Community perspective on the INSIGHT Strategic Timing of AntiRetroviral Treatment (START) trial.

Determining when to start antiretroviral treatment (ART) is vitally important for people living with HIV. Yet the optimal point at which to start to maximize clinical benefit remains unknown. In the absence of randomized studies, current guidelines rely on conflicting observational data and expert opinion, and consequently diverge on this point. In the USA, ART is recommended irrespective of CD4 cell count. The World Health Organization now recommends starting ART at a CD4 cell count of 500 cells/µL, while the threshold for the UK and South Africa remains at 350 cells/µL. The Strategic Timing of AntiRetroviral Treatment (START) study, one of the largest clinical trials on the treatment of HIV infection, will answer this question. START compares two treatment strategies: immediate treatment at a CD4 cell count of 500 cells/µL or higher versus deferring treatment until the CD4 cell count decreases to 350 cells/µL or until AIDS develops. START includes seven substudies, five of which will clarify the relative contributions of HIV and ART in common comorbidities. START is fully enrolled and expected to be completed in 2016. HIV advocates support the study's design and have been involved from inception to enrolment. The trial will produce rigorous data on the benefits and risks of earlier treatment. It will inform policy and treatment advocacy globally, benefitting the health of HIV-positive people.

Src family kinases and HER2 interactions in human breast cancer cell growth and survival.

Evidence from murine fibroblast models and human breast cancer cells indicates that c-Src and human EGF receptor (HER1) synergize to enhance neoplastic growth of mammary epithelial cells. To investigate whether interactions between c-Src and other HER family members may also play a role in breast tumor progression, we characterized 13 human breast carcinoma cell lines and 13 tumor samples for expression of HER family members and c-Src and examined a subset of the cell lines for Src-dependent, heregulin (HRG)-augmented, anchorage-dependent and independent growth. By immunoblotting, we found that all cell lines overexpressed one or more HER family member, and 60% overexpressed c-Src. Seventy-five per cent of the tumor tissues overexpressed HER2, while 64% overexpressed c-Src. Colony formation in soft agar was enhanced by HRG in three of five cell lines tested, a response that correlated with the presence of a c-Src/HER2 heterocomplex. This result suggests that HRG may act through both HER2 and c-Src to facilitate anchorage-independent growth. In contrast, HRG had little effect on anchorage-dependent growth in any of the cell lines tested. PP1, a Src family kinase inhibitor, reduced or ablated HRG-dependent and independent soft agar growth or anchorage dependent growth, and triggered apoptosis in all cell lines tested. The apoptotic effect of PP1 could be partially or completely reversed by HRG, depending on the cell line. These results suggest that while Src family kinases may cooperate with HRG to promote the survival and growth of human breast tumor cells, they also function independently of HER2/HRG in these processes.

Expression of the bHLH gene NSCL-1 suggests a role in regulating cerebellar granule cell growth and differentiation.

We report that the neuronal-specific basic helix-loop-helix (bHLH) gene NSCL-1 is expressed at multiple and distinct stages of cerebellar granule cell differentiation. During embryonic development, NSCI-1 expression is initially evenly distributed in the cerebellar primordium and then becomes restricted to the ventricular zone. At the early steps of granule cell development, NSCL-1 is not expressed in rhombic lip cells, but instead in migrating granule cell precursors. Its expression culminates during postnatal proliferation of the external germinal layer, and remains only transiently in the newly formed internal granular layer, and at a much lower level. Thus, NSCL-1 expression is linked to the onset of granule cell differentiation, but is not involved in the maintenance of the differentiated state. These findings suggest that NSCL-1 does not behave as a specification factor, but rather as a factor promoting expansion of progenitor external germinal layer (EGL) cells. Gel mobility shift assays show that NSCL-1 only binds DNA as a heterodimeric complex with the ME1a E-protein. We also provide the first evidence that NSCL-1 functions as a transcriptional activator when heterodimerized with the ME1a E-protein. Taken together, these results suggest that NSCL-1 participates in the regulatory network controlling gene expression during cerebellar granule cell differentiation.

Two pathways for insulin metabolism in adipocytes.

