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K E Sussman

Publications and source records attributed to K E Sussman.

At least 19 recordsLinked to original sources
PubMed1993

GLUT-4 phosphorylation and its intrinsic activity. Mechanism of Ca(2+)-induced inhibition of insulin-stimulated glucose transport.

In this study, we examined the influence of high levels of cytosolic calcium on phosphorylation status and function of GLUT-4 in isolated rat adipocytes. Intracellular calcium was elevated by exposing adipocytes to either extracellular ATP (1.6 mM) or thapsigargin (100 nM). Both agents increased cytosolic calcium 2-3 fold. While basal glucose uptake was unaffected, both ATP and thapsigargin reduced insulin-stimulated glucose transport by 40-70% (p < 0.05). Neither ATP nor thapsigargin affected GLUT-4 content or its translocation from the low density microsomes to the plasma membrane (PM). In contrast, GLUT-4 immunoprecipitated from the PM of adipocytes exposed to either ATP or thapsigargin was phosphorylated to a greater extent than the GLUT-4 isolated from control cells. ATP and thapsigargin also abolished insulin-stimulated dephosphorylation of GLUT-4. At the same time, GLUT-4 intrinsic activity was significantly reduced in adipocytes with high levels of cytosolic calcium (p < 0.05). Preincubation of adipocytes with cAMP antagonist, RpcAMP (10(-4) M), and calcium channel blocker, nitrendipine (30 microM), improved the ability of insulin to dephosphorylate GLUT-4 and restored insulin-stimulated GLUT-4 intrinsic activity. We conclude that elevated levels of cytosolic calcium interfere with insulin's ability to dephosphorylate GLUT-4, thus reducing its intrinsic activity.

Adenosine Triphosphate↗
PubMed1993

Inverse relationship between GLUT-4 phosphorylation and its intrinsic activity.

In this study, we examined the effect of phosphorylation on GLUT-4 function in isolated rat adipocytes. Adipocytes labeled with 32P for 2 h were incubated with parathyroid hormone (PTH) (20 ng/ml) for 60 min and then exposed to insulin (25 ng/ml) for an additional 30 min. 32P-GLUT-4 was immunoprecipitated from the plasma membrane and low density microsomal fractions, and its degree of phosphorylation was determined by autoradiography and densitometry. Results were expressed as 32P-GLUT-4 specific activity (phosphorylation/unit of protein). GLUT-4 intrinsic activity was measured using [14C]2-deoxyglucose uptake in plasma membrane vesicles. PTH significantly increased GLUT-4 phosphorylation and eliminated the insulin-stimulated dephosphorylation of GLUT-4. Western blotting revealed normal distribution of GLUT-4 before and after insulin stimulation in control and PTH-treated cells, suggesting that phosphorylation of GLUT-4 does not interfere with its recruitment to the plasma membrane. In contrast, intrinsic activity of phosphorylated GLUT-4 was significantly reduced (p < 0.01). Preincubation of adipocytes with calcium channel blocker (nitrendipine) and cyclic AMP antagonist (RpcAMP) restored GLUT-4 intrinsic activity in the PTH-treated cells. In several experiments, GLUT-4 was phosphorylated in vitro in plasma membrane vesicles isolated from normal adipocytes exposed to insulin. This in vitro phosphorylation reduced GLUT-4 intrinsic activity by approximately 35% (p < 0.01). We conclude that phosphorylation of GLUT-4 significantly impairs the ability of insulin to stimulate its intrinsic activity.

Adipose Tissue↗
PubMed1992

Calcium-induced inhibition of phosphoserine phosphatase in insulin target cells is mediated by the phosphorylation and activation of inhibitor 1.

In this study, we examined the mechanism of inhibition of phosphoserine phosphatase (PSPase) activity by elevated [Ca2+]i in insulin target cells. In in vitro studies, isolated rat adipocytes were incubated with either 40 mM K+ or parathyroid hormone (PTH) (20 ng/ml) for 1 h. In in vivo studies, rats were injected with PTH (three hourly injections of 40 micrograms intraperitoneally) prior to isolation of either adipocytes or skeletal muscle. Under these conditions, intracellular [Ca2+]i changed from 100 +/- 8.7 to 263 +/- 10.5 nM. There was a concomitant 30% decrease in adipocyte PSPase activity and a 35% decrease in skeletal muscle PSPase activity, assayed using 32P-labeled phosphorylase "a" as a substrate. The inhibition of PSPase was accompanied by a 60% increase in adipocytes (p less than 0.05) and a 118% increase (p less than 0.01) in skeletal muscle inhibitor 1 (I1) activities, respectively. Since I1 is active only in the phosphorylated state, we studied the effect of [Ca2+]i on I1 phosphorylation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of heat treated extracts immunoprecipitated with I1 antibody revealed significant increase in 32P incorporation (45-60%, p less than 0.05) into I1 protein in cells with elevated [Ca2+]i. Nitrendipine, a calcium channel blocker, completely prevented increases in I1 phosphorylation and activity in cells exposed to K+ but was only partially effective in the PTH-treated cells. In contrast, a cyclic AMP antagonist, RpcAMP, prevented both the K(+)-and the PTH-induced increases in I1 phosphorylation and activity, even though it failed to block the elevations in [Ca2+]i in these cells. We conclude that [Ca2+]i-induced and cAMP-mediated phosphorylation and activation of I1 results in inhibition of PSPase activity in insulin target cells. The inhibition of PSPases may cause inappropriate serine dephosphorylation of substrates of insulin action resulting in insulin resistance.

