{beta}-Cell function and islet morphology in normal, obese, and obese {beta}-cell mass-reduced Göttingen minipigs

M. O. Larsen,1 C. B. Juhl,4 N. Pørksen,5 C. F. Gotfredsen,2 R. D. Carr,6 U. Ribel,3 M. Wilken,7 and B. Rolin3

1Department of Pharmacology Research I, 2Department of Pharmacology Research IV, and 3Department of Pharmacology Research III, Novo Nordisk A/S, Maaloev; 4Medical Department M and 5Medical Department C, Aarhus University Hospital, Aarhus; and 6Discovery Management and 7Department of Assay and Cell Technology, Novo Nordisk A/S, Bagsvaerd, Denmark

Submitted 2 August 2004 ; accepted in final form 2 October 2004


    ABSTRACT
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Herein, we bridge {beta}-cell function and morphology in minipigs. We hypothesized that different aspects of {beta}-cell dysfunction are present in obesity and obesity with reduced {beta}-cell mass by using pulsatile insulin secretion as an early marker. Measures for {beta}-cell function (glucose and arginine stimulation plus baseline and glucose-entrained pulsatile insulin secretion) and islet morphology were studied in long-term (19–20 mo) obese (n = 5) and obese {beta}-cell-reduced [nicotinamide + streptozotocin (STZ), n = 5] minipigs and normal controls, representing different stages in the development toward type 2 diabetes. Acute insulin response (AIR) to glucose and arginine were, surprisingly, normal in obese (0.3 g/kg glucose: AIR = 246 ± 119 vs. 255 ± 61 pM in control; 67 mg/kg arginine: AIR = 230 ± 124 vs. 214 ± 85 pM in control) but reduced in obese-STZ animals (0.3 g/kg glucose: AIR = 22 ± 36, P < 0.01; arginine: AIR = 87 ± 92 pM, P < 0.05 vs. control). Baseline pulsatile insulin secretion was reduced in obese (59 ± 16 vs. 76 ± 16% in control, P < 0.05) and more so in obese-STZ animals (43 ± 13%, P < 0.01), whereas regularity during entrainment was increased in obese animals (approximate entropy: 0.85 ± 0.14 vs. 1.13 ± 0.13 in control, P < 0.01). {beta}-Cell mass (mg/kg body wt) was normal in obese and reduced in obese-STZ animals, with pancreatic fat infiltration in both groups. In conclusion, obesity and insulin resistance are not linked with a general reduction of {beta}-cell function, but dynamics of insulin secretion are perturbed. The data suggest a sequence in the development of {beta}-cell dysfunction, with the three groups representing stages in the progression from normal physiology to diabetes, and assessment of pulsatility as the single most sensitive marker of {beta}-cell dysfunction.

{beta}-cell reduction; obesity; pulsatile insulin secretion; in vivo model


TYPE 2 DIABETES IS A CONDITION characterized by several metabolic defects (58), two of which, {beta}-cell dysfunction and peripheral insulin resistance, are considered as hallmarks of the disease in humans (22, 49, 54, 57). Both of these defects can be further separated into several different contributory abnormalities. With regard to {beta}-cell function, there is a decrease in insulin-secretory capacity (48) and first-phase insulin secretion (9, 56), and impairment of pulsatile insulin secretion (21, 27, 50), each of which may reflect distinct aspects of pathophysiology. Furthermore, {beta}-cell mass has been reported to be reduced in type 2 diabetes (10, 14, 25). A reduction in {beta}-cell mass per se can reduce insulin secretory capacity and first-phase insulin secretion (7, 31, 41), and the resulting hyperglycemia seems to be a causative factor in the dysfunction of {beta}-cells (35, 39). Pulse mass of both baseline and entrained pulsatile insulin secretion is reduced by a reduction of {beta}-cell mass, whereas regularity of the pulsatile pattern has been reported to be normal (24, 29). Thus not all of the abnormalities seen in type 2 diabetes can be explained by a reduced {beta}-cell mass.

Causes and manifestations of insulin resistance, such as reduced glucose uptake, increased hepatic glucose production, dyslipidemia, etc., are numerous (12, 26, 38, 42, 51, 57). Obesity is thought to be a major cause of insulin resistance (5, 26), thereby contributing to an increased demand on insulin secretion that may, eventually, result in {beta}-cell exhaustion and failure (54). Alternatively or additionally, dyslipidemia associated with obesity is thought to cause {beta}-cell lipotoxicity and thereby impair {beta}-cell function (2, 4). Because the majority of obese individuals are not diabetic (6, 11, 19, 44, 45), it is believed that a {beta}-cell defect is also required to trigger diabetes in insulin-resistant states such as obesity.

