A combination of ß-hydroxybutyrate and citrate ameliorates disease progression in a rat model of polycystic kidney disease.

Our research has shown that interventions producing a state of ketosis are highly effective in rat, mouse, and cat models of polycystic kidney disease (PKD), preventing and partially reversing cyst growth and disease progression. The ketone ß-hydroxybutyrate (BHB) appears to underlie this effect. In addition, we have demonstrated that naturally formed microcrystals within kidney tubules trigger a renoprotective response that facilitates tubular obstruction clearance in healthy animals but, alternatively, leads to cyst formation in PKD. The administration of citrate prevents microcrystal formation and slows PKD progression. Juvenile Cy/+ rats, a nonorthologous PKD model, were supplemented from 3 to 8 wk of age with water containing titrated BHB, citrate, or in combination to find minimal effective and optimal dosages, respectively. Adult rats were given a reduced BHB/citrate combination or equimolar control K/NaCl salts from 8 to 12 wk of age. In addition, adult rats were placed in metabolic cages following BHB, citrate, and BHB/citrate administration to determine the impact on mineral, creatinine, and citrate excretion. BHB or citrate alone effectively ameliorates disease progression in juvenile rats, decreasing markers of cystic disease and, in combination, producing a synergistic effect. BHB/citrate leads to partial disease regression in adult rats with established cystic disease, inhibiting cyst formation and kidney injury. BHB/citrate confers benefits via multiple mechanisms, increases creatinine and citrate excretion, and normalizes mineral excretion. BHB and citrate are widely available and generally recognized as safe compounds and, in combination, exhibit high promise for supporting kidney health in polycystic kidney disease.NEW & NOTEWORTHY Combining ß-hydroxybutyrate (BHB) and citrate effectively slows and prevents cyst formation and expansion in young Cy/+ rats using less BHB and citrate than when used alone, demonstrating synergy. In adult rats, the combination causes a partial reversal of existing disease, reducing cyst number and cystic area, preserving glomerular health, and decreasing markers of kidney injury. Our results suggest a safe and feasible strategy for supporting kidney health in polycystic kidney disease (PKD) using a combination of BHB and citrate.

Ketone body ß-hydroxybutyrate (BHB) preserves mitochondrial bioenergetics.

The ketogenic diet is an emerging therapeutic approach for refractory epilepsy, as well as certain rare and neurodegenerative disorders. The main ketone body, ß-hydroxybutyrate (BHB), is the primary energy substrate endogenously produced in a ketogenic diet, however, mechanisms of its therapeutic actions remain unknown. Here, we studied the effects of BHB on mitochondrial energetics, both in non-stimulated conditions and during glutamate-mediated hyperexcitation. We found that glutamate-induced hyperexcitation stimulated mitochondrial respiration in cultured cortical neurons, and that this response was greater in cultures supplemented with BHB than with glucose. BHB enabled a stronger and more sustained maximal uncoupled respiration, indicating that BHB enables neurons to respond more efficiently to increased energy demands such as induced during hyperexcitation. We found that cytosolic Ca2+ was required for BHB-mediated enhancement of mitochondrial function, and that this enhancement was independent of the mitochondrial glutamate-aspartate carrier, Aralar/AGC1. Our results suggest that BHB exerts its protective effects against hyperexcitation by enhancing mitochondrial function through a Ca2+-dependent, but Aralar/AGC1-independent stimulation of mitochondrial respiration.

Ketone bodies supplementation restores the barrier function, induces a metabolic switch, and elicits beta-hydroxybutyrate diffusion across a monolayer of iPSC-derived brain microvascular endothelial cells.

Glucose constitutes the main source of energy for the central nervous system (CNS), its entry occurring at the blood-brain barrier (BBB) via the presence of glucose transporter 1 (GLUT1). However, under food intake restrictions, the CNS can utilize ketone bodies (KB) as an alternative source of energy. Notably, the relationship between the BBB and KBs and its effect on their glucose metabolism remains poorly understood. In this study, we investigated the effect of glucose deprivation on the brain endothelium in vitro, and supplementation with KBs using induced pluripotent stem cell (iPSC)-derived brain microvascular endothelial cell-like cells (iBMECs). Glucose-free environment significantly decreased cell metabolic activity and negatively impacted the barrier function. In addition, glucose deprivation did not increase GLUT1 expression but also resulted in a decrease in glucose uptake and glycolysis. Supplementation of glucose-deprived iBMECs monolayers with KB showed no improvement and even worsened upon treatment with acetoacetate. However, under a hypoglycemic condition in the presence of KBs, we noted a slight improvement of the barrier function, with no changes in glucose uptake. Notably, hypoglycemia and/or KB pre-treatment elicited a saturable beta-hydroxybutyrate diffusion across iBMECs monolayers, such diffusion occurred partially via an MCT1-dependent mechanism. Taken together, our study highlights the importance of glucose metabolism and the reliance of the brain endothelium on glucose and glycolysis for its function, such dependence is unlikely to be covered by KBs supplementation. In addition, KB diffusion at the BBB appeared induced by KB pre-treatment and appears to involve an MCT1-dependent mechanism.

