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Na+K+-ATPase activity and its αII subunit gene expression in rat skeletal muscle: Influence of diabetes, fasting, and refeeding
Na+/K+-ATPase Activity and its
T
HE UBIQUITOUS cellular enzyme Na’/K’-adenosine triphosphatase (ATPase) (EC 3.6.1.37) is responsible for the maintenance of intracellular sodium and potassium concentrations.‘~.’ The function of this enzyme is to transport three ions of sodium from the intracellular space to the extracellular environment and, in return, to allow two ions of potassium to enter the cell. Na’/K’-ATPase consists of two structurally different subunits.‘.’ The p subunit (MT -35,000) seems to be important for inserting the Na’/K’-ATPase into the plasma membrane and maintaining its structural integrity.3 The a subunit (Mr - 112,000) is the functionally active part of the enzyme. There are at least three isoforms of the a subunit, designated aI, aII (also known as a+), and aIII.‘.‘,* The a11 subunit, the most prominent isozyme in muscle,‘~’ possesses the greatest sensitivity to ouabain.“’ Its activity has been shown to be regulated by insulin” and certain metabolic changes, such as alterations in concentrations of intracellular sodium’*-” or free fatty acids (FFA).“‘* Whereas much is known concerning the factors modulating Na’/K’-ATPase activity, knowledge of the influence of diabetes and fasting on pretranslational regulation of the enzyme is lacking. The aim of this study is to compare skeletal muscle Na’/K’ATPase activity with gene expression of its catalytic a11 subunit under conditions known to influence Na’/K’ATPase activity, such as diabetes, fasting, and refeeding. MATERIALS
AND METHODS
Materials Streptozotocin (STZ) and sodium orthovanadate were purchased from Sigma (St Louis, MO), insulin from Eli Lilly (Indianap-
From the Medical Research Service and Depafiment of Medicine, Veterans Affairs Medical Center and Deparhnent of Medicine, University of Colorado Health Sciences Center, Denver, CO: and the Jichi Medical Schoof, Tochigi Ken, Japan. Supported by the Research Service of the Veterans Affairs and The Diabetes Research Foundation of Colorado. Address reprint requests to Boris Draznin. MD, Section of Endocrinology (IIIH), VAMC, IO.55 Clermont St, Denver. CO 80220. Copyright 0 1992 by W.B. Saunders Company 0026-0495/92/4101-0011$03.00/O 56
olis, IN), and phlorizin from Aldrich (Milwaukee, WI). Deoxycytidine-5’-triphosphate, tetra-triethylammonium salt, [,“P], specific activity 3,000 Ci/mmol, was purchased from ICN Biomedicals (Costa Mesa, CA). cDNA probes for rat Na’/K+-ATPase (~1 and a11 subunit(s) were kindly provided by Drs Shull and Lingrel (Cincinnati, OH), human p subunit cDNA by Dr Kawakami (Tochigi Ken, Japan), mouse p-actin cDNA by Dr Spiegelman (Boston, MA), and human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by Dr Terada (Denver, CO). Monoclonal antibodies for Na’/K’-ATPase a1 (McKl) and a11 (McB2) subunits were a gift from Dr K.J. Sweadner (Boston, MA).
Experimental Design Male Sprague-Dawley rats weighing 175 to 230 g were used in these experiments. Animals (eight to 10 per group) were randomly allocated into one of the following groups: 48-hour fasting (water ad libitum), 48-hour fasting followed by 48- or 72-hour refeeding, and diabetic animals. At least three independent experiments were performed with each group. Diabetes was induced by intravenous injection of STZ (65 mgikg body wieght [SW] dissolved in 0.01 mol/L citrate buffer, pH 4.5). Diabetic animals were further subdivided into four groups: untreated for 14 days, and treated for 7 days with either insulin (daily subcutaneous injections of 3 to 6 U NPH), vanadate (sodium orthovanadate 0.2 mg/mL in drinking water),“.‘” or phlorizin (0.4 g/kg BW/d prepared as a 40% solution in propylene glycol and injected subcutaneously every 8 hours to ensure continuous inhibition of renal tubular reabsorption of glucose).” The control group of animals was allowed food and water ad libitum. Control animals compared with the diabetic ones were injected with citrate buffer instead of STZ. All animals were killed by decapitation and skeletal muscle was removed for either RNA extraction or measurements of Na’/K’-ATPase activity. Blood was collected for glucose (Beckman glucose analyzer, Beckman Instruments, Fullerton, CA) and insulin determinations by radioimmunoassay.
RNA Extraction and Northern Blot Analysis Total cellular RNA was purified by guanidinium isothiocyanate method” from freshly isolated skeletal muscle (abdominal muscles in diabetes experiments and hind leg muscles in fasting experiments). Twenty micrograms of denatured total cellular RNA was electrophoresed in 1.4% agarose and 2.7% formaldehyde gels and blotted onto a nitrocellulose membrane and cross-linked by baking at 80°C for 2 hours in a vacuum oven. DNA probes for Na’/K’ATPase al, ~11, and l3 subunits, @-actin, and GAPDH were labeled Metabolism, Vol41,
No 1 (January),
1992: pp 56-63
57
NA+/K+-ATPASE ACTIVITY AND all GENE EXPRESSION
with [‘*P]dCTP by the random-primer method or nick translation using Boehringer-Mannheim (Indianapolis, IN) labeling kits. Hybridization was performed in 0.007 mol/L Tris-HCL, pH 8.0, 10% dextran sulfate, 40% deionized formamide, 4x SSC (1 x SSC = 0.15 mol/L NaCl + 0.015 mol/L sodium citrate), 0.8 x Denhardt’s solution, 20 ug/mL denatured salmon-sperm DNA, and 32Plabeled cDNA for approximately 14 to 18 hours at 42°C. The hybridized blots were washed four times in 2x SSC and 0.1% sodium dodecyl sulfate (SDS) for 5 minutes at room temperature and subsequently twice in 0.5~ SSC and 0.1% SDS for 30 minutes at 55°C. Autoradiographic exposures of the blots to Kodak X-O mat AR film (Eastman Kodak, Rochester, NY) were performed with two intensifying screens at -70°C. The levels of RNA transcripts were measured by scanning autoradiograms vertically with a video densitometer (model 620, Bio-Rad, Richmond, CA). Relative shifts in gene transcript concentrations were quantified by obtaining a ratio of the densitometric area of each peak of Na’/K’-ATPase gene transcript compared with area of each p-actin peak and GAPDH peak. Western Blot Analysis Skeletal muscle (0.5 to 1.0 g) was homogenized in 2.5 mL of buffer (5 mmol/L EGTA, 15 mmol/L Tris, 1 mmol/L phenylmeth-
ylsulfonyl flouride [PMSF], 1 pg/mL each of leupeptin, pepstain A, antipain, and soybean trypsin inhibitor [STI]) using Tissumizer (Tekmar, Cincinnati, OH). Homogenate was centrifuged at 8,000 x g
for 20 minutes at 4°C to discard all debris, nuclei, and mitochondria. Supernatant was further centrifuged at 100,OOQx g for 60 minutes at 4°C. The pellet dissolved in 200 uL of the above buffer was used as the crude cell membrane fraction. The supernatant represented the cytosolic fraction. Either 10 pg of protein of a membrane fraction or 100 ug of protein of a cytosolic fraction were applied to 7.5% polyacrylamide SDS gel electrophoresis, without heating of the samples,23,” and blotted onto a nitrocellulose membrane. The blots were blocked with 5% dried milk in TBS-T (20 mmol/L Tris, 137 mmol/L NaCl, pH 7.6,0.1% Tween 20) overnight. The blots were then incubated with an antibody (McKl was diluted in l:lOO, McB2 in 1:500) for 1 hour, washed, incubated with biotinylated anti-mouse second antibody for 20 minutes, and washed again. The biotinylated second antibody was detected by streptavidin alkaline phosphatase conjugate, nitro-blue tetrazolium, and 5-bromo-4-chloro-3-indoyly phosphate (Amersham Blotting detection kit, Arlington Heights, IL).
