The Journal of Nutrition Vol. 128 No. 2 February 1998, pp. 180-184

Chromium Picolinate Modulates Rat Vascular Smooth Muscle Cell Intracellular Calcium Metabolism¹

Jerry W. Moore, Margaret A. Maher², William J. Banz³, and Michael B. Zemel⁴

Departments of Nutrition and Medicine, The University of Tennessee, Knoxville, TN 37996-1900

	ABSTRACT

Abstract Introduction Methods Results Discussion References

We have previously shown that insulin attenuates vasoconstriction, accelerates both vascular relaxation and [Ca²⁺]_i recovery from pressor agonist-induced Ca²⁺ loads, and stimulates Ca²⁺-ATPase gene expression in rat and human vascular smooth muscle cells (VSMC). Moreover, these functions are impaired in VSMC from both insulin resistant and insulinopenic rats, suggesting that hypertension in insulin resistant states may result, in part, from impaired insulin-regulation of VSMC Ca²⁺ transport. Accordingly, we have now evaluated the effect of improving cellular insulin sensitivity with chromium picolinate (CrPic) on regulation of VSMC Ca²⁺ transport. Cultured VSMC from rats were grown from passage to confluence in the presence or absence of 1 µmol/L CrPic, maintained in a quiescent medium for 24 h and incubated with or without insulin (10⁸ mol/L) for the final 2 h. Cells were then harvested and RNA and protein extracted for Northern and Western blot analysis, respectively. Insulin caused a significant stimulation of plasmalemmal Ca²⁺-ATPase mRNA and protein (P < 0.05). A comparable stimulation of the mRNA and protein levels was caused by CrPic in the absence of insulin (P < 0.05), while the CrPic + insulin treatment caused a greater percentage stimulation of the Ca²⁺-ATPase mRNA level than either separate treatment (P < 0.05). Fluorometric analysis of the rate of [Ca²⁺]_i recovery following stimulation with arginine vasopressin support these findings: insulin caused an 83% increase, CrPic caused a 35% increase and insulin + CrPic caused a 133% increase in [Ca²⁺]_i recovery rate. These data suggest that CrPic may be an effective modality to reduce VSMC [Ca²⁺]_i loads and thereby reduce peripheral vascular resistance in insulin resistant states.

	INTRODUCTION

Abstract Introduction Methods Results Discussion References

Previous data from this laboratory demonstrate that insulin attenuates vasoconstriction (Zemel et al. 1990 and 1991), accelerates both vascular relaxation (Zemel et al. 1992) and intracellular free calcium ([Ca²⁺]_i)⁵ recovery from pressor agonist-induced Ca²⁺ loads (Kim and Zemel 1993) and stimulates Ca²⁺-ATPase expression in vascular smooth muscle cells (Kim and Zemel 1995, Zemel et al. 1993). Further, these functions are impaired in both insulin resistant (Abel and Zemel 1993a, Shehin et al. 1989) and insulinopenic (Abel and Zemel 1993b, Reddy et al. 1990) rats, and Ca²⁺-ATPase activity is impaired in erythrocytes of both type I and type II diabetic patients (Zemel et al. 1988). These data suggest that the common association between insulin resistance and hypertension may result, in part, from a failure of insulin to appropriately regulate vascular smooth muscle cell Ca²⁺ transport, resulting in increased [Ca²⁺]_i and exaggerated vascular reactivity (Zemel et al. 1988). Consequently, strategies directed at improving peripheral insulin sensitivity would also be expected to reduce vascular smooth muscle [Ca²⁺]_i and blood pressure (Zemel 1995). Consistent with this notion, the insulin-sensitizing agents pioglitazone, ciglitazone, phenformin and metformin have recently been demonstrated to reduce blood pressure in animal models of hypertension and in clinical studies (Dubey et al. 1993, Morgan et al. 1992, Pershadsingh et al. 1993).

Accordingly, the present study was conducted to determine whether utilizing chromium picolinate, an agent which increases peripheral insulin sensitivity, would increase vascular smooth muscle Ca²⁺-ATPase transcription and, consequently, accelerate [Ca²⁺]_i recovery from pressor-agonist induced loads. If so, this would provide a physiological mechanism to explain how insulin sensitizing agents may reduce blood pressure, and provide a basis for conducting clinical trials with chromium picolinate (chromium tripicolinate) in mild hypertension.

