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Nonalcoholic fatty liver disease is common in developed countries and is associated with obesity, metabolic syndrome, and type 2 diabetes. T deficiency is a risk factor for developing these metabolic deficiencies, but its role in hepatic steatosis has not been well studied. We investigated the effects of T on the pathogenesis of hepatic steatosis in rats fed a high-fat diet (HFD). Adult male rats were randomly placed into four groups and treated for 15 weeks: intact rats on regular chow diet (RCD), intact rats on liquid HFD (I+HFD), castrated rats on HFD (C+HFD), and castrated rats with T replacement on HFD (C+HFD+T). Fat contributed 71% energy to the HFD but only 16% of energy to the RCD. Serum T level was undetectable in castrated rats, and T replacement led to 2-fold higher mean serum T levels than in intact rats. C+HFD rats gained less weight but had higher percentage body fat than C+HFD+T. Severe micro- and macrovesicular fat accumulated in hepatocytes with multiple inflammatory foci in the livers of C+HFD. I+HFD and C+HFD+T hepatocytes demonstrated only mild to moderate microvesicular steatosis. T replacement attenuated HFD-induced hepatocyte apoptosis in castrated rats. Serum glucose and insulin levels were not increased with HFD in any group. Immunoblots showed that insulin-regulated proteins were not changed in any group. This study demonstrates that T deficiency may contribute to the severity of hepatic steatosis and T may play a protective role in hepatic steatosis and nonalcoholic fatty liver disease development without insulin resistance.

Despite altered regulation of insulin signaling, Pten(+/-) heterodeficient standard diet-fed mice, approximately 4 months old, exhibit normal fasting glucose and insulin levels. We report here a stable isotope flux phenotyping study of this "silent" phenotype, in which tissue-specific insulin effects in whole-body Pten(+/-)-deficient mice were dissected in vivo. Flux phenotyping showed gain of function in Pten(+/-) mice, seen as increased peripheral glucose disposal, and compensation by a metabolic feedback mechanism that 1) decreases hepatic glucose recycling via suppression of glucokinase expression in the basal state to preserve hepatic glucose production and 2) increases hepatic responsiveness in the fasted-to-fed transition. In Pten(+/-) mice, hepatic gene expression of glucokinase was 10-fold less than wild-type (Pten(+/+)) mice in the fasted state and reached Pten(+/+) values in the fed state. Glucose-6-phosphatase expression was the same for Pten(+/-) and Pten(+/+) mice in the fasted state, and its expression for Pten(+/-) was 25% of Pten(+/+) in the fed state. This study demonstrates how intra- and interorgan flux compensations can preserve glucose homeostasis (despite a specific gene defect that accelerates glucose disposal) and how flux phenotyping can dissect these tissue-specific flux compensations in mice presenting with a "silent" phenotype.

Background: Low-birth-weight (LBW) infants have high energy requirements and are dependent on high fat intake to maintain adequate postnatal growth. Fat energy is transported in plasma as triglycerides, which are either derived from the diet or from de novo lipogenesis (DNL). It is our hypothesis that DNL plays an important physiologic role in adapting to exclusive breast milk (EBM) feeding or to parenteral nutrition (PN)., Methods: We studied hepatic de novo lipogenesis in 14 LBW (<34-week gestation) appropriate for gestational age and receiving either EBM feedings or full PN support. Stable isotope tracer [2-(13)C] acetate was administered for 72 hours to achieve an estimated 10% enrichment of daily fat intake. Fatty acids were extracted from plasma for gas chromatography-mass spectrometry analyses., Results: Percent new synthesis of palmitate was 13.1% +/- 2.5% in the EBM group and 14.9% +/- 0.7% in the PN group (NS), stearate was 11.1% +/- 2.7% in the EBM group and 10.6% +/- 14% in the PN group (NS) and cholesterol was 12.7% +/- 2.1% in the EBM group and 17.4% +/- 4.6% in the PN group (NS) after 72 hours of tracer administration (mean +/- SEM). The plasma lipid fatty acid composition in palmitate, oleate, and stearate with intake of 3.6 +/- 0.6 g/kg/d of IV lipids (ILs) was similar to EBM-feeding infants taking 6.3 +/- 0.13 g/kg/d of fat., Conclusions: De novo lipogenesis is active in stable LBW infants maintaining standard postnatal growth. Hepatic DNL permits newborn infants to meet the fat energy needs of peripheral tissues for growth and storage and to maintain plasma fatty acid composition in adaptation to different dietary fat intake.

