Eur. J. Biochem. 193, 367-374 (1990) 6 FEBS 1990

Diabetes-induced changes in guanine-nucleotide-regulatory-proteinmRNA detected using synthetic oligonucleotide probes Susanne L. GRIFFITHS, John T. KNOWLER and Miles D. HOUSLAY Molecular Pharmacology Group, Department of Biochemistry, University of Glasgow, Scotland (Received June 6, 1990) - EJB 90 0638

Synthetic oligonucleotide probes were designed to detect the a-subunits of the guanine-nucleotide-regulatory proteins (G-proteins) Gi-l, Gi-2, Gi-3 and G, (G, is inhibitory and G, is stimulatory). Each probe detected a single major mRNA species in Northern blots of RNA extracted from a variety of tissues. A probe was designed to identify the two forms of G-protein 8-subunits, PI and 82. This probe hybridised with a single 1.8-kb transcript (82) in RNA from all tissues studied except for brain, where a less-abundant 3.4-kb transcript (81) was also detected. These probes were used to assess whether the induction of diabetes, using streptozotocin, altered the levels of mRNA coding for specific G-protein components. In hepatocytes, diabetes caused a significant reduction in the number of transcripts coding for a-G,, a-Gi-2 and a-Gi-3; mRNA for a-Gi-I was undetectable. In adipocytes, diabetes increased dramatically the mRNA coding for a-Gi-1 and a-Gi-3, whilst no significant changes occurred in the fractions coding for a-Gi-2 and a-G,. No significant changes in the mRNA coding for G-protein a-subunits were observed in either brain, heart, skeletal muscle or kidney. Diabetes did not cause any significant changes in the mRNA coding for 82 in any tissue or cell population studied. Such results on the relative levels of mRNA encoding G-protein components was obtained by comparing equal amounts of total RNA from tissues of control and diabetic animals. G-protein mRNA levels were expressed relative to ribosomal 28s RNA levels and, in some instances, relative to transcripts for a structural protein called CHO-B. The total cellular levels of both RNA and DNA were assessed in the various tissues and cells studied. Major falls in RNA levels/cell appeared to occur in hepatocytes and to a lesser extent in adipocytes and skeletal muscle. Thus major reductions in G-protein transcripts occurred in hepatocytes. The detected changes in G-protein mRNA are discussed in relation to the available evidence on G-protein expression. We suggest that diabetes causes tissue-specific changes in the levels of mRNA for particular G-protein species ; this may have consequences for the functioning of cellular signal-transduction mechanisms in the affected tissues.

synthesis [9]. However, whilst diabetes elicits substantial reductions in the levels of hepatic albumin and its mRNA [lo], and the mRNA for insulin-sensitive glucose transporters in both adipocytes [ l l , 121 and skeletal muscle [Ill, the level and rate of synthesis of liver phosphoenolpyruvate carboxykinase [13] and its mRNA is increased as is the level of mRNA for laminin B1 in kidney [14]. There are indications that alterations in the expression of G-proteins can occur in diabetic and insulin-resistant states [15 - 181 and diabetes-induced alterations in the coupling between stimulatory receptors and adenylyl cyclase have been noted in adipocytes [16, 19,201 and by some [21-241 but not other [25 - 271 investigators in liver. Thus, because of the central importance of G-proteins to many cell-signalling events including, possibly, certain actions of insulin itself [4, 28, 291, we have assessed the effect of streptozotocin-inCorrespondence to M. D. Houslay, Molecular Pharmacology duced diabetes on transcripts for G-protein components in Group, Department of Biochemistry, University of Glasgow, Glasgow various rat tissues. This has taken advantage of the known G I 2 SQQ, Scotland cDNA sequences of various G-protein a-subunits which has Abbreviations. G-protein, gunanine-nucleotide-regulatorypro- permitted us to design and synthesize oligodeoxynucleotides tein; Gi, adenylyl-cyclase-inhibitory guanine-nucleotide-regulatory protein; G,, adenylyl-cyclase-stimulatory guanine-nucleotide-regula- which distinguish betwen the individual a-subunit mRNAs. We report here on the use of these probes to examine the tory protein. Enzymes. Adenylyl cyclase (EC 4.6.1 .l); collagenase (EC effects of streptotocin-induced diabetes on the levels of mRNA for certain G-protein a-subunits in various rat tissues. 3.4.24.3); polynucleotide 5’-hydroxyl-kinase (EC 2.7.1.78).

