Low Density Microsomal Membrane Fraction Research Articles

Reversal of insulin resistance in diabetic rat adipocytes by insulin therapy. Restoration of pool of glucose transporters and enhancement of glucose-transport activity.

To determine the role of insulin in reversing the insulin resistance associated with depletion of the intracellular pool of glucose transporters, streptozocin-induced diabetic rats were treated with 5 U/day s.c. of insulin for 0, 8, or 14 days. At each time point, adipose cells were isolated, and 3-O-methylglucose transport was measured in the absence and presence of 1000 microU/ml insulin. With the cytochalasin B-binding assay, concentrations of glucose transporters in the plasma and the low-density microsomal membrane fractions were determined. Eight-day insulin therapy enhanced glucose transport rate (mean +/- SE) from 0.2 +/- 0.0 to 1.1 +/- 0.1 fmol X cell-1 X min-1 in the basal state and from 0.8 +/- 0.1 to 5.5 +/- 0.4 fmol X cell-1 X min-1 in the insulin-stimulated state in untreated and treated diabetic rats, respectively; this is a 3-fold increment of glucose transport rate in both states compared with control rats. After 14-day insulin therapy, glucose-transport activity declined toward normal but still remained approximately 1.5- and 4-fold higher than control and diabetic rats, respectively. Despite the persistent enhancement of glucose transport rate, concentration of glucose transporters in the intracellular pool was restored only to its prediabetic state. Likewise, the increased concentration of glucose transporters in the plasma membranes after insulin stimulation was similar to that of control rats. Thus, we suggest that 8-14 days of insulin therapy reversed the insulin resistance in diabetic rat adipocytes by at least two mechanisms: restoration of the intracellular pool of glucose transporters and enhancement of glucose-transport activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Photolabeling of the adipocyte hexose carrier with an aryl azide derivative of maltose

A nitrophenyl azide derivative of maltose, N-(4-azido-2-nitrophenyl)-maltosylamine (NAP-maltosylamine), was synthesized as a potential photoaffinity label for the hexose carrier of the rat adipocyte. This derivative inhibited 3-O-methylglucose uptake with a Ki of 1.3 mM in the dark, while that of maltose was 10.0 mM. Carbon-14-labeled maltose and NAP-maltosylamine entered adipocytes via the hexose carrier, the latter in a concentrative fashion. Photolysis of NAP-[14C]maltosylamine in the presence of an adipocyte low density microsomal membrane fraction labeled several electrophoretic bands. Among these are a 45 kDa band which showed features expected of the hexose carrier: its labeling was decreased 40% by d- but not l-glucose and pretreatment of intact adipocytes with insulin decreased labeling of the 45 kDa band by 10–40%, as predicted by the translocation theory of insulin-stimulated transport activation. These studies show the suitability of using carbon-1-modified sugar photoaffinity labels as probes for the hexose carrier and possibly of its regulation in rat adipocytes.

Internalization of insulin receptors in the isolated rat adipose cell. Demonstration of the vectorial disposition of receptor subunits.

