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Keywords:

  • horse;
  • insulin;
  • C-peptide;
  • insulin resistance

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Manufacturers' addresses
  9. References

Reasons for performing study: Hyperinsulinaemia is detected in horses with insulin resistance (IR) and has previously been attributed to increased pancreatic insulin secretion. Connecting peptide (C-peptide) can be measured to assess pancreatic function because it is secreted in equimolar amounts with insulin and does not undergo hepatic clearance.

Hypothesis: A human double antibody radioimmunoassay (RIA) detects C-peptide in equine serum and concentrations would reflect responses to different stimuli and conditions.

Methods: A validation procedure was performed to assess the RIA. Six mature mares were selected and somatostatin administered i.v. as a primed continuous rate infusion, followed by 50 nmol human C-peptide i.v. Insulin and C-peptide concentrations were measured in horses (n = 6) undergoing an insulin-modified frequently sampled i.v. glucose tolerance test, and in horses with insulin resistance (n = 10) or normal insulin sensitivity (n = 20).

Results: A human RIA was validated for use with equine sera. Endogenous C-peptide secretion was suppressed by somatostatin and median (range) clearance rate was 0.83 (0.15–1.61) ml/min/kg bwt. Mean ± s.d. C-peptide-to-insulin ratio significantly (P = 0.004) decreased during the glucose tolerance test from 3.60 ± 1.95 prior to infusion to 1.03 ± 0.18 during the first 20 min following dextrose administration. Median C-peptide and insulin concentrations were 1.5- and 9.5-fold higher, respectively in horses with IR, compared with healthy horses.

Conclusions: Endogenous C-peptide secretion decreases in response to somatostatin and increases after dextrose infusion. Results suggest that relative insulin clearance decreases as pancreatic secretion increases in response to dextrose infusion. Hyperinsulinaemia in insulin resistant horses may be associated with both increased insulin secretion and decreased insulin clearance.

Potential relevance: Both C-peptide and insulin concentrations should be measured to assess pancreatic secretion and insulin clearance in horses.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Manufacturers' addresses
  9. References

Insulin is a peptide hormone containing A and B chains that is synthesised by the β-cells of the pancreatic islets of Langerhans (Wahren et al. 2000; Wilcox 2005). Upon translation of insulin mRNA, pre-proinsulin is generated and this molecule is comprised of a signal peptide, the B chain, the connecting peptide (C-peptide) and the A chain (Wilcox 2005). Proinsulin is synthesised by removal of the signal peptide from pre-proinsulin in the ribosomes of the rough endoplasmic reticulum of β-cells. Proinsulin is then transported to the Golgi apparatus, where it forms soluble, zinc-containing hexamers. Enzymes acting outside the Golgi convert proinsulin to insulin by cleaving the C-peptide from the molecule during the formation of immature storage vesicles. C-peptide has an important role in insulin synthesis by linking the A and B chains of insulin in a manner that facilitates folding and interchain disulphide bond formation (Wahren et al. 2000). Proteolytic removal of C-peptide from proinsulin allows the carboxy terminal of the B-chain of the insulin molecule to assume a conformation that facilitates interaction with the insulin receptor. Insulin and C-peptide are cosecreted in equimolar amounts when mature granules release their contents into the portal circulation (Wilcox 2005).

Compensatory hyperinsulinaemia associated with insulin resistance typically predates the development of human type II DM (Brunton et al. 2006). Similarly, horses suffering from decreased insulin sensitivity triggered by endotoxaemia or obesity exhibit an augmented pancreatic insulin response to exogenous glucose challenge, indicated by higher acute insulin response to glucose (AIRg) values (Hoffman et al. 2003; Toth et al. 2008). In man, overproduction of insulin by pancreatic β-cells eventually induces β-cell exhaustion, impaired insulin secretion and relative insulin deficiency (Brunton et al. 2006). As glucose levels rise, β-cell function further deteriorates, with diminishing sensitivity to glucose, ultimately resulting in the development of type II DM (Wilcox 2005). Insulin-resistant horses and ponies rarely develop pancreatic β-cell insufficiency and usually maintain a state of compensated IR (Johnson et al. 2005; Treiber et al. 2005). Nevertheless, DM has been described in a Spanish Mustang in association with IR (Johnson et al. 2005) and hyperglycaemia was also registered in 2 ponies suffering from insulin resistance, which indicates failed compensation (Treiber et al. 2006). Therefore, estimation of the ability of pancreatic β-cells to secrete insulin is important for the accurate assessment of glucose homeostasis.