The action of many hormones is mediated by heterotrimeric GTP-binding proteins (G-proteins) composed of a, P and y subunits [l, 21. One of the best-understood examples of G-protein function is the hormone-sensitive adenylyl cyclase system, where the G-proteins known as Gi and G, serve to transduce information from receptors which inhibit or stimulate, respectively, the activity of this enzyme [l, 3, 41. Thus, changes in the concentration or activity of a G-protein(s) may be expected to alter the responsiveness of adenylyl cyclase to regulatory hormones and neurotransmitters as has been established in Albright hereditary osteodystrophy where expression of G, is attenuated [5, 61. Streptozotocin-induced diabetes in rats is a widely used animal for type 1 diabetes [7, 81, where reduced levels of insulin lead to alterations in overall levels and rates of protein

368 MATERIALS AND METHODS Guanidiniuin thiocyanate and formamide were from Fluka. All general (A.R. grade) and other biochemicals were from Sigma or BDH Chemicals, U.K. Radiochemicals were obtained from Amersham International plc, UK. Male Sprague-Dawley rats (200-250 g) were used throughout and diabetes was induced [30] using a single intraperitoneal injection of streptozotocin (70 mg/kg; 0.3 ml/ animal) in saline as described before [15]. Animals were sacrificed after 7 days if the concentration of glucose in the urine was greater than 12 mM. In order to isolate adipocytes from diabetic animals it was necessary to sacrifice animals after only 2 days [30]. Hepatocytes and adipocytes were prepared using collagenase digestion as described previously [I 5, 161.

R N A isolation Three different methods were used which were found to be optimal for the individual rat tissues. (a) A modified guanidinium thiocyanate method [31] was used to isolate total cellular RNA from brain, heart and adipocytes. In brief, brain and heart, dissected from animals immediately after sacrifice, were ground to a fine powder in liquid nitrogen and 1 g powder was homogenised in 10 ml guanidinium thiocyanate solution (4 M guanidinium thiocyanate, 25 mM sodium citrate, pH 7, 0.5% sacrosyl, 0.1 M 2-mercaptoethanol). Adipocytes (isolated from a single animal) were also homogenised in 10 ml of the same buffer. All homogenates were subsequently extruded through 18- and 23gauge needles then extracted once against an equal volume of phenol/chloroform/isoamyl alcohol (25 : 24: 1) and twice against equal volumes of chloroform before being precipitated with ethanol. The precipitates were resuspended in a further 4 ml guanidinium thiocyanate solution then precipitated again with ethanol. (b) Skeletal muscle (3 g powdered tissue) was homogenised in 30 m13 M LiCl and 6 M urea [32],extruded through needles as above. The RNA was precipitated overnight at 4"C, resuspended in 4 ml guanidinium thiocyanate solution then extracted and precipitated as above. (c) Hepatocytes (1 ml packed cells) and kidney (1 g powdered tissue) were hoinogenised in 6 ml buffer A (4 M guanidinium thiocyanate, 25 mM sodium acetate, pH 6, 0.1 M 2-mercaptoethanol), extruded through needles then centrifuged at 10000 x g for 10 min to remove particulate material. Homogenates were then layered over 5.5 ml 5.7 M CsCl and 25 mM sodium acetate, pH 6, in 14 mm x 89 mm Beckman pollyallomer ultracentrifuge tubes and centrifuged overnight (20 h) in a Sorvall TH641 rotor at 174000 x g (32000 rpm) at 20 'C [33].Total RNA was recovered from the bottom of the tubes, resuspended in buffer A and extracted and precipitated as described in (a). All preparations were finally resuspended in water. The concentration of the precipitated RNA was determined by absorbance at 260 nm, where A260 = 1corresponds to 40 pg/ ml. Equal amounts of each sample determined by absorbance were subjected to electrophoresis in the presence of ethidium bromide. Samples that showed no detectable degradation of the 28s and 18s ribosomal KNA species were used for Northern blot analysis. Plohry

