Photocopying Permitted by License Only Evidence for Mechanistic Alterations of Ca 2+ Homeostasis in Type 2 Diabetes Mellitus

Altered cytosolic Ca2+ is implicated in the aetiology of many diseases including diabetes but there are few studies on the mechanism(s) of the altered Ca2+ regulation. Using human lymphocytes, we studied cytosolic calcium (Cai) and various Ca2+ transport mechanisms in subjects with Type 2 diabetes mellitus and control subjects. Ca2+-specific fluorescent probes (Fura-2 and Fluo-3) were used to monitor the Ca2+ signals. Thapsigargin, a potent and specific inhibitor of the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA), was used to study Ca2+- store dependent Ca2+ fluxes. Significant (P < 0.05) elevation of basal Cai levels was observed in lymphocytes from diabetic subjects. Cai levels were positively correlated with fasting, plasma glucose and HbAlc. There was also a significant (P < 0.05) reduction in plasma membrane calcium (PMCA) ATPase activity in diabetic subjects compared to controls. Cells from Type 2 diabetics exhibited an increased Ca2+ influx (as measured both by Fluo-3 fliorescence and C45a assays) as a consequence of of thapsigargin-mediated Ca2+ store depletion. Upon addition of Mn2+ (a surrogate of Ca2+), the fura-2 fluorescence decayed in an exponential fashion and the rate and extent of this decline was steeper and greater in cells from type 2 diabetic patients. There was also a significant (P < 0.05) difference in the Na+/Ca2+ exchange activity in Type 2 diabetic patients, both under resting conditions and after challenging the cells with thapsigargin, when the internal store Ca2+ sequestration was circumvented. Pharmacological activation of protein kinase C (PKC) in cells from patients resulted in only partial inhibition of Ca2+ entry. We conclude that cellular Ca2+ accumulation in cells from Type 2 diabetes results from (a) reduction in PMCA ATPase activity, (b) modulation of Na+/Ca2+ exchange and (3) increased Ca2+ influx across the plasma membrane.

Altered cytosolic Ca 2+ is implicated in the aetiology of many diseases including diabetes but there are few studies on the mechanism(s) of the altered Ca 2+ regulation. Using human lymphocytes, we studied cytosolic calcium (Ca/) and various Ca 2+ transport mechanisms in subjects with Type 2 diabetes mellitus and control subjects. Ca2+-specific fluorescent probes (Fura-2 and Fluo-3) were used to monitor the Ca 2+ signals. Thapsigargin, a potent and specific inhibitor of the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA), was used to study Ca 2+store dependent Ca 2+ fluxes. Significant (P<0.05) elevation of basal Ca/ levels was observed in lymphocytes from diabetic subjects. Ca/levels were positively corr41ted with fasting, plasma glucose and HbAlc. There was also a significant (P<0.05) reduction in plasma membrane calcium (PMCA) ATPase activity in diabetic subjects compared to controls. Cells from Type 2 diabetics exhibited an increased Ca 2+ influx (as measured both by Fluo-3 fliorescence and 45Ca assays) as a consequence of fhapsigargin-mediated Ca 2+ store depletion. Upon addition of Mn 2+ (a surrogate of Ca2+), the fura-2 fluorescence decayed in an exponential fashion and the rate and extent of this decline was steeper and greater in cells from type 2 diabetic patients. There was also a significant (P<0.05) difference in the Na+/Ca 2+ exchange activity in Type 2 diabetic patients, both under resting conditions and after challenging the cells with thapsigargin, when the internal store Ca 2+ sequestration was circumvented. Pharmacological activation of protein kinase C (PKC) in cells from patients resulted in only partial inhibition of Ca 2+ entry. We conclude that cellular INTRODUCTION Recent reports indicate that Asian Indians as a race have a high prevalence of diabetes. 1 Indeed, India has the largest number of diabetic patients in the world and these numbers are expected to further increase in the next few [21 decades. Type 2 diabetes mellitus (non-insulin-dependent diabetes mellitus, NIDDM) is the most common form representing over 85% of all diabetes cases, TM and is commonly accompanied by elevated blood pressure. Type 2 diabetes is characterised by relative insulin deficiency and insulin resistance, but the cellular and molecular mechanism(s) of these defects remain unclear.
There is increasing evidence that Ca 2 + plays an important regulatory role in the cascade of insulin-generated signals [4][5][6] and fl-cell function. I7 Elevated or sustained levels of cytosolic calcium (Ca/) has been shown to diminish cellular sensitivity to insulin and might participate in the pathogenesis of insulin resistance in Type 2 diabetes and in various diabetes-related metabolic derangements, ks-11] Altered cellular Ca 2+ homeostasis could also well be an important mechanism for abnormal glucose metabolism and elevated blood pressure in Type 2 diabetes patients. Although the potential importance of the observed increases in cellular Ca 2 + is clear, the mechanism(s) responsible for this elevation of Ca/has not been identified. Therefore, mechanistic Ca 2 + turnover studies could provide a basis for better understanding at the cellular and molecular level of the long recognised clinical linkage between cardiovascular and metabolic syndromes. These intermediate phenotypes are also essential tools to fill the gap between gene polymorphism and complex diseases.
It is conceivable that the signal transduction defects of the Ca 2 + messenger system in the blood cells would reflect similar disturbances in target tissues affected in diabetes and diabetesassociated complications. Additionally, measurements of intracellular cations using circulating blood cells have been shown to be highly reproducible and the phenotypic characteristics persist even in cell culture models. [12] Lymphocytes were used in this study because they are readily accessible, their cytosolic calcium regulation is well understood 13' 14] and more importantly they could potentially provide genomic DNA for studying the underlying genetic mechanisms of diabetic complications.

