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Am J Physiol Endocrinol Metab 292: E1183-E1190, 2007. First published December 19, 2006; doi:10.1152/ajpendo.00168.2006
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Glucose intolerance and reduced islet blood flow in transgenic mice expressing the FRK tyrosine kinase under the control of the rat insulin promoter

Cecilia Annerén,1,2 Michael Welsh,1 and Leif Jansson1

1Department of Medical Cell Biology, Uppsala University, Uppsala; and 2Department of Cell and Molecular Medicine, Medical Nobel Institute, Karolinska Institutet, Stockholm, Sweden

Submitted 7 April 2006 ; accepted in final form 8 December 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The FRK tyrosine kinase has previously been shown to transduce beta-cell cytotoxic signals in response to cytokines and streptozotocin and to promote beta-cell proliferation and an increased beta-cell mass. We therefore aimed to further evaluate the effects of overexpression of FRK tyrosine kinase in beta-cells. A transgenic mouse expressing kinase-active FRK under control of the insulin promoter (RIP-FRK) was studied with regard to islet endocrine function and vascular morphology. Mild glucose intolerance develops in RIP-FRK male mice of at least 4 mo of age. This effect is accompanied by reduced glucose-stimulated insulin secretion in vivo and reduced second-phase insulin secretion in response to glucose and arginine upon pancreas perfusion. Islets isolated from the FRK transgenic mice display a glucose-induced insulin secretory response in vitro similar to that of control islets. However, islet blood flow per islet volume is decreased in the FRK transgenic mice. These mice also exhibit a reduced islet capillary lumen diameter as shown by electron microscopy. Total body weight and pancreas weight are not significantly affected, but the beta-cell mass is increased. The data suggest that long-term expression of active FRK in beta-cells causes an in vivo insulin-secretory defect, which may be the consequence of islet vascular abnormalities that yield a decreased islet blood flow.

beta-cells; insulin secretion

DIABETES MELLITUS IS A DISEASE of insufficient insulin delivery. Whereas a reduced number of beta-cells due to immune-mediated destruction causes type 1 diabetes, type 2 diabetes has a more complex etiology. Studies have demonstrated both insufficient insulin secretion and peripheral insulin resistance as contributing factors, and to complicate matters further, peripheral insulin resistance can cause insulin secretory defects and vice versa (20). However, a functional impairment of the beta-cells seems to be a prerequisite for the initiation of the disease (7, 20).

The FRK tyrosine kinase (also named GTK/RAK/BSK/IYK) is a cytoplasmic tyrosine kinase expressed in epithelial cells, particularly those in the gastrointestinal tract (32), including the pancreas and beta-cells (35). Although FRK expression levels are not very high in beta-cells, FRK is nevertheless likely to have an impact on beta-cell function, since tyrosine kinases do not require high levels of expression to exert effects. To assess a role of FRK in beta-cells, a transgenic mouse expressing kinase-active FRK under the control of the insulin promoter (RIP-FRK, previously named CBA-GTK) was generated. This transgenic mouse shows significant alterations in beta-cell function, such as increased beta-cell mass and increased beta-cell replication upon partial pancreatectomy but also increased susceptibility to beta-cell toxins such as streptozotocin and cytokines (1, 2). However, the consequences of these altered beta-cell characteristics for the long-term islet function have not been elucidated.

The vasculature of the pancreatic islets is both anatomically and physiologically autonomous from that of the exocrine parts of the gland (8, 17). The regulation of islet blood perfusion is exerted via complex interactions between nervous, endocrine, paracrine, and metabolic signals to maintain a high blood flow, adapted to the immediate needs of the endocrine cells (8, 12). Previous studies have consistently shown that type 2 diabetes-like conditions are associated with increased islet blood perfusion (4, 6, 9, 11, 29). It should be noted that abnormalities of the islet vasculature, including an altered capillary angioarchitecture, have been found in some, but not all, type 2 diabetes models (8, 24). To what extent an islet blood flow increase affects the endocrine function is at present unknown. We have speculated that an increased shear stress on endothelial cells may affect and alter their expression of paracrine substances, which may then influence also more specific organ functions (10).

