The conscious Göttingen minipig as a model for studying rapid pulsatile insulin secretion in vivo

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1 Diabetologia (2002) 45: DOI /s The conscious Göttingen minipig as a model for studying rapid pulsatile insulin secretion in vivo M.O. Larsen 1, 2, M. Elander 1, 2, J. Sturis 1, M. Wilken 3, R.D. Carr 1, B. Rolin 1, N. Pørksen 4 1 Department of Pharmacological Research I, Novo Nordisk A/S, Bagsvaerd, Denmark 2 Department of Pharmacology and Pathobiology, the Royal Veterinary and Agricultural University, Copenhagen, Denmark 3 Department of Assay and Cell technology, Novo Nordisk A/S, Bagsvaerd, Denmark 4 Medical Department M, Aarhus University Hospital, Aarhus, Denmark Abstract Aims/hypothesis. Pulsatile secretion is important for insulin action and suitable animal models are important tools for examining the role of impaired pulsatile insulin secretion as a possible link between beta-cell mass, function and morphology and insulin resistance. This study examines the vascular sampling site, insulin kinetics, pulsatility and the response to glucose pulse entrainment to evaluate the Göttingen minipig as a model for studying pulsatile insulin secretion. Methods. Basal and glucose entrained insulin secretion was examined in normal minipigs and evaluated by autocorrelation, cross correlation and deconvolution. Results. Cross correlation showed a relation between oscillations in insulin concentrations in the portal and jugular vein in anaesthetised animals (p<0.001 in all animals), confirming the usefulness of jugular vein sampling for pulse detection. Jugular vein sampling in conscious animals showed obvious oscillations allowing estimates of burst shape and insulin kinetics. Glucose entrainment improved the pulsatile pattern (autocorrelation: 0.555±0.148 entrained vs 0.350±0.197 basal, p=0.054). Deconvolution analysis resolved almost all insulin release as secretory bursts (69±20 basal vs 99.5±1.2% entrained, p<0.01) with a pulse interval (min) of 6.6±2.2 (basal) and 9.4±1.5 (entrained) (p<0.05) and a pulse mass (pmol/l per pulse) which was higher after entrainment (228±117 vs 41.2±18.6 basal, p<0.001). Conclusion/interpretation. The ability to fit kinetic parameters directly by deconvolution of peripheral endogenous insulin concentration time series in combination with the suitability of jugular vein sampling, rapid kinetics and entrainability makes the Göttingen minipig ideal for mechanistic studies of insulin pulsatility and its effects on insulin action. [Diabetologia (2002) 45: ] Keywords Pulsatile insulin secretion, insulin kinetics, deconvolution, in vivo model, insulin action. It is well documented that insulin concentrations show large amplitude oscillations in the peripheral circulation with a periodicity of 5 to 15 min [1, 2, 3], and it has been shown that these oscillations are of importance for insulin action in liver [4], muscle [5, 6] and adipose tissues [7]. Since Type II (non-insulin-depen- Received: 22 March 2002 / Revised: 3 June 2002 Published online: 5 September 2002 Springer-Verlag 2002 Corresponding author: M. O. Larsen, PhD, Department of Pharmacological Research I, Pharmacology, Research and Development, Novo Allé, 6A1.005, 2880 Bagsvaerd, Denmark. mmla@novonordisk.com dent) diabetes mellitus is characterized by defects in both insulin secretion and action, impaired pulsatile secretion could be an important link between beta-cell malfunction and impaired insulin action [8]. When addressing the cause of these impairments in humans, studies are often influenced by long-term impaired metabolism, and therefore demonstrate that defects could be secondary. This problem can, to some extent, be circumvented by studying first degree relatives of diabetic patients who are at risk of the disease but are still not affected by the effects of hyperglycaemia [9]. However, animal models are a valuable research tool for examining the relation between beta-cell mass, function and morphology on the one hand and insulin

