Ketosis prevents abdominal aortic aneurysm rupture through C-C chemokine receptor type 2 downregulation and enhanced extracellular matrix balance.

Abdominal aortic aneurysms (AAAs) are prevalent with aging, and AAA rupture is associated with increased mortality. There is currently no effective medical therapy to prevent AAA rupture. The monocyte chemoattractant protein (MCP-1)/C-C chemokine receptor type 2 (CCR2) axis critically regulates AAA inflammation, matrix-metalloproteinase (MMP) production, and extracellular matrix (ECM) stability. We therefore hypothesized that a diet intervention that can modulate CCR2 axis may therapeutically impact AAA risk of rupture. Since ketone bodies (KBs) can trigger repair mechanisms in response to inflammation, we evaluated whether systemic ketosis in vivo could reduce CCR2 and AAA progression. Male Sprague-Dawley rats underwent surgical AAA formation using porcine pancreatic elastase and received daily ß-aminopropionitrile to promote AAA rupture. Rats with AAAs received either a standard diet, ketogenic diet (KD), or exogenous KBs (EKB). Rats receiving KD and EKB reached a state of ketosis and had significant reduction in AAA expansion and incidence of rupture. Ketosis also led to significantly reduced aortic CCR2 content, improved MMP balance, and reduced ECM degradation. Consistent with these findings, we also observed that Ccr2-/- mice have significantly reduced AAA expansion and rupture. In summary, this study demonstrates that CCR2 is essential for AAA expansion, and that its modulation with ketosis can reduce AAA pathology. This provides an impetus for future clinical studies that will evaluate the impact of ketosis on human AAA disease.

Ketones and the Heart: Metabolic Principles and Therapeutic Implications.

The ketone bodies beta-hydroxybutyrate and acetoacetate are hepatically produced metabolites catabolized in extrahepatic organs. Ketone bodies are a critical cardiac fuel and have diverse roles in the regulation of cellular processes such as metabolism, inflammation, and cellular crosstalk in multiple organs that mediate disease. This review focuses on the role of cardiac ketone metabolism in health and disease with an emphasis on the therapeutic potential of ketosis as a treatment for heart failure (HF). Cardiac metabolic reprogramming, characterized by diminished mitochondrial oxidative metabolism, contributes to cardiac dysfunction and pathologic remodeling during the development of HF. Growing evidence supports an adaptive role for ketone metabolism in HF to promote normal cardiac function and attenuate disease progression. Enhanced cardiac ketone utilization during HF is mediated by increased availability due to systemic ketosis and a cardiac autonomous upregulation of ketolytic enzymes. Therapeutic strategies designed to restore high-capacity fuel metabolism in the heart show promise to address fuel metabolic deficits that underpin the progression of HF. However, the mechanisms involved in the beneficial effects of ketone bodies in HF have yet to be defined and represent important future lines of inquiry. In addition to use as an energy substrate for cardiac mitochondrial oxidation, ketone bodies modulate myocardial utilization of glucose and fatty acids, two vital energy substrates that regulate cardiac function and hypertrophy. The salutary effects of ketone bodies during HF may also include extra-cardiac roles in modulating immune responses, reducing fibrosis, and promoting angiogenesis and vasodilation. Additional pleotropic signaling properties of beta-hydroxybutyrate and AcAc are discussed including epigenetic regulation and protection against oxidative stress. Evidence for the benefit and feasibility of therapeutic ketosis is examined in preclinical and clinical studies. Finally, ongoing clinical trials are reviewed for perspective on translation of ketone therapeutics for the treatment of HF.

Clinicopathologic Appearance of Advanced Ketoacidosis With Basal Vacuolation in Renal Tubules.

