Acute ketamine administration corrects abnormal inflammatory bone markers in major depressive disorder.

Patients with major depressive disorder (MDD) have clinically relevant, significant decreases in bone mineral density (BMD). We sought to determine if predictive markers of bone inflammation-the osteoprotegerin (OPG)-RANK-RANKL system or osteopontin (OPN)-play a role in the bone abnormalities associated with MDD and, if so, whether ketamine treatment corrected the abnormalities. The OPG-RANK-RANKL system plays the principal role in determining the balance between bone resorption and bone formation. RANKL is the osteoclast differentiating factor and diminishes BMD. OPG is a decoy receptor for RANKL, thereby increasing BMD. OPN is the bone glue that acts as a scaffold between bone tissues matrix composition to bind them together and is an important component of bone strength and fracture resistance. Twenty-eight medication-free inpatients with treatment-resistant MDD and 16 healthy controls (HCs) participated in the study. Peripheral bone marker levels and their responses to IV ketamine infusion in MDD patients and HCs were measured at four time points: at baseline, and post-infusion at 230 min, Day 1, and Day 3. Patients with MDD had significant decreases in baseline OPG/RANKL ratio and in plasma OPN levels. Ketamine significantly increased both the OPG/RANKL ratio and plasma OPN levels, and significantly decreased RANKL levels. Bone marker levels in HCs remained unaltered. We conclude that the OPG-RANK-RANKL system and the OPN system play important roles in the serious bone abnormalities associated with MDD. These data suggest that, in addition to its antidepressant effects, ketamine also has a salutary effect on a major medical complication of depressive illness.

The role of adipokines in the rapid antidepressant effects of ketamine.

We previously found that body mass index (BMI) strongly predicted response to ketamine. Adipokines have a key role in metabolism (including BMI). They directly regulate inflammation and neuroplasticity pathways and also influence insulin sensitivity, bone metabolism and sympathetic outflow; all of these have been implicated in mood disorders. Here, we sought to examine the role of three key adipokines-adiponectin, resistin and leptin-as potential predictors of response to ketamine or as possible transducers of its therapeutic effects. Eighty treatment-resistant subjects who met DSM-IV criteria for either major depressive disorder (MDD) or bipolar disorder I/II and who were currently experiencing a major depressive episode received a single ketamine infusion (0.5 mg kg-1 for 40 min). Plasma adipokine levels were measured at three time points (pre-infusion baseline, 230 min post infusion and day 1 post infusion). Overall improvement and response were assessed using percent change from baseline on the Montgomery-Asberg Depression Rating Scale and the Hamilton Depression Rating Scale. Lower baseline levels of adiponectin significantly predicted ketamine's antidepressant efficacy, suggesting an adverse metabolic state. Because adiponectin significantly improves insulin sensitivity and has potent anti-inflammatory effects, this finding suggests that specific systemic abnormalities might predict positive response to ketamine. A ketamine-induced decrease in resistin was also observed; because resistin is a potent pro-inflammatory compound, this decrease suggests that ketamine's anti-inflammatory effects may be transduced, in part, by its impact on resistin. Overall, the findings suggest that adipokines may either predict response to ketamine or have a role in its possible therapeutic effects.

Klotho: a humeral mediator in CSF and plasma that influences longevity and susceptibility to multiple complex disorders, including depression.

Klotho is a hormone secreted into human cerebrospinal fluid (CSF), plasma and urine that promotes longevity and influences the onset of several premature senescent phenotypes in mice and humans, including atherosclerosis, cardiovascular disease, stroke and osteoporosis. Preliminary studies also suggest that Klotho possesses tumor suppressor properties. Klotho's roles in these phenomena were first suggested by studies demonstrating that a defect in the Klotho gene in mice results in a significant decrease in lifespan. The Klotho-deficient mouse dies prematurely at 8-9 weeks of age. At 4-5 weeks of age, a syndrome resembling human ageing emerges consisting of atherosclerosis, osteoporosis, cognitive disturbances and alterations of hippocampal architecture. Several deficits in Klotho-deficient mice are likely to contribute to these phenomena. These include an inability to defend against oxidative stress in the central nervous system and periphery, decreased capacity to generate nitric oxide to sustain normal endothelial reactivity, defective Klotho-related mediation of glycosylation and ion channel regulation, increased insulin/insulin-like growth factor signaling and a disturbed calcium and phosphate homeostasis accompanied by altered vitamin D levels and ectopic calcification. Identifying the mechanisms by which Klotho influences multiple important pathways is an emerging field in human biology that will contribute significantly to understanding basic physiologic processes and targets for the treatment of complex diseases. Because many of the phenomena seen in Klotho-deficient mice occur in depressive illness, major depression and bipolar disorder represent illnesses potentially associated with Klotho dysregulation. Klotho's presence in CSF, blood and urine should facilitate its study in clinical populations.