In humans, evaluation of {beta}-cell function can be linked to insulin action but not {beta}-cell mass or islet morphology. In addition, prospective mechanistic studies of the development of type 2 diabetes in humans are not feasible.

In animal models, it is possible to study both functional aspects of the {beta}-cell and morphology of islets in the same animals; in addition, obesity may be induced. The Göttingen minipig shares many metabolic similarities with humans (8, 53) and has been characterized experimentally with respect to both glucose tolerance (34) and {beta}-cell function (24, 29) in the context of type 2 diabetes. Obesity has been shown to be coincident with mild peripheral insulin resistance in the same model (30). In addition, we (28) have recently presented data on unique insulin secretory patterns, also in this preparation.

The present study was undertaken to test the hypothesis that 1) impaired insulin pulsatility is an early marker of {beta}-cell dysfunction, 2) obesity alone would be related to {beta}-cell dysfunction, and 3) the major hallmarks of {beta}-cell dysfunction are displayed only in the presence of obesity in coexistence with a reduced {beta}-cell mass, thereby representing a more advanced stage toward diabetes development. To evaluate this hypothesis, we have used obese minipigs as a model of mild insulin resistance and obese minipigs pretreated with a combination of nicotinamide (NIA) and streptozotocin (STZ) (34) to obtain a moderate reduction of {beta}-cell mass, representing a more advanced stage in the development toward type 2 diabetes. In addition, we have studied the relation between major aspects of {beta}-cell function and islet morphology in each of the three experimental groups.


    METHODS
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Animals

Adult male Göttingen minipigs 11–14 mo of age were obtained from the barrier unit at Ellegaard Göttingen minipigs (Dalmose, Denmark). Animals were housed in single pens under controlled conditions (temperature was kept between 18 and 22°C, relative air humidity was 30–70% with 4 air changes per h) with a 12:12-h light-dark cycle and allowed free access to water. All pigs were studied ≥2 wk after surgical implantation of central venous catheters (see below) and were trained carefully in all experimental procedures before the start of experiments. In total, 28 animals were included in the study. Data on pulsatile insulin secretion from some of the control animals (n = 8 during baseline and n = 6 during entrained conditions) have previously been reported (28).

Principles of laboratory animal care were followed, and the type of study was approved by the Animal Experiments Inspectorate, Ministry of Justice, Denmark.

Experimental Groups

Five animals were fed Special Diet Services (SDS) ad libitum from weaning (8 wk of age) until 12 mo of age and were then fed 240 g of a commercial swine fodder (Antonio, Slangerup, Denmark) and ~300 g of a high-fat diet (Danish pastry; Ganløse Bageren, Ganlose, Denmark) twice daily for 7 mo to induce obesity (obese animals). Another five animals had their {beta}-cell mass reduced followed by high-fat feeding (240 g of Antonio and 300 g of high-fat diet twice daily) for 8 mo to combine reduced {beta}-cell mass and obesity (obese-STZ animals). Twenty-six animals were kept as normal controls and were fed after a standard regimen twice daily: 140 g of SDS minipig diet (SDS; Witham, Essex, UK) and 240 g of Antonio (control animals).

Experimental Diet and Feeding Scheme

The compositions of samples of the experimental diets are summarized in Table 1. Analysis of the high-fat diet was performed at Steins Laboratorium (Brorup, Denmark) whereas composition of SDS and commercial fodder was provided by the supplier.


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Table 1. Nutrient composition of diets

 
Scan for Body Composition

A dual-energy X-ray absorptiometry scanning (QDR-1000 W; Hologic, Zaventem, Belgium) for body composition was performed (43) in anesthetized animals before and after the study period (evaluation of pulsatile insulin secretion and insulin secretion test). Anesthesia was induced using a combination of zolazepam, tiletamine, xylazine, ketamine, and methadone intramuscularly, as previously described (33). Analysis of body composition was performed using the Whole Body Analysis protocol in the Hologic software package.

Surgical Implantation of Central Venous Catheters

Two central venous catheters (Certo 455; B. Braun Melsungen, Melsungen, Germany) were inserted surgically via the jugular vein under general anesthesia, as described previously (34). Postsurgical analgesia was maintained by injection of 0.03 mg/kg buprenorfine (0.3 mg/ml Anorfin; GEA, Frederiksberg, Denmark) and 4 mg/kg carprofen (50 mg/ml Rimadyl vet.; Pfizer, Ballerup, Denmark) intramuscularly before the end of anesthesia and for 3 days postsurgery by injection of 4 mg/kg carporfen once daily intramuscularly. At the start of the study period, all animals had recovered fully from the surgical procedure, as evaluated by normal behavior and eating patterns.