The internalization of the insulin receptor in the isolated rat adipose cell and the spatial orientation of the alpha (Mr = 135,000) and beta (Mr = 95,000) subunits of the receptor in the plasma membrane have been examined. The receptor subunits were labeled by lactoperoxidase/Na125I iodination, a technique which side-specifically labels membrane proteins in intact cells and impermeable membrane vesicles. Internalization was induced by incubating cells for 30 min at 37 degrees C in the presence of saturating insulin. Plasma, high density microsomal (endoplasmic reticulum-enriched), and low density microsomal (Golgi-enriched) membrane fractions were prepared by differential ultracentrifugation. Receptor subunit iodination was analyzed by immunoprecipitation with anti-receptor antibodies, sodium dodecyl sulfate/polyacrylamide gel electrophoresis, and autoradiography. When intact cells were surface-labeled and incubated in the absence of insulin, the alpha and beta receptor subunits were clearly observed in the plasma membrane fraction and their quantities in the microsomal membrane fractions paralleled plasma membrane contamination. Following receptor internalization, however, both subunits were decreased in the plasma membrane fraction by 20-30% and concomitantly and stoichiometrically increased in the high and low density microsomal membrane fractions, without alterations in either their apparent molecular size or proportion. In contrast, when the isolated particulate membrane fractions were directly iodinated, both subunits were labeled in the plasma membrane fraction whereas only the beta subunit was prominently labeled in the two microsomal membrane fractions. Iodination of the subcellular fractions following their solubilization in Triton X-100 again clearly labeled both subunits in all three membrane fractions in identical proportions. These results suggest that 1) insulin receptor internalization comprises the translocation of both major receptor subunits from the plasma membrane into at least two different intracellular membrane compartments associated, respectively, with the endoplasmic reticulum and Golgi-enriched membrane fractions, 2) this translocation occurs without receptor loss or alterations in receptor subunit structure, and 3) the alpha receptor subunit is primarily, if not exclusively, exposed on the extracellular surface of the plasma membrane while the beta receptor subunit traverses the membrane, and this vectorial disposition is inverted during internalization.

Internalization of insulin and its receptor in the isolated rat adipose cell: Time-course and insulin concentration dependency

The time-course and insulin concentration dependency of internalization of insulin and its receptor have been examined in isolated rat adipose cells at 37°C. The internalization of insulin was assessed by examining the subcellular distribution of cell-associated [ 125I]insulin among plasma membrane, and high-density (endoplasmic reticulum-enriched) and low-density (Golgi-enriched) microsomal membrane fractions prepared by differential ultracentrifugation. The distribution of receptors was measured by the steady-state exchange binding of fresh [ 125I]insulin to these same membrane fractions. At 37°C, insulin binding to intact cells is accompanied initially by the rapid appearance of intact insulin in the plasma membrane fraction, and subsequently, by its rapid appearance in both the high-density and low-density microsomal membrane fractions. An apparent steady-state distribution of insulin per mg of membrane protein among these subcellular fractions is achieved within 30 min in a ratio of 1:1.54:0.80, respectively. Concomitantly, insulin binding to intact cells is associated with the rapid disappearance of approx. 30% of the insulin receptors initially present in the plasma membrane fraction and appearance of 20–30% of those lost in the low-density microsomal membrane fraction. However, the number of receptors in the high-density microsomal membrane fraction does not change. This redistribution of receptors also appears to reach a steady-state within 30 min. Both processes are insulin concentration-dependent, correlating with receptor occupancy in the intact cell, and are partially inhibited at 16°C. While the steady-state subcellular distributions of insulin and its receptor do not correlate with that of acid phosphatase, chloroquine markedly increases the levels of insulin associated with all three membrane fractions in apparent proportion to the distribution of this lysosomal marker enzyme activity, without more than marginally potentiating insulin's effects on the distribution of receptors. These results demonstrate that insulin, initially bound to the plasma membrane of the isolated rat adipose cell, is rapidly translocated by a receptor-mediated process into at least two intracellular compartments associated with the cell's high- and low-density microsomes. Furthermore, insulin simultaneously induces the translocation of its own receptor from the plasma membrane into the latter compartment. These translocations appear to represent the internalization and partial dissociation of the insulin-receptor complex through insulin-induced receptor cycling.

Insulin-induced internalization of the insulin receptor in the isolated rat adipose cell. Detection of both major receptor subunits following their biosynthetic labeling in culture.