33mer oligodeoxynucleotides, complementary to bases of the mRNAs encoding amino acids 125 - 135 of the cc-subunits

of rat Gi-l, Gi-2, Gi-3 and 147- 157 of a-G,, were made on an Applied Biosystems DNA synthesiser. This region corresponds to a divergent area of the coding sequence of rat cDNA clones [34, 351. A 29mer oligodeoxynucleotide, complementary to bases of the mRNAs encoding amino acids 82 - 91 of the bovine and human Pl- and P2-subunits was also synthesized [36 - 381. This region contains one nucleotide substitution in the sequence of the bovine /I1 cDNA [36]. The oligodeoxynucleotides were end-labelled [39] with P ~J - ~ ~ P I Ausing T P T4 polynucleotide kinase (BoehringerMannheim), to an average specific activity of 7 x 10' dpm/ pg. The DNA probes were labelled with [ E - ~ ~ P I ~ C byTthe P random priming method of Feinberg and Vogelstein [40]. The 28s ribosomal RNA probe in pBR322 has been described before by one of us [41]. A cDNA called CHO-B, in pBR322 and encoding a structural protein [42], was kindly provided by J. E. Darnell, Rockerfeller University, N.Y. Northern blotting Total RNA was denatured by incubation at 65°C with 2 M formaldehyde and 50% deionised formamide, resolved in 1YOagarose gels containing 2.2 M formaldehyde and transferred to Hybond N nylon filters (Amersham) [43]. Filters were prehybridised at 37°C for 4 h in hybridisation solution (30% deionised formamide, 0.75 M NaCl, 50 mM sodium dihydrogen phosphate, 5 mM EDTA, pH 7.4, Ficoll400, bovine serum albumin and poly(vinylpyrro1idone) each at 2 mg/ ml, 0.1YOSDS and 200 pg/ml denatured salmon testes DNA) then hybridised at 42°C overnight (16 h) in hybridisation solution containing either oligonucleotide probes, 5 x lo6 cpm/ml, or DNA probes, 2 x lo6 cpm/ml. Blots hybridised with oligonucleotide probes were washed to a final stringency of 75 mM sodium chloride, 7.5 mM trisodium citrate, pH 7, 0.1 Yn SDS at 55"C for 30 min, and with DNA probes the temperature was increased to 65'C and 15 mM sodium chloride, 1.5 mM trisodium citrate, pH 7, 0.1% SDS was used. Autoradiographic localisation of the bound probes was performed by exposure to presensitised Xray film [44] at -80°C. To determine the sizes of the transcripts recognised by the various probes used in this study, total cellular RNA or a RNA ladder (BRL) was run in gel lanes adjacent to those used for the hybridisation studies. Such sample lanes were transferred to Hybond N and the positions of the RNA species were located by staining the filters with methylene blue [39]. These filters were not used for hybridisation. Quantification of mRNA encoding G-protein a- m d b-subunits

Analysis of hybridisation of the probes to specific mRNAs was performed by scanning densitometry of autoradiographs of Northern blots employing a Bio-Rad gel scanner, driven by an Olivetti M24 microcomputer, using the Bio-Rad I-D software package. Multiple (at least three) scans were made of each band and the results averaged. This does not provide absolute values for mRNA concentrations but is valid as a comparison of different samples examined in the same blot. Thus individual blots contained at least three different RNA samples from both control and diabetic tissues. In each instance, film was preflashed and we determined that over the absorbance ranges encountered there was a linear relationship between the amount of bound probe and the absorbance measured, assessed by hybridising the probes with filters containing increasing the amounts of total RNA (0.5-50 pg/

369 Table 1. Comparison qf nucleotide sequences of G-protein subunit synthetic oligonucleotide probes The probes are the reverse complement of the cDNA sequence encoding amino acids 125-135 of the a-Gi subunits, 147-157 of the G, a-subunit, or 82-91 of the bovine and human b1 and 82 subunits. The nucleotide at position 12 in the Go probe is italicized, since in the bovine PI sequence there is a single nucleotide substitution in this region. The table of mismatched bases was constructed by comparing the number of identical nucleotides, over the relevant region of each cc-subunit sequence, with each oligonucleotide sequence: Y.-G,-I 5’-GCTGTCCTTCCACAGTCTCTTTATGACGCCGGC-3’ 2-Gi-2 5’-ATGGTCAGCCCAGAGCCTCCGGATGACGCCCGA-3’ a-Gj-3 S’-GCCATCTCGCCATAAACGTTTAATCACGCCTGC-3’ a-G, S’-CTCATCCTCCCACAGAGCCTTGGCATGCTCATA-3’ 8-G 5’-TGGAC CTTGTTGGT GGTGTAGCTGTCCGA-3’ a-subunit probe