Chronic administration of anticonvulsants but not antidepressants impairs bone strength: clinical implications.

Major depression and bipolar disorder are associated with decreased bone mineral density (BMD). Antidepressants such as imipramine (IMIP) and specific serotonin reuptake inhibitors (SSRIs) have been implicated in reduced BMD and/or fracture in older depressed patients. Moreover, anticonvulsants such as valproate (VAL) and carbamazepine (CBZ) are also known to increase fracture rates. Although BMD is a predictor of susceptibility to fracture, bone strength is a more sensitive predictor. We measured mechanical and geometrical properties of bone in 68 male Sprague Dawley rats on IMIP, fluoxetine (FLX), VAL, CBZ, CBZ vehicle and saline (SAL), given intraperitoneally daily for 8 weeks. Distinct regions were tested to failure by four-point bending, whereas load displacement was used to determine stiffness. The left femurs were scanned in a MicroCT system to calculate mid-diaphyseal moments of inertia. None of these parameters were affected by antidepressants. However, VAL resulted in a significant decrease in stiffness and a reduction in yield, and CBZ induced a decrease in stiffness. Only CBZ induced alterations in mechanical properties that were accompanied by significant geometrical changes. These data reveal that chronic antidepressant treatment does not reduce bone strength, in contrast to chronic anticonvulsant treatment. Thus, decreased BMD and increased fracture rates in older patients on antidepressants are more likely to represent factors intrinsic to depression that weaken bone rather than antidepressants per se. Patients with affective illness on anticonvulsants may be at particularly high risk for fracture, especially as they grow older, as bone strength falls progressively with age.

The organization of the stress system and its dysregulation in depressive illness.

Stressors are imminent or perceived challenges to homeostasis. The stress response is an innate, stereotypic, adaptive response to stressors that has evolved in the service of restoring the nonstressed homeostatic set point. It is encoded in specific neuroanatomical sites that activate a specific repertoire of cognitive, behavioral and physiologic phenomena. Adaptive responses, though essential for survival, can become dysregulated and result in disease. A clear example is autoimmune disease. I postulate that depression, like autoimmunity, represents a dysregulated adaptive response: a stress response that has gone awry. The cardinal manifestation of the normal stress response is anxiety. Cognitive programs shift from complex associative operations to rapid retrieval of unconscious emotional memories acquired during prior threatening situations. These emerge automatically to promote survival. To prevent distraction during stressful situations, the capacity to seek and experience pleasure is reduced, food intake is diminished and sexual activity and sleep are held in abeyance. Monoamines, cytokines, glutamate, GABA and other central mediators have key roles in the normal stress response. Many central loci are involved. The subgenual prefrontal cortex restrains the amygdala, the corticotropin-releasing hormone/hypothalamic-pituitary-adrenal (CRH/HPA) axis and the sympathomedullary system. The function of the subgenual prefrontal cortex is moderately diminished during normal stress to disinhibit these loci. This disinhibition promotes anxiety and physiological hyperarousal, while diminishing appetite and sleep. The dorsolateral prefrontal cortex is downregulated, diminishing cognitive regulation of anxiety. The nucleus accumbens is also downregulated, to reduce the propensity for distraction by pleasurable stimuli or the capacity to experience pleasure. Insulin resistance, inflammation and a prothrombotic state acutely emerge. These provide increased glucose for the brain and establish premonitory, proinflammatory and prothrombotic states in anticipation of either injury or hemorrhage during a threatening situation. Essential adaptive intracellular changes include increased neurogenesis, enhancement of neuroplasticity and deployment of a successful endoplasmic reticulum stress response. In melancholic depression, the activities of the central glutamate, norepinephrine and central cytokine systems are significantly and persistently increased. The subgenual prefrontal cortex is functionally impaired, and its size is reduced by as much as 40%. This leads to sustained anxiety and activations of the amygdala, CRH/HPA axis, the sympathomedullary system and their sequella, including early morning awakening and loss of appetite. The sustained activation of the amygdala, in turn, further activates stress system neuroendocrine and autonomic functions. The activity of the nucleus accumbens is further decreased and anhedonia emerges. Concomitantly, neurogenesis and neuroplasticity fall significantly. Antidepressants ameliorate many of these processes. The processes that lead to the behavioral and physiological manifestations of depressive illness produce a significant decrease in lifespan, and a doubling of the incidence of premature coronary artery disease. The incidences of premature diabetes and osteoporosis are also substantially increased. Six physiological processes that occur during stress and that are markedly increased in melancholia set into motion six different mechanisms to produce inflammation, as well as sustained insulin resistance and a prothrombotic state. Clinically, melancholic and atypical depression seem to be antithesis of one another. In melancholia, depressive systems are at their worst in the morning when arousal systems, such as the CRH/HPA axis and the noradrenergic systems, are at their maxima. In atypical depression, depressive symptoms are at their worst in the evening, when these arousal systems are at their minima. Melancholic patients experience anorexia and insomnia, whereas atypical patients experience hyperphagia and hypersomnia. Melancholia seems like an activation and persistence of the normal stress response, whereas atypical depression resembles a stress response that has been excessively inhibited. It is important that we stratify clinical studies of depressed patients to compare melancholic and atypical subtypes and establish their differential pathophysiology. Overall, it is important to note that many of the major mediators of the stress response and melancholic depression, such as the subgenual prefrontal cortex, the amygdala, the noradrenergic system and the CRH/HPA axis participate in multiple reinforcing positive feedback loops. This organization permits the establishment of the markedly exaggerated, persistent elevation of the stress response seen in melancholia. Given their pronounced interrelatedness, it may not matter where in this cascade the first abnormality arises. It will spread to the other loci and initiate each of their activations in a pernicious vicious cycle.

