Bihormonal fully closed-loop system for the treatment of type 1 diabetes: a real-world multicentre, prospective, single-arm trial in the Netherlands.

BACKGROUND: Management of insulin administration for intake of carbohydrates and physical activity can be burdensome for people with type 1 diabetes on hybrid closed-loop systems. Bihormonal fully closed-loop (FCL) systems could help reduce this burden. In this trial, we assessed the long-term performance and safety of a bihormonal FCL system. METHODS: The FCL system (Inreda AP; Inreda Diabetic, Goor, Netherlands) that uses two hormones (insulin and glucagon) was assessed in a 1 year, multicentre, prospective, single-arm intervention trial in adults with type 1 diabetes. Participants were recruited in eight outpatient clinics in the Netherlands. We included adults with type 1 diabetes aged 18-75 years who had been using flash glucose monitoring or continuous glucose monitors for at least 3 months. Study visits were integrated into standard care, usually every three months, to evaluate glycaemic control, adverse events, and person-reported outcomes. The primary endpoint was time in range (TIR; glucose concentration 3·9-10·0 mmol/L) after 1 year. The study is registered in the Dutch Trial Register, NL9578. FINDINGS: Between June 1, 2021, and March 2, 2022, we screened 90 individuals and enrolled 82 participants; 78 were included in the analyses. 79 started the intervention and 71 were included in the 12 month analysis. Mean age was 47.7 (SD 12·4) years and 38 (49%) were female participants. The mean preintervention TIR of participants was 55·5% (SD 17·2). After 1 year of FCL treatment, mean TIR was 80·3% (SD 5·4) and median time below range was 1·36% (IQR 0·80-2·11). Questionnaire scores improved on Problem Areas in Diabetes (PAID) from 30·0 (IQR 18·8-41·3) preintervention to 10·0 (IQR 3·8-21·3; p<0·0001) at 12 months and on World Health Organization-Five Well-Being Index (WHO-5) from 60·0 (IQR 44·0-72·0) preintervention to 76·0 (IQR 60·0-80·0; p<0·0001) at 12 months. Five serious adverse events were reported (one cerebellar stroke, two severe hypoglycaemic, and two hyperglycaemic events). INTERPRETATION: Real-world data obtained in this trial demonstrate that use of the bihormonal FCL system was associated with good glycaemic control in patients who completed 1 year of treatment, and could help relieve these individuals with type 1 diabetes from making treatment decisions and the burden of carbohydrate counting. FUNDING: Inreda Diabetic.

Sphingolipid mediators in cardiovascular cell biology and pathology.

Sphingolipids have emerged as a new class of lipid mediators. In response to various extracellular stimuli, sphingolipid turnover can be stimulated in vascular cells and cardiac myocytes. Subsequent generation of sphingolipid molecules such as ceramide, sphingosine, and sphingosine-1-phosphate, is followed by regulation of ion fluxes and activation of various signaling pathways leading to smooth muscle cell proliferation, endothelial cell differentiation or apoptotic cell death, cell contraction, retraction, or migration. The importance of sphingolipids in cardiovascular signaling is illustrated by recent observations implicating them in physiological processes such as vasculogenesis as well as in frequent pathological conditions, including atherosclerosis and its complications.

Comparative genomic hybridization analysis of 38 breast cancer cell lines: a basis for interpreting complementary DNA microarray data.

Breast cancer cell lines provide a useful starting point for the discovery and functional analysis of genes involved in breast cancer. Here, we studied 38 established breast cancer cell lines by comparative genomic hybridization (CGH) to determine recurrent genetic alterations and the extent to which these cell lines resemble uncultured tumors. The following chromosomal gains were observed: 8q (75%), 1q (61%), 20q (55%), 7p (44%), 3q (39%), 5p (39%), 7q (39%), 17q (33%), 1p (30%), and 20p (30%), and the most common losses were: 8p (58%), 18q (58%), 1p (42%), Xp (42%), Xq (42%), 4p (36%), 11q (36%), 18p (33%), 10q (30%), and 19p (28%). Furthermore, 35 recurrent high-level amplification sites were identified, most often involving 8q23 (37%), 20q13 (29%), 3q25-q26 (24%), 17q22-q23 (16%), 17q23-q24 (16%), 1p13 (11%), 1q32 (11%), 5p13 (11%), 5p14 (11%), 11q13 (11%), 17q12-q21 (11%), and 7q21-q22 (11%). A comparison of DNA copy number changes found in the cell lines with those reported in 17 published studies (698 tumors) of uncultured tumors revealed a substantial degree of overlap. CGH copy number profiles may facilitate identification of important new genes located at the hotspots of such chromosomal alterations. This was illustrated by analyzing expression levels of 1236 genes using cDNA microarrays in four of the cell lines. Several highly overexpressed genes (such as RCH1 at 17q23, TOPO II at 17q21-q22, as well as CAS and MYBL2 at 20q13) were involved in these recurrent DNA amplifications. In conclusion, DNA copy number profiles were generated by CGH for most of the publicly available breast cancer cell lines and were made available on a web site (http://www.nhgri.nih.gov/DIR/CGB/++ +CR2000). This should facilitate the correlative analysis of gene expression and copy number as illustrated here by the finding by cDNA microarrays of several overexpressed genes that were amplified.

