Islet isolation and culture

Pancreatic islets were isolated from 2-month-old female mice of the BALB/c strain (CLEA Japan, Inc., Tokyo) using collagenase (Moskalewski, 1965) with a slight modification. Mice were starved overnight, then killed by decapitation. The pancreas was excised immediately, transferred to ice-cold Hanks' medium and cut into small pieces with scissors. The pieces were placed in 1.5 ml of Hanks' medium containing 350 U collagenase (CLS IV; Worthington Biochemical Co., Freehold, NJ, USA) and vigorously shaken (200 strokes/min) at 37°C for 20 min. The digest was diluted with 10 ml of ice-cold Hanks' medium. After centrifugation at 400 × g for 1 min, the supernatant was discarded, and 250 μg DNase (DN-25; Sigma Chemical Co., St. Louis, MO, USA) was added. The digest was then transferred to a glass dish with ice-cold Hanks' medium, and the pancreatic islets were collected with a glass pipette under the binocular microscope and pooled in another dish filled with RPMI 1640 medium containing 10 mM glucose (GIBCO BRL, Gaithersburg, MA, USA) supplemented with 25 mM HEPES (pH 7.4). Groups of 50 islets were transferred to 24-well plates and cultured free-floating in 2 ml of RPMI 1640 medium containing 10 mM glucose, 15% fetal bovine serum (GIBCO), 100 U/ml penicillin and 100 μg/ml streptomycin in a humidified atmosphere containing 5% CO₂ at 37°C. After pre-incubation without hormones for 24 hr, the islets were cultured in media with or without hormones for 4 days. Under these culture conditions fibroblastic overgrowth was not seen even after 4 days of culture. Ovine PRL (oPRL), bovine GH (bGH) and estradiol-17β (E₂) were obtained from Sigma.

The culture medium was replaced with fresh medium every 24 hr, and stored at −70°C, until determination of insulin concentration by an enzyme immunoassay (Glazyme insulin EIA kit; Wako, Tokyo) as described previously (Matsuda et al., 1994).

Preparation of cDNA samples

Total RNA was extracted from cultured pancreatic islets by the acid guanidinium-phenol-chloroform method (Chomczynski and Sacchi, 1987). Immediately after removing the culture medium, 0.5 ml of denaturing solution (4 M guanidinium thiocyanate, 0.5% sodium sarcosyl, 100 mM 2-mercaptoethanol, 25 mM sodium citrate, pH 7.0) containing 2 μg yeast tRNA as the carrier RNA was added to each well, then the islets were homogenized with Microson™ Ultrasonic Cell Disruptor MS-50 (Heat Systems Ultrasonics, Inc., Farmingdale, NY, USA) with 3/32” microprobe at output settings 12 for 3 × 5 s. The homogenate was mixed with 50 μl of sodium acetate (2 M, pH 4), and proteins and genomic DNA were removed after phenol-chloroform treatment. Total RNA was then precipitated with isopropanol.

After denaturation at 90°C for 5 min, the RNA sample was ice-chilled. Template cDNAs for PCR were generated from half of the sample by reverse transcription (RT) as described previously (Matsuda and Mori, 1996). A 20-μl reaction mixture containing 200 U Superscript™ reverse transcriptase (GIBCO BRL) and 100 nM oligo d(T)_12–18 (Pharmacia) as a primer was incubated successively at 23°C for 10 min, 42°C for 1 hr, 95°C for 10 min, then ice-chilled and divided into 4-μl aliquots.

Primers, competitors and standard DNAs for cPCR

Primers, competitors and standards for cPCR of mPRL-R and mouse β-actin (mBA) cDNA are shown in Fig. 1. Those for mPRL-R were prepared as described before (Matsuda and Mori, 1996).

Fig. 1

Schematic representation of portions of mBA mRNA and two forms of mPRL-R mRNAs (PRL-R_L1 and PRL-R_S3) with PCR primers and the PCR products. Coding regions of the mRNAs are shown with boxes, PCR primers with arrowheads, and PCR products with bold lines. Each PCR product has an identical restriction site for BamH I, Kpn I or Msp I as indicated. The two forms of mPRL-R mRNAs have a common sequence corresponding to the 5′ noncoding region, signal peptide (S), extracellular domain (EC), transmembrane domain (TM) and a part of the intracellular domain (IC). They differ in the rest of the 3′ coding and noncoding regions.

