Short-term K+ deprivation provokes insulin resistance of cellular K+ uptake revealed with the K+ clamp

Cheol S. Choi, Curtis B. Thompson, Patrick K. K. Leong, Alicia A. McDonough, and Jang H. Youn

Department of Physiology and Biophysics, University of Southern California Keck School of Medicine, Los Angeles, California 90089-9142


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

We aimed to test the feasibility of quantifying insulin action on cellular K+ uptake in vivo in the conscious rat by measuring the exogenous K+ infusion rate needed to maintain constant plasma K+ concentration ([K+]) during insulin infusion. In this "K+ clamp" the K+ infusion rate required to clamp plasma [K+] is a measure of insulin action to increase net plasma K+ disappearance. K+ infusion rate required to clamp plasma [K+] was insulin dose dependent. Renal K+ excretion was not significantly affected by insulin at a physiological concentration (~90 µU/ml, P > 0.05), indicating that most of insulin-mediated plasma K+ disappearance was due to K+ uptake by extrarenal tissues. In rats deprived of K+ for 2 days, plasma [K+] fell from 4.2 to 3.8 mM, insulin-mediated plasma glucose clearance was normal, but insulin-mediated plasma K+ disappearance decreased to 20% of control, even though there was no change in muscle Na-K-ATPase activity or expression, which is believed to be the main K+ uptake route. After 10 days K+ deprivation, plasma [K+] fell to 2.9 mM, insulin-mediated K+ disappearance decreased to 6% of control (glucose clearance normal), and there were 50% decreases in Na-K-ATPase activity and alpha 2-subunit levels. In conclusion, the present study proves the feasibility of the K+ clamp technique and demonstrates that short-term K+ deprivation leads to a near complete insulin resistance of cellular K+ uptake that precedes changes in muscle sodium pump expression.

skeletal muscle; Na-K-ATPase isoforms; hypokalemia


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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EXTRACELLULAR K+ level is tightly regulated in mammals (5, 13). This is critical for normal neural and cardiac functions because cellular K+ gradient is a major determinant of the membrane potential. The maintenance of K+ balance is dependent on kidneys and muscles working in concert (16). The kidneys have a remarkable capacity to actively secrete or reabsorb K+ to match K+ excretion to K+ intake (13, 24). However, the adaptation is slow; during the first 4-6 h after an acute K+ load, only about one-half of the K+ load appears in the urine (5, 10). Thus extrarenal tissues, especially muscle, have a significant role in the acute regulation of extracellular K+. After a meal, insulin stimulates cellular K+ uptake, at least in part mediated by recruitment of sodium pumps (Na-K-ATPase), and during exercise catecholamines (beta -adrenergic agonists) stimulate cellular K+ uptake, mediated by activation of sodium pump activity (7, 11).

Although insulin action on glucose metabolism has been studied extensively, relatively less attention has been given to the regulation of insulin action on cellular K+ uptake in vivo. Insulin action on K+ fluxes in vivo has been characterized mainly by observing decreases in plasma K+ levels (1, 3, 8) or measuring K+ uptake by specific organs, e.g., splanchnic bed (9), during insulin infusion. With these approaches plasma K+ levels fall during insulin infusion, which may affect the K+ influx and efflux rates and confound the measurements.

During dietary K+ deprivation K+ excretion does not fall to zero, leading to a progressive loss of extracellular K+ that is in part buffered by a shift in K+ from intracellular stores to the extracellular fluid (ECF) (7, 11). Because the ECF contains <5% of the body K+ stores, while >75% is stored in muscle intracellular fluid (ICF), a small percentage shift from muscle ICF will have a significant impact on extracellular K+ concentration ([K+]). The net shift of K+ from muscle ICF to ECF fluid during K+ deprivation could be mediated by either an increased K+ efflux rate or a decreased K+ influx rate. K+ deprivation in rats has been shown to be associated with a >50% decrease in the number of sodium pumps in various rat muscles, estimated with [3H]ouabain binding, a similar decrease in enzymatic activity, and a decrease in K+ influx into isolated soleus (by 86Rb uptake) (7). In addition, we have established that K+ deprivation provokes a decrease in the sodium pump alpha 2-catalytic isoform abundance in skeletal muscle, while the alpha 1 isoform is unaffected (26, 27). These findings all support the hypothesis that a decrease in active K+ uptake by Na-K-ATPase mediates the transfer of K+ from muscle ICF to ECF (7, 26, 27). To bridge the gap between the whole animal studies of [K+] in ICF and ECF and the cellular and molecular studies of Na-K-ATPase isoform expression, it is necessary to have an in vivo measure of K+ transport between the ICF and ECF compartments.