Na’JK’-ATPase
Activity
Skeletal muscle (0.5 to 1.0 g) was homogenized in 5 mL of ice cold buffer (300 mmol/L o-mannitol, 5 mmol/L EGTA, 15 mmol/L Tris, pH 7.4) using Tissumizer. Homogenate was then centrifuged at 8,000 x g for 20 minutes at 4°C. Supernatant was further centrifuged at 100,000 x g for 60 minutes at 4°C to pellet the crude preparation of cell membrane fraction. The pellet was dissolved in 200 (LL of buffer (5 mmol/L MgCl,, 100 mmol/L NH,Cl, 100 mmol/L imidazole, 150 mmol/L NaCl, 1 mmol/L EGTA, and 2 mmol/L NaN,, pH 7.3) and left overnight at 4°C. The measurements of the ouabain-sensitive Na’/K’-ATPase activity were performed as described by Simon et a1.25.26 Results are expressed as micromole NADH converted to NAD per milligram of protein per hour.
RESULTS
Effect of Diabetes The levels of glycemia and insulinemia in control and experimental animals are shown in Table 1. Untreated diabetic animals were overtly hyperglycemic and profoundly hypoinsulinemic (P < .OOl). All three modes of therapy administered for 7 days (a single daily injection of insulin, oral vanadate, or three daily injections of phlorizin) normalized blood glucose levels. Vanadate in drinking water (0.2 mg/mL) appeared to be the most consistent in lowering blood glucose levels, but differences between the treated groups were not significant. Diabetic animals treated with vanadate or phlorizin became normoglycemic, but remained hypoinsulinemic. This experimental protocol allows for differentiation of the role of hypoinsulinemia from that of hyperglycemia. Skeletal muscle Na’/K+-ATPase activity was significantly decreased on the 2nd day of diabetes (3.05 * 0.14 v 6.05 ? 0.44 pmol NADH/mg protein/h in controls, P < .Ol) and on the 14th day (4.20 + 0.8 Fmol NADH/mg protein/h, P < .05). The difference between the two diabetes groups was not significant (Fig 1). Insulin therapy partially restored the enzyme activity, which remained lower than in control rats (4.60 * 0.7 pmol NADH/mg protein/h). We then examined the effect of diabetes on Na’/K’ATPase a11 subunit mRNA levels. Two species of RNA were detected by hybridization with a cDNA clone: 3.4 and 5.3 kb (Fig 2). In contrast to the decreased Na+/K’-ATPase activity, we observed an increase in the autoradiographic signal for the Na+/K’-ATPase in the lanes with RNA isolated from diabetic animals (Fig 2). The results were quantitated by calculating the ratios of Na’/K’-ATPase a11 densitometric signals to those of either p-actin or GAPDH. Since B-actin mRNA has been shown to be influenced by fasting,” we used GAPDH as an additional control. In our hands, the ratios obtained with both internal controls were essentially identical. When RNA autoradiography was analyzed by densitometry and expressed as a percent of RNA abundance relative to control, the following data were obtained. The control levels of each species of RNA are expressed as 100% (Fig 3A and B). Despite the early and significant reduction in enzyme activity (Fig l), the levels of crI1 mRNA remained unchanged on the 2nd day of diabetes. After 14 days of diabetes, there was a dramatic increase (3.4-kb band, P < .OOl; 5.3-kb band, P < .Ol) in the abundance of Na’/K’-ATPase CXIIsubunit mRNA. These findings were
Table 1. Characteristics of Control and Experimental Animals
”
GlUCOSe
Insulin
hWdL)
WJlmL)
Controls
7
232 + 15
147 + 21
24.8 * 8.3
ST.?!-diabetic
6
185 * 14
454 + 25
8.2 t 6.0
Insulin-treated
5
222 *a
219 -c56
Vanadate-treated
7
188 2 7
74* 22
Statistical Analysis
Phlorizin-treated
6
194 * 9
160 + 52
9.6 2 2.3 9.0 2 1.9
The Student’s unpaired t test was used to compare the mean + SEM values among the groups.
Fasted
6
183 +3
81 r 9
8.3 2 1.4
Refed
6
211+4
97 + 5
13.6k 1.2
NISHIDA ET AL
*
-
To determine whether the increases in mRNA abundance were translated into more (~11protein being present, we used immunoblotting (Western blot) with a monoclonal (~11antibody, which was kindly supplied by Dr K. Sweadner. Both plasma membrane and cytosolic fractions were tested. There was no difference in the amount of ~11 protein in the plasma membrane fraction between the control and diabetic muscle. We observed consistent, but not significant increases in the cytosolic (~11protein in diabetic muscle (Fig 4) in three independent experiments. Although the cl11 isoform is the most abundant one in muscle, we tested the influence of diabetes on the (~1 isoform as well. There was no change in either a1 mRNA levels or a1 protein in any of the preparations studied (not shown), suggesting that the diabetes-induced alterations are specific for (~11isoform. We then assessed the influence of diabetes on the p subunit of Na’/K’-ATPase (Fig 5). We found no change in p subunit mRNA levels on the 2nd day of diabetes and a significant reduction (P < .OOl) in p mRNA abundance on the 14th day. Effect of Fasting
Fig 1. Nat/K+-ATPase activity in skeletal muscle of diabetic rats. Results are expressed as mean ? SEM. lf c.05 v control; DM, diabetic animals.
true for both the 3.4-kb and 5.3-kb species of message (Fig 3A and B). The increases ranged from 150% to 300%. Treatment of diabetic animals with insulin reduced the levels of Na’/K’-ATPase a11 subunit mRNA toward normal (P < .Ol), whereas therapy with either vanadate or phlorizin (which normalized the levels of glycemia, Table l), did not affect Na’/K’-ATPase (~11mRNA levels (Fig 3A and B). Thus, it appears that it is the presence of insulin and not the correction of hyperglycemia which restores Na+iK’-ATPase activity and genekxpression.
