Stress-induced inhibition of nonsense-mediated RNA decay regulates intracellular cystine transport and intracellular glutathione through regulation of the cystine/glutamate exchanger SLC7A11.

SLC7A11 encodes a subunit of the xCT cystine/glutamate amino-acid transport system and has a critical role in the generation of glutathione and the protection of cells from oxidative stress. Expression of SLC7A11 promotes tumorigenesis and chemotherapy resistance, but while SLC7A11 has been previously noted to be upregulated in hypoxic cells, its regulation has not been fully delineated. We have recently shown that nonsense-mediated RNA decay (NMD) is inhibited by cellular stresses generated by the tumor microenvironment, including hypoxia, and augments tumorigenesis. Here we demonstrate that the inhibition of NMD by various cellular stresses leads to the stabilization and upregulation of SLC7A11 mRNA and protein. The inhibition of NMD and upregulation of SLC7A11 augments intracellular cystine transport and increases intracellular levels of cysteine and glutathione. Accordingly, the inhibition of NMD protects cells against oxidative stress via SLC7A11 upregulation. Together our studies identify a mechanism for the dynamic regulation of SLC7A11, through the stress-inhibited regulation of NMD, and add to the growing evidence that the inhibition of NMD is an adaptive response.

Hypoxic activation of ATR and the suppression of the initiation of DNA replication through cdc6 degradation.

Many severely hypoxic cells fail to initiate DNA replication, but the mechanism underlying this observation is unknown. Specifically, although the ataxia-telangiectasia-rad3 related (ATR) kinase has been shown to be activated in hypoxic cells, several studies have not been able to document down-stream consequences of ATR activation in these cells. By clearly defining the DNA replication initiation checkpoint in hypoxic cells, we now demonstrate that ATR is responsible for activating this checkpoint. We show that the hypoxic activation of ATR leads to the phosphorylation-dependent degradation of the cdc25a phosphatase. Downregulation of cdc25a protein by ATR in hypoxic cells decreases CDK2 phosphorylation and activity, which results in the degradation of cdc6 by APC/C(Cdh1). These events do not occur in hypoxic cells when ATR is depleted, and the initiation of DNA replication is maintained. We therefore present a novel mechanism of cdc6 regulation in which ATR can have a central role in inhibiting the initiation of DNA replication by the regulation of cdc6 by APC/C(Cdh1). This model provides insight into the biology and therapy of hypoxic tumors.

Hypoxia inhibits G1/S transition through regulation of p27 expression.

Mammalian cellular responses to hypoxia include adaptive metabolic changes and a G1 cell cycle arrest. Although transcriptional regulation of metabolic genes by the hypoxia-induced transcription factor (HIF-1) has been established, the mechanism for the hypoxia-induced G1 arrest is not known. By using genetically defined primary wild-type murine embryo fibroblasts and those nullizygous for regulators of the G1/S checkpoint, we observed that the retinoblastoma protein is essential for the G1/S hypoxia-induced checkpoint, whereas p53 and p21 are not required. In addition, we found that the cyclin-dependent kinase inhibitor p27 is induced by hypoxia, thereby inhibiting CDK2 activity and forestalling S phase entry through retinoblastoma protein hypophosphorylation. Reduction or absence of p27 abrogated the hypoxia-induced G1 checkpoint, suggesting that it is a key regulator of G1/S transition in hypoxic cells. Intriguingly, hypoxic induction of p27 appears to be transcriptional and through an HIF-1-independent region of its proximal promoter. This demonstration of the molecular mechanism of hypoxia-induced G1/S regulation provides insight into a fundamental response of mammalian cells to low oxygen tension.

University of Miami Division of Clinical Pharmacology Therapeutic Rounds: the water-intolerant patient and perioperative hyponatremia.