In the present study we exploited the theory behind the "glucose clamp" technique (2), which is used extensively to measure insulin-stimulated glucose uptake in vivo, to develop a K+ clamp to assess insulin-stimulated net cellular K+ uptake. The results indicate that plasma K+ could be clamped during insulin infusion in conscious rats by exogenous K+ infusion and that K+ infusion rates required to clamp plasma K+ were insulin dose dependent. In addition, using this novel method, we demonstrate that in rats on a K+ restricted diet for only 2 days, insulin-stimulated cellular K+ uptake was severely reduced, preceding a significant decrease in Na-K-ATPase expression, whereas insulin-stimulated glucose uptake was normal. Thus the implementation of the K+ clamp revealed a dissociation between insulin sensitivity of glucose vs. K+ uptake during early K+ deprivation.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
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Animals. Male Sprague-Dawley rats, ~8 wk old, were obtained from Simonsen Laboratories (Gilroy, CA). Animals were housed under controlled temperature (22 ± 2°C) and lighting (12:12-h light-dark cycle, 0600-1800 and 1800-0600 h) with free access to water and regular rat chow. For studies of the effects of K+ deprivation, animals were initially fed with a control diet (TD 88238; Harlan Teklad, Madison, WI) for 5 days and subsequently fed with a K+-deficient diet (TD 88239; Harlan Teklad) for 0 (control), 2, or 10 days.

Catheterization. At least 4 days before experiments, animals were placed in individual cages with wire floors. The distal one-third of each rat's tail was drawn through a hole placed low on the side of the cage and secured there with a rubber stopper. This arrangement was required to protect tail blood vessel catheters during experiments (15, 29, 30). Animals were free to move about and allowed unrestricted access to food and water. Two tail vein infusion catheters were inserted the day before the experiment, and a one tail artery blood sampling catheter was inserted 6 h before the start of experiments (i.e., ~0700 h). Catheters were placed percutaneously during local anesthesia with lidocaine while rats were restrained in a towel. Animals were returned to their cages after catheter placement with tails secured as previously described and free to move about during the experiments. Patency of the arterial catheter was maintained by a slow (0.016 ml/min) infusion of heparinized saline (10 U/ml).

Experimental protocols. Food was removed from the cage at ~0700. Around 1300, basal blood samples were taken from the tail artery. Experiments began with a constant infusion of saline or porcine insulin (5 or 50 mU · kg-1 · min-1; Novo Nordisk, Princeton, NJ) through a tail vein catheter for 2 or 2.5 h. Insulin infusate was prepared in saline, and the saline loads provided to the saline- and insulin-infused groups were equivalent. During the infusions, blood samples (60 µl) were collected at 10-min intervals for the immediate measurement of plasma glucose and K+ concentrations. Unless indicated otherwise, dextrose (20%) and KCl (150 mM) solutions were separately infused (via a Y connector) through a tail vein catheter at variable rates to maintain plasma glucose and K+ at basal levels (simultaneous glucose and K+ clamp). In some experiments, [3-3H]glucose (HPLC purified; DuPont, Boston, MA) was infused at a rate of 0.2 µCi/min throughout the clamp for the estimation of insulin-stimulated whole body glucose fluxes. In such experiments, blood samples (60 µl) for measurements of plasma [3H]glucose concentrations were taken every 10 min during the final 30 min of the clamp.

At the end of the clamp, animals were anesthetized with pentobarbital sodium and skeletal muscles were rapidly dissected out, frozen using liquid N2-cooled aluminum blocks, and stored at -70°C for later analysis. Urine was collected from the urinary bladder as well as the floor of the cage, as previously described (30). To avoid the contamination of urine passed during the experiments by fecal K+, a mesh screen was placed underneath the wire floor to separate feces from urine passed. The urine passed before the experiment was collected and removed by paper towels placed on the mesh screen. However, because the urinary bladder was not emptied at the beginning of the experiment, the urine in the bladder was included in the final urine collection. Although this would overestimate urinary K+ excretion during the experiment, the error appears to be small, because the amount of K+ in the bladder is typically ~10% of the total amount collected at the end of the experiment. Animals were killed by an overdose of pentobarbital sodium immediately after muscles and urine were collected.