.
CONTROL 1234
DM
* 5
6
7
DM + INS
t 8
9
To further assess the differential influence of insulinemia and glycemia on Na’/K’-ATPase, we examined enzyme activity and gene expression in skeletal muscle of fasted and refed animals. Levels of both glycemia and insulinemia were significantly reduced by 48 hours of fasting (Table 1). Subsequent refeeding for an additional 48 hours led to a partial restoration of the levels of glycemia and insulinemia. which nonetheless remained below normal. There was a significant reduction (P < .05) in skeletal muscle Na’/K’ATPase activity induced by 48 hours of fasting (Fig 6). Refeeding for either 48 or 72 hours restored enzyme activity towards normal. The Na’/K’-ATPase a11 subunit mRNA data are shown for control. fasted, and refed animals (Fig 7A and B). The same two species of RNA (3.4 and 5.3 kb) were identified in experiments with total or polyadenylated messenger RNA. Densitometric analysis of the autoradiograms (Fig 8A and B) showed that a 48-hour fast significantly increased Na’/ K’-ATPase a11 subunit gene expression (P < ,001) whereas
10
11 12
13
c 14
5.3 Kb 384 Kb
Actin
Fig 2. Representative Northern blot (cdl subunit) with total RNA isolated from skeletal muscle of control and diabetic (DM) animals. Each lane represents RNA isolated from an individual animal. DM + Ins, diabetic animals treated with insulin.
NA+/K+-ATPASE ACTIVITY
IA -
AND all GENE EXPRESSION
3.4 kb
1 B - 5.3 kb
Fig 3. Densitometric analysis of autoradiographic signals for (A) 3.4-Lb band and (5) 5.3-kb band of oil subunit of Na+/K+-ATPase. The intensity of signals in control animals is expressed as 100%. lf < .Ol v untreated diabetic rats (control); DM, diabetic animals (untreated for either 2 or 14 days); DM + Ins, insulin-treated rats; DM + Van, vanadete-treated rats; DM + Phl, phlorizin-treated rats.
refeeding restored the levels of mRNA to normal for both species of message. Similar to diabetes, it appears that fasting, another hypoinsulinemic state, is associated with a decrease in Na’/K’-ATPase activity and an increase in its catalytic (~11subunit mRNA levels. DISCUSSION
Two novel observations surfaced during the course of this investigation. First, while on the 2nd day of diabetes only enzyme activity was impaired, 14 days of diabetes or 48
59
hours of fasting decreased skeletal muscle Na’/K’-ATPase activity and increased mRNA levels of its catalytic CUII subunit. Second, these changes occurred as a consequence of hypoinsulinemia rather than being related to levels of glycemia. Both diabetes and fasting, two hypoinsulinemic states with opposite levels of glycemia, induced identical alterations in Na’/K’-ATPase activity and a11 mRNA levels. Moreover, in diabetic animals, only insulin therapy and not correction of hyperglycemia with either vanadate or phlorizin restored gene expression to normal levels. The fact that hypoinsulinemia caused a decrease in Na’/K’-ATPase activity was not unanticipated. From the pioneering work of Clausen et a11.zR-3” and Moore et al:‘“’ it has been well recognized that insulin stimulates Na’/K’ATPase in skeletal muscle. Insulin and catecholamines are the principal mediators of acute hormonal control of Na’/K+-ATPase.” Reduced activity of this enzyme has been found both in diabetes and starvation’.3”.“-‘S and was confirmed in the present study. The precise mechanism whereby insulin stimulates Na’/ K’-ATPase activity remains unclear. It is believed that insulin shifts the affinity of the enzyme for Na’ to lower values in adipocytes and muscle cells. ‘J’J”-~~ Previous studies using [3H]ouabain binding as a measure of the number of Na’/K’ pumps available at the plasma membrane found no effect of insulin on translocation of these pumps to the plasma membrane.‘~‘8~“~35 Moreover, insulin’s effect on Na’/ K’-ATPase was not inhibited by cycloheximide.3h Thus, mobilization of Na’lK’ pumps is not likely to explain the stimulatory effect of insulin on Na’/K*-ATPase activity. I.3”.3? Rosic et al” have suggested that the effect of insulin on the Na’/K’ pump may be indirect, secondary to stimulation of a large increase in Na’/K’ exchange and resultant Na’ influx. However, another study showed that amiloride did not prevent the effect of insulin on Na’/K’ transport in rat soleus muscle, suggesting that intracellular alkalinization is not the major factor mediating insulin’s effect.“’ The mechanisms whereby fasting and diabetes reduce Na’/K’-ATPase activity over an extended time interval may include not only lower insulin levels, but could conceivably involve reduction in thyroid hormone concentrations. Thyroid hormones have been shown to stimulate both Na’/K’-ATPase activity and gene expression.‘.6.3y They appear to be an important hormonal stimulus for the long-term regulation of Na+/K+-ATPase.‘.h.3”.30However, in contrast to the present observations relative to the effects of fasting and diabetes on (~11 isoform, thyroid hormones influence enzyme activity and gene expression in the same direction.6.39 Recent observations of isoform-specific regulation of Na’/K’-ATPase by thyroid status in skeletal muscle40 indicated that hypothyroidism was accompanied by decreased levels of a11 and l3 subunit mRNA. If the observed changes were related to decreased levels of thyroid hormones, one would have expected a decrease in the levels of both a11 and l3 subunit mRNA for the Na’/K’-ATPase. Our findings of a decrease in l3 subunit mRNA levels are consistent with the consequences of lower thyroid hormone concentrations. It is likely that the changes observed in muscle a11 isoform isolated from fasted and
NISHIDA ET AL
60
M --CONTROL-
Fig 4. Western blot of ull subunit of Na+/ K+-ATPase. Lanes 1 to 4 represent cytosolic fractions (100 pg protein per lane) isolated from muscle of 14 day diabetic animals. Lanes 5 to g represent cytosolic fractions from control animals. Lane 10 is an example of the membranous fraction (10 pg protein) from control animals.
cause and effect relationship between insulinemia and Na’/K’-ATPase activity. The changes may be related to a decrease in body weight or muscle mass commonly observed in diabetes and fasting. The precise contribution of these variables must be further evaluated. What is the mechanism that augments cl11gene expression in the presence of reduced pump activity? Brodie and Sampson,“’ Wolitzky and Fambrough,” Pressley,” and other?.“ have demonstrated that Nat/K’-ATPase can be upregulated as a result of increasing demand for sodium transport. They have suggested that the concentrations of intracellular Na’ may function as a driving force to augment Na’/K’-ATPase synthesis. Muscle from both fasted and diabetic animals has been shown to contain increased concentrations of intracellular Na+.” creating a strong demand for its outward transport. Thus, the sequence of
diabetic animals are related to decreased levels of insulin, whereas the changes in 0 subunit mRNA may be related to decreased levels of thyroid hormones. Another important feature of these findings is that cxlI gene expression seems to respond to the changes in the activity of the enzyme rather than being a target of insulin action. Reduced activity of the enzyme was found on the second day of diabetes without any change in the levels of mRNA of either of the subunits. It is evident that hypoinsulinemia has resulted both in decreased Na’/K’-ATPase activity and concomitant enhanced expression of the (rII subunit of the Na’/K’-ATPase gene. Whether these divergent effects represent an independent expression of two metabolic events occurring as the result of hypoinsulinemia or whether these changes are inextricably related remains to be elucidated. The present data do not establish a direct
CONTROL
A 1
2
3
DM + INS
DM 4
5
6
7
a
9
10
11
12
13
Fig 5. Representative Northern blots of total RNA probed with p subunit cDNA (A). The RNA was isolated from M-day diabetic animals (DM) either untreated or treated with insulin (DM + Ins). Densitometric anelysis of autoradiographic signals for fJ subunit (B). lP c.001. The difkamnce between DM-14 d and DM 14 is not significant.