During minimal model analysis of the frequently sampled i.v. glucose tolerance test (FSIGTT), serum insulin concentrations measured during the first 10 min after exogenous glucose administration provide an estimate for AIRg (Tiley et al. 2007). The AIRg reflects the ability of pancreatic β-cells to secrete insulin after glucose challenge. However, it has been demonstrated in man that approximately 60% of insulin secreted into the portal vein is removed by the liver (Wilcox 2005), therefore serum insulin concentrations measured in peripheral blood provide only a crude estimate of pancreatic function (Hovorka et al. 1998; Kjems et al. 2000). It is therefore preferable to measure serum C-peptide concentrations when assessing pancreatic function, because this peptide is not extracted by the liver (Hovorka et al. 1998; Kjems et al. 2000). Higher AIRg values have been associated with IR in horses (Hoffman et al. 2003; Toth et al. 2008), but it has not been determined whether this occurs as a result of increased pancreatic insulin secretion or reduced hepatic insulin extraction. This could be investigated by simultaneously measuring insulin concentrations in blood samples obtained from both the portal vein and hepatic vein, but the measurement of C-peptide concentrations from peripheral blood samples is a more practical alternative.

A literature search revealed only one report describing C-peptide measurements in horses; but the authors did not state whether the test was validated for the horse (Johnson et al. 2005). Characteristics of C-peptide kinetics in horses, including clearance rate and plasma half life have not been reported to date. C-peptide metabolism has been established in dogs by blocking endogenous C-peptide secretion using a somatostatin infusion (Polonsky et al. 1983). Exogenous C-peptide was then administered to investigate hepatic metabolism and metabolic clearance rate of C-peptide.

The aims of the present study were: 1) to measure C-peptide concentrations in equine serum using a human double antibody C-peptide radioimmunoassay (RIA); 2) to establish clearance rate of C-peptide; and 3) to describe C-peptide kinetics during the FSIGTT in horses. It was hypothesised that the human double antibody C-peptide RIA would be able to detect equine C-peptide and that concentrations would increase as the pancreas responds to exogenous glucose during the FSIGTT.

Materials and methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Manufacturers' addresses
  9. References

Horses

Six mares from the University of Tennessee teaching and research herd were evaluated during the first 2 phases of the study. Horses were admitted to the teaching hospital in pairs and housed separately in 3.7 × 3.7 m stalls. To eliminate differences attributable to sex, only mares were selected. Horses were aged 6–13 years (mean 9.3 years; median 9 years); breeds included Quarter Horse/Tennessee Walking Horse crossbreds (n = 3), Quarter Horse (n = 2) and Standardbred (n = 1). Horses were weighed at the time of admission and weights ranged from 461–523 kg (mean 497 kg; median 501 kg). Body condition score (on a scale of 1–9) ranged from 4–6 (Henneke et al. 1983). Daily physical examinations were performed throughout the study period. Grass hay and water were provided ad libitum, and each horse acclimated to its new environment for approximately 72 h before experiments started. The study protocol was approved by the University of Tennessee Institutional Animal Care and Use Committee.

Experimental design

The study was designed initially to be conducted in 2 phases. The aim of the first phase was to evaluate C-peptide clearance in horses after suppression of pancreatic insulin secretion by somatostatin, while the second phase involved measurement of C-peptide concentrations in equine sera obtained during the FSIGTT in the same 6 horses.