Analysis. Plasma glucose was analyzed during the clamps using 10 µl plasma by a glucose oxidase method on a Beckman glucose analyzer II (Beckman, Fullerton, CA). Plasma and urine K+ levels were determined by flame photometry using a Radiometer FLM 3 as previously reported (26). Plasma insulin levels during the clamps were measured by radioimmunoassay using a Porcine Insulin RIA kit from Linco Research (St. Charles, MO). For the determination of plasma [3H]glucose, plasma was deproteinized with ZnSO4 and Ba(OH)2, dried to remove 3H2O, resuspended in water, and counted in scintillation fluid (Ready Safe; Beckman, Fullerton, CA).

Na-K-ATPase alpha -subunit immunoreactivity was determined as previously described (27). In brief, frozen skeletal muscles [tibialis anterior (TA) and extensor digitorum longus (EDL)] were defrosted and homogenized with a Polytron homogenizer 1:20 (wt/vol) in 5% sorbitol, 25 mM histidine-imidazole (pH 7.4), 0.5 mM Na2EDTA, and proteolytic enzyme inhibitors: 0.5 mM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, and 1 mM 4-aminobenzamidine dichloride. Protein concentration was determined by the method of Lowry et al. (20) after trichloroacetic acid precipitation. Homogenate protein was resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and blotted onto Immobilon-P membranes (Millipore, Bedford, MA). Blots were incubated overnight with either anti-alpha 1-C464.6 (1:100 dilution, provided by M. Kashgarian, Yale Univ.) or anti-alpha 2-McB2 (1:100, provided by K. Sweadner, Harvard Univ.); antibody-antigen complexes were detected with 125I-protein A, quantitated by scanning densitometry, and linearity was verified by assaying samples at both 50 and 100 µg protein/lane on the same blot.

Na-K-ATPase activity. TA muscles were homogenized using a procedure specific for the measurement of Na-K-ATPase activity in muscle (4), distinct from that used for the immunoblot assay. In brief, whole TA muscle was dissected, weighed, and minced with scissors in ice-cold homogenization medium (250 mM mannitol, 30 mM L-histidine, 5 mM Tris-EDTA, and 0.1% sodium deoxycholate; brought to pH 6.8 with NaOH at room temperature). The minced suspension was homogenized [with Ultra Turrax T25 (IKA-Labortechnik) at 22,000 rpm] for 15 s twice before it was transferred to a glass homogenizer tube and further homogenized for six full strokes with a motor-driven Teflon pestle (Dyna-Mix, Fisher Scientific at setting 5). The homogenate was centrifuged at 3,000 g for 20 min (at 4°C) and frozen at -80°C overnight. The next day the Na-K-ATPase activity in the supernatant was measured as the ouabain-sensitive ATPase activity according to the method of Lo et al. (19). Homogenate was preincubated ±2 mM ouabain for 30 min, and reaction was started with the addition of ATP. Na-K-ATPase activity was calculated as the difference in Pi generated over 15 min in the absence (total activity) and presence (Mg-ATPase activity) of 2 mM ouabain and expressed as micromoles Pi liberated per milligram protein per hour.

