Impact of pulsatility on the ensemble orderliness (approximate entropy) of neurohormone secretion.

Regular patterns of neurohormone secretion are driven by underlying pulsatile and subordinate (feedback sensitive) dynamics. Measures of time-series orderliness, e.g., the approximate entropy (ApEn) statistic (Pincus SM. Proc Natl Acad Sci 88: 2297-2301, 1991), vividly discriminate pathological and physiological patterns of hormone release. To investigate how specific pulsatility features impact regularity estimates, we have examined the sensitivity of the ApEn metric to systematic variations in the frequency, amplitude, and half-life of simulated neurohormone pulse trains (Veldhuis JD, Carlson ML, and Johnson ML. Proc Natl Acad Sci 84: 7686-7690, 1987) and compared the impact of a high vs. low baseline luteinizing hormone (LH) pattern regularity state mimicking the normal female luteal phase and the young male, respectively. Shortening the interpulse interval length elevated ApEn in both pulsatility models, thereby signifying greater ensemble series irregularity. The frequency sensitivity of ApEn was robust to several complementary renditions of ApEn and to variations in experimental uncertainty, basal (nonpulsatile) LH secretion, and secretory burst amplitude. ApEn rose with increasing hormone half-life, especially in the face of low baseline variability emulated by midluteal LH secretion profiles. High variability of secretory burst amplitude, pulse duration, or interpeak intervals increased ApEn in the more orderly femalelike construct; in the highly irregular malelike LH pulse model, these variability changes had little effect on ApEn. In summary, the ensemble regularity statistic, ApEn, quantifies unequal pattern orderliness in neurohormone pulse trains with minimal dependence on mean pulse amplitude, interpulse baseline, or (subthreshold) sample uncertainty. Thus ApEn monitors changing secretory event frequency and interpulse variability with sensitivity to starting pattern regularity, providing a mechanistic linkage between model evolution and statistical change.

The use of regularity as estimated by approximate entropy to distinguish saltatory growth.

A nonlinear dynamics metric, approximate entropy (ApEn), is investigated as a diagnostic method for distinguishing between mathematical models, and the underlying mechanistic hypotheses that purport to describe the same time series experimental observations. ApEn measures the occurrence of pattern regularity within a time series, and is used here to investigate growth patterns in daily length growth. The notion investigated is that ApEn distributions for competing time series patterns expressed as mathematical formulations can be modelled by Monte Carlo and bootstrap methods and compared to the ApEn values for an original experimental data series. If the ApEn values for the different models do not overlap, then it is expected that ApEn can be utilized to distinguish these models and hypotheses, and to provide statistical assessment for the underlying biological patterns in experimental data. The conclusion is that the ApEn metric is successful as a time series diagnostic tool. It is a model-independent statistic that clearly differentiates saltatory growth from slowly varying continuous models of growth and serves to further document the saltatory nature of growth. This is a unique application of approximate entropy, illustrating the broad applicability of ApEn to biological time series, with the specific example of discriminating a saltatory growth process in longitudinal growth data. Future investigations of regularity in longitudinal time series in human biology with ApEn statistics are suggested.

Seizures induce phase shifts of rat circadian rhythms.

RATIONALE: Epileptic seizures may alter neuroendocrinological cycles. Light pulses induce phase shifts in circadian rhythms. Using hippocampal-kindled rats to ensure maximal clinical expression, we determined if seizures likewise induce phase shifts. METHODS: We monitored the circadian rhythm of temperature (CRT) with intraperitoneal radiotelemetry in rats (n=21) isolated from time cues and light for 3-week trials. Seizures were triggered with hippocampal electrical stimulation at different circadian phases. Optimized, least-error phase shifts were calculated from preictal and postictal CRTs. Induced seizures were referenced to CRT (t(max)=00:00, 24-h circadian cycle). RESULTS: Phase shifts (individual responses=57) differed across the circadian cycle. Rather than forming a clear phase-response curve, phase shifts were especially variable between 00:00 and 06:00 h. CONCLUSIONS: This study demonstrates that electrically-induced seizures induce advances and delays in CRT in a phase-dependent fashion but in a pattern different from typical light-induced phase shifts. Disorders of circadian regulation may contribute to some of the altered endogenous cycles associated with epilepsy.

Decrease in beta-cell mass leads to impaired pulsatile insulin secretion, reduced postprandial hepatic insulin clearance, and relative hyperglucagonemia in the minipig.

