Beta-cell Volume Research Articles

Glucose-inducible hypertrophy and suppression of anion efflux in rat beta cells.

Hypertrophy of beta cells from obese fa/fa rats is associated with increased sensitivity to basal glucose. Exposure to glucose in culture distorts insulin secretion more in beta cells from large than small islets from fa/fa rats. The aim of the present study is to investigate whether increased beta cell volume is associated with both glucose hypersensitivity and altered activity of the glucose-sensitive anion conductance. Beta cells from fa/fa rats had increased volume compared with those from lean rats after 24 h culture. Three-day exposure to 25 mM glucose in culture induced 10-15% hypertrophy in beta cells from lean rats and basal secretion from intact islets was increased tenfold. Estimates of ion channel activity were made from measurement of radiolabeled ion efflux. Taurine efflux, a marker of glucose-regulated anion channel activity, was reduced after high glucose exposure but no alterations in glucose-dependent K+ efflux were detected. The reverse hemolytic plaque assay was used to determine the contributions of the number of secreting cells (recruitment) versus secretion per cell in beta cells from enlarged (>250 microm diameter), intermediate (125-250 microm) and small (<125 microm) islets from lean and obese rats exposed to conditions mimicking hyperglycemia. After overnight culture, basal secretion was twofold greater from beta cells of large fa/fa islets compared with all other groups. Recruitment at low glucose was increased in all lean or fa/fa beta cells derived from >125 microm islets. When beta cells from small islets were exposed to supra-physiological glucose for 3 days, recruitment was increased at basal glucose and blunted at high glucose. Glucose exposure converts the recruitment profile of beta cells from small islets to resemble that of beta cells from large islets while inducing cellular hypertrophy and reduced anion conductance. However, hypertrophy alone did not predict functional characteristics of overnight-cultured beta cells from fa/fa rats.

Gradual loss of pancreatic beta-cell insulin, glucokinase and GLUT2 glucose transporter immunoreactivities during the time course of nutritionally induced type-2 diabetes in Psammomys obesus (sand rat).

The Psammomys obesus (sand rat) is a well-established model of nutritionally induced non-insulin-dependent type-2 diabetes. When fed a high-energy (HE) diet, the diabetes-prone animals develop hyperinsulinaemia and hyperglycaemia. Within 1 week, all animals become hyperinsulinaemic. However, a loss of immunostaining for insulin as well as for the GLUT2 glucose transporter in the plasma membrane and the glucokinase in the cytoplasm of the pancreatic beta cells became evident only when the animals subsequently developed hyperglycaemia. After 1 week of HE diet feeding, the pancreatic beta-cell volume was reduced by one-third in hyperglycaemic Psammomys. Insulin immunostaining as well as GLUT2 glucose transporter immunostaining in the plasma membrane and glucokinase immunostaining in the cytoplasm were reduced by more than 50%. After 3 weeks of HE diet feeding, all changes observed after 1 week were even more pronounced, with reductions in the range of 70-95%. The reduction of the total beta-cell volume of the pancreas due to beta-cell death and the diminution of insulin content of the remaining beta cells in the islets during the HE diet feeding was accompanied by a parallel fall of the pancreas insulin content. For all changes observed, there was a significant correlation with the increase of the blood glucose concentration (r>0.9) but not with the increase of the plasma insulin concentration (r>0.2). Thus, increasing glycaemia appears to be the factor responsible for the deterioration of the pancreatic beta-cell function and the resulting loss of the insulin secretory capacity in Psammomys. The final result of this development is an irreversible diabetic state due to the feeding of the HE diet.

Glucagon-like peptide 1 receptor signaling influences topography of islet cells in mice.

