S. Solomon

Insulin dynamically regulates calmodulin gene expression by sequential o-glycosylation and phosphorylation of sp1 and its subcellular compartmentalization in liver cells.

O-Glycosylation and phosphorylation of Sp1 are thought to modulate the expression of a number of genes in normal and diabetic state. Sp1 is an obligatory transcription factor for constitutive and insulin-responsive expression of the calmodulin gene (Majumdar, G., Harmon, A., Candelaria, R., Martinez-Hernandez, A., Raghow, R., and Solomon, S. S. (2003) Am. J. Physiol. 285, E584-E591). Here we report the temporal dynamics of accumulation of total, O-GlcNAc-modified, and phosphorylated Sp1 in H-411E hepatoma cells by immunohistochemistry with monospecific antibodies, confocal microscopy, and matrix-assisted laser desorption and ionization-time of flight mass spectrometry. Insulin elicited sequential and reciprocal post-translational modifications of Sp1. The O-glycosylation of Sp1 and its nuclear accumulation induced by insulin peaked early (∼30 min), followed by a steady decline of O-GlcNAc-modified Sp1 to negligible levels by 240 min. The accumulation of phosphorylated Sp1 in the nuclei of insulin-treated cells showed an opposite pattern, increasing steadily until reaching a maximum around 240 min after treatment. Analyses of the total, O-GlcNAc-modified, or phosphorylated Sp1 by Western blot and mass spectrometry corroborated the sequential and reciprocal control of post-translational modifications of Sp1 in response to insulin. Treatment of cells with streptozotocin (a potent inhibitor of O-GlcNAcase) led to hyperglycosylation of Sp1 that failed to be significantly phosphorylated. The mass spectrometry data indicated that a number of common serine residues of Sp1 undergo time-dependent, reciprocal O-glycosylation and phosphorylation, paralleling its rapid translocation from cytoplasm to the nucleus. Later, changes in the steady state levels of phosphorylated Sp1 mimicked the enhanced steady state levels of calmodulin mRNA seen after insulin treatment. Thus, O-glycosylation of Sp1 appears to be critical for its localization into the nucleus, where it undergoes obligatory phosphorylation that is needed for Sp1 to activate calmodulin gene expression.

Perifusion of Isolated Rat Adipose Cells: MODULATION OF LIPOLYSIS BY ADENOSINE

Incubation of isolated rat epididymal fat cells is associated with the accumulation of adenosine in the incubation medium. To more clearly define the effect of adenosine on lipolysis, isolated rat epididymal adipocytes were studied with the perifusion system. Various combinations of epinephrine, adenosine, and adenosine deaminase were perifused through the adipocytes. Exogenous adenosine, 0.001-10.0 muM, had no discernible influence upon unstimulated lipolysis; but exogenous adenosine inhibited epinephrine-sensitive lipolysis in a concentration-dependent manner. Cells perifused with 0.3 muM epinephrine plus 0.001 muM adenosine did not show any impairment of the lipolytic response to 0.3 muM epinephrine alone. Adenosine, 0.01 muM, inhibited the response to epinephrine by 50%; response to 0.3 muM epinephrine plus 0.1 muM adenosine was similar to the basal rate. Perifusion with adenosine deaminase significantly increased basal lipolysis to 30% of the epinephrine response. Adenosine deaminase and epinephrine were synergistic in stimulating lipolysis to 180% of the response to epinephrine alone. Isolated fat cells were incubated for 30 min, and the cell-free used medium was perifused through fresh fat cells. Epinephrine in used medium was less effective in promoting lipolysis than epinephrine in fresh buffer. High-pressure liquid chromatography identified adenosine in the used medium. Bovine serum albumin possessed adenosine deaminase activity but accounted for negligible conversion of adenosine to inosine. Adenosine is shown to have a modulating effect upon basal and hormone-stimulated lipolysis in the perifusion system. Sufficient endogenous adenosine (<0.01 muM) is present to maximally affect basal lipolysis. Hormone-stimulated lipolysis, although inhibited somewhat by endogenous adenosine, requires the addition of exogenous adenosine for complete inhibition.

