The distribution of GLUT2 was investigated in liver sections, taken a t the same tim e as the mammary tissue, to ensure th at the anti-GLUT2 C-terminal peptide antibodies would react against the intact GLUT2 protein. Non-specific IgG was used as a control antibody. The method of staining has been described in Section 6.3. The
Fig. 6.1 Localisation of GLUTl in rat neocortex (a) GLUTl f t tj ■ Æ-.* J) *- h- (b) Rabbit IgG ( * ‘ c ■
-:M
The GLUTl glucose transporter was localised in the rat brain neocortex by immunocytochemistry using the silver-enhanced immunogold technique, detailed in Section 6.3. GLUTl detected using antibodies raised to the GLUTl C-terminal peptide (a) was restricted to microvessels, while non-specific rabbit IgG (b) gave no detectable labelling. (40x mag.)
results in Fig. 6.2(a) show that the GLUT2 staining was restricted to the sinusoidal surfaces of the liver cells adjacent to the blood supply, as reported by Thorens et al. (1990), both in the hepatocyte plasma membrane and in the endothelial cells. The non-specific IgG (Fig. 6.2(b)) did not stain the tissue. These results confirm that the anti-GLUT2 antibodies react with the liver isoform of the glucose transporter, and that the antibodies raised against the GLUT2 C-terminal peptide recognise the intact transporter protein in its native state. These antibodies could therefore be used to look for GLUT2 in the mammary gland by immunocytochemistry.
6.3.3 3T3-L1 adipoqrtes
The distribution of GLUT4 in 3T3-L1 adipocytes, in both the basal and insulin- stimulated state, was investigated by Dr. Kan, to ensure that the antibodies raised against the C-terminal peptide of GLUT4 would recognise the intact protein. Antibodies at a dilution of lOpg/ml were found to stain a translocatable GLUT4 protein in paraformaldehyde-fixed, Triton X-100 permeabilised 3T3-L1 adipocytes, which had been grown on coverslips. GLUT4 was detected using an fluoroisothiocyanate-conjugated anti-rabbit IgG second antibody, diluted 1:50, and immunofluorescence, visualised using a Bio-Rad confocal microscope. These results confirmed that the anti-GLUT4 C-terminal peptide antibodies recognise and bind to the intact GLUT4 protein (results not shown).
6.3.4 Lactating ra t mammary gland
The distribution of GLUTl, GLUT2 and GLUT4 in the lactating rat mammary gland was investigated using antibodies to the C-terminal peptides of these
Fig. 6.2 Localisation of GLUT2 in rat liver (a) GLUT2
(b) Non-specific IgG
The GLUT2 glucose transporter was localised In the rat liver using the method described In Section 6.3. GLUT2 was detected using antibodies raised against the GLUT2 C-termlnal peptide (a). Non-specific rabbit IgG (b) was used as a control antibody. GLUT2 was localised in the membranes adjacent to the sinusoidal space. (40x mag.)
transporter isoforms. From the results described in Sections 6.3.1-6.3.3, all of these antibodies had been shown to react to the native form of the glucose transporters, by immunocytochemistry of tissues known to express the specific isoforms. Both GLUTl and GLUT4 had been shown to be present in manunary gland using Western blotting in Section 4.2. The method used for the mammary gland immunocytochemistry is described in Section 6.3.
The photographs in Fig. 6.3(a) show that there is detectable expression of GLUTl in the lactating mammary gland. The extent of labelling shown in Fig. 6.3(a) was characteristic of that obtained using the affinity-purified anti-GLUTl C-terminal peptide antibodies. The intensity of staining was never very high despite the high concentration of primary antibody used. Using antiserum raised against the purified GLUTl protein the intensity of staining was no higher than when using the antibodies against the C-terminal peptide (results not shown). The most intense labelling appeared around the periphery of the alveoli, presumed to be the basal epithelial cell plasma membrane, schematically illustrated in Fig. 1.2. The cytoplasm also appeared more densely stained using the specific antibodies, than when the non-specific IgG was used, although it was not possible to distinguish any intra-cellular structures. No intense staining was seen in the space between the alveoli. The pre-immune serum, control rabbit IgG (Fig. 6.3(b)), anti-GLUT4 antibodies or anti-GLUT2 antibodies gave no detectable labelling. Unfortunately, it was not possible to distinguish adipocytes in the mammary tissue. However, from the sections it can be seen that the majority of cells in the tissue are secretory epithelial cells, which implies that contamination with other cell types is minimal.