Using selected conditions, the appropriate collagenase, albumin and cell treatment, a preparation of isolated adipocytes was developed with no extracellular insulin degrading activity. Cell mediated insulin degradation rates were 0.68% +/- 0.05%/100,000 cell/h using trichloracetic acid precipitability as a measure. Chloroquine (CQ) increased cell-associated radioactivity and decreased degradation while dansylcadaverine (DC), PCMBS and bacitracin (BAC) decreased degradation with no effect on binding. Extraction and chromatography of the cell-associated radioactivity showed 3 peaks, a large molecular weight peak, a small molecular weight peak and an insulin-sized peak. CQ, DC and BAC all decreased the small molecular weight peak while CQ and DC also increased the peak of large molecular weight radioactivity. Cell mediated insulin degradation in the presence of combinations of inhibitors suggested two pathways in adipocytes, one affected by inhibitors of the insulin degrading enzyme (IDE) (bacitracin and PCMBS) and the other altered by cell processing inhibitors (DC, CQ and phenylarsenoxide). Chloroquine altered the pattern of the insulin-sized cell-associated HPLC assayed degradation products, further supporting two pathways of degradation; one a chloroquine-sensitive and one a chloroquine-insensitive pathway.

The GAP-43 gene is a direct downstream target of the basic helix-loop-helix transcription factors.

The GAP-43 promoter region contains seven E-boxes (E1 to E7) that are organized in two clusters, a distal cluster (E3 to E7) and a proximal cluster (E1 and E2). Deletion analysis and site-directed mutagenesis of the GAP-43 promoter region showed that only the most proximal E1 E-box significantly modulates GAP-43 promoter activity. This E-box is conserved between the rat and human GAP-43 promoter sequences in terms of flanking sequence, core sequence (CAGTTG), and position. We found that endogenous E-box-binding proteins present in neuronal N18 cells recognize the E1 E-box and activate the GAP-43 promoter. The transcriptional activity of the GAP-43 promoter was repressed not only by the negative regulator Id2 protein, but also by two class A basic helix-loop-helix proteins, E12 and ME1a. In vitro analyses showed that both ME1a and E12 bind to the E1 E-box as homodimers. By Northern analyses, we established an inverse correlation between the level of E12 and ME1a mRNAs and GAP-43 mRNA in various neuronal cell lines as well as in ME1a-overexpressing PC12 cells. Therefore, we have identified a cis-acting element, the E1 E-box, located in the GAP-43 promoter region that modulates either positively or negatively the expression of the GAP-43 gene depending on which E-box-binding proteins occupy this site. Together, these data indicate that basic helix-loop-helix transcription factors regulate the expression of the GAP-43 gene and that the class A ME1a and E12 proteins act as down-regulators of GAP-43 expression.

Vanadate action on renal phosphate transport.

Insulin stimulates reabsorption of phosphate (Pi) in the renal proximal tubule. Previous studies have shown that vanadate can mimic the action of insulin on various tissues. In the present study, we tested the action of vanadate on renal Pi transport both in control rats and in rats made diabetic by injection of streptozotocin. Vanadate was administered orally for 4 days by inclusion in drinking water (0.7 mg/ml). By the 4th day, vanadate treatment of control rats did not change acid-base status, plasma glucose or the filtered load of Pi, but the urinary excretion of Pi was reduced to 2.5 +/- 0.9 compared with 17.6 +/- 3.5 mumol/mg creatine (P < 0.02) in untreated control rats. However, Na+/Pi cotransport by isolated brush border membrane vesicles was not different between the two groups. Findings in parathyroidectomized rats were similar. By the 4th day of vanadate treatment of diabetic rats, there was reversal of polyuria, polydipsia and hyperglycemia with no change in acid-base status. The filtered load of Pi was decreased by vanadate, and urinary Pi excretion also tended to decrease but not significantly. The values for Pi excretion were 21.4 +/- 7.6 in vanadate treated diabetics and 36.1 +/- 4.5 mumol/mg creatinine in untreated diabetics. In contrast to vanadate, daily injections of insulin did not change the filtered load of Pi but reduced urinary Pi excretion in diabetic rats to 15.6 +/- 2.2 mumol/mg creatinine (P < 0.02). These findings suggest that vanadate stimulated tubular Pi reabsorption in control rats but not in diabetic rats. Vanadate treatment of diabetic rats may tend to decrease tubular Pi reabsorption in contrast to the action of insulin.

Protein degradation in cultured fetal hepatocytes. Absence of an inhibitory effect of insulin.

