Knockdown of mechanosensitive adaptor Hic-5 ameliorates post-traumatic osteoarthritis in rats through repression of MMP-13.

Osteoarthritis (OA) is the most common joint disease associated with articular cartilage destruction. Matrix metalloproteinase-13 (MMP-13) has an essential role in OA pathogenesis by degradation of collagen II, a major component of articular cartilage. Hydrogen peroxide-inducible clone-5 (Hic-5; TGFB1I1), a transforming growth factor-ß-inducible mechanosensor, has previously been reported to promote OA pathogenesis by upregulating MMP-13 expression in mouse osteoarthritic lesions. In our current study, immunohistochemical analysis showed that Hic-5 protein expression was increased in human OA cartilage compared with normal cartilage. Functional experiments demonstrated that Hic-5 and MMP-13 expression was increased by mechanical stress, and mechanical stress-induced MMP-13 expression was suppressed by Hic-5 siRNA in human chondrocytes. Moreover, intracellular localization of Hic-5 shifted to the nucleus from focal adhesions in human chondrocytes subjected to mechanical stress, and nuclear Hic-5 increased MMP-13 gene expression. In vivo, intra-articular injection of Hic-5 siRNA decreased the Osteoarthritis Research Society International score and MMP-13 protein expression in articular cartilage of OA rats. Our findings suggest that Hic-5 regulates transcription of MMP-13 in human chondrocytes, and Hic-5 may be a novel therapeutic target for OA because OA progression was suppressed by intra-articular injection of Hic-5 siRNA in rats.

Hypercholesterolemic Dysregulation of Calpain in Lymphatic Endothelial Cells Interferes With Regulatory T-Cell Stability and Trafficking.

BACKGROUND: Although hypercholesterolemia reportedly counteracts lymphocyte trafficking across lymphatic vessels, the roles of lymphatic endothelial cells (LECs) in the lymphocyte regulations remain unclear. Previous studies showed that calpain-an intracellular modulatory protease-interferes with leukocyte dynamics in the blood microcirculation and is associated with hypercholesterolemic dysfunction in vascular endothelial cells. METHODS: This study investigated whether the calpain systems in LECs associate with the LEC-lymphocyte interaction under hypercholesterolemia using gene-targeted mice. RESULTS: Lipidomic analysis in hypercholesterolemic mice showed that several lysophospholipids, including lysophosphatidic acid, accumulated in the lymphatic environment. Lysophosphatidic acid enables the potentiation of calpain systems in cultured LECs, which limits their ability to stabilize regulatory T cells (Treg) without altering Th1/Th2 (T helper type1/2) subsets. This occurs via the proteolytic degradation of MEKK1 (mitogen-activated protein kinase kinase kinase 1) and the subsequent inhibition of TGF (transforming growth factor)-ß1 production in LECs. Targeting calpain systems in LECs expanded Tregs in the blood circulation and reduced aortic atherosclerosis in hypercholesterolemic mice, concomitant with the reduction of proinflammatory macrophages in the lesions. Treg expansion in the blood circulation and atheroprotection in calpain-targeted mice was prevented by the administration of TGF-ß type-I receptor inhibitor. Moreover, lysophosphatidic acid-induced calpain overactivation potentiated the IL (interleukin)-18/NF-κB (nuclear factor κB)/VCAM1 (vascular cell adhesion molecule 1) axis in LECs, thereby inhibiting lymphocyte mobility on the cells. Indeed, VCAM1 in LECs was upregulated in hypercholesterolemic mice and human cases of coronary artery disease. Neutralization of VCAM1 or targeting LEC calpain systems recovered afferent Treg transportation via lymphatic vessels in mice. CONCLUSIONS: Calpain systems in LECs have a key role in controlling Treg stability and trafficking under hypercholesterolemia.

Intracerebroventricular injection taurine changes free amino acid concentrations in the brain and plasma in chicks.