1-phenyl-2-decanoylamino-3-morpholino-1-propanol chemosensitizes neuroblastoma cells for taxol and vincristine.

In this study, we show that an inhibitor of glycosphin-golipid biosynthesis, D,L-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP), increases the chemosensitivity of neuroblastoma tumor cells for Taxol and vincristine. At noneffective low doses of Taxol or vincristine, the addition of a noneffective dose of PDMP resulted in 70% cytotoxicity, indicating synergy. Such an effect was not observed for etoposide (VP16). PDMP caused an early (6 h) increase in ceramide (Cer) levels, but the excess Cer was metabolically removed in the long-term (96 h). However, upon incubation with PDMP in combination with Taxol, but not with etoposide, Cer levels remained elevated at 96 h. These results suggest that neuroblastoma cells are normally able to metabolically remove excess Cer, but lose this capacity upon exposure to microtubule modulating anticancer agents (Taxol or vincristine). In addition, PDMP treatment resulted in a decreased efflux of [14C]Taxol and [3H]vincristine from neuroblastoma cells, similar to treatment with PSC833 or MK571, suggesting an effect of PDMP on the transporter proteins P-glycoprotein and/or multidrug resistance protein. PDMP did not further reduce [14C]Taxol or [3H]vincristine efflux in PSC833-treated cells, although it did further diminish cell survival under these conditions. We conclude that a combined administration of nontoxic concentrations of PDMP and either Taxol or vincristine results in highly sensitized neuroblastoma cells. This appears to involve a sustained elevation of Cer levels, possibly in concert with increased drug accumulation.

Apparently complete restoration of normal daily adrenocorticotropin, cortisol, growth hormone, and prolactin secretory dynamics in adults with Cushing's disease after clinically successful transsphenoidal adenomectomy.

ACTH production in Cushing's disease is characterized by a markedly elevated rate of basal (nonpulsatile) secretion, an increased mass of ACTH released per burst and an unremarkable pulse frequency. In addition, the ACTH secretory process and that of GH and PRL exhibit profoundly disordered patterns. Whether some or all of these disturbances can be reversed or normalized by transsphenoidal microadenomectomy remains unknown. We therefore investigated the detailed dynamics of ACTH, GH, and PRL in eight patients (aged 38.9+/-4.2 yr) with pituitary-dependent Cushing's disease who were in long-term (8.2+/-1.7 yr) clinical remission following transsphenoidal surgery and eight controls matched for age, gender, and body mass index. To this end, blood was sampled at 10-min intervals for 24 h for the later assay of ACTH, cortisol, GH, and PRL. Secretory activity was quantitated by deconvolution methods, and the pattern orderliness (regularity) of hormone release was determined by the approximate entropy (ApEn) statistic. The joint synchrony of ACTH and cortisol secretion was monitored by the cognate bivariate statistic, cross-ApEn. Diurnal properties of the hormonal release were appraised by cosinor analysis. Based on deconvolution analysis, postsurgical patients exhibited a normal frequency, half-life, duration, and mass of ACTH and cortisol secretory bursts. Accordingly, the 24-h production rates of both ACTH (2.5+/-0.7 microg/L in patients vs. 2.9+/-0.7 microg/L in controls; P = 0.755) and cortisol (49+/-11 micromol/L in patients vs. 73+/-15 micromol/L in controls; P = 0.217) were normal also. The acrophase of the diurnal rhythm of ACTH (patients, 0817 h +/- 37 min; controls, 0850 h +/- 38 min; P = 0.629) and cortisol (patients, 1000 h +/- 24 min; controls, 0855 h +/- 30 min; P = 0.175) was also restored by surgery. ApEn values of ACTH (patients, 1.168 +/- 0.090; controls, 0.864+/-0.122; P = 0.133) and cross-ApEn of ACTH-cortisol (patients, 1.396+/-0.087; controls, 1.170+/-0.076; P = 0.140) secretion were both normal in this cohort, denoting restoration of the secretory process regularity. Cortisol ApEn was slightly higher in patients (patients, 1.034+/-0.084; controls, 0.831+/-0.038; P = 0.048). Both GH and PRL time series manifested full reconstitution of pulsatile, 24-h rhythmic, and entropic properties. In summary, clinically successful transsphenoidal microadenomectomy in adults with Cushing's disease can fully normalize virtually all quantitative features of regulated ACTH, cortisol, GH, and PRL secretion. Further studies will be needed to establish the consistency of these findings in larger cohorts of adults with Cushing's disease and in children with this disorder and to delineate the significance, if any, of a residual, minimally detectable disruption of orderly cortisol secretion in this patient population.

