In ruminants, most dietary carbohydrates undergo microbial digestion in the forestomach, making gluconeogenesis the sole source of blood glucose.1 Consequently, ruminants have adapted to supply most of their cellular energy needs with volatile fatty acids and have lower circulating blood glucose concentrations, compared with nonruminant species.1 Results of a study1 of fermentation characteristics of the foregut of New World camelids indicate that anaerobic fermentation and volatile fatty acids production occur in a similar manner. However, camelids are well-known to have higher blood glucose concentrations than those of domestic ruminants,1–5 suggesting that gluconeogenesis may be more robust in these species or that there is less insulin secretion or sensitivity, compared with domestic ruminants. These high blood glucose concentrations are thought to be normal in camelids, although a number of hyperglycemic disorders have been described, including stress hyperglycemia,2,4,6 hyperosmolar disorder,7 and diabetes mellitus.8 Interestingly, camelids are also susceptible to disorders of fat metabolism, such as hepatic lipidosis,9,10 hyperketonemia,9,11 high concentrations of circulating nonesterified fatty acids,9 and hyperlipemia,12 which may also relate to the intrinsic insulin resistance and reduced glucose tolerance of New World camelids.5,9,13 Thus, further understanding of camelid insulin and glucose dynamics may help clarify the pathogenesis and improve the prevention and treatment of metabolic disorders in these species.
Intravenous glucose and insulin tolerance testing have been performed in New World camelids, and results indicate that healthy llamas and alpacas have lower glucose tolerance and insulin sensitivity in comparison with other species.3,4,14 The techniques of HEC and HGC enable more precise measurement of insulin secretion and sensitivity and are generally considered to be the gold-standard methods for assessment of insulin sensitivity and glucose-induced insulin secretion, respectively, in many species.15–21 To the authors’ knowledge, results generated by these techniques have not been reported for New World camelids.
The purpose of the study reported here was to use these clamping techniques to assess insulin secretion and sensitivity in 8 healthy alpacas and 8 healthy llamas. The objectives were to determine whether there are differences between these 2 species and gain insights that may be of benefit in the clinical management of their metabolic disorders.
Materials and Methods
Animals—The procedures were performed between June and September 2006. Eight llamas and 8 alpacas from the Oregon State University research herd were used. Llamas consisted of 6 females and 2 castrated males; mean ± SD age was 14.4 ± 4.8 years (range, 7 to 21 years). Alpacas consisted of 6 females and 2 castrated males; mean ± SD age was 6.6 ± 2.0 years (range, 4 to 10 years). Physical examinations were performed on all animals, with no abnormalities found, and all were fed similar diets of grass hay and pasture. The Oregon State University Institutional Animal Care and Use Committee approved all procedures.
In each camelid, the HEC and HGC procedures were performed in a crossover design with a minimum of a 48-hour washout period between clamping procedures. Forty-eight hours prior to the start of the clamping procedures, 14-gauge IV catheters were placed into both jugular veins of each animal. The right jugular catheter was subsequently used for all administered medications, and the left catheter was used for all blood samples. Camelids were then rested in a stall for 24 hours, and food was withheld for 12 hours before performing the clamping procedure. Two camelids were tied side by side in a stall for the duration of the clamping procedure to maintain a calm state during testing. Four clamping procedures were staggered and performed simultaneously because each took 3 to 4 hours. Prior to the start of the clamping procedures, 3 baseline blood glucose concentrations were determined 10 minutes apart with a handheld glucose metera on blood samples obtained from the left jugular catheter. To ensure validity of the glucose meter readings, 99 additional plasma samples obtained during the clamping procedures were assayed by means of an automated biochemical analyzerb and compared with values obtained for whole blood samples assayed via the handheld glucose meter.a A strong correlation (R2 = 0.91) between blood glucose concentrations measured with the handheld glucose metera and corresponding plasma concentrations indicated that the handheld glucose meter provided accurate measurements.