G,-I Gi-2 G,-3

Gs

Nucleotide mismatch with G,-I

G,-2

Gi-3

G S

0 30.3 39.4 57.6

30.3 0 48.5 57.6

39.4 48.5 0 63.1

57.6 51.6 63.1 0

[2, 35,471. Thus, we have identified a region of least similarity which lies between residues 65 and 150 in the various Gprotein a-subunits. On this basis, we have synthesized a series of oligodeoxynucleotides which hybridise specifically with mRNAs for different rat G-protein a-subunits : the region encoding amino acids 125-135 for the a-G, probes, and amino acids 147 - 157 for the sc-G, probe. Table 1 shows the nucleotide sequences and percentage dissimilarity between these probes; the specificity is shown in Fig. 1A. Designing a probe for the P-subunit mRNAs was more difficult since the only known mammalian nucleotide sequences are for /?-subunit cDNAs isolated from bovine [27, 281 and human [28,29] libraries. By using the programs Fetch and Gapshow, from the UWGCG package, we were able to align the respective P-subunit sequences and define regions of similarity between them. The longest stretch was 29 nucleoD N A assay tides in length and encodes amino acids 82 - 90 plus the first The concentration of DNA, in crude tissue homogenates, two nucleotides of codon 91. Three of the four sequences are was determined by the method of Labarca and Paigen [45] identical over this stretch a single substitution, A for C, at position 18 in this region of the bovine P l sequence. We using bacteriophage 1- DNA as a standard. thus designed a probe complementary to this region and its sequence is given in Table 1 with the position of the substiImmunoblotting of G-protein fl-subunits tution indicated. Using these synthetic, radioactively labelled oligonucAdipocyte plasma membranes, from normal and diabetic animals (vide supra), were prepared as described in [16]. After leotides to probe Northern blots of total RNA we have estabSDSjPAGE the gels were immunoblotted with an antiserum lished (Fig. 1A) that the a-subunits of G,-2, GI-3 and G, are (HA1) specific for G-protein fl-subunits as described pre- expressed, to a greater or lesser degree, in all of the tissues viously in [46]. Detection of the 35-kDa G-protein p-subunit studied here, thus confirming and extending previous studies was carried out as before [16, 461 using either anti-IgG anti- which used full-length gene probes to identify transcripts [35, bodies coupled to horseradish peroxidase or an ‘251-labelled 481. A major mRNA species was identified by hybridising Northern blots with each probe and the molecular masses of second antibody. the mRNAs agree with those published previously [35]: aG,-2,2.35 kb; a-G,-3, 3.4 kb; a-G,, 1.85 kb. Northern blots of brain RNA probed with the a-G, probe sometimes contained RESULTS AND DISCUSSION a minor cross-reacting species of approximately 3.75 kb (data In this study we analysed mRNA levels for G,, the three not shown). The identity of this mRNA is not known and it types of Gi which have been identified [35] and the two forms may be either a precursor of the mature RNA or an as yet of P-subunit, fl1 and fl2 [27 - 291. unidentified a-subunit mRNA. We consider it unlikely to repThe cDNA and deduced amino acid sequences of particu- resent transcripts for the G,-like protein a-GQlf, found in olfaclar G-protein a-subunits from different species share a striking tory epithelium [48], as when the nucleotide sequence of the level of similarity. In contrast the different G-protein a-sub- a-GOIfcDNA was compared with the comparable region of units within a species can be distinguished readily on the basis a-G,, from which our a-G, probe was derived, we found a of protein sequence with the identity ranging over 40 - 94% 10-nucleotide difference. lane). The resulting autoradiographs were scanned and the amount of radioactive probe bound was quantified by liquid scintillation counting of the excised and extracted band. In order to assess for the possibility of any variations in the amounts of total RNA in individual samples applied to the gels, we also hybridised the blots with the 28s ribosomal RNA probe. This was carried out by stripping the blots which had been probed with the G-protein probes, using techniques described by the manufacturer (Amersham International), then rehybridising them with the 32P-labelled 28s rRNA probe. Levels of probe bound were quantified by liquid scintillation counting of the excised bands. This demonstrated that the loading variation was small and allowed for any correction.

