Effects of long-term cranberry supplementation on endocrine pancreas in aging rats

Abstract

The effects of long-term cranberry consumption on age-related changes in endocrine pancreas are not fully understood. Here we treated male Fischer 344 rats with either 2% whole cranberry powder supplemented or normal rodent chow from 6 to 22 month old. Both groups displayed an age-related decline in basal plasma insulin concentrations, but this age-related decline was delayed by cranberry. Cranberry supplementation led to increased β-cell glucose responsiveness during the oral glucose tolerance test. Portal insulin concentration was 7.6-fold higher in rats fed cranberry, coupled with improved β-cell function. However, insulin resistance values were similar in both groups. Total β-cell mass and expression of pancreatic and duodenal homeobox 1 and insulin within islets were significantly enhanced in rats fed cranberry relative to controls. Furthermore, cranberry increased insulin release of an insulin-producing β-cell line, revealing its insulinotropic effect. These findings suggest that cranberry is of particular benefit to β-cell function in normal aging rats.

Figure 1.

Body weight, body composition, and energy balance. Male Fisher-344 rats were randomly assigned to either a 2% cranberry diet (CB) or a normal diet (N) and maintained from 6 to 22 months of age (16 months of cranberry diet). Body weight (A) and food intake (B) were recorded monthly over the course of first 11 months of treatment. Efficiency of conversion of ingested food to unit of body weight (feed efficiency) was calculated by full body weight that included contents in the gastrointestinal track (grams) divided by weekly gross energy intake (kilocalories) calculated by the average nutrient composition of diet (C). At the time of sacrifice after overnight fasting, final body weight (D) and abdominal fat deposits (E) including epididymal white adipose tissue and intrascapular brown adipose tissue were determined. Values are means ± standard error of 10–12 rats per each group. *p < .05, **p < .01, and ***p < .001 versus age-matched N rats according to unpaired t test.

Figure 2.

Basal blood glucose, plasma insulin levels, and portal plasma insulin concentration. At each experimental point (4, 12, and 16 months of treatment), animals were fasted overnight. Blood glucose from snipped tail was measured using a portable glucose meter (A). Peripheral blood samples were taken from snipped tail for measuring plasma insulin concentration (B). At the time of sacrifice (16 months of treatment), after overnight fasting, blood sample from portal vein was collected for measuring portal insulin concentration (C). Values are means ± standard error of 10–12 rats per group. *p < .05 versus age-matched N rats according to unpaired t test.

Figure 3.

Blood glucose levels and insulin release after glucose challenge. Oral glucose tolerance tests were performed at 6 months (A and C) and 12 months (B and D) of cranberry treatment, respectively. After overnight fasting, D-glucose (2 g/kg body weight) was administrated orally. Blood samples were taken from snipped tail at different time points during Oral Glucose Tolerance Test for measurement of blood glucose (A and B) and plasma insulin (C and D). Values are means ± standard error of seven rats per group. *p < .05 and **p < .01 versus age-matched N rats according to unpaired t test.

Figure 4.

Pancreatic weight and total pancreatic β-cell mass. At the end of experiment (16 months of treatment), each pancreas was excised, cleared of extraneous lymph nodes and fat, and then weighed (A) and cassetted with the same anatomic orientation. After fixative, the pancreas was processed for paraffin embedding using a standard protocol. Two sets of five serial sections (5–7 μm) were obtained at intervals of 250 μm and immunostained by using an ABC kit for insulin, followed by background staining with Methyl Green. Using point-counting morphometric technique, the relative volumes of β-cells (percent relative to pancreas) were quantified (B). Absolute β-cell mass (C) was then calculated by multiplying the relative β-cell volume times the tissue weight. Values are means ± standard error of 10 rats per group. *p < .05 versus aged-matched controls according to unpaired t test.

Figure 5.

Pancreatic islet morphological configurations. The sizes of pancreatic islets per tissue block were measured at a 200× magnification using AxioVision software. Distributions of islet size (A and B) were obtained from individual data of islet sizes. After logarithmical transformation, the distribution of islet sizes was statistically compared by Kolmogorov–Smirnov test. For practical reason, the raw data of islet size are given and expressed as means ± standard error from 1,022 islets in CB rats and 1,056 islets in N rats (G). p Value was determined by unpaired t test based on logarithmically transformed variables. **p < .01 versus N rats. Newly formed islets by neogenesis were similarly frequent noted in ductal epithelia and duct-like tissue in both groups of rats. Representative images of β-cell neogenesis are shown in CB rats (arrows in C) and N rats (arrows in D). Numerous enlarged islets (>40,000 μm²) were present in the pancreatic tissue of CB rats in which tightly packed β-cells positioned centrally (E) in comparison of the islet from N rats (F). Original magnification of images, 200×.

Figure 6.

Expression of insulin, PDX-1, and glucagon within islet cells. Triple immunofluorescence and confocal analysis with antibodies against insulin (green), glucagon (blue), and PDX-1 (red; upper panel) revealed that expression levels of insulin (A), glucagon (B), and PDX-1 (C), expressed as mean density within islets. Values are means ± standard error of 62–106 islets. **p < .01 versus N rats according to unpaired t test.

Figure 7.

Insulin secretion in vitro. The insulin-producing β-cell line, INS-1 cells, was cultured in RPMI medium containing 10% heat-inactivated fetal calf serum, 5.5 mM glucose, 2 mM L-glutamine, 100 U/mL penicillin and 100 μg/mL streptomycin, 1 mM sodium pyruvate, and 50 μM 2-mercaptoethanol for 72 hours. After preincubation in oxygenized Krebs–Ringer bicarbonate HEPES buffer at 3.3 or 27.7 mmol/L glucose, cells were shifted to 1 mL of the same buffer, and insulin release was then measured by using static incubation for a 60-minute period. At the end of the incubation, supernatants were collected. The amount of insulin secretion into the medium was measured with a commercially available enzyme-linked immunosorbent assay kit, normalized to total DNA content, and represented as nanograms per microgram DNA per 60 minute. Values are means ± standard error of eight independent samples. *p < .05, **p < .01, and ***p < .001 according to unpaired t test.