Significant publications in diabetes pharmacotherapy and technology in 2020.

INTRODUCTION: The most significant articles on diabetes pharmacotherapy and technology in the peer-reviewed literature from 2020, as determined by a panel of pharmacists with expertise in diabetes care and education, are summarized. AREAS COVERED: Members of the Association of Diabetes Care and Education Specialists Pharmacy Community of Interest were selected to review articles published in prominent peer-reviewed journals in 2020 that most impacted diabetes pharmacotherapy and technology. A list of 37 nominated articles were compiled (22 in diabetes pharmacotherapy and 15 in diabetes technology). Based on discussion among the authors, the articles were ranked based on significant contribution, impact, and diversity to diabetes pharmacotherapy and technology. The top 10 highest ranked publications (n = 6 for diabetes pharmacotherapy and n = 4 in diabetes technology) are summarized in this article. EXPERT OPINION: With the significant number of publications in diabetes care and education, it can be challenging and overwhelming to remain current with published literature. This review article may be helpful in identifying key articles in diabetes pharmacotherapy and technology from the year 2020.

Evaluation of the Use of a Diabetes Risk Test to Identify Prediabetes in an Employee Wellness Screening.

BACKGROUND: The American Diabetes Association (ADA) Diabetes Risk Test (DRT) is a screening tool to identify people at risk for developing diabetes. Individuals with a DRT score of 5 or higher may have prediabetes or diabetes and should see a healthcare provider. OBJECTIVE: To determine how many additional employees are identified as being at risk for developing diabetes during an employee wellness screening by using a more stringent DRT cutoff score of 4 instead of 5. METHODS: During an annual employee wellness screening event, a hemoglobin A1C (A1c) was drawn for participants with a DRT score of > 4 or by request regardless of risk score. A1C values were classified as normal (<5.7%), prediabetes (>5.7 and <6.5%) or diabetes (>6.5%). Risk scores and A1C values were analyzed using descriptive statistics. Cost of additional laboratory testing was also reviewed. RESULTS: An A1C was collected for 158 participants. Fourteen of 50 (28%) participants with a DRT of 4 had A1c values in the prediabetes range and no history of diabetes or prediabetes. Using the lower DRT score of 4 resulted in an additional expenditure of $305 with $85.40 resulting in the identification of an otherwise unaware person at risk for developing diabetes. CONCLUSION: Using a DRT cutoff score of 4 as part of an employee wellness screening program resulted in additional laboratory costs to identify persons at risk for developing diabetes but also allowed for earlier education to slow or stop the progression to diabetes which may reduce healthcare costs over time.

Analysis of Omega-3 Fatty Acid Content in Fish Oil Products.

BACKGROUND: Omega-3 fatty acids eicosapentaenoic acid (EPA) and docosehexaenoic acid (DHA), often found in fish oil supplements, have been linked to cardiovascular benefits in proper doses. OBJECTIVES: Quantify serving sizes and EPA and DHA content of fish oil products and determine which products contain appropriate amounts of EPA and DHA per serving to lower cholesterol. METHODS: Products were identified through the National Institutes of Health's Dietary Supplement Label Database using the search term "fish oil." Product labels were reviewed for EPA and DHA content. The number of units, such as capsules, gummies, or milliliters, necessary to obtain a total of at least 2,000 mg of EPA and DHA was also evaluated. Descriptive statistics were used to report findings. RESULTS: Of 493 products identified, 231 products were analyzed. Two (0.9%) products, both of which were liquid formulations, contained at least 2,000 mg of EPA and DHA in the standard serving size listed on the labeling. The total amount of EPA and DHA per serving ranged from 60.2 mg to 2684 mg with an average of 697 mg. The number of servings necessary to achieve 2,000 mg of EPA and DHA ranged from 1 to 34 servings with an average of 5 servings. CONCLUSIONS: Serving sizes of fish oil products rarely result in adequate EPA and DHA intake to provide cholesterol-lowering benefit. Instruction by a trained healthcare professional, such as a pharmacist, is important to ensure patients are taking an appropriate serving of fish oil to obtain cardiovascular benefit.

A Systematic Review of the Efficacy and Safety of Over-the-Counter Medications Used in Older People for the Treatment of Primary insomnia.

