Hormonal effects on glucose and ketone metabolism in a perfused liver of an elasmobranch, the North Pacific spiny dogfish, Squalus suckleyi.

Hormonal influence on hepatic function is a critical aspect of whole-body energy balance in vertebrates. Catecholamines and corticosteroids both influence hepatic energy balance via metabolite mobilization through glycogenolysis and gluconeogenesis. Elasmobranchs have a metabolic organization that appears to prioritize the mobilization of hepatic lipid as ketone bodies (e.g. 3-hydroxybutyrate [3-HB]), which adds complexity in determining the hormonal impact on hepatic energy balance in this taxon. Here, a liver perfusion was used to investigate catecholamine (epinephrine [E]) and corticosteroid (corticosterone [B] and 11-deoxycorticosterone [DOC]) effects on the regulation of hepatic glucose and 3-HB balance in the North Pacific Spiny dogfish, Squalus suckleyi. Further, hepatic enzyme activity involved in ketogenesis (3-hydroxybutyrate dehydrogenase), glycogenolysis (glycogen phosphorylase), and gluconeogenesis (phosphoenolpyruvate carboxykinase) were assessed in perfused liver tissue following hormonal application to discern effects on hepatic energy flux. mRNA transcript abundance key transporters of glucose (glut1 and glut4) and ketones (mct1 and mct2) and glucocorticoid function (gr, pepck, fkbp5, and 11ßhsd2) were also measured to investigate putative cellular components involved in hepatic responses. There were no changes in the arterial-venous difference of either metabolite in all hormone perfusions. However, perfusion with DOC increased gr transcript abundance and decreased flow rate of perfusions, suggesting a regulatory role for this corticosteroid. Phosphoenolpyruvate carboxykinase activity increased following all hormone treatments, which may suggest gluconeogenic function; E also increased 3-hydroxybutyrate dehydrogenase activity, suggesting a function in ketogenesis, and decreased pepck and fkbp5 transcript abundance, potentially showing some metabolic regulation. Overall, we demonstrate hormonal control of hepatic energy balance using liver perfusions at various levels of biological organization in an elasmobranch.

Effects of poly(3-hydroxybutyrate) [P(3HB)] coating on the bacterial communities of artificial structures.

The expanding urbanization of coastal areas has led to increased ocean sprawl, which has had both physical and chemical adverse effects on marine and coastal ecosystems. To maintain the health and functionality of these ecosystems, it is imperative to develop effective solutions. One such solution involves the use of biodegradable polymers as bioactive coatings to enhance the bioreceptivity of marine and coastal infrastructures. Our study aimed to explore two main objectives: (1) investigate PHA-degrading bacteria on polymer-coated surfaces and in surrounding seawater, and (2) comparing biofilm colonization between surfaces with and without the polymer coating. We applied poly(3-hydroxybutyrate) [P(3HB)) coatings on concrete surfaces at concentrations of 1% and 6% w/v, with varying numbers of coating cycles (1, 3, and 6). Our findings revealed that the addition of P(3HB) indeed promoted accelerated biofilm growth on the coated surfaces, resulting in an occupied area approximately 50% to 100% larger than that observed in the negative control. This indicates a remarkable enhancement, with the biofilm expanding at a rate roughly 1.5 to 2 times faster than the untreated surfaces. We observed noteworthy distinctions in biofilm growth patterns based on varying concentration and number of coating cycles. Interestingly, treatments with low concentration and high coating cycles exhibited comparable biofilm enhancements to those with high concentrations and low coating cycles. Further investigation into the bacterial communities responsible for the degradation of P(3HB) coatings identified mostly common and widespread strains but found no relation between the concentration and coating cycles. Nevertheless, this microbial degradation process was found to be highly efficient, manifesting noticeable effects within a single month. While these initial findings are promising, it's essential to conduct tests under natural conditions to validate the applicability of this approach. Nonetheless, our study represents a novel and bio-based ecological engineering strategy for enhancing the bioreceptivity of marine and coastal structures.