A protocol has been developed for maintaining isolated rat adipose cells in primary tissue culture. Using this protocol, cells remain fully viable and responsive to insulin for at least 24 h, as assessed by measuring 3-0-methylglucose transport, lipogenesis from [U-14C]glucose, and the incorporation of [35S]methionine into total membrane protein. The acute insulin-induced internalization of its own receptor was then examined by biosynthetically labeling cells in culture with either [35S]methionine or [3H]glucosamine, maximally inducing receptor internalization with a 30-min incubation in the presence of saturating insulin, and preparing plasma and low-density microsomal membrane fractions by differential ultracentrifugation. Receptors were immunoprecipitated with anti-receptor antiserum, and the receptor subunits separated by NaDodSO4-PAGE under reducing conditions and analyzed by autoradiography. When cells not acutely treated with insulin are examined, both the 135K alpha- and 95K beta-receptor subunits are prominently labeled in the plasma membrane fraction, but only faintly labeled in the low-density microsomal membrane fraction. Following the induction of maximal acute receptor internalization, both subunits are decreased by 20-30% in the plasma membrane fraction and concomitantly increased in the low-density microsomal membrane fraction. However, the relative molecular weights and labeling intensities of the two subunits remain constant and correspond to those observed in the biosynthetically labeled human lymphocyte receptor. A minor band of Mr congruent to 190K is also labeled, but its labeling intensity is similar in the two membrane fractions from basal cells and does not change in response to insulin.(ABSTRACT TRUNCATED AT 250 WORDS)

Proposed mechanism of insulin-resistant glucose transport in the isolated guinea pig adipocyte. Small intracellular pool of glucose transporters.

A marked resistance to the stimulatory action of insulin on glucose metabolism has previously been shown in guinea pig, compared to rat, adipose tissue and isolated adipocytes. The mechanism of insulin resistance in isolated guinea pig adipocytes has, therefore, been examined by measuring 125I-insulin binding, the stimulatory effect of insulin on 3-0-methylglucose transport and on lipogenesis from [3-3H]glucose, the inhibitory effect of insulin on glucagon-stimulated glycerol release, and the translocation of glucose transporters in response to insulin. The translocation of glucose transporters was assessed by measuring the distribution of specific D-glucose-inhibitable [3H]cytochalasin B binding sites among the plasma, and high and low density microsomal membrane fractions prepared by differential centrifugation from basal and insulin-stimulated cells. At a glucose concentration (0.5 mM) where transport is thought to be rate-limiting for metabolism, insulin stimulates lipogenesis from 30 to 80 fmol/cell/90 min in guinea pig cells and from 25 to 380 fmol/cell/90 min in rat cells with half-maximal effects at approximately 100 pM in both cell types. Insulin similarly stimulates 3-O-methylglucose transport from 0.40 to 0.70 fmol/cell/min and from 0.24 to 3.60 fmol/cell/min in guinea pig and rat fat cells, respectively. Nevertheless, guinea pig cells bind more insulin per cell than rat cells, and insulin fully inhibits glucagon-stimulated glycerol release. In addition, the differences between guinea pig and rat cells in the stimulatory effect of insulin on lipogenesis and 3-O-methylglucose transport cannot be explained by the greater cell size of the former compared to the latter (0.18 and 0.09 micrograms of lipid/cell, respectively). However, the number of glucose transporters in the low density microsomal membrane fraction prepared from basal guinea pig cells is markedly reduced compared to that from rat fat cells (12 and 70 pmol/mg of membrane protein, respectively) and the translocation of intracellular glucose transporters to the plasma membrane fraction in response to insulin is correspondingly reduced. These results suggest that guinea pig adipocytes are markedly resistant to the stimulatory action of insulin on glucose transport and that this resistance is the consequence of a relative depletion in the number of intracellular glucose transporters.

Mechanism of insulin-resistant glucose transport activity in the enlarged adipose cell of the aged, obese rat.