CONTEXT.­: Basal vacuolization (BV) in renal tubules is a histopathologic hallmark of advanced ketoacidosis that enables us to retrospectively diagnose these cases. OBJECTIVE.­: To clarify the pathologic background and serologic findings of ketoacidosis with BV, and to reveal the pathologic findings by each pathologic background. DESIGN.­: We examined 664 serial autopsy cases. A systemic histopathologic examination and measurement of serum ß-hydroxybutyrate concentration were performed for the cases with BV. The extent of steatosis and fibrosis in the organs and the degree of coronary artery stenosis were semiquantitatively investigated. Immunohistochemistry for adipophilin was also performed to analyze its usefulness for the pathologic diagnosis. RESULTS.­: Basal vacuolization was found in 16 cases, all of which showed a pathologic serum ß-hydroxybutyrate concentration. The main background of ketoacidosis was considered as alcohol abuse in 6 cases, diabetes in 5, malnutrition in 3, and hypothermia and infection in 1 case each. Severe hepatic fibrosis was observed only in the alcohol-abuser group. Moreover, cardiac steatosis was more severe in patients with possible alcohol abuse than in those with other causes. Immunohistochemistry for adipophilin showed immunoreactivity consistent with BV in 13 of 16 cases. There was no correlation between ß-hydroxybutyrate concentration and either the postmortem or storage interval. CONCLUSIONS.­: Basal vacuolization may be a useful finding for detecting ketoacidosis cases in a postmortem investigation. Serum ß-hydroxybutyrate was a stable and reliable compound for the definitive diagnosis of ketoacidosis in such cases. The present study showed that pathologic changes in some organs may vary by each pathologic background of ketoacidosis with BV.

Association of Agenesis of the Dorsal Pancreas With HNF1B Heterozygote Mutation: A Case Report.

Background: Agenesis of the dorsal pancreas (ADP) is a rare disease, the pathogenic mechanism of which is partially related to variants of hepatocyte nuclear factor 1B (HNF1B) gene. Case Presentation: We report a case of ADP, which presented with acute ketoacidosis, hyperuricemia, and liver dysfunction. In this case, the HNF1B score was estimated as 16 and a heterozygous variant of HNF1B in exon 2 (c.513G>A-p.W171X) was identified through gene sequencing. Conclusions: A good understanding of the clinical comorbidities of ADP is essential for avoiding missed diagnosis to a great extent. Moreover, estimation of HNF1B score is recommended before genetic testing.

Systematic Review - Neuroprotection of ketosis in acute injury of the mammalian central nervous system: A meta-analysis.

To evaluate the neuroprotection exerted by ketosis against acute damage of the mammalian central nervous system (CNS). Search engines were interrogated to identify experimental studies comparing the mitigating effect of ketosis (intervention) versus non-ketosis (control) on acute CNS damage. Primary endpoint was a reduction in mortality. Secondary endpoints were a reduction in neuronal damage and dysfunction, and an 'aggregated advantage' (composite of all primary and secondary endpoints). Hedges' g was the effect measure. Subgroup analyses evaluated the modulatory effect of age, insult type, and injury site. Meta-regression evaluated timing, type, and magnitude of intervention as predictors of neuroprotection. The selected publications were 49 experimental murine studies (period 1979-2020). The intervention reduced mortality (g 2.45, SE 0.48, p < .01), neuronal damage (g 1.96, SE 0.23, p < .01) and dysfunction (g 0.99, SE 0.10, p < .01). Reduction of mortality was particularly pronounced in the adult subgroup (g 2.71, SE 0.57, p < .01). The aggregated advantage of ketosis was stronger in the pediatric (g 3.98, SE 0.71, p < .01), brain (g 1.96, SE 0.18, p < .01), and ischemic insult (g 2.20, SE 0.23, p < .01) subgroups. Only the magnitude of intervention was a predictor of neuroprotection (g 0.07, SE 0.03, p 0.01 per every mmol/L increase in ketone levels). Ketosis exerts a potent neuroprotection against acute damage to the mammalian CNS in terms of reduction of mortality, of neuronal damage and dysfunction. Hematic levels of ketones are directly proportional to the effect size of neuroprotection.

BCKDK regulates the TCA cycle through PDC in the absence of PDK family during embryonic development.

