Beta-hydroxybutyrate Production Research Articles

Ketone production and excretion even during mild hyperglycemia and the impact of sodium-glucose co-transporter inhibition in type 1 diabetes

AimsWe aimed to determine if ketone production and excretion are increased even at mild fasting hyperglycemia in type 1 diabetes (T1D) and if these are modified by ketoacidosis risk factors, including sodium-glucose co-transporter inhibition (SGLTi) and female sex. MethodsIn secondary analysis of an 8-week single-arm open-label trial of empagliflozin (NCT01392560) we evaluated ketone concentrations during extended fasting and clamped euglycemia (4–6 mmol/L) and mild hyperglycemia (9–11 mmol/L) prior to and after treatment. Plasma and urine beta-hydroxybutyrate (BHB) concentrations and fractional excretion were analyzed by metabolomic analysis. ResultsForty participants (50 % female), aged 24 ± 5 years, HbA1c 8.0 ± 0.9 % (64 ± 0.08 mmol/mol) with T1D duration of 17.5 ± 7 years, were studied. Increased BHB production even during mild hyperglycemia (median urine 6.3[3.5–13.6] vs. 3.5[2.2–7.0] µmol/mmol creatinine during euglycemia, p < 0.001) was compensated by increased fractional excretion (0.9 % [0.3–1.6] vs. 0.4 % [0.2–0.9], p < 0.001). SGLTi increased production and attenuated the increased BHB fractional excretion (decreased to 0.3 % during mild hyperglycemia, p < 0.001), resulting in higher plasma concentrations (increased to 0.21 [0.05–0.40] mmol/L, p < 0.001), particularly in females (interaction p < 0.001). ConclusionsEven mild hyperglycemia is associated with greater ketone production, compensated by urinary excretion, in T1D. However, SGLTi exaggerates production and partially reduces compensatory excretion, particularly in women.

1219-P: Enhanced Response in Serum Ketone Level in Men Compared with Women by Administration of SGLT2 Inhibitor

SGLT2 inhibitors (SGLT2is) reduce hyperglycemia and insulin levels via increased urinary glucose excretion (UGE). Reduced insulin triggers decreased tissue glucose disposal and increased lipid use followed by ketone production. The metabolic response to starvation, the production of beta-hydroxybutyrate (BHB), was reported to be influenced by sex and menopausal status in healthy persons. However, little is known of sex- and menopausal status-specific effects of SGLT2is on BHB. This study investigated the effect of the SGLT2i tofogliflozin on BHB according to sex and menopausal status in type 2 diabetes mellitus (T2DM). Analyzed were 190 T2DM patients taking tofogliflozin as initial monotherapy in a Phase 3 study. Baseline characteristics were: men (66%); premenopausal (7%) and postmenopausal women (27%), age (mean 58y), and mean HbA1c (7.8%), BMI (26kg/m2), BHB (69µmol/L) and eGFR (83mL/min/1.73m2). Sex- and menopausal status-specific differences were analyzed by multivariate analysis. Longer duration of diabetes and lower baseline HDL-C and adiponectin were noted in men while age and HbA1c, BMI, BHB and eGFR were identical between sexes. Increases in BHB at weeks 4 and 52 were: men, median +128%* and +64*, respectively (+ p&amp;lt;0.01 vs. baseline); women, +48* and +58*, respectively. Mean increases in the fasting free fatty acid-to-insulin ratio (FIR) at week 52 were +88%* for men and +53* for women. Reductions in HbA1c and fasting insulin and increases in UGE and free fatty acids at week 52 were similar between sexes. Multivariate analyses showed a greater increase in BHB at week 52 in men than in women, especially postmenopausal women. Further, FIR increased greater in men than in women. Finally, premenopausal women had a greater increase in BHB than postmenopausal women. Results suggest that the different degrees of BHB elevation between sexes via tofogliflozin may provide clinical insights into sex- and menopause-specific metabolic responses to increased lipid use via SGLT2is in T2DM. Disclosure A. Yoshida: Employee; Self; Kowa Pharmaceutical Co. Ltd. Y. Matsubayashi: None. T. Nojima: Employee; Self; Kowa Co., Ltd. H. Suganami: Employee; Self; Kowa Co., Ltd. K. Kaku: Advisory Panel; Self; Astellas Pharma Inc., AstraZeneca, Boehringer Ingelheim Pharmaceuticals, Inc., Novo Nordisk A/S. Speaker's Bureau; Self; Eli Lilly and Company, Merck Sharp &amp; Dohme Corp., Mitsubishi Tanabe Pharma Corporation, Ono Pharmaceutical Co., Ltd., Taisho Pharmaceutical Co., Ltd., Takeda Pharmaceutical Company Limited. H. Sone: Research Support; Self; Astellas Pharma Inc., Boehringer Ingelheim Pharmaceuticals, Inc., Daiichi Sankyo Company, Limited, Kowa Pharmaceutical Europe Co. Ltd., Kyowa Hakko Kirin Co., Ltd., Novo Nordisk Inc., Ono Pharmaceutical Co., Ltd., Taisho Pharmaceutical Co., Ltd., Takeda Pharmaceutical Company Limited, Teijin Pharma Limited.