OBJECTIVE: The purpose of this systematic review is to evaluate the available evidence for safety and efficacy of over-the-counter (OTC) sleep aids used for the treatment of insomnia in older people.
DATA SOURCES: PubMed, EBSCO, and International Pharmaceutical Abstracts.
STUDY SELECTION: Five studies were included that involved humans 65 years of age and older being evaluated on OTC sleep aids in the outpatient setting.
DATA EXTRACTION: Data extraction from each study included primary and secondary efficacy endpoints, such as differences in the mean total sleep time, sleep latency, sleep efficiency, and number of awakenings, along with safety endpoints, such as psychomotor ability, cognitive ability, and adverse effect profiles. Both subjective and objective measures of changes in sleep and adverse effects were included.
DATA SYNTHESIS: Diphenhydramine had a statistically significant increase in sedation and decrease in number of awakenings but was not shown to be any less or more safe than compared products. Despite lacking safety issues, valerian was found to have no effect on subjective or objective sleep outcomes. Overall, melatonin had the most evidence and was found to have a statistically significant positive impact on sleep measures without safety issues.
CONCLUSION: Diphenhydramine and melatonin appear to be efficacious in improving some sleep measures while causing minimal adverse effects. However, there are very few studies that examine the use of over-the-counter sleep aids in those 65 years of age and older with primary insomnia. Additional studies are needed in this population.

Addressing Lifestyle Management During Visits Involving Patients with Prediabetes: NAMCS 2013-2015.

BACKGROUND: The gap between treatment guidelines and clinical practice in prediabetes management has been identified in previous studies. The knowledge related to addressing lifestyle change during office visits in clinical practice to manage prediabetes is limited. OBJECTIVE: To describe patterns of lifestyle management addressed during office-based visits involving patients with prediabetes and identify factors associated with addressing lifestyle management during physician office visits in the USA. DESIGN: Cross-sectional study PARTICIPANTS: US National Ambulatory Medical Care Survey (NAMCS) data from 2013 to 2015 were combined to identify office-based visits involving patients with prediabetes. MAIN MEASURES: The major outcome is lifestyle management including diet/nutrition, exercise, and/or weight reduction. Patient and physician characteristics were collected for analysis. The prevalence and patterns of addressing lifestyle management during visits were estimated and described. Multivariate logistic regression model identified significant factors associated with lifestyle management. The patient visit weight was applied to all analyses to achieve nationally representative estimates. KEY RESULTS: Among 4039 office-based visits involving patients with prediabetes between 2013 and 2015, 22.8% indicated lifestyle management was addressed during the visits. Diet/nutrition, exercise, and weight reduction accounted for 86.1%, 62.6%, and 34.1% of the visits with lifestyle management addressed, respectively. Lifestyle management was more likely to be addressed during the visits involving patients with hyperlipidemia (OR = 1.74, 95% CI 1.24-2.46) and obesity (OR = 4.03, 95% CI 2.91-5.56), seeing primary physicians (vs. other specialties, OR = 1.46, 95% CI 1.03-2.08), and living in the southern region (vs. northeast, OR = 1.96, 95% CI 1.20-3.19). CONCLUSIONS: The prevalence of addressing lifestyle management during office visits involving patients with prediabetes remained low in the USA. Patients' clinical characteristics, geographic region, and physician's specialty were associated with addressing lifestyle management during the visits.

Comparison of light transmission aggregometry and multiple electrode aggregometry for the evaluation of patients with mucocutaneous bleeding.

INTRODUCTION: The "gold standard" diagnostic test for assessing in vitro platelet function, light transmission aggregometry (LTA), has limitations to application because of sample requirements. Whole blood or multiple electrode aggregometry (MEA) using the Multiplate® analyzer (Roche Diagnostics) requires smaller blood volumes and less sample manipulation than LTA, making it an attractive clinical testing option. Direct comparisons of MEA with LTA for diagnosis of platelet aggregation abnormalities are few. METHODS: Ninety-nine patients (66 F/33 M; median age 26 [range 2-86] years), referred for initial laboratory evaluation of mucocutaneous bleeding, had parallel MEA/LTA testing. Concentrations of ADP, arachidonic acid (AA), collagen, and thrombin receptor-activating peptide (TRAP) that produced threshold responses in normal controls were used for testing patients. RESULTS: Twenty-nine of the 99 patients (30%) had at least one abnormal agonist response by LTA; 15 of these patients had >1 abnormal agonist response. Thirty-six patients (36%) had at least one abnormal agonist response by MEA; 27 had >1 abnormal agonist response. Sensitivity/specificity of MEA relative to LTA: ADP, 0.70/0.72; AA, 0.71/0.85; collagen, 0.85/0.71; TRAP 0.25/0.84. Negative predictive values (NPVs) for MEA relative to LTA: ADP, 0.90; AA, 0.93; collagen, 0.97; TRAP, 0.96. CONCLUSIONS: Specific abnormal results of MEA testing did not adequately predict specific abnormalities in LTA testing using threshold agonist concentrations. However, favorable NPVs suggest that MEA may be useful in screening patients for platelet aggregation abnormalities; those with normal MEA results not requiring further diagnostic testing by LTA.