Competitor and standard for cPCR of mBA cDNA were prepared as follows. The RT-PCR product generated from mouse liver RNA using mBAs-1 and mBAa-1 as the PCR primers was TA-subcloned into pT7Blue(R) (Novagen, Madison, WI, USA). A mutant mBA cDNA was generated by mutagenesis with PCR using mBAs-1 and mBAa-2B, and mBAs-2B and mBAa-1 as primers and subcloned into pT7Blue(R). These plasmids were digested with EcoR I and Pst I, and the inserts were purified by using glass beads. Their concentrations were estimated by absorbance at 260 nm (1 OD unit was estimated to be 50 μg/ml). The DNA which contains the sequence for the 3′ part of mBA mRNA was used as the standard, and the DNA which was the same as the standard except for a BamH I site instead of a Kpn I site was used as the competitor. Competitive PCR of mBA cDNA was performed using mBAs-1 and mBAa-1 as primers. The sequences of primers were as follows: mBAs-1; 5′-TCTAGACTTCGA-GCAGGAGATGGCC-3′, mBAa-1; 5′-CTAGAAGCACTTGCGGTGCA-CGATG-3′, mBAs-2B; 5′-CTGGTGGATCCACCATGTAC-3′, mBAa-2B; 5′-GTGGATCCACCAGACAACAC-3′ (BamH I sites are underlined).

All restriction enzymes were products of Takara Shuzo Co., Ltd., Tokyo.

Competitive PCR and determination of the amount of target cDNAs

The cDNA standard was competitively amplified in a 40-μl reaction mixture containing PCR buffer (50 mM KCI, 1.5 mM MgCl₂, 10 mM Tris-CI, pH 8.0), 200 mM each of dNTPs, and 500 nM each of primers, mPRLR-4 and mLTHR-1, or mBAs-1 and mBAa-1. Deoxycytidine 5′-[α-³²P]triphosphate ([α-³²P]dCTP; 148 kBq/tube) (Amersham, UK) was also added to the mixture to radio-label the products. The reaction mixture was overlaid with mineral oil (Sigma), and hot start PCR amplification proceeded in a thermal cycler (PTC-100; MJ Research, Inc., USA). After denaturation at 90°C for 5 min, 1 U of TaKaRa Taq DNA polymerase (Takara) was added to the reaction mixture, then 40 cycles of PCR were performed. Each cycle consisted of denaturation at 94°C for 1 min, annealing at 62°C for 1 min, and extension at 72°C for 1 min. After the last extension, the reaction mixture was heated at 98°C for 10 min to denature the PCR product and inactivate DNA polymerase, and left at room temperature for the PCR product to randomly anneal while cooling.

Two step cPCR amplifications were performed on cDNA samples to determine the amounts of both mPRL-R and mBA cDNAs in one tube at a time. The first amplification was performed in a 20-μl reaction mixture containing 5 nM each of the two pairs of primers by 40 cycles of PCR. An additional amplification by 20 cycles of PCR was carried out in a 40-μl reaction mixture containing 500 nM each of the two pairs of primers and [α-³²P]dCTP with 20 PCR cycles. The reaction mixture was heated at 98°C for 10 min and left at room temperature.

The PCR product was digested with BamH I and/or Kpn I, resolved by 4% polyacrylamide gel electrophoresis in TBE buffer and stained with ethidium bromide. Gel strips containing each pair of PCR products cleaved by the restriction enzyme(s) was excised under UV light and transferred into a glass vial to measure Cerenkov radiation using a scintillation counter (LS6000IC; Beckman Instruments, Inc., Fullerton, CA, USA). PCR product without restriction enzyme digestion was also resolved by electrophoresis, and a gel strip equivalent to the cleaved fragments at the same position served as the background. The radiation intensity of the gel corresponding to the fragments cleaved by BamH I or Kpn I was proportional to the amount of homodimers of the PCR product bearing the restriction site. Sometimes the gels were exposed to Kodak X-OMAT film after electrophoresis.