Reduction of {beta}-Cell Mass

{beta}-Cell mass was reduced by intravenous administration of NIA (67 mg/kg, Sigma N-3376; Sigma-Aldrich, Steinheim, Germany) followed by STZ (125 mg/kg, Sigma S-0130) after an 18-h overnight fast in conscious animals, as previously described (34). Animals were offered SDS fodder 2 h after treatment and were observed frequently during the first 48 h after administration of NIA plus STZ, and blood glucose was monitored regularly to detect and treat any episodes of hypoglycemia due to sudden hyperinsulinemia caused by necrosis of {beta}-cells.

Insulin Secretion Test

The insulin secretion test was performed in 10 control, 5 obese and 4–5 obese-STZ animals. The test was performed after an 18-h overnight fast in nonrestrained, freely moving animals in their usual pens to reduce the amount of stress experienced by the animals during testing.

The test consisted of three elements: 1) a bolus of glucose (0.3 g/kg, 500 g/l; SAD, Copenhagen, Denmark; n = 5 obese-STZ), 2) a bolus of glucose (0.6 g/kg) followed by an infusion of glucose (2 g·kg–1·h–1, 200 g/l; SAD) for 40 min to maintain hyperglycemia at ~20–30 mM (n = 4 obese-STZ), and 3) a bolus of arginine (67 mg/kg) 30 min after the 0.6 g/kg glucose bolus (n = 4 obese-STZ).

All compounds were given intravenously through a central venous catheter, and blood samples were obtained from the central venous catheters at –15, –10, –5, 1, 3, 5, 7, and 10 min relative to each of the bolus injections. Parts 1 and 2 of the experiment were separated by ≥50 min.

Dynamics of Baseline Insulin Secretion

Baseline pulsatile insulin secretion was studied in fasted (18 h) conscious animals (n = 12 control, 5 obese, and 5 obese-STZ animals). Blood samples (0.8 ml) were taken from a jugular vein catheter every minute for 40 min. Before each blood sample was collected, 1.5 ml of blood, corresponding to the catheter dead space, was withdrawn and returned aseptically after each sample. Catheters were flushed with 0.8 ml of sterile saline (0.9%, SAD) after each blood sample.

Dynamics of Glucose-Entrained Insulin Secretion

Entrainment of pulsatile insulin secretion was studied in fasted (18 h) conscious animals (n = 10 control, 5 obese, and 5 obese-STZ animals). Blood samples (0.8 ml) were taken from a jugular vein catheter every minute for 40 min as described above. Every 10th min, starting at t = 0 min, a bolus of glucose (4 mg·kg–1·min–1 glucose, 200 mg/ml; SAD) 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 at 500 kallikrein inhibitor units (KIU)/ml full blood (10,000 KIU/ml Trasylol; Bayer, Lyngby, Denmark) and were kept on ice until centrifugation. Samples were centrifuged (4°C, 10 min, 3,000 rpm), and plasma was separated and stored at –20°C until analysis. Plasma glucose was analyzed using the immobilized glucose oxidase method, 10 µl of plasma in 0.5 ml of buffer (EBIO plus autoanalyzer and solution; Eppendorf, Hamburg, Germany).

Plasma insulin was analyzed 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 and detecting antibody OXI-005 raised against the B-chain of bovine insulin (1)], as previously described (34). The minimal detectable concentration was 3.2 pM, the upper limit was 1,200 pM (no sample dilution), and the inter- and intra-assay variations at three concentration levels were 15.3 and 3.2% (at 342 pM), 9.9 and 7.6% (at 235 pM), and 14.6 and 4.4% (at 87 pM). Recovery at high, medium, and low concentration levels was 97.1, 97.9, and 101%, respectively.

Histological Examination of Pancreas

Histological examination was performed in obese (n = 5), obese-STZ (n = 5), and control animals [n = 4 + 6 (6 of these animals did not have their insulin secretion determined)]. After euthanasia with pentobarbitone (20 ml per animal, 200 mg/ml; Pharmacy of the Royal Veterinary and Agricultural University, Copenhagen, Denmark) at the end of the study period, the pancreas was immediately isolated in toto and fixed in paraformaldehyde (Bie & Berntsen) for 24 h. The following day, the pancreas was embedded in 3% agar solution (cat. no. 303289, Meco-Benzon, Copenhagen, Denmark) at 45°C. After being cooled, the pancreas was cut into 3-mm slices (15), and every fifth tissue slice, starting at slice 1, 2, 3, 4, or 5, determined from a table of random numbers, was retained for sectioning into ~80 cubes of roughly equal size. Those cubes were arranged according to size, as practiced in the smooth fractionator method, with the largest cubes in the middle and the smallest cubes on the ends (3, 17). Every eighth cube, starting at cube 1, 2, 3,... 8, determined from a table of random numbers, was retained and placed in cassettes for dehydration and paraffin infiltration in a tissue processor (Leica TP 1050, Copenhagen, Denmark). The 10–12 pancreas cubes were embedded in paraffin in each of four blocks, and two sections 3 µm thick were cut from each of two blocks on a Leica RM 2165 microtome.