This study was performed to assess a role of FRK on the long-term function of beta-cells by maintaining FRK transgenic male mice (2) individually caged for more than 4 mo. We observe that, despite an increased beta-cell mass in the RIP-FRK mice, they exhibit beta-cell dysfunction in vivo with impaired insulin secretion, causing mild glucose intolerance. Furthermore, an altered islet vascular morphology was seen, and this was associated with a decreased islet blood perfusion. Thus, our findings suggest that changes in islet vascular physiology may influence glucose tolerance.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals. RIP-FRK transgenic mice, expressing a constitutively active FRK (FRK with a Y504F mutation and a COOH-terminal myc-epitope) under the control of the rat insulin I promoter, were generated as previously described (2). Homozygous FRK transgenic and control (mice of the parental CBA/C57Bl6 strain used for generating the transgene) male mice were weaned at 3 wk of age and put in single-mouse housing. The mice were fed a standard diet. Experiments were performed on 4- to 6-mo-old mice. All mice were housed in a temperature-controlled room with a 12:12-h light-dark cycle and had continuous access to standard pelleted chow (Type R3; Ewos, Sollentuna, Sweden) and water throughout the study. All experiments were approved by the local animal ethics committee.

RT-PCR. Selective expression of the transgene in islets was verified by RT-PCR and Southern blot analysis. Total RNA, extracted from isolated islets or liver tissue using Qiagen's RNeasy mini kit with on-column DNase digestion, was subjected to RT-PCR as follows: 1–4 µg of RNA were converted to cDNA by using SuperScript First-Strand Synthesis System (Invitrogen). Reactions not catalyzed by RT were run in parallel. cDNA (0.1–1 µl) was added to each 50 µl of PCR reaction using Taq Polymerase (Promega) and primers corresponding to nucleotide 1502–1522 in the FRK cDNA (5'-GGTAGACATGGCGGCACAGGT-3') and the last 18 nucleotides of the myc-epitope in the transgenic FRK cDNA (5'-TGAGATGAGTTTTTGTTC-3').

Intravenous glucose tolerance test. The fed-state glucose and insulin tolerance test was assessed as follows. Mice were injected intravenously with 250 µl of 30% (wt/vol) glucose. Blood glucose was determined (Pen Sensor; MediSense, Waltham, MA; Svenska MediSense, Stockholm, Sweden) on venous blood samples collected from the tail immediately before the glucose injections and after 10, 30, 60, and 120 min. In separate mice, blood was collected from the tail immediately before and 10 and 30 min after glucose administration, and the insulin concentrations in these blood samples were measured by ELISA (Mercodia, Uppsala, Sweden).

Insulin tolerance test. Mice were injected intraperitoneally with 0.75 E insulin/kg body wt (Actrapid Human; Novo Noridisk, Gentofte, Denmark), and blood glucose was determined immediately before the insulin injections and after 10, 30, 60, and 120 min.

Morphology. Pancreata from transgenic and control mice were fixed overnight in 10% (vol/vol) neutral buffer formalin, dehydrated in ethanol, and embedded in paraffin. The samples were sectioned (5 µm) and mounted on glass slides. The sections were deparaffinated and rehydrated. Endogenous peroxidases were inhibited with 5% H2O2. Nonspecific binding was blocked by incubation with 10% pig serum in PBS-T (0.2% NP-40 + 0.1% Triton X-100 in PBS) for 60 min and thereafter incubated with a polyclonal FRK antibody (34) diluted in blocking buffer in a humified chamber overnight at 4°C. The slides were then incubated with secondary antibody (biotinylated donkey anti-rabbit antibody 1:200) for 30 min followed by diaminobenzidine staining using the StrepABComplex system (Dakopatts).

For electron microscopy, pieces of the dorsal pancreas were fixed in 2.5% (vol/vol) glutaraldehyde and embedded in Epon 812. Ultrathin sections were contrasted with uranyl acetate and lead citrate. Electron microscopy was carried out with a Hitachi H-7100 transmission electron microscope at an accelerating voltage of 75 kV. Endothelial cells within islets (identified by surrounding endocrine cells) were then evaluated. Cytoplasmic thickness and lumen diameter were measured on the photographs, whereas the numbers of fenestrations, caveolae, and cytoplasmic vesicles were scored as number per length unit. All electron microscopic photographs were analyzed blindly with respect to origin.