2 1390 M.O. Larsen et al.: The conscious Göttingen minipig as a model for studying rapid pulsatile insulin secretion in vivo resistance on the other, because carefully controlled studies can involve surgical [10, 11, 12] or toxicological [10, 13, 14, 15, 16, 17] induction of diabetes and long-term maintenance on experimental diets can induce insulin resistance and lipotoxicity [18, 19, 20, 21]. An animal model should ideally allow peripheral sampling to assess pulsatility, so that invasive surgery to access portal sampling can be avoided. Furthermore, it should be possible to estimate insulin kinetics during study conditions to accommodate changes in kinetics due to metabolic changes (i.e. fasting vs post prandial and normal vs diabetic metabolism). To facilitate pulse detection, insulin kinetics should be very rapid so that any influence from recirculating insulin is minimized. Apart from these characteristics, the animal model should show patterns of pulsatility similar to what is seen in humans, both in the basal state and in response to the recently introduced glucose pulse entrainment method [22]. Several animal models have been used to study pulsatile insulin secretion, including the rat [23], dog [24] and baboon [2, 25]. Different experimental set-ups might require different properties of the model to be used. However, the small size of the rat does not favour long-term frequent sampling to facilitate pulse detection and, therefore a larger animal would often be preferable. Several non-human primates have a size that would overcome this problem, but studies of pulsatile insulin secretion should preferably be done in conscious, unstressed animals which can be difficult to obtain in primates. Furthermore, there are special considerations of animal welfare and ethics when using primates, thereby favouring the use of other species when possible. The pig shares many similarities with humans with regard to nutrition requirements and physiology of digestion and metabolism [26, 27, 28, 29], therefore it is very relevant for studies of fasting and postprandial glucose metabolism. The Göttingen minipig offers many advantages in this context because of its well described biology, including general characteristics such as clinical chemistry and haematology [30, 31, 32] as well as its glucose and lipid metabolism in normal animals [17, 18, 33, 34] and after induction of diabetes [16, 17] or challenge with high-fat diets [18, 34, 35, 36]. In addition, fully grown animals have a size that allows sufficient volumes of blood to be drawn but are still sufficiently small to allow easy handling [37, 38]. These animals can be trained to allow experiments to be done on them in a conscious and unstressed state. The aim of our study was to evaluate the Göttingen minipig as an experimental model for studying pulsatile insulin secretion in conscious animals, with special focus on the above mentioned desired characteristics of sampling site, insulin kinetics, pulsatility and response to glucose pulse entrainment in the model. Materials and methods Animals. Adult male Göttingen minipigs, 11 to 14 months of age, were obtained from the barrier unit at Ellegaard Göttingen minipigs ApS, Dalmose, Denmark. They were housed in single pens under controlled conditions (temperature was kept between 18 and 22 C, relative air humidity was 30 70% with four air changes per hour) with a 12-h light to 12-h dark cycle. They were fed twice daily with 140 g of SDS minipig diet (SDS, Essex, England) and 240 g of a commercial swine fodder ( Svinefoder 22, Slangerup, Denmark), and allowed free access to water. At the time of experiments, the pigs weighed 23±4 kg (range 19 to 31).They were studied either during anaesthesia (protocol 1), or at least 2 weeks after surgery, and were trained carefully in all experimental procedures