Sections were deparaffinized in xylen, brought to 99% ethanol, treated with 0.5% H2O2 for 20 min to block endogenous peroxidase activity, and rinsed with Tris-buffered saline (TBS). Sections were then immersed in 0.01 M citrate buffer, pH 6, preheated to 90°C, and submitted to antigen retrieval by microwave oven treatment for 3 x 5 min of heating at 80% (Polar Patent, Umea, Sweden). The slides were subsequently cooled, still in the citrate buffer, by immersion of the jar in running tap water and rinsed in TBS + 0.01% Triton X-100 (TBS-T; Sigma, St. Louis, MO), and the tissue sections were "ringed" with a DAKO-pen (DAKO, Copenhagen, Denmark). Sections were then incubated with primary antibodies to insulin and a mixture of antibodies to glucagon, somatostatin, and pancreatic polypeptide to visualize {beta}- and non-{beta}-endocrine cells. Furthermore, sections were counterstained with Mayer's hematoxylin as previously described (34).

Non-{beta}- and {beta}-endocrine cell mass was evaluated stereologically in two sections cut 250 µm apart from each of two blocks in an Olympus BX-50 microscope (Olympus, Copenhagen, Denmark) with a video camera and monitor at a total on-screen magnification of x960. The sections were analyzed by point counting of frames after systematic uniform random sampling using a PC-controlled motorized stage and the CAST-GRID software (Olympus).

Initially, the tissue sections were circumscribed using a x1.25 objective, and the counting of endocrine and exocrine structures took place within this area. The volume fractions of {beta}- or non-{beta}-cells were estimated by point-counting stereological techniques (16) at a total on-screen magnification of x1,026 obtained with a x20 objective, a grid of 4 x 64 points, and step lengths of maximum 900 x 600 µm controlled by the CAST-GRID software. The sections were examined with the origin of the sections blinded to the observer. Mean values of estimated volume fractions were calculated from area-weighted mean values.

Mass of endocrine cells is expressed both as percentage of pancreas volume, milligrams total (per pancreas and animal), and as milligrams per kilogram of body weight.

The presence of glycogen inclusion bodies in {beta}-cells was tested by periodic acid Schiff (PAS) staining of pancreas sections.

Evaluation of Results

Fasting levels and insulin secretion test. Acute insulin response (AIR) to glucose and arginine was calculated as the average increase in insulin levels during the first 10 min after intravenous stimulation [AIR = (mean insulin 1–10 min) – (baseline before dosing)].

Fasting glucose and insulin levels were used as a measure of insulin sensitivity: IS = 1/(FPG x FPI) (where FPG and FPI are fasting plasma glucose and insulin, respectively), based on the homeostasis model (40), and the disposition index (DI; IR x AIR, where IR = 1/IS) was calculated to obtain a relation between {beta}-cell function and insulin sensitivity.

Detection and quantification of pulsatile insulin secretion by deconvolution. The plasma insulin concentration time series were analyzed by deconvolution for detection and quantification of insulin secretory bursts, as previously described (28). In short, deconvolution of venous insulin concentration data was performed with a multiparameter technique (55), which requires the following assumptions. The venous plasma insulin concentrations measured in samples collected at 1-min intervals were assumed to have resulted from five determinable and correlated parameters: 1) a finite number of discrete insulin secretory bursts occurring at specific times and having 2) individual amplitudes (maximal rate of secretion attained within a burst); 3) a common half-duration (duration of an algebraically Gaussian secretory pulse at half-maximal amplitude), which is superimposed upon a 4) basal time-invariant insulin secretory rate; and 5) a biexponential insulin disappearance model in the systemic circulation. The insulin disappearance kinetics were estimated on the basis of model fitting to observed insulin concentration profiles previously reported in normal animals (28). Assuming the fitted insulin disappearance values, we estimated the number, locations, amplitudes, and half-duration of insulin secretory bursts, as well as a nonnegative basal insulin secretory rate, for each data set by nonlinear least squares fitting of the multiparameter convolution integral for each insulin time series. Secretory rates were expressed as mass units of insulin (pmol) released per unit distribution volume (liter) per unit time (min). The mass of hormone secreted per burst (time-integral of the calculated secretory burst) was thus computed as picomoles insulin released per liter of systemic distribution volume. All data analysis was performed in a blinded manner.

Autocorrelation analysis. The periodic nature of individual insulin profiles was assessed by autocorrelation analysis. Because trends can distort the subsequent correlation analyses, they were removed by subtracting from each profile its best fit line, calculated as an 11-point centered, unweighted, moving average process. All autocorrelation analyses were performed on the series from which this procedure was first performed. In the autocorrelation analyses, 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. For autocorrelation analysis, group statistical analysis of the correlation coefficients was performed after using Fishers z transformation (13).