One-sided cPCR to determine the S/L ratio

The ratio of the short form of mPRL-R cDNA (PRL-RS3) to the long form (PRL-RL1), was determined by one-sided cPCR as described (Matsuda and Mori, 1996). Briefly, the cDNA was amplified by means of PCR using 100 μM of the common primer, mPRLR-2, and 500 μM each of identical primers, mLTHR-L1A and mLTHR-S3A (Fig. 1). The ratio of the two cDNAs was estimated from that of two PCR products which were distinguished from each other by the presence of unique restriction sites.

Statistical analysis

All results are expressed as the mean ± S.E. Statistical significance of difference between means was assessed with Student's t-test. The number of samples is expressed as ‘n’ in parenthesis.

Competitive PCR of the standard with the competitor

Mouse PRL-R standard DNA and the competitor were combined in various proportions so that the sum of both would be 10⁵ molecules in each PCR-reaction tube. These samples were then amplified by cPCR. PCR amplification of mPRL-R generated a 653-bp product, a portion of which was cleaved by the appropriate restriction enzymes into fragments of 329 bp and 324 bp. Figure 2A shows the relationship between the initial ratio of the standard to the competitor and the ratio of the PCR product cleaved by BamH I to that cleaved by Kpn I. The square root of the ratio of the cleaved PCR products corresponded to the initial ratio of the standard to the competitor. These findings demonstrated that both the standard and the competitor were amplified with equal efficiency, the PCR products annealed randomly and the restriction enzyme completely digested the homodimer PCR product bearing the restriction site. Thus, these observations enabled us to estimate the amount of mPRL-R cDNA by cPCR using a competitor bearing a single mutation.

Fig. 2

Competitive PCR of the standard cDNAs with competitors. (A) A mixture of mPRL-R standard DNA and its competitor (the sum was about 10⁵ molecules per tube) combined in various proportions was amplified by cPCR. Note that the ratio of PCR product cleaved by BamH I to that cleaved by Kpn I nearly equals the square of the initial ratio of the standard to the competitor. Two sets of the experiment were performed. (B) A mixture of mBA standard DNA and its competitor (the sum was about 10⁷ molecules per tube) combined in various proportions was amplified by cPCR. The ratio of PCR product cleaved by Kpn I to that cleaved by BamH I nearly equals the square of the initial ratio of the standard to the competitor. Two sets of the experiment were performed.

We also established a method to estimate the amount of mBA cDNA by cPCR using a competitor bearing two point mutations (Fig. 2B). In this case, PCR amplification of mBA generated a 471-bp product, of which a portion was cleaved by the appropriate restriction enzymes into fragments of 248 bp and 223 bp.

Competitive PCR of a cDNA sample with the competitor

We performed cPCR amplification of both mPRL-R and mBA of a pancreatic islet cDNA sample to ensure that the method could allow us to estimate the amount of mPRL-R and mBA cDNAs. After cDNA was made from about 200 islets and was divided into 20 aliquots, serially diluted competitors were added to each aliquot. Competitive PCR amplification was performed, then the amounts of mPRL-R and mBA cDNAs were estimated. Figure 3A shows an example of cPCR product after cleavage with restriction enzymes and gel electrophoresis. Using the two step amplification, nearly the same amount of the two PCR products corresponding to mPRL-R and mBA were obtained with low background by cPCR (data not shown). The square root of the ratio of the PCR products cleaved by each restriction enzyme was proportional to the amount of the competitors added to the reaction tube (Fig. 3B, C). These results demonstrated that the amounts of both mPRL-R and mBA cDNA in pancreatic islet cDNA could be estimated from the ratio of the cPCR products cleaved by the restriction enzymes at least when the ratio of the target cDNA to the competitor was between 0.25 and 4.