EFFECT OF SHEARING DURING PREGNANCY ON PRODUCTIVE PERFORMANCE IN THE POST-PARTUM PERIOD OF EWES ON EXTENSIVE HUSBANDRY

The use of shearing during pregnancy has been described as a tool for improving productivity in sheep and for minimizing perinatal mortality in lambs through the increase of fetal development. This study assessed the effect of shearing around 74 days of gestation on the productive performance of ewes and lambs during the first month of life. Forty Corriedale ewes were inseminated in autumn in Southern Brazil. All ewes were kept together at the same pasture under extensive husbandry conditions. The ewes were randomly separated into two treatment groups: twenty animals were completely sheared at 74 ± 6 days of pregnancy, and twenty were kept without sheared during pregnancy, composing the control group. Ewes and their lambs were evaluated at three different times during the experiment: at birth, between 15 and 21 days post-partum and between 22 and 45 days post-partum. Ewes had their body condition score, body weight, placental weight, milk production and serum concentrations of beta-hydroxybutyrate measured, while lambs had hematocrit, hemoglobin, and plasma lactate and glucose, as well as body weight at birth and until wean determined. Values of hematocrit and hemoglobin were lower and body weight at birth and at wean was higher in the group of lambs born from sheared ewes. Placenta weight was higher in sheared ewes. Body condition score and beta-hydroxybutyrate showed no differences between groups. Milk production of sheared ewes (1.26 L/day) was higher than in control group (0.93 L/day). Shearing ewes at 74 days of pregnancy was efficient for the better development of lambs at post-birth, reducing perinatal mortality rates.

EFEITO DA ESQUILA DURANTE A GESTAÇÃO SOBRE O DESEMPENHO PRODUTIVO NO PERÍODO PÓS-PARTO EM OVELHAS MANTIDAS EM MANEJO EXTENSIVO

The use of shearing during pregnancy has been described as a tool for improving productivity in sheep and for minimizing perinatal mortality in lambs through the increase of fetal development. This study assessed the effect of shearing around 74 days of gestation on the productive performance of ewes and lambs during the first month of life. Forty Corriedale ewes were inseminated in autumn in Southern Brazil. All ewes were kept together at the same pasture under extensive husbandry conditions. The ewes were randomly separated into two treatment groups: twenty animals were completely sheared at 74 ± 6 days of pregnancy, and twenty were kept without sheared during pregnancy, composing the control group. Ewes and their lambs were evaluated at three different times during the experiment: at birth, between 15 and 21 days post-partum and between 22 and 45 days post-partum. Ewes had their body condition score, body weight, placental weight, milk production and serum concentrations of beta-hydroxybutyrate measured, while lambs had hematocrit, hemoglobin, and plasma lactate and glucose, as well as body weight at birth and until wean determined. Values of hematocrit and hemoglobin were lower and body weight at birth and at wean was higher in the group of lambs born from sheared ewes. Placenta weight was higher in sheared ewes. Body condition score and beta-hydroxybutyrate showed no differences between groups. Milk production of sheared ewes (1.26 L/day) was higher than in control group (0.93 L/day). Shearing ewes at 74 days of pregnancy was efficient for the better development of lambs at post-birth, reducing perinatal mortality rates. Keywords: beta-hydroxybutyrate; lambs; milk yield; metabolism; placenta weight.