Calculations and statistical analysis. Whole body glucose uptake was determined as the ratio of the [3H]glucose infusion rate (dpm/min) to the specific activity of plasma glucose (dpm/µmol) during the final 30 min of the clamps. Data are expressed as means ± SE. The significance of the differences in mean values between groups was evaluated using the unpaired two-tailed t-test or one-way ANOVA. Differences were considered significant at P < 0.05.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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K+ clamp. We first verified that plasma K+ levels could be clamped at basal levels during insulin infusion by exogenous K+ infusion. Insulin was infused at two constant rates, i.e., at a submaximal effective rate of 5 mU · kg-1 · min-1 from 0-150 min, followed by a maximal effective rate of 50 mU · kg-1 · min-1 from 150-270 min (Fig. 1). Plasma insulin was raised from basal levels of 10-15 µU/ml (15) to 89 ± 3 and 2,273 ± 106 µU/ml during the submaximal and maximal insulin infusion, respectively. Plasma glucose was clamped at the basal levels, and plasma K+ levels were either allowed to fall or clamped at the basal levels by exogenous K+ infusion. When plasma K+ was allowed to fall, insulin decreased plasma K+ in a dose-dependent manner (Fig. 1A) from a basal level of 4.2 ± 0.1 to 3.6 ± 0.1 mM after 150 min of submaximal insulin, to 3.2 ± 0.1 mM after an additional 120 min with maximal insulin infusion. To clamp plasma K+ during insulin infusion, K+ infusion rate had to be increased rapidly and it reached a steady state within 1 h. The K+ infusion rate was clearly dependent on insulin levels (Fig. 1B). During the maximal effective insulin infusion, new steady state of K+ infusion was established after 1 h, but toward the end of the time course K+ infusion started to decline, presumably due to excessive accumulation of intracellular K+ (after insulin stimulation of K+ uptake for ~4 h) that would increase the driving force for passive K+ exit. Glucose infusion rates were also insulin dose dependent as expected and were similar with vs. without K+ clamp (Fig. 1D), confirming that insulin action on glucose metabolism is independent of K+ levels or fluxes (6, 7, 12).


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Fig. 1.   Plasma K+ (A) and glucose (C) concentrations and K+ (B) and glucose (D) infusion rates during the hyperinsulinemic (5 mU · kg-1 · min-1, 0-150 min; 50 mU · kg-1 · min-1, 150-270 min) euglycemic clamps with () or without () clamping K+ levels. In D solid line represents the experiment with K+ clamp and broken line represents the one without K+ clamp. K+inf, K+ infusion rate; Ginf, glucose infusion rate. Values are means ± SE for 3 experiments.

Effect of insulin on renal K+ excretion. K+ infusion rates required to clamp plasma K+ are by definition equivalent to the insulin-mediated increase in net rate of K+ flux out of the plasma (net plasma K+ disappearance). There are two potential mechanisms by which insulin could increase net plasma K+ disappearance, i.e., via renal excretion or via extrarenal cellular uptake. To estimate the relative importance of the renal vs. extrarenal mechanisms, we determined the amount of renal K+ excretion during a saline or insulin infusion (Fig. 2). Insulin was infused at a submaximal effective rate of 5 mU · kg-1 · min-1 (plasma insulin = ~90 µU/ml). Plasma glucose and K+ levels were clamped during the insulin infusion so that they were matched between the saline- and insulin-infusion experiments (Fig. 2, A and B). Renal K+ excretion during the experiment, measured in the urine passed during the experiment plus urine in the urinary bladder at the end of the experiment, showed a tendency to increase with insulin infusion (P > 0.05; Fig. 2C). However, the insulin-mediated increase in urinary K+ excretion (renal mechanism) accounted for only a small portion (26%) of the K+ infused to clamp plasma K+ during the insulin infusion (Fig. 2D). The rate of renal K+ clearance, calculated from the urinary K+ excretion and plasma K+ concentrations, was not significantly altered by insulin (0.81 ± 0.09 ml/min with saline vs. 0.99 ± 0.06 ml/min with insulin infusion; P > 0.05). Thus these data indicate that most of insulin-mediated plasma K+ disappearance was due to K+ uptake by extrarenal tissues. Thus the K+ infusion rate required to clamp plasma K+ during insulin infusion appears to be a good measure of insulin's action to increase cellular K+ uptake.


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Fig. 2.   Plasma K+ (A) and glucose (B) concentrations matched between the saline () and insulin (5 mU · kg-1 · min-1; open circle ) infusion experiments. Plasma K+ and glucose levels were clamped during the insulin infusion so that they are matched to those with saline infusion. C: renal K+ excretion in the saline (open bar) and insulin infusion experiment (solid bar). D: insulin-mediated K+ flux (i.e., the total amount of K+ infused during the K+ clamp; open bar) compared with renal K+ excretion (solid bar). Values are means ± SE for 4 or 5 experiments.