Most insulin is secreted in discrete pulses at an interval of approximately 6 min. Increased insulin secretion after meal ingestion is achieved through the mechanism of amplification of the burst mass. Conversely, in type 2 diabetes, insulin secretion is impaired as a consequence of decreased insulin pulse mass. beta-cell mass is reported to be deficient in type 2 diabetes. We tested the hypothesis that decreased beta-cell mass leads to decreased insulin pulse mass. Insulin secretion was examined before and after an approximately 60% decrease in beta-cell mass achieved by a single injection of alloxan in a porcine model. Alloxan injection resulted in stable diabetes (fasting plasma glucose 7.4 +/- 1.1 vs. 4.4 +/- 0.1 mmol/l; P < 0.01) with impaired insulin secretion in the fasting and fed states and during a hyperglycemic clamp (decreased by 54, 80, and 90%, respectively). Deconvolution analysis revealed a selective decrease in insulin pulse mass (by 54, 60, and 90%) with no change in pulse frequency. Rhythm analysis revealed no change in the periodicity of regular oscillations after alloxan administration in the fasting state but was unable to detect stable rhythms reliably after enteric or intravenous glucose stimulation. After alloxan administration, insulin secretion and insulin pulse mass (but not insulin pulse interval) decreased in relation to beta-cell mass. However, the decreased pulse mass (and pulse amplitude delivered to the liver) was associated with a decrease in hepatic insulin clearance, which partially offset the decreased insulin secretion. Despite hyperglycemia, postprandial glucagon concentrations were increased after alloxan administration (103.4 +/- 6.3 vs. 92.2 +/- 2.5 pg/ml; P < 0.01). We conclude that an alloxan-induced selective decrease in beta-cell mass leads to deficient insulin secretion by attenuating insulin pulse mass, and that the latter is associated with decreased hepatic insulin clearance and relative hyperglucagonemia, thereby emulating the pattern of islet dysfunction observed in type 2 diabetes.

A construct of interactive feedback control of the GH axis in the male.

Growth hormone (GH) secretion is controlled by GH-releasing hormone (GHRH), the GH release-inhibiting hormone somatostatin (SRIF), and autofeedback connections. The ensemble network produces sexually dimorphic patterns of GH secretion. In an effort to formalize this system, we implemented a deterministically based autonomous feedback-driven construct of five principal dose-responsive regulatory interactions: GHRH drive of GH pituitary release, competitive inhibition of GH release by SRIF, GH autofeedback via SRIF with a time delay, delayed GH autonegative feedback on GHRH, and SRIF inhibition of GHRH secretion. This formulation engenders a malelike pattern of successive GH volleys due jointly to positive time-delayed feedback of GH on SRIF and negative feedback of SRIF on GH and GHRH. The multipeak volley is explicated as arising from a reciprocal interaction between GH and GHRH during periods of low SRIF secretion. The applicability of this formalism to neuroendocrine control is explored by initial parameter sensitivity analysis and is illustrated for selected feedback-dependent experimental paradigms. The present construct is not overparameterized and does not require an ad hoc pulse generator to achieve pulsatile GH output. Further evolution of interactive constructs could aid in exploring more complex feedback postulates that confer the vivid sexual dimorphism of female GH profiles.

Secretory process regularity monitors neuroendocrine feedback and feedforward signaling strength in humans.

The present experiments examine the neuroregulatory hypothesis that the degree of sample-by-sample regularity of hormone output by an interlinked hypothalamopituitary target-organ system monitors the strength of feedback and/or feedforward signaling. To test this postulate and assess its generality, we implemented a total of nine thematically complementary perturbation experiments. In particular, we altered feedback or feedforward signaling selectively in two distinct neuroendocrine systems; namely, the growth hormone (GH) insulin-like growth factor type I (IGF-I) and the luteinizing hormone-testosterone axes. Four experimental paradigms comprised preferential reduction vs. enhancement of IGF-I or testosterone feedback signal strength; and, conversely, five others entailed selective attenuation vs. augmentation of GH-releasing hormone and gonadotropin-releasing hormone feedforward signal intensity. In these independent interventions, quantitation of subordinate (nonpulsatile) secretory pattern reproducibility via the approximate entropy statistic unmasked salient changes (P values typically <10(-3)) in the conditional regularity of serial hormone output with high consistency (96-100%). In particular, approximate entropy quantified degradation of secretory subpattern orderliness under either muted feedback restraint or heightened feedforward drive. Assuming valid interpretation of the biological constraints imposed, these experimental observations coincide with earlier reductionist mathematical predictions, wherein increased irregularity of coupled parameter output mirrors attenuated feedback and/or augmented feedforward coupling within an integrative system.