During the second phase, it was noted that C-peptide-to-insulin ratio was markedly lower in one of the 6 horses in the study and further investigation determined that this horse suffered from IR. To investigate this finding, a third phase was added to the study to evaluate C-peptide-to-insulin ratios in horses with normal and decreased insulin sensitivity. Resting (baseline samples from FSIGTTs) blood samples were collected from 20 healthy mares with insulin sensitivity (SI) values ≥1.5 × 10-4 l/min/mu detected in a previous study (Toth et al. 2010), and from 10 horses with IR. Quarter Horse/Tennessee Walking Horse (n = 8), Quarter Horse (n = 10), Thoroughbred (n = 1) and Tennessee Walking Horse (n = 1) breeds were represented in the healthy group, compared with Paso Fino (n = 3), Arabian (n = 2), Morgan (n = 1), Kentucky Mountain Horse (n = 1), Missouri Fox Trotter (n = 1), Spotted Saddle Horse (n = 1) and Quarter Horse/Tennessee Walking Horse (n = 1) breeds in the insulin resistant group, which contained 5 geldings and 5 mares. Mean ± s.d. age for healthy mares (n = 20) was 11.1 ± 3.7 years, compared with 14.7 ± 3.2 years for horses with IR (n = 10). Insulin resistance was defined by both the maintenance of plasma glucose concentrations above the pre-injection value for 45 min during the combined glucose-insulin test (Frank et al. 2006) and SI values <1.0 × 10-4 l/min/mu from minimal model analysis of FSIGTT data. C-peptide and insulin concentrations were measured in these 30 horses and C-peptide-to-insulin ratios were calculated.

C-peptide kinetics

On the first day of the study, 14 gauge polypropylene catheters (Abbocath-T)1 were inserted into the right and left jugular veins. Patency of i.v. catheters was maintained by infusion of 5 ml of saline solution containing heparin (4 u/ml) every 6 h. Tests were performed 24 h after catheter insertion. Blood samples (n = 3) were collected at -100, -95 and -90 min to measure the baseline endogenous C-peptide concentration and then, at time = -90 min, a 500 µg bolus of (D-Trp8)-somatostatin-142 was administered followed by a continuous rate infusion (CRI) of 500 µg/h somatostatin for 330 min. Blood samples (n = 8) were collected for 90 min after initiation of the somatostatin infusion at time = -75, -60, -45, -30, -15, -10, -5, 0 min. At 0 min, a 50 nmol bolus of biosynthetic human C-peptide3 was administered i.v. Further blood samples (n = 32) were collected at 1, 2, 3, 4, 5, 6, 7, 8, 10, 12, 14, 16, 19, 22, 23, 24, 25, 27, 30, 35, 40, 50, 60, 70, 80, 90, 100, 120, 150, 180, 210 and 240 min. Blood samples were collected from the second i.v. catheter using an injection cap and infusion set1 (butterfly; length, 30 cm; internal diameter, 0.014 cm). At each time point, 3 ml blood was withdrawn from the catheter and discarded. A 4 ml blood sample was collected subsequently, and the catheter flushed with 5 ml of saline solution containing heparin. Blood was transferred to a tube without anticoagulant. Samples were allowed to clot at 22°C for 1h and serum was harvested via low-speed (1000 ×g) centrifugation. Serum samples were stored at -20°C until analysed.

FSIGTT procedure

This procedure was performed during the second phase of the study. Each horse was weighed and a 14 gauge polypropylene catheter was inserted into the left jugular vein one day before the FSIGTT. Patency of the i.v. catheter was maintained by infusion of 5 ml of saline solution containing heparin into the catheter every 6 h. During tests, horses were allowed access to grass hay and water ad libitum. An injection cap and infusion set (butterfly, length, 30 cm; internal diameter, 0.014 cm) were attached to the catheter. The FSIGTT procedure first described for use in horses by Hoffman et al. (2003) was followed. Briefly, a 300 mg/kg bwt bolus of 50% (wt/vol) dextrose (Dextrose 50% injection)1 solution was administered to each horse via the infusion line and catheter, followed by infusion of 20 ml saline solution containing heparin. Blood samples were collected via the catheter 10, 5 and 0 min before and 1, 2, 3, 4, 5, 6, 7, 8, 10, 12, 14, 16 and 19 min after infusion of dextrose. At 20 min, 30 mu/kg bwt regular insulin (Humulin R)4 was administered followed by another infusion of 20 ml saline solution containing heparin. Blood samples were subsequently collected via the catheter at 22, 23, 24, 25, 27, 30, 35, 40, 50, 60, 70, 80, 90, 100, 120, 150 and 180 min after the dextrose bolus infusion. At each time point, 3 ml of blood was withdrawn from the infusion line and discarded. A 6 ml blood sample was then collected, followed by infusion of 5 ml of saline solution containing heparin. Half the volume of the blood sample was transferred to a tube containing sodium fluoride and potassium oxalate, which was immediately cooled on ice and then refrigerated. The remaining blood was transferred to a tube containing no anticoagulant. These samples were allowed to clot at 22°C for 1 h and serum was harvested via low-speed (1000 × g) centrifugation. Plasma and serum samples were stored at -20°C until further analysed.