The effects of increasing cell size on glucose transport activity and metabolism and on the concentrations of glucose transport systems in both the plasma and low density microsomal membranes in isolated adipose cells from the aging rat model of obesity have been examined. Glucose transport activity was assessed by measuring l-arabinose transport and the concentration of glucose transport systems estimated by measuring specific d-glucose-inhibitable cytochalasin B-binding. Basal glucose transport activity increases from 0.3 to 1.4 fmol/cell/min with a 10-fold increase in cell size, but remains constant per unit cellular surface area and is accompanied by a constant 5 pmol of glucose transport systems/mg of membrane protein in the plasma membrane fraction. Maximally insulin-stimulated glucose transport activity, on the other hand, remains constant at 2.3 fmol/cell per min with increasing cell size, but markedly decreases per unit cellular surface area and is accompanied by a decrease from 30 pmol of glucose transport systems/mg of plasma membrane protein to the basal level. These diminished effects of insulin on glucose transport activity and the number of glucose transport systems in the plasma membrane fraction in enlarged cells are paralleled by an 80% decrease in the basal number of glucose transport systems/mg of membrane protein in the low density microsomal membrane fraction, the source of those glucose transport systems appearing in the plasma membrane in response to insulin. The effects of cell size on the metabolism of a low concentration of [1-(14)C]glucose (0.56 mM) directly parallel those on glucose transport activity and the concentration of glucose transport systems in the plasma membrane fraction, and are not associated with significant alterations in the cell's sensitivity to insulin. Thus, adipose cellular enlargement is accompanied by the development of a marked "insulin resistance" at the glucose transport level, which may be the consequence of a relative depletion of glucose transport systems in the intracellular pool.

A possible mechanism of insulin resistance in the rat adipose cell with high-fat/low-carbohydrate feeding. Depletion of intracellular glucose transport systems.

The effects of high-fat/low-carbohydrate feeding on glucose transport activity and on the concentrations of glucose transport systems in the plasma and low-density microsomal membranes in isolated rat adipose cells have been examined. Glucose transport activity was assessed by measuring 3-O-methylglucose transport and the concentration of glucose transport systems estimated by measuring specific D-glucose-inhibitable cytochalasin B-binding. Basal glucose transport activity is not significantly influenced by high-fat/low-carbohydrate relative to low-fat/high-carbohydrate feeding and is accompanied by a constant 10 pmol of glucose transport systems/mg of membrane protein in the plasma membrane fraction. In contrast, maximally insulin-stimulated glucose transport activity decreases from 4.72 to 2.29 fmol/cell/min and is accompanied by a decrease from 44 to 26 pmol of glucose transport systems/mg of plasma membrane protein. These diminished effects of insulin on glucose transport activity and the concentration of glucose transport systems in the plasma membrane fraction are paralleled by a 48% decrease in the basal number of glucose transport systems/mg of membrane protein in the low-density microsomal membrane fraction, the source of those glucose transport systems appearing in the plasma membrane in response to insulin. Thus, the "insulin-resistant" glucose transport of the adipose cell with high-fat/low-carbohydrate feeding may be the consequence of a depletion of glucose transport systems in the intracellular pool.

A possible mechanism of insulin resistance in the rat adipose cell in streptozotocin-induced diabetes mellitus. Depletion of intracellular glucose transport systems.

The effects of insulin-dependent diabetes mellitus on glucose transport activity and on the concentrations of glucose transport systems in the plasma and low density microsomal membranes in adipose cells isolated from streptozotocin-induced diabetic rats have been examined. Glucose transport activity was assessed by measuring 3-O-methylglucose transport and the concentration of glucose transport systems estimated by measuring specific D-glucose-inhibitable cytochalasin B-binding. Basal glucose transport activity decreases from 0.19 to 0.12 fmol/cell per min with the induction of diabetes, but remains constant per unit cellular surface area and is accompanied by a constant 6 pmol of glucose transport systems/mg of membrane protein in the plasma membrane fraction. Maximally insulin-stimulated glucose transport activity decreases from 3.16 to 1.05 fmol/cell per min and from 0.26 to 0.12 amol/micrometers 2 per min, and is accompanied by a decrease from 25 to 15 pmol of glucose transport systems/mg of plasma membrane protein. These diminished effects of insulin on glucose transport activity and the concentration of glucose transport systems in the plasma membrane fraction are paralleled by a 45% decrease in the basal number of glucose transport systems per milligram of membrane protein in the low density microsomal membrane fraction, the source of those glucose transport systems appearing in the plasma membrane in response to insulin. Thus, the "insulin resistant" glucose transport of the adipose cell in the streptozotocin-induced diabetic rat appears to be the consequence of a depletion of glucose transport systems in the intracellular pool.