Basal, pulsatile, entropic, and 24-hour rhythmic features of secondary hyperprolactinemia due to functional pituitary stalk disconnection mimic tumoral (primary) hyperprolactinemia.

Under physiological conditions, PRL secretion is regulated precisely by various stimulating and inhibiting factors. Hyperprolactinemia may arise as a primary consequence of a PRL-secreting pituitary adenoma. Secondary hyperprolactinemia (SH) may emerge in patients with hypothalamic disease, hypophyseal stalk compression, or suprasellar extension of a (nonlactotrope) pituitary adenoma. The latter may reflect diminished delivery of dopamine or other inhibitory factors to normal lactotropes. We hypothesized that diurnal and ultradian rhythms of PRL secretion would differ in secondary (e.g. hypothalamic) and primary (e.g. tumoral states) hyperprolactinemia (PH), assuming that the underlying pathophysiologies differ. To test this clinical postulate, we investigated the patterns of 24-h PRL release in eight patients with SH associated with functional hypothalamo-pituitary disconnection and in eight patients with PH attributable to microprolactinoma. Data in each group were compared with values in healthy gender-matched controls. PRL time series were obtained by repetitive 10-min blood sampling, followed by high- precision immunofluorometric assay. PRL concentration profiles were analyzed by the complementary tools of model-free discrete peak detection, waveform-independent deconvolution analysis, cosinor regression, and the approximate entropy metric to quantitate pulsatile, basal, 24-h rhythmic, and pattern-dependent (entropic) PRL secretion. Patients with tumoral hyperprolactinemia (PH) showed a 2-fold higher 24-h mean serum PRL concentration than patients with SH (62 +/- 13 microg /L vs. 30 +/- 6.9 microg/L, respectively, P = 0.029). Estimated PRL pulse frequency (events/24 h) was similar in the two patient groups (18.5 +/- 0.7 vs. 17.6 +/- 0.8; P = 0.395) but elevated over that in euprolactinemic controls (P < 0.0001 for both). Deconvolution analysis disclosed a mean daily PRL secretion rate of 790 +/- 170 microg in PH patients vs. 380 +/- 85 microg in SH patients (P = 0.030). Nonpulsatile PRL secretion comprised nearly 70% of total secretion in both patient groups and 50% in controls (P < 0.0001). Cosinor analysis revealed similar acrophases in all three study cohorts. The mean skewness of the statistical distribution of the individual PRL sample secretory rates was reduced, compared with controls (P < 10 (-5) for each), but equivalent in SH and PH patients (0.83 +/- 0.12 vs. 0.78 +/- 0.08, respectively), denoting a loss of the normal spectrum of low- and higher-amplitude secretion rates. Approximate entropy, a regularity statistic, was markedly elevated in both patient groups over controls (P < 10 (-6) for each) and was slightly higher in PH patients than in SH patients (1.639 +/- 0.029 vs. 1.482 +/- 0.067, P = 0.048). In summary, patterns of PRL secretion in PH and SH states exhibit an equivalently increased frequency of PRL pulses, a comparably marked rise in nonpulsatile (basal) PRL secretion. Despite overlap, the regularity of PRL release patterns is disrupted even more profoundly in PH (tumoral), compared with SH. Assuming that the orderliness of serial PRL output monitors normal integration within a feedback-controlled neurohormone axis, then the more disorderly patterns of tumoral PRL secretion point to greater regulatory disruption in PH. The latter may reflect abnormal secretory behavior associated with lactotrope neoplastic transformation and/or isolation of the tumor cell mass from normal hypothalamic controls.

Growth hormone (GH) secretion in patients with an inactivating defect of the GH-releasing hormone (GHRH) receptor is pulsatile: evidence for a role for non-GHRH inputs into the generation of GH pulses.