Evidence for treatable inborn errors of metabolism in a cohort of 187 Greek patients with autism spectrum disorder (ASD)

We screened for the presence of inborn errors of metabolism (IEM) in 187 children (105 males; 82 females, ages 4–14 years old) who presented with confirmed features of autism spectrum disorder (ASD). Twelve patients (7%) manifested increased 3-hydroxyisovaleric acid (3-OH-IVA) excretion in urine, and minor to significant improvement in autistic features was observed in seven patients following supplementation with biotin. Five diagnoses included: Lesch Nyhan syndrome (2), succinic semialdehyde dehydrogenase (SSADH) deficiency (2), and phenylketonuria (1) (2.7%). Additional metabolic disturbances suggestive of IEMs included two patients whose increased urine 3-OH-IVA was accompanied by elevated methylcitrate and lactate in sera, and 30 patients that showed abnormal glucose-loading tests. In the latter group, 16/30 patients manifested increased sera beta hydroxybutyrate (b-OH-b) production and 18/30 had a paradoxical increase of sera lactate. Six patients with elevated b-OH-b in sera showed improved autistic features following implementation of a ketogenic diet (KD). Five patients showed decreased serum ketone body production with glucose loading. Twelve of 187 patients demonstrated non-specific MRI pathology, while 25/187 had abnormal electroencephalogram (EEG) findings. Finally, family history was positive for 22/187 patients (1st or 2nd degree relative with comparable symptomatology) and consanguinity was documented for 12/187 patients. Our data provide evidence for a new biomarker (3-OH-IVA) and novel treatment approaches in ASD patients. Concise 1 sentence take-home message: Detailed metabolic screening in a Greek cohort of ASD patients revealed biomarkers (urine 3-hydroxyisovaleric acid and serum b-OH-b) in 7% (13/187) of patients for whom biotin supplementation or institution of a KD resulted in mild to significant clinical improvement in autistic features.

Interrelations between C4 Ketogenesis, C5 Ketogenesis, and Anaplerosis in the Perfused Rat Liver

We investigated the interrelations between C(4) ketogenesis (production of beta-hydroxybutyrate + acetoacetate), C(5) ketogenesis (production of beta-hydroxypentanoate + beta-ketopentanoate), and anaplerosis in isolated rat livers perfused with (13)C-labeled octanoate, heptanoate, or propionate. Mass isotopomer analysis of C(4) and C(5) ketone bodies and of related acyl-CoA esters reveal that C(4) and C(5) ketogenesis share the same pool of acetyl-CoA. Although the uptake of octanoate and heptanoate by the liver are similar, the rate of C(5) ketogenesis from heptanoate is much lower than the rate of C(4) ketogenesis from octanoate. This results from the channeling of the propionyl moiety of heptanoate into anaplerosis of the citric acid cycle. C(5) ketogenesis from propionate is virtually nil because acetoacyl-CoA thiolase does not favor the formation of beta-ketopentanoyl-CoA from propionyl-CoA and acetyl-CoA. Anaplerosis and gluconeogenesis from heptanoate are inhibited by octanoate. The data have implications for the design of diets for the treatment of long chain fatty acid oxidation disorders, such as the triheptanoin-based diet.

Effects of Dehydroepiandrosterone Administration on Rat Hepatic Metabolism Following Thermal Injury

Severe burns cause dramatic alterations in liver and whole-body metabolism. Recently, there has been interest in using dehydroepiandrosterone (DHEA) as a treatment for trauma patients, and enhanced survival and immune function have been reported using DHEA in animal trauma models. The specific effects of DHEA on hepatic metabolism following burn injury have not been explored. Male rats received either (1) a burn covering approximately 20% of the total body surface area or a sham burn or (2) burn injury followed by two intraperitoneal injections of DHEA or vehicle. After 4 days, the livers were isolated and perfused in vitro, and 28 metabolite fluxes were measured. Metabolic flux analysis was used to obtain the intracellular metabolic flux distribution and provide an overview of the metabolic state of the livers in each experimental group. Burn injury decreased the uptake of lactate and the production of beta-hydroxybutyrate and increased the deamination of glutamine to glutamate and asparagine to aspartate. DHEA, compared to vehicle treatment, decreased pentose phosphate pathway (PPP) fluxes and the uptake of several amino acids in burned rats. Furthermore, DHEA treatment restored liver metabolism in burned rats to a state that was very similar to that of the sham control group. DHEA administration appears to normalize hepatocellular metabolism in burned rats but also decreases the PPP flux, which may impair the liver's ability to recycle endogenous antioxidants. DHEA treatment combined with exogenous antioxidants should receive further consideration in the management of burn and trauma patients.