Fig. 6.3 Localisation of GLUTl in lactating rat mammary gland (a) GLUTl
(b) Non-specific IgG
S M :
*■■ ■ - ' '
The GLUTl glucose transporter was localised in mammary gland using the method detailed in Section 6.3. The distribution of GLUTl was detected using antibodies raised against the GLUTl C-terminal peptide (a). The most intense labelling is restricted to the interstitial surface of the secretory alveoli, probably corresponding to the basal plasma membrane of the epithelial cells, there was also a higher staining in general over the surface of the cytoplasm, compared to the non-specific rabbit IgG (b). (40x mag.)
6.4 Conclusion
The distribution of GLUTl observed in the rat brain using the immunogold silver- staining method, shown in Fig. 6.1, agreed with that reported by Bagley et al.
(1989) suggesting that the methodology was working in these fixed rat tissues. The anti-GLUTl antibodies were used at a concentration of 50pg protein/ml and antisera at a dilution of 1:200, which is higher than the concentration required to detect GLUTl in the brain, as reported by Bagley et al. (1989), but was the concentration required to detect GLUTl in mammary gland, as described in Section 6.3.4. Using immunogold silver-staining, low levels of GLUTl were detected in the rat mammary gland, apparently concentrated around the outside of the alveoli, corresponding to the basal plasma membrane of the secretory epithelial cells. The level of staining was far lower than in the rat brain microvessels. The concentration of GLUTl in bovine brain microvessels is approximately 12pmol/mg protein, using Scatchard analysis of cytochalasin B binding, or llpmol GLUTl/mg protein using quantitative Western blotting (Pardridge et aL, 1990), and in the mammary plasma membranes approximately 20pmol/mg protein (Table 5.1). Therefore the level of staining in the mammary gland would be expected to be higher, however, this is not the case. It is possible that the bovine membranes were less highly purified than the rat plasma membranes, which could in part account for the difference in intensity upon immunocytochemistry. However it was also found, during work described in Section 7.5, that separation of the litter from the lactating female down-regulates the mammary GLUTl protein within 24hr. The rats used in the immunocytochemistry experiments had been separated from their litters for up to 6hr, therefore there could be some down-regulation, decreasing the level of GLUTl detected in the mammary cells. The distribution of GLUTl in the
mammary gland served to exclude the possibility th at a significant proportion of the GLUTl iso form within the mammary homogenate identified by Western blotting originated from a source other than the epithelial cell. As well as the apparent localisation of GLUTl to the basal plasma membrane of the epithelial cells, there was a generally higher level of staining over the cytoplasm observed using antibodies against GLUTl compared to non-specific IgG. It was not feasible to precisely locate the transporter within the cells, although the higher cytoplasmic staining could infer the presence of an intracellular GLUTl. The immunogold silver- staining method has also been used by Erdmann & Binas (1991) to localise a growth inhibitor in bovine mammary tissue, indicating th at the method itself works well in mammary tissue. Further investigation of GLUTl distribution in mammary epithelial cells at both the light microscope and transmission electron microscopic level is needed to precisely localise the GLUTl.
GLUT4 was not detected in either adipose tissue within the mammary gland or in the mammary epithelial cells. Unfortunately, a control tissue for the GLUT4 isoform taken from the same rats as the brain, liver and mammary gland was not available a t the time of this study, therefore the immunoreactivity of the antibodies to the native GLUT4 protein under these fixation conditions could not be checked. However, the results described in Section 6.3.3 indicate th at the anti- GLUT4 antibodies detect GLUT4 in fixed immortal cell lines. Also preliminary work with the anti-GLUT4 antibodies had shown that they are selectively removed by incubation with an adipocyte membrane fraction in a competitive ELISA assay (personal communication, Mr. L. Fryer), indicating th at the antibodies are capable of binding to the native GLUT4 transporter. Therefore, the lack of GLUT4 detection in the mammary gland supports the suggestion th at this isoform is not
expressed in the mammary epithelial cells.
GLUT2 was also not detected in lactating ra t mammary gland, confirming the results of Western blotting in Section 4.2. Two glucose transporter isoforms, GLUT2 (predominant) and GLUTl (increased levels after fasting), are expressed in hepatocytes, however only GLUT2 is readily detected in liver (Hacker e t a/., 1991). The presence of GLUT2 on the sinusoidal membrane of hepatocytes demonstrated in Section 6.3.2 is consistent with the results obtained by Thorens at al. (1990), further confirming the specificity of the anti-GLUT2 antibodies.
CHAPTER 7
EXPRESSION OF GLUCOSE TRANSPORTERS DURING PREGNANCY.