Materials
'Lymphoprep' was obtained from GIBCO Life Technologies (Gaithersburg, MD, USA). Thapsigargin, ionomycin, Fura-2 AM and Fluo-3 AM, phorbol 12-myristate 13- Organization study group on diabetes. I15 Informed consent was obtained from all study subjects. Blood samples and blood pressure measurements were taken between 0700 and 0900h after an overnight fast. Blood (20ml) drawn from each subject in acid citrate dextrose buffer was used for lymphocyte Ca 2 + studies. An additional 10ml blood treated with EGTA was used for measurement of blood chemistry parameters. The study protocol was approved by the ethical committee of Centre for Biotechnology, Anna University. All biochemical studies were done on Corning Express plus Auto Analyzer (Corning, USA). Fasting plasma glucose (glucose oxidase method) was estimated using kits from Boehringer Mannheim, Germany. Glycosylated haemoglobin (HbAlc) were estimated by high-pressure liquid chromatography (HPLC) method using the Variant machine (Bio Rad, Hercules, USA).
Lipid profile and serum creatinine were assayed using commercial kits (Boehringer Mannheim, Germany).

Isolation of Lymphocytes from Whole Blood
Lymphocytes were isolated by the method of density gradient centrifugation t161 with modifications described earlier, t131 Blood was diluted with an equal volume of HEPES buffered solution (HBS) and layered onto lymphoprep (2:1 vol/vol). After density gradient centrifugation (45min at 1700rpm), lymphocytes were collected from the interface, diluted 1:1 with HBS. The lymphocyte pellet that resulted from a second centrifugation at 1600 rpm for 10 min was resuspended in Hepes buffer.
The membranes were preincubated with or without thapsigargin for 5 min prior to the start of the reaction and introduced into the ATP containing assay medium. The oxidation of NADH was monitored at 340nm in a Hitachi Spectrophotometer (model U-3210, Japan), which directly indicated the hydrolysis of ATP by the Ca 2 + ATPase. Free Ca 2 + in the medium was adjusted by EGTA and CaC12 additions, using a computer program. Ca 2+ dependent hydrolysis of ATP was determined by subtracting the rate of activity in the presence of EGTA from the rate in the presence of Ca 2 +. 45Ca2+ Uptake in Intact Lymphocytes Lymphocytes were preincubated with (for Na +/Ca 2+ exchange assay) or without (for Ca 2+ entry assay) 0.1mmol/1 ouabain in Na + containing medium for 30 min. To initiate Ca 2 + uptake, aliquots of cells (1 x 108 cells/ml) were diluted into Na + containing medium or Na + free medium (NaC1 is iso-osmotically replaced by NMDG) containing 10 gCi/ml 45CAC12. Ouabain pretreatment was used to increase cytosolic Na + through inhibition of the Na +/K + AT-Pase. [141 In experiments where the cells were treated with thapsigargin (Tg), 100 nmol/1 Tg was included in the assay medium. After one minute incubation at 37C, Ca 2 + uptake was stopped by addition of 5 ml of ice-cold stopping buffer (40mmol/1 Hepes, 100mmol/1 MgCI2, 5 mmol/1 LaCI3). Extracellular radioactivity was removed by rapid filtration of cells on 0.45 g filters with two additional washes of stopping buffer. Each data point represents the mean of 'n' experiments derived from triplicate or quadruplicate measurements. Background counts (medium without cells) were substracted from all experimental time points.