Forsythin inhibits ß-hydroxybutyrate-induced oxidative stress in bovine macrophages by regulating p38/ERK, PI3K/Akt, and Nrf2/HO-1 signaling pathways.

Ketosis is a metabolic disease of dairy cows in the perinatal period, ß-hydroxybutyrate (ß-HB) is the main component of ketosis. High levels of ß-HB can trigger oxidative stress and inflammatory response in dairy cows, leading to decreased milk yield and multiple postpartum diseases. Forsythin (FOR), the major constituent of the herbal medicine Forsythia, has anti-inflammatory, anti-oxidant, and antiviral effects. FOR was demonstrated to have an antioxidant effect on PC12 cells. However, the effects of FOR on ß-HB-stimulated bovine macrophages (BMs) has not been reported. Thus, the aim of the present study was to investigate the effects of FOR on ß-HB-stimulated BMs. Firstly, the CCK8 test confirmed that FOR (50, 100, 200 µg/mL) has no effect on BMs activity, and we selected these concentrations for subsequent experiments. Secondly, through detecting the oxidation indexes ROS, MDA and antioxidant indexes CAT and SOD, we confirmed the antioxidant effect of FOR on BMs. Next, qRT-PCR confirmed that FOR dramatically reduced the mRNA levels of IL-1ß and IL-6. Furthermore, the western blotting confirmed that FOR observably down-regulated ß-HB-stimulated phosphorylation of p38, ERK and Akt and up-regulated expression of Nrf2, and HO-1. Above results suggested that FOR plays antioxidant effects on ß-HB-induced BMs through p38, ERK and PI3K/Akt, Nrf2 and HO-1 signaling pathways. Therefore, we speculated that FOR may be a potential medicine to alleviate ß-HB-induced inflammatory response and provide a preliminary reference for the research and development of FOR.

Ketone Body Exposure of Cardiomyocytes Impairs Insulin Sensitivity and Contractile Function through Vacuolar-Type H+-ATPase Disassembly-Rescue by Specific Amino Acid Supplementation.

The heart is metabolically flexible. Under physiological conditions, it mainly uses lipids and glucose as energy substrates. In uncontrolled diabetes, the heart switches towards predominant lipid utilization, which over time is detrimental to cardiac function. Additionally, diabetes is accompanied by high plasma ketone levels and increased utilization of energy provision. The administration of exogenous ketones is currently being investigated for the treatment of cardiovascular disease. Yet, it remains unclear whether increased cardiac ketone utilization is beneficial or detrimental to cardiac functioning. The mechanism of lipid-induced cardiac dysfunction includes disassembly of the endosomal proton pump (named vacuolar-type H+-ATPase; v-ATPase) as the main early onset event, followed by endosomal de-acidification/dysfunction. The de-acidified endosomes can no longer serve as a storage compartment for lipid transporter CD36, which then translocates to the sarcolemma to induce lipid accumulation, insulin resistance, and contractile dysfunction. Lipid-induced v-ATPase disassembly is counteracted by the supply of specific amino acids. Here, we tested the effect of ketone bodies on v-ATPase assembly status and regulation of lipid uptake in rodent/human cardiomyocytes. 3-ß-hydroxybutyrate (3HB) exposure induced v-ATPase disassembly and the entire cascade of events leading to contractile dysfunction and insulin resistance, similar to conditions of lipid oversupply. Acetoacetate addition did not induce v-ATPase dysfunction. The negative effects of 3HB could be prevented by addition of specific amino acids. Hence, in sedentary/prediabetic subjects ketone bodies should be used with caution because of possible aggravation of cardiac insulin resistance and further loss of cardiac function. When these latter maladaptive conditions would occur, specific amino acids could potentially be a treatment option.