Elevated Growth Hormone Levels and Insulin Resistance in Patients with Cirrhosis of the Liver

Carbohydrate intolerance is frequently seen in patients with hepatic cirrhosis. To study the role of the counter regulitory hormones, glucagon, cortisol and growth hormone in this disease, these hormones were measured in 11 patients with hepatic cirrhosis and six controls during a 4-hour oral glucose tolerance test (OGTT) and in five normal and cirrhotic subjects during steady-state plasma insulin and glucose concentrations (SSPGI) achieved with the euglycemic clamp technique. Fasting plasma glucose was 103 ± 4.3 mgldl in cirrhotics and 88 ± 3.3 mg/dl in controls (p <0.00l). Immunoreactive insulin (IRI) was 24.3 pU/ml in cirrhotics and 12.7 ± 2.2 pUlml in controls (p < 0.001); immunoreactive glucagon (IRG) was 263 f 30 pg/ml in cirrhotics and 122 ± 17.5 pg/ml in controls (p < 0.001); serum growth hormone (GH) was 4.4 ± 0.9 ng/ml in cirrhotics and 0.5 ± 0.1 ng/ml in controls (p < 0.001). During OGTT, the 2-hour glucose concentration was 201 ± 9.7 mgldl in cirrhotic subjects and 147 ± 10.0 mg/dl in controls (p < 0.001). IRG levels were suppressed by 20% of basal values in patients with cirrhosis, while controls showed 10% suppression after an oral glucose load. At 60 minutes, the serum GH was 14.7 ± 3.9 ng/ml in cirrhotics and 0.3 ± 0.1 nglml in controls (p < 0,001). The normal suppressive effect of hyperglycemia on GH secretion in controls was sharply contrasted by a paradoxical elevation of serum GH in the cirrhotic group. In the euglycemic clamp studies, plasma glucose and insulin were maintained at steady state concentrations that were almost identical in both groups. The amount of glucose metabolized (M) was 3.6 ± 0.4 mg/kg/min in cirrhosis and 6.8 ± 0.2 mg/kg/min in controls (p < 0.001). IRG levels during SSPGI were 99 ± 15 pg/ml in cirrhotics and 73 f 19 pg/ml in controls, and this difference was not statistically significant. There was no significant difference in the plasma cortisol concentrations in the two groups. GH, however, peaked at 18 ± 2.1 ng/ml in the cirrhotics during SSPGI, while it was only 1.6 ± 0.2 ng/ml in controls ( p < 0.001). It was concluded, therefore, that patients with hepatic cirrhosis have significant insulin resistance as shown by a reduction in the insulinmediated glucose utilization rates. The paradoxical elevations of serum GH concentrations may play a role in the insulin resistance and glucose intolerance of cirrhosis.

Insulin Degradation by Mononuclear Cells

Mononuclear cells from peripheral blood possess insulin receptors that are altered in number or binding affinity in certain metabolic diseases as obesity. The monocyte, and not the lymphocyte, is the cell with the capacity to specifically bind insulin. Furthermore, this binding appears to mirror the receptor status on such insulin target tissues as liver, muscle, and fat. Since liver, muscle, and fat also degrade insulin, mononuclear cells from the blood of normal volunteers were examined for insulin-degrading activity. Intact cells were incubated with 125I-insulin and the amount of degraded insulin was measured by the trichloroacetic acid-precipitation technique. Insulin-degrading activity increased when the number of cells and the time of incubation were increased. Total insulin binding behaved in a similar fashion. Very little degradation was seen at 4° or 15°. The Km for insulin-degrading activity was 7.03 × 10−8 M. Homogenized mononuclear cells degraded two to five times more insulin than did intact cells and also demonstrated cell concentration, time, and temperature dependence for degradation. The Km of degradation for homogenized mononuclear cells was 2.2 × 108 M. Subcellular fractionation revealed significant degrading activity in the 100,000 × g supernatant, but little activity in the 100,000 × g pellet. A purified lymphocyte preparation did not bind insulin and contained little insulin-degrading activity.

Does Therapy With Anti–TNF-α Improve Glucose Tolerance and Control in Patients With Type 2 Diabetes?

Type 2 diabetes is associated with insulin resistance (IR). IR is partially caused by the cytokine tumor necrosis factor-α (TNF-α), which is produced in inflammatory fat tissue in muscle, liver, and adipose tissue. Inflamed abdominal fat releases adipokines and inflammatory cytokines, one of which is TNF-α. Past experimental studies using anti–TNF-α therapy in type 2 diabetic patients have been limited in both time and dosage because of the toxicity of these agents. We designed a retrospective study in which anti–TNF-α agents were used in larger therapeutic doses for up to 10 years in patients with rheumatoid arthritis (RA) and Crohns disease (CR). We assessed the effects of this treatment on control of type 2 diabetes. Eight Veterans Affairs patients with RA or CR and type 2 diabetes and a matched group of control patients with both …

The insulin-like activity of cyclic nucleotides and their inhibition by caffeine on the isolated fat cells

Abstract In the isolated fat cell system, the cyclic nucleotides of adenosine, cytidine, inosine, thymidine and uridine stimulated glucose conversion to CO2 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.

Composite pheochromocytoma-ganglioneuroma: a rare experiment of nature.