Pancreas perfusions. The mice were anesthetized with an intraperitoneal injection of 0.02 ml/g body wt avertin [a 2.5% (vol/vol) solution of 10 g of 97% 2,2,2-tribromoethanol (Sigma-Aldrich) in 10 ml of 2-methyl-2-butanol (Kemila, Stockholm, Sweden)]. The pancreas and duodenum were then prepared for perfusion according to a previously described technique (16). Perfusion was performed with Krebs-Ringer-bicarbonate-HEPES (KRBH) supplemented with 2% dextran T70 (Pharmacia-Upjohn, Uppsala, Sweden) and 2% bovine serum albumin (fraction V; ICN Biomedicals). Either 2.8 or 16.7 mmol/l D-glucose or 5.5 mmol/l D-glucose plus 10 mmol/l L-arginine (Sigma-Aldrich) were added to the KRBH. Perfusion took place at a constant flow rate of 1 ml/min, and the medium was continuously gassed with oxygen (O2/CO2, 95:5) and kept at a temperature of 37°C. The experiments started with perfusion with 2.8 mmol/l glucose (15 min), followed by 16.7 mmol/l glucose (20 min), 2.8 mmol/l glucose (15 min), 5.5 mmol/l glucose plus 10 mmol/l arginine (10 min), and finally 2.8 mmol/l glucose. Aliquots of 1 ml of the effluent fluid were collected from the portal vein and stored at –20°C. The insulin concentrations in these samples were measured by ELISA (Mercodia, Uppsala, Sweden). Any experiments where the insulin concentrations did not return to basal values during perfusion with low glucose (2.8 mmol/l) concentrations were excluded from the study (n = 2). To calculate total insulin release, planimetry of the individual perfusion curves was performed, and area under the curve was calculated. Separate measurements were made for the first phase (0–5 min) and second phase (5 min and onward) of stimulated insulin secretion.

Blood flow measurements. The experiments were performed according to a protocol previously described in detail (9). Briefly, nonfasted mice were anesthetized with an intraperitoneal injection of avertin (see Pancreas perfusions), heparinized, and placed on a heated operating table. Polyethylene catheters were inserted via the right carotid artery into the ascending aorta and into the femoral artery. The cranial catheter was connected to a pressure transducer (PDCR 75/1; Druck, Groby, UK), thereby allowing constant monitoring of the mean arterial blood pressure. Some of the animals were subjected to a subdiaphragmatic vagotomy; i.e., the left and right vagal nerves were cut at the level of the cardia region. The rationale for this was that in most animal models of type 2 diabetes the changed islet blood flow can be prevented by this measure due to loss of parasympathetic nervous control (11). After a stable blood pressure was achieved, 1.5–2.0 x 105 nonradioactive microspheres (Dye-Trak; Triton Technology, Los Angeles, CA) with a mean diameter of 10 µm were injected for 10 s via the catheter placed with its tip in the ascending aorta. Starting 5 s before the microsphere injection and continuing for a total of 60 s, an arterial blood sample was collected from the catheter in the femoral artery at a rate of ~0.20 ml/min. The exact withdrawal rate was confirmed in each animal by weighing the sample. After the reference sample was obtained, another blood sample was drawn for measurement of blood glucose. The whole pancreas and both adrenal glands as well as samples from the duodenum, colon, and left kidney were removed, blotted, weighed, and treated with a freeze-thawing technique to visualize the microspheres, as previously described (18). The blood flow values were calculated according to the formula Qorg = Qref x Norg/Nref, where Qorg is organ blood flow (ml/min), Qref is the withdrawal rate of the reference sample (ml/min), Norg is the number of microspheres present in the organ, and Nref is the number of microspheres in the reference sample. A difference of less than 10% in blood flow values between the adrenal glands was used to confirm adequate mixing of the spheres in the circulation.