HEC protocol—After baseline blood glucose concentration determination, an IV bolus of human recombinant regular insulinc (1 μU/kg) was administered to rapidly attain supraphysiologic blood insulin concentrations. This was immediately followed by the initiation (time 0 = start of infusion) of a constant rate infusion of insulin via a volumetric infusion pump.d The constant rate infusion of insulin was given at a rate of 6 mU/kg/min by mixing human recombinant regular insulinc in 500 mL of saline (0.9% NaCl) solution, along with 2 mL of homologous blood, which was added to prevent absorption of insulin to plastic surfaces.15 The insulin infusion was continued for 240 minutes. A higher rate of insulin infusion was chosen in comparison with studies15–21 in horses and other species, given the knowledge that New World camelids have poor suppression of the plasma glucose concentration following insulin administration.3,22 A 20% dextrose solution was infused simultaneously through the right catheter via another volumetric infusion pumpd at a rate that maintained blood glucose concentration within 150 ± 5 mg/dL. The level of glycemia was selected as the target on the basis of the knowledge that New World camelids typically have higher circulating blood glucose concentrations in comparison with other species.1,2,4
Blood glucose concentrations were monitored at 10-minute intervals with the handheld glucose meter.a If blood glucose concentration deviated by > 5 mg/dL from the target, the GIF was adjusted empirically on the basis of reported methods.15 Additional plasma samples were obtained at 20-minute intervals by centrifugation of blood samples immediately after collection. Plasma was harvested and stored at −80°C for subsequent measurement of insulin concentrations via an RIA.23,e Once the HEC was completed, the insulin infusion was discontinued and the camelid was given hay to eat. Blood glucose concentration was monitored every 10 to 60 minutes until blood glucose concentrations were stable, and the dextrose infusion was discontinued.
HGC protocol—After baseline blood glucose determination, infusion of approximately 4 to 6 mg of dextrose/kg/min as a 20% solution was started via the right jugular catheter to rapidly achieve and maintain hyperglycemia. Blood glucose concentrations were monitored at 10-minute intervals for 180 minutes, and the dextrose infusion was adjusted empirically, on the basis of reported methods,15 to maintain a blood glucose concentration between 300 and 320 mg/dL. During the HGC, urine samples were collected from as many animals as possible by free catch for determination of urine glucose concentration. Additional plasma samples were obtained at 20-minute intervals by centrifugation of blood samples immediately after collection. Plasma was harvested and stored at −80°C for subsequent measurement of insulin concentrations by use of an RIA.23,e
Calculations—The first 120 minutes of testing via the HEC technique and the first 90 minutes of testing via the HGC technique were considered an equilibration period. Data from 120 to 240 minutes for the HEC procedure and data from 90 to 180 minutes for the HGC were used for calculations.
To assess steady-state conditions for blood glucose, a space correction was calculated at 10-minute intervals for consecutive measurements of glucose concentration by use of the following equation15:
where G1 and G2 are glucose concentrations at the beginning and end of a time interval, T is the time interval (10 minutes), bw is body weight in kg, and (0.19 × bw) is the glucose space in liters.15 The formula reduces to the following:
The GIF during the HEC protocol was calculated for each camelid as milligrams of glucose per kilogram per minute. In addition, M was also calculated as follows15:
The GIF during the HGC protocol was calculated for each camelid as milligrams of glucose per kilogram per minute. To calculate M during the HGC, ideally a correction for urinary glucose loss should be made in addition to the space correction.15 Attempts were made to calculate urine glucose concentration by collecting urine from as many animals as possible at the end of the HGC.15 Unfortunately, only 3 samples could be collected (mean urinary glucose concentration, 430.3 mg/dL). Due to this low sample number, the small resulting correction calculation for urinary glucose loss was not performed (mean urinary glucose correction value, 0.03 mg/kg/min). Thus, during the HGC, M was calculated as follows:
The M:I was also calculated during the HGC. The M:I is used as an indication of the quantity of glucose metabolized per unit of insulin in plasma and thus is used as a measure of insulin sensitivity.15,17
Statistical analysis—Values are expressed as mean ± SD. For the HEC and HGC techniques, values were compared between llamas and alpacas. Mean resting glucose and insulin concentrations were compared via a Student t test. When comparing the difference between the results obtained from llamas and alpacas, the groups were assessed with a 2-way ANOVA for repeated measures. Differences between mean values were detected by means of a Tukey test. Statistical analyses were conducted via computer software programs.f,g For all analyses, values of P ≤ 0.05 were considered significant.
Results
HEC protocol—Hyperinsulinemia was attained approximately 20 minutes after the HEC technique was initiated, and steady-state conditions for blood glucose and plasma insulin concentrations were achieved in each camelid (Figure 1). Results for baseline blood glucose and plasma insulin concentrations, GIF, M, and insulin concentration during 120 to 240 minutes of the HEC protocol were summarized (Table 1).
Mean ± SD serum insulin concentrations (dotted lines) and blood glucose concentrations (solid lines) during testing with the HEC technique in 8 llamas (white squares) and 8 alpacas (black triangles). Insulin infusion and onset of testing were designated as time 0. The first 120 minutes was considered an equilibration period. Glucose concentrations from 120 to 240 minutes during the HEC were not significantly different between llamas and alpacas. Insulin concentrations from 120 to 240 minutes during the HEC procedure were significantly (P = 0.01) greater in llamas, compared with those in alpacas.