Glucagon-like peptide 1 (GLP-1) amplifies glucose-induced insulin release in vivo and in vitro. Activation of GLP-1 receptor (GLP-1R) signaling leads to differentiation of exocrine cells towards a beta-cell phenotype in vitro and stimulation of islet cell proliferation in vitro and in vivo, suggesting a potential role for GLP-1 in the modulation of islet growth and differentiation. To determine whether basal levels of GLP-1R signaling are essential for islet development, we examined islet cell composition and topography in GLP-1R-/- mice. Total beta-cell volume and number are not altered, but the topography of beta cells is markedly different in GLP-1R-/- mice compared with GLP-1R+/+ controls. The distribution of beta cells is shifted from large to small and medium-sized islets in the absence of GLP-1R signaling (large islets: 50 +/- 3% in GLP-1R+/+ vs 28 +/- 4% in GLP- 1R-/-, P < 0.01 and medium islets: 32 +/- 2% in GLP- 1R+/+ vs 48 +/- 3% in GLP-1R-/-, P < 0.001). Furthermore, GLP-1R-/- islets exhibit abnormalities in cell topography, with two to threefold more centrally located alpha cells detected in GLP-1R-/- islets. These alterations in alpha- and beta-cell topography indicate that basal levels of GLP-1 signaling in the normal rodent are involved in the normal cellular organization of the endocrine pancreas.

Recombinant human betacellulin promotes the neogenesis of beta-cells and ameliorates glucose intolerance in mice with diabetes induced by selective alloxan perfusion.

Betacellulin (BTC), a member of the epidermal growth factor family, is expressed predominantly in the human pancreas and induces the differentiation of a pancreatic acinar cell line (AR42J) into insulin-secreting cells, suggesting that BTC has a physiologically important role in the endocrine pancreas. In this study, we examined the in vivo effect of recombinant human BTC (rhBTC) on glucose intolerance and pancreatic morphology using a new mouse model with glucose intolerance induced by selective alloxan perfusion. RhBTC (1 microg/g body wt) or saline was injected subcutaneously every day from the day after alloxan treatment. The intraperitoneal glucose tolerance test revealed no difference between rhBTC-treated and rhBTC-untreated glucose-intolerant mice at 2-4 weeks. However, glucose tolerance was significantly improved and body weight was significantly increased in rhBTC-treated mice compared with untreated mice at 8 weeks. Islet-like cell clusters, consisting mainly of beta-cells, were increased in the pancreas and were localized in contact with the ductal lining cells and sometimes with acinar cells. In conclusion, administration of rhBTC improved glucose tolerance in this mouse model by increasing beta-cell volume, primarily through accelerated neogenesis from ductal lining cells.

Morphometric study of the beta-cell volume of the canine pancreas with consideration of the axis of tissue transection.

The pancreatic beta-cell volume was assessed in 12 clinically normal crossbred dogs using immunohistochemical labelling for insulin and computer-assisted morphometric analysis. The beta-cell volume was less in the right lobe compared with the body and left lobe, but the beta-cell volume did not differ between the body and the left lobe as reported previously. The use of immunohistochemistry, computer-assisted morphometry, the inclusion here of extra-islet beta-cells and the exclusion of non-beta-endocrine cells in the investigation of the beta-cell volume may have contributed to this discrepancy. As most beta-cells form irregularly shaped three-dimensional bodies (islets of Langerhans), the effect of the plane of tissue transection (sagittal, transverse, oblique) on the calculation of the beta-cell volume was also examined. The beta-cell volume in the sagittal plane of the right lobe was lower than the volume in the oblique plane of the same lobe but was not different from the volume in the transverse plane. However, there were no differences in beta-cell volume in different planes in the body or left lobe. When quantifying beta-cell volume in health or disease, variation in the volume of beta-cells in different locations of the pancreas is an important consideration and the plane of transection may also influence results.

Fas and Fas ligand expression in inflamed islets in pancreas sections of patients with recent-onset Type I diabetes mellitus.