Determinations of islet volume and mass. The frozen and thawed preparations used for the blood flow measurements referred to above were also used for determinations of islet volume and mass, as previously described in detail. Briefly, the volume was determined by randomly placing a grid over the pancreatic pieces and then determining the volume with a point sampling technique (33).

Islet insulin secretion. Islets were isolated from control or FRK transgenic mice and immediately put in 100 µl of preincubation medium (KRH buffer, 3.3 mM glucose) for 30 min. The medium was then carefully replaced with 100 µl of the same buffer containing 1.7 mM glucose and incubated further for 1 h at 37°C in 5% CO2. The medium was carefully removed (for insulin analysis) and replaced by a medium containing 16.7 mM glucose for an additional 1-h incubation. Medium and islets were then collected for protein and insulin (by ELISA) analysis.

Statistical analysis. Means ± SE for the number of observations are given. Each observation is based on the value obtained from one animal. Probabilities (P) of chance differences between experimental groups were calculated with Student's unpaired t-test. P < 0.05 was considered to be statistically significant for all comparisons.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
FRK transgene expression. Rat insulin promoter-1 was used to target expression of the FRK transgene to beta-cells (2). To validate appropriate targeting of the transgene product, expression was assessed in liver and pancreas. Expression of transgenic FRK mRNA was detected in mRNA from pancreatic islets but not in mRNA from the liver of transgenic mice (Fig. 1). Immunohistochemistry of pancreatic sections from control and FRK-transgenic mice confirmed slightly elevated FRK immunoreactivity in the islets, and not in the exocrine tissue, of the transgenic mice (results not shown).


Figure 1
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  		Fig. 1. Expression of RIP-FRK mRNA and protein in islets of Langerhans. RNA isolated from control or RIP-FRK mouse liver or pancreas was subjected to RT reactions or not prior to PCR. PCR was performed using one primer recognizing the FRK sequence and the other the myc-tag sequence present in the transgene mRNA.

 
Animal weight. The weight of the FRK transgenic mice was not increased (Table 1). Pancreas weight was also not significantly affected, although there was a trend toward an increased pancreas weight in the FRK transgenic animals, as has been shown previously (1, 2) (P = 0.08, Student's unpaired t-test).


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  		Table 1. Values recorded in male control and FRK mice aged 4–6 mo

 
Glucose homeostasis. To address whether long-term expression of the FRK transgene might influence glucose homeostasis, male mice were housed individually from 3 wk of age and subjected to an intravenous glucose tolerance test when aged 4–6 mo. In contrast to our previously reported results from younger male and female mice (1, 2), which did not display any differences between groups, the present study shows that older, individually housed FRK transgenic male mice have an impaired glucose tolerance compared with control mice, with elevated glucose values at 10, 60, and 120 min. The basal blood glucose concentrations, however, were similar in the control and FRK transgenic mice (Fig. 2A). Blood insulin concentrations were also measured. In the control mice, glucose significantly increased the blood insulin concentration at 10 min, which was restored to a basal level at 30 min. In the FRK transgenic mice, however, glucose failed to increase the blood insulin level (Fig. 2B). Insulin tolerance tests were also performed. Injected insulin readily decreased the blood glucose concentration in the control mice (P < 0.05 by one-way ANOVA, Dunnett's method at 30 min), whereas no decrease could be observed in the RIP-FRK transgenic mice (Fig. 2C). In conclusion, we observe that expression of the FRK transgene in beta-cells causes impaired glucose tolerance and reduced insulin sensitivity under the present conditions.


Figure 2
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  		Fig. 2. A: blood glucose concentrations at different times after iv injection of 250 µl of a 30% glucose solution in control or FRK transgenic mice. *P < 0.05, **P < 0.01, ***P < 0.001 vs. control mice. Values are based on 16 mice in each group. B: serum insulin concentrations at different time points after iv injection of 250 µl of a 30% glucose solution in control or FRK transgenic mice. *P < 0.05 vs. insulin concentration at the time before glucose injection (0 min). Values are based on 10 mice each group. C: blood glucose concentrations at different times after ip injection of insulin in control or FRK transgenic mice. Values are means ± SE for 3–6 experiments. *P < 0.05 vs. 0 time point before insulin injection by one-way ANOVA; +P < 0.05 vs. corresponding control value by Student's t-test.