View larger version (17K): [in this window] [in a new window]   Fig 2. Effects of chromium picolinate (1 µmol/L) and insulin (108 mol/L) on the rate of intracellular calcium recovery to baseline following arginine vasopressin stimulation of rat A7r5 vascular smooth muscle cells. Nonmatching superscripts denote significantly different group means (P < 0.05; n = 8 per group; values are means ± SD).

	MATERIALS AND METHODS

Abstract Introduction Methods Results Discussion References

Vascular smooth muscle cells.  Vascular smooth muscle cells (VSMC) were obtained from a clonal stock of A7r5 cells from the American Type Culture Collection (ATCC, Rockville, MD). Cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 50 g/L fetal bovine serum (FBS), 50 g/L supplemented calf serum, 50 g/L penicillin/streptomycin and 0.5 g/L fungizone (Life Technologies, Gaitherburg, MD) in a 95% humidity, 5% CO₂, 37°C atmosphere. Cells were grown to confluence prior to passage, and passages 4-12 were used for these experiments. For each experiment, cells were grown to confluence in the presence or absence of 0.5, 1 or 2 µmol/L chromium picolinate (Nutrition 21, San Diego, CA) and maintained in a quiescent medium containing 2 g/L FBS for 24 h and incubated with or without insulin (10⁸ or 10⁷ mol/L) for the final 2 h. The cells were rinsed twice with Hank's balanced salt solution (HBSS, Life Technologies) and harvested for northern blot, western blot or intracellular calcium measurements, as described below.

RNA isolation and Northern blot analysis.  Northern blot analysis of Ca²⁺-ATPase mRNA was conducted as previously described (Kim and Zemel 1995). Briefly, cells were harvested by the addition of ice-cold 4 mol/L guanidine isothiocyanate/2 g/L -mercaptoethanol (Life Technologies), homogenized using a PT-3000 Polytron (Brinkmann, Westbury, NY), layered onto a cesium chloride (Life Technologies) gradient, and centrifuged at 140,000 × g for 18 h (Sorvall, Newtown, CT). The RNA pellet was resuspended and assessed for quality and purity spectrophotometrically (absorbance at 260 and 280 nm). The RNA was denatured with 65 g/L formaldehyde/0.5 g/L formamide and its integrity assessed by running on a 10 g/L agarose gel containing formaldehyde (0.66 mol/L) in a 20 mol/L MOPS/5 mol/L sodium acetate 1 mol/L EDTA, pH 7.0 buffer at 75 V. The gels were rinsed, stained with 1 mg/L ethidium bromide, visualized under UV light, and photographed. The separated RNA was then transferred to nylon filters (Genescreen Plus, Dupont, Boston, MA) by Northern blot for ~16 h. An oligonucleotide probe from plasmalemmal Ca²⁺-ATPase (Oncogene Science, Uniondale, NY) was 3-end labeled with dATP-³²P (NEN, Boston, MA), hybridized, and the blot was exposed to Reflection X-ray film (Dupont, Boston, MA). The membranes were stripped and reprobed with -actin as a loading control, and the data was expressed as Ca²⁺-ATPase:-actin ratio.

View larger version (16K): [in this window] [in a new window]   Fig 1. Representative fluorometric tracing of the intracellular Ca2+ response to arginine vasopressin (AVP) in rat A7r5 vascular smooth muscle cells. Arrows indicate addition of AVP (100 nmol/L), peak response to AVP and recovery phase used to calculate recovery rates shown in Fig. 2.

Western blot analysis.  Ca²⁺-ATPase protein was measured by Western blot, as previously described (Kim and Zemel 1995). Briefly, cells were harvested and protein extracted by solubulizing in a 4% (wt/v) SDS/200 mmol/L dithiothreitol/100 mmol/L Tris buffer, heated for 5 min at 100°C and sonicated. The supernatant was assayed for total protein using a modified Lowry method. The samples were loaded onto a 6% resolving/5% stacking discontinuous acrylamide/bisacrylamide gel using the Mini-PROTEAN II cell (Bio-Rad, Hercules, CA). One gel was stained with Coomassie blue in order to visualize the protein bands and the other was transferred to polyvinylidene difluoride (PVDF) membranes (Life Technologies) by electroblot using the Mini-PROTEAN II apparatus. Membranes were probed with a chemiliuminescent-labeled plasmalemmal Ca²⁺-ATPase probe (Affinity Bioreagents, Neshanic Station, NJ) using the PhotoBlot system (Life Technologies).