OBJECTIVE To present a rare case of composite pheochromocytoma-ganglioneuroma (Pheo-GN) of the adrenal medulla, review the related literature, and discuss the clinical features, pathologic findings, behavior, and management of such tumors. METHODS A case report of a patient with composite Pheo-GN of the adrenal gland is presented. Using the online database PUBMED, we searched and analyzed all cases of composite pheochromocytoma reported in the English-language literature during the past 70 years. RESULTS On computed tomography, a 61-year-old man was incidentally found to have a 3.8-cm nonadenomatous right adrenal lesion. Adrenalectomy revealed a 5-cm mass consistent with composite Pheo-GN. To date, 45 cases of composite pheochromocytomas have been reported during the past 70 years, 71% of which coexisted with ganglioneuromas. These tumors occurred with approximately equal frequency in male and female patients, the majority of whom were from 40 to 60 years old. Only 14 cases have been reported in the United States. Bilateral tumors were found in 3 cases. The mean size was 4 to 6 cm. Preoperatively, functional evidence was found in 76.3% of all composite pheochromocytomas (and in 67% of Pheo-GN). Only one Pheo-GN was found to have liver metastatic lesions at the time of autopsy; the rest were not aggressive. CONCLUSION To our knowledge, this is the first literature review describing the characteristics and behavior of all reported cases of composite pheochromocytomas, with an emphasis on those with ganglioneuromas. Composite pheochromocytoma is a rare variant of a relatively uncommon disease diagnosed by pathologists only. Fortunately, the treatment of such an entity remains the same as for any pheochromocytoma.

Insulin Binding and Degradation by Muscles from Streptozotocin-diabetic Rats

Insulin degradation by muscle was examined in normal, streptozotocin-induced diabetic rats, and diabetic rats treated with insulin. Insulin degradation by the 100,000 × g supernatant fractions was identical in all three groups, but insulin metabolism by the intact epitrochlaris muscle was significantly increased in diabetic animals. Insulin treatment of the diabetic animals partially restored the activity toward normal. Specific binding of 125I-insulin to the intact muscles was also increased in the diabetic animals. Streptozotocin diabetes, therefore, increased the binding and degradation of insulin by intact muscle but did not alter the insulin degradation by the total soluble intracellular degradative activity.

The Glucose Intolerance of Acute Pancreatitis: Hormonal Response to Arginine

Patients with acute pancreatitis were studied by arginine infusion at 48–72 h, 7–10 days, and 18–21 days after onset of their illness. Plasma glucose, insulin, and glucagon values were determined. Acute pancreatitis was characterized by fasting hyperglycemia and hyperglucagonemia, associated with relative hypoinsulinemia. Arginine stimulation early in the disease (48–72 h) demonstrated hyperglycemia and hyperglucagonemia, which normalized by 18–21 days. Both phases of the normal biphasic insulin response to arginine were decreased during the initial arginine infusion. By 18–21 days, although the first phase was completely normal, the second phase of insulin secretion remained depressed. Acute pancreatitis is associated with damage to both the endocrine and exocrine pancreas. Glucose intolerance seen with this disease appears to be the result of hyperglucagonemia and relative hypoinsulinemia. Although the healing process at 3 wk is associated with return of plasma glucose and glucagon concentrations to normal, the impaired second phase insulin secretion persists.

Inhibitor of Calmodulin and cAMP Phosphodiesterase Activity in BB Rats

Diabetes mellitus in humans is associated with increased plasma and tissue levels of cAMP and decreased cAMP phosphodiesterase (PDE) activity. Calmodulin (CM) is a low-molecular-weight protein essential for activation of cAMP PDE. The inhibitor (INH) is a low-molecular-weight substance that inhibits the activity of CM in multiple systems, including PDE. Spontaneously diabetic BB rats (SDR) and their nondiabetic littermates (NDR) were used in this study. Holtzman rats were rendered diabetic by streptozocin (STZ). STZ-induced diabetic rats (STZ-DR) and BB rats werestudied with and without the benefit of insulin therapy. Calmodulin was assayed both by bioassay and by specific radioimmunoassay. The inhibitor was bioassayed by its ability to inhibit CM-activated PDE. Results showed that both spontaneous and STZ-induced diabetes are associated with a decrease in activity of the low-Michaelis constant (Km) cAMP PDE in the liver (39%, SDR; 70% STZ-DR). Calmodulin activity was also decreased in the livers of both animals (13%, SDR; 68%, STZ-DR). Similar data were obtained for NDRs. The inhibitor, on the other hand, was increased in the livers of untreated SDRs and STZ-DRs (155%, SDR; 125%, STZ-DR). No change was noted for NDRs. All these changes were restored toward normal after treatment with insulin. These data suggest that in diabetes the defect in the cAMP PDE-CM-INH system is demonstrated in both an environmental model, as illustrated by STZ-DRs, and a genetic model, as shown by SDRs and NDRs. The inhibitor activity, however, is not changed significantly in NDRs. We speculate that the inhibitor activity plays a role in dictating whether the genetic NDR will or will not become clinically diabetic.