before the experiments started (protocol 2 and 3). Principles of laboratory animal care were followed and the type of study was approved by the Animal Experiments Inspectorate, Ministry of Justice, Denmark. Protocol 1: relation between insulin concentration patterns in the jugular and portal vein in anaesthetized animals (n=3). A jugular vein catheter (Certo 455, B. Braun Melsungen AG, Melsungen, Germany) and a portal vein catheter (placed via the left gastroepiploic vein) (silastic laboratory tubing , Dow Corning, Midland, Mich., USA) were inserted surgically under general anaesthesia induced with propofol (Propofol Abbott, Abbott, Gentofte, Denmark) (5 mg/kg by intra venous injection) and atropine (Atropin DAK, 1 mg/ml, Nycomed, Roskilde, Denmark) (0.2 mg/pig by intra muscular injection) and maintained with α-chloralose (C8091, Sigma- Aldrich, Vallensbaek Strand, Denmark). A bolus of 150 ml (10 mg/ml) was infused over 30 min every 2 h). Local analgesia at the site of the laparotomy was obtained by infiltration of bupivacaine 2 mg/kg (Marcain 2.5 mg/ml, Astra, Albertslund, Denmark). Animals were ventilated artificially with pure oxygen (oxygen flow: 400 ml/min, tidal volume: 10 ml/kg and respiratory frequency: 14 breaths/min). Sterile 0.9% saline (SAD, Copenhagen, Denmark) was infused at ml kg 1 min 1 at all times. When both catheters were in place and functioning, pigs were left undisturbed for 30 min to minimize the effect of handling during surgery on insulin secretion. Blood samples were taken simultaneously from the portal and jugular vein catheters every minute for 60 min. Before each blood sample of 1 ml was collected, 1.5 ml blood, corresponding to the catheter dead space, was withdrawn, and returned aseptically after each sample and 1 ml of sterile saline (0.9%, SAD, Copenhagen, Denmark) was flushed in. After 60 min of sampling, animals were killed during anaesthesia using pentobarbitone (20 ml per animal by intra venous injection) (200 mg/ml, Pharmacy of the Royal Veterinary and Agricultural University, Copenhagen, Denmark). Surgical implantation of central venous catheters. For protocol 2 and 3, two central venous catheters (Certo 455, B. Braun Melsungen AG, Melsungen, Germany) were inserted surgically in the external jugular vein under general anaesthesia induced with a combination of zolazepam 0.83 mg/kg, tiletamine 0.83 mg/kg (Zoletil 50 vet., Boehringer Ingelheim, Copenhagen, Denmark), xylazine 0.90 mg/kg (Rompun vet, 20 mg/ml, Bayer, Lyngby, Denmark), ketamine 0.83 mg/kg (Ketaminol vet, 100 mg/ml, Rosco, Taastrup, Denmark) and methadone 0.20 mg/kg (Metadon DAK, 10 mg/ml, Nycomed, Roskilde, Denmark) given intra muscularly, and maintained with isoflurane (1 3%) (Forene, Abbot, Gentofte, Denmark) in 100% oxygen. Post surgical analgesia was maintained by injec-

3 M.O. Larsen et al.: The conscious Göttingen minipig as a model for studying rapid pulsatile insulin secretion in vivo 1391 tion of buprenorphine 0.03 mg/kg (Anorfin, 0.3 mg/ml, GEA, Frederiksberg, Denmark) and carprofen 4 mg/kg (Rimadyl vet., 50 mg/ml, Pfizer, Ballerup, Denmark) intra muscularly before the end of anaesthesia and for 3 days after surgery by injection of carporfen 4 mg/kg once daily intra muscularly. Post-surgical infection was prevented by intra muscular injection of dihydrostreptomycin sulphate (25 mg/kg) and benzylpenicillinprocain (20,000 IE/kg) (Streptocillin vet., 250 mg DHS IE benzylpenicillinprocain/ml, Boehringer Ingelheim, Copenhagen, Denmark) immediately after surgery and once daily for the following 2 days. All animals were allowed 2 to 3 weeks of recovery after the surgical procedure and had normal behaviour and eating patterns at the start of the study period. Protocol 2: basal secretion in overnight-fasted conscious animals (n=8). Blood samples (0.8 ml) were taken from a jugular vein catheter every min for 1 h. Before each blood sample was collected, 1.5 ml blood, corresponding