Microbially synthesized poly(hydroxybutyrate-co-hydroxyhexanoate) with low to moderate hydroxyhexanoate content: Properties and applications.

Plastic pollution is the biggest environmental concern of our time. Breakdown products like micro- and nano-plastics inevitably enter the food chain and pose unprecedented health risks. In this scenario, bio-based and biodegradable plastic alternatives have been given a momentum aiming to bridge a transition towards a more sustainable future. Polyhydroxyalkanoates (PHAs) are one of the few thermoplastic polymers synthesized 100 % via biotechnological routes which fully biodegrade in common natural environments. Poly(hydroxybutyrate-co-hydroxyhexanoate) [P(HB-co-HHx)] is a PHA copolymer with great potential for the commodity polymers industry, as its mechanical properties can be tailored through fine-tuning of its molar HHx content. We have recently developed a strategy that enables for reliable tailoring of the monomer content of P(HB-co-HHx). Nevertheless, there is often a lack of comprehensive investigation of the material properties of PHAs to evaluate whether they actually mimic the functionalities of conventional plastics. We present a detailed study of P(HB-co-HHx) copolymers with low to moderate hydroxyhexanoate content to understand how the HHx monomer content influences the thermal and mechanical properties and to link those to their abiotic degradation. By increasing the HHx fractions in the range of 2 - 14 mol%, we impart an extension of the processing window and application range as the melting temperature (Tm) and glass temperature (Tg) of the copolymers decrease from Tm 165 °C to 126 °C, Tg 4 °C to -5.9 °C, accompanied by reduced crystallinity from 54 % to 20 %. Elongation at break was increased from 5.7 % up to 703 % at 14 mol% HHx content, confirming that the range examined was sufficiently large to obtain ductile and brittle copolymers, while tensile strength was maintained throughout the studied range. Finally, accelerated abiotic degradation was shown to be slowed down with an increasing HHx fraction decreasing from 70 % to 55 % in 12 h.

Exercise-induced ß-hydroxybutyrate promotes Treg cell differentiation to ameliorate colitis in mice.

Increasing attention is being paid to the mechanistic investigation of exercise-associated chronic inflammatory disease improvement. Ulcerative colitis (UC) is one type of chronic inflammatory bowel disease with increasing incidence and prevalence worldwide. It is known that regular moderate aerobic exercise (RMAE) reduces the incidence or risk of UC, and attenuates disease progression in UC patients. However, the mechanisms of this RMAE's benefit are still under investigation. Here, we revealed that ß-hydroxybutyrate (ß-HB), a metabolite upon prolonged aerobic exercise, could contribute to RMAE preconditioning in retarding dextran sulfate sodium (DSS)-induced mouse colitis. When blocking ß-HB production, RMAE preconditioning-induced colitis amelioration was compromised, whereas supplementation of ß-HB significantly rescued impaired ß-HB production-associated defects. Meanwhile, we found that RMAE preconditioning significantly caused decreased colonic Th17/Treg ratio, which is considered to be important for colitis mitigation; and the downregulated Th17/Treg ratio was associated with ß-HB. We further demonstrated that ß-HB can directly promote the differentiation of Treg cell rather than inhibit Th17 cell generation. Furthermore, ß-HB increased forkhead box protein P3 (Foxp3) expression, the core transcriptional factor for Treg cell, by enhancing histone H3 acetylation in the promoter and conserved noncoding sequences of the Foxp3 locus. In addition, fatty acid oxidation, the key metabolic pathway required for Treg cell differentiation, was enhanced by ß-HB treatment. Lastly, administration of ß-HB without exercise significantly boosted colonic Treg cell and alleviated colitis in mice. Together, we unveiled a previously unappreciated role for exercise metabolite ß-HB in the promotion of Treg cell generation and RMAE preconditioning-associated colitis attenuation.

Relationship between serum ß-hydroxybutyrate and hepatic fatty acid oxidation in individuals with obesity and NAFLD.