GH secretion is regulated by the interaction of GHRH and somatostatin and is released in 10-20 pulses in each 24-h cycle. The exact roles in pulse generation played by somatostatin, GHRH, and the recently isolated GH-releasing peptide, Ghrelin, are not fully elucidated. To investigate the GHRH-mediated GH secretion in human, we investigated pulsatile, entropic, and 24-h rhythmic GH secretion in two young adults (male, 24 yr; female, 23 yr) from a Moroccan family with a novel inactivating defect of the GHRH receptor gene. Data were compared with values in age- and gender-matched controls. Plasma GH concentration were measured by a sensitive immunofluorometric assay, with a detection limit of 0.01 mU/L. All plasma GH concentrations in the female patient were measurable; in the male patient 30 of 145 samples were at or below the detection limit. GH secretion was pulsatile, with 21 and 23 secretory episodes/24 h in the male and female patients, respectively. The fraction of basal to total GH secretion was raised in both patients by 0.18 and 0.15, respectively. The total 24-h GH production rate was greatly diminished; in the male patient it was 6.9 mU/L (normal values for his age, 26--63 mU/L), and in the female patient it was 4.2 mU/L (normal values for her age, 96--390 mU/L). The nyctohemeral plasma GH rhythm was preserved (P < 0.001), with normal acrophases (0430 and 0218 h in the male and female, respectively). Approximate entropy was greatly elevated in both subjects (0.82 in the male and 1.17 in the female; upper normal values for age and gender, 0.24 and 0.59, respectively). Intravenous injection of 50 microg GHRH failed to increase the plasma GH concentration in both patients, but 100 microg GH-releasing peptide-2 elicited a definite increase (male patient, 0.13 to 1.74 mU/L; female patient, 0.29 to 0.87 mU/L). Both patients had a partial empty sella on magnetic resonance imaging scanning. In summary, the present studies in two patients with a profound loss of function mutation of the GHRH receptor favor the view that in the human the timing of GH pulses is primarily supervised by intermittent somatostatin withdrawal, and the amplitude of GH pulses is driven by GHRH. In addition, we infer that effectual GHRH input controls the GH cell mass and the orderliness of the secretory process.

Regulation of [Ca(2+)](i) homeostasis in MRP1 overexpressing cells.

Regulation of capacitative Ca(2+) entry was studied in two different multidrug resistance (MDR) protein (MRP1) overexpressing cell lines, HT29(col) and GLC4/ADR. MRP1 overexpression was accompanied by a decreased response to thapsigargin. Moreover, inhibition of capacitative Ca(2+) entry by D, L-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) was abolished in MRP1 overexpressing cells. Both PDMP and the MRP1 inhibitor MK571 greatly reduced InsP(3)-mediated (45)Ca(2+) release from intracellular stores in HT29 cells. Again, these effects were virtually abolished in HT29(col) cells. Our results point to a modulatory role of MRP1 on intracellular calcium concentration ([Ca(2+)](i)) homeostasis which may contribute to the MDR phenotype.

Hyperleptinemia in women with Cushing's disease is driven by high-amplitude pulsatile, but orderly and eurhythmic, leptin secretion.

The episodicity of 24 h leptin release was studied in seven women (mean age 39 years, range 22-56 years) with pituitary-dependent hypercortisolism and in seven age- and body mass index (BMI)-matched female controls. Pulsatile leptin release was quantified by model-free cluster analysis and deconvolution, the orderliness of leptin patterns by the approximate entropy statistic (ApEn), and nyctohemeral leptin rhythmicity by cosinor analysis. Blood samples were taken at 10 min intervals for 24 h. Both cluster and deconvolution analysis revealed 2.4-fold increased leptin secretion in patients, caused by combined and equal amplification of basal and pulsatile secretion. Cluster analysis identified 7.1+/-1.5 peaks per 24 h in patients and 6.0+/-0.5 in controls (not significant). The statistical distribution of the individual sample secretory rates was similarly skewed in patients and controls (0.55+/-0.12 vs 0.52+/-0.07). The acrophase (timing of the nyctohemeral leptin peak) in patients occurred at 2314 h (+/-76 min) and at 0058 h (+/-18 min) in controls (not significant). The approximate entropy of leptin release was equivalent in patients and controls (1.67+/-0.03 vs 1.61+/-0.05). The approximate entropy (ApEn) for cortisol in patients was 1.53+/-0.09 and in controls was 0.93+/-0.07 (P<0.0005). Cross-ApEn showed significant pattern synchrony between leptin and cortisol release, which (unexpectedly) was not disrupted by the cortisol excess (patients, 2.02+/-0.04; controls, 1.88+/-0.09; P=0.233). Insulin levels in fasting patients ('fasting insulin') were 27+/-5.7 mU/l vs 14+/-1.6 mU/l in controls (P=0.035). Leptin secretion correlated with fasting insulin levels (R(2)=0.34, P=0.028) and with the cortisol production rate (R(2)=0.33, P=0.033) when patients and controls were combined. In summary, Cushing's disease in women increases leptin production about twofold in an amplitude-specific way. The pulse-generating, nyctohemeral phase-determining, and entropy-control mechanisms that govern the 24 h leptin release are not altered. The increased secretion is not explained by BMI and is probably only partly explained by increased insulin production, suggesting a cortisol-dependent change in adipose leptin secretion.

