J. Endocrinol. 137, 465–472. Dupont, J., Dagou, C., Derouet, M., Simon, J., Taouis, M., 2004. Early steps of insulin receptor signaling in chicken and rat: apparent refractoriness in chicken muscle. Domest. Anim. Endocrinol. 29, 127–142. Dupont, J., Tesseraud, S., Derouet, M., Collin, A., Rideau, N., Crochet, S., Godet, E., Cailleau-Audouin, E., Metayer-Coustard, S., Duclos, M.J., Gespach, C., Porter, T.E., Cogburn, L.A., Simon, J., 2008. Insulin immuno-neutralization in chicken: effects on insulin signaling and gene expression in liver and muscle. J. Endocrinol. 197, 531–542. Gancz, A.Y., Wellehan, J.F.X., Boutette, J., Malka, S., Lee, S.E., Smith, D.A., Taylor, M., 2007. Diabetes mellitus concurrent with hepatic haemosiderosis in two macaws (Ara severa, Ara militaris). Avian Pathol. 36, 331–336. Hall, J.E., Brands, M.W., Mizelle, H.L., Gaillard, C.A., Hildebrandt, D.A., 1991. Chronic intrarenal hyperinsulinemia does not cause hypertension. Am. J. Physiol. 260, F663–669. Hinojosa-Laborde, C., Jespersen, B., Shade, R., 2015. Physiology lab demonstration: glomerular filtration rate in a rat. J. Vis. Exp. 101, e52425. Julian, D., Abbott, U.K., 1998. An avian model for comparative studies of insulin teratogenicity. Anat. Histol. Embryol. 27, 313–321. Juncos, L.A., Ito, S., 1993. Disparate effects of insulin on isolated rabbit afferent and efferent arterioles. J. Clin. Invest. 92, 1981–1985. Muggeo, M., Ginsberg, B.H., Roth, J., Neville, D.M., de Meyts, P., Kahn, C.R., 1979. The insulin receptor in vertebrates is functionally more conserved during evolution than insulin itself. Endocrinology 104, 1393–1402. Pal, L., Grossmann, R., Dublecz, K., Husveth, F., Wagner, L., Bartos, A., Kovacs, G., 2002. Effects of glucagon and insulin on plasma glucose triglyceride, and triglyceriderich lipoprotein concentrations in laying hens fed diets containing different types of fats. Poult. Sci. 81, 1694–1702. Patwardhan, V., Gokhale, M., Ghaskadbi, S., 2004. Acceleration of early chick embryo morphogenesis by insulin is associated with altered expression of embryonic genes. Int. J. Dev. Biol. 48, 319–326. Seki, Y., Sato, K., Kono, T., Abe, H., Akiba, Y., 2003. Broiler chickens (Ross strain) lack insulin-responsive glucose transporter GLUT4 and have GLUT8 cDNA. Gen. Comp. Endocrinol. 133, 80–87. Shanahan, M.F., 1984. Modulation of hexose transport in cultured skeletal muscle. Mol. Cell. Endocrinol. 38, 171–178. Shepherd, P.R., Kahn, B.B., 1999. Glucose transporters and insulin action – implications for insulin resistance and diabetes mellitus. N. Engl. J. Med. 341, 248–257. Stevens, L.A., Levey, A.S., 2009. Measured GFR as a confirmatory test for estimated GFR. JASN 20, 2305–2313. Sweazea, K.L., Braun, E.J., 2005. Glucose transport by English sparrow (Passer domesticus) skeletal muscle: have we been chirping up the wrong tree? J. Exp. Zool. A 303, 143–153. Sweazea, K.L., Braun, E.J., 2006. Glucose transporter expression in English sparrows (Passer domesticus). Comp. Biochem. Physiol. B 144, 263–270. Sweazea, K.L., McMurtry, J.P., Braun, E.J., 2006. Inhibition of lipolysis does not affect insulin sensitivity to glucose uptake in the mourning dove. Comp. Biochem. Physiol. B 144, 387–394. Thomas-Delloye, V., Marmonier, F., Duchamp, C., Pichon-Georges, B., Lachuer, J., Barre, H., Crouzoulon, G., 1999. Biochemical and functional evidences for a GLUT-4 homologous protein in avian skeletal muscle. Am. J. Physiol. 277, R1733–R1740. Tokushima, Y., Takahashi, K., Sato, K., Akiba, Y., 2005. Glucose uptake in vivo in skeletal muscles of insulin-injected chicks. Comp. Biochem. Physiol. B 141, 43–48. Tucker, B.J., Anderson, C.M., Thies, R.S., Collins, R.C., Blantz, R.C., 1992. Glomerular hemodynamic alterations during acute hyperinsulinemia in normal and diabetic rats. Kidney Internat. 42, 1160–1168. Welch, K.C., Allalou, A., Sehgal, P., Cheng, J., Ashok, A., 2013. Glucose transporter expression in an avian nectarivore: the ruby-throated hummingbird (Archilochus colubris). PLoS One 8, e77003.
