Intact lyophilized nuclei are obtainable from a variety of tissues, either in situ or in culture, by freezing at -156 degrees C, drying at -25 degrees C, and mechanical disassociation in glycerol at 2 degrees C. Centrifugal separation of nuclei is accomplished in an 85 : 15 by volume mixture of glycerol and 3-chloro-1,2 propanediol at 2 degrees C. The method gives homogeneous nuclear preparations in high yield with preservation of labile and water-soluble constituents.
Phosphoinositide (PI) 3-kinase is involved in insulin-mediated effects on glucose uptake, lipid deposition, and adiponectin secretion from adipocytes. Genetic disruption of the p85alpha regulatory subunit of PI 3-kinase increases insulin sensitivity, whereas elevated p85alpha levels are associated with insulin resistance through PI 3-kinase-dependent and -independent mechanisms. Adipose tissue plays a critical role in the antagonistic effects of growth hormone (GH) on insulin actions on carbohydrate and lipid metabolism through changes in gene transcription. The objective of this study was to assess the role of the p85alpha subunit of PI 3-kinase and PI 3-kinase signaling in GH-mediated insulin resistance in adipose tissue. To do this, p85alpha mRNA and protein expression and insulin receptor substrate (IRS)-1-associated PI 3-kinase activity were measured in white adipose tissue (WAT) of mice with GH excess, deficiency, and sufficiency. Additional studies using 3T3-F442A cells were conducted to confirm direct effects of GH on free p85alpha protein abundance. We found that p85alpha expression 1) is decreased in WAT from mice with isolated GH deficiency, 2) is increased in WAT from mice with chronic GH excess, 3) is acutely upregulated in WAT from GH-deficient and -sufficient mice after GH administration, and 4) is directly upregulated by GH in 3T3-F442A adipocytes. The insulin-induced increase in PI 3-kinase activity was robust in mice with GH deficiency, but not in mice with GH excess. In conclusion, GH regulates p85alpha expression and PI 3-kinase activity in WAT and provides a potential explanation for 1) the insulin hypersensitivity and associated obesity and hyperadiponectinemia of GH-deficient mice and 2) the insulin resistance and associated reduced fat mass and hypoadiponectinemia of mice with GH excess.
A new sequential gating perifusion technique was employed to investigate secretion vesicle margination and granule lysis in islets isolated from 2- and 18-month-old Fischer 344 rats. The technique is based on sequential perifusion (periods A, B, and C) of isolated islets with glucose (30, 165, or 300 mg/dl) in the presence of sodium isethionate, an inhibitor of granule lysis, followed thereafter by trifluoperazine, an inhibitor of secretion vesicle margination, and glucose (300 mg/dl) or isobutylmethylxanthine (IBMX; 400 μM). When glucose was employed during period A to marginate secretion vesicles to the plasma membrane, subsequent glucose- and IBMX-induced insulin release (period C) was depressed in islets from 18-monthold rats [maximal increase above the basal rate of release (Δmax), 9 ± 2 nUμm-min] compared to that in the 2-monthold animals (Δmax, –19 ± 3 nUμmmin). With glyburide (400 μM) used to induce secretion vesicle margination, glucose- and IBMX-induced insulin release was the same in young and old animals (Δmax, 14 ± 3 and 15 ± 3 nUμmi-min, respectively). Insulin release was then studied as a function of secretion vesicle margination at the plasma membrane by measuring somatostatin (SRIF) receptor recruitment. The islets from older animals must be stimulated with 300 mg/dl glucose to attain the same level of SRIF binding as in islets isolated from younger animals stimulated with 150–165 mg/dl glucose. Insulin release per unit SRIF binding was identical in young and old animals (65 and 69 nU/liter fmol SRIF binding), indicating normal lysis of marginated secretion granules. These studies implicate glucose- induced secretion vesicle margination as the site of impairment in age-related insulin release. (Endocrinology119: 827–832, 1986)
It may now be possible to identify certain intracellular events that impact specifically on secretion-granule fusion to the plasma membrane or on granule lysis. Secretion vesicles in isolated rat islets appear to translocate somatostatin (SRIF) receptors from the Golgi apparatus to the plasma membrane. We have proposed that secretion granule fusion to the plasma membrane can be determined by measuring recruitment of SRIF receptors to the surface membrane. Granule lysis can be assessed by measuring insulin release. To activate cyclic AMP (cAMP)-dependent pathways, we employed isobutylmethylxanthine (IBMX, 400 μM), glucagon (10 μM), and forskolin (20 μM), a diterpene activator of adenylate cyclase. These agents evoked rapid release of insulin (from 0.41 ± 0.02 to 1.88 ± 0.02; 0.41 ± 0.02 to 1.93 ± 0.08; and 0.41 ± 0.02 to 1.66 ± 0.03 μU/islet/ min, respectively, P < 0.001). There was no concomitant recruitment of SRIF receptors. Somatostatin (10 μg/ml), which inhibits cAMP-stimulated protein phosphorylation, suppresses insulin release evoked by IBMX, glucagon, or forskolin (inhibition: 80, 75, or 82%, respectively). In contrast, trifluoperazine (10 μM), an inhibitor of calmodulin, did not suppress insulin release induced through cAMP-dependent pathways. Trifluoperazine suppresses glucose-induced insulin release and the recruitment of SRIF receptors to the surface membrane, suggesting the possible role of calmodulin in promoting secretion-granule fusion with the plasma membrane. These data suggest that granule fusion to the plasma membrane may be a calmodulin-directed function. In contrast, the major site of action of CAMP in polypeptide hormone secretion may be in promoting lysis of secretion granules and associated hormone discharge.