Increased episodic release and disorderliness of prolactin secretion in both micro- and macroprolactinomas.

To quantify prolactin (PRL) secretion patterns, ten untreated (female) microprolactinoma patients and six (male) macroprolactinoma patients underwent repetitive blood sampling every 10 min over 24 h. PRL release activity was analyzed from plasma PRL concentration (immunofluorimetric assay) profiles via a model-independent discrete peak detection program (Cluster) and a waveform-independent deconvolution technique (Pulse). Diurnal variations were analyzed by cosinor analysis. The number of distinct PRL pulses (mean +/- S.E.M.) was increased in patients: microprolactinoma 18.6 +/- 0.6/24 h versus female controls 12.4 +/- 0.6 (P = 6.7 x 10-s), and macroprolactinoma 18.0 +/- 0.9 versus male controls 13.5 +/- 0.8/24 h (P = 0.003). In patients, PRL pulse height, amplitude, pulse area and interpeak nadir concentrations were each greatly elevated compared with gender-matched controls. By 2-component deconvolution analysis, the mean nadir PRL secretion rate in microprolactinoma patients was augmented 20-fold at 0.408 +/- 0.089 microgram/l per min versus in female controls 0.019 +/- 0.009 microgram/l per min (P < 0.001); and in macroprolactinoma by 130-fold at 2.067 +/- 0.693 micrograms/l per min versus male controls 0.016 +/- 0.001 microgram/l per min (P = 0.001). Corresponding 24 h mean PRL secretion rates were in women, 0.658 +/- 0.147 and 0.044 +/- 0.018 (P < 0.001), and in men, 3.309 +/- 1.156 and 0.035 +/- 0.010 micrograms/l per min (P = 0.001), being respectively 15- and 94-fold increased in tumors. The estimated PRL production per day was 160 +/- 15 and 187 +/- 20 micrograms in male and female controls respectively. PRL production was 2860 +/- 640 micrograms in female patients with microadenomas (P < 0.001), and 37,800 +/- 5900 micrograms in male macroadenoma patients (P = 0.001). Cosinor analysis of the plasma concentrations revealed a significant rhythm in nine of ten, patients with a microadenoma, and in five of six with a macroadenoma. The same method applied to pulse height and amplitude disclosed a significant rhythm for PRL pulse height, but not for pulse amplitude, suggesting preserved rhythmicity of baseline interpulse nadir PRL concentrations. Approximate entropy (ApEn), a scale- and model-independent regularity statistic, averaged 1.6559 +/- 0.028 in microprolactinoma patients versus 0.8128 +/- 0.079 in female controls (P = 1.7 x 10(-8)); ApEn in macroadenomas was 1.5674 +/- 0.054 versus male controls 0.8773 +/- 0.076 (P = 1.7 x 10(-5), signifying greater secretory irregularity in the patients. Compared with microadenomas, macroadenomas exhibited a higher mean plasma concentration, overall mean PRL secretion rate, nadir secretion rate and pulse area, but similar peak frequency. We conclude that PRL secretion by prolactinomas is characterized by increased plasma PRL episodicity of release, increased total (15- to 100-fold) and basal (20- to 130-fold) secretion rates, and increased disorderlines of minute-to-minute secretion. These abnormalities of secretory control are very similar to those for GH and ACTH identified earlier in acromegaly and Cushing's disease respectively, thus suggesting mechanistic generality of pituitary tumor secretory derangements, independent of the particular hormone.

Low prevalence of hypopituitarism after traumatic brain injury: a multicenter study.