Intracellular calcium measurements [Ca²⁺]_i.  [Ca²⁺]_i responses to and rates of recovery from arginine vasopressin (AVP) were assessed spectrofluorometrically, as previously described (Kim et al. 1995). Cells were pelleted by centrifugation at 200 × g for 5 min and the pellet was suspended in a HEPES-buffered salt solution (HBSS) consisting of (in mmol/L) NaCl, 138; CaCl₂, 1.8; MgSO₄, 0.8; NaH₂PO₄, 0.9; NaHCO₃, 4.0; D-glucose, 25; glutamine, 6.0; HEPES, 20; and 50 g/L bovine serum albumin at pH 7.4. Cell suspensions were prechilled on ice for 10 min, loaded with 10 µmol/L Fura 2/AM (Sigma Chemical, St. Louis, MO) and incubated in the dark in a shaking 37°C waterbath for 30 min. Cells were pelleted by centrifugation at × 100 g, washed twice with HBSS to remove the extracellular Fura-2 and resuspended at a concentration of approximately 10⁹ cells/L. [Ca²⁺]_i levels were determined using dual excitation (340 and 380 nm) and single emission (510 nm) fluorometry with a Hitachi F-2000 (Naperville, IL) spectrofluorometer. The Ca²⁺ signal was calibrated using maximal and minimal fluorescence ratios in response to 40 µmol/L digitonin and 100 mmol/L Tris/100 mmol/L EDTA pH 8.7, respectively. [Ca²⁺]_i was calculated by the Hitachi F2IC software using the method of Grynkiewicz et al. (1985). After a stable baseline was established, both [Ca²⁺]_i response to AVP and the rate of recovery from AVP were evaluated. The rate of [Ca²⁺]_i recovery was determined for the 10 s following maximal response to AVP, as previously described (Abel and Zemel 1993a, Kim and Zemel 1993 and 1995).

Statistical analysis.  All experiments were replicated eight times. Data were evaluated for normality of distribution and equality of variance prior to analysis for statistical significance using two-way analysis of variance and Tukey's test.

	RESULTS

Abstract Introduction Methods Results Discussion References

Figure 1 depicts a representative fluorometric tracing of the [Ca²⁺]_i response to 100 nmol/L AVP. Neither insulin nor chromium affected the magnitude of the rapid peak [Ca²⁺]_i response to AVP. However, the rate of [Ca²⁺]_i recovery to baseline following peak response was 2.83 nmol/L¹·s¹ (Fig. 2), and was increased to 3.82 nmol/L¹·s¹ in the presence of 1 µmol/L chromium picolinate, to 5.18 nmol/L¹·s¹ in the presence of insulin (10⁸ mol/L) and to 6.6 nmol/L¹·s¹ in the presence of both chromium picolinate and insulin (P < 0.05; Fig. 2). The concentration of chromium picolinate used (1 µmol/L) was based on that previously demonstrated to optimize biological response (Evans and Puchnik 1993). Use of a lower concentration (0.5 µmol/L) resulted in a significantly reduced augmentation of [Ca²⁺]_i recovery (P < 0.05), while a 2.0 µmol/L chromium picolinate concentration caused a stimulation not different than that found with the 1.0 µmol/L concentration (Fig. 2). Similarly, the concentration of insulin used (10⁸ mol/L) was based on our previous reports of the effects of insulin on vascular reactivity, vascular smooth muscle Ca²⁺-ATPase expression and [Ca²⁺]_i recovery, and increasing the concentration to 10⁷ mol/L elicited no further increase in [Ca²⁺]_i recovery (data not shown).

Ca²⁺-ATPase protein abundance (Fig. 3) and mRNA levels (Fig. 4) were markedly affected by both chromium picolinate and insulin. The combination of chromium picolinate and insulin exerted a more marked effect on Ca²⁺-ATPase mRNA than did either agent alone (Fig. 4), similar to their effects on [Ca²⁺]_i recovery.

View larger version (20K): [in this window] [in a new window]   Fig 3. Effects of chromium picolinate (1 µmol/L) and insulin (108 mol/L) on Ca2+-ATPase protein in rat A7r5 vascular smooth muscle cells. *Denotes significantly different than control (P < 0.05; n = 8/group; values are mean ± SD). Inset shows a representative autoradiograph.

View larger version (16K): [in this window] [in a new window]   Fig 4. Effects of chromium picolinate (1 µmol/L) and insulin (108 mol/L) on Ca2+-ATPase mRNA in rat A7r5 vascular smooth muscle cells. Nonmatching superscripts denote significantly different group means (P < 0.05; n = 8 per group; values are means ± SD).