Adipose Tissue↗
PubMed1992

Na+/K(+)-ATPase activity and its alpha II subunit gene expression in rat skeletal muscle: influence of diabetes, fasting, and refeeding.

We have examined the effects of diabetes, fasting, and refeeding on Na+/K(+)-adenosine triphosphatase (ATPase) activity and its catalytic alpha II subunit gene expression in skeletal muscle. Two hypoinsulinemic states, streptozotocin-induced diabetes and 48-hour fasting caused a significant decrease (P less than .05) in skeletal muscle Na+/K(+)-ATPase activity and a marked increase (P less than .01) in the levels of alpha II subunit mRNA. A decrease in enzyme activity was observed on the 2nd and the 14th day of diabetes, whereas an increase in alpha II mRNA levels was found only on the 14th day. The levels of alpha I mRNA were not affected, while the levels of mRNA of the structural beta subunit were decreased on the 14th day of diabetes. Correction of hyperglycemia with insulin restored enzyme activity and alpha II isoform mRNA levels toward normal in diabetic animals. Refeeding for 48 or 72 hours restored these parameters to normal in skeletal muscle of previously fasting rats. These observations suggest that a decrease in muscle Na+/K(+)-ATPase activity may lead to a compensatory increase in its alpha II subunit gene expression. The levels of insulin and not of glycemia appear to be critical in modulating Na+/K(+)-ATPase activity and gene expression.

Animals↗
PubMed1992

The diabetic foot problem--a failed system of health care?

It is evident that there is excess morbidity and mortality as a consequence of foot problems in patients with diabetes mellitus. Most of the data relative to foot lesions is taken from the study of subsets of diabetic patients undergoing lower extremity amputation. Such data probably do not provide accurate information relative to the incidence and/or prevalence of diabetic foot problems. Available evidence does suggest that diabetic foot care may be inadequate and the efficacy of various diagnostic and therapeutic modalities has not been proven. Advances have been made in obtaining a better understanding of the microbiology of lower extremity infected lesions both in hospitalized patients and in those subjects being followed in the outpatient setting. Attention should be directed at developing a systematic classification of foot lesions which can be universally applied. We need to understand and relate to those risk factors contributing to the development and progression of lower extremity lesions in the diabetic patient. In evaluating the efficacy of diverse diagnostic and therapeutic approaches, well-controlled clinical investigations need to be undertaken. Attention should be focussed upon both measures of process and outcome where appropriate. Finally, there needs to be recognition that the problem of the diabetic foot lesion represents a major public health challenge.

Diabetes Mellitus↗
PubMed1992

Role of cAMP in mediating effects of fasting on dephosphorylation of insulin receptor.

We studied the effect of fasting on phosphotyrosine phosphatase (PTPase) activities in particulate (PF) and cytosolic (CF) fractions of rat adipocytes and liver. PTPase activity was assessed using [32P]tyrosine insulin receptor (IR). In adipocytes, 48 h fasting significantly inhibited PTPase activity. Dephosphorylation of IR by PF and CF PTPases was reduced by 80 and 65%, respectively. Similar reductions of lesser magnitude were observed in fasted rat livers. The effect of fasting was completely reversed by either refeeding or by incubating "fasted" adipocytes for 2 h in tissue culture medium containing 5 mM glucose. Neither 20 mM glucose nor the presence of insulin influenced phosphatase activity. Because fasting is accompanied by elevated protein kinase C (PKC) and adenosine 3',5'-cyclic monophosphate (cAMP) levels, we examined their influence on adipocyte PTPases. Neither activation (1 microM 12-O-tetradecanoylphorbol-13-acetate) nor inhibition (20 microM sphingosine) of PKC affected PTPase activity. In contrast, cAMP (2 mM) significantly inhibited PTPase activity (80% inhibition at 2 h), and its effect was prevented by a cAMP antagonist RpcAMP. Fasting- and cAMP-induced inhibition of PTPase activity was restored by incubating PF with trypsin (4 micrograms/ml for 5 min), which separated the putative inhibitors from the phosphatases. We conclude that fasting-induced inhibition of PTPases is mediated by elevated cAMP levels, most likely by activating phosphatase inhibitors.