Nonalcoholic fatty liver disease (NAFLD) is characterized by excess lipid accumulation that can progress to inflammation (nonalcoholic steatohepatitis, NASH), and fibrosis. Serum ß-hydroxybutyrate (ß-HB), a product of the ketogenic pathway, is commonly used as a surrogate marker for hepatic fatty acid oxidation (FAO). However, it remains uncertain whether this relationship holds true in the context of NAFLD in humans. We compared fasting serum ß-HB levels with direct measurement of liver mitochondrial palmitate oxidation in humans stratified based on NAFLD severity (n = 142). Patients were stratified based on NAFLD activity score (NAS): NAS = 0 (no disease), NAS = 1-2 (mild), NAS = 3-4 (moderate), and NAS ≥ 5 (advanced). Moderate and advanced NAFLD is associated with reductions in liver 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2), serum ß-HB, but not 3-hydroxy-3-methylglutaryl-CoA lyase (HMGCL) mRNA, relative to no disease. Worsening liver mitochondrial complete palmitate oxidation corresponded with lower HMGCS2 mRNA but not total (complete + incomplete) palmitate oxidation. Interestingly, we found that liver HMGCS2 mRNA and serum ß-HB correlated with liver mitochondrial ß-hydroxyacyl-CoA dehydrogenase (ß-HAD) activity and CPT1A mRNA. Also, lower mitochondrial mass and markers of mitochondrial turnover positively correlated with lower HMGCS2 in the liver. These data suggest that liver ketogenesis and FAO occur at comparable rates in individuals with NAFLD. Our findings support the utility of serum ß-HB to serve as a marker of liver injury and hepatic FAO in the context of NAFLD.NEW & NOTEWORTHY Serum ß-hydroxybutyrate (ß-HB) is frequently utilized as a surrogate marker for hepatic fatty acid oxidation; however, few studies have investigated this relationship during states of liver disease. We found that the progression of nonalcoholic fatty liver disease (NAFLD) is associated with reductions in circulating ß-HB and liver 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2). As well, decreased rates of hepatic fatty acid oxidation correlated with liver HMGCS2 mRNA and serum ß-HB. Our work supports serum ß-HB as a potential marker for hepatic fatty acid oxidation and liver injury during NAFLD.

The therapeutic potential of ketones in cardiometabolic disease: impact on heart and skeletal muscle.

ß-Hydroxybutyrate (ßOHB) is the major ketone in the body, and it is recognized as a metabolic energy source and an important signaling molecule. While ketone oxidation is essential in the brain during prolonged fasting/starvation, other organs such as skeletal muscle and the heart also use ketones as metabolic substrates. Additionally, ßOHB-mediated molecular signaling events occur in heart and skeletal muscle cells, and via metabolism and/or signaling, ketones may contribute to optimal skeletal muscle health and cardiac function. Of importance, when the use of ketones for ATP production and/or as signaling molecules becomes disturbed in the presence of underlying obesity, type 2 diabetes, and/or cardiovascular diseases, these changes may contribute to cardiometabolic disease. As a result of these disturbances in cardiometabolic disease, multiple approaches have been used to elevate circulating ketones with the goal of optimizing either ketone metabolism or ketone-mediated signaling. These approaches have produced significant improvements in heart and skeletal muscle during cardiometabolic disease with a wide range of benefits that include improved metabolism, weight loss, better glycemic control, improved cardiac and vascular function, as well as reduced inflammation and oxidative stress. Herein, we present the evidence that indicates that ketone therapy could be used as an approach to help treat cardiometabolic diseases by targeting cardiac and skeletal muscles.

Pre-treatment with ß-hydroxybutyrate mitigates cisplatin-induced acute kidney injury.