370

A 28 Gi- 1 1.5 -

18 28

4.4 -

Gi-2 2.4 -

18 28

1.4

-

Pl

- Pz

-

18 . 28

-

Fig. 1 . Detection of tnR.YAs corresponding to the a- andb-subunits qf’Gil, Gi-2, Gi-3 and G, in rat tissues. mRNA for G-protein sc-subunits was localised as described in Materials and Methods. Each lane contains 10 pg total RNA from the different rat tissues, as indicatcd at the top of the figure. The Northern blots were hybridised with radiolabelled oligonucleotide probes complementary to the mRNA cncoding (A) the scsubunits of C3-1. (3-2.(3-3 or G,, as shown to the right of the figure. The positions of the 18s and 28s ribosomal KNA species are indicated to the left of each blot. The figure is a composite of autoradiographs which were exposed for 2-4 days. (B) Blots were hybridised with the P-subunit oligonucleotide probe and the positions of the B1 and 82 mRNAs are indicated to the right of the figure. RNA size markers (kb) are shown to the left. Autoradiographs were exposed for 7 days

Transcripts for a-Gi-l (3.45 kb) showed a very much more restricted distribution (Fig. 1A), being barely detectable in heart and not detectable in RNA prepared from either skeletal muscle or hepatocytes. This is consistent with our inability [41] to detect x-Gi-l using a specific anti-peptide antibody to probe hepatocytc membranes, which held true even when five times more hepatocyte RNA (50 pg) was used in similar experiments; indeed, the ability to detect transcripts for Gi-1 in rat liver appears to be controversial [3S, 491. Certainly, using both mRNA preparations and plasma membranes from whole liver rather than hepatocytes, we [SO] have found a-Gi-l transcripts and immunoreactive cc-Gi-l, respectively, indicating that the non-parenchymal cells of liver may contribute this G-protein (S. L. Griffiths, M. D. Houslay and M. Bushfield, unpublished results). Using our [{-subunit oligonucleotide probe we observed transcripts of 1.8 kb encoding the lower molecular mass form (82) at varying abundance in all tissues studied (Fig. 2B). In brain, howevcr, a weak hybridisation signal with RNA for the higher molecular mass form (Pl) was also seen (Fig. 1 B). It may be that this reflects the fact that /I2 predominates in rat tissues. although there is a possibility that our failure to observe transcripts in other tissues might be due to P-subunit variations between rat and the bovine and human forms over the region chosen to form our probe. Nevertheless, it would appear that brain may be particularly enriched in mRNA for

P1 compared to other sources. Our analysis of human postmortem brain RNA and sheep adipocyte RNA, using this probe, identified both transcripts, but again the lower molecular mass form (82) predominated (unpublished data). Decreases in total cellular RNA, caused by streptozotocininduced diabetes, have been noted in various tissues [I 1, 511. Here, we found that diabetes caused little change in the amount of RNA/DNA in most tissues, although, as noted before [I 11, we observed reductions in total RNA from skeletal muscle, adipocytes and hepatocytes (Table 1).The more-modest fall in adipocyte RNA seen here compared with an earlier study [ l l ]may be due to the 2-week period of diabetes used there [Ill rather than a 2-day period used in our study of adipocytes: a strategy we [I61 and others [30] havc found necessary for the successful isolation of metabolically active adipocytes from diabetic animals. The major reductions in RNA observed here were seen using skeletal muscle and hepatocytes (Table 1). This will reduce the total level of Gprotein transcripts found/cell and exaggerate the relative reduction of G-protein transcripts seen in hepatocytes (vidu infru; Fig. 2). In order to determine whether the relative levels of mRNA encoding G-protein subunits was altered in diabetes, we prepared RNA from six different tissues of both untreated (control) and diabetic rats. In these experiments, the amount of RNA loaded onto the gel tracks was increased to 20 pg for

371 A P2

-

r l

VI

W c

Protein level

c

G,

5

RNA level in pool

c

$

GI-3

L

a

0

20

40

60

80

100

P r o t e i n h R N A (%)

B P2

u

rn

c

c

:

RNA l e v e l in pool

Gs

a

5 c

GI-3

c 0

h GI-2 u GI-1

0

100

200

300

400

500

P r o t e i n h R N A (Yo)

