Orai calcium release-activated calcium modulator 1 (ORAI1) plays a role in endoplasmic reticulum stress in bovine mammary epithelial cells challenged with physiological levels of ketone bodies.

The Orai calcium release-activated calcium modulator 1 (ORAI1) is a key component of the store-operated Ca2+ entry mechanism regulating cellular Ca2+ balance in nonruminants. Alterations in ORAI1 abundance have been associated with endoplasmic reticulum (ER) stress and changes in lipid metabolism in hepatocytes, an important lipogenic organ in nonruminants. Objectives were to (1) determine abundance of ORAI1 and components of the ER stress response in mammary tissue of ketotic cows, and (2) the potential role of ORAI1 on mammary cell responses to high levels of ß-hydroxybutyrate (BHB). Healthy (n = 6, plasma BHB < 0.60 mmol/L) and clinically ketotic (n = 6, plasma BHB > 2.0 mmol/L) Holstein cows (days in milk = 10.13 ± 1.90) were used for mammary gland tissue and blood sample collection. Although milk production (22.5 ± 1.26, 33 ± 1.59, kg of milk/cow per day) and dry matter intake (19.5 ± 1.05, 21.9 ± 0.95, kg/d) were lower in ketotic cows, abundance of ORAI1 protein was greater and was associated with greater mRNA abundance of ER stress proteins (PERK, IRE1, ATF6, and GRP78) and lipogenic genes (FASN, SREBP1, and ACACA). Cellular mechanisms to establish links between BHB and mammary cell responses were evaluated using the immortalized cell line bovine mammary epithelial cells (MAC-T). First, a dose response study was performed with 0, 0.6, 1.2, 1.8, 2.4, or 4.8 mM BHB for 24 h. The mRNA abundance of FASN, SREBP1, and ACACA and lipid droplet formation peaked at 1.2 mM BHB. A subsequent study involved transfecting MAC-T with small interfering Orai 1 (siORAI1) or the ORAI1 inhibitor BTP2 for 24 h followed by a challenge with 1.2 mM BHB for 24 h. Transcription and protein abundance of FASN, SREBP1, ACACA, and ER stress proteins returned to basal levels when ORAI1 was silenced or inhibited. Furthermore, the Ca2+ ionophore ionomycin (raises the intracellular level of Ca2+) also increased abundance of ORAI1, FASN, SREBP1, ACACA, and ER stress proteins. Data suggest that the mammary gland experiences ER stress during ketosis, partly due to the greater supply of BHB originating from ketogenesis in the liver. Intracellular Ca2+ signaling and ORAI1 seem to mediate in part the BHB-induced ER stress in mammary cells.

Ketogenic diet modulates cardiac metabolic dysregulation in streptozocin-induced diabetic rats.

The ketogenic diet (KD) might improve cardiac function in diabetic cardiomyopathy, but the mechanisms remain unclear. This study investigated the effects of KD on myocardial fatty acid (FA), glucose, and ketone metabolism in diabetic cardiomyopathy. Echocardiograms, biochemistry, and micro-positron emission tomography were performed to evaluate cardiac function and glucose uptake in control rats and streptozotocin-induced diabetes mellitus (DM) rats with normal diet (ND) or KD for 6 weeks. Histopathology, adenosine triphosphate measurement, and Western blot were performed in the ventricular myocytes to analyze fibrosis, FA, ketone body, and glucose utilization. The ND-fed DM rats exhibited impaired left ventricular systolic function and increased chamber dilatation, whereas control and KD-fed DM rats did not. The KD reduced myocardial fibrosis and apoptosis in the DM rats. Myocardial glucose uptake in the micro-positron emission tomography was similar between ND-fed DM rats and KD-fed DM rats and was substantially lower than the control rats. Compared with the control rats,  ND-fed DM rats had increased phosphorylation of acetyl CoA carboxylase and higher expressions of CD-36, carnitine palmitoyltransferase-1ß, tumor necrosis factor-α, interleukin-1ß, interleukin6, PERK, and e-IF2α as well as more myocardial fibrosis and apoptosis (assessed by Bcl-2, BAX, and caspase-3 expression); these increases were attenuated in the KD-fed DM rats. Moreover, ND-fed DM rats had significantly lower myocardial adenosine triphosphate, BHB, and OXCT1 levels than the control and KD-fed DM rats. The KD may improve the condition of diabetic cardiomyopathy by suppressing FA metabolism, increasing ketone utilization, and decreasing endoplasmic reticulum stress and inflammation.

Ketone Body ß-Hydroxybutyrate Prevents Myocardial Oxidative Stress in Septic Cardiomyopathy.