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Novel role of insulin in the regulation of glucose excretion by mourning doves (Zenaida macroura) Karen L. Sweazea a,b,∗ , Eldon J. Braun c , Richard Sparr a a b c
School of Nutrition and Health Promotion, Arizona State University, 550 North 3rd Street, Phoenix, AZ 85004, USA School of Life Sciences, Arizona State University, 427 E Tyler Mall, Tempe, AZ 85287, USA Department of Physiology, College of Medicine, Arizona Health Sciences Center, University of Arizona, 1501 N. Campbell, Tucson, AZ 85724, USA
a r t i c l e
i n f o
Article history: Received 15 August 2016 Received in revised form 18 January 2017 Accepted 17 February 2017 Available online xxx Keywords: Avian physiology Insulin metabolism Kidney Glomerular filtration rate Glucose excretion
a b s t r a c t In mammals, insulin primarily lowers plasma glucose (PGlu ) by increasing its uptake into tissues. Studies have also shown that insulin lowers PGlu in mammals by modulating glomerular filtration rate (GFR). Birds have naturally high PGlu and, although insulin administration significantly decreases glucose concentrations, birds are resistant to insulin-mediated glucose uptake into tissues. Since prior work has not examined the effects of insulin on GFR in birds, the purpose of the present study was to assess whether insulin can augment renal glucose excretion and thereby lower PGlu . Therefore, the hypothesis of the present study was that insulin lowers PGlu in birds by augmenting GFR, as estimated by inulin clearance (CIn ). Adult mourning doves (Zenaida macroura) were used as experimental animals. Doves were anesthetized and the brachial vein was cannulated for administration of [14 C]-inulin and insulin and the brachial artery was cannulated for blood collections. Ureteral urine was collected via a catheter inserted into the cloaca. Ten minutes following administration of exogenous insulin (400 g/kg body mass, i.v.) plasma glucose was significantly decreased (p = 0.0003). Twenty minutes following insulin administration, increases in GFR (p = 0.016) were observed along with decreases in urine glucose concentrations (p = 0.008), glucose excretion (p = 0.028), and the fractional excretion of glucose (p = 0.003). Urine flow rate (p = 0.051) also tended to increase after administration of insulin. These data demonstrate a significant role for insulin in modulating GFR in mourning doves, which may in part explain the lower PGlu measured following insulin administration. © 2017 Elsevier GmbH. All rights reserved.
1. Introduction In mammals, postprandial euglycemia is maintained through the glucose-lowering action of the pancreatic hormone insulin. The typical mechanism by which insulin maintains euglycemia is through activation of signaling pathways in skeletal and cardiac muscle fibers as well as in adipose cells that promote glucose uptake through the insulin responsive glucose transporter, GLUT4 (for review see Shepherd and Kahn, 1999). In addition, insulin has been shown to increase the glomerular filtration rate (GFR) in mammals, affecting glucose excretion (Tucker et al., 1992); a similar action of insulin has not been described for birds. Thus, the purpose of the current study was to examine the role of insulin in renal glucose excretion of birds.