Cytokine production capabilities of human primary monocyte-derived macrophages from patients with diabetes mellitus type 2 with and without diabetic peripheral neuropathy.

INTRODUCTION: Monocytes from patients with diabetes mellitus type 2 (DM2) are dysfunctional, persistently primed, and prone to a proinflammatory phenotype. This may alter the phenotype of their differentiation to macrophages and result in diabetic peripheral neuropathy (DPN), nerve damage, nerve sensitization, and chronic pain. We have previously demonstrated that CD163 is a molecule that promotes an anti-inflammatory cellular phenotype in human primary macrophages, but this has not been proven in macrophages from patients with DM2 or DPN. Thus, we hypothesize that macrophages from patients with DM2 or DPN display an altered proinflammatory functional phenotype related to cytokine production and that the induction of CD163 expression will promote a more homeostatic phenotype by reducing their proinflammatory responsiveness. PATIENTS AND METHODS: We tested these hypotheses in vitro using blood monocyte-derived macrophages from healthy subjects and patients with DM2 with and without DPN. Cells were incubated in the presence or the absence of 5 µg/mL of lipopolysaccharide (LPS). The concentrations of interleukin-10, interleukin-6, tumor necrosis factor-alpha (TNF-α), TGF-ß, and monocyte chemoattractant protein-1 (MCP-1) were measured using ELISA assays. Macrophages were transfected with an empty vector plasmid or a plasmid containing the CD163 gene using mannosylated polyethylenimine nanoparticles. RESULTS: Our results show that nonstimulated DM2 or DPN macrophages have a constitutive primed proinflammatory state and display a deficient production of proinflammatory cytokines upon a proinflammatory challenge when compared to healthy macrophages. CD163 induction produced an anti-inflammatory phenotype in the healthy control group, and this effect was partial in DM2 or DPN macrophages. CONCLUSION: Our results suggest that diabetic macrophages adopt a complex phenotype that is only partially reversed by CD163 induction. Future experiments are focused on elucidating this differential responsiveness between healthy and diabetic macrophages.

Analysis of procoagulant phosphatidylserine-exposing platelets by imaging flow cytometry.

BACKGROUND: Upon platelet activation, a subpopulation of procoagulant platelets is formed, characterized by the exposure of the anionic aminophospholipid phosphatidylserine (PS) on the surface membrane. OBJECTIVE: To evaluate procoagulant PS-exposing platelets by imaging flow cytometry. METHODS: Platelet ultrastructure was examined by transmission electron microscopy, and a comprehensive analysis of procoagulant platelets was performed using imaging flow cytometry; platelets were fluorescently labeled for the markers glycoprotein (GP)IX, activated integrin αIIbß3, CD62P, and PS exposure. RESULTS: A subpopulation of platelets stimulated in suspension by the physiological agonists thrombin+collagen, and all platelets stimulated by the calcium ionophore A23187, had a distinct round morphology. These platelets were PS-exposing, larger in size, had an increased circularity index, and had reduced internal complexity compared with non-PS-exposing platelets. They expressed CD62P and αIIbß3 in an inactive conformation on the surface, and demonstrated depolarized inner mitochondrial membranes. For the first time, using imaging flow cytometry, a large proportion of PS-exposing platelets possessing platelet-associated extracellular vesicles (EVs) was observed, which demonstrated heterogeneous platelet marker expression that was different from free released EVs. CONCLUSIONS: Innovative imaging flow cytometry allowed detailed fluorescence-based, quantitative morphometric analysis of PS-exposing platelets; in becoming procoagulant, platelets undergo remarkable morphological changes, transforming into spherical "balloons," almost devoid of their normal internal architecture. Almost all PS-exposing platelets have associated EVs that are not detectable by traditional flow cytometry. While their functions have yet to be fully elucidated, the heterogeneity of platelet-associated and released EVs suggests that they may contribute to different aspects of hemostasis and of thrombosis.