Pyruvate dehydrogenase kinases (PDK1-4) inhibit the TCA cycle by phosphorylating pyruvate dehydrogenase complex (PDC). Here, we show that PDK family is dispensable for murine embryonic development and that BCKDK serves as a compensatory mechanism by inactivating PDC. First, we knocked out all four Pdk genes one by one. Surprisingly, Pdk total KO embryos developed and were born in expected ratios but died by postnatal day 4 because of hypoglycemia or ketoacidosis. Moreover, PDC was phosphorylated in these embryos, suggesting that another kinase compensates for PDK family. Bioinformatic analysis implicated branched-chain ketoacid dehydrogenase kinase (Bckdk), a key regulator of branched-chain amino acids (BCAAs) catabolism. Indeed, knockout of Bckdk and Pdk family led to the loss of PDC phosphorylation, an increase in PDC activity and pyruvate entry into the TCA cycle, and embryonic lethality. These findings reveal a regulatory crosstalk hardwiring BCAA and glucose catabolic pathways, which feed the TCA cycle.

Greater liver PNPLA3 protein abundance in vivo and in vitro supports lower triglyceride accumulation in dairy cows.

Fatty liver syndrome is a prevalent metabolic disorder in peripartum dairy cows that unfavorably impacts lactation performance and health. Patatin-like phospholipase domain-containing protein 3 (PNPLA3) is a lipase that plays a central role in human non-alcoholic fatty liver disease etiology but has received limited attention in bovine fatty liver research. Thus, we investigated the relationship between tissue PNPLA3 expression and liver triglyceride accumulation in vivo via a ketosis induction protocol in multiparous dairy cows peripartum, as well as in vitro via small interfering RNA knockdown of PNPLA3 mRNA expression in bovine primary hepatocytes. Results demonstrated a negative association (P = 0.04) between liver PNPLA3 protein abundance and liver triglyceride content in peripartum dairy cows, while adipose PNPLA3 protein abundance was not associated with liver triglyceride content or blood fatty acid concentration. Knockdown of PNPLA3 mRNA resulted in reduced PNPLA3 protein abundance (P < 0.01) and greater liver triglyceride content (P < 0.01). Together, these results suggest greater liver PNPLA3 protein abundance may directly limit liver triglyceride accumulation peripartum, potentially preventing bovine fatty liver or accelerating recovery from fatty liver syndrome.

Ingested Ketone Ester Leads to a Rapid Rise of Acetyl-CoA and Competes with Glucose Metabolism in the Brain of Non-Fasted Mice.

The role of ketone bodies in the cerebral energy homeostasis of neurological diseases has begun to attract recent attention particularly in acute neurological diseases. In ketogenic therapies, ketosis is achieved by either a ketogenic diet or by the administration of exogenous ketone bodies. The oral ingestion of the ketone ester (KE), (R)-3-hydroxybutyl (R)-3-hydroxybutyrate, is a new method to generate rapid and significant ketosis (i.e., above 6 mmol/L) in humans. KE is hydrolyzed into ß-hydroxybutyrate (ßHB) and its precursor 1,3-butanediol. Here, we investigate the effect of oral KE administration (3 mg KE/g of body weight) on brain metabolism of non-fasted mice using liquid chromatography in tandem with mass spectrometry. Ketosis (Cmax = 6.83 ± 0.19 mmol/L) was obtained at Tmax = 30 min after oral KE-gavage. We found that ßHB uptake into the brain strongly correlated with the plasma ßHB concentration and was preferentially distributed in the neocortex. We showed for the first time that oral KE led to an increase of acetyl-CoA and citric cycle intermediates in the brain of non-fasted mice. Furthermore, we found that the increased level of acetyl-CoA inhibited glycolysis by a feedback mechanism and thus competed with glucose under physiological conditions. The brain pharmacodynamics of this oral KE strongly suggest that this agent should be considered for acute neurological diseases.

SGLT2 Inhibitor-Induced Low-Grade Ketonemia Ameliorates Retinal Hypoxia in Diabetic Retinopathy-A Novel Hypothesis.