Fig. 2. The effect of streptozotocin induced diabetes on the levels of mRNA andprotein,for the cc- andp-subunits of the G-proteins. Northern blots of total RNA, isolated from different tissues from control (untreated) and diabetic rats, were probed with radiolabelled oligonucleotides as described. Values obtained from densitometric scans of the resulting autoradiographs were normalised relative to the amount of ribosomal RNA in the same blots. The corrected values (the mean & SEM) for RNA from diabetic tissues are given as a percentage of control values for hepatocytes (A) and adipocytes (B). Data for GIG, indicates that the induction of diabetes led to significant decreases in transcript numbers in hepatocytes with P < 0.0001 and to a lesser extent in kidney at 82% of control at P < 0.02, but with no significant differences in other tissues. Data for cc-Gi-l showed transcript numbers in adipocytes from diabetic animals significantly different at P < 0.001, but with no other significant differences. Data for c(-Gi-2,showed transcript numbers in hepatocytes from diabetic animals significantly different at P < 0.001, kidney at 85% of control (P< 0.05), but with no other significant differences. Data for cc-Gi-3 showed for both hepatocytes and adipocytes, of diabetic animals, significant differences at P < 0.001 and skeletal muscle at 78% of control (P < 0.05), but with no other significant differences and data for 82 indicated no significant differences for any data. In this analysis, changes in mRNA for specific G-protein components were determined relative to the whole RNA pool. However, alterations in total RNA/cell are evident for both adipocytes and hepatocytes (Table 2) and alterations in levels/cell will be altered accordingly, as referred to in the text. The number of separate animals examined were the same in both control and diabetic instances and data are given for kidney (n = 6), skeletal muscle ( n = 6), heart (n = 3), hepatocytes (n = 9), brain ( n = 3) and adipocytes ( n = 7). Values for P-subunits for all tissues refer to mRNA of the lower molecular mass form (835, P2). Brain, also exhibits the higher molecular mass form (836, P l ; see Fig. 2) which is unchanged by diabetes. Alterations in protein expression of G-protein components are also given both for adipocyte cc-subunits (adapted from [16]), adipocyte 8-subunits (this study) and hepatocyte G-protein components (adapted from [46, 571)

brain, heart, kidney and skeletal muscle, and to 30 pg for hepatocytes. We elected to use Northern rather than dot blotting to quantify the abundance of mRNAs in order to ensure that we assessed only the correct transcript under all conditions. This was especially important because, in some instances, and particularly when using the a-Gi-I probe, we found some non-specific hybridisation in the region of the 18s ribosomal RNA. This was also the case with the a-G, probe, where a higher molecular mass transcript was occasionally detected when hybridised with RNA from brain. Thus, by using Northern blotting procedures in each instance, we could be certain that only the transcripts of interest were detected and analysed. Using this method of analysis, we found that diabetes did not affect the size of the various G-protein transcripts (data not shown). However, when we probed equal amounts of total RNA from control and diabetic animals then, in certain instances, we identified alterations in the relative levels of G-protein mRNA occurring within the total RNA pool; being most apparent for adipocytes and hepatocytes (Fig. 2). Analysis using our a-G, probe showed diabetes induction caused a significant change in the relative levels of mRNA in hepatocytes only (Fig. 2). This we can expect to be exacerbated due to the reduced level of total cellular RNA found in these cells isolated from diabetic animals (Table 2), accounting for the reduced expression of this G-protein (Fig. 2). Indeed, as RNA/cell was reduced in both adipocytes and skeletal muscle from diabetic animals, then a-G, mRNA levels can also be expected to decrease by some 20 - 30%. This, at least in adipocytes, does not appear to reduce the level of expression of a-G, (Fig. 2 ) [16]. Interestingly, results obtained using our a-Gi-1 probe showed that whilst diabetes elicited no effect on the level of transcripts for this G-protein in either brain or kidney it caused a dramatic increase, of around fourfold, in the levels seen in adipocytes (Fig. 2). This increase would be attenuated by only some 20% due to the diabetes-induced reduction in total adipocyte RNA (Table 2). However, analysis of a-Gi-l protein, using anti-peptide antibodies, failed to show any corresponding increase in the levels of this G-protein in adipocytes of diabetic animals (Fig. 2) [16]. This observation is similar to the diabetes-induced increase in the number of transcripts for intestinal phosphoenolpyruvate carboxykinase which was not matched by any comparable increase at the protein level [52]. Our studies employing the a-Gi-2 probe showed that the levels of mRNA for this G-protein a-subunit were unaltered by the induction of diabetes in all tissues studied, except for hepatocytes where we observed a highly significant reduction of around 40% (Fig. 2). This is the major form of Gi found in hepatocytes, and such a fall, which would be amplified by the diabetes-induced reduction in total hepatocyte RNA (Table 2), is consistent with our observations [I 51 showing that diabetes does indeed cause a dramatic reduction of 50-90% in the level of Gi in hepatocytes from diabetic animals (Fig. 2). In that study we used an anti-peptide antibody that recognised a-Gi-l and a-Gi-2, but not a-Gi-3. Thus, as hepatocytes do not express a-Gi-l, the loss in Gi that was detected with this anti-peptide antibody can be attributed entirely to a reduction in the expression of a-Gi-2. Interestingly, a recent study [53] did not observe any decrease in a-Gi-2 in diabetic liver. However, in that instance, analyses were made of membranes prepared from whole liver rather than from hepatocytes, as analysed previously by us [I 51. Membranes from non-parenchymal cells