Increased glucose cycling between glucose and glucose-6-phosphate is characteristic of insulin resistance and hyperglycemia seen with Type II diabetes. Traditionally, glucose cycling is determined by the difference between hepatic glucose output measured with separate [2-3H]glucose and [6-3H]glucose infusions. We demonstrate a novel method for determining hepatic glucose recycling from an intraperitoneal glucose tolerance test (IPGTT). A single tracer, [1, 2-13C(2)]glucose (a M2 glucose isotopomer), was administered at 1mg/g body weight to 4-month-old C57BL/6 mice. Hepatic glucose recycling was monitored by the appearance of a plasma M1 isotopomer of glucose, which is produced by the action of the pentose cycle on the M2 glucose isotopomer in the liver. The initial M2 enrichment was 56% and decreased to 13% at the end of 3 h, and the M1 enrichment peaked at 2 h. The ratio of plasma M1/M2 glucose increased linearly with time to approximately 25%, and the regression of the M1/M2 ratio against time gives a slope, termed the in vivo glucose-dependent futile recycling rate constant k(HR). k(HR) estimates glucose/glucose-6-phosphate futile cycling, along with glucose recycling through the pentose cycle. These observations demonstrate complex substrate cycling during an IPGTT using a single stable isotope tracer., (Copyright 2003 Elsevier Science (USA))

The hypoglycemia seen in the fasting PPARalpha null mouse is thought to be due to impaired liver fatty acid beta-oxidation. The etiology of hypoglycemia in the PPARalpha null mouse was determined via stable isotope studies. Glucose, lactate, and glycerol flux was assessed in the fasted and fed states in 4-month-old PPARalpha null mice and in C57BL/6 WT maintained on standard chow using a new protocol for flux assessment in the fasted and fed states. Hepatic glucose production (HGP) and glucose carbon recycling were estimated using [U-(13)C(6)]glucose, and HGP, lactate, and glycerol turnover was estimated utilizing either [U-(13)C(3)]lactate or [2-(13)C]glycerol infused subcutaneously via Alza miniosmotic pumps. At the end of a 17-h fast, HGP was higher in the PPARalpha null mice than in WT by 37% (p < 0.01). However, recycling of glucose carbon from lactate back to glucose was lower in the PPARalpha null than in WT (39% versus 51%, p < 0.02). The lack of conversion of lactate to glucose was confirmed using an [U-(13)C(3)]lactate infusion. In the fasted state, HGP from lactate and lactate production were decreased by 65 and 55%, respectively (p < 0.05) in PPARalpha null mice. In contrast, when [2-(13)C]glycerol was infused, glycerol production and HGP from glycerol increased by 80 and 250%, respectively (p < 0.01), in the fasted state of PPARalpha null mice. The increased HGP from glycerol was not suppressed in the fed state. While little change was evident for phosphoenolpyruvate carboxykinase (PEPCK) expression, pyruvate kinase expression was decreased 16-fold in fasted PPARalpha null mice as compared with the wild-type control. The fasted and fed insulin levels were comparable, but blood glucose levels were lower in the PPARalpha null mice than in controls. In conclusion, PPARalpha receptor function creates a setpoint for a metabolic network that regulates the rate and route of HGP in the fasted and fed states, in part, by controlling the flux of glycerol and lactate between the triose-phosphate and pyruvate/lactate pools.

The synthesis of palmitate, stearate, and cholesterol in liver and nervous tissues (brain, cord, and nerve) of Sprague-Dawley rats was determined using deuterated water (D2O) and mass isotopomer analysis. Rats were given 4% deuterium in their drinking water after each receiving an intraperitoneal priming dose. Animals were killed at 1, 2, 4, and 8 wk for deuterium enrichment in body water and determination of mass isotopomer distribution in lipids from various tissues. In 1 wk, the enrichment in the body water reached a plateau of 2.6%, which is 65% of that in the drinking water. We observed the maximum incorporation number (N) in all lipids to be higher than those previously observed, being 22, 24, and 30 for liver palmitate, stearate, and cholesterol, respectively, and N may vary among tissues. Using a single exponential model, we found the half-time (t1/2) and the plateau levels of the newly synthesized lipids of the nervous tissues (t1/2 values ranging from 5 to 28 days) to be different from those of the liver (t1/2 values < or = 4 days) in this relatively long-term study. Mass isotopomer distribution analysis and D2O can be used not only to quantitate the replacement rate of many lipids in various compartments but may also be used to elucidate the tissue-specific synthetic pathways from N.