Citation: American Journal of Veterinary Research 74, 1; 10.2460/ajvr.74.1.96
Mean ± SD values obtained during HEC and HGC techniques performed on 8 alpacas and 8 llamas.
| HEC | HGC | |||||
|---|---|---|---|---|---|---|
| Variable | Llama | Alpaca | P value | Llama | Alpaca | P value |
| Baseline blood glucose (mg/dL) | 145.6 ± 27.9 | 131.8 ± 8.7 | 0.20 | 139.6 ± 17.7 | 131.0 ± 18.1 | 0.60 |
| Baseline insulin (μU/mL) | 5.4 ± 2.0 | 5.6 ± 2.2 | 0.90 | 4.5 ± 1.1 | 6.1 ± 2.0 | 0.06 |
| Steady-state glucose (mg/dL) | 146.9 ± 7.3 | 148.5 ± 12.7 | 0.39 | 365.5 ± 29.4 | 325.9 ± 17.8 | 0.001 |
| Steady-state insulin (μU/mL) | 3,504.4 ± 189.6 | 1,817.6 ± 589.3 | < 0.001 | 15.8 ± 8.0 | 10.4 ± 2.3 | 0.06 |
| Steady-state GIF (mg/kg/min) | 2.8 ± 0.9 | 2.9 ± 0.9 | 0.58 | 1.6 ± 0.9 | 0.9 ± 0.9 | 0.08 |
| Steady-state M (mg/kg/min) | 2.8 ± 1.0 | 2.9 ± 0.8 | 0.60 | 1.7 ± 0.8 | 0.9 ± 0.5 | 0.005 |
| Steady-state M:I | — | — | — | 13.4 ± 7.2 | 9.7 ± 6.3 | 0.21 |
— = Not applicable.
Statistical comparisons were made between llamas and alpacas, within HEC or HGC. Values of P ≤ 0.05 were considered significant.
Mean ± baseline blood glucose concentrations and plasma insulin concentrations obtained prior to testing were similar between the HEC technique performed in llamas and alpacas. During 120 to 240 minutes of the HEC procedure, blood glucose concentrations were similar between llamas and alpacas. However, plasma insulin concentrations during this same interval were significantly higher in llamas, compared with alpacas (Table 1; Figure 1). Mean ± SD GIF and M were also similar between llamas and alpacas (Figure 2).
Mean ± SD GIF required to maintain euglycemia during 30-minute periods of testing with the HEC technique in 8 llamas (white bars) and 8 alpacas (black bars). Insulin infusion and onset of testing with the HEC technique were designated as time 0. The first 120 minutes was considered an equilibration period. The GIFs required to maintain euglycemia between 120 and 240 minutes of testing were not significantly different between llamas and alpacas.
Citation: American Journal of Veterinary Research 74, 1; 10.2460/ajvr.74.1.96
HGC protocol—Hyperglycemia was attained approximately 40 to 60 minutes after the HGC technique was initiated, and steady-state conditions for blood glucose and plasma insulin concentrations were achieved in each camelid (Figure 3). Results for baseline blood glucose and plasma insulin concentrations, GIF, M, insulin concentration, and M:I after 90 to 180 minutes of starting the HGC procedure were summarized (Table 1).
Mean ± SD serum insulin concentrations (dotted lines) and whole-blood glucose concentrations (solid lines) during testing with the HGC technique in 8 llamas (white squares) and 8 alpacas (black triangles). Glucose infusion and onset of testing were designated as time 0. The first 90 minutes was considered an equilibration period. Glucose concentrations from 90 to 180 minutes during the HGC were significantly (P = 0.001) higher in llamas, compared with those in alpacas; however, insulin concentrations were similar.
Citation: American Journal of Veterinary Research 74, 1; 10.2460/ajvr.74.1.96
Mean ± SD baseline blood glucose concentrations and plasma insulin concentrations obtained prior to testing were similar between llamas and alpacas (Table 1). During 90 to 180 minutes of the HGC procedure, blood glucose concentrations were higher in llamas than in alpacas, although plasma insulin concentrations during this same interval were similar between llamas and alpacas (Figure 3). The higher blood glucose concentrations in llamas can be explained by the higher GIF in llamas, compared with that in alpacas during this time interval (Figure 4). The M:I during 90 to 180 minutes of the HGC procedure was similar between alpacas and llamas.
Mean ± SD GIF required to maintain hyperglycemia during 30-minute periods of testing with the HGC technique in 8 llamas (white bars) and 8 alpacas (black bars). Glucose infusion and onset of testing with the HGC technique were designated as time 0. The first 90 minutes was considered an equilibration period. The GIFs required to maintain hyperglycemia between 90 and 180 minutes of testing were higher in llamas, compared with those in alpacas.