Student pharmacist perceived benefit of a sequential training model for health testing.

PURPOSE: This article describes the health testing training process used at Presbyterian College School of Pharmacy and evaluates perceptions of the sequential training model among students in their first, second, and third professional years. EDUCATIONAL ACTIVITY AND SETTING: After observing student deficiencies in the knowledge and skills necessary for performing health tests, despite receiving didactic training within the core curriculum, faculty members searched for supplemental training programs used by other schools of pharmacy. No literature regarding structured programs was found. Consequently, faculty developed test-specific training modules for a variety of health screenings. Students who participated in the sequential self-learning followed by a live skills assessment were surveyed to determine their perceptions of the training. FINDINGS: During the 2014-2015 academic year, 78 students successfully completed health testing training modules. Of these students, 56 (72%) completed an attitudinal survey designed to assess their perceptions. Nearly 93% of respondents perceived improved confidence after completing the training. Regardless of the year in pharmacy school, 88% of respondents believe they would not have been adequately prepared to conduct the health test(s) without this training. SUMMARY: Student perception and acceptance of health testing training were positive. Using sequential training modules to teach and reinforce the skills necessary for performing health tests can improve student ability and confidence. Consequently, students have the opportunity to impact the health of the community while becoming practice ready in the area of health testing.

Metformin prescribing in low-income and insured patients with prediabetes.

OBJECTIVES: To describe prescribing patterns of metformin in low-income and Medicaid-insured patients with prediabetes and to identify common demographic characteristics and comorbid conditions of low-income and Medicaid-insured patients receiving metformin for treatment of prediabetes. DESIGN: Retrospective observational study. SETTING AND PARTICIPANTS: Patients (18-60 years old) who were enrolled in South Carolina Medicaid and diagnosed with prediabetes between January 2009 and December 2013. MAIN OUTCOME MEASURES: Metformin prescribing to treat prediabetes identified from pharmacy claims. RESULTS: Among 7102 patients who met the study criteria, 7.4% (n = 520) were prescribed metformin for prediabetes. Nearly 45% (n = 238) of eligible patients prescribed metformin initiated treatment within 30 days after diagnosis of prediabetes. Twenty-five percent of those prescribed metformin took 280 days or longer to initiate treatment after diagnosis of prediabetes. Older age, black race, managed care plan, comorbid hypertension and obesity, and longer enrollment period significantly increased the likelihood of metformin prescribing to treat prediabetes. CONCLUSION: Prevalence of metformin prescription to treat prediabetes is less than 8% in low-income and Medicaid-insured patients. Sociodemographic characteristics and comorbid conditions influenced metformin prescribing in the low-income population.

Telehealth for diabetes self-management education and support in an underserved, free clinic population: A pilot study.

OBJECTIVES: Primary study objectives were to (1) describe mean change in A1c from baseline of a free clinic population enrolled in telehealth diabetes self-management education and support (DSME/S) services and (2) to compare change in A1C and other clinical outcomes measures with free clinic patients enrolled in a traditional face-to-face DSME/S program. METHODS: An exploratory study design and comparative evaluation of telehealth DSME/S services in a free clinic population was used. Baseline clinical measures were collected upon referral. Diabetes educators met with patients individually over 2-3 months. Clinical outcomes measures were collected within 6 months of program completion. Data from the telehealth group was assessed individually and compared to a free clinic traditional DSME/S program population. RESULTS: Twelve patients completed a telehealth free clinic DSME/S pilot program with a mean ± SD change in A1C from baseline of -1.03 ± 1.53% (P = 0.050). Mean ± SD change in A1C from baseline in the free clinic population participating in traditional face-to-face DSME/S services was -1.42 ± 1.80% (P = 0.001). No significant differences in secondary outcomes measures, including body mass index and blood pressure, were revealed among the study populations. CONCLUSION: Expanding access to care in populations faced with challenges of socioeconomics, limited education, and lower health literacy is a step toward reducing health disparities and positively affecting care. Mean A1C can be improved with telehealth DSME/S services in an underserved, free clinic population.