Effects of Exogenous Ketone Supplementation on Blood Glucose: A Systematic Review and Meta-analysis.

Recently developed ketone (monoester or salt) supplements acutely elevate blood ß-hydroxybutyrate (BHB) exogenously without prolonged periods of fasting or carbohydrate restriction. Previous (small-scale) studies have found a blood glucose-lowering effect of exogenous ketones. This study aimed to systematically review available evidence and conduct meta-analyses of studies reporting on exogenous ketones and blood glucose. We searched 6 electronic databases on 13 December 2021 for randomized and nonrandomized trials of any length that reported on the use of exogenous ketones. We calculated raw mean differences (MDs) in blood BHB and glucose in 2 main analyses: 1) after compared with before acute ingestion of exogenous ketones and 2) following acute ingestion of exogenous ketones compared with a comparator supplement. We pooled effect sizes using random-effects models and performed prespecified subgroup analyses to examine the effect of potential explanatory factors, including study population, exercise, blood BHB, and supplement type, dosing, and timing. Risk of bias was examined using Cochrane's risk-of-bias tools. Studies that could not be meta-analyzed were summarized narratively. Forty-three trials including 586 participants are summarized in this review. Following ingestion, exogenous ketones increased blood BHB (MD = 1.73 mM; 95% CI: 1.26, 2.21 mM; P < 0.001) and decreased mean blood glucose (MD = -0.54 mM; 95% CI: -0.68, -0.40 mM; P < 0.001). Similarly, when compared with placebo, blood BHB increased (MD = 1.98 mM; 95% CI: 1.52, 2.45 mM; P < 0.001) and blood glucose decreased (MD = -0.47 mM; 95% CI: -0.57, -0.36 mM; P < 0.001). Across both analyses, significantly greater effects were seen with ketone monoesters compared with salts (P < 0.001). The available evidence indicates that acute ingestion of exogenous ketones leads to increased blood BHB and decreased blood glucose. Limited evidence on prolonged ketone supplementation was found.

Sodium Butyrate More Effectively Mitigates the Negative Effects of High-Concentrate Diet in Dairy Cows than Sodium ß-Hydroxybutyrate via Reducing Free Bacterial Cell Wall Components in Rumen Fluid and Plasma.

The present study was aimed at investigating the effects of sodium butyrate and sodium ß-hydroxybutyrate on lactation and health of dairy cows fed a high-concentrate (HC) diet. Eighty mid-lactation dairy cows with an average milk yield of 33.75 ± 5.22 kg/d were randomly allocated to four groups (n = 20 per group) and were fed either a low-concentrate (LC) diet, a HC diet, the HC diet with 1% sodium butyrate (HCSB), or the HC diet with 1% sodium ß-hydroxybutyrate (HCHB). The feeding trial lasted for 7 weeks, with a 2-week adaptation period and a 5-week measurement period, and the trial started from 96 ± 13 d in milk. Sodium butyrate supplementation delayed the decline in milk production and improved milk synthesis efficiency and milk fat content. Additionally, it decreased the proinflammatory cytokines and acute phase proteins (APPs) in plasma, the leucocytes in blood, the somatic cell count (SCC) in milk, and the gene expression of pattern recognition receptors (PRRs) and proinflammatory cytokines in the mammary gland, due to decreasing the contents of bacterial cell wall components (lipopolysaccharide, LPS; peptidoglycan, PGN; and lipoteichoic acid, LTA) in the rumen and plasma, compared with the HC diet. Sodium ß-hydroxybutyrate supplementation also improved milk yield, milk synthesis efficiency and milk fat content and partially reduced the adverse effects caused by the HC diet, but it had no effect on decreasing bacterial cell wall components in the rumen and plasma, compared with the HC diet. Collectively, both sodium butyrate and sodium ß-hydroxybutyrate mitigated the negative effects of HC diet on lactation and health of dairy cows, with sodium butyrate being more effective than sodium ß-hydroxybutyrate.

Ketones Elicit Distinct Alterations in Adipose Mitochondrial Bioenergetics.