Perioperative hyponatremia has been recognized as a serious in-hospital complication for many years. Because the kidney responds to changes in extracellular fluid tonicity by adjusting water excretion, a defect in any of several key elements of water excretion can lead to water retention and hyponatremia. Most cases of hyponatremia are caused by impaired renal water excretion in the presence of continued water intake. For the kidney to excrete excess free water and thereby protect the extracellular fluid against hyponatremia, there must be an adequate glomerular filtration rate (GFR), adequate delivery of glomerular filtrate to the diluting segments of the distal nephron, intact tubular diluting mechanisms, and appropriate inhibition of antidiuretic hormone (ADH) synthesis and release. Virtually all of the clinical disorders producing hyponatremia are based on abnormalities of these few mechanisms of water regulation. Finding the reason for impaired renal water excretion is the key to diagnosing the cause of hyponatremia. Impaired renal water excretion may be caused by impaired GFR (renal failure), impaired water delivery to the diluting segments of the distal nephron because of increased proximal reabsorption (decreased extracellular fluid volume and edematous states), impaired renal diluting mechanism (thiazide diuretics), the syndrome of inappropriate ADH (SIADH) due to a variety of causes including the perioperative state, and hypothyroidism or adrenal insufficiency. Any of the states that impair water excretion can produce hyponatremia in a patient with an initially normal serum sodium concentration if sufficient free water is supplied. Therefore, a patient who has one of the conditions listed above, including the perioperative state, may be considered "water intolerant" even if the serum sodium is normal. Such a patient is at risk for developing severe hyponatremia if given hypotonic IV fluids or a large oral water load. An understanding of the basic mechanisms leading to impaired water excretion and "water intolerance" is therefore an important key to avoiding perioperative hyponatremia.

Adverse selection among multiple competing health maintenance organizations.

This study examines risk selection among nine health plans competing for 16,182 employees of one large firm in 1989: one conventional fee-for-service plan, one group-model health maintenance organization (HMO), and seven network and independent practice model HMOs. We develop and compare measures of risk using weights based on HMO and fee-for-service expenditure data, respectively. We use a multiequation statistical model to develop two sets of utilization and expenditure weights for enrollees in each plan. One set of weights, based on discharge abstracts and outpatient records from the large group-model HMO, measures how much each of the nine groups of employees and dependents would have spent, had they been enrolled in a stringently managed plan with no consumer cost sharing. The other set of weights, based on fee-for-service claims data, measures how much each group would have spent, had it been enrolled in an unmanaged health plan with significant coinsurance and deductibles. Predicted annual expenditures per enrollee exhibit a 23% range from lowest (favorable selection) to highest (adverse selection) risk plans using the HMO weights and a 17% range using fee-for-service weights. The fee-for-service plan and group-model HMO with large enrollments have risk mixes near the center of the spectrum. Smaller HMOs exhibit the extreme forms of both favorable and adverse selection. The statistical methods adopted in this study can be used to risk-adjust capitation payments to competing health plans. As mergers among HMOs and group purchasing arrangements among employers increase the average enrollment in each plan from each payor, however, risk differences among plans will be attenuated and the need to risk-adjust payments will be less severe. Key words: health insurance; adverse selection; managed competition; health maintenance organization.

Insulin and glucose suppress hepatic glycogenolysis by distinct enzymatic mechanisms.

Both insulin and hyperglycemia can effectively suppress hepatic glucose output (HGO). We examined whether insulin and hyperglycemia specifically suppress liver net glycogen breakdown in a rat model in which glycogen is the major source of HGO. We further examined whether insulin and hyperglycemia act by similar or distinct enzymatic mechanisms. HGO, the rate of net glycogen loss, and glycogen phosphorylase and synthase activities were measured in fed, anesthetized rats infused with saline or insulin (7 mU/min/kg) while either maintaining plasma glucose at basal (7.8 +/- 0.2 mmol/L, euglycemic clamp [EC]) or at 10 mmol/L above basal (18 +/- 0.4 mmol/L, hyperglycemic clamp [HC]). During the basal period, the rate of HGO in each group was comparable to the rate of net glycogen breakdown, averaging 76 +/- 9 and 75 +/- 5 mumol/min/kg, respectively. Thus glycogen breakdown appeared to be a major source of ongoing HGO. Over the last 60 minutes of the experimental period, the rate of glycogenolysis averaged 69 +/- 8 mumol/min/kg in saline-treated rats; this could account for about 80% of the total HGO. During both EC and HC studies, HGO was suppressed (5.5 +/- 3 and -3.6 +/- 10 mumol/min/kg, respectively; P < .001 for each). Net glycogen breakdown decreased by 50% in EC rats (P < .05) and ceased in HC rats (P < .001). Glycogen synthase was predominantly in the active form in all three experimental groups (87% +/- 2%, 89% +/- 2%, and 95% +/- 3% in saline, EC, and HC rats, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

The role of glucose-6-phosphatase in the action of insulin on hepatic glucose production in the rat.