ß-Hydroxybutyrate (ß-HB), the primary circulating ketone body, plays a dual role as both a metabolic fuel and an endogenous signaling molecule, offering diverse systemic benefits. Recent studies have highlighted the renoprotective effects of exogenous ß-HB therapy in various animal models of kidney disease. In this investigation, our goal was to assess whether pre-treatment with exogenous ß-HB could alleviate kidney damage in a mouse model of cisplatin-induced acute kidney injury (AKI). Prior to cisplatin administration, intraperitoneal administration of ß-HB was carried out, and the groups were classified into four: Sham, ß-HB, cisplatin, and ß-HB + cisplatin. The tubular damage score and serum creatinine levels were significantly lower in the ß-HB + cisplatin group compared to the cisplatin group. Furthermore, the expression of phosphorylated NF-κB, inflammatory cytokines, and the quantity of F4/80-positive macrophages in the ß-HB + cisplatin group were reduced compared to those in the cisplatin group. Additionally, oxidative stress markers for DNA, protein, and lipid in the ß-HB + cisplatin group were markedly diminished compared to those in the cisplatin group. The number of TUNEL-positive and cleaved caspase 3-positive tubular cells in the ß-HB + cisplatin group was lower than in the cisplatin group. Pre-treating with exogenous ß-HB effectively mitigated kidney damage by suppressing inflammation, oxidative stress, and tubular apoptosis in cisplatin-induced AKI. Therefore, exogenous ß-HB as a pre-treatment emerges as a promising and novel strategy for preventing cisplatin-induced AKI.

Production of poly(3-hydroxybutyrate)/poly(lactic acid) from industrial wastewater by wild-type Cupriavidus necator H16.

The massive production of urban and industrial wastes has created a clear need for alternative waste management processes. One of the more promising strategies is to use waste as raw material for the production of biopolymers such as polyhydroxyalkanoates (PHAs). In this work, a lactate-enriched stream obtained by anaerobic digestion (AD) of wastewater (WW) from a candy production plant was used as a feedstock for PHA production in wild-type Cupriavidus necator H16. Unexpectedly, we observed the accumulation of poly(3-hydroxybutyrate)/poly(lactic acid) (P(3HB)/PLA), suggesting that the non-engineered strain already possesses the metabolic potential to produce these polymers of interest. The systematic study of factors, such as incubation time, nitrogen and lactate concentration, influencing the synthesis of P(3HB)/PLA allowed the production of a panel of polymers in a resting cell system with tailored lactic acid (LA) content according to the GC-MS of the biomass. Further biomass extraction suggested the presence of methanol soluble low molecular weight molecules containing LA, while 1 % LA could be detected in the purified polymer fraction. These results suggested that the cells are producing a blend of polymers. A proteomic analysis of C. necator resting cells under P(3HB)/PLA production conditions provides new insights into the latent pathways involved in this process. This study is a proof of concept demonstrating that LA can polymerize in a non-modified organism and paves the way for new metabolic engineering approaches for lactic acid polymer production in the model bacterium C. necator H16.

ß-hydroxybutyrate administered at reperfusion reduces infarct size and preserves cardiac function by improving mitochondrial function through autophagy in male mice.

Ischemia/reperfusion (I/R) injury after revascularization contributes ∼50% of infarct size and causes heart failure, for which no established clinical treatment exists. ß-hydroxybutyrate (ß-OHB), which serves as both an energy source and a signaling molecule, has recently been reported to be cardioprotective when administered immediately before I/R and continuously after reperfusion. This study aims to determine whether administering ß-OHB at the time of reperfusion with a single dose can alleviate I/R injury and, if so, to define the mechanisms involved. We found plasma ß-OHB levels were elevated during ischemia in STEMI patients, albeit not to myocardial protection level, and decreased after revascularization. In mice, compared with normal saline, ß-OHB administrated at reperfusion reduced infarct size (by 50%) and preserved cardiac function, as well as activated autophagy and preserved mtDNA levels in the border zone. Our treatment with one dose ß-OHB reached a level achievable with fasting and strenuous physical activity. In neonatal rat ventricular myocytes (NRVMs) subjected to I/R, ß-OHB at physiologic level reduced cell death, increased autophagy, preserved mitochondrial mass, function, and membrane potential, in addition to attenuating reactive oxygen species (ROS) levels. ATG7 knockdown/knockout abolished the protective effects of ß-OHB observed both in vitro and in vivo. Mechanistically, ß-OHB's cardioprotective effects were associated with inhibition of mTOR signaling. In conclusion, ß-OHB, when administered at reperfusion, reduces infarct size and maintains mitochondrial homeostasis by increasing autophagic flux (potentially through mTOR inhibition). Since ß-OHB has been safely tested in heart failure patients, it may be a viable therapeutic to reduce infarct size in STEMI patients.