OBJECTIVE: The rampant growth of obesity worldwide has stimulated explosive research into human metabolism. Energy expenditure has been shown to be altered by diets differing in macronutrient composition, with low-carbohydrate, ketogenic diets eliciting a significant increase over other interventions. The central aim of this study was to explore the effects of the ketone ß-hydroxybutyrate (ßHB) on mitochondrial bioenergetics in adipose tissue. METHODS: We employed three distinct systems-namely, cell, rodent, and human models. Following exposure to elevated ßHB, we obtained adipose tissue to quantify mitochondrial function. RESULTS: In every model, ßHB robustly increased mitochondrial respiration, including an increase of roughly 91% in cultured adipocytes, 113% in rodent subcutaneous adipose tissue (SAT), and 128% in human SAT. However, this occurred without a commensurate increase in adipose ATP production. Furthermore, in cultured adipocytes and rodent adipose, we quantified and observed an increase in the gene expression involved in mitochondrial biogenesis and uncoupling status following ßHB exposure. CONCLUSIONS: In conclusion, ßHB increases mitochondrial respiration, but not ATP production, in mammalian adipocytes, indicating altered mitochondrial coupling. These findings may partly explain the increased metabolic rate evident in states of elevated ketones, and may facilitate the development of novel anti-obesity interventions.

The Impact of Acute Ingestion of a Ketone Monoester Drink on LPS-Stimulated NLRP3 Activation in Humans with Obesity.

Activation of the NOD-like receptor pyrin-domain containing 3 (NLRP3) inflammasome is associated with chronic low-grade inflammation in metabolic diseases such as obesity. Mechanistic studies have shown that ß-hydroxybutyrate (OHB) attenuates activation of NLRP3, but human data are limited. In a randomized, double-blind, placebo-controlled crossover trial (n = 11) we tested the hypothesis that acutely raising ß-OHB by ingestion of exogenous ketones would attenuate NLRP3 activation in humans with obesity. Blood was sampled before and 30 min post-ingestion of a ketone monoester drink ((R)-3-hydroxybutyl (R)-3-hydroxybutyrate, 482 mg/kg body mass) or placebo. A 75 g oral glucose load was then ingested, and a third blood sample was obtained 60 min following glucose ingestion. NLRP3 activation was quantified by assessing monocyte caspase-1 activation and interleukin (IL)-1ß secretion in ex vivo lipopolysaccharide (LPS)-stimulated whole-blood cultures. LPS-stimulated caspase-1 activation increased following glucose ingestion (main effect of time; p = 0.032), with no differences between conditions. IL-1ß secretion did not differ between conditions but was lower 60 min post-glucose ingestion compared to the fasting baseline (main effect of time, p = 0.014). Plasma IL-1ß was detectable in ~80% of samples and showed a decrease from fasting baseline to 60 min in the ketone condition only (condition × time interaction, p = 0.01). In individuals with obesity, an excursion into hyperglycemia following ingestion of a glucose load augments LPS-induced activation of caspase-1 in monocytes with no apparent impact of raising circulating ß-OHB concentration via ingestion of exogenous ketones. Exogenous ketone supplementation may impact plasma IL-1ß, but these findings require confirmation in studies with larger sample sizes.

Suppression of enteroendocrine cell glucagon-like peptide (GLP)-1 release by fat-induced small intestinal ketogenesis: a mechanism targeted by Roux-en-Y gastric bypass surgery but not by preoperative very-low-calorie diet.

OBJECTIVE: Food intake normally stimulates release of satiety and insulin-stimulating intestinal hormones, such as glucagon-like peptide (GLP)-1. This response is blunted in obese insulin resistant subjects, but is rapidly restored following Roux-en-Y gastric bypass (RYGB) surgery. We hypothesised this to be a result of the metabolic changes taking place in the small intestinal mucosa following the anatomical rearrangement after RYGB surgery, and aimed at identifying such mechanisms. DESIGN: Jejunal mucosa biopsies from patients undergoing RYGB surgery were retrieved before and after very-low calorie diet, at time of surgery and 6 months postoperatively. Samples were analysed by global protein expression analysis and Western blotting. Biological functionality of these findings was explored in mice and enteroendocrine cells (EECs) primary mouse jejunal cell cultures. RESULTS: The most prominent change found after RYGB was decreased jejunal expression of the rate-limiting ketogenic enzyme mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase (mHMGCS), corroborated by decreased ketone body levels. In mice, prolonged high-fat feeding induced the expression of mHMGCS and functional ketogenesis in jejunum. The effect of ketone bodies on gut peptide secretion in EECs showed a ∼40% inhibition of GLP-1 release compared with baseline. CONCLUSION: Intestinal ketogenesis is induced by high-fat diet and inhibited by RYGB surgery. In cell culture, ketone bodies inhibited GLP-1 release from EECs. Thus, we suggest that this may be a mechanism by which RYGB can remove the inhibitory effect of ketone bodies on EECs, thereby restituting the responsiveness of EECs resulting in increased meal-stimulated levels of GLP-1 after surgery.