Insulin-mediated K+ uptake during K+ deprivation. Insulin stimulates K+ transport into insulin-sensitive tissues, such as skeletal muscle and liver (9, 13), via Na-K-ATPase pumps and perhaps via the bumetanide-sensitive Na-K-2Cl or K-2Cl cotransporters (11, 21, 28). We (26, 27) have shown that expression of Na-K-ATPase pump was substantially decreased in skeletal muscles in an isoform-specific manner (alpha 2 not alpha 1 decreased) during dietary K+ deprivation. If insulin-mediated cellular K+ uptake is largely mediated by Na-K-ATPase and if skeletal muscle is the major tissue for insulin-mediated K+ uptake in vivo, we predicted that in vivo insulin-mediated K+ uptake would be substantially reduced in K+-deprived rats secondary to reduced sodium pump number. To test this hypothesis, we performed the K+-clamp experiment in rats maintained on a K+-deficient diet for 2 days, when there is only minimal decreases in plasma [K+] and sodium pump expression, or for 10 days when there are large decreases in both plasma [K+] and sodium pump alpha 2-isoform expression (26). Basal plasma K+ levels were decreased from 4.2 ± 0.1 mM in controls to 3.8 ± 0.1 mM at day 2 and to 2.9 ± 0.1 mM at day 10 of K+-deficient feeding, respectively (P < 0.01, Fig. 3A; time 0). As predicted, insulin-mediated plasma K+ disappearance was substantially decreased in the hypokalemic rats K+ deprived for 10 days (Fig. 3B). However, not predicted was the finding that insulin action on K+ disappearance was drastically impaired in 2-day K+-deprived rats in which basal plasma K+ levels were decreased minimally. It is also noteworthy that the marked changes in insulin action on K+ flux occurred in the absence of any detectable change in the insulin action on glucose metabolism: there was no difference between control and hypokalemic groups in glucose infusion rates during the glucose and K+ clamps (Fig. 3D). In addition, there was no difference in peripheral glucose uptake determined by tracer [193 ± 4, 192 ± 7, and 185 ± 7 µmol · kg-1 · min-1 for day 0 (control), day 2, and day 10 of K+-deficient feeding, respectively]. Renal K+ excretion during the clamps was already reduced by 92% at day 2 and fell to near zero at day 10 of K+ restriction (P < 0.01 for both) illustrating two points, that 1) the kidneys have a remarkable capacity to adapt to low K+ intake and 2) insulin-stimulated K+ clearance during K+ deprivation is nearly all extrarenal.


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Fig. 3.   Plasma K+ (A) and glucose (C) concentrations and K+ (B) and glucose (D) infusion rates during the hyperinsulinemic (5 mU · kg-1 · min-1) glucose and K+ clamps in control (), 2-day (open circle ), and 10-day () K+-deprived rats. Values are means ± SE for 4 or 5 experiments.

Na-K-ATPase activity and isoform expression in skeletal muscle during K+ deprivation. To assess the relationship between the decrease in insulin-stimulated K+ uptake and decrease in Na-K-ATPase isoform abundance, immunoblots of constant amounts of protein from EDL and TA muscle homogenates from control and K+-restricted rats were probed with Na-K-ATPase isoform-specific antibodies. A typical immunoblot of TA muscle is shown in Fig. 4. As previously reported (26), muscle Na-K-ATPase alpha 1-abundance was unaltered by K+ deprivation. In contrast, after 10 days of K+-deficient diet, alpha 2-expression decreased 39% in EDL (not shown) as previously reported (27) and 42% in TA (Figs. 4 and 5) compared with levels in control rats. However, after 2 days of a K+-deficient diet, there was no significant change in alpha 2-expression detected in either muscle (Fig. 4 shows TA results). To assess the possibility that there was a significant decrease in Na-K-ATPase activity during K+ deprivation that could blunt K+ clearance during insulin stimulation, ouabain-sensitive Na-K-ATPase activity (expressed as µmol Pi · mg protein-1 · h-1) was measured in homogenates of TA muscle from control and K+-deprived rats. There was no significant difference between control (0.88 ± 0.09) and 2-day K+-deprived (0.70 ± 0.08) animals, but by 10 days of K+-deficient diet Na-K-ATPase activity fell ~50% (0.46 ± 0.07, P < 0.01 vs. control and P = 0.06 vs. 2-day low K+). This change in Na-K-ATPase activity follows the pattern of change in Na-K-ATPase alpha 2-protein expression. The side-by-side analysis of insulin-mediated whole body K+ uptake vs. Na-K-ATPase alpha -isoform properties (Fig. 5) illustrates that the 80% suppression of insulin-mediated plasma K+ disappearance observed after 2 days of a K+-deficient diet cannot be explained by the insignificant changes in sodium pump abundance or ATPase activity in skeletal muscle, the main route previously implicated in the shift in K+ from ICF to ECF during K+ deprivation (7, 26, 27).