Brain amino acid metabolism has been reported to regulate body temperature, feeding behavior and stress response. Central injection of taurine induced hypothermic and anorexigenic effects in chicks. However, it is still unknown how the amino acid metabolism is influenced by the central injection of taurine. Therefore, the objective of this study was to investigate the changes in brain and plasma free amino acids following central injection of taurine. Five-day-old male Julia layer chicks (n = 10) were subjected to intracerebroventricular (ICV) injection with saline or taurine (5 µmol/10 µL). Central taurine increased tryptophan concentrations in the diencephalon, and decreased tyrosine in the diencephalon, brainstem, cerebellum, telencephalon and plasma at 30 min post-injection. Taurine was increased in all the brain parts after ICV taurine. Although histidine and cystathionine concentrations were increased in the diencephalon and brainstem, several amino acids such as isoleucine, arginine, methionine, phenylalanine, glutamic acid, asparagine, proline, and alanine were reduced following central injection of taurine. All amino acid concentrations were decreased in the plasma after ICV taurine. In conclusion, central taurine quickly changes free amino acid concentrations in the brain and plasma, which may have a role in thermoregulation, food intake and stress response in chicks.

Central administration of neuropeptide Y reduces the cellular heat stress response and may enhance spleen antioxidative functions in heat-exposed chicks.

Previously it was found that mRNA expression of neuropeptide Y (NPY) was increased in the chicken brain under heat stress. NPY has also been reported as an anti-stress factor to regulate brain functions in heat-exposed chicks. However, to the best of our knowledge, there is no report on the action of central NPY in the immune organs under heat stress. The aim of this study was to examine whether central injection of NPY can regulate heat stress response in the spleen and liver. After intracerebroventricular (ICV) injection of NPY, chicks were exposed to control thermoneutral temperature (CT: 30 ± 1 °C) or high ambient temperature (HT: 35 ± 1 °C) chambers for 60 min. Central injection of NPY caused lowering in rectal temperature under CT, but not under HT. Moreover, ICV injection of NPY caused a significant lower mRNA expression of heat-shock protein-70 and higher expression of glutathione synthase in the spleen, but not liver. Furthermore, plasma uric acid concentrations were significantly increased by the ICV injection of NPY in chicks under HT. These results indicate that brain NPY may contribute to attenuate the intracellular heat stress response and enhance antioxidative status in the immune organ, spleen in chicks.

Intracerebroventricular injection of taurine induces hypothermia through modifying monoaminergic pathways in chicks.

Brain monoamines are reported to regulate body temperature and food intake. The objective of this study was to investigate the mechanism of brain monoamine metabolism in taurine-induced hypothermia and appetite suppression. In Experiment 1, 5-day-old male Julia layer chicks (n = 10) were subjected to intracerebroventricular (ICV) injection with saline or taurine (5 µmol/10 µL). In Experiment 2, the chicks were ICV injected with saline, taurine, fusaric acid (dopamine-ß-hydroxylase inhibitor: 558 nmol), or taurine with fusaric acid. In Experiment 3, the chicks were ICV injected with saline, taurine, para-chlorophenylalanine (PCPA, tryptophan hydroxylase inhibitor: 400 nmol), or taurine with PCPA. In Experiment 4, the chicks were ICV injected with saline, taurine, clorgyline (monoamine oxidase inhibitor: 81 nmol), or taurine with clorgyline. Central taurine lowered rectal temperature at 30 min post-injection and increased norepinephrine in the brainstem and its metabolite 3-methoxy-4-hydroxyphenylglycol in both the diencephalon and brainstem. Similarly, taurine treatment induced increases in serotonin (5-HT) and its metabolite 5-hydroxyindoleacetic acid in the diencephalon. Fusaric acid completely and PCPA partially, but not clorgyline, attenuated taurine-induced hypothermia. The anorexigenic effect of taurine was partially attenuated by PCPA, but not fusaric acid nor clorgyline. In conclusion, central taurine activates dopamine-ß-hydroxylase and tryptophan hydroxylase to produce norepinephrine and 5-HT, and then induces hypothermia, but 5-HT alone may be linked with taurine-induced anorexia in chicks.

Calpain-mediated proteolytic production of free amino acids in vascular endothelial cells augments obesity-induced hepatic steatosis.