Quantification of irregularity. The regularity of serum insulin concentration time series was assessed by application of approximate entropy (ApEn), which is a model-independent statistic (47) with the parameters m (number of datapoints in each pattern to be compared) and r (the tolerance width). ApEn measures the logarithmic likelihood that runs of patterns that are close (within r) for m adjacent observations will remain close (within the tolerance width r) on subsequent incremental comparisons. ApEn assigns a nonnegative number to a time series, with larger values indicating higher randomness and smaller values indicating more instances of recognizable patterns in the data set. ApEn is dependent on the input parameters m and r. To evaluate both the fine and the coarser patterns in the data, both a small (r = 0.2 x SD) and a large (r = 1 x SD) value of r was used, whereas m was set to 1 for all calculations. The analysis was performed using detrended data as described for autocorrelation analysis.

Statistics

Statistical evaluation of the results was performed by one-way ANOVA with Tukey's multiple comparison test as post hoc analysis or repeated-measures two-way ANOVA. When variances differed between groups, a Kruskal-Wallis test with Dunn's multiple comparison test as post hoc analysis was used instead of one-way ANOVA. All calculations were done using Excel (2000) and GraphPad Prism version 4.00 for Windows (GraphPad Software, San Diego CA). P values of 0.05 or less were considered significant. Data are presented as means (SD) in the text, whereas figures display means with SE.


    RESULTS
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Body Weight

At both the start and the end of the study period (evaluation of pulsatile insulin secretion and insulin secretion test) the obese [51.4 (5.7) and 56.0 (8.3) kg, respectively] and obese-STZ [44.6 (5.6) and 50.8 (6.5) kg, respectively] animals were significantly (P < 0.0001 by one-way ANOVA, P < 0.001 for both groups by post hoc analysis) overweight compared with control animals around the same age (32) [26.1 (6.0) and 34.6 (7.2) kg, respectively].

Body Composition

Relative lean mass (%) was reduced, whereas relative total and truncal fat masses (%) were significantly increased in both obese and obese-STZ animals compared with control animals (Table 2). None of these parameters changed significantly during the period when insulin secretion was studied in obese or obese-STZ animals (data not shown). Similarly, absolute lean and fat mass did not change in the obese or obese-STZ animals during the same period.


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Table 2. Body composition in obese, obese-STZ, and control Göttingen minipigs

 
Fasting Plasma Glucose and Fasting Plasma Insulin

Fasting plasma glucose (mM) did not change in either obese or obese-STZ animals during the period between evaluation of pulsatile insulin secretion and the insulin secretion test [a period of between 10 and 30 days in all animals; 3.2 (0.7) vs. 3.2 (0.5) mM in obese animals, 3.7 (0.8) vs. 3.9 (0.4) in obese-STZ animals, repeated-measures two-way ANOVA]. Similarly, fasting plasma insulin did not change in the two groups over this period of time [35 (13) vs. 46 (17) in obese animals and 49 (34) vs. 50 (24) in obese-STZ animals, repeated-measures two-way ANOVA].

When all three groups were compared, there was a significant difference in fasting plasma glucose (P < 0.05 by one-way ANOVA), and the post hoc analysis showed that this difference was due to a small increase in fasting plasma glucose (P < 0.05) in obese-STZ compared with the normal control animals [3.3 (0.5)], whereas fasting plasma glucose was not increased in obese animals.

When fasting plasma glucose and insulin were evaluated as a measure of insulin sensitivity, there was a trend toward reduced insulin sensitivity in both the obese [0.008 (0.004)] and obese-STZ animals [0.006 (0.003)] compared with normal controls [0.017 (0.016); P = 0.052 by Kruskal-Wallis test].

Insulin Response to Glucose and Arginine

The insulin response to the 0.3 g/kg glucose was not changed in obese animals, whereas obese-STZ animals showed a significantly reduced response (Fig. 1A).



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Fig. 1. Acute insulin response (AIR) to 0.3 g/kg glucose (A), 0.6 g/kg glucose followed by glucose infusion (2 g·kg–1·min–1; B) or 67 mg/kg arginine during hyperglycemia (C) in control (n = 10), obese (n = 5), or obese {beta}-cell-reduced [nicotinamide + streptozotocin (STZ)] animals (obese-STZ; n = 5). **P < 0.01 and *P < 0.05 vs. control by post hoc test. Values are means with SE.

 
The insulin response to a 0.6 g/kg glucose bolus followed by an infusion of glucose was not significantly increased in obese animals, whereas obese-STZ animals showed a significantly reduced insulin response (Fig. 1B). Similarly, the insulin response to arginine bolus injection during hyperglycemia (Fig. 1C) was not changed in obese but was decreased in obese-STZ animals compared with control animals.