Septic cardiomyopathy is a life-threatening complication of severe sepsis and septic shock. Oxidative stress and mitochondrial dysfunction have been identified as significant abnormalities in septic cardiomyopathy. However, specific treatments are rare. This study aims to investigate the impact of ß-hydroxybutyrate (ß-OHB) on septic cardiomyopathy and explore the underlying mechanism(s). We found that pretreatment of D-ß-hydroxybutyrate-(R)-1,3 butanediol monoester (ketone ester, 3 mg/g body weight, once daily) by gavage for three days elevated the levels of ketone bodies, especially that of ß-hydroxybutyrate (ß-OHB) in the circulation and mouse hearts, which exerted a protective effect against lipopolysaccharide (LPS, 20 mg/kg)-induced septic cardiomyopathy in mice. In addition, an LPS-stimulated macrophage-conditioned medium (MCM) was used to mimic the pathological process of septic cardiomyopathy. Mechanistically, ß-OHB alleviated myocardial oxidative stress and improved mitochondrial respiratory function through the antioxidant FoxO3a/MT2 pathway activated via histone deacetylase (HDAC) inhibition, which ultimately enhanced heart performance in septic cardiomyopathy. Our results, therefore, suggested an unappreciated critical role of ß-OHB in septic heart protection as well as highlighted the potential of ß-OHB as a simple remedy for the septic cardiomyopathy population.

Utility of Ketone Supplementation to Enhance Physical Performance: A Systematic Review.

Ingesting exogenous ketone bodies has been touted as producing ergogenic effects by altering substrate metabolism; however, research findings from recent studies appear inconsistent. This systematic review aimed to aggregate data from the current literature to examine the impact of consuming ketone supplements on enhancing physical performance. A systematic search was performed for randomized controlled trials that measured physical performance outcomes in response to ingesting exogenous ketone supplements compared with a control (nutritive or non-nutritive) in humans. A total of 161 articles were screened. Data were extracted from 10 eligible studies (112 participants; 109 men, 3 women ) containing 16 performance outcomes [lower-body power (n = 8) and endurance performance (n = 8)]. Ketone supplements were grouped as ketone esters (n = 8) or ketone salts/precursors (n = 8). Of the 16 performance outcomes identified by the systematic review, 3 reported positive, 10 reported null, and 3 reported negative effects of ketone supplementation on physical performance compared with controls. Heterogeneity was detected for lower-body power ( Q = 40, I2 = 83%, P < 0.01) and endurance performance (Q = 95, I2 = 93%, P < 0.01) between studies. Similarly high levels of heterogeneity were detected in studies providing ketone esters (Q = 111, I2 = 93%, P < 0.01), and to a lesser extent studies with ketone salts/precursors (Q = 25, I2 = 72%, P < 0.01). Heterogeneity across studies makes it difficult to conclude any benefit or detriment to consuming ketone supplements on physical performance. This systematic review discusses factors within individual studies that may contribute to discordant outcomes across investigations to elucidate if there is sufficient evidence to warrant recommendation of consuming exogenous ketone supplements to enhance physical performance.

Assessing ketosis: approaches and pitfalls.

Although the mechanism of action of the ketogenic diet (KD) remains unknown, ketones have been used as a marker of efficacy. Acetoacetate, acetone, and beta-hydroxybutyrate can be measured in urine, blood, and breath. The value of these measures is reviewed in this paper. Future brain measures hold promise as markers of efficacy, but research focused on the mechanisms of the KD should help to develop more reliable markers of efficacy.

Reduction in mitogenic response of bovine lymphocytes by ketone bodies.

The effect of toxic or subtoxic concentrations of ketone bodies (acetone, beta-hydroxybutyrate, acetoacetate) in blood on function of bovine lymphocytes was studied in vitro. Lymphocytes separated from peripheral bovine blood were distributed into the wells of Linbro Microtiter plates containing control medium and test medium with various concentrations of ketones and/or phytohemagglutinin. The mitogenic response of lymphocytes was measured by incorporation of [3H]thymidine into DNA of lymphocytes. Toxic and subtoxic concentrations of beta-hydroxybutyrate or the toxic concentration of acetoacetate significantly affected the mitogenic response of bovine lymphocytes. The reduction in the mitogenic response also occurred when lymphocytes were only preincubated for 2 hours or longer with beta-hydroxybutyrate or acetoacetate. The presence of the toxic concentration of acetone in the medium did not affect the mitogenic stimulation of lymphocytes.

Beta-hydroxybutyric acid--an indicator for an alcoholic ketoacidosis as cause of death in deceased alcohol abusers.