Type I (insulin-dependent) diabetes results mainly from T-cell-mediated autoimmune destruction of pancreatic beta cells. Cytotoxic T lymphocytes destroy target cells via a perforin-based or Fas-based mechanism. Our previous study indicated that the Fas-Fas ligand (FasL) pathway is required for the development of autoimmune diabetes in the NOD mouse. We now investigated whether or not the Fas-FasL system is involved in the beta-cell destruction in human Type I diabetes. We immunohistochemically analysed pancreas biopsy specimens of 13 recent-onset patients. Pancreatic islets were identified but showed various degrees of reduction in beta-cell volume in all patients. Out of 13 patients 6 had insulitis. In these 6 patients Fas was expressed in both the islets and infiltrating cells but not in either cell type in the 7 other patients without insulitis. Double immunostaining showed that Fas was positive in 92.2 to 97.7 % of beta cells but only in 17.6 to 46.7 % of alpha cells in Fas-positive, insulin-remaining islets. We found FasL was expressed exclusively in islet-infiltrating cells in patients with insulitis. Double immunostaining revealed that the most prevalent phenotype of FasL-positive cells was CD8, which was followed by macrophages and CD4. The interaction between Fas on beta cells and FasL on infiltrating cells might trigger selective apoptotic beta-cell death in inflamed islets, leading to immune-mediated Type I diabetes. [Diabetologia (1999) 42: 1332-1340]

Unbiased estimation of total β‐cell number and mean β‐cell volume in rodent pancreas

We describe a method for unbiased assumption-free estimation of the total number of beta-cells and the mean beta-cell volume in mouse and rat pancreas based on light microscopy. Such a method, which takes advantage of one of the most recent developments in stereology, the fractionator, has not previously been described. It relies on repeated fractionation of the tissue using systematic uniform random sampling combined with an unbiased counting principle. The method was applied to eight BALB/cBom male mice (56 days) and six Lewis/MOL male rats (47 days). In mice, the total number of beta-cells was 1.06 +/- 0.07 x 10(6) (mean +/- SEM) per pancreas with a mean beta-cell volume of 1280 +/- 17 microm3, while in rats the total beta-cell number was 2.76 +/- 0.42 x 10(6) per pancreas with a mean beta-cell volume of 1170 +/- 65 microm3. Furthermore, the results showed that in both species the biological variability in the total beta-cell volume is due to differences in the number of beta-cells rather than variability of the mean beta-cell volume. The method can be used to give a precise description of number and volume of beta-cells at different ages, and will make it possible to estimate the contributions of hyper/hypotrophia and hyper/hypoplasia to a given induced or spontaneous change in the total beta-cell mass.

Methylglyoxal causes swelling and activation of a volume-sensitive anion conductance in rat pancreatic beta-cells.

Membrane potential and whole-cell current were studied in rat pancreatic beta-cells using the 'perforated patch' technique and cell volume measured by a video-imaging method. Exposure of beta-cells to the alpha-ketoaldehyde methylglyoxal (1 mM) resulted in depolarization and electrical activity. In cells voltage-clamped at -70 mV, this effect was accompanied by the development of inward current noise. In voltage-pulse experiments, methylglyoxal activated an outwardly rectifying conductance which was virtually identical to the volume-sensitive anion conductance previously described in these cells. Two inhibitors of this conductance, 4,4'-dithiocyanatostilbene-2,2'-disulfonic acid (DIDS) and 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB), also inhibited the depolarization and inward current evoked by methylglyoxal. Methylglyoxal increased beta-cell volume to a relative value of 1.33 after 10 min with a gradual return towards basal levels following withdrawal of the alpha-ketoaldehyde. None of the effects of methylglyoxal was observed in response to t-butylglyoxal which, unlike methylglyoxal, is a poor substrate for the glyoxalase pathway. Methylglyoxal had no apparent effect on beta-cell K+ channel activity. It is suggested that the metabolism of methylglyoxal to D-lactate causes beta-cell swelling and activation of the volume-sensitive anion channel, leading to depolarization. These findings could be relevant to the stimulatory action of D-glucose, the metabolism of which generates significant quantities of L-lactate.

Evidence of a negative feedback system regulating the total beta-cell volume in nondiabetic rats that received pancreas transplants.