372 Table 2. The effect ofstreptozotocin-induced diabetes on t o t d cellular RNA and D N A RNA was isolated from the tissues of control and diabetic rats, and was quantified by absorbance at 260 nm, as described in Materials and DNA as a standard. Methods. DNA was quantified in crude tissue homogenates by a spectrophotometric assay [45] using bacteriophage i. Data are expressed as mean +_ SEM for ( n ) individual samples. Transcript ratio is given as the ratio of transcripts/cell (RNA/DNA) for diabetic animals relative to that seen using control animals. Adipocytes values represent RNA and DNA isolated/animal, i.e. the adipocytes isolated from two epididymal fat pads. Hepatocytes values are for the RNA and DNA isolated from 1 ml packed cells Tissue

RNA ( n )

DNA(n)

RNA/DNA

Kidney Diabetic kidney Skeletal muscle Diabetic skcletal muscle Heart Diabetic heart Adipoc yte Diabetic adipocyte Hepatocytc Diabetic hepatocytc Brain Diabetic brain

mg/g 1.33 f 0.21 ( 5 ) 1.37 +_ 0.08 (6) 0.34 -t 0.05 (6) 0.25 5 0.04 (6) 0.72 k 0.06 (6) 0.82 f 0.08 (6) 0.17 f 0.02 (8) 0.13 f 0.02 (10) 2.08 f 0.17 (12) 1.43 k0.12 (10) 1.35 0.19 (6) 1.11 kO.lO(12)

mg/g 10.48 f 1.35 (3) 9.39 & 0.32 (4) 2.47 f 0.13 (3) 2.55 f 0.09 (3) 10.45 0.35 (4) 10.66 k 0.72 (4) 2.09 & 0.40 (3) 1.91 i 0.15 (3) 5.65 -t 1.05 (4) 5.68 t- 0.42 (3) 2.77 f 0.35 (4) 2.48 f 0.1 8 (3)

mg/mg 0.13 0.15 0.14 0.10 0.07 0.08 0.24 0.068 0.37 0.25 0.49 0.45

constitute at least 40%) of preparations from whole liver [54] and we have been able to show (M. Bushfield and M. D. Houslay, unpublished results) [57] that diabetes does not reduce levels of a-Gi-2from such sources, which, of themselves, may obscure changes occurring in hepatocytes. The action of diabetes on hepatocytes may be an example of a specific effect of insulin withdrawal on gene expression in these cells, which is analogous to those reported for albumin and pyruvate kinase [lo, 55, 561. Indeed, when Northern blots were reprobed for albumin transcripts, we found a 58% decrease in the mRNA for albumin in hepatocytes from diabetic versus control rats (data not shown). Analysis of mRNA levels for a-Gi-3 showed that diabetes caused opposite effects in hepatocytes and adipocytes (Fig. 2); a striking elevation of around fourfold occurring in adipocytes and a significant reduction in hepatocytes. The levels in brain, heart, skeletal muscle and kidney were not significantly affected. In contrast to the studies on expression of a-Gi-1, we previously observed [I 6) an approximately 80% increase in the levels of a-Gi-3 in adipocyte membranes from diabetic animals (Fig. 2). This suggests that there are tissue-specific differences in the processing of mRNA for these two G-proteins. In this respect, it may be that in order to see enhanced expression of G-protein a-subunits there must be a corresponding increase in the expression of /?-subunits. This does not appear to happen in adipocytes as we observed transcript levels for p2 in cells prepared from both control and diabetic animals (Fig. 2) and, furthermore, probing membranes with a /?-subunit-specific antibody indicated that the induction of diabetes did not lead to any altered p-subunit expression in adipocytes (Fig. 2). Our inability here to identify any significant change in j-subunit mRNA levels is consistent with our analysis of /?-subunit expression in hepatocytes (Fig. 2) [46], where we have shown that diabetes does not elicit any change in the level of immunodetectable protein, despite the reduction in both a-Gi-2and x-G, protein (Fig. 2 ) [46, 571. Attributing functional changes to the altered expression of G-proteins in the adipocytes and hepatocytes of diabetic animals is problematic. This is not the least so because the true function of the various forms of Gi remains to be established [l, 351. Nevertheless. the diabetes-induced reduction in hepatocyte transcripts for G, and protein [I51 may account,