Liver fatty acid binding protein is required for high rates of hepatic fatty acid oxidation but not for the action of PPARalpha in fasting mice.

Liver fatty acid binding protein (L-FABP) has been proposed to limit the availability of long-chain fatty acids (LCFA) for oxidation and for peroxisome proliferator-activated receptor alpha (PPAR-alpha), a fatty acid binding transcription factor that determines the capacity of hepatic fatty acid oxidation. Here, we used L-FABP null mice to test this hypothesis. Under fasting conditions, this mutation reduced beta-hydroxybutyrate (BHB) plasma levels as well as BHB release and palmitic acid oxidation by isolated hepatocytes. However, the capacity for ketogenesis was not reduced: BHB plasma levels were restored by octanoate injection; BHB production and palmitic acid oxidation were normal in liver homogenates; and hepatic expression of key PPAR-alpha target (MCAD, mitochondrial HMG CoA synthase, ACO, CYP4A3) and other (CPT1, LCAD) genes of mitochondrial and extramitochondrial LCFA oxidation and ketogenesis remained at wild-type levels. During standard diet, mitochondrial HMG CoA synthase mRNA was selectively reduced in L-FABP null liver. These results suggest that under fasting conditions, hepatic L-FABP contributes to hepatic LCFA oxidation and ketogenesis by a nontranscriptional mechanism, whereas L-FABP can activate ketogenic gene expression in fed mice. Thus, the mechanisms whereby L-FABP affects fatty acid oxidation may vary with physiological condition.

Sheep rumen metabolic development in response to age and dietary treatments.

This study examined the time course of rumen metabolic development in the absence of solid feed consumption and the effect of delayed solid feed consumption on sheep rumen development. Twenty-seven lambs consumed milk replacer until slaughter at nine ages from 1 to 84 d (milk group). Three additional lambs consumed milk replacer from 1 to 48 d. From 49 d until slaughter at 84 d, these lambs were weaned onto solid feed (fed group). At slaughter, rumen contents were removed for VFA analysis and rumen epithelium was preserved for morphological examination. Rumen epithelial cells were isolated and incubated in media containing 2.5 mM U-[14C]-glucose or 10 mM 1-[14C]-butyrate. Rumen VFA concentrations did not change with age in lambs given milk replacer. At 84 d of age, intraruminal VFA concentrations were elevated in lambs consuming solid feed compared to 84-d-old lambs given milk replacer (P < .05). The number of ruminal papillae per square centimeter decreased (P < .05) while papillae length and width did not change significantly with age in rumen epithelium from lambs given milk replacer. At 84 d of age, rumen epithelium from lambs in the fed group had fewer and larger papillae/per square centimeter than rumen epithelium from lambs given milk replacer (P < .05). Rates of glucose and butyrate oxidation and acetoacetate and lactate production by rumen cells isolated from lambs given milk replacer did not change with age. Beta-hydroxybutyrate (BHBA) production was undetectable before 42 d of age in lambs given milk replacer and increased to levels found in conventionally raised adults by 84 d. At 84 d there were no differences in rates of glucose and butyrate oxidation or acetoacetate and lactate production by rumen cells between the two treatment groups. Thus, the change in substrate oxidation from glucose to butyrate, indicative of rumen metabolic maturation, does not occur in the absence of solid feed consumption. However, the development of rumen ketogenesis, as evidenced by increased BHBA production, does occur in the absence of solid feed consumption. Delaying the initiation of solid feed consumption results in rumen morphological development but does not stimulate rumen metabolic development. Increased intraruminal VFA concentrations, earlier exposure to VFA, or a longer period of exposure to VFA may be required to induce the genes responsible for rumen metabolic development.

Mechanism of the acceleration of CO2 production from pyruvate in liver mitochondria by HCO3-.