The aim of the present study was to investigate the long-term regulation of pancreatic beta-cell volume after pancreas transplantation into adult rats. A syngeneic pancreaticoduodenal transplantation was made in normoglycemic Wistar-Furth rats. By this means, the recipients doubled their pancreatic islet volume. Nine months after transplantation, the total beta-cell volume was measured in serial pancreatic sections immunostained for insulin from both the native and transplanted pancreata, and in the native pancreas of age-matched Wistar-Furth rats that did not receive transplants. No changes in the volume of individual beta-cells were seen. A 50% decrease in total beta-cell volume was observed in both the native and transplanted pancreas when compared with that of age-matched controls. However, the combined beta-cell volumes of the native and transplanted pancreas in the rats that received transplants were similar to those of the native pancreas in control animals. No signs of increased apoptosis in any of the glands could be seen during the first postoperative week or after 9 months. These findings provide evidence of a negative feedback system, which regulates the total beta-cell volume to the levels seen in age-matched rats that did not receive transplants. The underlying mechanisms for the decreased beta-cell volume are unknown, but may involve a diminished replicatory rate of the beta-cells.

Accelerated Loss of Islet β Cells in Sucrose-Fed Goto-Kakizaki Rats, a Genetic Model of Non-Insulin-Dependent Diabetes Mellitus

The Goto-Kakizaki (GK) rat is a spontaneously diabetic animal model of non-insulin-dependent diabetes mellitus, which is characterized by progressive loss of beta cells in the pancreatic islets with fibrosis. In the present study, we examined the effects of sucrose feeding on the islet pathology in this model. Six-week-old GK rats were fed with 30% sucrose for 6 weeks to induce severe hyperglycemia, and their condition was compared with that of nontreated rats. Age-matched normal Wistar rats were also given sucrose for the same periods and used for comparison. The sucrose-treated GK rats showed elevated blood glucose levels on oral glucose tolerance tests at 60 minutes and 120 minutes, representing 123% and 127% of values in untreated GK rats, respectively. At the end of the study, the mean beta-cell volume density in GK rats was 50% less than that in untreated Wistar rats. Sucrose feeding further reduced the volume densities of beta cells to only 50% of the levels of age-matched GK rats. Apoptotic cells were found in islet beta cells only in GK rats fed sucrose (mean 0.067%). There appeared to be more islets that immunohistochemically stained strongly positive with 8-hydroxy-deoxyguanosine as a marker of oxidative damage of DNA in GK rats fed sucrose compared with those not given sucrose. GK rats not fed sucrose showed significantly lower proliferative activity of beta cells measured by 5-bromo-2'-deoxyuridine uptake and intensified expression of Bcl-2 immunoreactivities at 6 weeks of age compared with those in age-matched Wistar rats. These two indices were reduced in both GK and Wistar rats with increasing age and were not affected by sucrose feeding in either group. The present study thus indicated that sucrose feeding promoted the apoptosis of beta cells in GK rats through increased oxidative stress without altering their proliferative activity.

Number and Volume of Islets Transplanted in Immunobarrier Devices

Immunobarrier devices may prevent immune destruction of transplanted islets, but there are concerns about survival within such devices. Islets were transplanted in diffusion chambers that employed two laminated polytetrafluoroethylene membranes held together with titanium rings. Five hundred syngeneic mouse islets placed in devices were transplanted into the epididymal fat pads of streptozotocin (STZ) diabetic mice (B6AF1). After 2 wk the devices were removed. Sections were made parallel to the membrane surface. Eight to 13 systematically selected sections of each device were analyzed by planimetry to determine the area of the device space and of the islets within that space. From these data we estimated total volume of the device, volume of islets, and number of islets in a device. The data were segregated into two groups: group I (blood glucose less than 100 mg/dL 2 wk after implantation), and group II (over 150 mg/dL). The volume (mean ± SE) of devices implanted for 2 wk was 2.1 ± 0.4 μL in group I and 2.2 ± 0.2 μL in group II. The islet volume and number within devices were 0.30 ± 0.06 and 0.17 ± 0.01 μL, or 340 ± 50 and 230 ± 20 islets in group I and group II, respectively. The volume of fibrous tissue in devices was about 0.50 μL. About 10% of the islet tissue had central necrosis. The beta cell volume in a membrane device needed for cure is comparable to that required with islets under the kidney capsule (0.25–0.80 μL). The mass of islets contained within membrane devices needed to cure diabetes is equivalent to that of a graft in an optimal transplant site such as under the kidney capsule.