to the catheter s dead space, was withdrawn and returned aseptically after each sample. Catheters were flushed with 0.8 ml of sterile saline (0.9%, SAD, Copenhagen, Denmark) after each blood sample. Protocol 3: entrainment of pulsatile insulin secretion in overnight-fasted conscious animals (n=6). Blood samples of 0.8 ml were taken from a jugular vein catheter every min for 1 h, as described above. Every 10 th min, starting at t=0 min, a bolus of glucose (4 mg kg 1 min 1, glucose 200 mg/ml SAD, Copenhagen, Denmark) was infused over 1 min via the other jugular vein catheter. Handling and analysis of blood samples. Blood samples were immediately transferred to vials containing EDTA (1.6 mg/ml final concentration) and aprotinin 500 KIU/ml full blood (Trasylol, KIU/ml, Bayer, Lyngby, Denmark) and kept on ice until centrifugation. Samples were centrifuged (4 C, 10 min, 3500 rpm), and plasma was separated and stored at 20 C until analysis. Plasma glucose was analysed using the immobilized glucose oxidase method, 10 µl of plasma in 0.5 ml buffer (EBIO plus auto analyser and solution, Eppendorf, Hamburg, Germany). Plasma insulin was analysed in a two-site immunometric assay with monoclonal antibodies as catching and detecting antibodies (Catching antibody HUI-018 raised against the A-chain of human insulin; detecting antibody OXI-005 raised against the B-chain of bovine insulin) [39] and using purified porcine insulin for calibration of the assay. The minimal detectable concentration was 3.2 pmol/l, the upper limit was 1200 pmol/l (no sample dilution) and the inter-assay and intra-assay variations were 15.3% and 3.2% (at 342 pmol/l), 9.9% and 7.6% (at 235 pmol/l) and 14.6% and 4.4% (at 87 pmol/l). Recovery at high, medium and low concentrations was 97.1%, 97.9% and 101% respectively. Detection and quantification of pulsatile insulin secretion by deconvolution. The plasma insulin concentration time series was analysed by deconvolution to detect and quantify insulin secretory bursts. Deconvolution of venous insulin concentration data was done with a multi-parameter technique [40], which depends on the following assumptions. The venous plasma insulin concentrations measured in samples collected at 1- min intervals were assumed to result from five determinable and correlated parameters: (i) a finite number of discrete insulin secretory bursts occurring at specific times and having; (ii) individual amplitudes (maximal rate of secretion attained within a burst); (iii) a common half-duration (duration of an algebraically Gaussian secretory pulse at half-maximal amplitude), which are superimposed upon a (iv) basal time-invariant insulin secretory rate; and (v) a bi-exponential insulin disappearance model in the systemic circulation. The insulin disappearance kinetics were estimated based on model fitting to observed insulin concentration profiles. Assuming slower or faster kinetics for first and second half-life (20% change) resulted in considerably impaired fit as evaluated by sum of squared residuals. In some data sets without entrainment (4 out of 8), different values could arise. In these cases, data was fitted to observed concentrations, but only allowing kinetics to be within the range of values observed in other data sets. Subsequently kinetics could still vary, all yielding comparable fits, and therefore values were fixed in agreement with values in other data sets (mean). Assuming the fitted insulin disappearance values, the number, locations, amplitudes and half-duration of insulin secretory bursts, as well as a non-negative basal insulin secretory rate, were estimated for each data set by non-linear leastsquares fitting of the multi-parameter convolution integral for each insulin time series. A modified Gauss-Newton quadratically convergent iterative technique was used with an inverse (sample variance) weighting function. Parameters were estimated until their values and the total