Fasted rats were intragastrically infused with either [2,3-13C]lactate or [1,2,3-13C]lactate. The infusate also contained 14C-labeled lactate and [3-3H]glucose. Glucose, alanine, glutamate, and glutamine were isolated from liver and blood. There was near complete equilibration of lactate and alanine, and the relative specific activities and relative enrichments were the same in blood and liver. Glucose was cleaved enzymatically to lactate. The compounds were examined by gas chromatography-mass spectroscopy. From the mass isotopomer spectra of the lactate, glutamate, and glutamine and their cleavage fragments the positional isotopomer composition of these compounds was obtained. The enrichment and isotopomer pattern in the lactate from cleaved glucose represents that in phosphoenolpyruvate (PEP). When [1,2,3-13C]lactate was infused the mass isotopomer spectrum of glutamates consisted only of compounds containing either one, two, or three 13C carbons per molecule (m1, m2, and m3). There was little 13C in C-4 and C-5 of glutamate. The rate of pyruvate decarboxylation is low, and 3-4% of the acetyl-CoA flux in the Krebs cycle is contributed by lactate carbon. The major isotopomers in lactate, alanine, and PEP were m3 and m2 with 13C in C-2 and C-3. The predominant isotopomer in PEP from [2,3-13C]lactate was m2 with 13C in C-2 and C-3. There was much more of m1 isotopomer with 13C in C-3 and C-2 than the m1 isotopomer with 13C in C-1. There was very little m3, the isotopomer with 13C in all three carbons. Most of the 13C in C-3 and C-4 of glucose and C-1 of glutamate was introduced via 13CO2 fixation. From the isotopomer distribution and the rate of glucose turnover we deduced, applying the analysis described in the "Appendix," the absolute rates of gluconeogenic pathways, recycling of PEP and the Cori cycle, and flux in the Krebs cycle. The flux from oxaloacetate (OAA)-->PEP was seven times that of OAA-->citrate, and about half of PEP was recycled to pyruvate via pyruvate kinase. The mass isotopomer patterns in glutamate and glutamine were similar but differed from those of lactate and glucose. It appears that the glutamates are derived from alpha-ketoglutarate from a different Krebs cycle pool than PEP. The flux from OAA to PEP in this pool was two to three times that of OAA to citrate.(ABSTRACT TRUNCATED AT 400 WORDS)

Rats were infused with glucose at 30 mg/min, containing 18% enriched [U-13C]glucose and [1-14C]- and [3-3H]glucose. The mass isotopomer patterns of 13C-labeled blood glucose and liver glycogen were determined by gas chromatography/mass spectroscopy. The contribution of the direct pathway to glycogen was calculated from the three tracers, and the values by all three were nearly identical, about 50%. The 14C specific activity in carbon 6 of glycogen glucose was about 6% that of carbon 1. The [3H]glucose/[1-14C]glucose ratio in glycogen was 80-90% that in blood glucose. The enrichment of 13C and the specific activity of 14C in glycogen formed by the indirect path were 20-25% of glycogen formed directly from glucose. The dilution is of two kinds: (i) an exchange of labeled carbon with unlabeled carbon in the tricarboxylic acid cycle and (ii) dilution by unlabeled nonglucose carbon. Methods to calculate the two types of dilution are presented. In control rats the dilution factor by exchange in the tricarboxylic acid cycle is 1.4, and the dilution by unlabeled carbon is 2.5- to 3.0-fold, with the overall dilution about 4-fold. In rats preinjected with glucagon, the dilution through the tricarboxylic acid cycle was unaffected but that by nonglucose carbon was decreased.

Hepatic steatosis is associated with increased insulin resistance and tricarboxylic acid (TCA) cycle flux, but decreased ketogenesis and pyruvate dehydrogenase complex (PDC) flux. This study examined whether hepatic PDC activation by inhibition of pyruvate dehydrogenase kinase 2 (PDK2) ameliorates these metabolic abnormalities. Wild-type mice fed a high-fat diet exhibited hepatic steatosis, insulin resistance, and increased levels of pyruvate, TCA cycle intermediates, and malonyl-CoA but reduced ketogenesis and PDC activity due to PDK2 induction. Hepatic PDC activation by PDK2 inhibition attenuated hepatic steatosis, improved hepatic insulin sensitivity, reduced hepatic glucose production, increased capacity for β-oxidation and ketogenesis, and decreased the capacity for lipogenesis. These results were attributed to altered enzymatic capacities and a reduction in TCA anaplerosis that limited the availability of oxaloacetate for the TCA cycle, which promoted ketogenesis. The current study reports that increasing hepatic PDC activity by inhibition of PDK2 ameliorates hepatic steatosis and insulin sensitivity by regulating TCA cycle anaplerosis and ketogenesis. The findings suggest PDK2 is a potential therapeutic target for nonalcoholic fatty liver disease. [ABSTRACT FROM AUTHOR]