C-peptide RIA

Serum concentrations of C-peptide were measured using a human double antibody RIA kit5 previously used in horses (Johnson et al. 2005). Samples were assayed in duplicate in accordance with instructions provided by the manufacturer. The accuracy of the assay was assessed by recovery and parallelism. Dilutional parallelism was evaluated by diluting 3 aliquots of 6 separate equine serum samples to 1:2, 1:4 and 1:8 dilutions of their initial concentration using sterile nanopure water. Once the concentration for the undiluted sample was obtained, expected (E) concentrations were calculated according to the dilutions used. Observed (O) concentrations were compared with expected values and a ratio was calculated, which was expressed as a percentage (%O/E). Spiking and recovery was assessed by adding 50 µl solutions containing 195.3, 71.2 and 21.8 ng/ml human C-peptide to 950 µl of 3 aliquots of 6 different equine serum samples. Expected values were calculated by calculating the contribution of the added human C-peptide plus the initial value for horse serum alone. For each spiked sample, observed concentrations were compared with expected values and %O/E was calculated. Since a human antibody RIA was used in this study, results are expressed as human equivalents of immunoreactive C-peptide (ir-C-peptide HE). C-peptide values were converted from ng/ml to pmol/ml using a conversion factor of 0.333.

Serum insulin concentrations

Insulin concentrations were determined by use of a radioimmunoassay (Coat-a-Count)5 previously validated for equine insulin (Freestone et al. 1991). Each sample was assayed in duplicate and intra-assay coefficients of variation of <10% were required for acceptance of assay results. Insulin values were converted from µu/ml to pmol/ml using a conversion factor of 6.945 × 10-3. Percentage differences between insulin and C-peptide concentrations were calculated to estimate hepatic insulin clearance by subtracting the insulin concentration from the C-peptide concentration and expressing the percent change relative to the C-peptide concentration.

Statistical analysis

Clearance of C-peptide was calculated using methods described previously (Wastney et al. 1999; Stefanovski et al. 2003) and computer software6. Serum C-peptide concentrations measured before and during the first 90 min of the somatostatin CRI were averaged for each horse and compared using mixed model analysis of variance (one-tailed test). Mixed model analysis of variance with repeated measures was used to compare C-peptide concentrations measured before and after dextrose administration using statistical software7. During the FSIGTT in the second phase of the study, C-peptide-to-insulin ratios were averaged in each horse for the 3 baseline samples and for samples obtained between 1 and 19 min (n = 13 samples) and subsequently compared using mixed model analysis of variance. Log transformation was used to correct for non-normally distributed data. No comparisons were made after the 19 min time point because of the confounding effect of exogenous insulin. During the third phase of the study, data did not fit a normal distribution, so median (range) values are reported and the nonparametric Mann-Whitney U test was applied to compare groups. Spearman correlation coefficients were also calculated to compare variables. Significance was defined at a value of P<0.05.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Manufacturers' addresses
  9. References

Validation procedures for the C-peptide RIA revealed a mean ± s.d. percent O:E ratio for 6 serially diluted samples of 119 ± 25% and parallelism. Observed-to-expected spike and recovery ratios for 6 samples with 3 spiking concentrations ranged from 112–136%, mean ± s.d. 123 ± 8%.