Enhancing poly(3-hydroxybutyrate) production in halophilic bacteria through improved salt tolerance.

Polyhydroxyalkanoates (PHA) have emerged as a promising bio-compound in the industrial application due to their potential to replace conventional petroleum-based plastics with sustainable bioplastics. This study focuses on Halomonas sp. YJPS3-3, a halophilic bacterium, and presents a novel approach to enhance PHA production by exploiting its salt tolerance toward PHA biosynthesis. Through gamma irradiation-induced mutants with enhanced salt tolerance from 15% NaCl to 20% NaCl, mutant halo6 showing a significant 11% increase in PHA yield, was achieved. Moreover, the mutants displayed not only higher PHA content but also remarkable cell morphology with elongation. In addition, this research unravels the genetic determinants behind the elevated PHA content and identifies a corresponding shift in fatty acid composition favoring PHA accumulation. This novel mutant obtained from gamma irradiation with enhanced salt tolerance in halophilic bacteria opens up new avenues not only for the bioplastic industry but also for applications in the production of high-value metabolites.

Metabolic engineering of Halomonas bluephagenesis for production of five carbon molecular chemicals derived from L-lysine.

5-Aminovaleric acid (5-AVA), 5-hydroxyvalerate (5HV), copolymer P(3HB-co-5HV) of 3-hydroxybutyrate (3HB) and 5HV were produced from L-lysine as a substrate by recombinant Halomonas bluephagenesis constructed based on codon optimization, deletions of competitive pathway and L-lysine export protein, and three copies of davBA genes encoding L-lysine monooxygenase (DavB) and 5-aminovaleramide amidohydrolase (DavA) inserted into its genome to form H. bluephagenesis YF117ΔgabT1+2, which produced 16.4 g L-1 and 67.4 g L-1 5-AVA in flask cultures and in 7 L bioreactor, respectively. It was able to de novo synthesize 5-AVA from glucose by L-lysine-overproducing H. bluephagenesis TD226. Corn steep liquor was used instead of yeast extract for cost reduction during the 5-AVA production. Using promoter engineering based on Pporin mutant library for downstream genes, H. bluephagenesis YF117 harboring pSEVA341-Pporin42-yqhDEC produced 6 g L-1 5HV in shake flask growth, while H. bluephagenesis YF117 harboring pSEVA341-Pporin42-yqhDEC-Pporin278-phaCRE-abfT synthesized 42 wt% P(3HB-co-4.8 mol% 5HV) under the same condition. Thus, H. bluephagenesis was successfully engineered to produce 5-AVA and 5HV in supernatant and intracellular P(3HB-co-5HV) utilizing L-lysine as the substrate.

Metabolic Messengers: ketone bodies.

Prospective molecular targets and therapeutic applications for ketone body metabolism have increased exponentially in the past decade. Initially considered to be restricted in scope as liver-derived alternative fuel sources during periods of carbohydrate restriction or as toxic mediators during diabetic ketotic states, ketogenesis and ketone bodies modulate cellular homeostasis in multiple physiological states through a diversity of mechanisms. Selective signalling functions also complement the metabolic fates of the ketone bodies acetoacetate and D-ß-hydroxybutyrate. Here we discuss recent discoveries revealing the pleiotropic roles of ketone bodies, their endogenous sourcing, signalling mechanisms and impact on target organs, and considerations for when they are either stimulated for endogenous production by diets or pharmacological agents or administered as exogenous wellness-promoting agents.

The PNPLA3 I148M variant increases ketogenesis and decreases hepatic de novo lipogenesis and mitochondrial function in humans.