Progression to Type 2 Diabetes and Its Effect on Health Care Costs in Low-Income and Insured Patients with Prediabetes: A Retrospective Study Using Medicaid Claims Data.

BACKGROUND: Prediabetes is a high-risk factor for progression to diabetes. Without lifestyle changes, such as weight loss and moderate physical activity, 15%-30% of people with prediabetes are projected to develop type 2 diabetes within 5 years. Progression to diabetes increases the financial burden significantly for patients and health care systems. Populations with low socioeconomic status are associated with a higher risk of diabetes. However, knowledge is limited about the effect of transition to diabetes on future costs incurred in low-income populations. OBJECTIVES: To (a) describe the characteristics of low-income and insured patients with prediabetes and (b) examine the effect of progression to type 2 diabetes on health care utilization and costs. METHODS: This study used South Carolina Medicaid claims data (2009-2014) to identify patients (aged ≥18 years) with newly diagnosed prediabetes. All patients were enrolled in Medicaid continuously for at least 1 year before and after the diagnosis of prediabetes and were followed for at least 1 year and up to 6 years. The time to progression to type 2 diabetes was measured by a Kaplan Meier curve, and risk factors associated with onset of type 2 diabetes were identified by Cox regression. Generalized linear models were applied to assess the effect of progression to type 2 diabetes on total health care costs during the first 3-year period. RESULTS: A total of 7,650 patients with prediabetes met the study criteria. During the follow-up period, 30.3% of the study population developed type 2 diabetes within 3 years. Older age, African-American race, fee-for-service plan, comorbid hypertension, obesity, and dyslipidemia were associated with higher risk for onset of type 2 diabetes. Compared with patients who did not progress to type 2 diabetes, the progression to type 2 diabetes increased total health care costs by 22.1% (P < 0.001), 39.1% (P < 0.001), and 47.6% (P < 0.001) during the first 3 years after adjusting for demographic and comorbid conditions. CONCLUSIONS: Age, race, type of Medicaid plan, and diabetes-related comorbidities were associated with risk for progression of prediabetes. Progression to type 2 diabetes significantly increased total health care costs in the first 3 years. Early detection and intervention to prevent or delay onset of type 2 diabetes are needed to control health care utilization and costs. DISCLOSURES: This study was funded by Small Pharmacy Awards for Research and Collaboration, Presbyterian College. The funding resource had no role in the design and conduct of the study, analysis or interpretation of the data, or the preparation or final approval of the manuscript before publication. The authors declare no conflicts of interest. Study concept and design were contributed by Wu, Ward, and Lu, along with Threatt. Wu took the lead in data collection, along with Ward and Lu, with assistance from Threat. Data interpretation was provided by Wu, Ward, Threatt, and Lu. The manuscript was written and revised by Wu, Ward, and Threatt, along with Lu.

Clinical Pharmacokinetics and Pharmacodynamics of Insulin Glargine 300 U/mL.

Concentrated insulin analogs have recently been approved and are available for clinical use in the management of diabetes mellitus. One new product is insulin glargine U-300 (Sanofi), a basal concentrated insulin of 300 U/mL. Several studies have been conducted and completed evaluating blood samples for the pharmacokinetics of insulin glargine U-300 and euglycemic clamp procedures for the pharmacodynamics. This concentrated insulin has a low within-day variability and high day-to-day reproducibility, allowing for a more constant and prolonged duration of action, compared with insulin glargine U-100 (100 U/mL). Insulin glargine U-300 is equally effective, when compared with insulin glargine U-100 for glycemic control in patients with type 1 and 2 diabetes mellitus. Insulin glargine U-300 has a similar efficacy profile to insulin glargine U-100 for glycemic control, yet with lower rates of nocturnal and severe hypoglycemia. Insulin glargine U-300 can be considered an acceptable basal insulin for patients with type 1 and 2 diabetes mellitus, and it has a potential role among patients who are naïve to insulin therapy or require titration of basal insulin. Titration of insulin glargine U-300 would result in less volume and a lower risk of hypoglycemia, compared with insulin glargine U-100. This article evaluates and summarizes the pharmacokinetics and pharmacodynamics of insulin glargine U-300, for patients with type 1 or 2 diabetes mellitus, and summarizes its application to clinical practice.