Fluorescent Dye Loading Ca/Monitoring
Lymphocytes were incubated with 5 gmol/1 Fura-2 or 5 gmol/1 Fluo-3 for a minimum of 30 min at 37C and delivered as 100 gl (0.5 x 106 cells/ml) aliquots. Prior to each experiment, cells were centrifuged for 5-10 sec at room temperature, resuspended in 100 gl of HBS, and injected into cuvettes containing 3 ml of assay buffer. Ca/ monitoring using Fluo-3 was monitored in a Hitachi spectrofluorimeter (model F-3010, Japan) by excitation wavelength set at 490nm and emission wavelength at 525nm. Calibration was achieved by exposing newly resuspended lymphocytes to digitonin and EGTA to get Fmax and Fmin values. [13] From the fluorescence values (F), the Ca/was calculated according to the method of Grynkiewicz et al. [18] utilizing the dissociation constant of the dye (Kd--400 nM), Fmax and Fmin values. Autofluorescence of the unloaded cells was substracted from the fluorescence values.

Mn 2+ Quench Experiments
Mn 2+ has been used as a Ca 2 + surrogate to study Ca 2+ entry mechanisms [13]. We thus monitored the rate of quenching of intracellular fura-2 by Mn 2 + as a measure of Ca 2 + entry across the plasma membrane. This analysis was performed by following the decay of fura 2 fluorescence at excitation wavelength 360 nm in lymphocytes suspended in Ca 2 +-HBS and incubated with 0.5 mmol/1 MnC12.

Data Analysis
Statistical analysis was performed with Student's test, one-way analysis of variance and correlation analyses. P K 0.05 was considered to be statistically significant. Data in the figures are expressed as Means 4-SE.