OBJECTIVE: Hypopituitarism after traumatic brain injury (TBI) is considered to be a prevalent condition. However, prevalence rates differ considerably among reported studies, due to differences in definitions, endocrine assessments of hypopituitarism, and confounding factors, such as timing of evaluation and the severity of the trauma. Aim To evaluate the prevalence of hypopituitarism in a large cohort of TBI patients after long-term follow-up using a standardized endocrine evaluation. Study design Cross-sectional study. PATIENTS AND METHODS: We included 112 patients with TBI, hospitalized for at least 3 days and duration of follow-up >1 year after TBI from five (neurosurgical) referral centers. Evaluation of pituitary function included fasting morning hormone measurements and insulin tolerance test (n=90) or, when contraindicated, ACTH stimulation and/or CRH stimulation tests and a GH releasing hormone-arginine test (n=22). Clinical evaluation included quality of life questionnaires. RESULTS: We studied 112 patients (75 males), with median age 48 years and mean body mass index (BMI) 26.7±4.8 kg/m(2). Mean duration of hospitalization was 11 (3-105), and 33% of the patients had a severe trauma (Glasgow Coma Scale <9) after TBI. The mean duration of follow-up was 4 (1-12) years. Hypopituitarism was diagnosed in 5.4% (6/112) of patients: severe GH deficiency (n=3), hypogonadism (n=1), adrenal insufficiency (n=2). Patients diagnosed with pituitary insufficiency had significantly higher BMI (P=0.002). CONCLUSION: In this study, the prevalence of hypopituitarism during long-term follow-up after TBI was low. Prospective studies are urgently needed to find reliable predictive tools for the identification of patients with a significant pre-test likelihood for hypopituitarism after TBI.

Specificity of FNR-type regulators in Paracoccus denitrificans.

The three FNR (fumarate and nitrate reductase regulatory protein)-type transcription activators of Paracoccus denitrificans, NarR, NnrR and FnrP, appear to have specific tasks in gene regulation during the switch from aerobic growth to denitrification. We here set out a series of experiments to get a fundamental understanding of the mechanism underlying this specificity. In one of these, we changed the nucleotide sequence of an NnrR box, the binding site for NnrR, into one found in FnrP-regulated promoters. As a result, we observed a change in regulation of that promoter from NnrR to FnrP. In a second series, we constructed hybrid promoters of NnrR-, NarR- and FnrP-regulated promoters and analysed their expression profiles in cells grown under various growth conditions. Our results indicate that the specificity of the FNR-type regulators is determined in part by the quality of the FNR box and in part by the sequences downstream of the FNR box. The latter suggests that specific sigma factors are involved in binding any of the Fnr-type regulators in P. denitrificans.

The involvement of sphingolipids in multidrug resistance.

Administration of most chemotherapeutic agents eventually results in the onset of apoptosis, despite the agents' variety in structure and molecular targets. Ceramide, the central molecule in cellular glycosphingolipid metabolism, has recently been identified as an important mediator of this process. Indeed, one of the events elicited by application of many cytotoxic drugs is an accumulation of this lipid. Treatment failure in cancer chemotherapy is largely attributable to multidrug resistance, in which tumor cells are typically cross-resistant to multiple chemotherapeutic agents. Different cellular mechanisms underlying this phenomenon have been described. Of these the drug efflux pump activity of P-glycoprotein and the multidrug resistance-associated proteins are the most extensively studied examples. Recently, an increased cellular capacity for ceramide glycosylation has been recognized as a novel multidrug resistance mechanism. Indeed, virtually all multidrug-resistant cells exhibit a deviating sphingolipid composition, most typically, increased levels of glucosylceramide. On the other hand, several direct molecular interactions between sphingolipids and drug efflux proteins have been described. Therefore, in addition to a role in the multidrug resistance phenotype by which ceramide accumulation and, thus, the onset of apoptosis are prevented, an indirect role for sphingolipids might be envisaged, by which the activity of these efflux proteins is modulated. In this review, we present an overview of the current understanding of the interesting relations that exist between sphingolipid metabolism and multidrug resistance.

Metabolism and apoptotic properties of elevated ceramide in HT29rev cells.

Ceramide (Cer) has been implicated in the regulation of apoptosis. In this study, we elevated cellular Cer levels in human colon-carcinoma (HT29(rev)) cells by incubating the cells in the presence of bacterial sphingomyelinase (bSMase) or, alternatively, in the presence of C2-Cer, a short-chain analogue of the sphingolipid. bSMase treatment did not induce apoptosis in these cells, as revealed by a lack of both DNA fragmentation and cleavage of poly(ADP-ribose)polymerase. In contrast, apoptosis did occur upon addition of C2-Cer. These findings led us to study whether differences in the metabolic fate of the excess of Cer, as generated by both treatments, contributed to the observed difference in apoptosis-inducing capacity. C2-Cer was rapidly taken up by HT29(rev) cells and accumulated due to the absence of substantial metabolic conversion. Upon addition of bSMase, hydrolysis of sphingomyelin resulted in a reduction of that pool to 20% compared with control values, accompanied by a multi-fold increase in Cer level. In spite of the continuous presence of active bSMase, the Cer increase turned out to be transient. Cer levels reached their maximum 1-2 h after addition of bSMase, followed by a significant decrease. Excessive Cer was mainly turned over via cerebrosides into complex glycolipids, including gangliosides. In the presence of glucosylceramide synthase- and/or ceramidase inhibitors, this conversion was significantly blocked and bSMase-generated Cer accumulated in the cells. However, even under these conditions apoptosis did not occur. In conclusion, the inability of bSMase to induce apoptosis of HT29(rev) cells does not appear to be due to rapid metabolic conversion of excessive Cer. Since apoptosis is induced upon addition of C2-Cer, we therefore propose that the intracellular target involved in the propagation of the apoptotic signal is reached by C2-Cer, but not by bSMase-generated Cer.

