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.

A Hypomethylating Ketogenic Diet in Apolipoprotein E-Deficient Mice: A Pilot Study on Vascular Effects and Specific Epigenetic Changes.

Hyperhomocysteneinemia (HHcy) is common in the general population and is a risk factor for atherosclerosis by mechanisms that are still elusive. A hypomethylated status of epigenetically relevant targets may contribute to the vascular toxicity associated with HHcy. Ketogenic diets (KD) are diets with a severely restricted amount of carbohydrates that are being widely used, mainly for weight-loss purposes. However, studies associating nutritional ketosis and HHcy are lacking. This pilot study investigates the effects of mild HHcy induced by nutritional manipulation of the methionine metabolism in the absence of dietary carbohydrates on disease progression and specific epigenetic changes in the apolipoprotein-E deficient (apoE-/-) mouse model. ApoE-/- mice were either fed a KD, a diet with the same macronutrient composition but low in methyl donors (low methyl KD, LMKD), or control diet. After 4, 8 or 12 weeks plasma was collected for the quantification of: (1) nutritional ketosis, (i.e., the ketone body beta-hydroxybutyrate using a colorimetric assay); (2) homocysteine by HPLC; (3) the methylating potential S-adenosylmethionine to S-adenosylhomocysteine ratio (AdoHcy/AdoMet) by LC-MS/MS; and (4) the inflammatory cytokine monocyte chemoattractant protein 1 (MCP1) by ELISA. After 12 weeks, aortas were collected to assess: (1) the vascular AdoHcy/AdoMet ratio; (2) the volume of atherosclerotic lesions by high-field magnetic resonance imaging (14T-MRI); and (3) the content of specific epigenetic tags (H3K27me3 and H3K27ac) by immunofluorescence. The results confirmed the presence of nutritional ketosis in KD and LMKD mice but not in the control mice. As expected, mild HHcy was only detected in the LMKD-fed mice. Significantly decreased MCP1 plasma levels and plaque burden were observed in control mice versus the other two groups, together with an increased content of one of the investigated epigenetic tags (H3K27me3) but not of the other (H3K27ac). Moreover, we are unable to detect any significant differences at the p < 0.05 level for MCP1 plasma levels, vascular AdoMet:AdoHcy ratio levels, plaque burden, and specific epigenetic content between the latter two groups. Nevertheless, the systemic methylating index was significantly decreased in LMKD mice versus the other two groups, reinforcing the possibility that the levels of accumulated homocysteine were insufficient to affect vascular transmethylation reactions. Further studies addressing nutritional ketosis in the presence of mild HHcy should use a higher number of animals and are warranted to confirm these preliminary observations.

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.

Genetic associations of lactose and its ratios to other milk solids with health traits in Austrian Fleckvieh cows.

The genetic correlations (ra) of milk lactose percentage (LP), lactose yield (LY), and ratios of LP to other milk solids with udder, metabolic, and fertility disorders have not been assessed in dairy cattle so far. To evaluate the potential role of milk lactose as indicator of cow health, 142,285 lactation records of 84,289 Austrian Fleckvieh cows were analyzed with univariate and bivariate animal models. Milk traits were on a 150-d basis and health traits were coded as binary (0/1). Other than LP and LY, 3 new phenotypes were defined and included in the present study, namely the lactose-to-fat, lactose-to-protein, and lactose-to-solids ratios. The most heritable trait was LP (0.566 ± 0.008) and heritability of LY was much lower (0.145 ± 0.005). Heritability estimates close to 0.50 were assessed for the ratios. The frequency of health disorders was higher in multiparous cows yielding milk with low LP (≤4.553%) compared with cows yielding milk with high LP (≥5.045%). Heritabilities of health traits were in the expected ranges, with the highest estimate for ovarian cysts (CYS; 0.037 ± 0.004) and the lowest for retained placenta (0.005 ± 0.001). Mastitis (MAS) genetically correlated with LY (0.518 ± 0.057); considering that the amount of synthesized lactose is the key regulator of milk volume, this result confirmed that high-producing cows are more genetically susceptible to MAS than low-producing animals. Similar to MAS, ketosis (KET) was also positively genetically associated with LY (0.420 ± 0.077) and a weak and unfavorable ra between KET and lactose-to-protein ratio was estimated (0.159 ± 0.077). The ra of LY with milk fever (MFV) and CYS were approximately 0.20. The ra of LP with MAS, KET, and MFV were negative (-0.142 on average), supporting the idea that LP is a potential health indicator. Genetic correlations between health traits ranged from zero (retained placenta with MAS and CYS) to 0.463 ± 0.090 (MAS and MFV). Results of the present study suggest that LP has potentiality to be used as indicator trait to improve udder health in Austrian Fleckvieh population.

