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Upon activation by phytohemagglutine (PHA), T-lymphocytes (T-cells) express receptors for growth factors, insulin, IGF-1 and IL2 and become insulin sensitive. Diabetic ketoacidosis (DKA) is associated with in vivo emergence of these growth factor receptors without incubation with PHA. As DKA consists of multiple metabolic alterations, in addition to hyperglycemia, we investigated the in vitro effect of different concentrations of glucose (5, 15, and 30 mM) in isolated CD4 of human T-cells at various time intervals (0, 24, 48, and 72 h). Hyperglycemia, but not euglycemia, resulted in de novo emergence of growth factor receptors in a dose- and time-dependent fashion. The activation was also associated with incremental changes in GLUT 4, IRS-1, proinflammatory cytokines, and oxidative stress components. We propose that activation of T-cells with development of insulin receptors in hyperglycemic conditions may serve as a mechanism for control of glucose entry into these cells, thus, protecting them against glucose toxicity.

Diabetic conditions are associated with hyperglycemia and hyperlipidemia, but the role of saturated fatty acids (SFA) vs. unsaturated fatty acids (UFA) in activation of T-lymphocytes and human aortic endothelial cells (HAEC) is not known. We investigated in vitro effects of various concentrations of SFA (palmitate) and UFA (oleic, linoleic, linolenic, and arachidonic) acids in activation of these cells. These cells in presence of palmitate, but not UFA, exhibited time, and concentration-dependent emergence of insulin receptors, GLUT 4 expression, generation of ROS, cytokines, lipid peroxidation, and IRS-1. We conclude that both T-lymphocytes and HAEC share common characteristics in exhibiting activation of these cells to palmitate, but not to UFA, by developing insulin receptors and becoming insulin responsive tissues, a hitherto unknown response to palmitate. We hypothesize that these events may serve as protective defense mechanisms against acute effects of glucotoxicity and lipotoxicity in these cells.

T-lymphocytes (T-cells) are unique in that unlike monocytes, they have no insulin receptors, and are insulin insensitive, but upon activation with antigens develop insulin, IGF-1, and IL-2 receptors, and become insulin sensitive tissues. In vivo activation of these cells has now been demonstrated in patients with diabetic ketoacidosis. We analyzed the genomics and proteomics of activated and non-activated CD4+ and CD8+ T-cells of normal subjects using Affymetrix microarray gene chips and proteomes by SELDI-TOF mass spectrometry analysis. Genes for IL-2, insulin, and IGF-1 receptors were increased at least 2-fold in activated vs non-activated T-cells. Using an expression array containing the entire human genome of 39,500 genes, we evaluated approximately 27,000 genes relevant in physiologic and cellular ontologies. Of these, approximately 10,500 genes were increased in activated cells, compared to about 7000, which were decreased, and approximately 9500, which were unchanged. Among activated ontologies were signal transduction pathways such as IRS-1, IRS-2, Akt, and glycolytic pathways. To our knowledge this is the first report of an hitherto unreported event. Possible implications of these processes are discussed in the light of their physiological significance.

Diabetic ketoacidosis (DKA) is an inflammatory state associated with immune responses in polymorphonuclear cells (PMN). Activation of subgroup of T-lymphocytes in PMN of DKA patients, however, is not known. We studied in vivo activation of CD4 and CD8 lymphocytes by measuring de novo growth factor receptor for insulin, IGF-1, and IL-2 in eight patients on admission and at resolution of DKA, and compared them with matched controls. The presence of these receptors was demonstrated in all patients' lymphocytes on admission, but not in control subjects. This event was associated with increased levels of thiobarbituric acid-reacting material and dichlorofluorescien, as markers of oxidative stress. Based on these new findings and works in the literature, we hypothesize that hyperglycemia/ketosis results in increased reactive oxygen species, leading to increased levels of cytokines and emergence of growth factor receptors. We propose DKA changes the T-lymphocytes to insulin sensitive tissues as a compensatory mechanism.

To study the mechanism of degradation of glucagon with purified insulin-glucagon protease, glucagon was reacted with the enzyme at various times of incubation. The proteolysis was followed by the production of flourescamine-reacting material as well as reaction with dansyl chloride, cleavage by acid hydrolysis, and identification by thin layer chromatography. For quantitative measurement of the degradation products, [ 14 C] dansyl derivatives were produced, identified by autoradiography, and counted. In the degradation products in addition to histidine, the dansyl derivatives of tyrosine, phenylalanine, two leucines, alanine and lysine were identified. For comparison, glucagon was also reacted with chymotrypsin and the degradation products consisted of threonine, serine, two leucines and valine. Thus, insulin-glucagon protease degrades glucagon in a manner distinct from that of chymotrypsin.

To assess the role of various modulators of insulin processing on cell-associated A14-125I-insulin intermediates in human fibroblasts, we have studied the effect of N-ethylmaleimide (NEM), chloroquine, bacitracin, dansylcadavarine, and phenylarsine oxide on generation of these intermediate products with the use of HPLC. NEM completely inhibited generation of intermediate peaks or iodotyrosine. Chloroquine inhibited conversion of A14-125I-insulin to iodotyrosine by about 75 percent and the remaining A14-125I-insulin was not susceptible to acid wash. Bacitracin, dansylcadavarine, and phenylarsine oxide, on the other hand, stimulated formation of intermediate products with concomitant inhibition of iodotyrosine formation. We conclude that there are at least three components of insulin degradation in human fibroblasts. These include the sulfhydryl group inhibitor-sensitive, the intracellular chloroquine-sensitive, and membrane site inhibitor-sensitive components.