The role of insulin to regulate protein turnover in fetal liver was investigated using primary cultures of fetal-rat hepatocytes. The basal rate of protein degradation (in the presence of insulin and amino acids) was the same in cultured fetal and adult hepatocytes (2.48 +/- 0.16 versus 2.46 +/- 0.06% of total protein degraded/h respectively). Incubation of cells in an unsupplemented media (without insulin or amino acids) resulted in a deprivation-induced increase in degradation in cells from both groups (P less than 0.05). Rates of proteolysis could be returned to their respective basal values by the addition of amino acids at 5 times their normal plasma concentrations. In adult cells, addition of insulin alone significantly inhibited protein degradation (P less than 0.05), whereas, in contrast, insulin was without effect on protein degradation in fetal hepatocytes. Both fetal and adults cells responded to dibutyryl cyclic AMP with an increase in protein degradation above that seen in the no-additions group. Results of experiments in which the effect of inhibitors of protein degradation (chloroquine, NH4Cl, amino acids and dinitrophenol) were tested suggested that lysosomes were responsible for 20-30% of total protein degradation in fetal hepatocytes. Impaired insulin processing in fetal hepatocytes was examined as a possible cause of the insulin-resistance in these cells. As determined by h.p.l.c. analysis, the same pattern of initial degradation products of insulin was found in fetal hepatocytes as had previously been found in adult hepatocytes. Incubation of cells with various doses of chloroquine resulted in an increase in cell-associated 125I-insulin and a decrease in insulin degradation in both fetal and adult cells. At the highest dose of chloroquine tested (500 microM), a slightly greater increase in insulin binding and a decrease in insulin degradation were observed in fetal cells as compared with adult cells. Rates of insulin internalization were also compared between fetal and adult cells. A 30% slower rate of insulin internalization was observed in fetal cells, as compared with adult cells. It was concluded that the absence of an effect of insulin on protein degradation in fetal hepatocytes is not the result of a major difference in insulin internalization and processing between fetal and adult hepatocytes.

Insulin degradation products from perfused rat kidney.

The kidney is a major site for insulin metabolism, but the enzymes involved and the products generated have not been established. To examine the products, we have perfused rat kidneys with insulin specifically iodinated on either the A14 or the B26 tyrosine. Labeled material from both the perfusate and kidney extract was examined by Sephadex G50 and high-performance liquid chromatography (HPLC). In perfusate from a filtering kidney, 22% of the insulin-sized material was not intact insulin on HPLC. With the nonfiltering kidney, 10.6% was not intact insulin. Labeled material from HPLC was sulfitolyzed and reinjected on HPLC. By use of 125I-iodo(A14)-insulin, almost all the degradation products contained an intact A-chain. By use of 125I-iodo(B26)-insulin, several different B-chain-cleaved products were obtained. The material extracted from the perfused kidney was different from perfusate products but similar to intracellular products from hepatocytes, suggesting that cellular metabolism by kidney and liver are similar. The major intracellular product had characteristics consistent with a cleavage between the B16 and B17 amino acids. This product and several of the perfusate products are also produced by insulin protease suggesting that this enzyme is involved in the degradation of insulin by kidney.

Hepatic metabolism of insulin.

The liver plays a major role in the metabolism of insulin, but the precise cellular mechanisms, the enzymes involved, and the products generated have only recently become clarified. The initial step in insulin degradation by the liver is binding to a cell membrane receptor, following which some insulin is degraded and the products released into the incubation medium, whereas some insulin is internalized and degraded intracellularly. Recently, it has been demonstrated that the degradation of insulin by hepatocytes produces products identical to those generated by the enzyme insulin protease. With both enzyme and intact hepatocytes, two A-chain cleavages and four major and three minor B-chain cleavages occur in intact insulin. It has also been demonstrated that internalized insulin is degraded in early endosomes, primarily by cleavages in the B chain and occurring prior to acidification of the endosome and thus prior to dissociation of insulin from its receptor. The initial cleavages in the B chain of insulin occur in the same sites as are cleaved by insulin protease, supporting a role for this enzyme, both in the extracellular and intracellular metabolism of insulin. These findings also indicate that lysosomes probably play a minor or secondary role for hepatic insulin metabolism.

Conversion of biosynthetic human proinsulin to partially cleaved intermediates by collagenase proteinases adsorbed to isolated rat adipocytes.