Exogenous Ketones Lower Blood Glucose Level in Rested and Exercised Rodent Models.

Diseases involving inflammation and oxidative stress can be exacerbated by high blood glucose levels. Due to tight metabolic regulation, safely reducing blood glucose can prove difficult. The ketogenic diet (KD) reduces absolute glucose and insulin, while increasing fatty acid oxidation, ketogenesis, and circulating levels of ß-hydroxybutyrate (ßHB), acetoacetate (AcAc), and acetone. Compliance to KD can be difficult, so alternative therapies that help reduce glucose levels are needed. Exogenous ketones provide an alternative method to elevate blood ketone levels without strict dietary requirements. In this study, we tested the changes in blood glucose and ketone (ßHB) levels in response to acute, sub-chronic, and chronic administration of various ketogenic compounds in either a post-exercise or rested state. WAG/Rij (WR) rats, a rodent model of human absence epilepsy, GLUT1 deficiency syndrome mice (GLUT1D), and wild type Sprague Dawley rats (SPD) were assessed. Non-pathological animals were also assessed across different age ranges. Experimental groups included KD, standard diet (SD) supplemented with water (Control, C) or with exogenous ketones: 1, 3-butanediol (BD), ßHB mineral salt (KS), KS with medium chain triglyceride/MCT (KSMCT), BD acetoacetate diester (KE), KE with MCT (KEMCT), and KE with KS (KEKS). In rested WR rats, the KE, KS, KSMCT groups had lower blood glucose level after 1 h of treatment, and in KE and KSMCT groups after 24 h. After exercise, the KE, KSMCT, KEKS, and KEMCT groups had lowered glucose levels after 1 h, and in the KEKS and KEMCT groups after 7 days, compared to control. In GLUT1D mice without exercise, only KE resulted in significantly lower glucose levels at week 2 and week 6 during a 10 weeks long chronic feeding study. In 4-month and 1-year-old SPD rats in the post-exercise trials, blood glucose was significantly lower in KD and KE, and in KEMCT groups, respectively. After seven days, the KSMCT group had the most significantly reduced blood glucose levels, compared to control. These results indicate that exogenous ketones were efficacious in reducing blood glucose levels within and outside the context of exercise in various rodent models of different ages, with and without pathology.

Beta-hydroxybutyrate infusion identifies acutely differentially expressed genes related to metabolism and reproduction in the hypothalamus and pituitary of castrated male sheep.

To identify molecular pathways that couple metabolic imbalances and reproduction, we randomly assigned 10 castrated male sheep to be centrally injected into the lateral ventricle through intracerebroventricular cannulas with 1 ml of ß-hydroxybutyric acid sodium salt solution (BHB; 12,800 µmol/l) or saline solution (CON; 0.9% NaCl). Approximately 2 h postinjection, sheep were humanely euthanized, and hypothalamus and pituitary tissues were harvested for transcriptome characterization by RNA sequencing. RNA was extracted from the hypothalamus and pituitary and sequenced at a high depth (hypothalamus: 468,912,732 reads; pituitary: 515,106,092 reads) with the Illumina Hi-Seq 2500 platform and aligned to Bos taurus and Ovis aries genomes. Of the total raw reads, 87% (hypothalamus) and 90.5% (pituitary) mapped to the reference O. aries genome. Within these read sets, ~56% in hypothalamus and 69% in pituitary mapped to either known or putative protein coding genes. Fragments per kilobase of transcripts per million normalized counts were averaged and ranked to identify the transcript expression level. Gene Ontology analysis (DAVID Bioinformatics Resources) was utilized to identify biological process functions related to genes shared between tissues, as well as functional categories with tissue-specific enrichment. Between CON- and BHB-treated sheep, 11 and 44 genes were differentially expressed (adj. P < 0.05) within the pituitary and hypothalamus, respectively. Functional enrichment analyses revealed BHB altered expression of genes in pathways related to stimulus perception, inflammation, and cell cycle control. The set of genes altered by BHB creates a foundation from which to identify the signaling pathways that impact reproduction during metabolic imbalances.

ß-Hydroxybutyrate Boosts Mitochondrial and Neuronal Metabolism but is not Preferred Over Glucose Under Activated Conditions.