Label-Free Quantitative Proteomics in a Methylmalonyl-CoA Mutase-Silenced Neuroblastoma Cell Line.

Methylmalonic acidemias (MMAs) are inborn errors of metabolism due to the deficient activity of methylmalonyl-CoA mutase (MUT). MUT catalyzes the formation of succinyl-CoA from methylmalonyl-CoA, produced from propionyl-CoA catabolism and derived from odd chain fatty acids ß-oxidation, cholesterol, and branched-chain amino acids degradation. Increased methylmalonyl-CoA levels allow for the presymptomatic diagnosis of the disease, even though no approved therapies exist. MMA patients show hyperammonemia, ketoacidosis, lethargy, respiratory distress, cognitive impairment, and hepatomegaly. The long-term consequences concern neurologic damage and terminal kidney failure, with little chance of survival. The cellular pathways affected by MUT deficiency were investigated using a quantitative proteomics approach on a cellular model of MUT knockdown. Currently, a consistent reduction of the MUT protein expression was obtained in the neuroblastoma cell line (SH-SY5Y) by using small-interfering RNA (siRNA) directed against an MUT transcript (MUT siRNA). The MUT absence did not affect the cell viability and apoptotic process in SH-SY5Y. In the present study, we evaluate and quantify the alterations in the protein expression profile as a consequence of MUT-silencing by a mass spectrometry-based label-free quantitative analysis, using two different quantitative strategies. Both quantitative methods allowed us to observe that the expression of the proteins involved in mitochondrial oxido-reductive homeostasis balance was affected by MUT deficiency. The alterated functional mitochondrial activity was observed in siRNA_MUT cells cultured with a propionate-supplemented medium. Finally, alterations in the levels of proteins involved in the metabolic pathways, like carbohydrate metabolism and lipid metabolism, were found.

Glucagon receptor inhibition normalizes blood glucose in severe insulin-resistant mice.

Inactivating mutations in the insulin receptor results in extreme insulin resistance. The resulting hyperglycemia is very difficult to treat, and patients are at risk for early morbidity and mortality from complications of diabetes. We used the insulin receptor antagonist S961 to induce severe insulin resistance, hyperglycemia, and ketonemia in mice. Using this model, we show that glucagon receptor (GCGR) inhibition with a monoclonal antibody normalized blood glucose and ß-hydroxybutyrate levels. Insulin receptor antagonism increased pancreatic ß-cell mass threefold. Normalization of blood glucose levels with GCGR-blocking antibody unexpectedly doubled ß-cell mass relative to that observed with S961 alone and 5.8-fold over control. GCGR antibody blockage expanded α-cell mass 5.7-fold, and S961 had no additional effects. Collectively, these data show that GCGR antibody inhibition represents a potential therapeutic option for treatment of patients with extreme insulin-resistance syndromes.

FGF21 is not required for glucose homeostasis, ketosis or tumour suppression associated with ketogenic diets in mice.

AIMS/HYPOTHESIS: Ketogenic diets (KDs) have increasingly gained attention as effective means for weight loss and potential adjunctive treatment of cancer. The metabolic benefits of KDs are regularly ascribed to enhanced hepatic secretion of fibroblast growth factor 21 (FGF21) and its systemic effects on fatty-acid oxidation, energy expenditure (EE) and body weight. Ambiguous data from Fgf21-knockout animal strains and low FGF21 concentrations reported in humans with ketosis have nevertheless cast doubt regarding the endogenous function of FGF21. We here aimed to elucidate the causal role of FGF21 in mediating the therapeutic benefits of KDs on metabolism and cancer. METHODS: We established a dietary model of increased vs decreased FGF21 by feeding C57BL/6J mice with KDs, either depleted of protein or enriched with protein. We furthermore used wild-type and Fgf21-knockout mice that were subjected to the respective diets, and monitored energy and glucose homeostasis as well as tumour growth after transplantation of Lewis lung carcinoma cells. RESULTS: Hepatic and circulating, but not adipose tissue, FGF21 levels were profoundly increased by protein starvation, independent of the state of ketosis. We demonstrate that endogenous FGF21 is not essential for the maintenance of normoglycaemia upon protein and carbohydrate starvation and is therefore not needed for the effects of KDs on EE. Furthermore, the tumour-suppressing effects of KDs were independent of FGF21 and, rather, driven by concomitant protein and carbohydrate starvation. CONCLUSIONS/INTERPRETATION: Our data indicate that the multiple systemic effects of KD exposure in mice, previously ascribed to increased FGF21 secretion, are rather a consequence of protein malnutrition.