To investigate the mechanism by which HCO3- accelerates pyruvate metabolism in guinea pig liver mitochondria, we measured continuously, at pH 7.4 and 37 degrees C, 13C16O2 production from [1-13C]pyruvate by mass spectrometry and NADH concentration by fluorescence and analyzed total malate, citrate, and beta-hydroxybutyrate produced by standard biochemical methods. When [1-13C]pyruvate is added to the mitochondrial suspension, 13C16O2 concentration rises steeply in the first seconds and then slows to a steady lower rate. Carbonic anhydrase (CA) eliminates this initial phase, which shows that decarboxylation of pyruvate produces CO2, not HCO3-, and it does this more rapidly than it can equilibrate without CA. HCO3- (25 mM) increased 13C16O2 production, O2 consumption and total malate and citrate production and decreased NADH concentration and total beta-hydroxybutyrate production. After obtaining the total amount of 13C16O2, malate, citrate, and beta-hydroxybutyrate produced, we calculated that the addition of 25 mM HCO3- to the suspension medium increased the amount of pyruvate decarboxylated by pyruvate dehydrogenase (PDH) 16% and increased the amount carboxylated by pyruvate carboxylase 300%. This supports our initial proposal that HCO3- accelerates the pyruvate carboxylation, which in turn consumes ATP directly and NADH and acetyl CoA secondarily, all of which increase PDH activity. However, we found no acceleration of pyruvate decarboxylation by 0.5 and 1 microM free Ca2+ concentration, unless the mitochondria were uncoupled and ATP was added.

Energy state in HT-29 cells is linked to differentiation.

The relationship between the energy source used by HT-29 cells and their state of differentiation was determined. Short chain fatty acids and acetoacetate were applied to the cells for 9 d, after which the medium was replaced with conventional culture medium for a further 9 d so that the permanence of the changes could be assessed (18 d). Glucose utilization and lactic acid, acetoacetate, and beta-hydroxybutyrate production by the cells were determined. Differentiation was assessed by the presence of the enzymes sucrase-isomaltase and carbonic anhydrase 1, as well as morphological changes of the cells. By tracing carbon from acetate, propionate, and butyrate through the cells, it was found that the carbon from the short-chain fatty acids was fluxed into acetoacetate. Significant amounts of acetoacetate were released by the propionate-treated culture after 9 d and the acetate-, propionate-, valerate-, and caproate-treated cultures after 18 d. A significant positive correlation was found between acetoacetate synthesis and differentiation. Acetoacetate applied to HT-29 cells also induced their differentiation. The acetate-, butyrate-, valerate-, isovalerate-, and caproate-treated cells underwent terminal differentiation, while the propionate- and isocaproate-treated cultures underwent programming events. We, therefore, conclude that HT-29 cells utilize short chain fatty acids in preference to glucose, metabolize these to ketones, thereby raising the energy state and effecting the observed morphological and functional changes in the cells.

Patterns of nutrient interchange and oxygen use among portal-drained viscera, liver, and hindquarters of beef steers from 235 to 525 kg body weight.

Our objective was to quantify changes in supply and use of nutrients and O2 by large-frame, multicatheterized beef steers as they grew from 235 to 525 kg BW. Steers consumed 5.25 to 9.87 kg DM/d of a 62% concentrate diet that provided 126 to 217 g N/d and 1 kg ADG. Steers were assigned to three groups (eight, nine, and eight steers each) that divided the BW range into thirds. Weights at first sampling for the three groups were 236, 319, and 445 kg, respectively. Each group was sampled twice. Groups were killed after the second sampling. Tissue weights and hindquarters (HQ) contents of fat, protein, and ash were measured. Blood flow, oxygen uptake, and net uptake or release of metabolites were regressed against functions of BW.75 to assess changes during growth. Blood flow in all tissues except liver and oxygen use by all tissues decreased per unit tissue weight as BW.75 and age increased. Changes with age per unit liver weight were as follows: decreased uptake of propionate and lactate, increased uptake of alpha-amino N and glutamine, decreased production of urea and glutamate, and increased production of acetate and beta-hydroxybutyrate. Glucose and urea production per unit liver weight was constant. Changes with age per unit HQ weight were as follows: increased uptake of glucose, decreased uptake of alpha-amino N and glutamate, decreased release of lactate, and increased release of glutamine. Weight of the portal-drained viscera (PDV) increased from 91 to 97 g/kg EBW as BW increased from 236 to 522 kg; PDV fat increased from 375 to 552 g/kg PDV tissues. Liver decreased from 16 to 12 g/kg EBW. Hindquarters decreased from 286 to 266 g/kg EBW; HQ protein was 200, 197, and 200 g/kg HQ tissue for Groups 1, 2, and 3, respectively. Corresponding fat was 131, 182, and 177 g/kg HQ tissue. Changes in net flux reflect changes in nutrient partitioning and tissue deposition as steers grew and aged.