The PNPLA3 I148M variant is the major genetic risk factor for all stages of fatty liver disease, but the underlying pathophysiology remains unclear. We studied the effect of this variant on hepatic metabolism in homozygous carriers and non-carriers under multiple physiological conditions with state-of-the-art stable isotope techniques. After an overnight fast, carriers had higher plasma ß-hydroxybutyrate concentrations and lower hepatic de novo lipogenesis (DNL) compared to non-carriers. After a mixed meal, fatty acids were channeled toward ketogenesis in carriers, which was associated with an increase in hepatic mitochondrial redox state. During a ketogenic diet, carriers manifested increased rates of intrahepatic lipolysis, increased plasma ß-hydroxybutyrate concentrations, and decreased rates of hepatic mitochondrial citrate synthase flux. These studies demonstrate that homozygous PNPLA3 I148M carriers have hepatic mitochondrial dysfunction leading to reduced DNL and channeling of carbons to ketogenesis. These findings have implications for understanding why the PNPLA3 variant predisposes to progressive liver disease.

Ketosis Suppression and Ageing (KetoSAge): The Effects of Suppressing Ketosis in Long Term Keto-Adapted Non-Athletic Females.

Most studies on ketosis have focused on short-term effects, male athletes, or weight loss. Hereby, we studied the effects of short-term ketosis suppression in healthy women on long-standing ketosis. Ten lean (BMI 20.5 ± 1.4), metabolically healthy, pre-menopausal women (age 32.3 ± 8.9) maintaining nutritional ketosis (NK) for > 1 year (3.9 years ± 2.3) underwent three 21-day phases: nutritional ketosis (NK; P1), suppressed ketosis (SuK; P2), and returned to NK (P3). Adherence to each phase was confirmed with daily capillary D-beta-hydroxybutyrate (BHB) tests (P1 = 1.9 ± 0.7; P2 = 0.1 ± 0.1; and P3 = 1.9 ± 0.6 pmol/L). Ageing biomarkers and anthropometrics were evaluated at the end of each phase. Ketosis suppression significantly increased: insulin, 1.78-fold from 33.60 (± 8.63) to 59.80 (± 14.69) pmol/L (p = 0.0002); IGF1, 1.83-fold from 149.30 (± 32.96) to 273.40 (± 85.66) µg/L (p = 0.0045); glucose, 1.17-fold from 78.6 (± 9.5) to 92.2 (± 10.6) mg/dL (p = 0.0088); respiratory quotient (RQ), 1.09-fold 0.66 (± 0.05) to 0.72 (± 0.06; p = 0.0427); and PAI-1, 13.34 (± 6.85) to 16.69 (± 6.26) ng/mL (p = 0.0428). VEGF, EGF, and monocyte chemotactic protein also significantly increased, indicating a pro-inflammatory shift. Sustained ketosis showed no adverse health effects, and may mitigate hyperinsulinemia without impairing metabolic flexibility in metabolically healthy women.

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.

Biotransformation of starch-based wastewater into bioplastics: Optimization of poly(3-hydroxybutyrate) production by Cupriavidus necator DSM 545 using potato wastewater hydrolysate.

Biodegradable biopolymers, such as polyhydroxyalkanoates (PHAs), have emerged as an alternative to petrochemical-based plastics. The present work explores the production of PHAs based on the biotransformation of potato processing wastewater and addresses two different strategies for PHA recovery. To this end, culture conditions for PHA synthesis by Cupriavidus necator DSM 545 were optimized on a laboratory scale using a response surface methodology-based experimental design. Optimal conditions rendered a PHB, poly(3-hydroxybutyrate), accumulation of 83.74 ± 2.37 % (5.1 ± 0.2 gL-1), a 1.4-fold increase compared to the initial conditions. Moreover, polymer extraction with non-halogenated agent improved PHB recovery compared to chloroform method (PHB yield up to 78.78 ± 0.57 %), while maintaining PHB purity. (99.83 ± 4.95 %). Overall, the present work demonstrated the potential valorization of starch-based wastewater by biotransformation into PHBs, a high value-added product, and showed that recovery approaches more eco-friendly than the traditional treatments could be applied to PHB recovery to some extent.