NA’/K+-ATPASE ACTIVITY AND (III GENE EXPRESSION
12
8 6
Fig 6. Na+/K’-ATPase activity in skeletal muscle of control, fasted, and refed animals. Results are expressed as mean + SEM. lp < 66 v fasted.
CQNTROL
61
events in fasting and diabetes may begin with absolute or relative hypoinsulinemia, and proceed to decreased Na+/K’ATPase activity, accumulation of intracellular Na+, and subsequent stimulation of gene expression. The precise mechanism whereby intracellular Na’ increases Na’/K’-ATPase gene expression is unknown. This ion-mediated mechanism of upregulation of Na’ pumps appears to differ from the upregulation of this enzyme occurring with thyroid hormones or aldosterone. Thyroid hormones or aldosterone may increase the rate of transcription in the absence of any change in intracellular Na’ levels.‘3,43Changes in intracellular Na’ concentrations may alter the affinity of regulatory proteins for the binding site on the Na’/K’-ATPase gene. Recently, Barlet-Bas et al“* have demonstrated in short-term experiments (2 to 3 hours) that an increase in intracellular Na’ recruited a latent pool of Na’ pumps to the surface of cortical collecting tubules. This effect was not abolished by either actinomycin D or cycloheximide, suggesting that at least within this time interval, intracellular Na’ does not stimulate the synthesis of Na’/K’-ATPase. It is conceivable that the influence of intracellular Na’ on Na’/K’-ATPase production may be analogous to the influence of iron on ferritin synthesis.44.45 Intracellular iron has been shown to stimulate synthetic rates of ferritin and cellular ferritin levels at the levels of transcription,4”.47 translation4* and assembly.46 Intracellular
FASTED
5.3 Kb 3.4 Kb
B
CONTROL
RE-FED 5.3Kb 3.4 Kb
Fig 7. Representative Northem blots with total RNA (probed with oil cDNA) isolated from skeletal muscle of control, fasted (A), and refed (B) animals. Each lane represents RNA isolated from an individual animal.
62
500
1
NISHIDA ET AL
A
- 3.4 kb
ACKNOWLEDGMENT The authors are greatly indebted to Dr Stewart Metz for his kind assistance with the insulin radioimmunoassay. We also wish to thank Gloria Smith for her excellent secretarial assistance.
400 -
REFERENCES
B -
500
5.3
kb
1 400
1
300 -
200 -
loo-
Fig 8. Densitometric analysis of autoradiographic signals for (A) the X4-kb band and (B) the 5.3-kb band of ofI subunit of Na+/K’ATPase. The intensity of signals in control animals is expressed as 100%. lP < .OO?v control.
Na’ may influence one or all of these steps in promoting the synthesis of Na’/K’-ATPase. Augmented (~11 subunit mRNA levels in diabetes and fasting are unlikely to result in a greater availability of Na’ pumps at the plasma membrane. The amount of a11 protein was not increased sufficiently (at least at 14 days of diabetes), and the levels of l3 subunit appear to be crucial for the enzyme assembly and incorporation into the plasma membrane.’ Studies of diabetes of longer duration as well as the interplay between the levels of insulinemia and thyroid hormones are necessary for better understanding of the transcriptional and translational regulation of Na’/K’ATPase.
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IS: Thyroidal regulation gene expression. J Biol
40. Horowitz B, Hensley B, Quintero M, et al: Differential regulation of Na,K-ATPase cd, &I, and B subunit mRNA and protein levels by thyroid hormones. J Biol Chem 265:14308-14314, 1990 41. Taormino JP, Fambrough DM: Pre-translational regulation of the (Na+ + K+)-ATPase in response to demand for ion transport in cultured chicken skeletal muscle. J Biol Chem 265:41164123,199O 42. Barlet-Bas C, Khadouri C, Marsy S, et al: Enhanced intracellular sodium concentration in kidney cells recruits a latent pool of Na-K-ATPase whose size is modulated by corticosteroids. J Biol Chem 265:7799-7803,199O 43. Barlet-Bas C, Khadouri independent in vitro induction of in renal target cells: Permissive Nat1 Acad Sci USA 85:1707-1711, 44. Drysdale JW: Human Acid Res Mol Biol35:127-155,
C, Marsy S, et al: SodiumNat/K’-ATPase by aldosterone effect of triiodothyronine. Proc 1988
ferritin 1988
gene expression.