It has been suggested that regulation of glucose-6-phosphatase by insulin plays a role in the suppression of hepatic glucose production during feeding. We used hepatic glucose production (measured with the D-[3-3H]glucose infusion method) as an indicator of substrate flux through glucose-6-phosphatase in vivo. Compared with saline controls, insulin (7 mU.min-1 x kg-1, euglycemic clamp) suppressed hepatic glucose production virtually completely in both fasted (32.4 +/- 2.4 vs. -6.1 +/- 14 mumol.min-1 x kg-1) and fed (64.6 +/- 6.4 vs. 5.5 +/- 5.2 mumol.min-1 x kg-1) rats. Whereas hepatic glucose production was totally suppressed, [glucose-6-phosphate] in liver cytosol declined by only 27 and 35% in fasted and fed rats, respectively. Addition of hyperglycemia (10 mM) to the insulin infusion likewise fully suppressed hepatic glucose production (26.9 +/- 1.4 vs. -9 +/- 10 mumol.min-1 x kg-1 and 80.8 +/- 10.1 vs. -3.6 +/- 12.6 mumol.min-1 x kg-1 in fasted and fed rats, respectively), but [glucose-6-phosphate] again declined only modestly (21 and 27% in fasted and fed rats, respectively). This disproportionate suppression of hepatic glucose production could not be explained by cooperative substrate effects inasmuch as microsomal glucose-6-phosphatase isolated from saline- and insulin-treated rats followed Michaelis-Menten kinetics (Hill coefficient approximated 1). Acute insulin treatment of fasted rats in vivo did not reproducibly inhibit glucose-6-phosphatase activity assayed subsequently in isolated microsomes incubated in the absence of insulin.(ABSTRACT TRUNCATED AT 250 WORDS)

An endogenous glycogen-associated compound modulates glucose-6-phosphatase activity in rat liver microsomes.

Liver glycogen is closely associated with the endoplasmic reticulum, which contains the glucose-6-phosphatase enzyme system that catalyses the final step of hepatic glucose production. To examine whether this structural association has functional consequences, microsomes were isolated from 48 h fasted (n = 6) and ad lib fed rats (n = 3). Microsomes from fed rats had a higher glycogen content and lower enzyme activity than fasted rats. Overall, glucose-6-phosphatase activity was inversely proportional to microsomal glycogen content. Partially purified rabbit or rat liver glycogen, at physiological relevant concentrations (10-100 mM glucose equivalents), added directly to either intact or Triton-disrupted microsomes from fasted rats significantly decreased glucose-6-phosphatase activity. Inhibitory activity was present in native liver glycogen prepared by sedimentation and could be dissociated from glycogen by ion-exchange and ultrafiltration. These findings suggest that a low molecular weight (< 5000 D) compound closely associated with glycogen can modulate glucose-6-phosphatase and may have a physiologic role in the regulation of hepatic glucose production.

Trends in cesarean section use in California, 1983 to 1990.

OBJECTIVE: Our aim was to assess recent trends in cesarean section use in California. STUDY DESIGN: California discharge abstract data on hospital deliveries in 1983 through 1990 (379,759 to 587,508 annual deliveries) were used to analyze time trends by indication, age, race, and payment source. RESULTS: California cesarean section rates increased annually from 21.8% in 1983 to 25.0% in 1987 and then decreased to 22.7% by 1990. Similar patterns were noted for all age and race or ethnicity groups. Primary cesarean section rates increased from 15.2% in 1983 to 17.9% in 1987, then decreased to 16.2% by 1990. Declines in repeat cesarean section rates continued throughout 1983 through 1990, accelerating after 1987. For both primary and repeat cesarean section rates, time trends after mid-1987 were significantly different than those for 1983 to 1987. CONCLUSION: After increasing from 1983 to 1987, California cesarean section rates declined from 1988 to 1990. Existing payment source differences in cesarean section use increased in magnitude from 1983 to 1990, with privately insured women consistently having the highest cesarean section rates.

Health plan switching in anticipation of increased medical care utilization.

We compare rates and days of maternity and nonmaternity hospital admission for the years 1981 through 1984 for three groups of employees and dependents from a large private employer: those continuously enrolled in a fee-for-service (FFS) plan (N = 147,700), those continuously enrolled in a health maintenance organization (HMO) (N = 30,957), and those switching from the FFS plan to the HMO (N = 2,144). The rate of maternity admissions for plan switchers increased by 106% (P < 0.001) in the post-switch year compared with the pre-switch year, while maternity rates for continuing FFS-plan enrollees declined by 12% (P < 0.001) and rates for continuing HMO enrollees remained unchanged. Nonmaternity admission rates for switchers decreased by 19% (P = 0.079), consistent with the expectation that HMOs reduce these rates substantially, while rates for FFS-plan stayers increased 4% (P < 0.001) and those for HMO stayers remained unchanged. We conclude that employees often switch health plans when anticipating increased needs for maternity care and therefore that pre-switch rates of utilization are unreliable measures of the true magnitude of risk selection between HMOs and FFS plans.