Tyrosine kinase receptor EGFR regulates the switch in cancer cells between cell survival and cell death induced by autophagy in hypoxia.

Autophagy is an intracellular lysosomal degradation pathway where its primary function is to allow cells to survive under stressful conditions. Autophagy is, however, a double-edge sword that can either promote cell survival or cell death. In cancer, hypoxic regions contribute to poor prognosis due to the ability of cancer cells to adapt to hypoxia in part through autophagy. In contrast, autophagy could contribute to hypoxia induced cell death in cancer cells. In this study, we showed that autophagy increased during hypoxia. At 4 h of hypoxia, autophagy promoted cell survival whereas, after 48 h of hypoxia, autophagy increased cell death. Furthermore, we found that the tyrosine phosphorylation of EGFR (epidermal growth factor receptor) decreased after 16 h in hypoxia. Furthermore, EGFR binding to BECN1 in hypoxia was significantly higher at 4 h compared to 72 h. Knocking down or inhibiting EGFR resulted in an increase in autophagy contributing to increased cell death under hypoxia. In contrast, when EGFR was reactivated by the addition of EGF, the level of autophagy was reduced which led to decreased cell death. Hypoxia led to autophagic degradation of the lipid raft protein CAV1 (caveolin 1) that is known to bind and activate EGFR in a ligand-independent manner during hypoxia. By knocking down CAV1, the amount of EGFR phosphorylation was decreased in hypoxia and amount of autophagy and cell death increased. This indicates that the activation of EGFR plays a critical role in the switch between cell survival and cell death induced by autophagy in hypoxia.

Palmitoylation supports the association of tetraspanin CD63 with CD9 and integrin alphaIIbbeta3 in activated platelets.

CD63 and CD9 are members of the tetraspanin superfamily of integral membrane proteins that function as organizers of multi-molecular signaling complexes involved in cell morphology, motility and proliferation. Tetraspanin complexes cluster dynamically in unique cholesterol-rich tetraspanin-enriched microdomains (TEMs). In resting platelets, CD63 is located in the membranes of lysosomes and dense granules. Following platelet activation and granule exocytosis, CD63 is expressed on the plasma membrane, co-localizes with the alphaIIbbeta3-CD9 complex and is incorporated into the Triton-insoluble actin cytoskeleton, dependent on fibrinogen binding to alphaIIbbeta3. In nucleated cell lines, the assembly and maintenance of TEMs depends on the palmitoylation of both tetraspanins and some partner proteins. This study investigated the role of palmitoylation in platelet TEM assembly and maintenance. [(3)H]-palmitate-labeled, washed human platelets were studied at rest, or following activation with thrombin (0.1 U/ml). CD63 and CD9 were separated by density gradient centrifugation, isolated by immunoprecipitation, and [(3)H]-palmitate was measured in each fraction. Palmitate levels increased in all fractions following thrombin activation. However, the relative inter-fraction distribution of the tetraspanins did not change. 2-bromopalmitate (2-BP), an inhibitor of protein palmitoylation as demonstrated by decreased [(3)H]-palmitate labeling of platelet proteins, blocked both thrombin-induced platelet aggregation and platelet spreading on immobilized fibrinogen in a dose-dependent manner. 2-BP also inhibited the activation-dependent association of CD63 with CD9, and the incorporation of CD63 into the Triton-insoluble actin cytoskeleton. In contrast, 2-BP had no effect on the incorporation of alphaIIbbeta3 into the activated platelet cytoskeleton. These results demonstrate that palmitoylation is required for platelet tetraspanin-tetraspanin and tetraspanin-integrin interaction and for complete platelet spreading on a fibrinogen substrate.

Hypoxia induces autophagic cell death in apoptosis-competent cells through a mechanism involving BNIP3.