 
Insulin secretion during pancreas perfusion. The reduced in vivo insulin secretion prompted us to investigate the secretory response of perfused pancreas. Isolated pancreata from control and FRK transgenic mice were sequentially perfused with high glucose and arginine, whereas the insulin contents of the effluents were determined (Fig. 3A). The first-phase insulin response was unchanged, whereas the second-phase insulin effluents in response to both stimuli were significantly reduced from the FRK transgenic pancreata (Fig. 3B). Furthermore, total insulin release, i.e., combined first and second phases, was lower during arginine stimulation in FRK transgenic mice (Fig. 3B).


Figure 3
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  		Fig. 3. A: insulin concentrations in the effluent medium collected from the perfused pancreas from FRK transgenic mice (n = 6) and control mice (n = 5). Insulin release was stimulated with 16.7 mmol/l D-glucose (15–35 min) and 5.5 mmol/l D-glucose + 10 mmol/l L-arginine (50–60 min). At all other time points, organs were perfused with KRBH buffer containing 2.8 mmol/l D-glucose. B: insulin release calculated as areas under the curve of the perfusion curves presented in A. Figs. 1 and 2 refer to the 1st (0 to 5 min) and 2nd phases (5 min and onward) of insulin release, respectively. 1+2 represents combined area under curve for both 1st and 2nd phases of insulin secretion stimulated with glucose (GLU) or arginine (ARG). Values are means ± SE. *P < 0.05, **P < 0.01 vs. FRK mice.

 
Insulin secretion of isolated islets. Islets isolated from control or FRK-transgenic mice were assessed for their in vitro insulin secretion to exclude any fundamental secretory defect as the cause of the findings in Fig. 3. Islets from both groups of animals displayed a comparable glucose-induced secretory response (Table 2). Likewise, the FRK transgenic islet insulin content was similar to that of control islets.


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  		Table 2. Insulin secretion from islets isolated from control or FRK mice

 
Islet morphology. Pancreatic islet volume and islet mass were both markedly increased in FRK transgenic mice (Table 1). The islet morphology appeared otherwise normal. Islet endothelial cell morphology was assessed by electron microscopy, and capillaries with a characteristically oval lumen were observed. When the lumen was measured, capillaries in the FRK transgenic islets showed a smaller lumen diameter (Fig. 4 and Table 3). This was done in EM sections, since capillary lumina are difficult to identify in conventional light microscopy sections. These were otherwise similar to those of the control islets with respect to fenestrations and cytoplasmic thickness (Table 3). The transgenic islets, however, contained endothelial cells with a higher density of cytoplasmic vesicles (Table 3).


Figure 4
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  		Fig. 4. Electron micrographs of endothelial cells in islets from control (A and C) and FRK mice (B and D), respectively. Magnifications in A and B are x5,000 and in C and D are x12,500, respectively. L, capillary lumen; RBC, erythrocytes; f, fenestrations; *, cytoplasmic vesicles. Capillaries of control islets show a larger lumen diameter.

 

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  		Table 3. Electron microscopy of islets from control or FRK mice

 
Organ blood flow. The altered endothelial morphology motivated islet blood flow measurements. Mean arterial blood pressure (Table 1) and hematocrit values (not shown) were similar in both groups of animals. Total pancreatic, duodenal, colonic, and adrenal blood flows were similar in control and FRK transgenic mice, whereas renal blood flow was decreased in the latter group (Table 1). The total islet blood flow per pancreas weight was not different between the control and FRK transgenic mice (6.4 ± 1.7 vs. 13.8 ± 3.7 µl·min–1·g pancreas–1, respectively; P = 0.104). However, when recalculated as blood perfusion per islet weight, the islet blood flow was significantly reduced (Fig. 5). To assess whether this difference was due to parasympathetic nervous control, mice were subjected to subdiaphragmatic vagotomy. This procedure decreased islet blood flow per islet weight in both control and FRK-transgenic mice (P < 0.01 for both comparisons), but the value in the latter group still remained lower (2.92 ± 0.83 vs. 0.96 ± 0.26 µl·min–1·g pancreas–1, respectively, P = 0.035), suggesting that the effect of the FRK transgene was not due to parasympathetic nervous control.