Number and volume of islets transplanted in immunobarrier devices.

Immunobarrier devices may prevent immune destruction of transplanted islets, but there are concerns about survival within such devices. Islets were transplanted in diffusion chambers that employed two laminated polytetrafluoroethylene membranes held together with titanium rings. Five hundred syngeneic mouse islets placed in devices were transplanted into the epididymal fat pads of streptozotocin (STZ) diabetic mice (B6AF1). After 2 wk the devices were removed. Sections were made parallel to the membrane surface. Eight to 13 systematically selected sections of each device were analyzed by planimetry to determine the area of the device space and of the islets within that space. From these data we estimated total volume of the device, volume of islets, and number of islets in a device. The data were segregated into two groups: group I (blood glucose less than 100 mg/dL 2 wk after implantation), and group II (over 150 mg/dL). The volume (mean +/- SE) of devices implanted for 2 wk was 2.1 +/- 0.4 microL in group I and 2.2 +/- 0.2 microL in group II. The islet volume and number within devices were 0.30 +/- 0.06 and 0.17 +/- 0.01 microL, or 340 +/- 50 and 230 +/- 20 islets in group I and group II, respectively. The volume of fibrous tissue in devices was about 0.50 microL. About 10% of the islet tissue had central necrosis. The beta cell volume in a membrane device needed for cure is comparable to that required with islets under the kidney capsule (0.25-0.80 microL). The mass of islets contained within membrane devices needed to cure diabetes is equivalent to that of a graft in an optimal transplant site such as under the kidney capsule.

Changes of islet size and islet size distribution resulting from protein-malnutrition in lean (Fa/Fa) and obese (fa/fa) Zucker rats.

Potential alterations in islet size and islet size distribution resulting from protein malnutrition were studied in lean (Fa/Fa) and obese (fa/fa) Zucker rats. The purpose was to investigate whether the distribution of enlarged islets in obese rats was altered by low-protein feeding. Four-week-old, male, lean and obese Zucker rats were fed either a diet containing 20% (w/w) protein (control diet) or a diet containing 5% (w/w) protein (low-protein diet) for 3 weeks. Pancreata were dissected at autopsy and immunostained for insulin. Islet size and distribution were determined by morphometric analysis. Bodyweight gain, food intake, and serum insulin and glucose were also measured. After 3 weeks on the diets, serum insulin was significantly lower in both lean (-75%) and obese (-54%) rats fed low protein compared with that in controls. However, obese rats were still hyperinsulinemic compared with lean rats. Protein malnutrition resulted in a shift in distribution of islets to smaller size both in lean and in obese rats, with an increase in the population of small islets (< or = 100 microns2) and a decrease in the population of large islets (> 20,000 microns2). In lean and obese rats fed low protein, beta-cell weight was significantly lower, beta cell volume fraction tended to decrease, and islet number per section area was significantly elevated when compared with controls. Taken together, these results show that protein deficiency alters the endocrine pancrease in both lean and obese Zucker rats. Although the decrease in islet size and the shift in distribution to smaller islets most likely contribute to the decrease in serum insulin concentration, these changes appear insufficient to normalize hyperinsulinemia in the obese Zucker rat.

Glucose-induced swelling in rat pancreatic beta-cells.

