Using [2H]water to quantify the contribution of de novo palmitate synthesis in plasma: enabling back-to-back studies.

An increased contribution of de novo lipogenesis (DNL) may play a role in cases of dyslipidemia and adipose accretion; this suggests that inhibition of fatty acid synthesis may affect clinical phenotypes. Since it is not clear whether modulation of one step in the lipogenic pathway is more important than another, the use of tracer methods can provide a deeper level of insight regarding the control of metabolic activity. Although [2H]water is generally considered a reliable tracer for quantifying DNL in vivo (it yields a homogenous and quantifiable precursor labeling), the relatively long half-life of body water is thought to limit the ability of performing repeat studies in the same subjects; this can create a bottleneck in the development and evaluation of novel therapeutics for inhibiting DNL. Herein, we demonstrate the ability to perform back-to-back studies of DNL using [2H]water. However, this work uncovered special circumstances that affect the data interpretation, i.e., it is possible to obtain seemingly negative values for DNL. Using a rodent model, we have identified a physiological mechanism that explains the data. We show that one can use [2H]water to test inhibitors of DNL by performing back-to-back studies in higher species [i.e., treat nonhuman primates with platensimycin, an inhibitor of fatty acid synthase]; studies also demonstrate the unsuitability of [13C]acetate.

siRNA-mediated inhibition of SREBP cleavage-activating protein reduces dyslipidemia in spontaneously dysmetabolic rhesus monkeys.

BACKGROUND: SREBP cleavage-activating protein (SCAP) is a cholesterol binding endoplasmic reticulum (ER) membrane protein that is required to activate SREBP transcription factors. SREBPs regulate genes involved in lipid biosynthesis. They also influence lipid clearance by modulating the expression of LDL receptor (LDLR) and proprotein convertase subtilisin/kexin type 9 (PCSK9) genes. Inhibiting SCAP decreases circulating PCSK9, triglycerides (TG), and LDL-cholesterol (LDL-C), both in vitro and in vivo. Type 2 diabetics with dyslipidemia are at high risk for cardiovascular diseases. These patients present a unique pathophysiological lipid profile characterized by moderately elevated LDL-C, elevated TG and reduced HDL-cholesterol (HDL-C). The spontaneous dysmetabolic rhesus monkey model (DysMet RhM) recapitulates this human dyslipidemia and therefore is an attractive preclinical model to evaluate SCAP inhibition as a therapy for this disease population. The objective to of this study was to assess the effect of SCAP inhibition on the lipid profile of DysMet RhM. METHOD: We assessed the effect of inhibiting hepatic SCAP on the lipid profile of DysMet RhM using an siRNA encapsulated lipid nanoparticle (siRNA-LNP). RESULTS: The SCAP siRNA-LNP significantly reduced LDL-C, PCSK9 and TG in DysMet RhM; LDL-C was reduced by ≥20%, circulating PCSK9 by 30-40% and TG by >25%. These changes by the SCAP siRNA-LNP agree with the predicted effect of SCAP inhibition and reduced SREBP tone on these endpoints. CONCLUSION: These data demonstrate that a SCAP siRNA-LNP improved the lipid profile in a clinically relevant preclinical disease model and provide evidence for SCAP inhibition as a therapy for diabetic dyslipidemic patients.

Pioglitazone increases whole body insulin sensitivity in obese, insulin-resistant rhesus monkeys.

Hyperinsulinemic-euglycemic clamps are considered the "gold standard" for assessing whole body insulin sensitivity. When used in combination with tracer dilution techniques and physiological insulin concentrations, insulin sensitization can be dissected and attributed to hepatic and peripheral (primarily muscle) effects. Non-human primates (NHPs), such as rhesus monkeys, are the closest pre-clinical species to humans, and thus serve as an ideal model for testing of compound efficacy to support translation to human efficacy. We determined insulin infusion rates that resulted in high physiological insulin concentrations that elicited maximal pharmacodynamic responses during hyperinsulinemic-euglycemic clamps. These rates were then used with [U-13C]-D-glucose, to assess and document the degrees of hepatic and peripheral insulin resistance between healthy and insulin-resistant, dysmetabolic NHPs. Next, dysmetabolic NHPs were treated for 28 days with pioglitazone (3 mg/kg) and again had their insulin sensitivity assessed, illustrating a significant improvement in hepatic and peripheral insulin sensitivity. This coincided with a significant increase in insulin clearance, and normalization of circulating adiponectin. In conclusion, we have determined a physiological clamp paradigm (similar to humans) for assessing glucose turnover in NHPs. We have also demonstrated that insulin-resistant, dysmetabolic NHPs respond to the established insulin sensitizer, pioglitazone, thus confirming their use as an ideal pre-clinical translational model to assess insulin sensitizing compounds.

Demonstration of diet-induced decoupling of fatty acid and cholesterol synthesis by combining gene expression array and 2H2O quantification.