RESULTS
Clinical characteristics of the control and Type 2 diabetes subjects are summarised in Table I. Diabetic patients had significantly (P K0.05) higher body mass index, fasting plasma glucose, cholesterol, HbAlc and systolic blood pressure than controls. Ca/was positively correlated with fasting plasma glucose (r 0.4752, p 0.05) and HblAc (r 0.5617, p 0.05).
The basal Ca/measurement performed using Fluo-3 in lymphocytes is shown in Figure 1. The  membranes from Type 2 diabetic patients exhibit lower levels (P < 0.05) of PMCA ATPase activity (Fig. 2). Because inhibition of either the PMCA or SERCA ATPases could lead to an increase in cytosolic Ca 2 +, the data suggests that there is a defect in PMCA ATPase mediated Ca 2 + extrusion mechanism.
Na + dependent 45Ca uptake assay was used to determine the Na +/Ca 2+ exchange activity, a maneuver that involves raising of the cytosolic Na + concentration and/or lowering of the external Na + concentration. The protocol for measuring Na + dependent Ca 2 + uptake assay is illustrated in Figure 3. Ouabain pretreatment was used to inhibit the Na-K-ATPase, increase Nai and decrease the inwardly directed Na gradient that inhibits Ca entry by 'forward mode' Na +/Ca 2+ exchange. A greater accumulation of Ca 2 + uptake seen in Na +-free condi-5000 2500 +Na lOO FIGURE 3 Protocol for measuring Na +-dependent Ca + influx. Lymphocytes were preincubated for 30 min at 37C in Na + medium with or without 0.1mmol/1 ouabain and resuspended in Na +-or Na +-free medium containing 45Ca2 +. Ca + uptake was measured after 60sec; each experiment was performed in quadruplicate and averaged. Data represent mean values of 6 individual experiments. + 2+ % Na -Ca exchange (inset) was calculated by subtracting medium from Na -free the values of Ca + uptake in Na + + medium.
tions is the evidence for Na +/Ca 2+ exchange activity (Fig. 3). Per cent Na +/Ca 2+ exchange was calculated by substracting the 45Ca 2+ uptake in Na +-containing medium from that obtained in Na + free conditions (inset). The composite results of % Na +/Ca 2+ exchange derived from a number of similar experiments with and without Tg and in control and Type 2 diabetes subjects are summarised in Figure 4. In control cells, the reversal of Na + gradient resulted in a net Ca 2+ uptake mediated by Na +/Ca 2 + exchange (70.4%) that increased to 92.2% in the presence of Tg. The same maneuver measured only 36% Na +/Ca 2+ exchange activity in ceils from Type 2 diabetes, but this significantly (P <0.05) increased to 137.2% in the presence of Tg. These results suggest that Na +/Ca 2+ exchange activity is depressed in Type 2 diabetes patients under resting, unstimulated conditions. However, augmented Na +/Ca 2+ exchange was noticed in cells from both control and Type 2 diabetic subjects after treatment with Tg. Indeed, cells from Type 2 diabetes incubated with Tg, showed a 3 fold increase in Na +/Ca 2+ exchange activity when compared to resting cells (Na +/Ca 2+ exchange activity recorded as an increase from 36% to 137%). This means that the internal stores undoubtedly play a crucial role in buffering increases in net Ca 2 + gain occurred through Percentage Na +/Ca + exchange in lymphocytes from control (A) and Type 2 diabetes (B) subjects in the presence and absence of thapsigargin (Tg). The methodology for these experiments is exactly the same as in Figure 3 and % Na +/Ca + exchange values (each representing mean of 6 separate cell preparations) are shown combined.
Na +/Ca 2+ exchange. In the presence of Tg, the internal store Ca 2 + buffering was circumvented.
Since the alterations in resting Ca/ could be a manifestation of changes occurring through many mechanisms, we next looked into the Ca 2+ entry (Ca 2+ uptake) processes across the plasma membrane. 45Ca 2+ uptake (one min) measurements in the presence and absence of Tg, in cells from control and Type 2 diabetic patients respectively, are depicted in Figure 5. Tg-mediated Ca 2 + uptake over the resting Ca 2 + uptake was significantly (P <0.05) higher in both control and Type 2 diabetic patient cells.
However, Tg-stimulated Ca 2+ entry was significantly higher (47.8%) in cells from Type 2 diabetics compared to control lymphocytes (23.2%) (insets). Measurement of Ca/in lymphocytes from control and Type 2 diabetes subjects also indicates a differential profile of storeoperated Ca 2 + influx (Figs. 6A and B). Cells from Type 2 diabetes exhibited an increased initial rate of Tg-evoked Ca 2+ influx when compared to the similar profile in control cells. As depicted in Figure 6C, mean Ca/levels at 60 sec after the addition of extracellular Ca (comparable to the one min 45Ca2+ uptake) were significantly (P < 0.05) different in cells from control (162 nmol/1) and patients (235 nmol/1). Figure 7 illustrates the role of Tg-evoked Mn 2 + influx as another experimental maneuver for studying store-operated Upon addition of Mn 2 +, the fura-2 fluorescence decayed in an exponential fashion and the rate and the extent of this decline was steeper and greater in cells from Type 2 diabetes. The initial rate of Mn 2 + entry is a parameter that reflects the opening state of the Ca 2 + influx pathway.
Phorbol esters, such as PMA activate PKC and alter multiple cellular responses. We incubated lymphocytes with PMA, and monitored the Ca/ responses after treatment with Tg (Figs. 6A and B). Though in general, PKC activation resulted in inhibition of Ca 2 + influx, the extent of this inhibition in cells from Type 2 diabetic subjects (38%) was substantially lower than control (68%) subjects (Fig. 6D). This was also supported by Mn