Severe cerebral edema in a patient with anasarca and hypernatremia.

We describe a woman whose fatal post-liver transplantation cerebral edema was unexpected and of unusual pathogenesis. Her severe cerebral edema is of considerable pathophysiologic interest: 1) it developed in the setting of marked anasarca and persistent hypernatremia, and 2) although hepatic function was poor, it was not considered sufficiently deranged to induce cerebral edema. Furthermore, there was no histologic evidence of hepatic rejection or antemortem hepatic necrosis. We postulate that an impairment of the blood brain barrier in association with a degree of hepatic dysfunction insufficient by itself to cause cerebral edema permitted the brain interstitial fluid volume to increase pari passu with ECF expansion. Cytotoxic cerebral edema and vascular engorgement may also have contributed to a life-threatening increase in intracranial pressure.

Detection of clinically suspected deep vein thrombosis using light reflection rheography.

Venography is the current standard for the diagnosis of deep vein thrombosis (DVT). Noninvasive tests have differing sensitivity and specificity, are technically demanding, and may be subject to variability in interpretation. Light reflection rheography (LRR) is a noninvasive method utilizing light-emitting diodes and a sensor to measure light reflected from the skin surface. The intensity of reflected light establishes a graphic pattern that indirectly quantifies parameters of venous function by measuring changes in the microcirculation. Seventy-two patients who underwent contrast venography at our institution were also evaluated with LRR. Twenty-four patients were found to have DVT as demonstrated by venography. Of these, 23 also had DVT detected by LRR. No evidence of thrombus was seen in 45 patients studied by venography; in this group, 35 had normal venous emptying indicated by LRR. Using LRR, a sensitivity of 96% was achieved in the evaluation of clinically suspected DVT. This sensitivity is comparable with other noninvasive tests. In addition, LRR is easy to operate, portable, inexpensive, and not technically demanding. Further investigation is needed to confirm these data and further define the role of LRR in the evaluation of clinically suspected DVT.

A method for risk-adjusting employer contributions to competing health insurance plans.

Biased selection can threaten the viability of multiple choice health systems unless payments to particular plans are adjusted to offset risk differences among employees. We report the results of a study designed to predict medical care utilization and expenditures for groups of fee-for-service plan (FFS) and health maintenance organization (HMO) enrollees, using characteristics commonly available in the personnel files of large employers. Simulation analyses indicate that the six-equation, maximum likelihood model predicts well for groups of 1,000 or more. Additional data are required to reduce prediction errors for smaller groups. This new methodology potentially allows risk-rating of employer contributions to competing health plans, based on the expected utilization of the individuals choosing each plan.

Services and charges by PPO physicians for PPO and indemnity patients. An episode of care comparison.

Physicians who participate in preferred provider organizations (PPOs) usually agree to various types of utilization review and sometimes discount their charges or agree to accept lower fees. This study was performed to determine whether they provided more or fewer services to their PPO patients than to their indemnity patients and whether the discounting resulted in lower expenditures for each episode of illness. In 1984, Metropolitan Life offered PPO coverage to Dade County (Florida) school board employees and dependents but only a standard indemnity plan to Dade County government employees and dependents. Episodes of care were examined for patients with chest pain, hypertension, joint pain, gastrointestinal or liver disorders, and lower back pain cared for by physicians who treated patients in both the PPO and indemnity employee groups. For PPO patients, charges per physician service were the same or lower, but total physician charges during an episode were higher. For services such as laboratory tests, diagnostic x-rays, and room and board, PPO and indemnity patients' charges were not significantly different.

Diagnostic variability in suspected pulmonary embolism.

Over a 12-month period, we observed adult patients with suspected pulmonary embolism referred for lung scanning to determine variability in the diagnostic process. Among 269 studies, 157 lung scans were judged necessary by predetermined criteria. Ninety-three of these 157 patients had inconclusive results (low probability, intermediate probability, or indeterminate). Of these 93 patients, 42 had pulmonary angiograms, ten of which were positive. Of the 51 patients with necessary but inconclusive scans, five were poor candidates for angiography, 15 had other indications for anticoagulation, seven refused the study, and 24 had physicians who considered further studies unwarranted. Patients with and without pulmonary angiography were demographically and clinically similar. Although confirmatory testing such as pulmonary angiography was used frequently (45%) after an inconclusive lung scan, the question of pulmonary embolism was often left unanswered (55%). Methods for linking clinical judgment to lung scan results are necessary to select proper patients for invasive confirmatory testing.