Studies of the biological activity of proinsulin have resulted in widely varying conclusions. Relative to insulin, the biological activity of proinsulin has been reported from less than 1% to almost 20%. Many of the assays in vitro for the biological potency of proinsulin have utilized isolated rat adipocytes. To examine further the interaction of proinsulin with rat adipocytes, we prepared specifically-labelled proinsulin isomers that were iodinated on tyrosine residues corresponding to the A14, A19, B16 or B26 residue of insulin. These were incubated with rat adipocytes and their metabolism was examined by trichloroacetic acid precipitation, by Sephadex G-50 chromatography, and by h.p.l.c. chromatography. By trichloroacetic acid-precipitation assay, there was little or no proinsulin degradation. By G-50 chromatography and subsequent h.p.l.c. analysis, however, we found that the labelled proinsulin isomers were converted rapidly and almost completely to materials which eluted differently on h.p.l.c. from intact proinsulin. This conversion was due primarily to proteolytic activity which adsorbed to the fat cells from the crude collagenase used to isolate the cells. Two primary conversion intermediates were found: one with a cleavage at residues 23-24 of proinsulin (the B-chain region of insulin), and one at residues 55-56 in the connecting peptide region. These intermediates had receptor binding properties equivalent to or less than intact proinsulin. These findings show that isolated fat cells can degrade proinsulin to intermediates due to their contamination with proteolytic activity from the collagenase used in their preparation. Thus the previously reported range in biological activities of proinsulin in fat cells may have arisen from such protease contamination. Finally, the present findings demonstrate that a sensitive assay for degradation of hormones is required to examine biological activities in isolated cells.

Insulin-like effects of vanadate in isolated rat adipocytes.

Vanadate has been shown to have a number of insulin-like effects in various cells, including isolated rat adipocytes. In the present study we compared the activities of vanadate and insulin in isolated fat cells using a number of different assays of insulin-like activity. Both insulin and vanadate stimulated [2-3H]glucose incorporation into fat cell lipid in a dose-dependent manner, but the maximal effect of vanadate was markedly greater than that of insulin. At 10(-2) M vanadate the effect was 3-4 times as great as the maximal effect of insulin. This effect was dependent on specific glucose transport. Combinations of insulin and vanadate were not more effective than vanadate alone. Vanadate also produced antilipolysis with an effect somewhat greater than that of insulin. Using [U-14C]glucose both vanadate and insulin stimulated 14CO2 production and [14C]glucose incorporation into lipid, and again the effect of vanadate was greater than that of insulin. Vanadate had a greater effect on 14CO2 production than on [14C]glucose incorporation into lipid. When [1-14C]glucose was used vanadate again had a significantly greater effect on 14CO2 production than did insulin, but when [6-14C]glucose was used the effects of vanadate and insulin were equal. These results demonstrate that vanadate has insulin-like effects in isolated fat cells, but it selectively stimulates certain pathways to a greater extent than does insulin. The greater effect of vanadate than insulin appears to be primarily on the pentose phosphate shunt, suggesting that this agent may be useful for examination of this intracellular pathway in fat cells.

Differential binding of monoiodinated insulins to muscle and liver derived receptors and activation of the receptor kinase.

The binding affinity of monoiodoinsulin analogues to receptors purified from rat skeletal muscle and liver were compared. Insulin iodinated at tyrosine B26 bound to both muscle and liver derived insulin receptors with higher affinity than the A14-iodoisomer or native insulin. The affinity of the B26-iodoanalogue was greater for muscle than for liver derived receptors; by Scatchard analysis the affinity ratio B26/A14 was 2.8 for muscle and 1.3 for liver. The affinity of muscle and liver derived receptors for A14-iodoinsulin was not different. Dose response curves of autophosphorylation and exogenous tyrosine kinase activation showed significantly increased sensitivity to the B26-iodoanalogue (compared to the A14-iodoisomer or native insulin) in muscle derived receptors, but not in liver. The difference in affinity between muscle and liver derived insulin receptors towards B26-monoiodotyrosyl-insulin likely reflects the observed structural difference between the insulin receptor alpha-subunits from muscle and liver.

Degradation products of insulin generated by hepatocytes and by insulin protease.