The ketone body, ß-hydroxybutyrate (ßOHB), is metabolised by the brain alongside the mandatory brain fuel glucose. To examine the extent and circumstances by which ßOHB can supplement glucose metabolism, we studied guinea pig cortical brain slices using increasing concentrations of [U-13C]D-ßOHB in conjunction with [1-13C]D-glucose under conditions of normo- and hypoglycaemia, as well as under high potassium (40 mmol/L K+) depolarization in normo- and hypoglycaemic conditions. The contribution of ßOHB to synthesis of GABA was also probed by inhibiting the synthesis of glutamine, a GABA precursor, with methionine sulfoximine (MSO). [U-13C]D-ßOHB at lower concentrations (0.25 and 1.25 mmol/L) stimulated mitochondrial metabolism, producing greater total incorporation of label into glutamate and GABA but did not have a similar effect in the cytosolic compartment where labelling of glutamine was reduced at 1.25 mmol/L [U-13C]D-ßOHB. At higher concentrations (2.5 mmol/L) [U-13C]D-ßOHB inhibited metabolism of [1-13C]D-glucose, and reduced total label incorporation and total metabolite pools. When glucose levels were reduced, ßOHB was able to partially restore the loss of glutamate and GABA caused by hypoglycaemia, but was not able to supplement levels of lactate, glutamine or alanine or to prevent the increase in aspartate. Under depolarizing conditions glucose was the preferred substrate over ßOHB, even in hypoglycaemic conditions where comparatively less ßOHB was incorporated except into aspartate isotopomers. Inhibition of glutamine synthesis with MSO had no significant effect on incorporation of label from [U-13C]D-ßOHB into GABA C2,1 indicating that the majority of this GABA was synthesized in GABAergic neurons from [U-13C]D-ßOHB rather than from Gln C4,5 imported from astrocytes.

Hypothalamic sensing of ketone bodies after prolonged cerebral exposure leads to metabolic control dysregulation.

Ketone bodies have been shown to transiently stimulate food intake and modify energy homeostasis regulatory systems following cerebral infusion for a moderate period of time (<6 hours). As ketone bodies are usually enhanced during episodes of fasting, this effect might correspond to a physiological regulation. In contrast, ketone bodies levels remain elevated for prolonged periods during obesity, and thus could play an important role in the development of this pathology. In order to understand this transition, ketone bodies were infused through a catheter inserted in the carotid to directly stimulate the brain for a period of 24 hours. Food ingested and blood circulating parameters involved in metabolic control as well as glucose homeostasis were determined. Results show that ketone bodies infusion for 24 hours increased food intake associated with a stimulation of hypothalamic orexigenic neuropeptides. Moreover, insulinemia was increased and caused a decrease in glucose production despite an increased resistance to insulin. The present study confirms that ketone bodies reaching the brain stimulates food intake. Moreover, we provide evidence that a prolonged hyperketonemia leads to a dysregulation of energy homeostasis control mechanisms. Finally, this study shows that brain exposure to ketone bodies alters insulin signaling and consequently glucose homeostasis.

ß-Hydroxybutyric acid inhibits growth hormone-releasing hormone synthesis and secretion through the GPR109A/extracellular signal-regulated 1/2 signalling pathway in the hypothalamus.

ß-Hydroxybutyric acid (BHBA) has recently been shown to regulate hormone synthesis and secretion in the hypothalamus. However, little is known about the effects of BHBA-mediated hormone regulation or the detailed mechanisms by which BHBA regulates growth hormone-releasing hormone (GHRH) synthesis and secretion. In the present study, we examined the expression of the BHBA receptor GPR109A in primary hypothalamic cell cultures. We hypothesised that BHBA regulates GHRH via GPR109A and its downstream signals. Initial in vivo studies conducted in rats demonstrated that GHRH mRNA expression in the hypothalamus was strongly inversely correlated with BHBA levels in the cerebrospinal fluid during postnatal development (r = -0.89, P < 0.01). Furthermore, i.c.v. administration of BHBA acutely decreased GHRH mRNA expression in rats. Further in vitro studies revealed a decrease in GHRH synthesis and secretion in primary hypothalamic cells after treatment with BHBA; this effect was inhibited when hypothalamic cells were pretreated with pertussis toxin (PTX). BHBA had no effect on GHRH synthesis and secretion in GT1-7 cells, which do not exhibit cell surface expression of GPR109A. Furthermore, BHBA acutely decreased the transcription of the homeobox gene for Gsh-1 in the hypothalamus in both in vivo and in vitro, and this effect was also inhibited by PTX in vitro. In primary hypothalamic cells, BHBA activated the extracellular signal-regulated kinase (ERK)1/2, p38 and c-Jun N-terminal kinase mitogen-activated protein kinase (MAPK) kinases, as shown by western blot analysis. Moreover, inhibition of ERK1/2 with U0126 attenuated the BHBA-mediated reduction in Gsh-1 expression and GHRH synthesis and secretion. These results strongly suggest that BHBA directly regulates GHRH synthesis and secretion via the GPR109A/ERK1/2 MAPK pathway, and also that Gsh-1 is essential for this function.