The liver is a crossroad for metabolism of lipid and carbohydrates, with acetyl-CoA serving as an important metabolic intermediate and a precursor for fatty acid and cholesterol biosynthesis pathways. A better understanding of the regulation of these pathways requires an experimental approach that provides both quantitative metabolic flux measurements and mechanistic insight. Under conditions of high carbohydrate availability, excess carbon is converted into free fatty acids and triglyceride for storage, but it is not clear how excessive carbohydrate availability affects cholesterol biosynthesis. To address this, C57BL/6J mice were fed either a low-fat, high-carbohydrate diet or a high-fat, carbohydrate-free diet. At the end of the dietary intervention, the two groups received (2)H(2)O to trace de novo fatty acid and cholesterol synthesis, and livers were collected for gene expression analysis. Expression of lipid and glucose metabolism genes was determined using a custom-designed pathway focused PCR-based gene expression array. The expression analysis showed downregulation of cholesterol biosynthesis genes and upregulation of fatty acid synthesis genes in mice receiving the high-carbohydrate diet compared with the carbohydrate-free diet. In support of these findings, (2)H(2)O tracer data showed that fatty acid synthesis was increased 10-fold and cholesterol synthesis was reduced by 1.6-fold in mice fed the respective diets. In conclusion, by applying gene expression analysis and tracer methodology, we show that fatty acid and cholesterol synthesis are differentially regulated when the carbohydrate intake in mice is altered.

Quantifying cholesterol synthesis in vivo using (2)H(2)O: enabling back-to-back studies in the same subject.

The advantages of using (2)H(2)O to quantify cholesterol synthesis include i) homogeneous precursor labeling, ii) incorporation of (2)H via multiple pathways, and iii) the ability to perform long-term studies in free-living subjects. However, there are two concerns. First, the t(1/2) of tracer in body water presents a challenge when there is a need to acutely replicate measurements in the same subject. Second, assumptions are made regarding the number of hydrogens (n) that are incorporated during de novo synthesis. Our primary objective was to determine whether a step-based approach could be used to repeatedly study cholesterol synthesis a subject. We observed comparable changes in the (2)H-labeling of plasma water and total plasma cholesterol in African-Green monkeys that received five oral doses of (2)H(2)O, each dose separated by one week. Similar rates of cholesterol synthesis were estimated when comparing data in the group over the different weeks, but better reproducibility was observed when comparing replicate determinations of cholesterol synthesis in the same nonhuman primate during the respective dosing periods. Our secondary objective was to determine whether n depends on nutritional status in vivo; we observed n of ∼25 and ∼27 in mice fed a high-carbohydrate (HC) versus carbohydrate-free (CF) diet, respectively. We conclude that it is possible to acutely repeat studies of cholesterol synthesis using (2)H(2)O and that n is relatively constant.

The use of stable-isotopically labeled oleic acid to interrogate lipid assembly in vivo: assessing pharmacological effects in preclinical species.

The use of stable isotopically labeled substrates and analysis by mass spectrometry have provided substantial insight into rates of synthesis, disposition, and utilization of lipids in vivo. The information to be gained from such studies is of particular benefit to therapeutic research where the underlying causes of disease may be related to the production and utilization of lipids. When studying biology through the use of isotope tracers, care must be exercised in interpreting the data to ensure that any response observed can truly be interpreted as biological and not as an artifact of the experimental design or a dilutional effect on the isotope. We studied the effects of dosing route and tracer concentration on the mass isotopomer distribution profile as well as the action of selective inhibitors of microsomal tri-glyceride transfer protein (MTP) in mice and diacylglycerol acyltransferase 1 (DGAT1) in nonhuman primates, using a stable-isotopically labeled approach. Subjects were treated with inhibitor and subsequently given a dose of uniformly ¹³C-labeled oleic acid. Samples were analyzed using a rapid LC-MS technique, allowing the effects of the intervention on the assembly and disposition of triglycerides, cholesteryl esters, and phospholipids to be determined in a single 3 min run from just 10 µl of plasma.

In vivo D2O labeling to quantify static and dynamic changes in cholesterol and cholesterol esters by high resolution LC/MS.

High resolution LC/MS-MS and LC/APPI-MS methods have been established for the quantitation of flux in the turnover of cholesterol and cholesterol ester. Attention was directed toward quantifying the monoisotopic mass (M0) and that of the singly deuterated labeled (M+1) isotope. A good degree of isotopic dynamic range has been achieved by LC/MS-MS ranging from 3-4 orders of magnitude. Correlation between the linearity of GC/MS and LC atmospheric pressure photoionization (APPI)-MS are complimentary (r² = 0.9409). To prove the viability of this particular approach, male C57Bl/6 mice on either a high carbohydrate (HC) or a high fat (HF) diet were treated with ²H2O for 96 h. Gene expression analysis showed an increase in the activity of stearoyl-CoA desaturase (Scd1) in the HC diet up to 69-fold (P < 0.0008) compared with the HF diet. This result was supported by the quantitative flux measurement of the isotopic incorporation of ²H into the respective cholesterol and cholesterol ester (CE) pools. We concluded that it is possible to readily obtain static and dynamic measurement of cholesterol and CEs in vivo by coupling novel LC/MS methods with stable isotope-based protocols.

Headspace analyses of ²H labeling of acetone: enabling studies of fatty acid oxidation in vivo.

We demonstrate that one can measure low levels of ²H labeling (e.g., <0.025% excess ²H) by exchanging hydrogen (deuterium) in water with acetone and subjecting samples to gas chromatography-pyrolysis-isotope ratio mass spectrometry. This analytical method circumvents the need to use typical off-line reduction methods that convert water to hydrogen gas prior to isotope ratio mass spectrometry or the need to purchase extra peripheral devices that would permit the direct analysis of water labeling. This method enables routine measurements of fatty acid oxidation in rodents; that is, one administers a ²H-labeled fatty acid(s) and then quantifies the production of ²H-labeled water.