Hypoxia (lack of oxygen) is a physiological stress often associated with solid tumors. Hypoxia correlates with poor prognosis since hypoxic regions within tumors are considered apoptosisresistant. Autophagy (cellular "self digestion") has been associated with hypoxia during cardiac ischemia and metabolic stress as a survival mechanism. However, although autophagy is best characterized as a survival response, it can also function as a mechanism of programmed cell death. Our results show that autophagic cell death is induced by hypoxia in cancer cells with intact apoptotic machinery. We have analyzed two glioma cell lines (U87, U373), two breast cancer cell lines (MDA-MB-231, ZR75) and one embryonic cell line (HEK293) for cell death response in hypoxia (<1% O(2)). Under normoxic conditions, all five cell lines undergo etoposide-induced apoptosis whereas hypoxia fails to induce these apoptotic responses. All five cell lines induce an autophagic response and undergo cell death in hypoxia. Hypoxia-induced cell death was reduced upon treatment with the autophagy inhibitor 3-methyladenine, but not with the caspase inhibitor z-VAD-fmk. By knocking down the autophagy proteins Beclin-1 or ATG5, hypoxia-induced cell death was also reduced. The pro-cell death Bcl-2 family member BNIP3 (Bcl-2/adenovirus E1B 19kDainteracting protein 3) is upregulated during hypoxia and is known to induce autophagy and cell death. We found that BNIP3 overexpression induced autophagy, while expression of BNIP3 siRNA or a dominant-negative form of BNIP3 reduced hypoxia-induced autophagy. Taken together, these results suggest that prolonged hypoxia induces autophagic cell death in apoptosis-competent cells, through a mechanism involving BNIP3.

Platelet tetraspanin complexes and their association with lipid rafts.

Tetraspanins are a superfamily of integral membrane proteins that facilitate the organization of membrane and intracellular signaling molecules into dynamic signaling microdomains, tetraspanin-enriched microdomains (TEMs). Four tetraspanin family members have been identified in platelets: CD9, CD151 and TSSC6, which are constitutively associated with alphaIIbbeta3, and CD63, which is present on granule membranes in resting platelets and associates with alphaIIbbeta3-CD9 following platelet activation. CD63 and CD9 associate with a type II phosphatidylinositol 4-kinase, PI4K55, in both resting and activated platelets. Immunoelectron microscopic studies showed co-localization of CD63 and PI4K55 on internal membranes of resting platelets and on the filopodia of thrombin-activated platelets. Because TEMs in malignant cell lines appear to be distinct from prototypic lipid rafts, this study examined whether CD63-PI4K55 and CD9-PI4K55 complexes were resident in platelet-lipid rafts, or formed distinct microdomains. CD63, CD9 and PI4K55 were recovered from low-density membrane fractions (LDMFs) of sucrose gradients following platelet lysis in Brij 35, but unlike lipid-raft proteins were not insoluble in Triton X-100, being absent from LDMFs of platelets lysed with Triton. Incubation of platelets with methyl-beta-cyclodextrin, to deplete cholesterol and disrupt lipid rafts, shifted the complexes to higher density sucrose gradient fractions, but did not disrupt the tetraspanin-PI4K55 complexes. These results demonstrate that tetraspanin complexes in platelets form cholesterol-associated microdomains that are distinct from lipid rafts. It is probable that TEMs and lipid rafts associate under certain conditions, resulting in the close proximity of distinct sets of signaling molecules, facilitating signal transduction.

Mitochondrial electron-transport-chain inhibitors of complexes I and II induce autophagic cell death mediated by reactive oxygen species.

Autophagy is a self-digestion process important for cell survival during starvation. It has also been described as a form of programmed cell death. Mitochondria are important regulators of autophagy-induced cell death and damaged mitochondria are often degraded by autophagosomes. Inhibition of the mitochondrial electron transport chain (mETC) induces cell death through generating reactive oxygen species (ROS). The role of mETC inhibitors in autophagy-induced cell death is unknown. Herein, we determined that inhibitors of complex I (rotenone) and complex II (TTFA) induce cell death and autophagy in the transformed cell line HEK 293, and in cancer cell lines U87 and HeLa. Blocking the expression of autophagic genes (beclin 1 and ATG5) by siRNAs or using the autophagy inhibitor 3-methyladenine (3-MA) decreased cell death that was induced by rotenone or TTFA. Rotenone and TTFA induce ROS production, and the ROS scavenger tiron decreased autophagy and cell death induced by rotenone and TTFA. Overexpression of manganese-superoxide dismutase (SOD2) in HeLa cells decreased autophagy and cell death induced by rotenone and TTFA. Furthermore, blocking SOD2 expression by siRNA in HeLa cells increased ROS generation, autophagy and cell death induced by rotenone and TTFA. Rotenone- and TTFA-induced ROS generation was not affected by 3-MA, or by beclin 1 and ATG5 siRNAs. By contrast, treatment of non-transformed primary mouse astrocytes with rotenone or TTFA failed to significantly increase levels of ROS or autophagy. These results indicate that targeting mETC complex I and II selectively induces autophagic cell death through a ROS-mediated mechanism.