Interferon-gamma-induced differentiation and apoptosis of HT29 cells: dissociation of (glucosyl)ceramide signaling.

Recently, (glyco)sphingolipids (SL) like ceramide (Cer) and glucosylceramide (GlcCer) have been shown to be involved in signaling pathways leading to differentiation and apoptosis in several cell types, including the colon adenocarcinoma cell line HT29. Intracellular levels of Cer can be modulated by ligands such as interferon-gamma (IFN gamma). In the present study we show that IFN gamma, depending on its concentration, has both differentiation- and apoptosis-inducing effects on HT29 cells. Since both phenomena have been related to SL-mediated signaling in other cell types, we next examined whether IFN gamma was able to induce changes in the SL levels of HT29 cells. Remarkably, no significant changes in these levels could be revealed, implying that SL are not involved in IFN gamma-induced differentiation and/or apoptosis of HT29 cells. This observation provides evidence for the hypothesis that SL-mediated signaling pathways might be more cell type specific than is generally assumed.

N-hexanoyl-sphingomyelin potentiates in vitro doxorubicin cytotoxicity by enhancing its cellular influx.

Anticancer drugs generally have intracellular targets, implicating transport over the plasma membrane. For amphiphilic agents, such as the anthracycline doxorubicin, this occurs by passive diffusion. We investigated whether exogenous membrane-permeable lipid analogues improve this drug influx. Combinations of drugs and lipid analogues were coadministered to cultured endothelial cells and various tumour cell lines, and subsequent drug accumulation in cells was quantified. We identified N-hexanoyl-sphingomyelin (SM) as a potent enhancer of drug uptake. Low micromolar amounts of this short-chain sphingolipid, being not toxic itself, enhanced the uptake of doxorubicin up to 300% and decreased its EC(50) toxicity values seven- to 14-fold. N-hexanoyl SM acts at the level of the plasma membrane, but was found not incorporated in (isolated) lipid rafts, and artificial disruption or elimination of raft constituents did not affect its drug uptake-enhancing effect. Further, any mechanistic role of the endocytic machinery, membrane leakage or ABC-transporter-mediated efflux could be excluded. Finally, a correlation was established between the degree of drug lipophilicity, as defined by partitioning in a two-phase octanol-water system, and the susceptibility of the drug towards the uptake-enhancing effect of the sphingolipid. A clear optimum was found for amphiphilic drugs, such as doxorubicin, epirubicin and topotecan, indicating that N-hexanoyl-SM might act by modulating the average degree of plasma membrane lipophilicity, in turn facilitating transbilayer drug diffusion. The concept of short-chain sphingolipids as amphiphilic drug potentiators provides novel opportunities for improving drug delivery technologies.

Modulation of carcinoembryonic antigen release by glucosylceramide--implications for HT29 cell differentiation.

Previous work suggested that glucosylceramide (GlcCer) plays a role in the regulation of cell differentiation of HT29 human colon tumor cells. In the present study, we investigated the role of GlcCer in the cellular release of carcinoembryonic antigen (CEA), a marker for cell differentiation. This was done by modulating the intracellular level of the glycolipid, according to two different approaches. The cells were treated with D,L-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP), which resulted in a specific lowering of the cellular GlcCer pool. Alternatively, by exogenous addition of a short-chain analog of the lipid, hexanoyl(C6)-GlcCer, the cellular pool was enhanced. The results demonstrate that PDMP causes an increase in the release of CEA, while exogenous C6-GlcCer suppresses its release. Furthermore, the enhanced release of CEA in the presence of PDMP, could be completely reversed upon exogenous addition of C6-GlcCer. Control experiments reveal that a potential interference of the well-known modulator of cell physiology, ceramide (Cer), can be excluded. Long-term depletion of GlcCer resulted in a change in a morphological feature of differentiation of the cells, i.e. an increase in apical membrane surface with microvilli brush borders, accompanied by an enhanced expression of the cytoskeletal protein villin. These results, together with the observations on modulation of the differentiation marker CEA by GlcCer, provide support for the conclusion that GlcCer interferes with the differentiation of HT29 cells.