Hyperketonemia induces upregulation of LFA-1 in monocytes, which is mediated by ROS and P38 MAPK activation.

Type 1 diabetic patients have hyperketonemia, elevated levels of pro-inflammatory and oxidative stress markers, and a higher incidence of vascular disease. This study examines the hypothesis that hyperketonemia increases reactive oxygen species (ROS) and is in part responsible for increased expression of adhesion molecules in monocytes. THP-1 monocytes were treated with acetoacetate (AA) or ß-hydroxybutyrate (BHB) (0-10 mmol/L) for 24 h. Results show that AA, but not BHB, increases ROS production in monocytes. Pretreatment of monocytes with N-acetylcysteine (NAC) inhibited AA-induced ROS production. AA treatment induced upregulation of LFA-1 and pretreatment of monocytes with NAC or an inhibitor to p38 MAPK inhibited this upregulation in monocytes. This suggests that physiological concentrations of AA can contribute to increased ROS and activation of p38 MAPK, which may be responsible for AA-induced upregulation of LFA-1 in monocytes. Thus, hyperketonemia contributes to the risk for cardiovascular disease in type 1 diabetes.

Ablation of steroid receptor coactivator-3 resembles the human CACT metabolic myopathy.

Oxidation of lipid substrates is essential for survival in fasting and other catabolic conditions, sparing glucose for the brain and other glucose-dependent tissues. Here we show Steroid Receptor Coactivator-3 (SRC-3) plays a central role in long chain fatty acid metabolism by directly regulating carnitine/acyl-carnitine translocase (CACT) gene expression. Genetic deficiency of CACT in humans is accompanied by a constellation of metabolic and toxicity phenotypes including hypoketonemia, hypoglycemia, hyperammonemia, and impaired neurologic, cardiac and skeletal muscle performance, each of which is apparent in mice lacking SRC-3 expression. Consistent with human cases of CACT deficiency, dietary rescue with short chain fatty acids drastically attenuates the clinical hallmarks of the disease in mice devoid of SRC-3. Collectively, our results position SRC-3 as a key regulator of ß-oxidation. Moreover, these findings allow us to consider platform coactivators such as the SRCs as potential contributors to syndromes such as CACT deficiency, previously considered as monogenic.

Genetic parameters for producer-recorded health data in Canadian Holstein cattle.

Health traits are of paramount importance for economic dairy production. Improvement in liability to diseases has been made with better management practices, but genetic aspects of health traits have received less attention. Dairy producers in Canada have been recording eight health traits (mastitis (MAST), lameness (LAME), cystic ovarian disease (COD), left displaced abomasum (LDA), ketosis (KET), metritis (MET), milk fever (MF) and retained placenta (RP)) since April 2007. Genetic analyses of these traits were carried out in this study for the Holstein breed. Edits on herd distributions of recorded diseases were applied to the data to ensure a sufficient quality of recording. Traits were analysed either individually (MAST, LAME, COD) or were grouped according to biological similarities (LDA and KET, and MET, MF and RP) and analysed with multiple-trait models. Data included 46 104 cases of any of the above diseases. Incidence ranged from 2.3% for MF to 9.7% for MAST. MET and KET also had an incidence below 4.0%. Variance components were estimated using four different sire threshold models. The differences between models resulted from the inclusion of days at risk (DAR) and a cow effect, in addition to herd, parity and sire effects. Models were compared using mean squared error statistic. Mean squared error favoured, in general, the sire and cow within sire model with regression on DAR included. Heritabilities on the liability scale were between 0.02 (MET) and 0.21 (LDA). There was a moderate, positive genetic correlation between LDA and KET (0.58), and between MET and RP (0.79).