	DISCUSSION

Abstract Introduction Methods Results Discussion References

Data from the present study demonstrate that chromium picolinate modulates vascular smooth muscle cell Ca²⁺ and exerts an additive effect with insulin on Ca²⁺ recovery. Moreover, chromium picolinate stimulated increases in plasmalemmal Ca²⁺-ATPase mRNA and protein and significantly increased the rate of [Ca²⁺]_i to baseline following AVP stimulation. Although numerous previous studies have demonstrated chromium potentiation of insulin action (Mertz 1993), this is, to the best of our knowledge, the first report of a chromium compound exerting an insulin-like action independent of insulin.

Trivalent chromium is an essential trace element in human and animal nutrition, serving to potentiate insulin action and maintain normal glucose tolerance (Mertz 1993). Impaired glucose tolerance may be induced in animals by feeding a chromium restricted diet, while chromium supplementation improves glucose tolerance in diabetic rats (Mertz 1993, Schwartz and Mertz 1959). In humans, chromium-responsive insulin resistance has been demonstrated in patients receiving total parenteral nutrition (Brown et al. 1986, Freund et al. 1979, JeeJeebhoy et al. 1977, Mertz 1993). Moreover, several studies have demonstrated improved glucose tolerance in response to chromium supplementation in patients with impaired glucose tolerance (Mertz 1993).

Although the precise mechanism of chromium action is not known, it clearly potentiates insulin action both in vitro and in vivo, an effect mediated by glucose tolerance factor (GTF), a complex whose active axis contains two molecules of nicotinic acid and amino acids coordinated to Cr³⁺ (Mertz 1993). In a previous study, we proposed that both nicotinic acid and chromium may be limiting in human diets as precursors of GTF (Urberg and Zemel 1987), because nicotinic acid accounts for only a small portion of the niacin in most human diets; although the acid is metabolized to the amide following absorption, it exhibits unique effects not associated with nicotinamide feeding. In support of this notion, elderly volunteers improved their glucose tolerance in response to a combined supplement of 4 µmol of chromium and 0.8 mmol of nicotinic acid, but not with either nutrient alone (Urberg and Zemel 1987). More recently, several investigators have utilized chromium tripicolinate, a stable complex of three molecules of picolinic acid and trivalent chromium (Evans and Puchnik 1993), in lieu of GTF-chromium or other dinicotinate complexes to provide biologically active chromium (Cefalu et al. 1997, Evans 1989, Evans and Puchnik 1993, Hasten et al. 1992, Kaats et al. 1996, Lindemann et al. 1995, Page et al. 1993). These studies demonstrate chromium picolinate to be effective in potentiating insulin action (Anderson et al. 1996a and 1996b, Cefalu et al. 1997, Evans 1989, Evans and Puchnik 1993, Hasten et al. 1992, Kaats et al. 1996, Lindemann et al. 1995, Page et al. 1993).

Accordingly, we have attempted to utilize chromium picolinate to potentiate a novel action of insulin in vascular smooth muscle cells. Previous studies from this laboratory have demonstrated that insulin attenuates vascular reactivity responses to pressor agonists in vitro (Zemel et al. 1990 and 1991), accelerates vascular smooth muscle relaxation and Ca²⁺-ATPase-mediated cellular Ca²⁺ efflux (Zemel et al. 1992) and inhibits responses of vascular smooth muscle cell intracellular Ca²⁺ to arginine vasopressin (Standley et al. 1991). Further, we have found insulin to accelerate intracellular Ca²⁺ recovery from agonist-induced Ca²⁺ loads in both cultured rat and human vascular smooth muscle cells (Kim and Zemel 1993 and 1995) and to stimulate gene expression for both plasmalemmal and sarcoplasmic reticulum Ca²⁺-ATPases (Kim and Zemel 1995, Zemel et al. 1993). These effects appear to result from a glucose-6-phosphate-dependent carbohydrate response element in the Ca²⁺-ATPase gene (Kim and Zemel 1995). Insulin has also been shown to exert vasodilatory effects in canine femoral artery and rabbit efferent arteriole preparations (Juncos et al. 1992, Kahn et al. 1993).