fitted variance both varied by less than 1 part in Asymmetric highly correlated variance spaces were calculated for each parameter by the Monte Carlo support-plane procedure. Secretory rates were expressed as mass units of insulin (pmol) released per unit distribution volume (l) per unit time (min). The mass of hormone secreted per burst (time-integral of the calculated secretory burst) was thus computed as pmol insulin released per litre of systemic distribution volume. Basal secretion was adjusted during the deconvolution analysis to allow accommodation of most troughs. Likewise the secretory burst half duration was adjusted to fit individual obvious secretory bursts, consisting of series of data points building up to a peak and down to a trough. All data analysis was done in a blinded manner. Auto-correlation and cross-correlation analyses. The periodic nature of individual insulin profiles was assessed by autocorrelation analysis and the relation between jugular and portal vein data was quantified by cross-correlation analysis. Because trends can distort the subsequent correlation analyses, they were removed by subtracting from each profile its best-fit line calculated by linear regression analysis. In the autocorrelation analysis, the correlation coefficients between the time series and a copy of itself at lags of 0, 1, 2, 3, etc. up to 25 min were calculated. Similarly, in the cross-correlation analysis, the coefficients of cross-correlation between portal and jugular insulin series at lags of 0 (i.e. simultaneous values of insulin), ±1 (i.e. portal leading jugular by 1 min or vice versa), ±2, ±3, etc. up to ±25 min were calculated. The largest coefficient of crosscorrelation and the lag at which it occurred were assessed in each case. For both auto-correlation and cross-correlation analysis, group statistical analysis of the correlation coefficients was done after using Fisher s z-transformation [41]. Data are presented as means ± standard deviation. A Students t-test was used for statistical analyses. Results Jugular versus portal sampling. In animals investigated in protocol 1, mean insulin concentration in the jugular vein was 11.5±5.6 pmol/l whereas mean portal concentration was 67.3±19.2 pmol/l and mean plasma glucose was 5.0±0.9 mmol/l. Cross correlation showed a relation between oscillations in insulin concentrations in the portal and jugular veins in all animals with a mean

4 1392 M.O. Larsen et al.: The conscious Göttingen minipig as a model for studying rapid pulsatile insulin secretion in vivo Fig. 1. A Portal ( ) and jugular ( ) vein insulin concentrations during anaesthesia (pig no. 15). B Jugular vein insulin concentrations in the basal state in a conscious animal (pig no. 7) Fig. 2A D. ugular vein plasma insulin concentrations in conscious animals during basal conditions (A, C) (A pig no. 1, C pig no. 2) or during entrainment of insulin pulses with glucose infusion (B, D) (B pig no. 11, D pig no. 12) lag time of 1 min in the jugular compared to the portal vein (cross correlation coefficient: 0.72, 0.42 and 0.49, p<0.001 in all animals). An example of simultaneously measured portal vein and jugular vein insulin concentrations together with an example of data from a conscious animal is shown in Fig. 1. Conscious animals, jugular sampling. In animals investigated in protocol 2, mean insulin concentration in the jugular vein was 26.7±12.6 pmol/l and mean glucose concentration was 3.5±0.5 mmol/l. The insulin concentration profiles obtained from the jugular vein showed obvious large amplitude oscillations (Fig. 2) with frequent peaks, allowing estimates of burst shape and insulin kinetics. Glucose pulse entrainment (protocol 3) further enhanced the pulsatile pattern, with large amplitude (increased from pmol/l to approximately 100 pmol/l) oscillations at approximately 10 min intervals, and in some cases (n=3), with undetectable concentrations (<3.2 pmol/l) between