Metabolism of alpha-ketoisocaproic acid in isolated perfused liver of cirrhotic rats.

To determine the hepatic fate of alpha-ketoisocaproate (KIC) in cirrhosis, six groups of isolated rat livers were perfused with 0, 0.5, 1 (with or without alpha-[1-14C]KIC), 2, and 5 mM KIC; control livers from healthy rats were studied in parallel under similar conditions. KIC was rapidly removed by the normal livers, whereas uptake was lower in the cirrhotic livers at all concentrations tested (at 2 mM, 4.04 +/- 0.33 vs. 6.32 +/- 0.58 mumol/min; P < or = 0.05). The transamination pathway, evaluated by leucine exchanges, was more important in the cirrhotic livers (25.4 vs. 6.8% in controls at 2 mM). The incorporation of alpha-[1-14C]KIC in proteins of cirrhotic liver was increased compared with controls (0.25 +/- 0.04% of alpha-[1-14C]KIC was incorporated in proteins excreted in perfusate vs. 0.20 +/- 0.04 in controls; P < or = 0.05). In addition, a line of evidence suggests that glutamine rather than glutamate is the N donor for leucine synthesis from KIC. The decarboxylation pathway evaluated by beta-hydroxybutyrate production and by 14CO2 release from alpha-[1-14C]KIC was reduced, respectively, by 40-85% (according to KIC dose) and by 24% at 90 min in cirrhotic livers compared with healthy livers. These results indicate a dramatic modification of KIC metabolism in the cirrhotic liver; its uptake by the liver is decreased and its incorporation into proteins is increased via an enhancement of transamination to leucine, probably as a consequence of an inhibition of branched-chain keto acid dehydrogenase.

Mechanisms of impaired hepatic fatty acid metabolism in rats with long-term bile duct ligation.

Hepatic metabolism of fatty acids is impaired in experimental animals with long-term bile duct ligation. To characterize the underlying defects, fatty acid metabolism was investigated in isolated hepatocytes and isolated liver mitochondria from rats subjected to long-term bile duct ligation or sham surgery. After starvation for 24 hr, the plasma beta-hydroxybutyrate concentration was decreased in rats with bile duct ligation as compared with control rats. Production of beta-hydroxybutyrate from butyrate, octanoate and palmitate by hepatocytes isolated from rats subjected to bile duct ligation was also decreased. Liver mitochondria from rats subjected to bile duct ligation showed decreased state 3 oxidation rates for L-glutamate, succinate, duroquinone, and fatty acids but not for ascorbate as substrate. State 3u oxidation rates (uncoupling with dinitrophenol) and activities of mitochondrial oxidases were also decreased in mitochondria from rats subjected to bile duct ligation. Direct assessment of the activities of the subunits of the electron transport chain revealed reduced activities of complex I, complex II and complex III in mitochondria from rats subjected to bile duct ligation. Activities of the beta-oxidation enzymes specific for short-chain fatty acids were all reduced in rats subjected to bile duct ligation. Mitochondrial protein content per hepatocyte was increased by 32% in rats subjected to bile duct ligation compared with control rats. Thus the studies directly demonstrate mitochondrial defects in fatty acid oxidation in rats subjected to bile duct ligation, which explain decreased ketosis during starvation.