Insulin-mediated vascular relaxation is impaired in vascular smooth muscle from both insulin resistant (Zucker obese) and insulinopenic (streptozotocin-treated) rats (Abel and Zemel 1993a, and 1993b, Reddy et al. 1990, Shehin et al. 1989). Consequently, we have proposed that insulin modulation of Ca²⁺ transport results in vascular relaxation and reduced vascular tone, while blunting of this modulation may be responsible for hypertension in insulin-resistant states. A corollary of this hypothesis is that strategies directed at improving peripheral insulin sensitivity will reduce vascular tone and blood pressure, and this has been confirmed in both animal models of hypertension and clinical studies of both the biguanide and thiazolidinedione classes of insulin-sensitizing agents (Dubey et al. 1993, Morgan et al. 1992, Pershadsingh et al. 1993). Consequently, we conducted the present study to determine whether chromium picolinate would similarly potentiate insulin action in vascular smooth muscle cells. Our results indicate that such a potentiation does occur, as demonstrated by enhanced increases in both intracellular Ca²⁺ recovery following arginine vasopressin stimulation compared to insulin-treatment alone, although chromium picolinate also exerted these effects independently of insulin.

Accordingly, we now propose that chromium picolinate be evaluated as a potentially effective clinical modality to reduce vascular smooth muscle intracellular Ca²⁺ loads following agonist stimulation and thereby reduce peripheral vascular resistance in insulin resistant states.

	FOOTNOTES

Manuscript received 28 July 1997. Initial reviews completed 21 August 1997. Revision accepted 17 October 1997.