Fig. 3

Estimation of the amount of mPRL-R and mBA cDNA by cPCR in isolated pancreatic islets. An islet cDNA sample was amplified by cPCR for mPRL-R and mBA with various amounts of the competitor. (A) The c PCR product of islet cDNA with the competitor for mPRLR and mBA (10⁵ and 10⁷ molecules, respectively) was resolved by polyacrylamide gel electrophoresis after digestion with no enzyme (lane 1), BamH I (lane 2), Kpn I (lane 3) and both enzymes (lane 4). PCR generated 653 bp mPRL-R and 471 bp mBA DNA fragments, and the products were cleaved by restriction enzymes into two pairs of fragments (329 bp and 324 bp, and 248 bp and 223 bp, respectively). (B) Relationship between the initial amount of the competitor for mPRL-R and the ratio of PCR product of mPRL-R cleaved by BamH I to that cleaved by Kpn I. The latter was proportional to the square of the former. The amount of mPRL-R cDNA in islet cDNA sample was estimated to be about 1 × 10⁶ molecules. (The competitor applied was double-strand DNA.) Vertical bars indicate S.E. (n = 3–4). (C) Relationship between the initial amount of the competitor for mBA and the ratio of PCR product of mBA cleaved by Kpn I to that cleaved by BamH I. The amount of mBA cDNA in this sample was estimated to be about 8 × 10⁷ molecules.

Effect of hormones on the amount of PRL-R mRNA in cultured islets

We examined the effects of oPRL, bGH or estrogen on the ratio of mPRL-R to mBA in cultured islets by cPCR. Complementary DNA from 50 islets was divided into 5 aliquots, mixed with both 10⁵ or 10⁶ molecules of the competitor for mPRL-R and 10⁷ or 10⁸ molecules of the competitor for mBA, then the amount of those cDNA was determined. The results indicated that mPRL-R and mBA cDNAs ranging from 4 × 10⁴ to 9 × 10⁵ and from 7 × 10⁶ to 5 × 10⁷ molecules, respectively, were generated from about 10 islets. The ratio of mPRL-R to mBA was increased significantly by adding oPRL (1 μg/ml) or bGH (1 μg/ml) to the culture medium, but the ratio was not affected by E₂ (100 ng/ml) (Fig. 4).

Fig. 4

Effects of hormones on expression of PRL-R mRNA in pancreatic islets in vitro. Complementary DNA samples were prepared from adult female mouse pancreatic islets cultured for 4 days with or without PRL, GH or E₂ and the ratio of mPRL-R to mBA cDNA was measured by cPCR. The ratio was significantly larger in the PRL- or GH-treated groups as compared with the control group without hormones (*P<0.05). Vertical bars indicate S.E. (n = 4–6).

S/L ratio of PRL-R mRNA in cultured islets

The S/L ratio was examined by one-sided cPCR, in pancreatic islets cultured with or without hormones. Figure 5A shows a one-sided cPCR product digested by BamH I which cleaves the short form and/or Msp I which cleaves the long form derivative. Long form mPRL-R mRNA was dominant in the pancreatic islets. Adding oPRL, bGH or E₂ to the culture medium did not affect the S/L ratio (Fig. 5B).

Fig. 5

Effects of hormones on the S/L ratio of mPRL-R mRNA in pancreatic islets in vitro. (A) Products of one-sided cPCR for two forms of mPRL-R cDNAs from pancreatic islets isolated from adult female mice were resolved on a 10% polyacrylamide gel after digestion with no enzyme (lane 1), Msp I (lane 2), BamH I (lane 3), and both enzymes (lane 4). PCR generated 232-bp and 228-bp fragments of the long and short forms of mPRL-R, respectively, and the products were cleaved by restriction enzymes into two pairs of fragments (170 bp and 62 bp by Msp I, and 175 bp and 53 bp by BamH I, respectively). (B) S/L ratio was measured in the cDNA sample from pancreatic islets cultured for 4 days with or without PRL, GH or E₂. There was no significant (P<0.05) difference in the S/L ratio between the groups. Vertical bars indicate S.E. (n = 4–6).

Effect of hormones on insulin secretion in cultured islets

The concentration of insulin was measured in islet culture media to which hormones were added (Fig. 6). Four days of culture with oPRL significantly increased the insulin secretion. Bovine GH tended to stimulate insulin secretion, but the difference from the control was not statistically significant. Insulin secretion was not affected by E₂.

Fig. 6

Effects of oPRL, bGH, or E₂ on insulin secretion from cultured adult female mouse pancreatic islets. The graph gives the 24-hr secretion values of insulin in oPRL-treated (•), bGH-treated (▵), E₂-treated (▴) and control (○) groups. The data are expressed as the amount of insulin relative to that secreted during 24-hr preculture period (Day 0). There is a significant difference in the value between the oPRL-treated and the control groups at Day 4 (*P<0.05). Vertical bars indicate S.E. (n = 6).