Free amino acids that accumulate in the plasma of patients with diabetes and obesity influence lipid metabolism and protein synthesis in the liver. The stress-inducible intracellular protease calpain proteolyzes various substrates in vascular endothelial cells (ECs), although its contribution to the supply of free amino acids in the liver microenvironment remains enigmatic. In the present study, we showed that calpains are associated with free amino acid production in cultured ECs. Furthermore, conditioned media derived from calpain-activated ECs facilitated the phosphorylation of ribosomal protein S6 kinase (S6K) and de novo lipogenesis in hepatocytes, which were abolished by the amino acid transporter inhibitor, JPH203, and the mammalian target of rapamycin complex 1 inhibitor, rapamycin. Meanwhile, calpain-overexpressing capillary-like ECs were observed in the livers of high-fat diet-fed mice. Conditional KO of EC/hematopoietic Capns1, which encodes a calpain regulatory subunit, diminished levels of branched-chain amino acids in the hepatic microenvironment without altering plasma amino acid levels. Concomitantly, conditional KO of Capns1 mitigated hepatic steatosis without normalizing body weight and the plasma lipoprotein profile in an amino acid transporter-dependent manner. Mice with targeted Capns1 KO exhibited reduced phosphorylation of S6K and maturation of lipogenic factor sterol regulatory element-binding protein 1 in hepatocytes. Finally, we show that bone marrow transplantation negated the contribution of hematopoietic calpain systems. We conclude that overactivation of calpain systems may be responsible for the production of free amino acids in ECs, which may be sufficient to potentiate S6K/sterol regulatory element-binding protein 1-induced lipogenesis in surrounding hepatocytes.

Central Taurine Attenuates Hyperthermia and Isolation Stress Behaviors Augmented by Corticotropin-Releasing Factor with Modifying Brain Amino Acid Metabolism in Neonatal Chicks.

The objective of this study was to determine the effects of centrally administered taurine on rectal temperature, behavioral responses and brain amino acid metabolism under isolation stress and the presence of co-injected corticotropin-releasing factor (CRF). Neonatal chicks were centrally injected with saline, 2.1 pmol of CRF, 2.5 µmol of taurine or both taurine and CRF. The results showed that CRF-induced hyperthermia was attenuated by co-injection with taurine. Taurine, alone or with CRF, significantly decreased the number of distress vocalizations and the time spent in active wakefulness, as well as increased the time spent in the sleeping posture, compared with the saline- and CRF-injected chicks. An amino acid chromatographic analysis revealed that diencephalic leucine, isoleucine, tyrosine, glutamate, asparagine, alanine, ß-alanine, cystathionine and 3-methylhistidine were decreased in response to taurine alone or in combination with CRF. Central taurine, alone and when co-administered with CRF, decreased isoleucine, phenylalanine, tyrosine and cysteine, but increased glycine concentrations in the brainstem, compared with saline and CRF groups. The results collectively indicate that central taurine attenuated CRF-induced hyperthermia and stress behaviors in neonatal chicks, and the mechanism likely involves the repartitioning of amino acids to different metabolic pathways. In particular, brain leucine, isoleucine, cysteine, glutamate and glycine may be mobilized to cope with acute stressors.

Adult-onset hypothyroidism causes mechanical hypersensitivity due to peripheral nerve hyperexcitability based on voltage-gated potassium channel downregulation in male mice.

Thyroid hormones play an important role in the central and peripheral nervous system functions. Approximately 50% of adult-onset hypothyroid patients have sensory symptoms including pain, possibly caused by peripheral neuropathy. However, the mechanism causing the pain has not been clarified. We generated an adult-onset hypothyroid model animal by administering 50 ppm propylthiouracil (PTU) for 5 weeks to male mice. Female mice were not tested in this study. Mechanical hypersensitivity, determined by the von Frey hair test, was observed during the PTU exposure and recovered after the exposure termination. The sciatic nerve compound action potential was also analyzed. Under single-pulse stimulation, no significant change in the threshold and conduction velocity was observed in the PTU-administered group. On the other hand, under train-pulse stimulation, the latency delay in the Aδ-fiber component was less in the PTU-administered group in Week 4 of PTU exposure, indicating relative hyperexcitability. Fluticasone, which is the anti-inflammatory agent with an ability to activate the voltage-gated potassium channel subfamily A (Kv1), restored the decrease in the latency change ratio by PTU exposure under the train-pulse stimulation supporting our hypothesis that Kv1 may be involved in the conductivity change. Kv1.1 protein level decreased significantly in the sciatic nerve of the PTU-administered group. These results indicate that adult-onset hypothyroidism causes mechanical hypersensitivity owing to hyperexcitability of the peripheral nerve and that reduction of Kv1.1 level may be involved in such alteration.

Intracrine activity involving NAD-dependent circadian steroidogenic activity governs age-associated meibomian gland dysfunction.