The Effects of Acute Hypercortisolemia on β-Hydroxybutyrate and Glycerol Metabolism During Insulin Deficiency

The present study was undertaken to determine whether an acute physiologic rise in plasma cortisol during selective insulin deficiency would have significant effects on glycerol and beta-hydroxybutyrate metabolism in conscious overnight-fasted dogs. Each experiment consisted of a two hour dye equilibration period, a 40 minute basal period, and a 3 hour experimental period. A continuous infusion of indocyanine green dye for blood flow estimation was initiated at the start of the equilibration period and continued throughout the experiment. In both of two protocols selective insulin deficiency was created during the experimental period by infusing somatostatin peripherally (0.8 microgram/kg-min) with basal replacement of glucagon intraportally (0.65 ng/kg-min). In the test protocol (CORTISOL, n = 5), 3.0 micrograms/kg-min of hydrocortisone was infused during the experimental period. In the control protocol (SALINE, n = 5), saline was infused. Net hepatic balances were determined using the (A-V) difference technique. During selective insulin deficiency alone (SALINE), the arterial blood glycerol level increased from 81 +/- 19 to 140 +/- 11 microM (p < 0.01) and net hepatic glycerol uptake (NHGlyU) tended to increase from 2.3 +/- 0.3 to 3.3 +/- 0.6 mumol/kg-min (0.05 < 0.1). The arterial plasma free fatty acid (FFA) level remained unchanged at 1041 +/- 35 microM. The arterial beta-hydroxybutyrate (BHOB) level increased slightly from 21 +/- 4 to 29 +/- 5 microM while net hepatic beta-hydroxybutyrate production (NHBP) remained unchanged (1.0 +/- 0.2 mumol/kg-min). During acute hypercortisolemia with selective insulin deficiency (CORTISOL), similar changes occurred in the arterial blood glycerol level and net hepatic glycerol uptake.(ABSTRACT TRUNCATED AT 250 WORDS)

Technical note: isolation and characterization of sheep ruminal epithelial cells

A system for the isolation and characterization of sheep ruminal epithelial cells has been developed. Ruminal papillae from the ventral cranial sac of 12- to 24-wk-old Dorset ram lambs were subjected to serial tryptic digestions. Initial digestions (two 15-min periods) in the series were used to remove keratinized cells of the stratum corneum, which were able to produce only small amounts of beta-hydroxybutyrate from butyrate (5.37 +/- .72 nmol/120 min/per milligram of dry cell weight). Later digest fractions, containing primarily cells from the stratum basale, exhibited high viabilities (75 to 95%) and proved capable of converting butyrate to beta-hydroxybutyrate at relatively high rates (33.6 +/- 6.7 nmol/120 min per milligram of dry cell weight). Neither acetate nor propionate underwent significant conversion to beta-hydroxybutyrate. However, addition of acetate inhibited (77.4% of control) and addition of propionate stimulated (200% of control) beta-hydroxybutyrate production. Acetate addition reduced the propionate-induced stimulation of beta-hydroxybutyrate production from butyrate (136% of control). These results are similar to those obtained from in vitro incubations of ruminal papillae and suggest that an isolated cell system may prove useful in the further study of ruminal epithelial metabolism.

Effects of extracellular pH, CO2, and HCO3- on ketogenesis in perfused rat liver

Effects of extracellular pH, CO2, and HCO3- on ketone body production from octanoate were studied in perfused livers from fasted rats. pH was adjusted to 7.1-7.5 by varying perfusate [HCO3-] and [CO2], where brackets denote concentration. At constant 25 mM [HCO3-], total production of beta-hydroxybutyrate (beta-OHB) + acetoacetate (AcAc) was constant from pH 7.1 to 7.5. However, the [beta-OHB]/[AcAc] ratio decreased from 1.60 to 1.00 when pH decreased from 7.3 to 7.1; there was no change at pH 7.4. At constant [CO2], decreasing pH from 7.4 to 7.1 did not alter either total ketogenesis or the [beta-OHB]/[AcAc] ratio. This suggests that high [CO2] rather than low pH was responsible for the alteration in the redox ratio. At constant pH of 7.4, variations in [HCO3-] between 15 and 25 mM did not influence total ketogenesis or the [beta-OHB]/[AcAc] ratio. However, increasing [HCO3-] from 25 to 35 mM decreased the [beta-OHB]/[AcAc] ratio from 1.76 to approximately 1, again without affecting total ketogenesis. At constant 1.75 mM [CO2], increasing [HCO3-] from 25 to 35 mM also reduced the [beta-OHB]/[AcAc] ratio from 1.63 to approximately 1, suggesting that the effect of high [HCO3-] on this redox ratio can be ascribed to HCO3- itself. It is concluded that high [CO2] or [HCO3-] decreases the mitochondrial [NADH]/[NAD+] ratio in hepatocytes, resulting in a decreased [beta-OHB]/[AcAc] ratio.