∗ Corresponding author at: School of Life Sciences, Arizona State University, 427 E Tyler Mall, Tempe, AZ 85287-4501, USA. E-mail address: [email protected] (K.L. Sweazea).
A prior study of 97 avian species found that plasma glucose (PGlu ) levels were 1.5–2 times those of mammals (n = 162) of comparable body mass (Braun and Sweazea, 2008). Data also show that many avian species are resistant to the glucose-lowering effects of insulin such that supraphysiological doses must be administered to achieve a significant reduction in PGlu (Sweazea and Braun, 2005; Sweazea et al., 2006; Braun and Sweazea, 2008). Plasma concentrations of insulin in doves are much lower (1.03 ± 0.1 ng/ml; Sweazea et al., 2006) than observed in mammals because of fewer insulin-producing -cells in the pancreas, a trend observed for several species of birds (Braun and Sweazea, 2008). In a study of Gallus gallus chicks, administration of porcine insulin significantly decreased PGlu levels and uptake was reportedly increased into tissues (Tokushima et al., 2005). However, studies of adult mourning doves (Zenaida macroura) showed that despite significantly decreasing PGlu levels, insulin failed to increase glucose uptake into skeletal muscle, heart, liver, kidney, adipose or brain cells (Sweazea et al., 2006). Moreover, extensor digitorum communis flight muscles of adult house sparrows (Passer domesticus) showed resistance
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Please cite this article in press as: Sweazea, K.L., et al., Novel role of insulin in the regulation of glucose excretion by mourning doves (Zenaida macroura). Zoology (2017), http://dx.doi.org/10.1016/j.zool.2017.02.006
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thesia induced by sodium pentobarbital (23.6 mg/kg body mass, i.p.), the left brachial artery was cannulated with heparinized PE-50 tubing to collect blood for analyses of inulin and glucose concentrations. The left brachial vein was cannulated with heparinized PE-50 tubing to facilitate the infusion of insulin and [14 C]-inulin. Ureteral urine samples were collected by a small cannula inserted into the cloaca with a funnel opening that was juxtaposed to the ureters to avoid fecal contamination. Animal protocols were approved by the Institutional Animal Care and Use Committees of the University of Arizona and Arizona State University. 2.2. Administration of [C14 ]-inulin and insulin
Fig. 1. Variations in (A) plasma and (B) urine glucose concentrations (mg/ml) as well as (C) urine flow rate (ml/min,) following insulin administration (400 g/kg body mass, i.v.). Data for individual birds are shown in gray symbols, with the same symbols representing the same bird in each graph. Black hexagons represent the mean ± SEM of the individual data. Time 0 (baseline) represents the last 10-min clearance period prior to the insulin dose. * p < 0.005 from baseline; # p < 0.05 from 10 min; n = 7.
to insulin-mediated glucose uptake (Sweazea and Braun, 2005). Similarly, a study of chickens found that the insulin signaling pathway is refractory in skeletal muscle (Dupont et al., 2004). These characteristics may be attributed to the absence of a GLUT4-like transport protein in adult birds (Carver et al., 2001; Duclos et al., 1993; Seki et al., 2003; Sweazea and Braun, 2006; Sweazea et al., 2006; Welch et al., 2013). Despite the lack of a GLUT4-like protein, exogenous insulin does significantly decrease plasma glucose concentrations in birds. The mechanism behind this glucose-lowering effect remains elusive. Since insulin has been shown to modulate renal excretion in mammals, the purpose of the present study was to examine whether insulin can similarly alter the glomerular filtration rate in birds. The hypothesis of the current study was that insulin lowers PGlu concentrations in doves through enhanced excretion. 2. Materials and methods 2.1. Animals Adult mourning doves (Zenaida macroura, n = 7) of both sexes (105–130 g body mass) were captured from the Tucson metropolitan area using walk-in style funnel traps and transported to the laboratory in ventilated plastic animal carriers. Following anes-