Compared with stearic acid, palmitic acid increased the yield of milk fat and improved feed efficiency across production level of cows.

The effects of dietary palmitic and stearic acids on feed intake, yields of milk and milk components, and feed efficiency of dairy cows were evaluated in an experiment with a crossover arrangement of treatments with a covariate period. Cows with a wide range of milk production (38 to 65 kg/d) were used to determine if response to fat supplementation varied according to production level. Thirty-two Holstein cows (143 ± 61 d in milk) were assigned randomly to a treatment sequence within level of milk production. Treatments were diets supplemented (2% of diet dry matter) with palmitic acid (PA; 97.9% C16:0) or stearic acid (SA; 97.4% C18:0). Treatment periods were 21 d and cows were fed a nonfat supplemented diet for 14 d immediately before the first treatment period. The final 4d of each period were used for sample and data collection. Milk production measured during the covariate period (preliminary milk yield) was used as the covariate. No interactions were detected between treatment and preliminary milk yield for the production response variables measured. Compared with SA, the PA treatment increased milk fat concentration (3.66 vs. 3.55%) and yield (1.68 vs. 1.59 kg/d), and 3.5% fat-corrected milk yield (47.5 vs. 45.6 kg/d). Treatment did not affect dry matter intake, milk yield, milk protein yield, body weight, or body condition score. Milk protein concentration was lower for PA compared with SA treatment (3.24 vs. 3.29%). The PA treatment increased feed efficiency (3.5% fat-corrected milk yield/dry matter intake) compared with SA (1.48 vs. 1.40). The increase in milk fat yield by PA was entirely accounted for by a 24% increase in 16-carbon fatty acid output into milk. Yields of de novo (3.2%) and preformed fatty acids (2.9%) were only slightly decreased by PA relative to SA. The PA treatment increased plasma concentration of nonesterified fatty acids (96.3 vs. 88.2 µEq/L) and glucose (56.6 vs. 55.7 mg/dL) compared with SA, but insulin and ß-hydroxybutyrate were not altered by the treatments. Results demonstrate that palmitic acid is more effective than stearic acid in improving milk fat concentration and yield as well as efficiency of feed conversion to milk. Responses were independent of production level and without changes in body condition score or body weight. Further studies are required to test the consistency of these responses across different types of diets.

ß-Hydroxybutyrate modulates N-type calcium channels in rat sympathetic neurons by acting as an agonist for the G-protein-coupled receptor FFA3.

Free fatty acids receptor 3 (FFA3, GPR41) and 2 (FFA2, GPR43), for which the short-chain fatty acids (SCFAs) acetate and propionate are agonist, have emerged as important G-protein-coupled receptors influenced by diet and gut flora composition. A recent study (Kimura et al., 2011) demonstrated functional expression of FFA3 in the rodent sympathetic nervous system (SNS) providing a potential link between nutritional status and autonomic function. However, little is known of the source of endogenous ligands, signaling pathways, or effectors in sympathetic neurons. In this study, we found that FFA3 and FFA2 are unevenly expressed in the rat SNS with higher transcript levels in prevertebral (e.g., celiac-superior mesenteric and major pelvic) versus paravertebral (e.g., superior cervical and stellate) ganglia. FFA3, whether heterologously or natively expressed, coupled via PTX-sensitive G-proteins to produce voltage-dependent inhibition of N-type Ca(2+) channels (Cav2.2) in sympathetic neurons. In addition to acetate and propionate, we show that ß-hydroxybutyrate (BHB), a metabolite produced during ketogenic conditions, is also an FFA3 agonist. This contrasts with previous interpretations of BHB as an antagonist at FFA3. Together, these results indicate that endogenous BHB levels, especially when elevated under certain conditions, such as starvation, diabetic ketoacidosis, and ketogenic diets, play a potentially important role in regulating the activity of the SNS through FFA3.