Carnitine Status and Lipid Utilization in Neonatal Piglets Fed Diets Low in Carnitine

Forty-eight newborn piglets were assigned to dietary regimens to examine carnitine status and lipid utilization by piglets nursing sows (Diet 1), fed a high-carnitine (1507 mumol carnitine/kg dry matter) casein-whey formula (Diet 2) or fed a low-carnitine (35 mumol carnitine/kg dry matter) egg white protein formula (Diet 3). Four piglets were killed at birth, and four per dietary group were killed at 3, 7, 14 and 21 d of age. Piglets fed Diet 3 had lower (P less than 0.01) carnitine in plasma and liver by d 7 and carnitine remained lower to d 21. Heart carnitine tended to be lower in piglets fed Diet 3, but the differences were not significant. Longissimus muscle carnitine was not affected. In vitro palmitate oxidation was lower (P less than 0.05) on d 7 in the liver from piglets fed Diet 3 but not on d 3, 14 and 21. Low dietary carnitine did not affect in vitro liver beta-hydroxybutyrate production or oxidation of palmitate by longissimus muscle. Plasma glucose and nonesterified fatty acids were higher (P less than 0.05) in piglets nursing sows than those fed Diets 2 or 3. Piglets receiving Diet 3 had reduced carnitine in plasma and certain tissues, and liver lipid oxidation was depressed on d 7 of age, compared with those receiving the other two diets. Feeding the low carnitine diet did not alter glucose status or plasma lipids and ketones.

Effect of cyclosporin A, azathioprine, and prednisolone on carbohydrate metabolism of rat hepatocytes

The effect of different immunosuppressive drugs (prednisolone, azathioprine, cyclosporin A) on liver carbohydrate metabolism in the rat was investigated. Daily administration of prednisolone (3 mg/kg body weight) and azathioprine (2 mg/kg body weight) intraperitoneally for 2 weeks caused significantly lower liver glycogen content than that in NaCl-treated controls. Liver glucose and lactate content, as well as plasma glucose, glucagon, and serum insulin concentration of these animals, remained unchanged. There were no differences in any of these parameters between cyclosporin A (15 mg/kg body weight)-treated and vehicle (olive oil/ethanol)-treated animals. Prednisolone caused significantly lower glucose production in isolated rat hepatocytes using Na-pyruvate as the substrate, whereas glucose production was unchanged in hepatocytes of azathioprine-treated rats using pyruvate or L-serine as substrates. Glucose production from pyruvate or serine was significantly inhibited by cyclosporin A compared to the vehicle, but did not differ from the effects of azathioprine and prednisolone. Lactate production was significantly lower in cyclosporin-treated animals than in those given either the vehicle or azathioprine. Cyclosporin A completely reversed the inhibition of hepatocyte glycogen consumption caused by the vehicle. However, glycogen production in the presence of cyclosporin A was comparable to the effects of prednisolone and azathioprine. Finally, hepatocyte ketone body production using pyruvate as the substrate was higher in the presence of all immunosuppressive drugs. In the presence of serine, acetoacetate production increased in rats treated with 50 mg/kg body weight cyclosporin A, and beta-hydroxybutyrate production in animals receiving 15 and 50 mg/kg body weight cyclosporin A.

Effect of L-carnitine on ketone bodies, redox state and free amino acids in the liver of hyperammonemic mice.

L-Carnitine stimulates urea synthesis in mice given a LD100 of ammonium acetate. Unprotected mice show decreased levels of hepatic ketone bodies and lowered NADH/NAD+ ratio in both cytosol and mitochondria. L-Carnitine enhances markedly the production of beta-hydroxybutyrate and raises the NADH/NAD+ ratio in mitochondria. The alterations induced by ammonium acetate in the free amino acid pool are prevented by L-carnitine. The results shown in this paper indicate that L-carnitine stimulates fatty acid oxidation as well as flux through the Krebs cycle in hyperammonemic mice and that these effects may be responsible for the increase in urea synthesis in these animals.