Citation: American Journal of Veterinary Research 74, 1; 10.2460/ajvr.74.1.96
Insulin RIA validation—A commercial RIAe used in veterinary diagnostic and research testing designed for quantification of insulin in serum and plasma was used. The assay reagents included porcine insulin antiserum and iodine 125–labeled human insulin, with phase separation achieved via double-antibody immunoprecipitation. For intra-assay precision, 3 camelid samples (mean, 3.45, 4.13, and 6.86 μU/mL) tested 10 times in the same assay had a mean percentage coefficient of variation of 7.9% (10.5%, 7.4%, and 5.7%, respectively). For interassay precision, the mean percentage coefficient of variation for 3 camelid pool samples (mean values, 2.60, 4.98, and 18.84 μU/mL) tested in 10 assays during a 2-week period was 16.4% (22.2%, 18.6%, and 8.4%, respectively). The assay sensitivity determined by the manufacturer is 1.61 μU/mL. For accuracy, 3 endogenous camelid serum samples were spiked with 5 quantities of exogenous insulin (5, 10, 25, 50, and 100 μU/mL) from a commercial source.h The mean percentage recovery for the 3 samples was 105% (individual values, 109%, 109%, and 98%). In addition, 3 endogenous camelid serum samples were diluted serially 4 times (to 1:16). The mean ratio of the actual result to the expected result was 102% (individual values, 105%, 102%, and 100%).
Discussion
To the authors’ knowledge, this is the first description of the use of the glucose clamp technique to examine glucose metabolism in these species, and the results indicated that New World camelids had profoundly lower insulin secretion and action in comparison with several other species. Our findings were in accordance with studies3,4,14 of IV glucose tolerance testing and IV insulin tolerance testing in New World camelids, in which healthy llamas and alpacas were also found to have lower insulin secretion and insulin sensitivity in comparison with other species. In the study reported here, no sexually intact males were available to study and it is possible that castration may have had an effect on individual sensitivity to insulin in the 4 geldings.
Glucose metabolism can be assessed with methods such as oral and IV glucose tolerance testing, but the use of HEC and HGC techniques is generally regarded as a more accurate method of assessing insulin sensitivity and glucose tolerance.15,18,20 The protocols used in the study reported here were based on methods used in another study,15 with adjustments made on the basis of prior knowledge of insulin and glucose metabolism in camelids.2–4 These adjustments included a higher rate of insulin infusion during the HEC procedure than that used in other species of other studies18,19,21 in an effort to compensate for the camelids’ relative insulin insensitivity and lack of a substantial endogenous pancreatic β-cell response.24 In addition, a higher blood glucose concentration of 150 ± 5% mg/dL was chosen for euglycemia during the HEC procedure, and a blood glucose concentration of 300 to 320 mg/dL was chosen for hyperglycemia during the HGC on the basis of prior knowledge that New World camelids have higher blood glucose concentrations than domestic ruminants.1–4 Clamping procedures were also prolonged in comparison with procedures performed in other species, to ensure that time-dependent changes in insulin secretion and action could be detected.15,18
In the present study, insulin-mediated glucose uptake was remarkably lower in alpacas and llamas than has been reported in other species. Interestingly, to maintain euglycemia in the setting of the extreme hyperinsulinemia attained during the HEC procedure, New World camelids required only a doubling in the GIF over 3 hours (Figure 2), compared with a 4-fold increase in glucose infusion required by clinically normal humans and cats.15,16 With the HEC technique, the rate of glucose required per kilogram of body weight to maintain euglycemia during the first 2 hours of the clamp was markedly lower than that required by other species.15,18
In both Old World camels17 and the New World camelids of the present study, despite comparable insulin infusion rates, the maximal insulin concentrations achieved during the HEC procedures were markedly higher in comparison with other species, which may be a result of the lower rate of removal of plasma insulin.17 It has been suggested that, in both Old World and New World camelids, insulin-degrading enzyme may be underexpressed in comparison with other species because of their naturally low basal insulin concentrations.17
When results from HEC performed on alpacas and llamas were compared, no significant difference was found between blood glucose concentrations during the clamping procedures. However, plasma insulin concentrations were markedly higher in llamas during the clamping procedures, compared with those in alpacas. The difference for this is unclear but may be related to the suggested higher volume of distribution of extracellular fluid in alpacas4 or may have been due to a decrease in the use, uptake, or clearance of insulin in llamas. Despite the markedly higher concentrations of plasma insulin in llamas, compared with alpacas, during the HEC, the GIFs were the same, indicating that llamas and alpacas have similar overall whole-body insulin sensitivity. The M:I obtained during the HGC can also be used as an indication of insulin sensitivity,15 and this value was also found to be similar between alpacas and llamas (Table 1).