pulses, strongly indicating the absence of basal insulin release (Fig. 2). Regularity statistics. The presence of regular oscillatory patterns in individual data sets was confirmed by autocorrelation showing oscillations in both basal and entrained situations, but with improved pulsatility during entrainment (autocorrelation coefficients: 0.555±0.148 vs 0.350±0.197 at basal, p=0.054) with a frequency of 10.1±1 basal versus 9.8±0.4 induced, p=0.51, whereas deconvolution showed periodicities of approximately 9.4±1.4 min during induction versus 6.6±2.2 min at basal conditions (p<0.05). Data from individual animals are shown in Table 1. Deconvolution. Insulin disappearance kinetic parameters were fitted to observed data whenever data presented obvious large amplitude oscillations. The data in protocol 3 resulted in an obvious on/off release pattern with peaks allowing fitting of the insulin kinetics in six out of six cases, whereas five out of eight cases allowed fitting in protocol 2. In cases

5 M.O. Larsen et al.: The conscious Göttingen minipig as a model for studying rapid pulsatile insulin secretion in vivo 1393 Fig. 3A, B. Jugular vein plasma insulin concentrations (dotted line) and deconvolved best-fit curves for insulin secretion (continuous line) in an animal during basal conditions (A) (pig no. 1) and during glucose entrainment of insulin pulses (B) (pig no. 11). Kinetics of insulin are derived directly from plasma insulin concentration profiles Table 1. Plasma concentrations, regularity statistics, kinetic and deconvolution parameters for individual animals Pig Protocol Plasma Regularity Kinetics Deconvolution concentrations statistic T 1 / 2 1 T 1 / 2 2 Interval Amplitude Mass Basal % pulse FPG FPI Auto- (min) (min) (min) (pmol l 1 (pmol l 1 (pmol l 1 (mmol/l) (pmol/l) correlation min 1 ) pulse 1 ) min 1 ) 1 Basal 3.1±0.2 17± Basal 3.0±0.3 18± Basal 3.3±0.3 33± Basal 3.4±0.4 20± Basal a 3.0±0.4 23± Basal a 3.6±0.4 19± Basal a 3.8±0.1 30± Basal a 4.4±0.2 54± Entrained 3.5±0.3 45± Entrained 4.2±0.3 82± Entrained 3.8±0.2 9± Entrained 3.4±0.1 21± Entrained 3.2±0.9 52± Entrained 3.4±0.2 19± a Insulin kinetics pre-set at values in accordance with values obtained in animals where fitting was possible where insulin kinetics could not easily be fitted to observed data, a mean of the fitted parameters was used. This resulted in a two phased decay in both nonentrained (t 1 / 2 1=1.7±0.4 min, t 1 / 2 2=3.8±2.3 min) and entrained animals (t 1 / 2 1=0.7±0.3 min and t 1 / 2 2=3.4±1.2 min). Notably, an additional number of alternative half-lives were assumed, resulting in increased sum of squared residuals of the best fit curve. Examples of half-lives used to fit observations are shown in Fig. 3. Using the above described kinetic parameters, deconvolution analysis for detection and quantification of the pulsatile insulin release resolved non-entrained insulin release into serial secretory bursts with a pulse interval of 6.6±2.2 min and with a mean amplitude of 24.3±14.3 pmol l 1 min 1. The mean pulse mass was 41.2±18.6 pmol l 1 pulse 1. Most non-entrained secretion of insulin occurred in pulses (69±20%) and in two, no significant basal secretion was detected. In animals where glucose entrainment was carried out, significant pulsatile secretion of insulin was also detected with a pulse interval of 9.4±1.5 min (p vs basal conditions <0.05), equivalent to the entrainment interval, but indicating a few (~1 per 20 induced) break-through secretory bursts. In these animals the mean amplitude was 290±267 pmol/l (p<0.05 vs nonentrained amplitude) with a pulse mass of 228±117 (p<0.001 vs non-entrained pulse mass). Similar to what was seen without entrainment, but with further enhancement almost all insulin was secreted in pulses (99.5±1.2%, p<0.01) with little (n=3) or no (n=3) basal secretion detected. Examples of deconvolved insulin secretion curves in individual animals are shown in Fig. 4.