Diabetic retinopathy (DR) is a well-recognized microvascular complication of diabetes. Growing evidence suggests that, in addition to retinal vascular damage, there is significant damage to retinal neural tissue in DR. Studies reveal neuronal damage before clinically evident vascular lesions and DR is now classified as a neurovascular complication. Hyperglycemia causes retinal damage through complex metabolic pathways leading to oxidative stress, inflammation, vascular damage, capillary ischemia, and retinal tissue hypoxia. Retinal hypoxia is further worsened by high oxygen consumption in the rods. Persistent hypoxia results in increases in vascular endothelial growth factor (VEGF) and other pro-angiogenic factors leading to proliferative DR/macular edema and progressive visual impairment. Optimal glucose control has favorable effects in DR. Other treatments for DR include laser photocoagulation, which improves retinal oxygenation by destroying the high oxygen consuming rods and their replacement by low oxygen consuming glial tissue. Hypoxia is a potent stimulator of VEGF, and intravitreal anti-VEGF antibodies are effective in regressing macular edema and in some studies, retinal neovascularization. In this review, we highlight the complex pathophysiology of DR with a focus on retinal oxygen/fuel consumption and hypoxic damage to retinal neurons. We discuss potential mechanisms through which sodium-glucose cotransporter 2 (SGLT2) inhibitors improve retinal hypoxia-through ketone bodies, which are energetically as efficient as glucose and yield more ATP per molecule of oxygen consumed than fat, with less oxidative stress. Retinal benefits would occur through improved fuel energetics, less hypoxia and through the anti-inflammatory/oxidative stress effects of ketone bodies. Well-designed studies are needed to explore this hypothesis.

Oxidative stress, NF-κB signaling, NLRP3 inflammasome, and caspase apoptotic pathways are activated in mammary gland of ketotic Holstein cows.

Ketosis is a serious metabolic disorder characterized by systemic and hepatic oxidative stress, inflammation, and apoptosis, as well as reduced milk yield. Because of the paucity of data on mammary responses during ketosis, the aim of this study was to evaluate alterations in oxidative stress, NF-κB signaling, NLRP3 inflammasome, and caspase apoptotic pathways in mammary gland of dairy cows with ketosis. Blood, mammary gland tissue, and milk samples were collected from healthy cows [Control, blood concentration of ß-hydroxybutyrate (BHB) <0.6 mM, n = 10] and cows with subclinical ketosis (SCK, blood concentration of BHB >1.2 mM and <3 mM, n = 10) or clinical ketosis (CK, blood concentration of BHB >3 mM, n = 10) at median 8 d in milk (range = 6-12). Compared with Control, serum concentration of glucose was lower (3.91 vs. 2.86 or 2.12 mM) in cows with SCK or CK, whereas concentrations of fatty acids (0.25 vs. 0.57 or 1.09 mM) and BHB (0.42 vs. 1.81 or 3.85 mM) were greater. Compared with Control, the percentage of milk fat was greater in cows with SCK or CK. In contrast, the percentage of milk protein was lower in cows with SCK or CK. We detected no differences in milk lactose content across groups. Compared with Control, activities of glutathione peroxidase, superoxide dismutase, and catalase were lower in mammary gland tissue of cows with SCK or CK. In contrast, concentrations of hydrogen peroxide and malondialdehyde were greater in cows with SCK or CK. Compared with Control, mRNA abundances of TNFA, IL6, and IL1B were greater in mammary tissues of cows with SCK or CK. In addition, activity of IKKß and the ratio of phosphorylated inhibitor of κBα to IκBα, and of phosphorylated NF-κB p65 to NF-κB p65, were also greater in mammary tissues of cows with SCK or CK. Subclinical or clinical ketosis also led to greater activity of caspase 1 and protein abundance of caspase 1, NLRP3, Bax, caspase 3, and caspase 9. In contrast, abundance of the antiapoptotic protein was lower in SCK or CK cows. The data indicate that the mammary gland of SKC or CK cows undergoes severe oxidative stress, inflammation, and cell death.

N-acetylcysteine modulates non-esterified fatty acid-induced pyroptosis and inflammation in granulosa cells.