Canonically, hormones are produced in the endocrine organs and delivered to target tissues. However, for steroids, the concept of tissue intracrinology, whereby hormones are produced in the tissues where they exert their effect without release into circulation, has been proposed, but its role in physiology/disease remains unclear. The meibomian glands in the eyelids produce oil to prevent tear evaporation, which reduces with aging. Here, we demonstrate that (re)activation of local intracrine activity through nicotinamide adenine dinucleotide (NAD+)-dependent circadian 3ß-hydroxyl-steroid dehydrogenase (3ß-HSD) activity ameliorates age-associated meibomian gland dysfunction and accompanying evaporative dry eye disease. Genetic ablation of 3ß-HSD nullified local steroidogenesis and led to atrophy of the meibomian gland. Conversely, reactivation of 3ß-HSD activity by boosting its coenzyme NAD+ availability improved glandular cell proliferation and alleviated the dry eye disease phenotype. Both women and men express 3ß-HSD in the meibomian gland. Enhancing local steroidogenesis may help combat age-associated meibomian gland dysfunction.

Liver metabolomic analysis in broiler chicks: Profiling the metabolites after oral administration of l-citrulline.

Hypothermia is directly linked to metabolism; however, it is still unknown how the overall metabolism is altered by oral administration of hypothermic agent, l-citrulline (l-Cit). The present study aimed to determine the characteristics of liver metabolites of chicks orally administered l-Cit to provide a greater understanding of its metabolism. Capillary electrophoresis-time-of-flight mass spectrometry (CE-TOFMS) and liquid chromatography-time-of-flight mass spectrometry (LC-TOFMS) were conducted on liver samples after oral administration of l-Cit. A total of 361 liver metabolites were identified. Although a small number of samples were used for each group, a principal component analysis and heatmap patterns confirmed that the composition of metabolites could be segregated from each other. Of the 361 compounds detected in the liver, 41 compounds, including amino acids related to the Cit-arginine (Arg) cycle, argininosuccinic acid, Arg, ornithine, and Cit, as well as gamma aminobutyric acid, glycine, histidine, and nicotinamide adenine dinucleotide were abundant in l-Cit-treated livers. In contrast, 24 compounds containing fatty acids, amino acids, and cyclic adenosine monophosphate were lower in the l-Cit group. These data imply that the active Cit-Arg cycle, TCA cycle metabolism, and a low activity in fatty acid metabolism occur in l-Cit-treated broiler chicks.

Oral administration of L-citrulline changes the concentrations of plasma hormones and biochemical profile in heat-exposed broilers.

We examined the effects of oral administration of L-citrulline (L-Cit) on plasma metabolic hormones and biochemical profile in broilers. Food intake, water intake, and body temperature were also analyzed. After dual oral administration (20 mmol/head/administration) of L-Cit, broilers were exposed to a high ambient temperature (HT; 30 ± 1°C) chamber for 120 min. Oral administration of L-Cit reduced (p < .001) rectal temperature in broilers. Food intake was increased (p < .05) by heat stress, but it was reduced (p < .05) by L-Cit. Plasma levels of 3,5,3'-triiodothyronine, which initially increased (p < .0001) due to heat stress, were reduced (p < .01) by oral administration of L-Cit. Plasma insulin levels were increased by heat exposure (p < .01) and oral L-Cit (p < .05). Heat stress caused a decline (p < .05) in plasma thyroxine. Plasma lactic acid (p < .05) and non-esterified fatty acids (p < .01) were increased in L-Cit-treated heat-exposed broilers. In conclusion, our results suggest that oral L-Cit can modulate plasma concentrations of major metabolic hormones and reduces food intake in broilers.

Central GABAA receptor mediates taurine-induced hypothermia and possibly reduces food intake in thermo-neutral chicks and regulates plasma metabolites in heat-exposed chicks.

The aim of this study was to examine the central action of taurine on body temperature and food intake in neonatal chicks under control thermoneutral temperature (CT) and high ambient temperature (HT). Intracerebroventricular injection of taurine caused dose-dependent hypothermia and reduced food intake under CT. The mRNA expression of the GABAA receptors, GABAAR-α1 and GABAAR-γ, but not that of GABABR, significantly decreased in the diencephalon after central injection of taurine. Subsequently, we found that picrotoxin, a GABAAR antagonist, attenuated taurine-induced hypothermia. Central taurine significantly decreased the brain concentrations of 3-methoxy-4-hydroxyphenylglycol, a major metabolite of norepinephrine; however, the concentrations of serotonin, dopamine, and the epinephrine metabolites, 3,4-hydroxyindoleacetic acid and homovanillic acid, were unchanged. Although hypothermia was not observed under HT after central injection of taurine, plasma glucose and uric acid levels were higher, and plasma sodium and calcium levels were lower, than those in chicks under CT. In conclusion, brain taurine may play a role in regulating body temperature and food intake in chicks through GABAAR. The changes in plasma metabolites under heat stress suggest that brain taurine may play an important role in maintaining homeostasis in chicks.