Growth hormone and prolactin are secreted more irregularly in patients with Cushing's disease.

OBJECTIVE: To investigate whether the spontaneous secretion of growth hormone and prolactin in adult patients with pituitary-dependent Cushing's disease is decreased. PATIENTS: Fourteen adult patients (9 women, 5 men; age: 34 +/- 3.4 years, mean +/- SEM) with pituitary-dependent Cushing's disease and 14 controls matched for age, gender and body mass index were studied. METHODS: Blood samples were withdrawn at 10 minutes intervals starting at 0900 h for 24 h. GH and PRL release were quantified with deconvolution methods. The regularity of GH and PRL release was measured with approximate entropy statistics. RESULTS: The number of GH secretory events per 24 h was higher in patients than in controls: 19 +/- 1.3 vs. 14 +/- 1.5 peaks per 24 h, respectively (P = 0.020). GH secretion rate was about one quarter lower in patients (ns), and the 24 h secretion of PRL was unchanged. Total GH production correlated negatively with the urinary excretion of free cortisol (R = 0.729, P = 0.005) and with the plasma cortisol production rate (R = 0.613; P = 0.026). The orderliness of GH and PRL secretion was appraised with the approximate entropy statistic (ApEn). For GH secretion ApEn(1,20%) in patients was 0.952 +/- 0.084 vs. 0.404 +/- 0.047 in controls, P = 1.17 x 10-4, pointing to a markedly disordered secretion in patients. Similar results were obtained for PRL secretion: patients: 1.586 +/- 0.063 vs. 1.003 +/- 0.068 in controls, P = 3.67 x 10-5. No statistically significant differences in secretory dynamics were demonstrated between the 10 patients with a microadenoma and the four with a macroadenoma. CONCLUSION: The amount of GH released spontaneously into the circulation in adult patients with pituitary-dependent Cushing's disease is inversely related to the degree of cortisol hypersecretion. However, except for severe hypercortisolism, GH secretion is relatively preserved. In addition, secretion of GH and PRL is remarkably disordered in patients with Cushing's disease. Since we could not detect differences in GH and/or PRL secretory dynamics between patients with a microadenoma and those harboring a macroadenoma, we speculate that an intrapituitary paracrine mechanism and/or elevated cortisol feedback effects may be responsible for the evident disruption of GH and PRL secretion patterns.

Elevation of glucosylceramide in multidrug-resistant cancer cells and accumulation in cytoplasmic droplets.

Multidrug-resistant (MDR) cancer cells have been shown to have an accumulation of glucosylceramide (GlcCer). In this study, we aim at localizing, at subcellular level, where these lipids accumulate. Neutral lipids and phospholipid containing organelles have been identified using confocal fluorescence microscopy and microspectrofluorometry by monitoring the emission of the fluorescent probe Nile-red. Data from confocal fluorescence microscopy analysis shows accumulation of neutral lipids in cytoplasmic droplets of MDR human carcinoma MCF7R cells. Microspectrofluorometric measurements show an increase of the gold-yellow emission intensity in MCF7R cells, corresponding to neutral lipids. Similar observations were made in human MDR vincristine-HL60 and doxorubicin-KB selected cells. Total cellular glucosylceramide (GlcCer) measurements using [(3)H]-palmitic acid and thin layer chromatography show a significant increase of GlcCer in MCF7R cells. Moreover, MCF7R cells treated with fluorescent GlcCer-bodipy exhibit an accumulation of this lipid in cytoplasmic droplets. Treatment of MCF7R cells with 1-phenyl-2-palmitoylamino-3-morpholino-1-propanolol (PPMP), a potent inhibitor of GlcCer synthase, attenuates the Nile-red fluorescence emission emanating from these structures and reverses MDR. Moreover, Golgi compartments stained with fluorescent PPMP-bodipy, show an increase in the Golgi compartments density. Treatment of MCF7R cells with cyclosporine A (CSA), tamoxifen (TMX) and 3'-azido-3'deoxythymidine (AZT) leads to the same effect observed in the presence of PPMP. Treatment of MCF7 and MCF7R with the beta-glucosidase inhibitor conduritol beta-epoxide (CBE) significantly increases resistance to daunorubicin only in MCF7R cells. These data demonstrate also that: (i) CSA, an inhibitor of MDR, has an additional target in addition to P-glycoprotein; and (ii) TMX (used in breast cancer treatment and prevention) and AZT (used in the treatment of HIV) could have side effects by disturbing lipid metabolism and inhibiting many cellular functions required in normal cells.