Early molecular changes of nutritionally-induced insulin resistance are still enigmatic. It is also unclear if acute overnutrition alone can alter insulin signaling in humans or if the macronutrient composition of the diet can modulate such effects.To investigate the molecular correlates of metabolic adaptation to either high-carbohydrate (HC) or high-fat (HF) overfeeding, we conducted overfeeding studies in 21 healthy lean (BMI < 25) individuals (10 women, 11 men), age 20-45, with normal glucose metabolism and no family history of diabetes. Subjects were studied first following a 5-day eucaloric (EC) diet (30% fat, 50% CHO, 20% protein) and then in a counter balanced manner after 5 days of 40% overfeeding of both a HC (20% fat, 60% CHO) diet and a HF (50% fat, 30% CHO) diet. At the end of each diet phase, in vivo insulin sensitivity was assessed using the hyperinsulinemic-euglycemic clamp technique. Ex vivo insulin action was measured from skeletal muscle tissue samples obtained 15 minutes after insulin infusion was initiated.Overall there was no change in whole-body insulin sensitivity as measured by glucose disposal rate (GDR, EC: 12.1 ± 4.7; HC: 10.9 ± 2.7; HF: 10.8 ± 3.4). Assessment of skeletal muscle insulin signaling demonstrated increased tyrosine phosphorylation of IRS-1 (p < 0.001) and increased IRS-1-associated phosphatidylinositol 3 (PI 3)-kinase activity (p < 0.001) following HC overfeeding. In contrast, HF overfeeding increased skeletal muscle serine phosophorylation of IRS-1 (p < 0.001) and increased total expression of p85α (P < 0.001).We conclude that acute bouts of overnutrition lead to changes at the cellular level before whole-body insulin sensitivity is altered. On a signaling level, HC overfeeding resulted in changes compatible with increased insulin sensitivity. In contrast, molecular changes in HF overfeeding were compatible with a reduced insulin sensitivity.
Insulin stimulates tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) and Shc inRat1 fibroblasts overexpressing wild type insulin receptors.We investigated the relative role of IRS-1 and Shc in insulin activation of guanine nucleotide releasing factor (GNRF) and p21"GTP formation.The time course of insulinstimulated tyrosine phosphorylation of IRS-1 was rapid, whereas Shc phosphorylation was relatively slow.Growth factor receptor bound protein-2 (Grb2) associated with IRS-1 rapidly and gradually dissociated after 5 min, whereas Grb2 association with Shc was slower and reached a maximum at 10 min after insulin stimulation.Thus, the kinetics of Grb2 association with IRS-1 and Shc corresponded closely to the time course of tyrosine phosphorylation of IRS-1 and Shc, respectively.Importantly, 3-13-fold more Grb2 was associated with Shc than with IRS-1.In addition, the kinetics of insulinstimulated GNRF activity and p21"GTP formation corresponded more closely to the time course of Shc phosphorylation than to the kinetics of IRS-1 phosphorylation.Furthermore, immunoprecipitation of Shc proteins from cell lysates of insulin-stimulated cells removed 67% of the GNRF activity, whereas precipitation of IRS-1 had a negligible effect on GNRF activity.Thus, although both IRS-1 and Shc associate with Grb2, the current results indicate that Shc plays a more important role than IRS-1 in insulin stimulation of GNRF activity and subsequent p21"GTP formation.Insulin binding to the extracellular a-subunits activates the intrinsic tyrosine kinase activity of the cytoplasmic portion of the insulin receptor P-subunit (1).One early molecular event linking the receptor kinase to insulin's biologic actions is tyrosine phosphorylation of IRS-1' (2).Current evidence suggests that IRS-1 acts as a multisite "docking" protein by binding to downstream signal-transducing molecules (3).The physical in-
Insulin's interaction with its receptor initiates a multitude of cellular effects on metabolism, growth, and differentiation. We recently described an insulin-mediated inhibition of nuclear protein phosphatase 2A (PP-2A), which is associated with an increase in phosphorylation of the transcription factor cAMP response element-binding protein. To clarify the role of nuclear PP-2A inhibition in the insulin signaling cascade, we examined the regulation of this phosphatase activity by insulin in Rat-1 fibroblasts overexpressing normal (HIRc) or mutant human insulin receptors (delta CT cells, deletion of a 43-amino acid C-terminal domain). The delta CT cells represent an excellent model of impaired metabolic and intact mitogenic action of insulin. Insulin inhibited nuclear PP-2A activity and enhanced cAMP response element-binding protein phosphorylation in HIRc cells, but not in delta CT cells. The delta CT cells exhibited normal ras activation and blunted mitogen-activating protein kinase phosphorylation and activation in response to insulin (16-fold in HIRc cells vs. 3-fold in delta CT cells), indicating that the mitogen-activating protein kinase pathway is important for the regulation of nuclear PP-2A activity by insulin. We conclude that insulin inhibits nuclear PP-2A activity, and that the carboxy-terminal domain of the insulin receptor is important for this effect.