We analyzed the postmortem blood of a total of 100 fatal cases for beta-hydroxybutyric acid (BHBA). In 25 cases of sudden and unexpected death of alcoholics we found pathologically increased levels of BHBA of 1260 to 47200 (median 8000) micromol/L. This led us to the diagnosis of an alcoholic ketoacidosis (AKA) as cause of death in these cases. The control group of 69 postmortem cases revealed that BHBA concentrations below 500 can be regarded as normal, and values up to 2500 micromol/L as elevated. Our study shows that BHBA values over 2500 micromol/L could lead to death, if no medical attention is sought. During storage we did not find any indication of postmortem formation or decomposition of BHBA in blood in vitro or in the corpses. In our opinion, BHBA should be considered the diagnostic marker of choice for the postmortem determination of alcoholic ketoacidosis (AKA) as the cause of death. The classical indications of such deaths are: unexpected death of a chronic alcoholic; none or only traces of ethanol in the blood; increased acetone blood concentration; and neither autopsy, histology, microbiology, nor toxicology reveal the cause of death. In six further cases a diabetic ketoacidosis (DKA) was diagnosed as the cause of death.

Management of pitfalls for the successful clinical use of hypothermia treatment.

Therapeutic hypothermia is a promising method for controlling intracranial pressure (ICP) in severely brain-injured patients. However, clinical data regarding the effect of brain hypothermia on overall outcome of these patients is limited. This may be because there are specific pitfalls associated with the clinical management of induced hypothermia in patients with severe traumatic brain injury (TBI). These pitfalls may be avoided by preventing specific risk factors when cooling is induced and with rewarming. However, these risk factors have not been well systematically discussed in the literature. In this paper, three categories of clinical issues regarding the management of brain hypothermia are discussed: (1) stress-induced secondary brain injury mechanisms; (2) technical aspects of intensive care unit (ICU) cooling management; and (3) rewarming rates and methods. For patients with a Glasgow Coma Scale (GCS) score of less than 8, management of stress-induced insulin-resistant hyperglycemia, and unstable systemic circulation due to impaired cardiac contractility are especially important. For example, in our experience, posttraumatic hyperglycemia, exacerbated by cooling, may be ameliorated by the administration of a ketone body with mannitol. Prevention of selective free radical damage to neurons is also an important target for successful brain hypothermia treatment. Taken together, it is clear that several orchestrated steps should be initiated to enhance the protective effects of hypothermia therapy and prevent these possible pitfalls.

Diabetes-associated endometrial disruption in the ketonuric, diabetic Chinese hamster.

The effects of the ketonuric-diabetic (KD) condition on uterine endometrial morphology were studied in the genetically diabetic Chinese hamster. All KD hamsters were matched for age, body weight and cyclic state with nondiabetic control animals. All KD hamsters were reproductively acyclic as compared to controls. The uterine epithelium of KD hamsters was devoid of surface microvilli, secretory activity, and exhibited a height reduction as compared to controls. The basal lamina underlying the luminal epithelium was 2-5 times as thick in the KD condition than control. The endometrial stroma was disrupted in the KD hamsters, with the endometrium composed of rounded cells surrounded by a thickened intercellular matrix. This was in marked contrast to the stroma of controls which consisted of an orderly array of fibroblasts and collagen fibers. Stromal vessels had a thickened basement membrane in diabetic animals. The results of this study indicate that epithelial and stromal disruption are associated with the KD state in the Chinese hamster and are probably causally associated with reproductive failure.

Dieta cetogênica para epilepsia intratável em crianças e adolescentes: relato de seis casos / Ketogenic diet for intractable epilepsy in children and adolescents: report of six cases

OBJETIVO: Descrever a introdução e o manejo da dieta cetogênica em um grupo de seis crianças e adolescentes com epilepsia refratária. MÉTODOS: Os autores reviram o prontuário médico de cada paciente menor de 15 anos submetido à dieta cetogênica entre abril de 1999 e julho de 2003 e compararam os resultados terapêuticos e efeitos adversos e benéficos com a literatura pertinente. RESULTADOS: A dieta cetogênica foi introduzida para seis pacientes, com idade mediana de sete anos (faixa: 1,8-12,2). A duração média da aplicação da dieta foi 9,7 meses (faixa: 7 dias-4 anos). Observou-se uma redução igual ou maior que 50 por cento da freqüência das crises epilépticas em metade dos casos. As complicações observadas foram leucopenia, constipação, desidratação, priapismo e recorrência das crises epilépticas. CONCLUSÕES: A dieta cetogênica foi eficaz e segura em três pacientes de uma série de seis casos com epilepsia intratável. A complicação mais comum foi leucopenia.