Thioredoxin-interacting protein (Txnip) is a critical regulator of hepatic glucose production.

Thioredoxin-interacting protein (Txnip) has been recently described as a possible link between cellular redox state and metabolism; Txnip binds thioredoxin and inhibits its disulfide reductase activity in vitro, while a naturally occurring strain of Txnip-deficient mice has hyperlipidemia, hypoglycemia, and ketosis exacerbated by fasting. We generated Txnip-null mice to investigate the role of Txnip in glucose homeostasis. Txnip-null mice were hypoglycemic, hypoinsulinemic, and had blunted glucose production following a glucagon challenge, consistent with a central liver glucose-handling defect. Glucose release from isolated Txnip-null hepatocytes was 2-fold lower than wild-type hepatocytes, whereas beta-hydroxybutyrate release was increased 2-fold, supporting an intrinsic defect in hepatocyte glucose metabolism. While hepatocyte-specific gene deletion of Txnip did not alter glucose clearance compared with littermate controls, Txnip expression in the liver was required for maintaining normal fasting glycemia and glucose production. In addition, hepatic overexpression of a Txnip transgene in wild-type mice resulted in elevated serum glucose levels and decreased ketone levels. Liver homogenates from Txnip-null mice had no significant differences in the glutathione oxidation state or in the amount of available thioredoxin. However, overexpression of wild-type Txnip in Txnip-null hepatocytes rescued cellular glucose production, whereas overexpression of a C247S mutant Txnip, which does not bind thioredoxin, had no effect. These data demonstrate that Txnip is required for normal glucose homeostasis in the liver. While available thioredoxin is not changed in Txnip-null mice, the effects of Txnip on glucose homeostasis are abolished by a single cysteine mutation that inhibits binding to thioredoxin.

Hepatic glycogen synthase deficiency: an infrequently recognized cause of ketotic hypoglycemia.

The glycogen storage diseases comprise several inherited diseases caused by abnormalities of enzymes that regulate the synthesis or degradation of glycogen. In contrast to the classic hepatic glycogen storage diseases that are characterized by fasting hypoglycemia and hepatomegaly, the liver is not enlarged in GSD0. Patients with GSD0 typically have fasting ketotic hypoglycemia without prominent muscle symptoms. Most children are cognitively and developmentally normal. Short stature and osteopenia are common features, but other long-term complications, common in other types of GSD, have not been reported in GSD0. Until recently, the definitive diagnosis of GSD0 depended on the demonstration of decreased hepatic glycogen on a liver biopsy. The need for an invasive procedure may be one reason that this condition has been infrequently diagnosed. Mutation analysis of the GYS2 gene (12p12.2) is a non-invasive method for making this diagnosis in patients suspected to have this disorder. This mini-review discusses the pathophysiology of this disorder, use of mutation analysis to diagnose GSD0, and the clinical characteristics of all reported cases of GSD0.

Targeted disruption of the pituitary adenylate cyclase-activating polypeptide gene results in early postnatal death associated with dysfunction of lipid and carbohydrate metabolism.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a hormone belonging to the glucagon superfamily of hormones. These hormones are known to play important roles in metabolism and growth. PACAP is a neuropeptide that causes accumulation of cAMP in a number of tissues and affects the secretion of other hormones, vasodilation, neural and immune functions, as well as the cell cycle. To determine whether PACAP is essential for survival and to evaluate its function(s), we have generated mice lacking the PACAP gene via homologous recombination. We found that most PACAP null mice died in the second postnatal week in a wasted state with microvesicular fat accumulation in liver, skeletal muscle, and heart. Gas chromatography-mass spectrometry showed that fatty acid beta-oxidation in liver mitochondria of PACAP(-/-) mice was not blocked based on the distribution of 3-hydroxy-fatty acids (C6-16) in the plasma. Instead, increased metabolic flux through the beta-oxidation pathway was suggested by the presence of ketosis. Also, serum triglycerides and cholesterol were significantly higher (2- to 3-fold) in PACAP null mice than littermates. In the fed state, both serum insulin and blood glucose were normal in 5-d-old null mice compared with their littermates. In contrast, fasted PACAP null pups had a significant increase in insulin, but a decrease in blood glucose compared with littermates. Glycogen in the liver was reduced. These results suggest PACAP is a critical hormonal regulator of lipid and carbohydrate metabolism.