Experimental procedures were well tolerated throughout the study and no abnormalities were detected when daily physical examinations were performed. Evaluation of baseline insulin concentrations and SI values revealed that one of the 6 horses included in the study was insulin resistant; therefore data obtained from this horse was excluded from further analysis. The mean resting insulin concentration for this horse was 58.3 µu/ml and mean SI was determined to be 0.23 × 10-4 l/min/mu. Resting serum ir-C-peptide HE concentration was 0.24 pmol/ml for this horse.

Data from one horse could not be fitted to the compartmental model to determine clearance rate. Median (n = 4 horses) clearance rate of ir-C-peptide HE was 0.83 ml/min/kg bwt (range: 0.15-1.61 ml/min/kg bwt). Mean ± s.d. ir-C-peptide HE concentration for 4 horses decreased (P = 0.030; one-tailed test) from baseline 0.23 ± 0.12 pmol/ml at time = -100, -95 and -90 min to 0.11 ± 0.06 pmol/ml during the first 90 min of somatostatin infusion, prior to exogenous C-peptide administration (average obtained from 8 samples). When 50 nmol biosynthetic human C-peptide was administered at time = 0, mean ir-C-peptide HE concentration peaked at 2.96 ± 0.67 pmol/ml at 1 min and then gradually decreased throughout the experiment (Fig 1).

image

Figure 1. Mean±s.e. concentration of human equivalents of immunoreactive C-peptide in serum collected from horses (n=4) after administration of 50 nmol biosynthetic human C-peptide at 0 min. Endogenous C-peptide secretion was suppressed by infusion of 500 µg biosynthetic human somatostatin at -90 min followed by 500 µg/h for 330 min.

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Measurements of ir-C-peptide HE were successfully performed from blood samples collected during the FSIGTT in all 5 horses. For 100 min after i.v. dextrose administration, ir-C-peptide HE concentrations remained significantly elevated (P = 0.042 at 100 min) relative to baseline (Fig 2). Mean ± s.d. ir-C-peptide HE-to-insulin ratio was 3.6 ± 1.95 preceding glucose administration, and this ratio decreased significantly (P = 0.004) to 1.03 ± 0.18 during the first 20 min following dextrose administration (Fig 3).

image

Figure 2. Mean±s.e. concentrations of human equivalents of immunoreactive C-peptide measured during the frequently sampled i.v. glucose tolerance test in 5 healthy horses. Dextrose (300 mg/kg bwt) was infused i.v. at time=0 followed by 30 mu/kg bwt insulin after 20 min.

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image

Figure 3. Mean±s.e. human equivalents of immunoreactive C-peptide-to-insulin ratio from 3 baseline (pre-injection) samples and 13 samples collected during the first 19 min of the frequently sampled i.v. glucose tolerance test in 5 healthy horses.

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In the third phase of the study, median resting serum insulin (P<0.001) and C-peptide (P = 0.007) concentrations differed significantly between healthy and IR horses (Table 1). Median SI for insulin resistant horses was approximately 5% that of healthy horses. Median C-peptide and insulin concentrations were 1.5- and 9.5-fold higher, respectively in horses with IR, when compared with healthy horses. Insulin concentrations were 74% lower than C-peptide concentrations in healthy horses and 5% higher in horses with IR. Accordingly, the median C-peptide-toinsulin ratio for horses with IR was lower (P<0.001) than that of horses with normal insulin sensitivity. Serum insulin and C-peptide concentrations were negatively correlated (r = -0.73 and -0.50; P<0.001 for both) and C-peptide-to-insulin ratio was positively correlated (r =0.76) with SI.

Table 1. Median (range) sensitivity to insulin (SI) values, resting serum C-peptide and insulin concentrations in 20 healthy horses and 10 horses with insulin resistance (IR)
 Healthy horses (n = 20)Horses with IR (n = 10)P
  • *

    Percentage difference between resting (baseline samples from frequently sampled i.v. glucose tolerance tests) serum C-peptide and insulin concentrations as an estimate of hepatic insulin clearance; calculated by subtracting the insulin concentration from the C-peptide concentration and expressing the percent change relative to the C-peptide concentration.