Results of testing by the HGC technique, in both llamas and alpacas, revealed that during steady-state hyperglycemia, the mean insulin concentrations were substantially lower than those observed in horses and other species.15,18 This corroborates findings of reduced pancreatic β-cell response to hyperglycemia in a study4 of New World camelids, compared with other species evaluated under similar conditions. It is also possible that the finding of a low insulin response to glucose in camelids is related to poor specificity of the insulin RIA for camelid insulin, given that the insulin RIAs used in these studies have not been specifically validated in this species. However, the active region of insulin is remarkably conserved among species, and the antibody used in the RIA is against this conserved region.17 In further support of the low pancreatic response to hyperglycemia in camelids, the GIF required to maintain hyperglycemia was markedly lower than that required in other species18 during the HGCs, indicating a remarkably low rate of glucose clearance in these animals.
Overall, it is apparent that insulin-mediated glucose uptake in camelids is low in comparison with other species. This might support a hypothesis that insulin-dependent GLUT-4 is less important in glucose transport in camelids. Recently, western blotting was used to measure GLUT-1 and GLUT-4 protein content in skeletal muscle tissue of Old World camels.25 Glucose transporter type-4 was found to be present in the skeletal muscle of camels,25 suggesting that the reduced insulin-stimulated glucose uptake in camelids may be due to an interruption in GLUT-4 translocation into the myocyte plasma membrane, rather than a decrease in overall quantity of GLUT-4. That study25 also found GLUT-1 to be the predominant glucose transporter in camels and other ruminants, suggesting that GLUT-1 may be of much greater importance in the whole-body usage of glucose in forestomach-fermenting herbivores than in monogastric omnivores.25 Glucose uptake by GLUT-1 occurs via facilitated diffusion along a glucose concentration gradient. Thus, a basal hyperglycemia could promote the supply of glucose to the cell in this manner. Interestingly, high blood glucose concentrations are also found in reindeer, another ruminant species that, similar to camelids, have evolved to exist in a harsh environment.17 On the basis of these observations, it is possible that there is an evolutionary advantage in limiting glucose uptake during severe feeding restrictions.17
It appears that high basal blood glucose concentrations in New and Old World camelids are associated with a combination of low pancreatic production of insulin and poor insulin responsiveness, and it is likely that the long-term basal supply of cells with glucose in this species is through insulin-independent GLUT-1.25 Glucose and insulin metabolism in camelids has similarities to non–insulin-dependent diabetes mellitus in humans, in which insulin-stimulated glucose usage is reduced and may be compensated for by insulin-independent glucose uptake via GLUT-1.25 Although high basal plasma glucose concentrations in camelids may be a regulatory necessity to maintain glucose requirements of muscle cells, disorders such as hyperosmolar disorder, hepatic lipidosis, ketosis, and hyperlipemia9–11 develop when excessive glucose or lipid constituents accumulate in the blood. These disorders of fat and glucose metabolism may have different etiologies; however, their simultaneous occurrence in some camelids as well as their linked hormonal control suggests that the conditions may be associated, with the result that this species is susceptible to disorders of energy balance.5,13
ABBREVIATIONS
| GIF | Glucose infusion rate |
| GLUT | Glucose transporter type |
| HEC | Hyperinsulinemic euglycemic clamping |
| HGC | Hyperglycemic clamping |
| M | Mean glucose infusion rate corrected for changes in glucose space |
| M:I | Ratio of mean glucose infusion rate corrected for changes in glucose space to mean insulin concentration |
| RIA | Radioimmunoassay |
Precision QID glucose monitoring system, Abbott Laboratories Inc, Bedford, Mass.
Hitachi 717 biochemical analyzer, Boehringer Mannheim Diagnostics, Division of Boehringer Mannheim Corp, Indianapolis, Ind.
Novolin R, human insulin injection, recombinant DNA origin USP, Novo Nordisk Pharmaceutical Industries, Clayton, NC.
FLO-GARD Volumetric Infusion Pump, Baxter International Inc, Deerfield, Ill.
Porcine Insulin RIA, Millipore Corp, Billerica, Mass.
GraphPad Prism, version 4.00, GraphPad Software Inc, San Diego, Calif.
SigmaStat, version 2.0, SPSS Inc, Chicago, Ill.
045K14702, Sigma-Aldrich Corp, St Louis, Mo.
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