Adipose Tissue↗
PubMed1991

High levels of cytosolic free calcium inhibit dephosphorylation of insulin receptor and glycogen synthase.

Treatment of adipocytes with depolarizing concentrations of K+ (40 mM) for 60 min increased [Ca2+]i from 158 +/- 28 nM to 328 +/- 38 nM. This significantly reduced (up to 80% inhibition) dephosphorylation of insulin receptor (IR), EGF receptor (EGF-R) and glycogen synthase (GS). The calcium channel blocker, nitrendipine (30 microM), or Ca2+ free medium completely prevented K(+)-induced inhibition of phosphoprotein phosphatase (PPTase). This effect of high [Ca2+]i was completely reversible when the cells were returned into the non-depolarizing medium. Trypsin treatment (4 micrograms/ml) of the membrane fraction containing inhibited PPTase activity, restored dephosphorylation activity to normal suggesting that elevated [Ca2+]i may inhibit PPTase by promoting its association with the inhibitors. These observations indicate that dephosphorylation of IR and GS can be regulated by [Ca2+]i.

Adipose Tissue↗
PubMed1991

Regulation of GLUT-4 phosphorylation by intracellular calcium in adipocytes.

Sustained elevations in cytosolic calcium concentrations ([Ca2+]i) have been shown to render insulin target cells resistant to insulin action. In this study we examined the mechanisms of the detrimental effect of high levels of [Ca2+]i on insulin-induced 2-deoxyglucose (2-DOG) uptake. To elevate [Ca2+]i, we incubated rat adipocytes with either 40 mM potassium (K+) or 20 ng/ml PTH for 1 h for in vitro experiments and injected rats with PTH (injections of 50 micrograms, ip, every hour for 3 h) for in vivo studies. Adipocytes with elevated [Ca2+]i demonstrated a 30% decrease in insulin-stimulated 2-DOG uptake. A calcium channel blocker (nitrendipine) and a cAMP antagonist (RpcAMP) each partially restored insulin-stimulated glucose transport, but together they completely restored 2-DOG uptake. Concomitantly, we found a significant increase in phosphorylation of GLUT-4 in adipocytes with elevated [Ca2+]i. This change in GLUT-4 phosphorylation was also attenuated by nitrendipine and RpcAMP. These observations confirm that elevated [Ca2+]i diminishes insulin-stimulated glucose transport and suggest that increased phosphorylation of GLUT-4 in adipocytes with high [Ca2+]i may alter its intrinsic activity.

Adipose Tissue↗
PubMed1991

Effect of diabetes on cytosolic free Ca2+ and Na(+)-K(+)-ATPase in rat aorta.

We examined Na(+)-K(+)-ATPase activity and the levels of alpha I-, alpha II-, and beta-subunit mRNA and protein in aortic cells of diabetic rats. Diabetes was induced by streptozocin. Na(+)-K(+)-ATPase activity was significantly reduced on the 2nd day of diabetes (9.4 +/- 1.3 vs. 17.5 +/- 2.1 mumol NADH.mg-1 protein.h-1, P less than 0.05) and remained depressed on days 7 and 14. The levels of 5.3-kilobase (kb) mRNA band of the catalytic alpha II-subunit of Na(+)-K(+)-ATPase were also decreased on the 2nd day of diabetes, whereas the second band, 3.4 kb, was not affected. Both bands were significantly decreased on days 7 and 14. This was followed by a reduction in the levels of alpha II-protein (day 14). The levels of alpha I- and beta-subunit mRNA and alpha I- protein were not affected by diabetes. A decrease in Na(+)-K(+)-ATPase activity was accompanied by a significant (P less than 0.001) increase in the cytosolic free Ca2+ concentrations [( Ca2+]i) in diabetic aortic cells (221 +/- 18 nM on the 7th day and 242 +/- 17 nM on the 14th day vs. 153 +/- 7 nM in controls). These findings are consistent with the hypothesis that decreased Na(+)-K(+)-ATPase activity and gene expression in vascular smooth muscle cells with accompanied rises in [Ca2+]i may be an important pathogenetic factor in the development of hypertension and atherosclerosis in diabetes.

Animals↗
PubMed1991

Differential effects of diabetes on adipocyte and liver phosphotyrosine and phosphoserine phosphatase activities.