The ketone body ß-hydroxybutyric acid influences agouti-related peptide expression via AMP-activated protein kinase in hypothalamic GT1-7 cells.

ß-Hydroxybutyric acid (BHBA) acts in the brain to influence feeding behaviour, but the underlying molecular mechanisms are unclear. GT1-7 hypothalamic cells expressing orexigenic agouti-related peptide (AGRP) were used to study the AMP-activated protein kinase (AMPK) pathway known to integrate dietary and hormonal signals for food intake regulation. In a 25 mM glucose culture medium, BHBA increased intracellular calcium concentrations and the expression of monocarboxylate transporter 1 (MCT1 (SLC16A1)). Phosphorylation of AMPK-α (PRKAA1 and PRKAA2) at Thr(172) was diminished after 2 h but increased after 4 h. Its downstream target, the mammalian target of rapamycin, was increasingly phosphorylated on Ser(2448) after 2 h but not changed after 4 h of BHBA treatment. After 4 h, BHBA treatment also increased Agrp mRNA expression. This increase was prevented by preincubation with the AMPK inhibitor Compound C. The inhibition of MCT1 activity by p-hydroxymercuribenzoate suppressed BHBA-stimulated AMPK phosphorylation but did not prevent BHBA-induced Agrp mRNA expression. This finding demonstrates that BHBA triggers the AMPK pathway resulting in orexigenic signalling under 25 mM glucose culture conditions. Under conditions of 5.5 mM glucose, however, BHBA marginally increased intracellular calcium but significantly decreased AMPK phosphorylation and Agrp mRNA expression, demonstrating that under physiological conditions BHBA reduces central orexigenic signalling.

Mitochondrial biogenesis and increased uncoupling protein 1 in brown adipose tissue of mice fed a ketone ester diet.

We measured the effects of a diet in which D-ß-hydroxybutyrate-(R)-1,3 butanediol monoester [ketone ester (KE)] replaced equicaloric amounts of carbohydrate on 8-wk-old male C57BL/6J mice. Diets contained equal amounts of fat, protein, and micronutrients. The KE group was fed ad libitum, whereas the control (Ctrl) mice were pair-fed to the KE group. Blood d-ß-hydroxybutyrate levels in the KE group were 3-5 times those reported with high-fat ketogenic diets. Voluntary food intake was reduced dose dependently with the KE diet. Feeding the KE diet for up to 1 mo increased the number of mitochondria and doubled the electron transport chain proteins, uncoupling protein 1, and mitochondrial biogenesis-regulating proteins in the interscapular brown adipose tissue (IBAT). [(18)F]-Fluorodeoxyglucose uptake in IBAT of the KE group was twice that in IBAT of the Ctrl group. Plasma leptin levels of the KE group were more than 2-fold those of the Ctrl group and were associated with increased sympathetic nervous system activity to IBAT. The KE group exhibited 14% greater resting energy expenditure, but the total energy expenditure measured over a 24-h period or body weights was not different. The quantitative insulin-sensitivity check index was 73% higher in the KE group. These results identify KE as a potential antiobesity supplement.

Hyperketonemia decreases mitochondrial membrane potential and its normalization with chromium (III) supplementation in monocytes.

Altered cellular mitochondrial membrane potential (MMP) has been implicated in the increased insulin resistance and the risk for diabetes. Hyperketonemia is increasingly being identified in type 2 diabetic patients in addition to those with type 1 diabetes. No previous study has examined the effect of hyperketonemia and trivalent chromium on cellular mitochondrial membrane potential (MMP) in any cell type. Using a U937 monocyte cell culture model, this study examined the hypothesis that hyperketonemia decreases and trivalent chromium normalizes the cellular MMP level. Cells were cultured with control and ketone bodies [acetoacetate (AA), ß-hydroxybutyrate (BHB)] in the absence or the presence (0.5-100 µM) of Cr(3+) at 37°C for 24 h. The MMP was determined using DiOC6 and flow cytometry. The results show a significant decrease in MMP in cells treated with AA, but not in the cells treated with BHB. The effect of AA on cellular MMP was prevented in chromium (III)-pretreated cells. Thus, hyperketonemia decreases the MMP, and supplementation with chromium (III) normalizes altered MMP, which may play a role in the improvement in glucose metabolism seen after chromium (III) supplementation in some studies with diabetic animals and patients.