Following cannulations, birds were administered a priming dose of [14 C]-inulin (1 Ci) followed by sustained infusion of 0.01 Ciinulin in 0.9% saline at a rate of 0.2 ml/min. Clearance of inulin (CIn ) can be used to estimate the glomerular filtration rate (GFR) as it is freely filtered, but not metabolized, reabsorbed or secreted by the renal tubules. Therefore, measurement of plasma and urine inulin concentrations is considered a gold standard for estimating filtration (Stevens and Levey, 2009; Hinojosa-Laborde et al., 2015). Baseline urine and blood samples were collected for three 10-min clearance periods. As these pre-insulin values were not different, the last 10-minute period was used to establish the baseline values (time 0) prior to administration of insulin. Following the baseline collections, porcine insulin (400 g/kg body mass in 0.9% saline) was administered intravenously and plasma and urine samples were collected at 10 and 20 min post-infusion. Porcine insulin was chosen as it has been used routinely in prior studies of birds (Julian and Abbott, 1998; Pal et al., 2002; Tokushima et al., 2005) as well as in veterinary medicine to treat rare cases of avian hyperglycemia, such as reported in macaws (Gancz et al., 2007). Moreover, the insulin receptor is highly conserved across species (Muggeo et al., 1979). Samples were weighed (assuming a specific gravity of 1) to measure urine flow rate (ml/min). Blood samples were centrifuged to separate the formed elements from plasma and urine samples were centrifuged to remove solid uric acid prior to freezing at −20 ◦ C until further analysis. 2.3. Quantification of plasma and urine glucose and inulin Urine and plasma glucose concentrations were quantified by the glucose oxidase method using a commercially available assay kit (Cat. 10009582; Cayman Chemical Company, Ann Arbor, MI, USA). An aliquot of 10 l urine and/or plasma was used for [14 C]-inulin determination. Samples were dark-adapted for 3 h prior to measuring radioactive inulin by liquid scintillation. GFR was calculated using the clearance formula: GFR = urine inulin concentration (counts/minute) x urine flow rate (ml/min)/plasma inulin concentration (counts/minute). 2.4. Statistical analyses Data in the figures represent changes within individual birds in gray as well as the mean ± SEM of the individual data shown in black. Data in the table are expressed as baseline mean ± SEM and average percent changes from baseline ± SEM. Raw data were analyzed by one-way repeated measures ANOVA and Holm–Sidak posthoc analyses. Percent changes in values were analyzed using paired t-tests. Statistical analyses were completed using SigmaStat 3.0 (Systat Software Inc., San Jose, CA, USA). A probability of ≤ 0.05 was considered statistically significant for all comparisons.
Please cite this article in press as: Sweazea, K.L., et al., Novel role of insulin in the regulation of glucose excretion by mourning doves (Zenaida macroura). Zoology (2017), http://dx.doi.org/10.1016/j.zool.2017.02.006
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Fig. 3. (A) Variations in glucose excretion (mg/min) by the kidney following insulin administration (400 g/kg body mass, i.v.). Data for individual birds are shown in gray symbols, with the same symbols representing the same bird in each graph. Black hexagons represent the mean ± SEM of the individual data. Time 0 (baseline) represents the last 10-min clearance period prior to the insulin dose. Glucose excretion calculated as urine flow rate (ml/min) × UGlu (mg/ml). (B) Variations in the fractional excretion of glucose from baseline following insulin administration (400 g/kg body mass, i.v.). Fractional excretion of glucose calculated as (UGlu /PGlu ) × (UInulin /PInulin )−1 . Data for individual birds are shown in gray symbols, with the same symbols representing the same bird in each graph. Black hexagons represent the mean ± SEM of the individual data. * p < 0.005 from baseline; # p < 0.05 from 10 min; n = 7.