The enzymatic mechanisms for insulin breakdown by hepatocytes have not been established, nor have the degradation products been identified. Several lines of evidence have suggested that the enzyme insulin protease is involved in insulin degradation by hepatocytes. To identify the products of insulin generated by insulin protease and to compare them with those produced by hepatocytes, we have incubated insulin specifically iodinated at either the B-16 or the B-26 tyrosines with insulin protease and with isolated hepatocytes, separated the products on high performance liquid chromatography (HPLC), and identified the B-chain cleavages. Insulin-sized products were obtained by Sephadex G-50 filtration. These insulin-sized products were injected on reverse-phase HPLC, and the peaks of radioactivity were identified. The product patterns generated by the enzyme and by hepatocytes were essentially identical with both isomers. The products were also sulfitolized to prepare the S-sulfonate derivatives of the B-chain and B-chain peptides. Again, the patterns on HPLC generated by the enzyme and by hepatocytes with both isomers were identical. Each of the original product peaks was also sulfitolized and injected separately on HPLC to relate B-chain peptides with product peaks. Again, the peptide compositions of the product peaks for both enzyme and hepatocytes were essentially identical. To identify the cleavage sites in the B-chain of insulin produced by insulin protease, the peptides from the degradation of [125I]iodo(B-26)insulin were purified and submitted to automated Edman degradation to identify the cycle in which radioactivity appeared. Seven peptides with cleavages on the amino side of the B26 residue were identified, and the cleavage sites were determined. Cleavages were found between B-9 and B-10 (Ser-His), B-10 and B-11 (His-Leu), B-14 and B-15 (Ala-Leu), B-13 and B-14 (Glu-Ala), B-16 and B-17 (Tyr-Leu), B-24 and B-25 (Phe-Phe), and B-25 and B-26 (Phe-Tyr). Peptides were also isolated from [125I]iodoinsulin incubated with isolated hepatocytes, and the cleavage sites in several of these were determined. These agreed exactly with the cleavage sites identified generated by the enzyme. The major peptides generated by the degradation of [125I]iodo(B-16)insulin were also isolated and sequenced, again showing identical cleavage sites.(ABSTRACT TRUNCATED AT 400 WORDS)

Identification of A chain cleavage sites in intact insulin produced by insulin protease and isolated hepatocytes.

The degradation of insulin by the enzyme insulin protease and by isolated hepatocytes results in proteolytic cleavages in both the A and B chains of intact insulin. Previous studies have shown that one of the A chain cleavages is between A13 leucine and A14 tyrosine and that a second cleavage occurs carboxyl to the A14 residue. In the present study we have used insulin specifically iodinated on the A19 tyrosine and examined the A chain cleavages by the enzyme and by hepatocytes. Insulin degradation products were purified by HPLC and sequenced by automated Edman degradation. Only two A chain cleavage sites were identified, one the previously reported A13-A14 and the other between A14 tyrosine and A15 glutamine. These data thus identify the second A chain cleavage site and further support the role of insulin protease in hepatic metabolism of insulin.

Immunohistochemical differentiation of Schwann and epithelial basal lamina in the rabbit cornea.

Transmission electron microscopy has shown that at the point of entry of axons into corneal epithelium, Schwann cell lamina densa merges with lamina densa of epithelial origin. Staining properties and the thickness of lamina densa from these sources are similar. Immunostaining with monoclonal antibodies to laminin and type IV collagen revealed that while Schwann cell basal lamina fluoresced to both of these probes, epithelial basal lamina was visualized only with anti-laminin. Monoclonal antibodies to neurofilament protein (70 and 200 kDa) were used to visualize the neural tissue. Segments of the deep stromal and subepithelial innervation appeared similar to those seen following gold chloride impregnation. The results suggest that intercalation of lamina densa from these tissue sources and regulation of the production of basal lamina components by both the epithelial and the Schwann cells probably occur.

HPLC analysis of insulin degradation products from isolated hepatocytes. Effects of inhibitors suggest intracellular and extracellular pathways.

Isolated rat hepatocytes were incubated with A14-[125I]monoiodotyrosyl insulin for 30 min, and labeled material was extracted from the cells and incubation media. The medium and the cell extract were chromatographed on a Sephadex G-50 column, and radioactivity eluting in the position of intact insulin was concentrated and analyzed on HPLC. The HPLC analysis of the cell extract showed two major products eluting from the column at 19 and 23 min, whereas medium extracts showed one prominent product eluting at 14 min. Inclusion of chloroquine in the incubation blocked the formation of cellular products at 19 and 23 min and caused the accumulation of a product eluting at 41 min while not affecting the media products. After sulfitolysis all cellular products contained an intact A-chain. Dansylcadaverine increased media products and altered the cell-extracted product pattern such that it had a major peak at 14 min, similar to media. These results suggest that two pathways for insulin degradation exist within hepatocytes. The extracellular process forms products that are essentially unchanged by chloroquine and dansylcadaverine. The intracellular process is altered by chloroquine and apparently inhibited by dansylcadaverine.

Induction of metallothionein synthesis in human peripheral blood leukocytes.