Hic-5 is required for activation of pancreatic stellate cells and development of pancreatic fibrosis in chronic pancreatitis.

Accumulated evidence suggests that activated pancreatic stellate cells (PSCs) serve as the main source of the extracellular matrix proteins accumulated under the pathological conditions leading to pancreatic fibrosis in chronic pancreatitis (CP). However, little is known about the mechanisms of PSC activation. PSCs have morphologic and functional similarities to hepatic stellate cells, which are activated by hydrogen peroxide-inducible clone-5 (Hic-5), a TGF-ß1-induced protein. In this study, we investigated whether Hic-5 activates PSCs, which promote pancreatic fibrosis development in CP. Hic-5-knockout and wild type mice were subjected to caerulein injection to induce CP. Hic-5 expression was strongly upregulated in activated PSCs from human CP tissue and from mouse pancreatic fibrosis in caerulein-induced CP. Hic-5 deficiency significantly attenuated mouse pancreatic fibrosis and PSC activation in the experimental murine CP model. Mechanistically, Hic-5 knock down significantly inhibited the TGF-ß/Smad2 signaling pathway, resulting in reduced collagen production and α-smooth muscle actin expression in the activated PSCs. Taken together, we propose Hic-5 as a potential marker of activated PSCs and a novel therapeutic target in CP treatment.

Pineal Neurosteroids: Biosynthesis and Physiological Functions.

Similar to the adrenal glands, gonads, and placenta, vertebrate brains also produce various steroids, which are known as "neurosteroids." Neurosteroids are mainly synthesized in the hippocampus, hypothalamus, and cerebellum; however, it has recently been discovered that in birds, the pineal gland, a photosensitive region in the brain, produces more neurosteroids than other brain regions. A series of experiments using molecular and biochemical techniques have found that the pineal gland produces various neurosteroids, including sex steroids, de novo from cholesterol. For instance, allopregnanolone and 7α-hydroxypregnenolone are actively produced in the pineal gland, unlike in other brain regions. Pineal 7α-hydroxypregnenolone, an up-regulator of locomotion, enhances locomotor activity in response to light stimuli in birds. Additionally, pineal allopregnanolone acts on Purkinje cells in the cerebellum and prevents neuronal apoptosis within the developing cerebellum in juvenile birds. Furthermore, exposure to light during nighttime hours can cause loss of diurnal variations of pineal allopregnanolone synthesis during early posthatch life, eventually leading to cerebellar Purkinje cell death in juvenile birds. In light of these new findings, this review summarizes the biosynthesis and physiological functions of pineal neurosteroids. Given that the circadian rhythms of individuals in modern societies are constantly interrupted by artificial light exposure, these findings in birds, which are excellent model diurnal animals, may have direct implications for addressing problems regarding the mental health and brain development of humans.

Neuroprotective actions of cerebellar and pineal allopregnanolone on Purkinje cells.

The brain produces steroids de novo from cholesterol, so-called "neurosteroids." The Purkinje cell, a cerebellar neuron, was discovered as a major site of the biosynthesis of neurosteroids including sex steroids, such as progesterone, from cholesterol in the brain. Allopregnanolone, a progesterone metabolite, is also synthesized in the cerebellum and acts on the Purkinje cell to prevent cell death of this neuron. Recently, the pineal gland was discovered as an important site of the biosynthesis of neurosteroids. Allopregnanolone, a major pineal neurosteroid, acts on the Purkinje cell for the survival of this neuron by suppressing the expression of caspase-3, a crucial mediator of apoptosis. This review summarizes the discovery of cerebellar and pineal allopregnanolone and its neuroprotective action on Purkinje cells.

Potential Role of Amino Acids in the Adaptation of Chicks and Market-Age Broilers to Heat Stress.