We examined the activities of particulate and cytosolic phosphotyrosine phosphatase (PTPase) and phosphoserine phosphatase (PSPase) in adipocytes and livers of diabetic rats. PTPase activity was assessed with [32P]tyrosine-phosphorylated insulin receptor (IR), whereas PSPase activity was assayed with [32P]serine-phosphorylated glycogen synthase. Diabetes increased adipocyte particulate PTPase activity and enhanced IR dephosphorylation by 75% on the 2nd, 93% on the 14th, and 108% on the 30th day. In contrast, cytosolic PTPase activity decreased by 78% on the 14th and 45% on the 30th day (no change on the 2nd day). Similar changes were observed with PSPase (increased activity in particulate and decreased in cytosolic). Insulin therapy for 14 or 30 days restored PTPase and PSPase activities in both fractions. Vanadate, despite rapid normalization of glycemia, restored these activities only after 30 days of therapy. Diabetes-related changes in liver PTPase activity were observed on the 14th day only. At this time, it was increased in both particulate and cytosolic fractions. There was spontaneous normalization of the liver PTPase activity at 30 days of diabetes. In contrast, liver cytosolic PSPase activity was significantly inhibited and not normalized by the 30th day of disease without therapy. In summary, diabetes appears to induce tissue-specific changes in PTPase and PSPase activities resulting in significant alterations in dephosphorylation of IR and glycogen synthase. Moreover, there appears to be a differential regulation of PTPase and PSPase activities in diabetes, particularly in the liver.

Adipose Tissue↗
PubMed1989

In vivo administration of interleukin-1 inhibits glucose-stimulated insulin release.

Recombinant interleukin-1 beta (IL-1 beta) was administered intraperitoneally for 3 days to normal C57BL/6ByJ (B6) mice. The islets from IL-1-treated and control animals were isolated and glucose-stimulated insulin secretion studied in the perifusion system. The total islet insulin content and the ultrastructure of the islets isolated from the animals treated with IL-1 did not differ from those seen in control animals. However, glucose-stimulated insulin release was significantly impaired after 3 days of in vivo administration of IL-1, either 3 micrograms/animal/day or 0.3 micrograms/animal/day. The administration of IL-1 inhibited an acute phase of glucose-induced insulin release, whereas neither basal insulin secretion nor insulin release from 10-30 min of perifusion with glucose was impaired. There was an only partial (27%) and non-significant restoration of the insulin secretory response to glucose stimulation 4 days after discontinuation of IL-1 treatment. We conclude that IL-1 administered in vivo is capable of adversely affecting pancreatic islet response to glucose stimulation. After 3 days of administration, these changes are confined to the process of insulin release, with the islet cell morphology and total insulin content being unaffected.

Animals↗
PubMed1988

Diabetes--the road ahead.

Considerable progress has been made in the acquisition of knowledge relative to diabetes and its complications. However, areas exist wherein even greater progress can be anticipated. These include: (1) defining the genetics of diabetes; (2) the development of suitable markers for early identification of those persons at risk for developing diabetes; (3) the formulation of intervention strategies for preventing or ameliorating the phenotypic expression of diabetes; (4) improving the treatment of diabetes to achieve better control; (5) the possible use of adjunct agents to prevent the chronic complications associated with long-term diabetes; and (6) the development of possible approaches to achieving a cure for diabetes including application of the new knowledge in molecular biology to gene therapy.

Diabetes Complications↗
PubMed1988

Cyclosporin-induced inhibition of insulin release. Possible role of voltage-dependent calcium transport channels.

The exposure of normal pancreatic islets to cyclosporin-A (1 microgram/ml) for 24 hr resulted in significant inhibition of glucose-induced (16.7 mM) insulin release from 197 +/- 14 microU/10 islets/15 min (control) to 103 +/- 14 microU/10 islets/15 min (Cy-A-treated islets; P less than 0.001). Cy-A did not alter insulin release in the presence of non-stimulatory (1.7 mM) or submaximally effective glucose concentrations (9.2 mM). In parallel experiments, Cy-A reduced glucose-stimulated increases in cytosolic free calcium concentrations, [Ca2+]i (217 +/- 15 nM without and 137 +/- 3 nM with Cy-A in the presence of 16.7 mM glucose, P less than 0.01). To better define the site of Cy-A action, we studied its effect on insulin release and increases in [Ca2+]i induced by either K+ (50 mM), which promotes Ca2+ influx via voltage-dependent Ca2+ channels, or by forskolin (20 microM), dibutyryl cyclic AMP (1 mM) or arachidonic acid (49 microM), all of which stimulate mobilization of intracellular Ca2+ stores. Cy-A significantly inhibited K+-induced changes (203 +/- 13 nM without and 77 +/- 6 nM with Cy-A, respectively, P less than 0.001), but not those induced by forskolin, dibutyryl cyclic AMP or arachidonic acid. These observations suggest that Cy-A inhibits insulin release by interfering with Ca2+ influx via voltage-dependent calcium channels.

Animals↗