In the perinatal period of dairy cows, negative energy balance (NEB) is likely to occur, which increases the level of non-esterified fatty acids (NEFA) in the follicular fluid, hinders the proliferation of granulosa cells (GCs), and thus endangers the development of oocytes and the fecundity of dairy cows. We found that there were oxidative stress and inflammatory response in the serum of cows with perinatal ketosis. Whether the oxidative stress induced by NEFA is involved in the pyroptosis and inflammation of GCs remains unclear. After NEFA treatment, the expression of NLRP3 and caspase-1 and the release of inflammatory cytokines IL-1ß were increased in a dose-dependent manner, indicating that NEFA may contribute to pyroptosis. Besides, NEFA stimulation induced oxidative stress, resulting in the phosphorylation of NF-κB, and increased the production of interleukin (IL)-6 and nitric oxide (NO), indicating that NEFA may induce inflammation in GCs. However, the NEFA-mediated effects were observably reversed when the GCs were pre-treated with antioxidant and radical scavenger, N-acetylcysteine (NAC). Taken together, our results reveal that NEFA can induce pyroptosis and inflammation through NLRP3 inflammasome and TLR4/NF-κB pathway, respectively, and NAC can alleviate these conditions.

COVID-19 infection may cause ketosis and ketoacidosis.

The present study included 658 hospitalized patients with confirmed COVID-19. Forty-two (6.4%) out of 658 patients presented with ketosis on admission with no obvious fever or diarrhoea. They had a median (interquartile range [IQR]) age of 47.0 (38.0-70.3) years, and 16 (38.1%) were men. Patients with ketosis were younger (median age 47.0 vs. 58.0 years; P = 0.003) and had a greater prevalence of fatigue (31.0% vs. 10.6%; P < 0.001), diabetes (35.7% vs. 18.5%; P = 0.007) and digestive disorders (31.0% vs. 12.0%; P < 0.001). They had a longer median (IQR) length of hospital stay (19.0 [12.8-33.3] vs. 16.0 [10.0-24.0] days; P < 0.001) and a higher mortality rate (21.4% vs. 8.9%; P = 0.017). Three (20.0%) out of the 15 patients with diabetic ketosis developed acidosis, five patients (26.7%) with diabetic ketosis died, and one of these (25.0%) presented with acidosis. Two (7.4%) and four (14.3%) of the 27 non-diabetic ketotic patients developed severe acidosis and died, respectively, and one (25.0%) of these presented with acidosis. This suggests that COVID-19 infection caused ketosis or ketoacidosis, and induced diabetic ketoacidosis for those with diabetes. Ketosis increased the length of hospital stay and mortality. Meanwhile, diabetes increased the length of hospital stay for patients with ketosis but had no effect on their mortality.

Estimating the combined costs of clinical and subclinical ketosis in dairy cows.

Clinical ketosis (CK) and subclinical ketosis (SCK) are associated with lower milk production, lower reproductive performance, an increased culling of cows and an increased probability of other disorders. Quantifying the costs related to ketosis will enable veterinarians and farmers to make more informed decisions regarding the prevention and treatment of the disease. The overall aim of this study was to estimate the combined costs of CK and SCK using assumptions and input variables from a typical Dutch context. A herd level dynamic stochastic simulation model was developed, simulating 385 herds with 130 cows each. In the default scenario there was a CK probability of almost 1% and a SCK probability of 11%. The herds under the no risk scenario had no CK and SCK, while the herds under the high-risk scenario had a doubled probability of CK and SCK compared to the default scenario. The results from the simulation model were used to estimate the annual cash flows of the herds, including the costs related to milk production losses, treatment, displaced abomasum, mastitis, calf management, culling and feed, as well as the returns from sales of milk and calves. The difference between the annual net cash flows of farms in the no risk scenario and the default scenario provides the estimate of the herd level costs of ketosis. Average herd level costs of ketosis (CK and SCK combined) were €3,613 per year for a default farm and €7,371 per year for a high-risk farm. The costs for a single CK case were on average €709 (with 5 and 95 percentiles of €64 and €1,196, respectively), while the costs for a single SCK case were on average €150 (with 5 and 95 percentiles of €18 and €422, respectively) for the default farms. The differences in costs between cases occurred due to differences between cases (e.g., cow culled vs cow not culled, getting another disease vs not getting another disease).