Increased average air temperatures and more frequent and prolonged periods of high ambient temperature (HT) associated with global warming will increasingly affect worldwide poultry production. It is thus important to understand how HT impacts poultry physiology and to identify novel approaches to facilitate improved adaptation and thereby maximize poultry growth, health and welfare. Amino acids play a role in many physiological functions, including stress responses, and their relative demand and metabolism are altered tissue-specifically during exposure to HT. For instance, HT decreases plasma citrulline (Cit) in chicks and leucine (Leu) in the embryonic brain and liver. The physiological significance of these changes in amino acids may involve protection of the body from heat stress. Thus, numerous studies have focused on evaluating the effects of dietary administration of amino acids. It was found that oral l-Cit lowered body temperature and increased thermotolerance in layer chicks. When l-Leu was injected into fertile broiler eggs to examine the cause of reduction of Leu in embryos exposed to HT, in ovo feeding of l-Leu improved thermotolerance in broiler chicks. In ovo injection of l-Leu was also found to inhibit weight loss in market-age broilers exposed to chronic HT, giving rise to the possibility of developing a novel biotechnology aimed at minimizing the economic losses to poultry producers during summer heat stress. These findings and the significance of amino acid metabolism in chicks and market-age broilers under HT are summarized and discussed in this review.

Alleviation of murine osteoarthritis by deletion of the focal adhesion mechanosensitive adapter, Hic-5.

Excessive mechanical stress is a major cause of knee osteoarthritis. However, the mechanism by which the mechanical stress begets osteoarthritis development remains elusive. Hydrogen peroxide-inducible clone-5 (Hic-5; TGFß1i1), a TGF-ß inducible focal adhesion adaptor, has previously been reported as a mediator of mechanotransduction. In this study, we analyzed the in vivo function of Hic-5 in development of osteoarthritis, and found that mice lacking Hic-5 showed a significant reduction in development of osteoarthritis in the knee. Furthermore, we found reduced expression of catabolic genes, such as metalloproteinase-13 and a disintegrin and metalloproteinase with thrombospondin type 1 motif 5 in osteoarthritic lesions in mice lacking Hic-5. During osteoarthritis development, Hic-5 is detected in chondrocytes of articular cartilage. To investigate the role of Hic-5 in chondrocytes, we isolated chondrocytes from articular cartilage of wild type and Hic-5-deficient mice. In these primary cultured chondrocytes, Hic-5 deficiency resulted in suppression of catabolic gene expression induced by osteoarthritis-related cytokines such as tumor necrosis factor α and interleukin 1ß. Furthermore, Hic-5 deficiency in chondrocytes suppressed catabolic gene expression induced by mechanical stress. Revealing the regulation of chondrocyte catabolism by Hic-5 contributes to understanding the pathophysiology of osteoarthritis induced by mechanical stress.

Light-at-night exposure affects brain development through pineal allopregnanolone-dependent mechanisms.

The molecular mechanisms by which environmental light conditions affect cerebellar development are incompletely understood. We showed that circadian disruption by light-at-night induced Purkinje cell death through pineal allopregnanolone (ALLO) activity during early life in chicks. Light-at-night caused the loss of diurnal variation of pineal ALLO synthesis during early life and led to cerebellar Purkinje cell death, which was suppressed by a daily injection of ALLO. The loss of diurnal variation of pineal ALLO synthesis induced not only reduction in pituitary adenylate cyclase-activating polypeptide (PACAP), a neuroprotective hormone, but also transcriptional repression of the cerebellar Adcyap1 gene that produces PACAP, with subsequent Purkinje cell death. Taken together, pineal ALLO mediated the effect of light on early cerebellar development in chicks.

L-Leucine increases the daily body temperature and affords thermotolerance in broiler chicks.

OBJECTIVE: Heat stress poses an increasing threat for poultry production. Some amino acids have been found to play critical roles in affording thermotolerance. Recently, it was found that in ovo administration of L-leucine (L-Leu) altered amino acid metabolism and afforded thermotolerance in heat-exposed broiler chicks. METHODS: In this study, two doses (35 and 70 µmol/egg) of L-Leu were administered in ovo on embryonic day 7 to determine their effect on rectal temperature (RT), body weight (BW) and thyroid hormones at hatching. Changes in RT, BW, and thermotolerance in post-hatched chicks were also analyzed. RESULTS: It was found that in ovo administration of L-Leu dose-dependently reduced RT and plasma thyroxine (T4) level just after hatching. In post-hatched neonatal broiler chicks, however, the higher dose of L-Leu administered in ovo significantly increased RT without affecting BW gain. In chicks that had been exposed to heat stress, the RT was significantly lowered by in ovo administration of L-Leu (high dose) in comparison with the control chicks under the same high ambient temperature (HT: 35°C±1°C, 120 min). CONCLUSION: In ovo administration of L-Leu in a high dose contributed to an increased daily body temperature and afforded thermotolerance under HT in neonatal broiler chicks.