DISCUSSION
Several studies have demonstrated elevated Ca/ in cells from diabetic and/or hypertensive individualsI10, [21][22][23][24][25][26][27][28] and a reduction in Ca/ associated with a combination drug therapy. L291 The fine tuning of the cell Ca 2 + is primarily performed by two high Ca 2 + affinity pumps under the control of SERCA and PMCA ATPases. A calmodulin-stimulated ATP-dependent Ca 2 + uptake and a corresponding PMCA ATPase have been described in lymphocytes. L3l Balasubramanyam et al. [13] have also demonstrated fluorimetrically a well defined Ca 2+ extrusion process in freshly isolated human peripheral blood lymphocytes, which is mediated by Ca pump of the plasma membrane. The present study shows that there is an impairment in Ca 2 + turnover in Type 2 diabetic subjects with a significant reduction of PMCA ATPase activity. In diabetic patients as well as in experimental diabetes mellitus, there are conflicting results on cellular Ca 2+ ATPase with the reports of decreased [31][32][33][34][35][36][37] and increased [22,[38][39] activities. Our results support the work of Spieker et al. [36] who demonstrated decreased Ca 2 +-ATPase activity in erythrocytes from both Type 1 and Type 2 diabetic patients.
Similar inhibition of PMCA activity related to higher Ca/levels in platelets from hypertensive individuals has also been reported. 4' 41] The mechanism(s) of inhibition of PMCA ATPase in Type 2 diabetes might be attributed to increased protein phosphatase activity, modulation of protein kinase A or C, or both. [421 While alterations in lipid composition in patients (changes in both membrane fluidity and acidic phospholipid content) have been shown to affect the plasma membrane Ca 2 + pump, high glucose in uncontrolled diabetes would also probably leads to glycosylation of the PMCA pump and its inhibition. 431 Apart from PMCA ATPase, the other important transport process that can mediate net Ca 2 + extrusion across the plasma membrane is Na + / Ca 2+ exchange. Our findings also suggest a possible role for Na +/Ca 2+ exchange in Ca 2 + regulation in lymphocytes and its modulation in diabetes states. A primary increase in Na + permeability, 44j an increase in intracellular Na + caused by a circulating inhibitor of the sodium pump 451 or an increase in Na + due to enhanced activity of the Na +/H + exchanger could all promote Na +-dependent Ca 2 + influx via Na +/Ca 2+ exchange leading to the observed increase in Ca/. Pharmacologically, ouabain is routinely used in Na +-dependent Ca 2 + uptake studies. Lymphocytes exposed to 0.   HbAlc levels, indicating the degree of metbolic control over a longer period of time, may exert a lasting effect on cellular Ca/and/or other intermediary mechanisms. Interestingly, not only can higher Ca/ levels contribute to insulin resistance, but diabetes can also lead to significant alterations in cellular Ca 2 + handling.
The origin of the cellular Ca 2+ shifts in diabetes and/or hypertension is not clear. Genetic defects in cell membrane Ca 2 + transport or transport of Ca 2 + across the intracellular membrane systems may contribute to alterations in cellular Ca 2 + homeostsis. There is general agreement that genetic factors could be the primary determinants of impaired insulin secretion and action. Altered cellular and sub-cellular Ca 2 + homeostasis can cause abnormal insulin secretion, increased vascular resistance, and altered response of vascular smooth muscle cells to Ca 2 + mobilising vasoactive hormones. [6" 611 The vascular changes accompanied by an appreciable rise in systemic blood pressure may in turn, cause hypertension. Finally, as suggested by Wehling and Theisen, I621 hypertension and insulin resistance may have further deleterious effects on membrane phospholipid content and cellular Ca 2+ homeostasis, creating a vicious cycle. This work has several implications for future studies. This is the first report of Ca 2 + turnover studies in Indian Type 2 diabetes patients. We have identified defects in Ca 2 + transport activities, viz., ATPase(s), Na +/Ca 2+ exchanger and store-operated Ca 2+ channels anc! these can be studied further using both proteomic and genomic approaches and lymphoblasts would be an ideal cell culture model for such studies.
The fact that ion transport abnormalities are persistent in in vitro culture [63,64] indicates that these phenotypic expressions may be genetically determined. More importantly, a recent study has already reported sequence variants of the SERCA 3 gene in white Caucasians with Type 2 diabetes I651 which needs to be studied in different ethnic diabetic populations. Until a true genetic marker is identified, cellular elements may serve as intermediate phenotypes for screening high-risk diabetic patients and as targets for the development of new therapeutic compounds. Continued research efforts on molecular and genetic studies of families of diabetic patients are warranted to identify the initial defect and its genetic component of altered Ca 2 + homeostasis.