Furthermore, when the DI was evaluated, based on the response to 0.3 g/kg glucose, a reduction of DI was seen both in obese [1.8 (0.6)] and especially in obese-STZ [0.7 (0.4)] vs. control animals [4.2 (3.3); P < 0.01 by Kruskal-Wallis test]. When the relation between AIR to 0.3 g/kg glucose and insulin sensitivity was evaluated, it was seen that both insulin sensitivity but especially {beta}-cell function was reduced in some of the obese and all of the obese-STZ animals compared with normal controls (Fig. 2).



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Fig. 2. Relation between {beta}-cell function (AIR to 0.3 g/kg glucose) and insulin sensitivity [1/(fasting plasma glucose) x (fasting plasma insulin)] in control ({blacktriangledown}), obese ({square}) and obese-STZ ({circ}) Göttingen minipigs.

 
Insulin Concentration Time Series

As previously described (28), visual inspection of insulin concentration time series clearly indicates large-amplitude oscillations in control animals, both at baseline conditions and during entrainment.

In contrast, obesity was associated with apparently irregular oscillations during baseline conditions, with small peaks superimposed on a relatively high basal level. When nadir concentrations were compared, these were sharp in most of the control animals, whereas in the obese animals they appeared blunter. Reduced {beta}-cell mass in combination with obesity resulted in an apparent reduction of pulse amplitude.

During entrainment, control animals revealed an on/off pattern with no or little basal insulin in most animals (7 of 10 animals), whereas in three animals some basal secretion was seen (Fig. 3B). In obese animals, entrainment resulted in plateau concentrations between pulses consisting of 4–5 min with steady insulin concentrations of 25–50 pM (Fig. 3D). Entrainment was markedly impaired in two and modestly impaired in one of the obese-STZ animals (Fig. 3F), indicating further deterioration of {beta}-cell function.



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Fig. 3. Plasma insulin concentrations during basal conditions (left) and entrainment (right). A and B: control animals. C and D: obese animals. E and F: obese-STZ animals.

 
Characterization of Pulsatile Insulin Secretion by Deconvolution

During baseline and entrained conditions, no significant differences were found in the pulse interval (Fig. 4, A and B). During baseline conditions, no difference in total insulin secretion was seen between any of the groups (Fig. 4C), whereas during entrainment nonsignificant reductions in total insulin secretion were seen in both obese and obese-STZ animals compared with control animals (Fig. 4D).



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Fig. 4. Parameters from deconvolution during basal (left) and entrained conditions (right) in control (basal, n = 12; entrainment, n = 10), obese (basal and entrainment, n = 5), and obese-STZ animals (basal and entrainment, n = 5). A and B: pulse interval. C and D: total secretion. E and F: basal secretion. G and H: pulse mass. I and J: %secretion as pulses. ANOVA, one-way analysis of variance; KW, Kruskal-Wallis test. *P < 0.05 vs. control; **P < 0.01 vs. control by post hoc test. Values are means with SE.

 
During baseline conditions, basal insulin secretion was different across groups (Fig. 4E), with a nonsignificant tendency toward an increase in obese (2-fold) and a significant increase in obese-STZ animals (3-fold) compared with controls. Similarly, during entrainment, basal secretion was different across the groups (Fig. 4F) due to an increase in obese-STZ animals compared with controls.

Pulse mass during baseline conditions was not significantly affected by obesity or obesity and {beta}-cell reduction, although a tendency for a reduction was seen in both groups (Fig. 4G). During entrainment, pulse mass was different across the groups (Fig. 4H) due to a significant reduction in obese-STZ animals, whereas there was only a nonsignificant reduction in obese animals compared with control values.

As a consequence of the tendency for reduced pulse mass, percent pulsatile secretion during baseline conditions was different across groups (Fig. 4I) due to a reduction in obese animals and even more so in obese-STZ animals compared with control animals. Similarly, during entrainment, percent pulsatile insulin secretion was different across groups (Fig. 4J). This parameter was unchanged in obese compared with control animals, whereas it was reduced in obese-STZ animals, both compared with control animals and obese animals.

Regularity Statistics

During baseline conditions, no significant differences were found in autocorrelation function [0.21 (0.10) in obese, 0.33 (0.03) in obese-STZ, and 0.42 (0.16) in control animals] or ApEn [r = 1.0: 0.51 (0.16) in obese, 0.63 (0.11) in obese-STZ, and 0.55 (0.10) in control animals; r = 0.2: 1.06 (0.12) in obese, 1.14 (0.08) in obese-STZ, and 1.15 (0.07) in control animals] between any of the groups.