45. Theil EC: Regulation of ferritin mRNAs. J Biol Chem 265:4771-4774,199O
and transferrin
Prog Nucl receptor
46. Cairo G, Bardella L, Schiaffonati L, et al: Multiple mechanisms of iron-induced ferritin synthesis in HeLa cells. Biochem Biophys Res Commun 133:314-321,1985 47. White K, Munro HN: Induction of ferritin subunit synthesis by iron is regulated at both transcriptional and translational levels. J Biol Chem 263:8938-8942,198s 48. Aziz N, Munro HN: Both subunits of rat liver ferritin are regulated at translational levels by iron induction. Nucl Acids Res 14:915-927, 1986
T
HE UBIQUITOUS cellular enzyme Na’/K’-adenosine triphosphatase (ATPase) (EC 3.6.1.37) is responsible for the maintenance of intracellular sodium and potassium concentrations.‘~.’ The function of this enzyme is to transport three ions of sodium from the intracellular space to the extracellular environment and, in return, to allow two ions of potassium to enter the cell. Na’/K’-ATPase consists of two structurally different subunits.‘.’ The p subunit (MT -35,000) seems to be important for inserting the Na’/K’-ATPase into the plasma membrane and maintaining its structural integrity.3 The a subunit (Mr - 112,000) is the functionally active part of the enzyme. There are at least three isoforms of the a subunit, designated aI, aII (also known as a+), and aIII.‘.‘,* The a11 subunit, the most prominent isozyme in muscle,‘~’ possesses the greatest sensitivity to ouabain.“’ Its activity has been shown to be regulated by insulin” and certain metabolic changes, such as alterations in concentrations of intracellular sodium’*-” or free fatty acids (FFA).“‘* Whereas much is known concerning the factors modulating Na’/K’-ATPase activity, knowledge of the influence of diabetes and fasting on pretranslational regulation of the enzyme is lacking. The aim of this study is to compare skeletal muscle Na’/K’ATPase activity with gene expression of its catalytic a11 subunit under conditions known to influence Na’/K’ATPase activity, such as diabetes, fasting, and refeeding. MATERIALS
AND METHODS
Materials Streptozotocin (STZ) and sodium orthovanadate were purchased from Sigma (St Louis, MO), insulin from Eli Lilly (Indianap-
From the Medical Research Service and Depafiment of Medicine, Veterans Affairs Medical Center and Deparhnent of Medicine, University of Colorado Health Sciences Center, Denver, CO: and the Jichi Medical Schoof, Tochigi Ken, Japan. Supported by the Research Service of the Veterans Affairs and The Diabetes Research Foundation of Colorado. Address reprint requests to Boris Draznin. MD, Section of Endocrinology (IIIH), VAMC, IO.55 Clermont St, Denver. CO 80220. Copyright 0 1992 by W.B. Saunders Company 0026-0495/92/4101-0011$03.00/O 56
olis, IN), and phlorizin from Aldrich (Milwaukee, WI). Deoxycytidine-5’-triphosphate, tetra-triethylammonium salt, [,“P], specific activity 3,000 Ci/mmol, was purchased from ICN Biomedicals (Costa Mesa, CA). cDNA probes for rat Na’/K+-ATPase (~1 and a11 subunit(s) were kindly provided by Drs Shull and Lingrel (Cincinnati, OH), human p subunit cDNA by Dr Kawakami (Tochigi Ken, Japan), mouse p-actin cDNA by Dr Spiegelman (Boston, MA), and human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by Dr Terada (Denver, CO). Monoclonal antibodies for Na’/K’-ATPase a1 (McKl) and a11 (McB2) subunits were a gift from Dr K.J. Sweadner (Boston, MA).
Experimental Design Male Sprague-Dawley rats weighing 175 to 230 g were used in these experiments. Animals (eight to 10 per group) were randomly allocated into one of the following groups: 48-hour fasting (water ad libitum), 48-hour fasting followed by 48- or 72-hour refeeding, and diabetic animals. At least three independent experiments were performed with each group. Diabetes was induced by intravenous injection of STZ (65 mgikg body wieght [SW] dissolved in 0.01 mol/L citrate buffer, pH 4.5). Diabetic animals were further subdivided into four groups: untreated for 14 days, and treated for 7 days with either insulin (daily subcutaneous injections of 3 to 6 U NPH), vanadate (sodium orthovanadate 0.2 mg/mL in drinking water),“.‘” or phlorizin (0.4 g/kg BW/d prepared as a 40% solution in propylene glycol and injected subcutaneously every 8 hours to ensure continuous inhibition of renal tubular reabsorption of glucose).” The control group of animals was allowed food and water ad libitum. Control animals compared with the diabetic ones were injected with citrate buffer instead of STZ. All animals were killed by decapitation and skeletal muscle was removed for either RNA extraction or measurements of Na’/K’-ATPase activity. Blood was collected for glucose (Beckman glucose analyzer, Beckman Instruments, Fullerton, CA) and insulin determinations by radioimmunoassay.
RNA Extraction and Northern Blot Analysis Total cellular RNA was purified by guanidinium isothiocyanate method” from freshly isolated skeletal muscle (abdominal muscles in diabetes experiments and hind leg muscles in fasting experiments). Twenty micrograms of denatured total cellular RNA was electrophoresed in 1.4% agarose and 2.7% formaldehyde gels and blotted onto a nitrocellulose membrane and cross-linked by baking at 80°C for 2 hours in a vacuum oven. DNA probes for Na’/K’ATPase al, ~11, and l3 subunits, @-actin, and GAPDH were labeled Metabolism, Vol41,
No 1 (January),
1992: pp 56-63
57
NA+/K+-ATPASE ACTIVITY AND all GENE EXPRESSION
with [‘*P]dCTP by the random-primer method or nick translation using Boehringer-Mannheim (Indianapolis, IN) labeling kits. Hybridization was performed in 0.007 mol/L Tris-HCL, pH 8.0, 10% dextran sulfate, 40% deionized formamide, 4x SSC (1 x SSC = 0.15 mol/L NaCl + 0.015 mol/L sodium citrate), 0.8 x Denhardt’s solution, 20 ug/mL denatured salmon-sperm DNA, and 32Plabeled cDNA for approximately 14 to 18 hours at 42°C. The hybridized blots were washed four times in 2x SSC and 0.1% sodium dodecyl sulfate (SDS) for 5 minutes at room temperature and subsequently twice in 0.5~ SSC and 0.1% SDS for 30 minutes at 55°C. Autoradiographic exposures of the blots to Kodak X-O mat AR film (Eastman Kodak, Rochester, NY) were performed with two intensifying screens at -70°C. The levels of RNA transcripts were measured by scanning autoradiograms vertically with a video densitometer (model 620, Bio-Rad, Richmond, CA). Relative shifts in gene transcript concentrations were quantified by obtaining a ratio of the densitometric area of each peak of Na’/K’-ATPase gene transcript compared with area of each p-actin peak and GAPDH peak. Western Blot Analysis Skeletal muscle (0.5 to 1.0 g) was homogenized in 2.5 mL of buffer (5 mmol/L EGTA, 15 mmol/L Tris, 1 mmol/L phenylmeth-
ylsulfonyl flouride [PMSF], 1 pg/mL each of leupeptin, pepstain A, antipain, and soybean trypsin inhibitor [STI]) using Tissumizer (Tekmar, Cincinnati, OH). Homogenate was centrifuged at 8,000 x g
for 20 minutes at 4°C to discard all debris, nuclei, and mitochondria. Supernatant was further centrifuged at 100,OOQx g for 60 minutes at 4°C. The pellet dissolved in 200 uL of the above buffer was used as the crude cell membrane fraction. The supernatant represented the cytosolic fraction. Either 10 pg of protein of a membrane fraction or 100 ug of protein of a cytosolic fraction were applied to 7.5% polyacrylamide SDS gel electrophoresis, without heating of the samples,23,” and blotted onto a nitrocellulose membrane. The blots were blocked with 5% dried milk in TBS-T (20 mmol/L Tris, 137 mmol/L NaCl, pH 7.6,0.1% Tween 20) overnight. The blots were then incubated with an antibody (McKl was diluted in l:lOO, McB2 in 1:500) for 1 hour, washed, incubated with biotinylated anti-mouse second antibody for 20 minutes, and washed again. The biotinylated second antibody was detected by streptavidin alkaline phosphatase conjugate, nitro-blue tetrazolium, and 5-bromo-4-chloro-3-indoyly phosphate (Amersham Blotting detection kit, Arlington Heights, IL).