Summary Corticosterone synthesis in isolated rat adrenal cells in response to ACTH and dibutyryl cyclic-AMP (dcAMP) and formation of cyclic-AMP (cAMP) from prelabeled 8- 14 C-adenine have been studied in the presence of varying Ca ++ concentrations. At physiologic concentrations of ACTH steroidogenesis is proportional to Ca ++ . In the absence of Ca ++ , 10 4 higher concentrations of ACTH is necessary. cAMP formation is detectable only at supraphysiologic concentrations of ACTH. Maximum corticosterone formation in response to dcAMP is dependent upon Ca ++ concentration. Action of Ca ++ may be both prior to and following elaboration of the second messenger; other compounds besides cAMP may be the mediators of ACTH action at physiologic concentrations.

Corticosterone production in isolated adrenal cells (IAC) of rat has been measured in response to ACTH or ribonucleoside 3′,5′-cyclic phosphate of adenosine (c-AMP), guanosine (c-GMP), inosine (c-IMP) and N 6 -2′-0 dibutyryl adenosine monophosphate (dc-AMP) in the presence and absence of caffeine. Caffeine inhibited ACTH-induced steroidogenesis in a manner independent of its effect on PDE. Study of PDE in whole adrenal homogenate showed hydrolysis of c-AMP, c-GMP and c-IMP but not of dc-AMP and other cyclic nucleotides. No PDE activity was demonstrable in IAC. High sensitivity of IAC to minute quantities of ACTH and various cyclic nucleotides may be due in part to lack of PDE activity in these preparations.

The effects of the second generation sulfonylurea, glyburide, on the pyruvate dehydrogenase multienzyme complex (PDC) of rat myocardial tissue were examined using rat ventricular slices and isolated mitochondria. Therapeutic concentrations (10(-7) to 10(-6)M) of glyburide produced a 30% increase in the decarboxylation of [1(-14)C] pyruvate by the PDC of ventricular tissue. Addition of glyburide to intact rat heart mitochondria stimulated activity of the PDC in a time- and concentration-dependent manner. Half-maximal stimulation of the enzyme occurred with 6 X 10(-5)M glyburide and maximal activation of the enzyme was achieved with 1 X 10(-4)M glyburide. At the height of stimulation, PDC activities were 6-fold greater than those observed under control conditions with succinate alone. When mitochondria were disrupted by sonication or freeze-thawing, glyburide produced no stimulation of pyruvate decarboxylation. We conclude that glyburide directly stimulates the decarboxylation of pyruvate by the PDC of the myocardium. Furthermore, the presence of intact mitochondria is necessary for the stimulatory action of glyburide on the PDC.

Membranes of isolated adrenocortical cells have binding sites for [ 3 H] d-α-tocopherol which exhibit specificity, saturability, time and temperature dependence, and reversibility of binding. The apparent equilibrium association constants (4 × 10 −5 M and 7 × 10 −6 M) for binding suggest that these binding sites are physiologically significant. Stability data indicate that binding sites are at least partly protein in constitution.

Binding and degradation of A14125I-Insulin as well as the effect of insulin on pyruvate dehydrogenase (PDH) activation were studied in non-stimulated and phytohemagglutinin (PHA)-stimulated thymic-derived lymphocytes (T-lymphocytes) of man under varying conditions of time, temperature, and cell concentration. The nonstimulated viable T-lymphocytes exhibited neither binding, degradation, nor PDH activation in response to insulin. With PHA stimulation, a time and temperature-dependent binding was noted in T-lymphocytes which paralleled the appearance of cell-associated insulin degrading activity. Concomitant with the emergence of insulin binding and degrading activities in these cells, PDH activation was observed which was responsive to as little as 5.0 microU/ml of insulin. We conclude that in PHA-activated T-lymphocytes of man the process of insulin binding and degradation is closely related to insulin sensitive activation of PDH. These activated cells may serve as a useful model in which to study insulin binding and processing, as well as effects of insulin on postreceptor events.

In the isolated fat cell system, the cyclic nucleotides of adenosine, cytidine, inosine, thymidine and uridine stimulated glucose conversion to CO 2 and lipids. The dibutyryl cyclic 3′,5′-AMP and cyclic guanosine 3′,5′-nucleotide as well as caffeine, however, exhibiyted a dose-related inhibitory effect on glucose oxidation. Thus, two groups of cyclic nucleotides have been identified, one mimicking and one opposing the insulin action in regard to glucose oxidation. Based on the above data, it is suggested that the mediator of lipolytic hormones may resemble the dibutyryl analog of cyclic AMP rather than cyclic AMP, and that a separate group of cyclic nucleotides resembling cyclic 3′,5′-thymidine monophosphate may act as mediators of insulin action.