Figure 5
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  		Fig. 5. Pancreatic islet blood flow per islet weight in control and FRK mice, respectively. Values are means ± SE for 8–9 experiments. *P < 0.05 vs. control mice.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In the current study, we describe glucose intolerance in individually housed transgenic male mice at 4–6 mo of age expressing a kinase-active mutant of the cytoplasmic tyrosine kinase FRK under the control of the insulin promoter. This promoter construct is commonly used for targeting transgene expression to beta-cells (14). The present data confirm the anticipated pattern of expression of the transgene in islet cells compared with liver or the exocrine pancreas. It should be noted, however, that recent studies have demonstrated inappropriate expression of rat insulin promoter transgenes in a subset of cells, particularly in the hypothalamus (26). The present data do not exclude erroneous expression of the FRK transgene in such cells as discussed further below.

The FRK transgenic mice display an impaired secretory response in vivo and in the perfused pancreas, as demonstrated by lowered second-phase release in response to glucose and arginine. This secretory defect is unusual compared with animal models of type 2 diabetes in that the second phase is primarily reduced and that the impairment is seen to a similar extent in both the glucose and arginine responses. Thus, in most animal models there is a defect in phase 1 insulin secretion that is more pronounced during glucose than during arginine stimulation (13, 21, 23). Islets isolated from transgenic mice displayed a normal glucose-responsive insulin secretion, suggesting that the deficient insulin secretion is dependent on the intact pancreatic microenvironment, but its exact nature is at present unknown.

The glucose intolerance currently described is likely to stem from the in vivo secretory defect observed to which the reduced peripheral insulin sensitivity may contribute. In line with one possible interpretation of the findings, the presently observed insulin-secretory defect is primary and could be the consequence of transgene expression in the islets of Langerhans, and thus the reduced peripheral insulin sensitivity ensues as a consequence of this. It awaits confirmation whether this represents a changed sensitivity in liver or skeletal muscle. Previous experiments on younger FRK transgenic mice showed no signs of impaired glucose tolerance, however (2). Thus, the present experimental protocol with older single-caged male mice appears to contribute to a tendency toward glucose intolerance due to a more sedentary environment than when caged several together, which may make the animals more prone to acquire the observed reduced peripheral insulin sensitivity. In conclusion, the observed phenotype, with impaired glucose intolerance and reduced peripheral insulin sensitivity, may be a consequence of both genes (i.e., the expression of the transgenes in the islets) and the environment. It cannot be excluded, however, that erroneous or leaky expression of the transgene at other sites than in beta-cells, e.g., in the hypothalamus, could contribute to the reduced peripheral insulin resistance.

We currently observe that the total islet blood flow is not affected by the FRK transgene. However, when related to the islet mass, islet blood flow was decreased. Previous studies on islet blood perfusion have shown that conditions with increased functional demands on the islets, either temporarily, such as after partial pancreatectomy (19), continuous glucose infusion (5, 28), and dietary manipulations (31), or more permanently, as in type 2 diabetes-like conditions (3, 6, 15, 29), are associated with increases in islet blood perfusion. Whether, and to what extent, this blood flow increase affects islet function is at present unknown. However, recent experiments on pregnant rats, in which the increased demands of insulin are met by growth of the islets (27), showed that, even if total islet blood flow was increased, islet blood perfusion was actually decreased when corrected for islet mass (30), i.e., a finding similar to that in the present study, with the exception that the rats were not glucose intolerant. This finding in rats demonstrates that decreased islet blood perfusion is not necessarily associated with an impaired islet endocrine function. It should also be noted that in pregnant rats the blood flow decrease is not affected by the vagal system, i.e., findings similar to those in the present study. We and others (3, 11, 29) have previously demonstrated on several occasions that changes in islet blood flow during impaired glucose tolerance are usually associated with the vagus system. The observed decrease in islet blood flow may be an adaptive response to the increased islet mass. Thus, the total islet blood flow is relatively unchanged by the transgene. This adaptation seems structural in nature, suggested by the electron microscopy findings, which revealed a smaller endothelial cell diameter in the transgenic islets and the unchanged blood flow difference seen after vagotomy. The latter also argues against the possibility that hypothalamic misexpression of the transgene is the cause of the reduction in islet blood flow. The FRK transgenic beta-cells replicate at an elevated rate in response to partial pancreatectomy (1), and the pancreas beta-cell volume is elevated in this transgenic mouse (2), as confirmed in the present study. The increased beta-cell mass may thus be counteracted by a vasculature with capillaries of smaller diameter and lower blood flow. However, as the mice grow old and insulin sensitivity decreases, particularly when individually housed, the preceding adaptation of the microvasculature may at that point yield insufficient blood flow to adequately maintain glucose homeostasis. Thus, the question of whether the decreased islet blood flow is instrumental in conveying the glucose tolerance in the current setting remains an intriguing, but unproven, possibility. Figure 6 illustrates our current view of the appearance of glucose intolerance in the RIP-FRK male mouse, emphasizing the concerted effects of structural changes occurring in the islets as a consequence of transgene expression and environmental effects related to aging and single-mouse housing.