Na’JK’-ATPase
Activity
Skeletal muscle (0.5 to 1.0 g) was homogenized in 5 mL of ice cold buffer (300 mmol/L o-mannitol, 5 mmol/L EGTA, 15 mmol/L Tris, pH 7.4) using Tissumizer. Homogenate was then centrifuged at 8,000 x g for 20 minutes at 4°C. Supernatant was further centrifuged at 100,000 x g for 60 minutes at 4°C to pellet the crude preparation of cell membrane fraction. The pellet was dissolved in 200 (LL of buffer (5 mmol/L MgCl,, 100 mmol/L NH,Cl, 100 mmol/L imidazole, 150 mmol/L NaCl, 1 mmol/L EGTA, and 2 mmol/L NaN,, pH 7.3) and left overnight at 4°C. The measurements of the ouabain-sensitive Na’/K’-ATPase activity were performed as described by Simon et a1.25.26 Results are expressed as micromole NADH converted to NAD per milligram of protein per hour.
RESULTS
Effect of Diabetes The levels of glycemia and insulinemia in control and experimental animals are shown in Table 1. Untreated diabetic animals were overtly hyperglycemic and profoundly hypoinsulinemic (P < .OOl). All three modes of therapy administered for 7 days (a single daily injection of insulin, oral vanadate, or three daily injections of phlorizin) normalized blood glucose levels. Vanadate in drinking water (0.2 mg/mL) appeared to be the most consistent in lowering blood glucose levels, but differences between the treated groups were not significant. Diabetic animals treated with vanadate or phlorizin became normoglycemic, but remained hypoinsulinemic. This experimental protocol allows for differentiation of the role of hypoinsulinemia from that of hyperglycemia. Skeletal muscle Na’/K+-ATPase activity was significantly decreased on the 2nd day of diabetes (3.05 * 0.14 v 6.05 ? 0.44 pmol NADH/mg protein/h in controls, P < .Ol) and on the 14th day (4.20 + 0.8 Fmol NADH/mg protein/h, P < .05). The difference between the two diabetes groups was not significant (Fig 1). Insulin therapy partially restored the enzyme activity, which remained lower than in control rats (4.60 * 0.7 pmol NADH/mg protein/h). We then examined the effect of diabetes on Na’/K’ATPase a11 subunit mRNA levels. Two species of RNA were detected by hybridization with a cDNA clone: 3.4 and 5.3 kb (Fig 2). In contrast to the decreased Na+/K’-ATPase activity, we observed an increase in the autoradiographic signal for the Na+/K’-ATPase in the lanes with RNA isolated from diabetic animals (Fig 2). The results were quantitated by calculating the ratios of Na’/K’-ATPase a11 densitometric signals to those of either p-actin or GAPDH. Since B-actin mRNA has been shown to be influenced by fasting,” we used GAPDH as an additional control. In our hands, the ratios obtained with both internal controls were essentially identical. When RNA autoradiography was analyzed by densitometry and expressed as a percent of RNA abundance relative to control, the following data were obtained. The control levels of each species of RNA are expressed as 100% (Fig 3A and B). Despite the early and significant reduction in enzyme activity (Fig l), the levels of crI1 mRNA remained unchanged on the 2nd day of diabetes. After 14 days of diabetes, there was a dramatic increase (3.4-kb band, P < .OOl; 5.3-kb band, P < .Ol) in the abundance of Na’/K’-ATPase CXIIsubunit mRNA. These findings were
Table 1. Characteristics of Control and Experimental Animals
”
GlUCOSe
Insulin
hWdL)
WJlmL)
Controls
7
232 + 15
147 + 21
24.8 * 8.3
ST.?!-diabetic
6
185 * 14
454 + 25
8.2 t 6.0
Insulin-treated
5
222 *a
219 -c56
Vanadate-treated
7
188 2 7
74* 22
Statistical Analysis
Phlorizin-treated
6
194 * 9
160 + 52
9.6 2 2.3 9.0 2 1.9
The Student’s unpaired t test was used to compare the mean + SEM values among the groups.
Fasted
6
183 +3
81 r 9
8.3 2 1.4
Refed
6
211+4
97 + 5
13.6k 1.2
NISHIDA ET AL
*
-
To determine whether the increases in mRNA abundance were translated into more (~11protein being present, we used immunoblotting (Western blot) with a monoclonal (~11antibody, which was kindly supplied by Dr K. Sweadner. Both plasma membrane and cytosolic fractions were tested. There was no difference in the amount of ~11 protein in the plasma membrane fraction between the control and diabetic muscle. We observed consistent, but not significant increases in the cytosolic (~11protein in diabetic muscle (Fig 4) in three independent experiments. Although the cl11 isoform is the most abundant one in muscle, we tested the influence of diabetes on the (~1 isoform as well. There was no change in either a1 mRNA levels or a1 protein in any of the preparations studied (not shown), suggesting that the diabetes-induced alterations are specific for (~11isoform. We then assessed the influence of diabetes on the p subunit of Na’/K’-ATPase (Fig 5). We found no change in p subunit mRNA levels on the 2nd day of diabetes and a significant reduction (P < .OOl) in p mRNA abundance on the 14th day. Effect of Fasting
Fig 1. Nat/K+-ATPase activity in skeletal muscle of diabetic rats. Results are expressed as mean ? SEM. lf c.05 v control; DM, diabetic animals.
true for both the 3.4-kb and 5.3-kb species of message (Fig 3A and B). The increases ranged from 150% to 300%. Treatment of diabetic animals with insulin reduced the levels of Na’/K’-ATPase a11 subunit mRNA toward normal (P < .Ol), whereas therapy with either vanadate or phlorizin (which normalized the levels of glycemia, Table l), did not affect Na’/K’-ATPase (~11mRNA levels (Fig 3A and B). Thus, it appears that it is the presence of insulin and not the correction of hyperglycemia which restores Na+iK’-ATPase activity and genekxpression.
.
CONTROL 1234
DM
* 5
6
7
DM + INS
t 8
9
To further assess the differential influence of insulinemia and glycemia on Na’/K’-ATPase, we examined enzyme activity and gene expression in skeletal muscle of fasted and refed animals. Levels of both glycemia and insulinemia were significantly reduced by 48 hours of fasting (Table 1). Subsequent refeeding for an additional 48 hours led to a partial restoration of the levels of glycemia and insulinemia. which nonetheless remained below normal. There was a significant reduction (P < .05) in skeletal muscle Na’/K’ATPase activity induced by 48 hours of fasting (Fig 6). Refeeding for either 48 or 72 hours restored enzyme activity towards normal. The Na’/K’-ATPase a11 subunit mRNA data are shown for control. fasted, and refed animals (Fig 7A and B). The same two species of RNA (3.4 and 5.3 kb) were identified in experiments with total or polyadenylated messenger RNA. Densitometric analysis of the autoradiograms (Fig 8A and B) showed that a 48-hour fast significantly increased Na’/ K’-ATPase a11 subunit gene expression (P < ,001) whereas
10
11 12
13
c 14
5.3 Kb 384 Kb
Actin
Fig 2. Representative Northern blot (cdl subunit) with total RNA isolated from skeletal muscle of control and diabetic (DM) animals. Each lane represents RNA isolated from an individual animal. DM + Ins, diabetic animals treated with insulin.