Modulation of Cellular Biochemistry, Epigenetics and Metabolomics by Ketone Bodies. Implications of the Ketogenic Diet in the Physiology of the Organism and Pathological States.

Ketone bodies (KBs), comprising ß-hydroxybutyrate, acetoacetate and acetone, are a set of fuel molecules serving as an alternative energy source to glucose. KBs are mainly produced by the liver from fatty acids during periods of fasting, and prolonged or intense physical activity. In diabetes, mainly type-1, ketoacidosis is the pathological response to glucose malabsorption. Endogenous production of ketone bodies is promoted by consumption of a ketogenic diet (KD), a diet virtually devoid of carbohydrates. Despite its recently widespread use, the systemic impact of KD is only partially understood, and ranges from physiologically beneficial outcomes in particular circumstances to potentially harmful effects. Here, we firstly review ketone body metabolism and molecular signaling, to then link the understanding of ketone bodies' biochemistry to controversies regarding their putative or proven medical benefits. We overview the physiological consequences of ketone bodies' consumption, focusing on (i) KB-induced histone post-translational modifications, particularly ß-hydroxybutyrylation and acetylation, which appears to be the core epigenetic mechanisms of activity of ß-hydroxybutyrate to modulate inflammation; (ii) inflammatory responses to a KD; (iii) proven benefits of the KD in the context of neuronal disease and cancer; and (iv) consequences of the KD's application on cardiovascular health and on physical performance.

Silibinin inhibits in vitro ketosis by regulating HMGCS2 and NF-kB: elucidation of signaling molecule relationship under ketotic conditions.

Ketosis is a condition where ketone bodies are produced as an alternative energy source, due to insufficient glucose for energy production so that the body switches from carbohydrate metabolism to mostly fat metabolism. In this study, we examined the anti-ketosis effects of silibinin, a major active component of silymarin. We induced ketosis in FL83B mouse hepatocytes in vitro by culturing in low glucose media and compared results to hepatocytes maintained in high-glucose conditions. We quantified ß-hydroxybutyrate (BHB) levels with a colorimetric assay. In low-glucose conditions, silibinin reduced the amount of BHB produced, compared to high-glucose conditions; thus, silibinin exhibited an anti-ketotic effect. Ketone body formation during beta oxidation is mediated by 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2). The nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) regulates the transcription of HMGCS2, and plays a vital role in BHB levels. We showed that silibinin inhibited the expression of HMGCS2 and NF-kB at transcriptional and translational levels. Silibinin also inhibited the nuclear translocation of NF-kB and its DNA binding activity. To elucidate the relationship between HMGCS2 and NF-kB, we tested inhibited and over-expressed NF-kB. We found that NF-kB acted as a positive regulator for HMGCS2 under ketosis treatment conditions.

Dehydration and insulinopenia are necessary and sufficient for euglycemic ketoacidosis in SGLT2 inhibitor-treated rats.

Sodium-glucose transport protein 2 (SGLT2) inhibitors are a class of anti-diabetic agents; however, concerns have been raised about their potential to induce euglycemic ketoacidosis and to increase both glucose production and glucagon secretion. The mechanisms behind these alterations are unknown. Here we show that the SGLT2 inhibitor (SGLT2i) dapagliflozin promotes ketoacidosis in both healthy and type 2 diabetic rats in the setting of insulinopenia through increased plasma catecholamine and corticosterone concentrations secondary to volume depletion. These derangements increase white adipose tissue (WAT) lipolysis and hepatic acetyl-CoA content, rates of hepatic glucose production, and hepatic ketogenesis. Treatment with a loop diuretic, furosemide, under insulinopenic conditions replicates the effect of dapagliflozin and causes ketoacidosis. Furthermore, the effects of SGLT2 inhibition to promote ketoacidosis are independent from hyperglucagonemia. Taken together these data in rats identify the combination of insulinopenia and dehydration as a potential target to prevent euglycemic ketoacidosis associated with SGLT2i.