Hypoglycemia and resistance to ketoacidosis in a subject without functional insulin receptors.

Humans with congenital absence of the islets of Langerhans and mice rendered null for the insulin receptor rapidly develop severe hyperglycemia and ketoacidosis and, if untreated, die in the early neonatal period. In contrast, children with homozygous or compound heterozygous mutations of the insulin receptor gene, although hyperglycemic postprandially, survive for many months without developing ketoacidosis. Paradoxically, they often develop hypoglycemia. The rarity of the condition and the difficulties of undertaking metabolic studies in ill infants have limited the physiological information that might explain the clinical features. We studied a boy with Donohue's syndrome who represents a further example of the null phenotype, with two different and novel nonsense mutations in the alpha-subunit of the receptor. He survived for 8 months without developing ketoacidosis, and fasting hypoglycemia was a frequent problem. Despite the complete absence of insulin receptors, evidence for persistent insulin-like effects on fat and liver was seen; fasting plasma beta-hydroxybutyrate and nonesterified fatty acid levels were low, fell further during the early postprandial period, and failed to rise in response to hypoglycemia. The inverse relationships between plasma insulin and insulin-like growth factor-binding protein-1 levels were maintained, suggesting persistent hepatic effects of insulin. GH levels measured over a 6.5-h period were low throughout. Thus, the differences between congenital insulin deficiency vs. insulin receptor deficiency in humans may be explained by persistent insulinomimetic activity of the grossly elevated plasma insulin presumably being mediated through the type 1 insulin-like growth factor receptor. As GH plays a critical role in the regulation of ketogenesis during insulinopenia in humans, but not in rodents, this may contribute to the distinct phenotype of human vs. mouse insulin receptor knockouts.

Succinyl CoA: 3-oxoacid CoA transferase (SCOT): human cDNA cloning, human chromosomal mapping to 5p13, and mutation detection in a SCOT-deficient patient.

Succinyl CoA: 3-oxoacid CoA transferase (SCOT; E.C.2.8.3.5) mediates the rate-determining step of ketolysis in extrahepatic tissues, the esterification of acetoacetate to CoA for use in energy production. Hereditary SCOT deficiency in humans causes episodes of severe ketoacidosis. We obtained human-heart SCOT cDNA clones spanning the entire 1,560-nt coding sequence. Sequence alignment of the human SCOT peptides with other known CoA transferases revealed several conserved regions of potential functional importance. A single approximately 3.2-kb SCOT mRNA is present in human tissues (heart > leukocytes >> fibroblasts), but no signal is detectable in the human hepatoma cell line HepG2. We mapped the human SCOT locus (OXCT) to the cytogenetic band 5p13 by in situ hybridization. From fibroblasts of a patient with hereditary SCOT deficiency, we amplified and cloned cDNA fragments containing the entire SCOT coding sequence. We found a homozygous C-to-G transversion at nt 848, which changes the Ser 283 codon to a stop codon. This mutation (S283X) is incompatible with normal enzyme function and represents the first documentation of a pathogenic mutation in SCOT deficiency.

Genetic parameters for common health disorders of Holstein cows.

Observations on 7416 Canadian Holstein cows were examined to estimate genetic parameters for the most common diseases of dairy cows. Mastitis, ovarian cyst, ketosis, milk fever, abomasal displacement, and culling that is due to reproductive failure or leg problems were analyzed as binomial traits, assuming an underlying threshold model that included fixed and random effects. Sire and residual components of variance were estimated by REML to provide heritability estimates from paternal half-sibs. A multiple-trait mixed model was also used to estimate genetic and environmental correlations between production and disease traits. Heritabilities of disease traits were relatively low and ranged from 0 to .15, except for displaced abomasum (h2 = .28). Evidence of genetic antagonism existed between incidence of mastitis and milk production. Incidence of milk fever was genetically associated with cows of lower genetic potential for production. Genetic associations between displaced abomasum and production traits were small, and estimates of genetic correlations between ovarian cyst and milk production were inconsistent across lactations. Ketosis was antagonistically associated genetically with production of milk and fat but was favorably associated with production of protein. The long-term cumulative effect of genetic selection against diseases might be useful to diminish their incidence.