Modulation of dose intensity in aerodigestive tract cancers: strategies to reduce toxicity.

Advances in diagnostic and therapeutic radiology and a better understanding of cell biology are being applied in practical ways to modulate treatment morbidity. Conformal radiotherapy targets the cancer precisely and can be combined with new systemically administered radiosensitizers. The successes of conventional chemoradiation programs support continued study of newer ways to deliver systemic radiosensitizing chemotherapy. However, chemoradiation creates a narrower therapeutic window compared to irradiation alone and increased treatment intensity, even with conformal chemoradiation techniques, can potentially result in frequent complications, detrimental treatment delays, and decreased quality of life. Treatment schedules employing a "best tolerated time" modelfor systemic administration of radiosensitizing chemotherapy, based on the concept of chronotolerance, offer attractive ways to address the challenging problem of normal tissue toxicity associated with conformal chemoradiation. This approach may be beneficial in the elderly and those medically unfit to tolerate traditional dose-intense combined-modality schedules. Further evaluation of this concept is warranted, based on existing data.

Orchestrated transcription of key pathways in Arabidopsis by the circadian clock.

Like most organisms, plants have endogenous biological clocks that coordinate internal events with the external environment. We used high-density oligonucleotide microarrays to examine gene expression in Arabidopsis and found that 6% of the more than 8000 genes on the array exhibited circadian changes in steady-state messenger RNA levels. Clusters of circadian-regulated genes were found in pathways involved in plant responses to light and other key metabolic pathways. Computational analysis of cycling genes allowed the identification of a highly conserved promoter motif that we found to be required for circadian control of gene expression. Our study presents a comprehensive view of the temporal compartmentalization of physiological pathways by the circadian clock in a eukaryote.

Episodes of severe hypoglycemia in type 1 diabetes are preceded and followed within 48 hours by measurable disturbances in blood glucose.

This study quantifies blood glucose (BG) disturbances occurring before and after episodes of severe hypoglycemia (SH). For 6-8 months, 85 individuals with type 1 diabetes and a history of SH (age, 44+/-10 yr; 41 women and 44 men; duration of diabetes, 26+/-11 yr; hemoglobin A1c, 7.7+/-1.1%) used Lifescan One Touch BG meters for self-monitoring three to five times daily and recorded the date and time of SH episodes in diaries. For each subject, the timing of SH episodes was located in the temporal stream of SMBG readings recorded by the meter, and characteristics, including the Low BG index (LBGI), were computed in 24-h increments. In the 24-h period before the SH episode LBGI rose (P < 0.001), average BG was lower (P = 0.001), and BG variance increased (P = 0.001). In the 24 h after SH, LBGI and BGvariance remained elevated (P < 0.001), but average BG returned to baseline. These disturbances disappeared in 48 h. On the basis of LBGI we identified subjects at low, moderate, and high risk of SH, who reported, on the average, 1.7, 3.4, and 7.4 SH episodes (P < 0.005) during the study. In addition, we designed an algorithm that predicted 50% of all SH episodes that occurred in this subject group. We conclude that episodes of SH are preceded and followed by quantifiable BG disturbances, which could be used to devise warnings of imminent SH.

Dual epileptic foci in a single patient express distinct temporal patterns dependent on limbic versus nonlimbic brain location.

How timing information is transferred from the suprachiasmatic nucleus to other regions of the brain to mediate activity, either physiological or pathological, is largely unclear. A patient with medically refractory epilepsy and a well-documented, long-term seizure diary provided a unique means to demonstrate how susceptibility to chronobiological modulation varies with brain region. Evaluation for epilepsy surgery disclosed two independent epileptic foci, one limbic and the other nonlimbic. Seizures from both foci occurred periodically with a dominant period of 24 hours but were out of phase with each other. Temporal lobe seizures occurred maximally in the light portion of the daily light-dark cycle, and parietal lobe seizures occurred nocturnally and out of phase with limbic seizures. These data suggest that neuronal excitation and inhibition, depending on the anatomical system involved in epilepsy, may be differently affected by circadian modulation.

Nuclear scintigraphic assessment of intestinal dysfunction after combined treatment with 9-amino-20(S)-camptothecin (9-AC) and irradiation.