Transcript ratio RNA/DNA

1.11 0.71 1.14

0.84 0.68 0.92

at least in part, for the loss of guanine-nucleotide-mediated inhibition of adenylate cyclase [15]. That diabetes causes transcript numbers for both G, and G i forms to fall in hepatocytes, suggests that complex changes in the ratio of these two Gproteins may occur over the period of diabetes examined. This may account for the fact that some investigators have noted reduction [21, 22, 581, enhancement [15, 23, 241 or no change [25 - 271 in the efficiency of glucagon to activate adenylyl cyclase in liver: a process known to be highly sensitive to the G,/Gi ratio [l]. The consensus of reports identifying enhanced coupling of the B-adrenoceptor to adenylyl cyclase in adipocytes of diabetic animals [16, 19, 201 may reflect the fact that expression and transcript numbers for G, are not altered, whereas Gi function and expression of some forms are [16]. Routinely, equal amounts of RNA, as determined by absorbance, were loaded onto gels for subsequent Northern analysis. We made an independent assessment of this by hybridising blots with a 28s rRNA probe. In addition, we also used a cDNA probe for a structural protein called CHO-B [32], the mRNA for which, in adipocytes and skeletal muscle from streptozotocin-diabetic rats, is considered to be unaffected by diabetes [ll]. Although CHO-B mRNA was detected in Northern blots of all tissues, the autoradiographic signals were too weak to provide meaningful data in kidney, adipocytes and brain. When signals for G-protein subunit mRNAs were expressed relative to that obtained for CHO-B mRNA on the Northern blots, very similar results were obtained. Thus, for hepatocytes, mRNA levels for a-Gi-2, a-Gi-3, a-G, and p2 in diabetic animals were some 40 & 8 % (P < 0.005, n = 4), 66 5 13% (P< 0.05; n = 4), 35 A 15%; (P< 0.005; n = 3) and 97 & 12% (not significant; n = 4), respectively, of those of controls (100%); expressed relative to CHO-B mRNA, values for transcript levels for a-Gs,a-Gi1, a-Gi-3 and p2, in both heart and skeletal muscle, were not significantly altered in diabetic animals ( n = 4 in each instance). Altered levels of G-protein a-subunits and their corresponding mRNAs have been reported when differentiation is induced in tissue culture cells by factors such as dibutyrylCAMP [59, 60) and it is possible that G-protein expression is hormonally regulated. Indeed, analysis of the 5’ non-coding sequence of various G-protein r-subunit genes [61, 621 has

373 revealed the existence of consensus binding sites for steroid receptor binding and candidate binding domains for the transcription factors AP-1 and AP-2, which mediate some actions of phorbol esters and CAMP [63]. However, it remains to be determined which factors effect the tissue-specific changes in the expression of G-protein genes in diabetes. This could be related to the fact that not all tisues are equally responsive to insulin. Furthermore, diabetes is associated with changes in the plasma levels of other hormones and metabolites which may affect particular cells in very different ways. For example, there is evidence that G-protein gene expression can indeed be regulated by steroid [64, 651 and thyroid [66] hormones. It will then be of much interest to elucidate factors controlling the expression of G-proteins, and to evaluate any potential role of fl-subunit expression in determining the final levels of expressed a-subunits. This work was supported by grants from the Agriculture and Food Research Council, Medical Research Council and the California Metabolic Research Foundation. We wish to thank Dr G. J. Murphy for various hepatocyte samples and Ms June Fotheringham for preparing some of the RNA samples used in this study.

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Diabetes-induced changes in guanine-nucleotide-regulatory-protein mRNA detected using synthetic oligonucleotide probes.

Synthetic oligonucleotide probes were designed to detect the alpha-subunits of the guanine-nucleotide-regulatory proteins (G-proteins) Gi-1, Gi-2, Gi-...
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