CD63 modulates spreading and tyrosine phosphorylation of platelets on immobilized fibrinogen.

CD63 is a member of the tetraspanin superfamily of integral membrane proteins. Present on a variety of cells, tetraspanins can form lateral associations with integrins and may act as 'organizers' of multimolecular networks that modulate integrinmediated signaling, cell morphology, motility and migration. In resting platelets, CD63 is present on the membranes of dense granules and lysosomes but relocates to the plasma membrane following platelet activation and exocytosis where it associates with the platelet integrin alphaIIBbeta3-CD9 complex and with the actin cytoskeleton in an alphaIIBbeta 3-dependent manner. D545, a monoclonal antibody directed at the second extracellular loop of CD63,was used to investigate the role of CD63 in platelet adhesion, spreading and tyrosine phosphorylation. Using immunofluorescence microscopy and confocal imaging, we have demonstrated that D545 does not alter adhesion of platelets to immobilized fibrinogen, but instead platelet spreading. In the presence of buffer or non-specific mouse IgG, activated platelets showed fully spread morphology, F-actin reorganization, redistribution of vinculin and extensive tyrosine phosphorylation, all of which were inhibited by D545. D545 also inhibited the phosphorylation of focal adhesion kinase in thrombin-activated adherent platelets. These results suggest that CD63 may modulate alphaIIBbeta3-dependent cytoskeletal reorganization. To identify signaling enzymes associated with CD63 that could affect this pathway, lipid kinase assays were performed on D545 immunoprecipitates. CD63 co-immunoprecipitated with a lipid kinase which, on the basis of enzymatic properties(stimulated by nonionic detergents, inhibited by adenosine), is consistent with PI 4-kinase type II. The CD63-PI 4-kinase complex was not activation-dependent as the constituents were co-purified from both resting and activated platelets. The linkage of CD63 with PI 4-kinase may result in the recruitment of this signaling enzyme to specific membrane locations in the platelet where it influences phosphoinositide-dependent signaling and platelet spreading.

BNIP3 plays a role in hypoxic cell death in human epithelial cells that is inhibited by growth factors EGF and IGF.

Hypoxic regions within solid tumors are often resistant to chemotherapy and radiation. BNIP3 (Bcl-2/E1B 19 kDa interacting protein) is a proapoptotic member of the Bcl-2 family that is expressed in hypoxic regions of tumors. During hypoxia, BNIP3 expression is increased in many cell types and upon forced overexpression BNIP3 induces cell death. Herein, we have demonstrated that blockage of hypoxia-induced BNIP3 expression using antisense oligonucleotides against BNIP3 or blockage of BNIP3 function through expression of a mutant form of BNIP3 inhibits hypoxia-induced cell death in human embryonic kidney 293 cells. We have also determined that hypoxia-mediated BNIP3 expression is regulated by the transcription factor, hypoxia-inducible factor-1alpha (HIF-1alpha) in human epithelial cell lines. Furthermore, HIF-1alpha directly binds to a consensus HIF-1alpha-responsive element (HRE) in the human BNIP3 promoter that upon mutation of this HRE site eliminates the hypoxic responsiveness of the promoter. Since BNIP3 is expressed in hypoxic regions of tumors but fails to induce cell death, we determined whether growth factors block BNIP3-induced cell death. Treatment of the breast cancer cell line MCF-7 cells with epidermal growth factor (EGF) or insulin-like growth factor effectively protected these cells from BNIP3-induced cell death. Furthermore, inhibiting EGF receptor signaling using antibodies against ErbB2 (Herceptin) resulted in increased hypoxia-induced cell death in MCF-7 cells. Taken together, BNIP3 plays a role in hypoxia-induced cell death in human epithelial cells that could be circumvented by growth factor signaling.