Non-Esterified Fatty Acids Over-Activate the TLR2/4-NF-Κb Signaling Pathway to Increase Inflammatory Cytokine Synthesis in Neutrophils from Ketotic Cows.

BACKGROUND/AIMS: Dairy cows with clinical ketosis display a negative energy balance and high blood concentrations of non-esterified fatty acids (NEFAs), the latter of which is an important pathological factor of ketosis in cows. The aims of this study were to investigate the inflammatory status of ketotic cows and to determine whether and through what underlying mechanism high levels of NEFAs induce an inflammatory response. METHODS: Proinflammatory factors and the nuclear factor kappa B (NF-κB) signaling pathway were evaluated in neutrophils from clinical ketotic and control cows, using methods including western blotting, quantitative real-time polymerase chain reaction, and enzyme-linked immunosorbent assay. In vitro, the effects of NEFAs on the NF-κB signaling pathway in cow neutrophils were also evaluated using the above experimental techniques. RESULTS: Ketotic cows displayed low blood concentrations of glucose and high blood NEFA and ß-hydroxybutyrate concentrations. Importantly, Toll-like receptor 2 (TLR2) and TLR4 expression and IκBα and NF-κB p65 phosphorylation levels in neutrophils (PMNs) were significantly higher in ketotic cows than in control cows, indicating over-activation of the TLR2/4-induced NF-κB inflammatory pathway in PMNs in ketotic cows. The blood concentrations of the inflammatory cytokines interleukin-6 (IL-6), IL-1ß, and tumor necrosis factor-α (TNF-α) were also significantly increased in ketotic cows. Interestingly, we found that NEFAs were positively correlated with proinflammatory cytokines. In vitro, after pharmacological inhibition of TLR2 and TLR4 expression in cow neutrophils, TLR2 and TLR4 expression was significantly decreased, and the phosphorylation level of NF-κB p65 was also reduced. Cow neutrophils were treated with different concentrations of NEFAs and pyrrolidine dithiocarbamate (PDTC; an NF-κB inhibitor). High concentrations of NEFAs (0.5 and 1 mM) significantly increased TLR2 and TLR4 expression, IκBα and NF-κB p65 phosphorylation levels, NF-κB p65 transcriptional activity, and IL-6, IL-1ß, and TNF-α synthesis in cow neutrophils. The inhibition of NF-κB by PDTC suppressed the NEFA-induced synthesis of proinflammatory cytokines. CONCLUSIONS: High concentrations of NEFAs can over-activate the TLR2/4-mediated NF-κB signaling pathway to induce the over-production of proinflammatory cytokines, thereby increasing inflammation in cows with clinical ketosis.

Diazoxide toxicity in a child with persistent hyperinsulinemic hypoglycemia of infancy: mixed hyperglycemic hyperosmolar coma and ketoacidosis.

BACKGROUND: Persistent hyperinsulinemic hypoglycemia of infancy (PHHI), also known as congenital hyperinsulinism, has been known to go into spontaneous remission, with patients developing diabetes in later life. A temporary phase of hyperglycemia is, however, rarely reported. CASE PRESENTATION: We describe a 16-month-old child, a known case of diazoxide responsive PHHI, presenting with mixed hyperglycemic hyperosmolar coma and ketoacidosis with rhabdomyolysis while on diazoxide treatment. The patient required temporary cessation of diazoxide and initiation of insulin infusion, followed by a relapse of hypoglycemia again necessitating diazoxide therapy. CONCLUSIONS: Hyperosmolar coma with ketoacidosis is a rare side-effect of diazoxide therapy, documented even in patients with persistent hyperinsulinemic hypoglycemia of infancy.

Dietary Manipulations That Induce Ketosis Activate the HPA Axis in Male Rats and Mice: A Potential Role for Fibroblast Growth Factor-21.