1. Changes in relative cell volume in response to hypotonic solutions and glucose were studied in single isolated rat pancreatic beta-cells using a video-imaging technique. beta-cell electrical activity was recorded under similar conditions using the perforated patch technique. 2. Exposure of beta-cells to hypotonic solutions (10 and 33% hypotonicity) caused an immediate increase in cell volume to relative values of 1.09 and 1.33, respectively. This was followed by a gradual regulatory volume decrease. 3. Raising the concentration of glucose from 4 to 20 mM or 12 mM (with substitution of mannitol) increased beta-cell volume by 12 and 10%, respectively. This effect of glucose persisted when CO2+ was added to inhibit insulin release. Glucose-induced volume increases were sustained for the duration of exposure to elevated hexose concentration. The addition of 16 mM 3-O-methylglucose, which is transported into the beta-cell but not metabolized, produced only a transient 5% increase in beta-cell volume. 4. Exposure of beta-cells to a 15% hypotonic solution resulted in a transient depolarization and electrical activity. Raising the glucose concentration to 20 or 12 mM caused a sustained depolarization and generation of electrical activity. However, the addition of 16 mM 3-O-methylglucose had no effect on beta-cell membrane potential. The glucose-induced increase in volume and induction of electrical activity, when measured in single beta-cells simultaneously, showed comparable kinetics. 5. The secretion of insulin from intact pancreatic islets was stimulated by exposure to hypotonic solutions (10-33% hypotonicity). A 15% hypotonic solution stimulated insulin release to a peak value comparable to that elicited by raising the glucose concentration from 4 to 20 mM. Whereas hypotonic solutions caused a transient stimulation of insulin release, the effect of glucose was sustained. 6. It is suggested that glucose increases the volume in rat pancreatic beta-cells by a mechanism dependent upon metabolism of the sugar. The extent of cell swelling evoked by raised glucose concentrations is sufficient to depolarize the cells and induce electrical and secretory activity and may involve activation of a volume-sensitive anion conductance.

Caloric restriction in obese pre-diabetic rats prevents beta-cell depletion, loss of beta-cell GLUT 2 and glucose incompetence.

Pre-diabetic male Zucker diabetic fatty rats (ZDF) become diabetic between 8 and 10 weeks of age. At that time their beta cells exhibit high basal insulin secretion, absent insulin response to glucose and loss of GLUT 2 glucose transporter. Beta-cell volume, which is increased at the onset of non-insulin-dependent diabetes, declines precipitously by age 18 weeks. To determine if expression of this diabetic phenotype was dependent upon the increased food intake of these rats, they were diet-matched to lean littermates for 12 weeks beginning at 6 weeks of age. Untreated control ZDF rats received an unrestricted diet for 3 months. All of the controls became hyperglycaemic by 8 weeks of age, whereas all diet-matched rats remained euglycaemic throughout the 3 months, despite the fact that at 18 weeks of age their mean body weight equaled that of obese rats on an unrestricted diet. In the former rats glucose-stimulated insulin secretion was absent at 12 weeks of age and GLUT-2-positive beta cells had fallen below 30%. The volume fraction of their beta cells was 2.6 times normal at this age but by 18 weeks of age it had declined by 75%. Diet restriction for 3 months prevented the loss of glucose-stimulated insulin secretion and the reduction of beta-cell GLUT-2 and beta-cell volume fraction. However, neither the elevated basal insulin secretion nor the exaggerated arginine-stimulated insulin secretion of the obese rats was reversed or prevented by caloric restriction.(ABSTRACT TRUNCATED AT 250 WORDS)

Caloric restriction in obese pre-diabetic rats prevents beta-cell depletion, loss of beta-cell GLUT 2 and glucose incompetence