Figure 6
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  		Fig. 6. Model explaining the RIP-FRK phenotype. Islets with vasculature are shown. FRK expands beta-cell mass without concomitantly increasing vascular "capacity". At 3 mo of age, mice have sufficient capacity to maintain glucose homeostasis. When single-caged male mice age, an increased functional demand ensues, and eventually glucose intolerance develops as a consequence of reduced phase secretion. The low capacity of the vascular bed could be the cause of the poor secretory function at this stage.

 
The correct integrity of the islet microvasculature is essential for normal islet function, as it not only provides the means for transport of nutrients and oxygen but also ensures adequate paracrine interactions within the individual islets (8, 25). A recent study with beta-cell-specific vascular endothelial growth factor (VEGF)-A gene ablation resulted in glucose intolerance and diabetes (22). The density of the microvasculature was decreased, but more severely, and capillaries were dysfunctional and exhibited an abnormal morphological appearance (22). The importance of normal vascular function for glucose tolerance is demonstrated by that study, but our current data do not support a role of VEGF-A in the FRK-induced vascular abnormalities, since we currently observe a decreased capillary lumen diameter without changes in fenestrations. Our present data are thus in line with the possibility that increased beta-cell replication, as a consequence of the FRK transgene expression, is not paralleled by an appropriate compensation in the size of the microvasculature. To directly address the possibility that the reduced insulin secretion is a consequence of a defective islet vasculature, the RIP-FRK mouse can be crossed with RIP transgenic mice exhibiting improved islet vasculature, i.e., RIP-VEGF, to monitor islet blood flow and insulin secretion.

In addition to the effects on islet blood flow, we also noted a decrease in total renal blood flow in the FRK transgenic mouse. No macroscopic or microscopic changes could be seen in the kidneys, so the mechanisms behind this finding are at present unclear.

In summary, this study presents an animal model, the FRK transgenic mouse, in which disturbed insulin-secretory response can be related to decreased peripheral insulin sensitivity and impaired islet blood flow resulting in glucose intolerance. Furthermore, the animals present a morphologically abnormal microvasculature in the islets, which suggests a link between the islet blood vessels and their endocrine function.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
Financial support was received from the Swedish Research Council (72X-109, 31X-10822), the Swedish Diabetes Association, the Juvenile Diabetes Research Foundation, the EFSD/Novo Nordisk for Type 2 Diabetes Research Grant, the NOVO Nordic Fund, and the Swedish Cancer Foundation and the Family Ernfors Fund.


   ACKNOWLEDGMENTS
 
The skilled technical assistance of Birgitta Bodin, Ing-Britt Hallgren, Eva Törnelius, Kärstin Flink, and Astrid Nordin is gratefully acknowledged.


   FOOTNOTES
 

Address for reprint requests and other correspondence: L. Jansson; Dept. of Medical Cell Biology; Biomedical Centre; Box 571, Husargatan 3; SE-75123, Uppsala, Sweden (e-mail: leif.jansson{at}medcellbiol.uu.se)

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.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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