Ileal apical Na+-dependent bile acid transporter ASBT is upregulated in rats with diabetes mellitus induced by low doses of streptozotocin

Abstract

Increased intestinal bile acid absorption and expansion of the bile acid pool has been implicated in the hypercholesterolemia associated with diabetes mellitus. However, the molecular basis of the increase in bile acid absorption in diabetes mellitus is not fully understood. The ileal apical Na(+)-dependent bile acid transporter (ASBT) is primarily responsible for active reabsorption of the majority of bile acids. Current studies were designed to investigate the modulation of ASBT function and expression in streptozotocin (STZ)-induced diabetes mellitus in rats and to examine the effect of insulin on rat ASBT promoter by insulin. Diabetes mellitus was induced in Sprague-Dawley rats by intraperitoneal injection of low doses of STZ (20 mg/kg body wt) on five consecutive days. Human insulin (10 U/day) was given to a group of diabetic rats for 3 days before euthanasia. RNA and protein were extracted from mucosa isolated from the small intestine and ASBT expression was assessed by real-time quantitative RT-PCR and Western blotting. Our data showed that ASBT mRNA and protein expression were significantly elevated in diabetic rats. Insulin treatment of diabetic rats reversed the increase in ASBT protein expression to control levels. Consistently, ileal Na(+)-dependent [(3)H]taurocholic uptake in isolated intestinal epithelial cells was significantly increased in diabetic rats. In vitro studies utilizing intestinal epithelial Caco-2 cells demonstrated that ASBT expression and promoter activity were significantly decreased by insulin. These studies demonstrated that insulin directly influences ASBT expression and promoter activity and that ASBT function and expression are increased in rats with STZ-induced diabetes mellitus. The increase in ASBT expression may contribute to disturbances in cholesterol homeostasis associated with diabetes mellitus.

Fig. 1.

Multiple low doses of streptozotocin (STZ) are not associated with weight loss. Sprague-Dawley rats obtained from Charles River were given 5 ip injections (○) with multiple low doses (20 mg·kg body wt⁻¹·day⁻¹). Control rats (●) were injected with vehicle alone (citrate buffer, pH 4.5). Glucose was measured at the indicated day (A), and insulin level was assessed after the animals were euthanized (B). Body weight was measured at the indicated day (C), demonstrating that the diabetic rats did not lose weight throughout the experiment. Values are expressed as means ± SE from 4 animals. *P ≤ 0.05 compared with control.

Fig. 2.

Apical Na⁺-dependent bile acid transporter (ASBT) expression is increased in diabetes mellitus. Distal ileum was removed from control and diabetic animals, and mucosa was scraped as mentioned in materials and methods. Total RNA was extracted, and the relative expression of ASBT mRNA (A) and organic solute transporter (Ost) α and β mRNA (B) was assessed by real-time RT-PCR using SYBR green. The level of mRNA expression for each target gene was normalized to the expression of 18S mRNA in the same sample. In C, immunofluorescence staining for actin (red) in optimal cutting temperature (OCT)-embedded sections of rat ileum from control and diabetic rats showing long villi but no apparent change in the size of epithelial cells. In D, ileal segments were embedded in OCT media, sectioned to 9 μm thickness. The tissues were then mounted on special slides suitable for laser capture microdissection as mentioned in materials and methods. Ileal sections were stained with hematoxylin-eosin (H&E), and epithelial cells were visualized under the microscope. Equal number of cells was cut by laser from tissues from control and diabetic rats. Cells were collected, and total RNA was extracted. The relative expression of ASBT mRNA normalized to 18S mRNA was evaluated by real-time RT-PCR. Values represent means ± SE from at least 3–5 animals. *P ≤ 0.05 compared with control.

Fig. 3.

ASBT protein expression is upregulated in diabetic rats. Total protein extracts were prepared from mucosal scrapings from rat colon and small intestine. Equal amounts of protein were separated on 10% SDS-PAGE and electrotransferred to nitrocellulose blots. Blots were then probed with anti-rat ASBT antibodies, and bands corresponding to ASBT fusion proteins were visualized as described in materials and methods. Blot in A shows that ASBT expression is detected in rat ileum but not jejunum or colon. In B, a representative blot demonstrating an increase in ASBT protein expression in diabetic rat ileum compared with control is shown. Insulin treatment to diabetic rats reversed the increase in ASBT protein expression to the level of control rats. In C, immunofluorescence staining for ASBT (green) and villin (red) with blue-stained nuclei in OCT-embedded sections of rat ileum from control, diabetic, and insulin-treated diabetic rats is shown. Data are representative of 3 different experiments.

Fig. 4.

ASBT function is elevated in rats with diabetes mellitus. Intestinal epithelial cells were isolated from ileal segments, washed, and immediately resuspended in uptake buffer containing 10 μM of [³H]taurocholic acid (TC) in the presence or absence of Na⁺. Uptake was terminated at the indicated time point by two washes in ice-cold PBS buffer and was expressed as pmol/mg protein. Na⁺-dependent [³H]TC uptake was linear up to 20 min of incubation with uptake buffer (A). Na⁺-dependent [³H]TC uptake was evaluated in isolated epithelial cells from control and diabetic rats (B). Results are presented as means ± SE of uptake values obtained from 3 different animals. Where not shown, error bars are smaller than symbol. *P ≤ 0.05 compared with control.

Fig. 5.

Rat ASBT promoter activity is decreased by insulin. A: intestinal Caco-2 cells were plated on Transwell inserts and were exposed to 50 nM insulin for 24 h from the basolateral compartment. Total RNA was extracted, and ASBT mRNA expression was assessed by real-time RT-PCR normalized to actin mRNA level (internal control). Control cells were treated with vehicle only. B: Caco-2 cells were transiently cotransfected by electroporation (Amexa) with rat ASBT or the promoter or human SLC26A3 transporter along with expression vector of human insulin receptor and CMV β for β-galactosidase to normalize for the transfection efficiency. Cells were plated on Transwell, treated with 100 nM insulin for 24 h, and harvested for firefly luciferase and β-galactosidase assays to assess the promoter activity. Control cells are cells treated with vehicle only. C: Caco-2 cells were transfected with rat ASBT promoter along with different amounts of the expression vector of human insulin receptor. Cells were plated on Transwell, treated with 100 nM insulin from 24 h, and harvested for firefly luciferase and β-galactosidase assays. Control cells are transfected with rat ASBT promoter and β-galactosidase vectors only without insulin receptor expression vector. Results are presented as % of control and represent means ± SE for 6–9 determinations performed on 3 separate occasions. *P ≤ 0.05 compared with control.