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Fig. 4.   Effect of K+-deficient feeding for 2 or 10 days on Na-K-ATPase alpha 1- and alpha 2-catalytic subunit abundance in tibialis anterior (TA) skeletal muscle. Representative autoradiograms of immunoblots of homogenate samples from TA were measured as described in METHODS. The antibody-antigen complexes were detected with 125I-protein A. Because the control and low-K+ diet samples were run on the same gel and processed identically, comparisons should be made horizontally (low-K+ diet vs. control), not vertically (2 days vs. 10 days), because of the variable incubation and exposure times between different representative autoradiograms. The lanes labeled "1/2 sample" contain one-half of the same sample loaded in the lane to the left and were run to establish linearity of the signal with the amount of protein loaded.



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Fig. 5.   Insulin-mediated plasma K+ disappearance (K+inf, open bars), Na-K-ATPase alpha 2-subunit abundance, assessed by immunoblot of a constant amount of homogenate protein (cross-hatched bars), and Na-K-ATPase activity (solid bars) in TA muscles of control (day 0) rats, and rats fed a K+-deficient diet for 2 or 10 days. All results are expressed as percentage of mean control value. Values are means ± SE for 4 or 5 experiments. *P < 0.05 vs. control. #P < 0.05 vs. day 2.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The present study demonstrates that plasma K+ levels can be clamped during constant insulin infusion in conscious rats by exogenous K+ infusion and that the rate of K+ infusion required to clamp plasma K+ is insulin dose dependent. K+ infusion rate during the K+ clamp is equivalent to the insulin-mediated increase in net plasma K+ disappearance due to cellular uptake and urinary excretion. Because insulin at a physiological concentration had no significant effect on urinary K+ excretion, the K+ infusion rate during the K+ clamp provides a good measure of insulin action on cellular K+ uptake in vivo. In addition, the present study demonstrates that insulin-mediated plasma K+ disappearance was drastically reduced at as early as 2 days of K+-deficient diet, a point when plasma K+ fell only 9% and when there were no measurable changes in muscle sodium pump activity or expression, and that by 10 days of K+ restriction insulin-mediated K+ disappearance was reduced even further, accompanied by frank hypokalemia, reduced sodium pump activity and alpha 2-isoform abundance, and reduced cell K+ (Ref. 26 and this study). Thus the present study demonstrates the feasibility of this new K+ clamp technique as a tool for the quantification of insulin action on cellular uptake in vivo as well as the utility of the K+ clamp, which was key to establishing extreme insulin resistance of cellular K+ uptake very early in K+ deprivation.

Insulin action on K+ fluxes has been assessed previously in vivo by simply measuring the decreases in plasma K+ levels (1, 3, 8) and/or measuring K+ uptake by specific organs (9) during insulin infusion. The fall in plasma K+ in these studies, as such, may have affected K+ fluxes directly (by mass action) or indirectly (by altering hormone levels and/or membrane potentials), independent of insulin action, and may have confounded the results. The K+ clamp improves and simplifies the quantitative methodology to assess in vivo insulin action on cellular K+ uptake because it allows investigators to assess the insulin action relatively free from such confounding factors and, like the glucose-clamp technique, can be used in small rodents where measuring organ-specific K+ uptake by arteriovenous difference is impractical. The K+ clamp has the potential to allow quantification of the effects of any agent that increases plasma K+ disappearance including K+ levels per se (i.e., "autoregulation"), which has not been possible with conventional methodology.

The K+ infusion rates during the K+ clamp represent insulin-mediated increase in net, rather than absolute, rate of plasma K+ disappearance. Therefore, the K+ infusion rates will reflect both the effect of insulin to increase K+ disappearance from plasma by either transport into cells or renal excretion, as well as any effect of insulin to reduce plasma K+ appearance by decreasing cellular efflux. Theoretically, plasma K+ appearance and disappearance can be separately quantified by the tracer dilution technique with 42K+ or 86Rb+. However, this approach was not exploited in the current study because of the practical difficulties associated with the use of these tracers in vivo.