6 1394 M.O. Larsen et al.: The conscious Göttingen minipig as a model for studying rapid pulsatile insulin secretion in vivo Fig. 4A, B. Jugular vein plasma insulin concentrations (dotted line) and deconvolved best-fit curves for insulin secretion (continuous line) in an animal during basal conditions (A) (pig no. 6) and during glucose entrainment of insulin pulses (B) (pig no. 14) Discussion Pulsatile insulin secretion is impaired in Type II diabetes [42], impaired glucose tolerance [43] and in glucose tolerant first degree relatives of Type II diabetic patients [9], which indicates a very early defect in pulsatility in the pathogenesis of diabetes. In addition, insulin action is markedly impaired in diabetes and prediabetes [44]. The impaired pulsatile insulin release has been linked to impaired insulin action [4, 5, 6, 7] but the causes and implications need to be fully established. Mechanistic studies in animal models are an important tool for evaluating this relation in vivo. Because of the many similarities between pigs and humans with regard to nutrition physiology [26, 27, 28, 29], and the well-described biology [16, 17, 18, 30, 31, 32, 33, 34, 35, 36] and relatively small size of the Göttingen minipig [37, 38], this strain of pigs offers major advantages, allowing in vivo studies in the conscious, unrestrained animal during basal and post prandial conditions. In our study, the ability to predict pancreatic insulin release based on jugular vein sampling, even in animals with reduced insulin concentrations due to anaesthesia, means that extensive abdominal surgery to obtain portal vein sampling can be avoided. The endogenous insulin concentration pulses obtained from the jugular vein in conscious animals allowed good fit estimates of insulin kinetics in individual animals during pulse entrainment (and in 4/8 cases under basal conditions), indicating that induction of pulses (at least two) can be used to acquire useful insulin kinetics for deconvolution purposes. The insulin kinetics we reported are similar to those obtained based on intra-portally injected exogenous insulin (t 1 / 2 2~3 min) although a monoexponential model was used [16]. However, deconvolution of peripheral endogenous insulin concentration time series has allowed fitting of the insulin kinetic parameters, confirming the Göttingen minipig as a very suitable model for insulin pulsatility studies. The striking ability to discern insulin release into an on/off pattern is very likely due to the rapid insulin kinetics in this species, making the model very favourable for studies involving dynamic changes in insulin secretion and insulin kinetics. Both inspection and deconvolution of data show a highly dynamic pulsatile insulin secretion that, in terms of frequency, is similar to what has been reported in humans and dogs [24, 45]. The estimated periodicity of the pulsatile pattern depends on the analytical tool used. Thus, when based on deconvolution, the period appears shorter than what is obtained with autocorrelation. This finding is in agreement with published results in human studies, when comparing separate studies [3, 45] and when using the different techniques in the same study [46]. Although the periodic nature of the secretory pattern is clear, pulsatility outside this regular pattern also occurs and these pulses will not be evident when an analytical tool designed to detect periodicity is used. However, the rapid insulin kinetics seen in the Göttingen minipig result in even further amplification of peripheral oscillations in insulin concentrations favouring pulse detection. Entrainment of insulin pulses with glucose offers the advantage of allowing control of pulse frequency and amplitude, so that the impact of these parameters on insulin action can be addressed. In parallel with the findings in humans [22], glucose pulse induction in the Göttingen minipig was able to entrain oscillatory insulin release, showing an entrained on/off release pattern of insulin. In most animals, insulin concentrations were undetectable between insulin secretory bursts. We show that peripheral sampling allows a clear distinction of insulin secretory bursts by detecting 99.5% of insulin secretion as being pulsatile with undetectable insulin concentrations between pulses. Several animal models, including the rat [23], dog [24] and baboon [25], have been used to investigate insulin pulsatility. Although it is probably not possible to define one single optimal model in this field of research, the characteristics of the Göttingen minipig makes this species very relevant. Furthermore, pertubation of beta-cell function after reducing beta-cell mass using alloxan in the Göttingen minipig has been published [16]. In conclusion, the reported pertubation of pulsatility combined with the advantages of the model reported here, especially the ability to fit insulin kinetics based on endogenous hormone levels obtained from peripheral sampling and the very rapid insulin kinetics, holds promise for the Göttingen minipig as an excellent model in future studies of beta-cell

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