NA+/K+-ATPASE ACTIVITY
IA -
AND all GENE EXPRESSION
3.4 kb
1 B - 5.3 kb
Fig 3. Densitometric analysis of autoradiographic signals for (A) 3.4-Lb band and (5) 5.3-kb band of oil subunit of Na+/K+-ATPase. The intensity of signals in control animals is expressed as 100%. lf < .Ol v untreated diabetic rats (control); DM, diabetic animals (untreated for either 2 or 14 days); DM + Ins, insulin-treated rats; DM + Van, vanadete-treated rats; DM + Phl, phlorizin-treated rats.
refeeding restored the levels of mRNA to normal for both species of message. Similar to diabetes, it appears that fasting, another hypoinsulinemic state, is associated with a decrease in Na’/K’-ATPase activity and an increase in its catalytic (~11subunit mRNA levels. DISCUSSION
Two novel observations surfaced during the course of this investigation. First, while on the 2nd day of diabetes only enzyme activity was impaired, 14 days of diabetes or 48
59
hours of fasting decreased skeletal muscle Na’/K’-ATPase activity and increased mRNA levels of its catalytic CUII subunit. Second, these changes occurred as a consequence of hypoinsulinemia rather than being related to levels of glycemia. Both diabetes and fasting, two hypoinsulinemic states with opposite levels of glycemia, induced identical alterations in Na’/K’-ATPase activity and a11 mRNA levels. Moreover, in diabetic animals, only insulin therapy and not correction of hyperglycemia with either vanadate or phlorizin restored gene expression to normal levels. The fact that hypoinsulinemia caused a decrease in Na’/K’-ATPase activity was not unanticipated. From the pioneering work of Clausen et a11.zR-3” and Moore et al:‘“’ it has been well recognized that insulin stimulates Na’/K’ATPase in skeletal muscle. Insulin and catecholamines are the principal mediators of acute hormonal control of Na’/K+-ATPase.” Reduced activity of this enzyme has been found both in diabetes and starvation’.3”.“-‘S and was confirmed in the present study. The precise mechanism whereby insulin stimulates Na’/ K’-ATPase activity remains unclear. It is believed that insulin shifts the affinity of the enzyme for Na’ to lower values in adipocytes and muscle cells. ‘J’J”-~~ Previous studies using [3H]ouabain binding as a measure of the number of Na’/K’ pumps available at the plasma membrane found no effect of insulin on translocation of these pumps to the plasma membrane.‘~‘8~“~35 Moreover, insulin’s effect on Na’/ K’-ATPase was not inhibited by cycloheximide.3h Thus, mobilization of Na’lK’ pumps is not likely to explain the stimulatory effect of insulin on Na’/K*-ATPase activity. I.3”.3? Rosic et al” have suggested that the effect of insulin on the Na’/K’ pump may be indirect, secondary to stimulation of a large increase in Na’/K’ exchange and resultant Na’ influx. However, another study showed that amiloride did not prevent the effect of insulin on Na’/K’ transport in rat soleus muscle, suggesting that intracellular alkalinization is not the major factor mediating insulin’s effect.“’ The mechanisms whereby fasting and diabetes reduce Na’/K’-ATPase activity over an extended time interval may include not only lower insulin levels, but could conceivably involve reduction in thyroid hormone concentrations. Thyroid hormones have been shown to stimulate both Na’/K’-ATPase activity and gene expression.‘.6.3y They appear to be an important hormonal stimulus for the long-term regulation of Na+/K+-ATPase.‘.h.3”.30However, in contrast to the present observations relative to the effects of fasting and diabetes on (~11 isoform, thyroid hormones influence enzyme activity and gene expression in the same direction.6.39 Recent observations of isoform-specific regulation of Na’/K’-ATPase by thyroid status in skeletal muscle40 indicated that hypothyroidism was accompanied by decreased levels of a11 and l3 subunit mRNA. If the observed changes were related to decreased levels of thyroid hormones, one would have expected a decrease in the levels of both a11 and l3 subunit mRNA for the Na’/K’-ATPase. Our findings of a decrease in l3 subunit mRNA levels are consistent with the consequences of lower thyroid hormone concentrations. It is likely that the changes observed in muscle a11 isoform isolated from fasted and
NISHIDA ET AL
60
M --CONTROL-
Fig 4. Western blot of ull subunit of Na+/ K+-ATPase. Lanes 1 to 4 represent cytosolic fractions (100 pg protein per lane) isolated from muscle of 14 day diabetic animals. Lanes 5 to g represent cytosolic fractions from control animals. Lane 10 is an example of the membranous fraction (10 pg protein) from control animals.
cause and effect relationship between insulinemia and Na’/K’-ATPase activity. The changes may be related to a decrease in body weight or muscle mass commonly observed in diabetes and fasting. The precise contribution of these variables must be further evaluated. What is the mechanism that augments cl11gene expression in the presence of reduced pump activity? Brodie and Sampson,“’ Wolitzky and Fambrough,” Pressley,” and other?.“ have demonstrated that Nat/K’-ATPase can be upregulated as a result of increasing demand for sodium transport. They have suggested that the concentrations of intracellular Na’ may function as a driving force to augment Na’/K’-ATPase synthesis. Muscle from both fasted and diabetic animals has been shown to contain increased concentrations of intracellular Na+.” creating a strong demand for its outward transport. Thus, the sequence of
diabetic animals are related to decreased levels of insulin, whereas the changes in 0 subunit mRNA may be related to decreased levels of thyroid hormones. Another important feature of these findings is that cxlI gene expression seems to respond to the changes in the activity of the enzyme rather than being a target of insulin action. Reduced activity of the enzyme was found on the second day of diabetes without any change in the levels of mRNA of either of the subunits. It is evident that hypoinsulinemia has resulted both in decreased Na’/K’-ATPase activity and concomitant enhanced expression of the (rII subunit of the Na’/K’-ATPase gene. Whether these divergent effects represent an independent expression of two metabolic events occurring as the result of hypoinsulinemia or whether these changes are inextricably related remains to be elucidated. The present data do not establish a direct
CONTROL
A 1
2
3
DM + INS
DM 4
5
6
7
a
9
10
11
12
13
Fig 5. Representative Northern blots of total RNA probed with p subunit cDNA (A). The RNA was isolated from M-day diabetic animals (DM) either untreated or treated with insulin (DM + Ins). Densitometric anelysis of autoradiographic signals for fJ subunit (B). lP c.001. The difkamnce between DM-14 d and DM 14 is not significant.