Pathological parainflammation and endoplasmic reticulum stress in depression: potential translational targets through the CNS insulin, klotho and PPAR-γ systems.

Major depression and bipolar disorder are heterogeneous conditions in which there can be dysregulation of (1) the stress system response, (2) its capacity for counterregulation after danger has passed and (3) the phase in which damaging molecules generated by the stress response are effectively neutralized. The response to stress and depressed mood share common circuitries and mediators, and each sets into motion not only similar affective and cognitive changes, but also similar systemic manifestations. We focus here on two highly interrelated processes, parainflammation and endoplasmic reticulum (ER) stress, each of which can potentially interfere with all phases of a normal stress response in affective illness, including adaptive neuroplastic changes and the ability to generate neural stem cells. Parainflammation is an adaptive response of the innate immune system that occurs in the context of stressors to which we were not exposed during our early evolution, including overfeeding, underactivity, aging, artificial lighting and novel foodstuffs and drugs. We postulate that humans were not exposed through evolution to the current level of acute or chronic social stressors, and hence, that major depressive illness is associated with a parainflammatory state. ER stress refers to a complex program set into motion when the ER is challenged by the production or persistence of more proteins than it can effectively fold. If the ER response is overwhelmed, substantial amounts of calcium are released into the cytoplasm, leading to apoptosis. Parainflammation and ER stress generally occur simultaneously. We discuss three highly interrelated mediators that can effectively decrease parainflammation and ER stress, namely the central insulin, klotho and peroxisome proliferator-activated receptor-γ (PPAR-γ) systems and propose that these systems may represent conceptually novel therapeutic targets for the amelioration of the affective, cognitive and systemic manifestations of major depressive disorder.

Low early morning plasma cortisol in posttraumatic stress disorder is associated with co-morbid depression but not with enhanced glucocorticoid feedback inhibition.

BACKGROUND: Co-morbid major depressive disorder (MDD) in individuals with posttraumatic stress disorder (PTSD) confers a more severe clinical course and is associated with distinct biologic abnormalities. Although dysregulation in the hypothalamic pituitary adrenal (HPA) axis has been well established in PTSD, the impact of commonly co-occuring MDD has received scant attention. METHODS: Overnight (7p.m. to 7a.m.) plasma cortisol, adrenocorticotropic hormone (ACTH), dehydroepiandrosterone sulphate (DHEA-S) were measured at 30 min intervals in 9 participants with PTSD with MDD (PTSD+MDD), 9 with PTSD without MDD (PTSD-MDD) and 16 non-traumatized healthy controls. A low-dose dexamethasone suppression test was administered to evaluate feedback sensitivity to glucocorticoids. Linear mixed models with body mass index (BMI) and age as covariates and Bonferroni corrected post hoc tests assessed group differences. RESULTS: Compared to healthy controls, subjects with PTSD+MDD, but not those subjects with PTSD-MDD, exhibited lower basal plasma cortisol levels between 1:30 a.m. and 3:30 a.m. and at 4:30 a.m. and 6:30 a.m. (effect size d=0.75). Despite similar plasma ACTH levels between the three groups, the ACTH/cortisol ratio was higher in PTSD+MDD patients compared to controls. We obtained similar results when the patient and control groups were re-studied 1 week later, and when men and current smokers were excluded. Basal plasma DHEA-S levels, and cortisol and ACTH response to a low-dose dexamethasone suppression test were similar in all three groups. CONCLUSIONS: Lower early morning plasma cortisol levels and a high ACTH/cortisol ratio in subjects with PTSD and co-morbid MDD may not be due to enhanced peripheral sensitivity to glucocorticoids. A central abnormality in glucocorticoid regulation could explain HPA axis dysfunction in this subgroup.