Fig. 2. Variations in (A) glomerular filtration rate (ml/min), (B) filtered load of glucose (mg/min) and (C) glucose reabsorption (mg/min) following insulin administration (400 g/kg body mass, i.v.). Data for individual birds are shown in gray symbols, with the same symbols representing the same bird in each graph. Black hexagons represent the mean ± SEM of the individual data. Time 0 (baseline) represents the last 10-min clearance period prior to the insulin dose. Glomerular filtration rate was estimated by inulin clearance (UInulin (counts/minute) × urine flow rate (ml/min))/PInulin (counts/minute). Filtered load of glucose calculated as GFR (ml/min) × PGlu (mg/ml). Glucose reabsorption calculated as filtered load of glucose (mg/min) − glucose excretion (mg/min). * p < 0.02 from baseline; n = 7.
3. Results Results from the present study show that the infusion of exogenous insulin significantly decreases PGlu concentrations (p < 0.001, F-value = 25.619, df = 6; Fig. 1A). Holm–Sidak post hoc analyses show that PGlu was decreased within 10 min post insulin administration (p = 0.0003) and remained significantly decreased 20 min later (p = 0.00002). Urine glucose concentrations were significantly decreased with insulin administration (p = 0.008, F-value = 7.359, df = 6; Fig. 1B). Holm–Sidak post hoc analyses show that urine glucose concentrations were significantly decreased 20 min after administration as compared to baseline (p = 0.003). The observed trend for increased urine flow rate (p = 0.051, Chi-square = 6.0, df = 2; Fig. 1C) is supported by a significant increase in CIn (p = 0.040, F-value = 4.245, df = 6; Fig. 2A). The filtered load of glucose (PGlu x CIn ) did not significantly change although there was
considerable variability in the data (p = 0.335; Fig. 2B). The increase in CIn was not associated with an increase in systemic blood pressure as it did not change with insulin infusion (data not shown). The data show that renal glucose reabsorption was not significantly altered by insulin (p = 0.291, F-value 1.370, df = 6; Fig. 2C). In contrast, glucose excretion was significantly decreased (p = 0.028, F-value = 4.908, df = 6; Fig. 3A). Similarly, when the data for excretion of glucose are factored by GFR (fractional excretion of glucose), a significant decrease in fractional glucose excretion is observed by 20 min post-administration of insulin (p = 0.003, F-value = 9.706, df = 6; Fig. 3B). Examining the percent change in variables (Table 1) similarly shows that plasma and urine glucose as well as fractional excretion of glucose are significantly decreased following insulin administration whereas urine flow rate, glucose excretion and CIn are significantly increased. 4. Discussion The major findings of the present study are that exogenous insulin significantly lowers PGlu in mourning doves by increasing glomerular filtration leading to augmented renal flow rates. It is often assumed that insulin regulation of PGlu concentrations in birds results from enhanced insulin-mediated glucose uptake into tissues as observed in mammals. However, previous studies have shown that despite significantly lowering PGlu in mourning doves, insulin administration did not result in increased glucose uptake into any of the tissues examined (brain, skeletal muscle,
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Table 1 Insulin-mediated alterations of renal glucose handling in mourning doves (Zenaida macroura). Parameter
Baseline
% Change from baseline at 10 min (p-value, T-statistic)
% Change from baseline at 20 min (p-value, T-statistic)
318.0 ± 15.1 mg/dl
−30.55 ± 7.51 (0.007, 4.06)
−47.57 ± 13.3 (0.012, 3.56)
118.0 ± 35.4 mg/dl
−16.40 ± 7.32 (0.066, 2.24)
−156.3 ± 38.6 (0.007, 3.96)
0.041 ± 0.014 ml/min
38.17 ± 9.34 (0.006, −4.09)
41.50 ± 15.0 (0.033, −2.76)
0.224 ± 0.065 ml/min
46.10 ± 11.7 (0.008, −3.95)
45.66 ± 17.9 (0.043, −2.55)
6.96 ± 2.03 mg/min
31.57 ± 13.4 (0.057, −2.35)
21.65 ± 28.0 (0.478, −0.755)
6.45 ± 1.89 mg/min
31.45 ± 13.5 (0.059, −2.33)
24.41 ± 27.3 (0.414, −0.877)
0.508 ± 0.176 mg/min
29.27 ± 10.0 (0.027, −2.91)
−60.93 ± 48.2 (0.249, −1.28)
0.065 ± 0.012 AU
−38.29 ± 45.3 (0.427, 0.853)
−348.5 ± 257.19 (0.264, 1.23)
Plasma glucose
Urine glucose
Urine flow rate
Glomerular filtration rate
Filtered load of glucose
Glucose reabsorption
Glucose excretion
Fractional excretion
Bold font indicates significant difference from baseline (time 0). Data analyzed by paired t-tests. df = 6 for all comparisons. AU = arbitrary units.