Our results indicate that insulin at a physiological concentration (~90 µU/ml) had no significant effect on urinary K+ excretion. This is consistent with the finding of Rossetti et al. (23) that insulin increased urinary K+ excretion at a maximally effective concentration (370 µU/ml) but not at a physiological concentration (164 µU/ml) in rats. Taken together, these data indicate that most of insulin-mediated plasma K+ disappearance at a physiological insulin concentration is mediated by insulin's action to increase net K+ uptake by extrarenal tissues. Using arteriovenous balance measurements in humans, DeFronzo et al. (9) showed that skeletal muscle and the splanchnic bed are the major extrarenal tissues responsible for insulin-mediated K+ disappearance from the blood. However, the dynamics of insulin stimulation of K+ uptake in these tissues were quite different; during the first hour after insulin infusion, 70% of the decline in plasma K+ level could be accounted for by splanchnic uptake and 30% by peripheral tissue uptake. During the second hour there was a net splanchnic release of K+, coupled to a fall in plasma [K+], which led the investigators to conclude that the major sites of K+ disposal were peripheral tissues, mainly skeletal muscle. Assuming rats are similar to humans (i.e., if significant splanchnic K+ uptake occurs only during the first hour of insulin stimulation), the bulk of the insulin-mediated K+ flux at steady state during K+ clamp (e.g., 90-150 min after the start of insulin infusion) may represent K+ uptake by skeletal muscle. It is interesting to note that the K+ infusion rates during K+ clamp were more profoundly suppressed in K+-restricted rats after 1 h vs. during the first hour of the clamp. This may indicate that the effect of K+ deprivation to suppress insulin-mediated K+ uptake was more pronounced in skeletal muscle than in other extrarenal tissues such as the splanchnic bed.

We observed a dramatic decrease in insulin-stimulated cellular K+ uptake, coupled to a decrease in urinary K+ excretion after just 2 days of K+ restriction. This illustrates that homeostatic mechanisms to maintain extracellular K+ are activated very early during K+ restriction. The fall in plasma K+ during K+ deprivation is minimized by tapping the intracellular muscle stores, which contain >75% of the body's total K+. We and others have provided evidence that this may be mediated by a decrease in active K+ uptake by the sodium pump alpha 2-isoform: during K+ restriction, there is a decrease in high-affinity ouabain binding to muscle (reviewed in Ref. 7) and a decrease in alpha 2-protein levels (26, 27). However, the side-by-side analysis of insulin-mediated whole body K+ uptake vs. Na-K-ATPase alpha -isoform properties in muscle challenges the notion that a decrease in the number of alpha 2-type pumps is responsible for the decrease in net K+ uptake at 2 days when insulin-mediated net K+ uptake was suppressed 80% but there were insignificant changes in muscle sodium pump abundance or ATPase activity. Eventually, there were very large decreases in sodium pump activity and abundance between 2 and 10 days of K+ restriction, but they were associated with only a small further decrease in the magnitude of the insulin-stimulated K+ uptake. These data suggest that another mechanism must account for the early decrease in insulin-stimulated K+ uptake.

Potential mechanisms to reduce insulin-stimulated K+ uptake during hypokalemia include 1) sodium pumps are internalized to endosomal pools that are insensitive to insulin's action to translocate Na-K- ATPase pumps to the plasma membrane, 2) sodium pump activity per transporter is depressed in vivo, but the modification does not persist through homogenization and is not detected enzymatically, and 3) the abundance or activity of insulin-sensitive K+ uptake pathways other than Na-K-ATPase is decreased [e.g., bumetanide-sensitive Na-K-2Cl cotransporters (28) or KCC4 (21)]. The first possibility is intriguing because it is generally accepted that insulin increases active K+ uptake by shuttling endosomal alpha 2-isoform pumps to the sarcolemma (11), and a defect in this pathway would imply that insulin-stimulated shuttling of sodium pump alpha 2-isoform is impaired even though insulin-stimulated shuttling of GLUT4 glucose transporters is normal. This is feasible because, although the two responses do share signaling features and both transporters do cosegregate from intracellular membranes to the plasma membrane after in vivo insulin stimulation (11, 17, 25), the two transporters appear to be localized to distinct, separate intracellular endosomes (17). The third possibility warrants serious consideration because studies in fibroblasts conclude that insulin stimulation of K+ uptake (using 86Rb uptake) involves concerted stimulation of both a bumetanide-sensitive K+ uptake pathway, presumably a Na+-K+-2Cl- transporter, and the sodium pump, although these cells did not express the insulin-sensitive alpha 2-Na-K-ATPase subunit (25). Another possible candidate for mediating K+ fluxes in muscle is the recently cloned KCC4. KCC4 mRNA transcripts are most abundant in muscle (21), and it is homologous to KCC2, which has a key role in buffering extracellular K+ in the brain (22). Further studies are required to test these three possibilities. The K+ clamp will be a critical tool to evaluate the significance of changes at the molecular level that may be underlying the development of insulin resistance with respect to K+ homeostasis during K+ restriction and ensuing hypokalemia.