PURPOSE: The camptothecins (CPTs) are potent radiosensitizers of malignant tumors in vivo. The extent of normal tissue damage after combined CPT and radiation treatment is unknown. In this article, a jejunal absorption assay with (99m)Tc- pertechnetate (Na[(99m)TcO(4)]) was used to assess C3H/Kam mice given total body irradiation (TBI) of 4 Gy, 6 Gy, and 8 Gy, 2 mg/kg single intramuscular injection of 9-AC or a combination of 2 mg/kg 9-AC + 4 Gy TBI. We also correlated the absorption data with morphologic changes in the jejunal mucosa. MATERIALS AND METHODS: ((99m)TcO(4))(-) absorption from the intestinal lumen into the circulation was studied with dynamic gamma-scintigraphy combined with a multichannel analyzer to record the radiometry data in a time-dependent fashion. Jejunal cross sections were scored for the number of cells per villus and the percentage of apoptotic and mitotic cells in the crypt compartment. The jejunal microcolony assay was used to quantify jejunal crypt survival. RESULTS: A dose-dependent decrease in the absorption function was observed 3.5 days following TBI. The mean absorption rate was reduced to 89 +/- 16% of control in response to a sublethal 4 Gy TBI and dropped to 47. 5 (9.8% in response to 8 Gy TBI. The mean rate of intestinal absorption was delayed by single sublethal 2 mg/kg 9-AC injection to 62 (11% in comparison with control values. The combination of a single 4 Gy TBI with a 9-AC treatment decreased the ((99m)TcO(4))(-) jejunal absorption in an additive fashion producing absorption lifetime values more than twofold longer than controls. The decrease in ((99m)TcO(4))(-) absorption at 3.5 days after irradiation, 9-AC treatment or the combination of the two agents correlates with the number of cells per villus and the percentage of apoptotic cells in the crypt compartment. CONCLUSION: Dynamic enteroscintigraphy with (99m)Tc-pertechnetate is a sensitive functional assay for rapid evaluation of radiation and chemotherapy induced intestinal damage. Reduced intestinal absorptive function has a cellular basis and correlates directly with the numbers of cells lost per villus in a treatment-dependent manner.

Effects of circadian regulation and rest-activity state on spontaneous seizures in a rat model of limbic epilepsy.

PURPOSE: Circadian regulation via the suprachiasmatic nuclei and rest-activity state may influence expression of limbic seizures. METHODS: Male rats (n = 14) were made epileptic by electrical stimulation of the hippocampus, causing limbic status epilepticus and subsequent seizures. We monitored seizures with intrahippocampal electrodes in 12-12-h light/dark (LD) cycles and in continuous dark (DD). We used radiotelemetry monitoring of activity to measure state and body temperature to determine circadian phase. Cosinor analysis and chi2 tests determined whether seizures occurred rhythmically when plotted by phase. State was defined as inactive or active in 10-min epochs based on whether activity count was below or above a cut-off value validated from video observation. RESULTS: In LD, the peak seizure occurrence was 14:59 h after circadian temperature peak (95% confidence limit, 13:37-16:19). Phasic seizure occurrence persisted in DD for 14:05 (12:31-15:38), p < 0.0001, against uniform mean distribution. In LD, 14,787 epochs contained 1, 268 seizures; seizures preferentially occurred during inactive epochs (965 observed, 878 expected in proportion to the overall distribution of inactive versus active epochs; p < 0.001). In DD, 20, 664 epochs contained 1,609 seizures; seizures had no preferential occurrence by state (999 observed, 1,025 expected; p = 0.16). CONCLUSIONS: Limbic seizures occurred with an endogenous circadian rhythm. Seizures preferentially struck during inactivity during entrainment to the light-dark cycle.

Nuclear scintigraphic assessment of radiation-induced intestinal dysfunction.