Pre-diabetic male Zucker diabetic fatty rats (ZDF) become diabetic between 8 and 10 weeks of age. At that time their beta cells exhibit high basal insulin secretion, absent insulin response to glucose and loss of GLUT 2 glucose transporter. Beta-cell volume, which is increased at the onset of non-insulin-dependent diabetes, declines precipitously by age 18 weeks. To determine if expression of this diabetic phenotype was dependent upon the increased food intake of these rats, they were diet-matched to lean littermates for 12 weeks beginning at 6 weeks of age. Untreated control ZDF rats received an unrestricted diet for 3 months. All of the controls became hyperglycaemic by 8 weeks of age, whereas all diet-matched rats remained euglycaemic throughout the 3 months, despite the fact that at 18 weeks of age their mean body weight equaled that of obese rats on an unrestricted diet. In the former rats glucose-stimulated insulin secretion was absent at 12 weeks of age and GLUT-2-positive beta cells had fallen below 30%. The volume fraction of their beta cells was 2.6 times normal at this age but by 18 weeks of age it had declined by 75%. Diet restriction for 3 months prevented the loss of glucose-stimulated insulin secretion and the reduction of beta-cell GLUT-2 and beta-cell volume fraction. However, neither the elevated basal insulin secretion nor the exaggerated arginine-stimulated insulin secretion of the obese rats was reversed or prevented by caloric restriction. We conclude that in diabetic ZDF rats the glucose incompetence of beta cells and the reduction of beta-cell GLUT 2, which coincide with the onset of hyperglycaemia, and the subsequent loss of beta-cell volume, occur only when the caloric intake is excessive. The increased basal insulin secretion and exaggerated insulin response to arginine appear to be relatively independent of caloric intake.

Pancreatic β-Cells in Obesity: EVIDENCE FOR INDUCTION OF FUNCTIONAL, MORPHOLOGIC, AND METABOLIC ABNORMALITIES BY INCREASED LONG CHAIN FATTY ACIDS

To elucidate the mechanism of the basal hyperinsulinemia of obesity, we perfused pancreata from obese Zucker and lean Wistar rats with substimulatory concentrations of glucose. Insulin secretion at 4.2 and 5.6 mM glucose was approximately 10 times that of controls, whereas beta-cell volume fraction was increased only 4-fold and DNA per islet 3.5-fold. We therefore compared glucose usage at 1.4, 2.8, and 5.6 mM. Usage was 8-11.4 times greater in Zucker islets at 1.4 and 2.8 mM and 4 times greater at 5.6 mM; glucose oxidation at 2.8 and 5.6 mM glucose was > 12 times lean controls. To determine if the high free fatty acid (FFA) levels of obesity induce these abnormalities, normal Wistar islets were cultured with 0, 1, or 2 mM long chain FFA for 7 days. Compared to islets cultured without FFA insulin secretion by FFA-cultured islets (2 mM) perifused with 1.4, 3, or 5.6 mM glucose was increased more than 2-fold, bromodeoxyuridine incorporation was increased 3-fold, and glucose usage at 2.8 and 5.6 mM glucose was increased approximately 2-fold (1 mM FFA) and 3-fold (2 mM FFA). We conclude that hypersecretion of insulin by islets of obese Zucker fatty rats is associated with, and probably caused by, enhanced low Km glucose metabolism and beta-cell hyperplasia, abnormalities that can be induced in normal islets by increased FFA.

Beta-cell proliferation in normal and streptozotocin-treated newborn rats: site, dynamics and capacity.

Regeneration of neonatal beta cells after streptozotocin (STZ)-induced destruction may be due to either replication from pre-existing intra-islet beta cells or extra-islet precursor cells. To further investigate this issue, beta-cell growth was analysed in normal and streptozotocin-treated newborn rats (100 micrograms/g body weight) at several time points during the first 20 days of life. Beta cells were identified by insulin immunostaining, non-isotopic in situ hybridization for rat preproinsulin mRNA, and electron microscopy. Their proliferative activity was recorded by bromodeoxyuridine-pulse labelling. Beta-cell size and total volume were determined by computerized morphometry. In normal rats, there was a threefold increase in total beta-cell volume during the first 5 days of life, with no further expansion till day 20. The bromodeoxyuridine labelling index of the intra-islet beta cells was smaller than that of the extra-islet beta cells (2-3% vs 15-20%). Comparison of the cell birth rate, calculated from the beta-cell labelling index, with the observed increase in beta-cell volume suggested that in normal neonatal rats proliferation of the intra-islet beta-cell population could account for only 10% of the observed expansion. Administration of streptozotocin at birth resulted in more than 90% reduction of the total beta-cell volume at day 2 which then increased to 39% of the normal value by day 20. During this period of partial regeneration, which restored normoglycaemia, the labelling index of intra-islet beta cells was higher than in normal rats (9% vs 2%, p < 0.001), whereas no change was seen in the extra-islet beta-cell labelling index.(ABSTRACT TRUNCATED AT 250 WORDS)