Insulin-mediated plasma K+ disappearance was assessed by clamping plasma K+ at the individual rat's own basal levels. Because plasma K+ levels were decreased with K+ deprivation, clamp K+ levels were lower in the K+-deprived groups. When we attempted to clamp plasma K+ at normal levels (~4.2 mM) during insulin infusion of K+-deprived rat (10 days), K+ infusion rates were substantially greater than those during the clamps at their own basal levels (data not shown). The difference can be attributed to the effect of elevated K+ level per se to increase its own plasma disappearance because raising and clamping plasma K+ to normal levels without insulin infusion required substantial K+ infusion rates in K+-deprived rats (data not shown). These preliminary findings suggest that elevation of plasma K+ per se has profound effects to increase cellular K+ uptake (to replenish the cellular K+ pool) and/or increase urinary K+ excretion. Thus the K+ infusion rates during acutely elevated K+ clamps would significantly overestimate insulin-mediated plasma K+ disappearance, and, along the same reasoning, K+ infusion rates during acutely lowered K+ clamps would significantly underestimate insulin-mediated K+ disappearance because of the increased gradient for cellular K+ efflux. Therefore, insulin-mediated plasma K+ disappearance should be assessed at basal K+ levels to avoid confounding effects of altered K+ levels. Future studies are warranted to evaluate the relative importance of (and/or interaction between) the effects of insulin vs. extracellular K+ level per se on K+ disposal in normal and altered states of chronic K+ balance.

Of the many homeostatic systems of the body, the K+ and glucose homeostatic systems are unique in that they share acute regulation by insulin. This feature suggests the potential for interaction between the two systems. Insulin resistance with respect to glucose metabolism is usually associated with hyperinsulinemia because pancreatic beta -cells increase insulin secretion to compensate for insulin resistance (14). It is conceivable that the resulting hyperinsulinemia would have a substantial impact on K+ homeostasis (e.g., provoking hypokalemia and/or increasing intracellular K+ pools), unless insulin's action on K+ uptake was dampened. Impaired insulin action on K+ fluxes has been reported in obesity (8) and in adolescents with type 1 diabetes (3), both of which are known to be associated with insulin resistance with respect to glucose metabolism. On the other hand, Alvestrand et al. (1) showed that insulin-mediated K+ uptake was not altered in uremia, whereas insulin-mediated glucose uptake was markedly impaired. Opposite to this situation is hypokalemia, examined in the present study, in which insulin action on K+ uptake but not glucose uptake was significantly reduced. Thus there is evidence for association as well as dissociation of resistance to insulin action on glucose vs. K+ fluxes. However, this issue has not been examined rigorously in part due to limited methodology. The K+ clamp technique, if combined with the glucose clamp technique, will be an invaluable tool for studying the potential interactions between the glucose and K+ systems via insulin action.


    ACKNOWLEDGEMENTS

We thank Dr. Yong K. Lee for technical assistance.


    FOOTNOTES

This study was supported by a research grant from the American Diabetes Association to J. H. Youn, and the National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-34316 and an American Heart Association Western States Affiliate Grant in Aid to A. A. McDonough.

Address for reprint requests and other correspondence: J. H. Youn, Dept. of Physiology and Biophysics, Univ. of Southern California School of Medicine, 1333 San Pablo Ave., MMR 626, Los Angeles, CA 90089-9142.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 23 March 2000; accepted in final form 7 September 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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