In response to an acute threat to homeostasis or well-being, the hypothalamic-pituitary-adrenocortical (HPA) axis is engaged. A major outcome of this HPA axis activation is the mobilization of stored energy, to fuel an appropriate behavioral and/or physiological response to the perceived threat. Importantly, the extent of HPA axis activity is thought to be modulated by an individual's nutritional environment. In this study, we report that nutritional manipulations signaling a relative depletion of dietary carbohydrates, thereby inducing nutritional ketosis, acutely and chronically activate the HPA axis. Male rats and mice maintained on a low-carbohydrate high-fat ketogenic diet (KD) exhibited canonical markers of chronic stress, including increased basal and stress-evoked plasma corticosterone, increased adrenal sensitivity to adrenocorticotropin hormone, increased stress-evoked c-Fos immunolabeling in the paraventricular nucleus of the hypothalamus, and thymic atrophy, an indicator of chronic glucocorticoid exposure. Moreover, acutely feeding medium-chain triglycerides (MCTs) to rapidly induce ketosis among chow-fed male rats and mice also acutely increased HPA axis activity. Lastly, and consistent with a growing literature that characterizes the hepatokine fibroblast growth factor-21 (FGF21) as both a marker of the ketotic state and as a key metabolic stress hormone, the HPA response to both KD and MCTs was significantly blunted among mice lacking FGF21. We conclude that dietary manipulations that induce ketosis lead to increased HPA axis tone, and that the hepatokine FGF21 may play an important role to facilitate this effect.

Elevated Apoptosis in the Liver of Dairy Cows with Ketosis.

BACKGROUND/AIMS: Dairy cows with ketosis are characterized by oxidative stress and hepatic damage. The aim of this study was to investigate hepatic oxidative stress and the apoptotic status of ketotic cows, as well as the underlying apoptosis pathway. METHODS: The blood aspartate aminotransferase (AST), alanine aminotransferase (ALT), glutamate dehydrogenase (GLDH) and gamma-glutamyl transferase (GGT) activities and the haptoglobin (HP), serum amyloid A (SAA) and serum apoptotic cytokeratin 18 neo-epitope M30 (CK18 M30) concentrations were determined by commercially available kits and ELISA kits, respectively. Liver histology, TUNEL and Oil red O staining were performed in liver tissue samples. TG contents were measured using an enzymatic kit; Caspase 3 assays were carried out using the Caspase 3 activity assay kit; oxidation and antioxidant markers were measured using biochemical kits; apoptosis pathway were determined by qRT-PCR and western blot. RESULTS: Ketotic cows displayed hepatic fat accumulation. The hepatic malondialdehyde (MDA) content was significantly increased, but the activities of catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) were markedly decreased in ketotic cows compared with control cows, indicating that ketotic cows displayed severe oxidative stress. Significantly higher serum levels of the hepatic damage markers AST, ALT, GGT and GLDH were observed in ketotic cows than in control cows. The blood concentration of the apoptotic marker CK18 M30 and the number of TUNEL-positive cells in the liver of ketotic cows were 1.19- and 2.61-fold, respectively, higher than the values observed in control cows. Besides, Caspase 3 activity was significantly increased in the liver of ketosis cows. Importantly, the levels of phosphorylated c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38MAPK) were significantly increased but the level of phosphorylated extracellular signal-regulated kinase1/2 (ERK1/2) was markedly decreased, which further promoted tumor protein 53 (p53) expression and inhibited nuclear factor E2-related factor 2 (Nrf2) expression. The apoptosis-related molecules p21, MDM2, Caspase 3, Caspase 9 and Bax were expressed at significantly higher levels in ketotic cows than in healthy cows, whereas the anti-apoptosis molecule Bcl-2 was expressed at significantly lower levels. CONCLUSIONS: Based on these results, ketotic cows display severe hepatic oxidative stress. The hepatic MAPK-p53-Nrf2 apoptotic pathway is over induced and partially mediated apoptotic damage in the liver.