kidney, liver, adipose; Sweazea et al., 2006). This lack of insulinmediated glucose uptake has also been observed in isolated skeletal muscles from house sparrows (Sweazea and Braun, 2005) as well as chickens (Dupont et al., 2004), which may be attributed to the absence of an insulin-responsive GLUT4 transport protein in birds (Duclos et al., 1993; Carver et al., 2001; Seki et al., 2003; Dupont et al., 2004; Sweazea and Braun, 2006; Welch et al., 2013; Coudert et al., 2015). While a separate study reported GLUT4 protein expression in pigeon (Columba livia) leg muscle plasma membrane fractions, examination of the images from the western blots shows only a weak band and no attempt was made to sequence the protein (Diamond and Carruthers, 1993). In contrast to adult birds, insulin has been shown to increase glucose uptake into cardiac and skeletal muscles of Gallus gallus chicks (Tokushima et al., 2005). Similarly, insulin-mediated glucose uptake has been observed in isolated skeletal muscle cells and cultured myotubes from embryonic quail and Muscovy ducklings (Shanahan, 1984; Thomas-Delloye et al., 1999). It should be noted, however, that the antibody used to detect GLUT4 in ducklings reportedly crossreacts with GLUT1 (Thomas-Delloye et al., 1999). While adult avian muscle lacks GLUT4 expression, a recent study found that chicken skeletal muscle expresses a GLUT12 transport protein which may exhibit insulin responsiveness (Coudert et al., 2015). Thus, it is possible that age or species variations exist in, not only insulinmediated glucose transport into tissues, but perhaps regulation of glucose excretion as well. Similar to rodents, percent glucose excretion was significantly augmented in response to insulin in the present study. However, the fractional excretion of glucose (the amount excreted compared to the amount filtered) was not significantly altered at 10 min following the administration of insulin and was actually decreased 20 min post-administration. Therefore, one mechanism by which exogenous insulin may regulate glucose excretion by the avian kidney is through augmenting GFR resulting in an increase in urine flow rate leading to the observed enhanced percent change in glucose excretion at 10 min post-infusion. This may be attributed to reduced glucose contact time with proximal tubule transport proteins, thereby resulting in glucose loss in the urine. In fact, studies of laboratory rodents show that exogenous insulin acutely pro-
motes dilation of the afferent and efferent renal arterioles, with greater dilation of the afferent arteriole, thereby increasing GFR (Tucker et al., 1992). Moreover, others have shown that insulin increases permeability of the filtration barrier in mammals by acting directly on the podocytes through GLUT isoforms 1, 2, 3, 4, and 8 (Coward and Fornoni, 2015). Insulin has also been shown to regulate the contractility of podocytes by altering calcium transport and inducing actin remodeling, thereby increasing permeability of the filtration barrier (Coward and Fornoni, 2015). Thus, it is likely that the increase in CIn observed in the present study following insulin administration may have been due to altered vascular resistance of the glomerular afferent and/or efferent arterioles or changes in permeability of the filtration barrier. Neither the filtered load nor the reabsorption of glucose were significantly changed in response to insulin. The tendency for percent change in filtered glucose to increase 10 min post-infusion was likely compensated by partial recovery of glucose in the proximal renal tubules as evidenced by the tendency for percent change of glucose reabsorption to increase 10 min post-infusion. These findings suggest that under normal physiological conditions the tubular maximum for glucose reabsorption by the proximal renal tubules is not exceeded. However, additional studies are needed to elucidate the precise mechanism of the proposed insulin-mediated alterations of reabsorption in the avian kidney. The acute increase in GFR in doves administered insulin is similar to what has been reported for rats and humans. However, an increase in glucose excretion was not observed in rats and humans. In contrast to these acute effects of exogenous insulin, studies on dogs have shown that chronic intrarenal insulin infusion (7 days) modestly increases GFR but has no effect on renal plasma flow (Hall et al., 1991). Contrary to the observations in the present study where CIn and glucose excretion were increased, studies on isolated rabbit afferent and efferent arterioles suggest that insulin could decrease single nephron filtration rate (SNGFR) (Juncos and Ito, 1993). An increased resistance at the efferent, or a decreased resistance at the afferent, arteriole could mediate the increased CIn observed in the present study in addition to changes in the permeability of the filtration barrier.