NA’/K+-ATPASE ACTIVITY AND (III GENE EXPRESSION
12
8 6
Fig 6. Na+/K’-ATPase activity in skeletal muscle of control, fasted, and refed animals. Results are expressed as mean + SEM. lp < 66 v fasted.
CQNTROL
61
events in fasting and diabetes may begin with absolute or relative hypoinsulinemia, and proceed to decreased Na+/K’ATPase activity, accumulation of intracellular Na+, and subsequent stimulation of gene expression. The precise mechanism whereby intracellular Na’ increases Na’/K’-ATPase gene expression is unknown. This ion-mediated mechanism of upregulation of Na’ pumps appears to differ from the upregulation of this enzyme occurring with thyroid hormones or aldosterone. Thyroid hormones or aldosterone may increase the rate of transcription in the absence of any change in intracellular Na’ levels.‘3,43Changes in intracellular Na’ concentrations may alter the affinity of regulatory proteins for the binding site on the Na’/K’-ATPase gene. Recently, Barlet-Bas et al“* have demonstrated in short-term experiments (2 to 3 hours) that an increase in intracellular Na’ recruited a latent pool of Na’ pumps to the surface of cortical collecting tubules. This effect was not abolished by either actinomycin D or cycloheximide, suggesting that at least within this time interval, intracellular Na’ does not stimulate the synthesis of Na’/K’-ATPase. It is conceivable that the influence of intracellular Na’ on Na’/K’-ATPase production may be analogous to the influence of iron on ferritin synthesis.44.45 Intracellular iron has been shown to stimulate synthetic rates of ferritin and cellular ferritin levels at the levels of transcription,4”.47 translation4* and assembly.46 Intracellular
FASTED
5.3 Kb 3.4 Kb
B
CONTROL
RE-FED 5.3Kb 3.4 Kb
Fig 7. Representative Northem blots with total RNA (probed with oil cDNA) isolated from skeletal muscle of control, fasted (A), and refed (B) animals. Each lane represents RNA isolated from an individual animal.
62
500
1
NISHIDA ET AL
A
- 3.4 kb
ACKNOWLEDGMENT The authors are greatly indebted to Dr Stewart Metz for his kind assistance with the insulin radioimmunoassay. We also wish to thank Gloria Smith for her excellent secretarial assistance.
400 -
REFERENCES
B -
500
5.3
kb
1 400
1
300 -
200 -
loo-
Fig 8. Densitometric analysis of autoradiographic signals for (A) the X4-kb band and (B) the 5.3-kb band of ofI subunit of Na+/K’ATPase. The intensity of signals in control animals is expressed as 100%. lP < .OO?v control.
Na’ may influence one or all of these steps in promoting the synthesis of Na’/K’-ATPase. Augmented (~11 subunit mRNA levels in diabetes and fasting are unlikely to result in a greater availability of Na’ pumps at the plasma membrane. The amount of a11 protein was not increased sufficiently (at least at 14 days of diabetes), and the levels of l3 subunit appear to be crucial for the enzyme assembly and incorporation into the plasma membrane.’ Studies of diabetes of longer duration as well as the interplay between the levels of insulinemia and thyroid hormones are necessary for better understanding of the transcriptional and translational regulation of Na’/K’ATPase.
I. Clausen T: Regulation of active Na*/K’ transport in skeletal muscle. Physiol Rev 66542-580, 1986 2. Sweadner KJ: Isozymes of the Na+/K’-ATPase. Biochim Biophys Acta 988:185-220, 1989 3. McDonough AA, Geering K, Farley RA: The sodium pump needs its B subunit. FASEB J 4: 1598-1605. 1990 4. Shull GE, Greeb J, Lingrel JB: Molecular cloning of three distinct forms of the Nat/K’-ATPase o-subunit from rat brain. Biochemistry 25:8125-8132. 1986 5. Emanuel JR, Garetz S, Stone L, et al: Differential expression of Na’/K’-ATPase CX-and B-subunit mRNAs in rat tissues and cell lines. Proc Natl Acad Sci USA 84:9030-9034, 1987 6. McDonough AA, Brown TA, Horowitz B. et al: Thyroid hormone coordinately regulates Na+/K’-ATPase cy- and B-subunit mRNA levels in kidney. Am J Physiol 254:C323-C329, 1988 7. Shull MM, Lingrel JB: Multiple genes encode the human Na‘ /K’-ATPase catalytic subunit. Proc Nat1 Acad Sci USA 84:40394043. 1987 8. Shull MM, Pugh DC. Lingrel JB: Characterization of the human Na,K-ATPase (~2 gene and identification of intragenic restrict ion fragment length polymorphisms. J Biol Chem 264:1753217543.1989 9. Young RM, Lingrel JB: Tissue distribution of mRNAs encoding the a isoforms and B subunit of rat Na+,K*-ATPase. Biochem Biophys Res Commun 145:52-58, 1987 10. Sweadner KJ: Two molecular forms of (Na’ + K’)-stimulated ATPase in brain. J Biol Chem 254:6060-6067. 1979 Il. Lytton J: Insulin affects the sodium affinity of the rat adipocyte (Na’ + K’)-ATPase. J Biol Chem 260:10075-10080. 1985 12. Wolitzky BA. Fambrough DM: Regulation of the (Na’ + K’)-ATPase in cultured chick. J Biol Chem 261:9990-9995). 1986 13. Pressley TA: Ion concentration-dependent regulation of Na,K-pump abundance. J Membr Biol 105:187-195. 1988 14. Brodie C, Sampson SR: Regulation of the sodium-potassium pump in cultured rat skeletal myotubes by intracellular sodium ions. J Cell Physiol 140:131-137. 1989 15. Owens K, Kennett FF, Weglicki WB: Effects of fatty acid intermediates on Na+-K’-ATPase activity of cardiac sarcolemma. Am J Physiol242:H456-H461, 1982 16. Ng LL, Hockaday TDR: Non-esterified fatty acids may regulate human leucocyte sodium pump activity. Clin Sci 71:737742, 1986 17. Huang W-H, Wang Y, Askari A: Mechanism of the control of (Nat + K’)-ATPase by long-chain Acyl coenzyme A. J Biol Chem 264:2605-2608,1989 18. Oishi K, Zheng B, Kuo JF: Inhibition of Na,K-ATPase and sodium pump by protein kinase C regulators sphingosine, lysophosphatidylcholine, and oleic acid. J Biol Chem 265:70-75. 1990 19. Meyerovitch J, Farfel 2, Sack J, et al: Oral administration of vanadate normalizes blood glucose levels in streptozotocin-treated rats. J Biol Chem 262:6658-6662, 1987 20. Shechter Y: Insulin-mimetic effects of vanadate: Possible implications for future treatment of diabetes. Diabetes 39:1-5, 1990 21. Rossetti L, Smith D, Schulman Gi, et al: Correction of hyperglycemia with phlorizin normalizes tissue sensitivity to insulin in diabetic rats. J Clin Invest 79:1510-1515, 1987
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