Radiation-induced damage to the intestine can be measured by abnormalities in the absorption of various nutrients. Changes in intestinal absorption occur after irradiation because of loss of the intestinal absorptive surface and a consequent decrease in active transport. In our study, the jejunal absorption of (99m)Tc-pertechnetate, an actively transported gamma-ray emitter, was assessed in C3H/Kam mice given total-body irradiation with doses of 4, 6, 8 and 12.5 Gy and correlated with morphological changes in the intestinal epithelium. The absorption of (99m)Tc-pertechnetate from the intestinal lumen into the circulation was studied with a dynamic gamma-ray-scintigraphy assay combined with a multichannel analyzer to record the radiometry data automatically in a time-dependent regimen. The resulting radioactivity-time curves obtained for irradiated animals were compared to those for control animals. A dose-dependent decrease in absorptive function was observed 3.5 days after irradiation. The mean absorption rate was reduced to 78.8 +/- 9.3% of control levels in response to 4 Gy total-body irradiation (mean +/- SEM tracer absorption lifetime was 237 +/- 23 s compared to 187 +/- 12 s in nonirradiated controls) and to 28.3 +/- 3.7% in response to 12.5 Gy (660 +/- 76 s). The decrease in absorption of (99m)Tc-pertechnetate at 3.5 days after irradiation correlated strongly (P < 0.001) with TBI dose, with the number of cells per villus, and with the percentage of cells in the crypt compartment that were apoptotic or mitotic. A jejunal microcolony assay showed no loss of crypts and hence no measured dose-response effects after 4, 6 or 8 Gy TBI. These results show that dynamic enteroscintigraphy with sodium (99m)Tc-pertechnetate is a sensitive functional assay for rapid evaluation of radiation-induced intestinal damage in the clinically relevant dose range and has a cellular basis.

Hypothalamic neuronal loss and altered circadian rhythm of temperature in a rat model of mesial temporal lobe epilepsy.

PURPOSE: Numerous dysfunctions in endogenous hypothalamic function have been associated with mesial temporal lobe epilepsy (MTLE). One endogenous activity is the circadian rhythm of temperature (CRT). In this study we examined whether hypothalamically mediated function is altered in the electrically induced, self-sustained, limbic status epilepticus model of MTLE. We then wished to determine whether there was a structural basis for regulatory alterations. METHODS: We measured CRT with peritoneal temperature telemetry obtained in light-entrained (LD) and in free-running, constant-dark (DD) conditions. CRT from epileptic and controls of normal animals and kindled animals were quantized by fast Fourier transform-nonlinear least squares analysis to determine rhythmic complexity. RESULTS: The circadian component of CRT was preserved in all animals. In DD, CRTs of epileptic animals were more complex than those of normal animals. CRT of kindled animals showed no increased complexity after electrically induced seizures. Neuronal density was decreased in regions of the anterior and posterior hypothalamus but not in the suprachiasmatic nuclei from the epileptic rats. CONCLUSIONS: Alterations in CRT due to the epileptic state were independent of isolated seizures. Altered circadian thermoregulation in epileptic rats corresponded to regional hypothalamic neuronal loss. Structural changes of the hypothalamus may explain alterations in endogenous rhythms in MTLE.

Growth hormone (GH) receptor blockade with a PEG-modified GH (B2036-PEG) lowers serum insulin-like growth factor-I but does not acutely stimulate serum GH.

B2036-PEG, a GH receptor (GH-R) antagonist, is an analog of GH that is PEG-modified to prolong its action. Nine mutations alter the binding properties of this molecule, preventing GH-R dimerization and GH action. A potential therapeutic role of B2036-PEG is to block GH action, e.g. in refractory acromegaly. A phase I, placebo-controlled, single rising-dose study was performed in 36 normal young men (ages, 18-37 yr; within 15% ideal body weight). Four groups received a single s.c. injection of either placebo (n = 3 in each group, total n = 12) or B2036-PEG (0.03, 0.1, 0.3, or 1.0 mg/kg; n = 6 each dose). B2036-PEG and GH concentrations were measured 0, 0.25, 0.5, 1, 3, 6, 9, 12, 24, 36, 48, 72, 96, 120, and 144 h after dosing. Serum insulin-like growth factor-I was measured before and 1-7 days after dosing. All doses were well tolerated, with no serious or severe adverse reactions. B2036-PEG, at 1.0 mg/kg, reduced insulin-like growth factor-I by 49 +/- 6% on day 5 (P < 0.001 vs. placebo). GH was measured by two independent methods: 1) modified Nichols chemiluminescence assay (empirically corrected for B2036-PEG cross-reactivity); and 2) direct GH two-site immunoassay, using monoclonal antibodies that did not react with B2036-PEG. There was good agreement between the two methods. GH did not change substantially at any B2036-PEG dose, suggesting that B2036-PEG does not interact with hypothalamic GH-Rs to block short-loop feedback. B2036-PEG may thus block peripheral GH action without enhancing its secretion.