Glucocorticoid receptor gene polymorphisms in premenopausal women with major depression.

Glucocorticoid receptor gene polymorphisms are associated with glucocorticoid hypersensitivity and visceral obesity. Perturbations in HPA axis sensitivity to glucocorticoids implicated in the pathogenesis of major depression may result from functional alterations in the glucocorticoid receptor gene. We 1) examined the prevalence of genotype distribution of specific polymorphisms of the glucocorticoid receptor gene (Bcl1, N363S, rs33388, rs33389) in a subset of women from the P.O.W.E.R. Study (which enrolled 21- to 45-year-old premenopausal women with major depression and healthy controls) and 2) explored whether such polymorphisms were associated with visceral obesity and insulin resistance. Women with major depression had a higher body mass index, a higher waist:hip ratio, and more body fat than did controls. No differences were observed in plasma and urinary cortisol or in insulin sensitivity. The G/G genotype of the Bcl1 polymorphism was significantly more common (p<0.03) in women with major depression (n=52) than in controls (n=29). In addition, GG homozygotes (depressed n=10; controls n=2) had higher waist:hip ratios than did non-GG carriers (p<0.02). N363S, rs33388, and rs33389 polymorphisms were not different between groups. In conclusion, premenopausal women with both major depression and the GG genotype of the Bcl1 polymorphism had greater abdominal obesity compared with non-GG carriers.

Potential uses of corticotropin-releasing hormone antagonists.

Corticotropin-releasing hormone (CRH), its natural homologs urocortins (UCN) 1, 2, and 3, and several types of CRH receptors (R), coordinate the behavioral, endocrine, autonomic, and immune responses to stress. The potential use of CRH antagonists is currently under intense investigation. Selective antagonists have been used experimentally to clarify the role of CRH-related peptides in anxiety and depression, addictive behavior, inflammatory disorders, acute and chronic neurodegeneration, and sleep disorders, as well as preterm labor.

Development of a human mitochondria-focused cDNA microarray (hMitChip) and validation in skeletal muscle cells: implications for pharmaco- and mitogenomics.

Mitochondrial research has influenced our understanding of human evolution, physiology and pathophysiology. Mitochondria, intracellular organelles widely known as 'energy factories' of the cell, also play fundamental roles in intermediary metabolism, steroid hormone and heme biosyntheses, calcium signaling, generation of radical oxygen species, and apoptosis. Mitochondria possess a distinct DNA (mitochondrial DNA); yet, the vast majority of mitochondrial proteins are encoded by the nuclear DNA. Mitochondria-related genetic defects have been described in a variety of mostly rare, often fatal, primary mitochondrial disorders; furthermore, they are increasingly reported in association with many common morbid conditions, such as cancer, obesity, diabetes and neurodegenerative disorders, although their role remains unclear. This study describes the creation of a human mitochondria-focused cDNA microarray (hMitChip) and its validation in human skeletal muscle cells treated with glucocorticoids. We suggest that hMitChip is a reliable and novel tool that will prove useful for systematically studying the contribution of mitochondrial genomics to human health and disease.

Marked suppression of gastric ulcerogenesis and intestinal responses to stress by a novel class of drugs.