Ketogenic propensity is differentially related to lipid-induced hepatic and peripheral insulin resistance.

AIM: Determine the ketogenic response (ß-hydroxybutyrate, a surrogate of hepatic ketogenesis) to a controlled lipid overload in humans. METHODS: In total, nineteen young, healthy adults (age: 28.4 ± 1.7 years; BMI: 22.7 ± 0.3 kg/m2 ) received either a 12 h overnight lipid infusion or saline in a randomized, crossover design. Plasma ketones and inflammatory markers were quantified by colorimetric and multiplex assays. Hepatic and peripheral insulin sensitivity was assessed by the hyperinsulinemic-euglycemic clamp. Skeletal muscle biopsies were obtained to quantify gene expression related to ketone body metabolism and inflammation. RESULTS: By design, the lipid overload-induced hepatic (50%, p < 0.001) and peripheral insulin resistance (73%, p < 0.01) in healthy adults. Ketones increased with hyperlipidemia and were subsequently reduced with hyperinsulinemia during the clamp procedure (Saline: Basal = 0.22 mM, Insulin = 0.07 mM; Lipid: Basal = 0.78 mM, Insulin = 0.51 mM; 2-way ANOVA: Lipid p < 0.001, Insulin p < 0.001, Interaction p = 0.07). In the saline control condition, ketones did not correlate with hepatic or peripheral insulin sensitivity. Conversely, in the lipid condition, ketones were positively correlated with hepatic insulin sensitivity (r = 0.59, p < 0.01), but inversely related to peripheral insulin sensitivity (r = -0.64, p < 0.01). Hyperlipidemia increased plasma inflammatory markers, but did not impact skeletal muscle inflammatory gene expression. Gene expression related to ketone and fatty acid metabolism in skeletal muscle increased in response to hyperlipidemia. CONCLUSION: This work provides important insight into the role of ketones in human health and suggests that ketone body metabolism is altered at the onset of lipid-induced insulin resistance.

Association between hyperketolactia and production in early-lactating dairy cows.

Study aims were to investigate associations of hyperketolactia (HYKL) status of Holstein dairy cows between 6 and 60 d in milk (DIM), defined by milk acetone (mACE) and ß-hydroxybutyrate (mBHB) content, with daily milk yield and composition. Milk samples (∼5.0 million) were collected over a 5-yr period (2014-2019) within the milk recording system in Poland. Concentrations of mACE and mBHB determined by Fourier-transform infrared spectroscopy were used to categorize samples into 4 ketolactia groups. Based on threshold values of ≥0.15 mmol/L mACE and ≥0.10 mmol/L mBHB, ketolactia groups were normoketolactia (NKL; mACE <0.15 mmol/L and mBHB <0.10 mmol/L), BHB hyperketolactia (HYKLBHB; mACE <0.15 mmol/L and mBHB ≥0.10 mmol/L), ACE hyperketolactia (HYKLACE; mACE ≥0.15 mmol/L and mBHB <0.10 mmol/L), and ACE and BHB hyperketolactia (HYKLACEBHB; mACE ≥0.15 mmol/L and mBHB ≥0.10 mmol/L). To investigate ketolactia association with production outcomes, a linear model was developed, including ketolactia group, DIM, parity, their interactions, year-season as fixed effects, and random effects of herd and cow. Among all milk samples, 31.2% were classified as HYKL, and of these, 52.6%, 39.6%, and 7.8% were HYKLACEBHB, HYKLBHB, and HYKLACE, respectively. Ketolactia groups differed for all traits studied in all parities and DIM. Among HYKL groups, lowest milk yield was found in HYKLACEBHB cows, except for 6 to 30 DIM in first- and second-lactation cows. Milk yield of HYKLBHB cows was higher than that of NKL cows until 20 to 30 DIM, and then it was lower than NKL cows. Milk yield of HYKLACE cows was mostly lower than NKL cows. Energy-corrected milk (ECM) yield of HYKLACEBHB cows was higher than that of NKL cows until 30 to 35 DIM for second lactation and third lactation or greater, and in the whole study period for first lactation. The yield of ECM for HYKLBHB cows was mostly higher than that of NKL cows, whereas HYKLACE cows had higher ECM than NKL cows until 15 to 25 DIM and then was lower for the HYKLACE group. Milk composition differed among HYKL groups. Highest milk fat (MF) and lowest milk lactose (ML) contents were observed in HYKLACEBHB cows. Cows in HYKLACEBHB and HYKLBHB groups had higher MF and lower milk protein (MP; except in 6-8 DIM in first lactation) and ML content than NKL cows. Milk fat content was higher in HYKLACE than NKL cows in first lactation and during the first 30 to 40 DIM in older cows. Lactose content was lower in HYKLACE than in NKL cows within 30 to 40 DIM; afterward it was higher in NKL cows. Lower MP content was found in HYKLACE than in NKL cows, except during 6 to 9 DIM for cows in first lactation and third lactation or greater. In conclusion, HYKL is associated with altered milk production in all parities, but a range of these negative relations depends on ketone status addressing both ACE and BHB contents. Further research is needed to ascertain underpinning biochemical defects of HYKL from elevated ACE, alone or in combination with BHB, during early lactation.