Similarly, during entrainment, no significant differences were found in autocorrelation function [0.64 (0.10) in obese, 0.59 (0.15) in obese-STZ, and 0.50 (0.21) in control animals], whereas ApEn was different between groups (P < 0.05 for r = 1.0, P < 0.01 for r = 0.2 by one-way ANOVA) due to a reduction in obese animals compared with control animals [ApEn r = 1.0: 0.37 (0.07) vs. 0.49 (0.07); ApEn r = 0.2: 0.85 (0.14) vs. 1.13 (0.13)] and to obese-STZ animals [ApEn r = 1.0: 0.54 (0.12) and ApEn r = 0.2: 1.05 (0.13)].

{beta}-Cell Mass and Pancreas Morphology

Results from the histological analysis of pancreata are summarized in Table 3. Absolute pancreas weight did not differ between the groups, whereas relative pancreas weight was different across groups due to a reduction in both obese and obese-STZ compared with control animals.


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Table 3. Results from histological examination of pancreata from obese, obese-STZ, and control Göttingen minipigs

 
{beta}-Cell volume (expressed as percentage of pancreas) was different across groups due to an increase in obese animals, whereas it was reduced in obese-STZ animals. {beta}-Cell mass (expressed as mg/kg) was different across groups due to a reduction in obese-STZ animals.

Figure 5 shows sections of pancreata from the three experimental groups. The pancreas of the control animals had many large- and intermediate-sized irregular islets and small clusters of islet cells (Fig. 5A, inset). The number of fat cells in the exocrine pancreas was very limited, and no presumed fat globules could be seen in the {beta}-cells (Fig. 5A). The pancreas of the obese animals showed many intra- and interlobular fat cells and small clusters thereof (Fig. 5B). There was a clear expansion of the number of well-staining {beta}-cells, with many very large and large compact islets with a core of {beta}-cells. Many of the {beta}-cells contained a number of small, pale inclusion bodies (fat globules) in their cytoplasm (Fig. 5B).



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Fig. 5. Pancreatic sections from minipigs. A–C: {beta}-cell fat globules seen in insulin-stained sections from control (A), obese (B), and obese-STZ animals (C). Pictures were taken with x100 oil immersion objective. Insets: islets and inter- and intralobular fat seen with overall pancreas morphology. Pictures were taken with x4 objective. Bar in B, 25 µm; bar in inset in B, 500 µm. D: periodic acid Schiff-stained section negative for glycogen. Picture was taken with x100 oil immersion objective.

 
The obese-STZ animals showed a very high number of intra- and interlobular fat cells and small clusters thereof (Fig 5C, inset). There were still some large- and intermediate-sized islets with fewer well-staining {beta}-cells. Many islet remnants were devoid of {beta}-cells as seen from the staining of non-{beta}-cells (not shown). Several of the {beta}-cells contained quite a number of larger inclusion bodies (fat globules) in their cytoplasm. There was a relative increase of non-{beta}-cells, and their location was more central than peripheral (not shown). Figure 4D shows that the large inclusion bodies were not glycogen, as they were negative after PAS staining.


    DISCUSSION
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The present study was designed specifically to assess {beta}-cell function in vivo, with a focus on high-frequency oscillatory insulin release in control, obese, and obese-STZ minipigs, possibly representing stages toward the development of type 2 diabetes. In addition, we have assessed other major aspects of in vivo {beta}-cell function and islet morphology in each group.

The main conclusion to be drawn from the present study is that relatively long-term obesity (up to ~2 yr of age) was not commensurate with major indicators of {beta}-cell dysfunction in vivo in this model. However, we did observe a reduction in the proportion of insulin secreted in pulses in obese compared with control animals, whereas pulsatile responses to entrainment appeared to be more orderly than that seen in control animals. These findings indicate that obesity, in itself, is linked to subtle changes in the coordination of pulsatile insulin secretion, but whether this is a cause or a consequence of obesity cannot be determined from the present data. The apparently increased regularity seen in obese animals could represent an adaptation to insulin resistance, although experimental induction of insulin resistance has been reported to disturb high-frequency pulsatile insulin secretion (20). However, one may speculate that the improved regularity in the obese pigs is a result of increased levels of circulating free fatty acids (FFA). In support of this, an inverse relation between disorderliness and circulating FFA levels has been reported previously in humans, indicating a stabilizing role of FFA on pulsatile insulin secretion (50). Long-chain fatty acids have been reported to potentiate glucose-stimulated insulin secretion (59). However, as FFA levels were not measured in this study, this possible explanation remains to be fully explored.

Considering the hyperbolic relation between insulin sensitivity and insulin secretion (22, 23, 36), obese animals would have been expected to adapt to the apparent insulin resistance, as was seen in two animals (Fig. 2), whereas failure to compensate may indicate impaired {beta}-cell function, as was seen in some of the obese and all of the obese-STZ animals. Therefore, the DI could be viewed as a tool to detect early {beta}-cell failure in the minipig.