	LITERATURE CITED

Abstract Introduction Methods Results Discussion References

• Abel M. A., Zemel M. B. Impaired recovery of vascular smooth muscle intracellular calcium following agonist stimulation in insulin resistant (Zucker obese) rats. Am. J. Hypertens. 1993a; 6:500-504 [Medline]
• Abel, M. A. & Zemel, M. B. (1993b) Impaired recovery of vascular smooth muscle cell intracellular Ca²⁺ following angiotension II stimulation in streptozotocin-induced diabetic rats. FASEB J. 7: A747 (abs.).
• Anderson R. A., Bryden N. A., Polansky M. M., Gautschi K. Dietary chromium effects on tissue chromium concentrations and chromium absorption in rats. J. Trace Elem. Exp. Med. 1996a; 9:11-25
• Anderson, R. A., Polansky, M. M., Bryden, N. A. & Zawadzki, J. (1996b) Selective uptake of chromium in type II diabetes mellitus. FASEB J. 10: A220 (abs.).
• Brown R. O., Forloines-Lynn S., Cross R. E., Heizer W. D. Chromium deficiency after long-term total parenteral nutrition. Dig. Dis. Sci. 1986; 31:661-664 [Medline]
• Cefalu, W. T., Bell-Farrow, A. D., Wang, Z. Q., McBride, D. G., Stegner, J., Terry, J. G. & Morgan, T. M. (1997) The effect of chromium supplementation on carbohydrate metabolism and body fat distribution. Diabetes 46: 55A (abs.).
• Dubey R. K., Zhang H. Y., Reddy S. R., Boegehold M. A., Kotchen T. A. Pioglitazone attenuates hypertension and growth of renal arteriolar smooth muscle in rats. Am. J. Physiol. 1993; 265:R726-R732 [Abstract/Free Full Text]
• Evans G. W. The effect of chromium picolinate on insulin controlled parameters in humans. Int. J. Biosocial. Med. Res. 1989; 11:163-180
• Evans G. W., Puchnik D. J. Composition and biological activity of chromium-pyridine carboxylate complexes. J. Inorganic Biochem. 1993; 49:177-187
• Freund H., Atamian S., Fischer F.E.P. Chromium deficiency during total parenteral nutrition. J. Am. Med. Assoc. 1979; 241:496-498 [Abstract]
• Grynkiewicz G., Poenie M., Tsien R. Y. A new generation of Ca²⁺ indicators with greatly improved fluorescent properties. J. Biol. Chem. 1985; 260:3440-3450 [Abstract/Free Full Text]
• Hasten D. L., Rome E. P., Franks B. D., Hegsted M. Effects of chromium picolinate on beginning weight training students. Int. J. Sports Nut. 1992; 2:343-350
• JeeJeebhoy K. N., Chu R. C., Marliss E. B., Greenberg G. R., Bruce-Robertson A. Chromium deficiency, glucose intolerance and neuropathy reversed by chromium supplementation in a patient receiving long-term total parenteral nutrition. Am. J. Clin. Nutr. 1977; 30:531-538 [Abstract/Free Full Text]
• Juncos, L. A., Ito, S. & Carretero, O. A. (1992) Disparate effects of insulin on isolated rabbit afferent and efferent arterioles. Hypertension 20: 403 (abs.).
• Kaats G. R., Blum K., Fisher J. A., Adelman J. A. Effects of chromium picolinate supplementation on body composition: a randomized, double-masked, placebo-controlled study. Curr. Ther. Res. 1996; 57:747-756
• Kahn A. M., Seider C. L., Allen J. C., O'Neil R. G. Insulin reduces contraction and intracellular calcium concentration in vascular smooth muscle. Hypertension 1993; 22:735-742 [Abstract/Free Full Text]
• Kim Y.-C., Zemel M. B. Insulin increases vascular smooth muscle recovery from intracellular calcium loads. Hypertension 1993; 22:74-77 [Abstract/Free Full Text]
• Kim Y.-C., Zemel M. B. Insulin stimulation of intracellular free Ca²⁺ recovery and Ca²⁺-ATPase gene expression in cultured vascular smooth-muscle cells: role of glucose 6-phosphate. Biochem. 1995; 311:555-559
• Lindemann M. D., Wood C. M., Harper A. F., Kornegay E. T., Anderson R. A. Dietary chromium picolinate additions improve gain feed and carcass characteristics in growing-finishing pigs and increase litter size in reproducing sows. J. Anim. Sci. 1995; 73:457-465 [Abstract]
• Mertz W. Chromium in human nutrition: a review. J. Nutr. 1993; 123:626-633
• Morgan, D. A., Ray, C. A., Balon, T. W. & Mark, A. L. (1992) Metformin increases insulin sensitivity and lowers arterial pressure in spontaneously hypertensive rats. Hypertension 20: 421 (abs.).
• Page T. G., Southern L. L., Ward T. L., Thompson D. L. Jr. Effect of chromium picolinate on growth and serum and carcass traits of growing-finishing pigs. J. Anim. Sci. 1993; 71:656-662 [Abstract]
• Pershadsingh H. A., Szollosi J., Benson S., Hyun W. C., Feverstein B. G., Kurtz T. W. Effects of ciglitazone on blood pressure and intracellular calcium metabolism. Hypertension 1993; 21:1020-1023 [Abstract/Free Full Text]
• Reddy S., Shehin S., Sowers J. R., Dardas G., Zemel M. B. Aortic ⁴⁵Ca flux and blood pressure regulation in streptozotocin-induced diabetic rats. J. Vasc. Med. Biol. 1990; 2:47-50
• Schwartz K., Mertz W. Chromium III and the glucose tolerance factor. Arch. Biochem. Biophys. 1959; 85:292-295
• Shehin S. E., Sowers J. R., Zemel M. B. Impaired vascular smooth muscle ⁴⁵Ca efflux and hypertension in Zucker obese rats. J. Vasc. Med. Biol. 1989; 1:278-282
• Standley P. R., Zhang F., Ram J. L., Zemel M. B., Sowers J. R. Insulin attenuates vasopressin-induced calcium transients and voltage-dependent calcium current in rat vascular smooth muscle cells. J. Clin. Invest. 1991; 88:1230-1236
• Urberg M., Zemel M. B. Evidence for synergism between chromium and nicotinic acid in the control of glucose tolerance in elderly humans. Metabolism 1987; 36:896-899 [Medline]
• Zemel M. B. Insulin resistance versus hyperinsulinemia in hypertension: insulin regulation of Ca²⁺ transport and Ca²⁺ regulation of insulin sensitivity. J. Nutr. 1995; 125:1738S-1743S
• Zemel, M. B., Bedford, B. A., Zemel, P. C., Marwah, O. & Sowers, J. R. (1988) Altercation transport in non-insulin-dependent diabetics: effects of dietary calcium. J. Hypertens. 6(suppl 4): S228-S230.
• Zemel M. B., Iannucci A., Moore J. W. Role of insulin in regulating vascular smooth muscle Ca²⁺-ATPase expression. J. Vasc. Med. Biol. 1993; 4:79-84
• Zemel M. B., Johnson B. A., Ambrozy S. A. Insulin-stimulated vascular relaxation: role of Ca²⁺-ATPase. Am. J. Hypertens. 1992; 5:637-641 [Medline]
• Zemel M. B., Reddy S., Shehin S., Lockette W., Sowers J. R. Vascular reactivity in Zucker obese rats. Role of insulin resistance. J. Vasc. Med. Biol. 1990; 2:81-85
• Zemel M. B., Reddy S., Sowers J. R. Insulin attenuation of vasoconstrictor responses to phenylephrine in Zucker lean and obese rats. Am. J. Hypertens. 1991; 4:537-539 [Medline]

0022-3166/98 $3.00 ©1998 American Society for Nutritional Sciences