The Impact of Ketone Body Metabolism on Mitochondrial Function and Cardiovascular Diseases.

Ketone bodies, consisting of beta-hydroxybutyrate, acetoacetate, and acetone, are metabolic byproducts known as energy substrates during fasting. Recent advancements have shed light on the multifaceted effects of ketone body metabolism, which led to increased interest in therapeutic interventions aimed at elevating ketone body levels. However, excessive elevation of ketone body concentration can lead to ketoacidosis, which may have fatal consequences. Therefore, in this review, we aimed to focus on the latest insights on ketone body metabolism, particularly emphasizing its association with mitochondria as the primary site of interaction. Given the distinct separation between ketone body synthesis and breakdown pathways, we provide an overview of each metabolic pathway. Additionally, we discuss the relevance of ketone bodies to conditions such as nonalcoholic fatty liver disease or nonalcoholic steatohepatitis and cardiovascular diseases. Moreover, we explore the utilization of ketone body metabolism, including dietary interventions, in the context of aging, where mitochondrial dysfunction plays a crucial role. Through this review, we aim to present a comprehensive understanding of ketone body metabolism and its intricate relationship with mitochondrial function, spanning the potential implications in various health conditions and the aging process.

Preimplantation embryo exposure to ketone bodies exerts sex-specific effects on mouse fetal and placental transcriptomes.

RESEARCH QUESTION: Does in vitro exposure of preimplantation mouse embryos to the ketone bodies ß-hydroxybutyrate (ßOHB) and acetoacetate (AcAc) impact post-transfer fetal and placental gene expression? DESIGN: Blastocysts cultured in vitro with or without 2 mmol/l ßOHB alone ('ßOHB') or combined with 0.8 mmol/l AcAc ('Keto') underwent embryo transfer. Transcriptional profiles of sexed placenta, liver and brain at gestational day 14.5 were examined via RNA sequencing and DAVID functional analysis. RESULTS: A sexually dimorphic response to in vitro ketone exposure was observed. Both ßOHB and Keto exposure down-regulated genes related to oxidative phosphorylation specifically in female liver. ßOHB down-regulated female placental steroid biosynthetic processes, while Keto treatment up-regulated genes relevant to blood vessel formation and cell migration in male placenta. Brain transcriptomes were minimally affected. X-linked genes and chromatin modifiers were identified as differentially expressed in both liver and placenta, alluding to a sex-specific regulatory mechanism. CONCLUSIONS: Transient preimplantation ketone exposure perturbs sex-specific fetal liver and placental gene expression, demonstrating a developmental programming effect that warrants future investigation of the postnatal metabolic health of male and female offspring.