SI (×10-4; l/min/mu)3.55 (1.64–6.62)0.19 (0.05–0.95)<0.001
Insulin (µu/ml)9.2 (2.8–24.3)82.2 (18.0–650.7)<0.001
Insulin (pmol/ml)0.06 (0.02–0.17)0.57 (0.12–4.52)<0.001
ir-C-peptide HE (pmol/ml)0.27 (0.12–0.43)0.41 (0.16–1.36)0.007
C-peptide to insulin ratio3.87 (1.30–9.50)0.95 (0.30–2.70)<0.001
Percentage difference*-74% (-23 to -89)5% (-63 to 232)<0.001

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Manufacturers' addresses
  9. References

Serum C-peptide concentrations were successfully measured in equine serum and release of C-peptide and insulin from the pancreas was suppressed in horses by i.v. administering somatostatin. This enabled determination of the C-peptide clearance rate in horses. Administration of dextrose during the FSIGTT was associated with higher concentrations of ir-C-peptide HE relative to baseline and a lower C-peptide-to-insulin ratio. Horses with IR had significantly lower C-peptide-to-insulin ratios relative to healthy horses, indicating that hyperinsulinaemia may result from both increased insulin secretion and reduced insulin clearance in insulin resistant horses.

Somatostatin is a peptide hormone that has 2 biologically active isoforms somatostatin-14 and somatostatin-28 containing 14 and 28 amino acids, respectively (Ludvigsen 2007). This peptide hormone is present in the brain, and is also secreted by the enteroendocrine D cells and δ-cells of the pancreas. Pancreatic somatostatin secretion is influenced by changes in blood glucose concentrations, and hormonal effects are exerted in an autocrine or paracrine fashion through binding to 1–5 somatostatin receptor subtypes (Low 2004).

Successful suppression of C-peptide secretion in horses was evident from the significantly lower ir-C-peptide HE concentrations detected after initiation of the somatostatin CRI. However, continued detection of ir-C-peptide HE in the blood during the same period suggests that suppression was incomplete. These findings are consistent with previous observations in man, as i.v. administration of somatostatin or its analogue octreotide, resulted in only partial suppression of C-peptide release to 48% and 27% of baseline concentrations, respectively (Hwu et al. 2001). In dogs, however, an i.v. bolus of 50 µg somatostatin followed by a CRI of 800 ng/kg bwt/min, suppressed C-peptide concentrations below the detection limit of the assay (Polonsky et al. 1983). Complications associated with somatostatin infusion such as gastrointestinal discomfort, diarrhoea or fainting observed in human subjects (Hwu et al. 2001) were not detected in this study.

The resting mean ± s.d. ir-C-peptide HE concentration of 0.23 ± 0.12 pmol/ml detected here in 4 healthy mares corresponds well with reported values for femoral artery (0.24 ± 0.04 pmol/ml) and hepatic vein (0.33 ± 0.06 pmol/ml) blood samples from fasted dogs (Polonsky et al. 1983). In man, reported values range from 0.14–0.35 pmol/ml (Faber et al. 1978). The results of the present study also compare favourably with plasma C-peptide concentrations of 0.18 pmol/ml and 0.64 ± 0.17 pmol/ml registered in one diabetic Spanish Mustang and 5 healthy horses, respectively (Johnson et al. 2005).

Median (range) clearance rate for ir-C-peptide HE measured in this study was 0.83 (0.15–1.61) ml/min/kg bwt, which was lower than the values of 4.4 and 4.7 ml/min/kg bwt detected in healthy and diabetic human subjects, respectively (Faber et al. 1978). A much higher clearance rate of mean ± 11.5 ± 0.8 ml/min/kg bwt has been detected in dogs (Polonsky et al. 1983). In man, C-peptide is metabolised by the kidney and partially excreted into the urine (Zavaroni et al. 1987). In the basal state, renal C-peptide metabolism is characterised by a high fractional extraction of approximately 26% and very low urinary clearance of only 14% (Zavaroni et al. 1987). These values indicate that most C-peptide reabsorbed by the kidney is metabolised by renal tissues. Differences in kidney function may therefore explain the wide variation in C-peptide clearance rate registered in different species.