Beta-cell proliferation in normal and streptozotocin-treated newborn rats: site, dynamics and capacity

Regeneration of neonatal beta cells after streptozotocin (STZ)-induced destruction may be due to either replication from pre-existing intra-islet beta cells or extra-islet precursor cells. To further investigate this issue, beta-cell growth was analysed in normal and streptozotocin-treated newborn rats (100 μg/g body weight) at several time points during the first 20 days of life. Beta cells were identified by insulin immunostaining, non-isotopic in situ hybridization for rat preproinsulin mRNA, and electron microscopy. Their proliferative activity was recorded by bromodeoxyuridine-pulse labelling. Beta-cell size and total volume were determined by computerized morphometry. In normal rats, there was a threefold increase in total beta-cell volume during the first 5 days of life, with no further expansion till day 20. The bromodeoxyuridine labelling index of the intraislet beta cells was smaller than that of the extra-islet beta cells (2–3% vs 15–20%). Comparison of the cell birth rate, calculated from the beta-cell labelling index, with the observed increase in beta-cell volume suggested that in normal neonatal rats proliferation of the intra-islet beta-cell population could account for only 10% of the observed expansion. Administration of streptozotocin at birth resulted in more than 90% reduction of the total beta-cell volume at day 2, which then increased to 39% of the normal value by day 20. During this period of partial regeneration, which restored normoglycaemia, the labelling index of intra-islet beta cells was higher than in normal rats (9% vs 2%, p<0.001), whereas no change was seen in the extra-islet beta-cell labelling index. Comparison of cell birth rate with the increase in beta-cell volume indicated that 50–60% of the observed beta-cell growth could result from the intra-islet beta-cell proliferation. These results suggest that replication from pre-existing, surviving beta cells plays an important role in regeneration of neonatal beta cells after destruction by streptozotocin.

Expression ofreg/PSP, a Pancreatic Exocrine Gene: Relationship to Changes in Islet β-Cell Mass

A cDNA termed reg was recently isolated by differential screening of a library prepared from regenerating islets isolated from pancreatic remnants of rats subjected to 90% pancreatectomy and nicotinamide treatment. This led to speculation that this gene may be involved in expansion of beta-cell mass. In the current study we have measured reg expression after implantation and resection of a solid insulinoma tumor into rats, maneuvers known, respectively, to reduce and reexpand the volume of beta-cells in the islet. Animals with an implanted insulinoma tumor became profoundly hypoglycemic. Islet beta-cells declined from the normal 75% of total islet volume to less than 30%, in concert with a marked reduction in the reg mRNA level. Removal of the tumor resulted in a sharp increase in beta-cell replication, as measured by [3H]thymidine incorporation and a return to normal beta-cell volume within 4 days of tumor resection. This was associated with a transient induction in reg expression compared to that in tumor-bearing animals, effectively returning the amount of reg mRNA to the levels found in normal animals within 48 h; at later time points after tumor removal (3-7 days) reg expression declined, but then rose toward normal. In situ hybridization analysis localized the initial induction in reg mRNA expression to the exocrine pancreas. Continuous infusion of insulin into normal rats for 4 days, a maneuver that does not significantly reduce beta-cell mass, resulted in dramatically reduced insulin mRNA in islets, but no change in the levels of reg mRNA. We conclude that the diminution in pancreatic beta-cell mass caused by subcutaneous implantation of an insulinoma is associated with reduced reg gene expression and that the increase in beta-cell replication after resection of the tumor is preceded by return of reg gene expression toward normal.