A primary reduction of {beta}-cell mass results in a corresponding reduction of pulse mass and amplitude but is not thought to cause dysfunction of regular pulsatile insulin secretion from the remaining {beta}-cell population during baseline or entrained conditions (24, 29). The impaired pulsatile insulin secretion in type 2 diabetes (21, 27, 50) may therefore have distinct and different causes. Insulin resistance could be a plausible mechanism for disturbed pulsatile insulin secretion, and this has previously been reported in obese humans (61), whereas others have reported increased, but more variable, pulsatility in obese subjects (52) or reduced relative amplitude of normal-frequency pulses (18). Furthermore, weight loss has been reported to improve pulsatile insulin secretion in humans (60). On the other hand, increased frequency of pulsatile insulin secretion has been suggested as a cause of insulin resistance in humans (46).

Compared with the obese group, the obese-STZ animals had a reduced insulin secretory response to glucose and arginine, and all methods for evaluation of pulsatile insulin secretion revealed a deterioration of insulin secretion compared with control and obese animals. Furthermore, the apparent increase in basal insulin secretion, especially during entrainment, has not been reported previously in lean {beta}-cell-reduced animals (24, 29) and could therefore represent a further deterioration in {beta}-cell function when obesity and reduced {beta}-cell mass are present concomitantly. This finding indicates a regulation of nonpulsatile insulin secretion that is different from regulation of pulsatile insulin secretion. The data do not exclude the presence of asynchronous pulsatile release from individual islets, where lack of intrapancreatic coordination would result in the overall release being apparent as nonpulsatile. However, during entrainment, large oscillations can be produced with 60% of insulin released as distinct secretory bursts. The remaining basal secretion, therefore, could be a result of constitutive insulin release and/or a population of {beta}-cells that is not entrainable.

As has been reported in humans (10), obesity resulted in increased {beta}-cell volume, whereas {beta}-cell mass (in mg/kg body wt) did not change, indicating that {beta}-cell mass is increased proportionally to the increase in body weight in this model. Furthermore, by visual inspection of the islets, obesity resulted in the formation of somewhat larger islets in the minipigs, similar to what has been reported in one study in humans (37), whereas a more recent study has shown only a minimal increase in islet size in obese humans (10). Both obese and obese-STZ animals showed intra- and interlobular fat infiltration, as well as intracellular fat globules in the {beta}-cells, that could possibly be involved in development of {beta}-cell dysfunction.

The magnitude of the impairment of pulsatile insulin secretion in obese and obese-STZ animals resulted in an impaired ability to fit insulin kinetic parameters to peripheral insulin oscillations in individual animals (28), which was not seen in lean {beta}-cell-reduced animals (29). However, data in the present study were in agreement with literature values (28) that were used herein. The close agreement between model-based deconvolution fitting (assuming literature values) and observed time series concentrations strongly supports the kinetic modeling used; but ideally, values based on insulin bolus injection in individual animals might have been a more appropriate data source for study of pulsatile insulin secretion in either obese or {beta}-cell-reduced obese animals. However, applying several changes in insulin kinetics did not affect relative contributions of pulsatile components.

In conclusion, obesity is only marginally associated with disturbed pulsatile insulin secretion, whereas further impairment was observed in the {beta}-cell-reduced obese group. The abnormalities included decreased pulsatile secretion and impaired first-phase insulin secretion (AIR), all of which represent features of {beta}-cell dysfunction in type 2 diabetes. Surprisingly, regularity of entrained insulin secretion appeared to be improved in obese animals, suggesting that it is a compensatory phenomenom secondary to elevated insulin resistance. Thus obesity and insulin resistance, as such, are not linked with a general reduction of {beta}-cell function, whereas dynamics of insulin secretion are, to some extent, perturbed.

The data suggest a sequential process in the development of {beta}-cell dysfunction in the development of diabetes from the normal state in the minipig model, with the three experimental groups representing different stages of the disease. We conclude that abnormalities of insulin pulsatility are the most sensitive single marker of {beta}-cell dysfunction in the {beta}-cell mass-reduced Göttingen minipig model.


    ACKNOWLEDGMENTS
 
We thank Lene Sejersen Winther, Helle Nygaard, Lotte Gotlieb Sørensen, Anne Grethe Juul, Annemette Petersen, Susanne Primdal, Karsten Larsen, Ejnar Eriksen, and Hans Rasmussen for excellent technical assistance.


    FOOTNOTES
 

Address for reprint requests and other correspondence: M. O. Larsen, Dept. of Pharmacology Research I, Pharmacology Research and Development, Novo Nordisk Park F6.1.30, Maaloev Dk-2760, Denmark (E-mail: mmla{at}novonordisk.com)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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