Concentrations of ir-C-peptide HE increased and remained significantly higher than baseline for 100 min during the FSIGTT. This observation was expected because insulin and C-peptide are cosecreted from the pancreas in equimolar amounts, and increased insulin concentrations are routinely demonstrated during the FSIGTT in horses (Hovorka et al. 1998; Hoffman et al. 2003). Similar observations were made in dogs during a 60 min i.v. glucose infusion, with elevated C-peptide and insulin concentrations detected (Polonsky et al. 1983). When i.v. glucose tolerance tests are performed in man, higher C-peptide concentrations are detected after glucose infusion (Kjems et al. 2001).

Characteristic biphasic C-peptide secretion occurs in man following glucose infusion (Kjems et al. 2001), but this was not observed in the present study. The probable explanation for this finding is that glucose concentrations are not sustained during the FSIGTT in horses, therefore concentrations may not remain high enough to stimulate second phase insulin secretion. It has been suggested that the second phase insulin secretion requires the augmenting action of glucose (Henquin et al. 2002). Indeed, prolonged elevation of glucose concentration during the hyperglycaemic clamp facilitates clearer determination of the second phase insulin response than the i.v. glucose tolerance test (Caumo and Luzi 2004). For this reason, the hyperglycaemic clamp is considered the gold standard method for the assessment of biphasic insulin response in vivo (Caumo and Luzi 2004). Additionally, the second phase insulin/C-peptide response may blend with the first phase in horses, resulting in a secretory pattern similar to that demonstrated in mice (Henquin et al. 2002). In isolated mouse pancreatic islets, second phase insulin response to glucose is flatter and lower than the first phase (Henquin et al. 2002).

One of the most intriguing findings of the present study was the significant decrease in ir-C-peptide HE-to-insulin ratio from a baseline of mean ± s.d. 3.6 ± 1.95 to 1.03 ± 0.18 after dextrose administration during the FSIGTT. Similar results have been obtained in human subjects as the C-peptide-to-insulin molar ratio decreased from a fasting level of 5.0 to values between 2.0 and 3.0 following β-cell stimulation (Faber et al. 1978). One possible explanation for this change in proportions is that hepatic insulin extraction may decrease following glucose challenge in horses, causing the peripheral blood C-peptide-to-insulin ratio to approach 1:1 after stimulation of pancreatic β-cells. It seems unlikely that altered hepatic extraction of C-peptide contributes to the shift in ratio, since very little or no hepatic C-peptide extraction has been demonstrated in dogs (Polonsky et al. 1983) or man (Polonsky et al. 1986). Reduced hepatic insulin extraction may be a normal physiological response to dextrose infusion because it would result in more insulin remaining in circulation to act on insulin-sensitive tissues that store glucose. Alternatively, renal clearance of C-peptide might increase in response to dextrose, which would lower serum concentrations and alter the C-peptide-to-insulin ratio. In human subjects, renal uptake, renal clearance and fractional extraction of C-peptide markedly increased when elevated plasma C-peptide concentrations were induced by consumption of amino acids (Zavaroni et al. 1987).

Horses with IR exhibited lower C-peptide-to-insulin ratios than healthy horses, indicating that insulin clearance is reduced in affected horses. Our findings suggest that the hyperinsulinaemia previously documented in insulin resistant horses and ponies (Kronfeld et al. 2005; Treiber et al. 2005; Firshman and Valberg 2007) may be a result of both enhanced insulin secretion and reduced insulin clearance. However, it is important to state that these conclusions are based upon the assumption that C-peptide clearance remains unaffected by IR. Reduced insulin clearance contributes to hyperinsulinaemia in dogs (Mittelman et al. 2000). When dogs were made insulin resistant by feeding a diet rich in fat, higher resting insulin concentrations were detected and the insulinto-C-peptide ratio was 2-fold higher than baseline at 3 and 12 weeks. Insulin dynamics were evaluated using frequently-sampled i.v. glucose tolerance testing and minimal model analysis, with insulin clearance estimated from exponential decay curves that were fitted to the decline in insulin concentrations following insulin injection. A 22% increase in insulin secretion and approximately 50% decrease in insulin clearance were detected. Insulin clearance was also measured directly in another phase of the same study using the euglycaemic hyperinsulinaemic clamp and somatostatin infusion. Insulin clearance was found to be 20% lower in fat-fed dogs using this method. Similarly, high insulin concentrations detected in nondiabetic insulin-resistant human subjects were found to be a result of both increased secretion and a decreased clearance of insulin (Jones et al. 1997).