When exposed to prolonged stress, rats develop gastric ulceration, enhanced colon motility with depletion of its mucin content and signs of physiological and behavioral arousal. In this model, we tested whether antidepressants (fluoxetine and bupropion), anxiolytics (diazepam and buspirone) or the novel nonpeptide corticotropin-releasing hormone (CRH) type-1 receptor (CRH-R1) antagonist, antalarmin, modify these responses. Fluoxetine, bupropion, diazepam and antalarmin all suppressed stress-induced gastric ulceration in male Sprague-Dawley rats exposed to four hours of plain immobilization. Antalarmin produced the most pronounced anti-ulcer effect and additionally suppressed the stress-induced colonic hypermotility, mucin depletion, autonomic hyperarousal and struggling behavior. Intraperitoneal CRH administration reproduced the intestinal but not the gastric responses to stress while vagotomy antagonized the stress-induced gastric ulceration but not the intestinal responses. We conclude that brain CRH-R1 and vagal pathways are essential for gastric ulceration to occur in response to stress and that peripheral CRH-R1 mediates colonic hypermotility and mucin depletion in this model. Nonpeptide CRH-R1 antagonists may therefore be prophylactic against stress ulcer in the critically ill and therapeutic for other pathogenetically related gastrointestinal disorders such as peptic ulcer disease and irritable bowel syndrome.

Organization of the stress system and its dysregulation in melancholic and atypical depression: high vs low CRH/NE states.

Stress precipitates depression and alters its natural history. Major depression and the stress response share similar phenomena, mediators and circuitries. Thus, many of the features of major depression potentially reflect dysregulations of the stress response. The stress response itself consists of alterations in levels of anxiety, a loss of cognitive and affective flexibility, activation of the hypothalamic-pituitary-adrenal (HPA) axis and autonomic nervous system, and inhibition of vegetative processes that are likely to impede survival during a life-threatening situation (eg sleep, sexual activity, and endocrine programs for growth and reproduction). Because depression is a heterogeneous illness, we studied two diagnostic subtypes, melancholic and atypical depression. In melancholia, the stress response seems hyperactive, and patients are anxious, dread the future, lose responsiveness to the environment, have insomnia, lose their appetite, and a diurnal variation with depression at its worst in the morning. They also have an activated CRH system and may have diminished activities of the growth hormone and reproductive axes. Patients with atypical depression present with a syndrome that seems the antithesis of melancholia. They are lethargic, fatigued, hyperphagic, hypersomnic, reactive to the environment, and show diurnal variation of depression that is at its best in the morning. In contrast to melancholia, we have advanced several lines of evidence of a down-regulated hypothalamic-pituitary adrenal axis and CRH deficiency in atypical depression, and our data show us that these are of central origin. Given the diversity of effects exerted by CRH and cortisol, the differences in melancholic and atypical depression suggest that studies of depression should examine each subtype separately. In the present paper, we shall first review the mediators and circuitries of the stress system to lay the groundwork for placing in context physiologic and structural alterations in depression that may occur as part of stress system dysfunction.

Differential gene expression profile of glucocorticoids, testosterone, and dehydroepiandrosterone in human cells.

Glucocorticoids are the major immunomodulating hormones in the human body. Recently, increasing interest in androgens as immunomodulators has emerged. In particular, Dehydroepiandrosterone (DHEA) has been suggested as beneficial in the treatment of some autoimmune disorders. However, the action and role of testicular and adrenal androgens on human immune cells remains unclear. This is the first study to provide large-scale gene expression data on the action of different steroids (DHEA, glucocorticoids, and testosterone) on human peripheral blood mononuclear cells using the recently developed genomic-scale technology of microarrays. Novel computational tools and techniques such as Principal Component Analysis (PCA) were used for analysis, clustering and visualization. We have demonstrated that each steroid has its distinct gene expression profile, although DHEA and testosterone co-regulated most genes in a similar direction while glucocorticoids frequently regulated the same genes in an opposite direction. Our data suggest an important and a complex regulatory role for androgens on human immune cells that should be considered in androgen replacement or treatment strategies.

Neuroendocrinology of stress.

Stimuli that are interpreted by the brain as extreme or threatening, regardless of their modality, elicit an immediate stereotypic response characterized by enhanced cognition, affective immobility, vigilance, autonomic arousal and a global catabolic state. The brain's ability to mobilize this so-called stress response is paralleled by activation of corticotropin-releasing hormone (CRH) in several nuclei, including the hypothalamus, amygdala and locus ceruleus, and stimulation of the locus ceruleus norepinephrine (LC/NE) system in the brain stem. These systems perpetuate one another, interact with several other transmitter systems in the brain and directly activate the hypothalamic-pituitary-adrenal (HPA) axis and the three components of the autonomic nervous system, namely the sympatho-adrenal, the cranio-sacral parasympathetic and the enteric nervous systems. The widespread body system responses to stress are discussed, and the implications of aberrant stress system activity on physical and mental health are outlined. Moreover, the promise of nonpeptide CRH type-1 receptor antagonists to directly target the stress system in the brain is highlighted.