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A limitation of the present study is the use of porcine insulin as opposed to an avian isoform. This may be one reason why such high doses of insulin are required before measuring a glucose-lowering effect in birds as chicken insulin binds to receptors (both avian and mammalian) with high affinity (Muggeo et al., 1979). However, it should be noted that studies using chicken insulin at high doses have shown the absence of changes in glucose uptake into tissues of several species of birds, including mourning doves (Sweazea et al., 2006; Sweazea and Braun, 2005). Porcine insulin also binds with relatively high affinity and the insulin receptor is highly conserved across species (Muggeo et al., 1979). Several prior studies have reported using porcine insulin in avian research (Julian and Abbott, 1998; Pal et al., 2002; Tokushima et al., 2005). Moreover, porcine insulin antibodies have been used to block the effects of insulin in prior studies of birds (Dupont et al., 2008; Patwardhan et al., 2004) as well as in veterinary medicine to treat rare cases of hyperglycemia in macaws (Gancz et al., 2007). Therefore, it is unlikely that the use of chicken insulin would have produced a different effect than the porcine insulin used in the current study. In conclusion, the data from the present study demonstrate that the glucose-lowering effect of exogenous insulin may be more complex than previously thought in birds and involves an increase in GFR with the subsequent disposal of glucose in the urine. To our knowledge, a mechanism of glucose regulation through augmented renal excretion by insulin has not been previously demonstrated in birds. However, it should be emphasized that these effects were observed with exogenous administration of insulin. Thus, when administering insulin at doses sufficient to reduce blood glucose in birds, it is important to consider the potential effects of insulin on renal excretion as well as changes in tissue uptake of glucose, which is the primary mechanism of insulin-mediated glucose regulation in mammals. Acknowledgements The authors thank Donna Fergusen and Jennifer Vranish for their assistance with the experiments. This work was supported by new faculty start-up funds provided by Arizona State University (to KLS). References Braun, E.J., Sweazea, K.L., 2008. Glucose regulation in birds. Comp. Biochem. Physiol. B 151, 1–9. Carver, F.M., Shibley Jr., I.A., Pennington, J.S., Pennington, S.N., 2001. Differential expression of glucose transporters during chick embryogenesis. Cell. Mol. Life Sci. 58, 645–652. Coudert, E., Pascal, G., Dupont, J., Simon, J., Cailleau-Audouin, E., Crochet, S., Duclos, M.J., Tesseraud, S., Coustard, S.M., 2015. Phylogenesis and biological characterization of a new glucose transporter in the chicken (Gallus gallus), GLUT12. PLoS ONE 10, e0139517. Coward, R., Fornoni, A., 2015. Insulin signaling: implications for podocyte biology in diabetic kidney disease. Curr. Opin. Nephrol. Hypertens. 24, 104–110. Diamond, D.L., Carruthers, A., 1993. Metabolic control of sugar transport by derepression of cell surface glucose transporters: an insulin-independent recruitment-independent mechanism of regulation. J. Biol. Chem. 25, 6437–6444.
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Please cite this article in press as: Sweazea, K.L., et al., Novel role of insulin in the regulation of glucose excretion by mourning doves (Zenaida macroura). Zoology (2017), http://dx.doi.org/10.1016/j.zool.2017.02.006