Weaknesses of the study reported here include the accuracy of the RIA assay because spike and recovery ratios were higher than the desired 100 ± 20% (Braggio et al. 1996) and the inability to measure equine specific C-peptide, due to the lack of a species-specific C-peptide RIA or biosynthetic equine C-peptide. However, amino acid sequences of equine and human C-peptide are homologous, differing only in 7 residues (Wahren et al. 2000), which suggests a high potential for cross reactivity. Measurement of ir-C-peptide HE may provide an additional tool for the assessment of glucose and insulin dynamics in horses, facilitating precise reconstruction of the prehepatic insulin secretion profile. Evaluation of C-peptide concentrations during glucose tolerance tests has been used to assess prehepatic insulin secretion in healthy human subjects and patients with type 2 diabetes mellitus (Kjems et al. 2000, 2001).

Estimation of pancreatic insulin secretion is important in horses because IR and hyperinsulinaemia have been associated with laminitis (Treiber et al. 2006; Asplin et al. 2007; Bailey et al. 2007; Carter et al. 2009). Insulin concentrations above 32 mu/l were used to predict incipient pasture-associated laminitis in ponies before animals were exposed to pastures rich in nonstructural carbohydrates (Carter et al. 2009). A process called decompensation has also been described in insulin resistant horses and ponies, and this occurs when increased insulin secretion can no longer offset reduced insulin sensitivity, which results in hyperglycaemia (Treiber et al. 2006). Measurement of C-peptide concentrations may therefore facilitate the assessment of decompensation in the future.

In conclusion, the present study demonstrated that C-peptide can be detected and quantified in equine serum using a double antibody human C-peptide RIA. The expected changes in serum ir-C-peptide HE and insulin concentrations were detected following the administration of somatostatin, exogenous C-peptide and dextrose. Results suggest that hepatic insulin extraction decreases in response to rising blood glucose concentrations. Evidence was found to suggest that hyperinsulinaemia can be attributed to both increased insulin secretion and decreased insulin clearance in horses with IR. Further studies are required to quantify equine-specific C-peptide and to investigate insulin and C-peptide clearance in horses with IR.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Manufacturers' addresses
  9. References

Supported by a grant from Waltham Foundation and the Charles and Julie Wharton Fellowship. The authors thank Nancy Rohrbach and the University of Tennessee Animal Science Endocrinology Laboratory for assisting with assay validation procedures.

Manufacturers' addresses

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Manufacturers' addresses
  9. References

1 Abbott Laboratories, North Chicago, Illinois, USA.

2 Bachem Americas, Torrence, California, USA.

3 GenScript Co., Piscataway, New Jersey, USA.

4 Eli Lilly and Co, Indianapolis, Indiana, USA.

5 Siemens Medical Solutions Diagnostics, Los Angeles, California, USA.

6 WinSAAM, Dr. Ray Boston, University of Pennsylvania, Kennett Square, Pennsylvania, USA.

7 SAS Institute Inc, Cary, North Carolina, USA.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Manufacturers' addresses
  9. References
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Author contributions The initiation, conception and planning for this study were by F.T., N.F., T.M.-J., R.J.G. and R.C.B. Its execution was by F.T., N.F., S.B.E. and R.C.B., with statistics by F.T., N.F., T.M.-J. and R.C.B. The paper was written by F.T., N.F., R.J.G. and R.C.B.