Major depression and the stress system: a life span perspective.

From a transactional developmental perspective, the authors review findings from studies of animals and humans regarding a proposed relation between stress system abnormalities and major depression. The stress system has evolved to promote successful adaptation across the life span, but disruptions in its functioning may increase the risk of pathological outcomes. Emphasis is placed on the role of prenatal and early postnatal experience in contributing to individual differences in postnatal stress reactivity, which may interact with cognitive and psychosocial vulnerabilities to increase susceptibility to later onset of depression. Findings regarding cognitive, psychosocial, and medical sequelae of depression are also reviewed, with a focus on the possible mediating role of the stress system. The authors highlight the importance of multidisciplinary, longitudinal studies in attempting to gain a deeper understanding of the complex developmental processes involved in the onset and course of depression.

Expression of corticotropin releasing hormone receptors type I and type II mRNA in suicide victims and controls.

Corticotropin-releasing hormone (CRH) is a key neuroendocrine factor implementing endocrine, immune and behavioral responses to stress. CRH exerts its action through two major receptors, CRH-R1 and CRH-R2. Recently novel non-peptidic antagonists directed against CRH-R1 or CRH-R2 have been proposed as promising agents in the treatment of depression, anxiety and eating disorder. However, so far the CRH-receptor system has not been widely studied in humans. Therefore, we employed quantitative TaqMan PCR to analyze the expression and distribution of both CRH-R1 and CRH-R2 in human brain tissue and peripheral organs. Furthermore the expression of CRH receptors was analyzed for the first time in pituitaries of suicide victims by in situ hybridization and quantitative PCR. Our data demonstrated a different expression pattern in humans as compared to rodents. Both CRH-R1 and CRH-R2 were expressed in high amounts in the brain with the strongest expression in the pituitary. As described in rodents, however the CRH-R1 in human was the predominant receptor in the brain (82.7 +/- 11.0%), whilst CRH-R2 was the predominant receptor in peripheral organs (77.0 +/- 15.8%). There was a shift in the ratio of CRH-R1/R2 in the pituitaries of suicide victims. In conclusion, both CRH-R1 and CRH-R2 are widely expressed in human tissues with a distribution substantially different from rodents. Strong expression of both CRH-R1 and CRH-R2 in human pituitaries suggests that particularly under stress, activation of the HPA axis can be maintained through both receptors.

Synchronicity of frequently sampled thyrotropin (TSH) and leptin concentrations in healthy adults and leptin-deficient subjects: evidence for possible partial TSH regulation by leptin in humans.

Leptin signals the status of energy reserves to the brain. Leptin stimulates biosynthesis of TRH in vitro and influences the activity of the hypothalamic-pituitary-thyroid axis in vivo in rodents. Because blood levels of both leptin and TSH display diurnal variation with a distinct nocturnal rise, we sought to determine whether a relationship exists between fluctuations in circulating leptin and TSH. We measured serum leptin and TSH levels every 7 min for 24 h in five healthy men and found that both leptin and TSH levels are highly organized and pulsatile. A similar pattern of leptin and TSH rhythms was observed, with TSH and leptin levels reaching a nadir in late morning and a peak in the early morning hours. Importantly, cosinor analysis on the absolute leptin and TSH levels revealed a statistically significant fit for a 24-h period and the two hormones showed similar probabilities of rhythm and superimposable peak values. Furthermore, this study shows a strong positive Pearson correlation between the 24-h patterns of variability of leptin and TSH in healthy subjects. Finally, the ultradian fluctuations in leptin levels showed pattern synchrony with those of TSH as determined by cross-correlation analysis, by cross-approximate enthropy and Bayessian analysis applied independently. To further explore whether these associations could reflect an underlying regulation of TSH secretion by leptin, we also studied frequently sampled leptin and TSH levels in four brothers, members of a family with leptin deficiency (one normal homozygote, two heterozygotes, and one leptin-deficient homozygote). Leptin levels of the homozygous leptin-deficient subject are detectable but bioinactive, and the rhythm of his TSH is disorganized. 24-h pattern of leptin and TSH variability in the heterozygous subjects, although significantly correlated, showed a weaker correlation compared with the strong correlation in the normal subjects. These data are consistent with the possibility that leptin may regulate TSH pulsatility and circadian rhythmicity, but interventional studies are needed to definitively prove whether leptin regulates the minute-to-minute oscillations and ultradian rhythm of TSH levels.