Metallothionein (MT), a low molecular weight, metal-binding protein, has recently been shown to protect murine mononuclear phagocytic cells from the cytotoxic effects of bacterial lipopolysaccharides (LPS), the endotoxic component of Enterobacteriaceae. MT appears to function intracellularly as an antioxidant since autolysis results from lipid peroxidation initiated by free radicals of O2. Since this activity is distinct from MT's capacity to specifically sequestrate heavy metals, we examined whether MT synthesis can be induced by direct membrane activation or through interaction with soluble leukocyte mediators. Normal human monocytes, polymorphonuclear neutrophils (PMN), and lymphocytes, isolated from heparinized whole blood, were incubated with and without LPS from Escherichia coli and Salmonella typhosa. MT in cell lysates was quantitated using a 203Hg-binding assay employing Sephadex G-10 "minicolumns." When incubated with monocytes, PMN, or lymphocytes, neither preparation of LPS (10-100 micrograms/ml) was capable of enhancing 203Hg-binding activity after 24 or 72 hr incubation. CdCl2 (2 micrograms/ml), however, increased binding activity in monocyte and lymphocyte cultures 4- and 15-fold, respectively. When monocytes and lymphocytes were cocultured with LPS, 203Hg-binding activity was not enhanced. Addition of human interleukin 1 (endogenous pyrogen) to these cultures had no significant effect. Leukocyte endogenous mediator (LEM), a product of LPS-activated PMN that possesses hypozincemic activity in vivo, did not induce MT synthesis. Collectively, these results demonstrate that leukocyte MT does not arise from direct LPS activation or from interaction with products secreted by LPS-activated cells. De novo synthesis of MT observed during endotoxemia and gram negative sepsis appears, therefore, to be induced by endogenously released corticosteroids.

Toxicologic interactions between ozone and bacterial endotoxin.

The effects of acute exposure of mice to bacterial lipopolysaccharide (LPS), the endotoxin of gram negative microorganisms, and ozone (O3) have been investigated. Intraperitoneal (ip) administration of 5 mg/kg LPS to CD-1 mice followed by exposure to 15 ppm O3 for 1.5 hr produced synergistic effects as measured by pulmonary edemagenesis and lethality assays. In contrast, ip administration of 0.1-1.6 mg/kg LPS to CD-1 mice over 5 consecutive days, a dose regimen resulting in LPS tolerance, protected against a lethal challenge of 20 ppm O3 for 3 hr. A statistically significant increase in catalase and glutathione peroxidase activity was measured in homogenates of lungs obtained from CD-1 mice receiving a tolerance-inducing regimen of LPS. These results demonstrate that two, distinct toxicologic interactions can occur between O3 and bacterial LPS. Synergism between these agents could explain, in part, the increased susceptibility of O3-exposed animals to respiratory infection with gram negative microorganisms. Protection resulting from LPS-induced increases in pulmonary antioxidant activity provides additional evidence that O3 and, possibly, LPS mediate their toxicity through oxidative mechanisms.

Insulin-stimulated conversion of D-[5-3H] glucose to 3HOH in the perifused isolated rat adipocyte.

Characteristics of basal and insulin-stimulated glucose utilization by perifused adipocytes have been investigated by measuring the formation of 3HOH from D-(5-3H) glucose. At a glucose concentration of 0.55 mmol/L, basal glucose utilization ranged from 0.5 to 1.0 nmol/min/10(6) cells. Perifused adipocytes showed a maximal response to insulin of a threefold to fourfold increase in the conversion of (5-3H) glucose to 3HOH with a half-maximal response at an insulin concentration of 20 microU/mL. The response to insulin was blocked by phlorizin and cytochalasin B, competitive inhibitors of glucose transport, consistent with an effect of insulin on glucose transport. Insulin increased the Vmax for glucose metabolism but had no effect on the apparent affinity for glucose utilization. The characteristics of glucose utilization and the stimulation of glucose metabolism by insulin in the perifused adipocyte are therefore similar to characteristics previously observed with incubated adipocytes. Because insulin can readily be removed from the system, perifused adipocytes are especially suited for studying the termination of insulin action. The termination of insulin-stimulated glucose metabolism occurred at the same rate in the presence of tracer (1 nmol/L) (5-3H)-glucose alone as when 0.55 mmol/L glucose or 2 mmol/L pyruvate were added to the perifusion buffer. The halftime for this process in both cases was approximately 40 minutes. These data suggest that the presence of metabolizable substrate is not required for the termination of the insulin response, but the time course suggests that termination requires more than simply insulin-receptor dissociation.