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Lipin Deficiency Impairs Diurnal Metabolic Fuel Switching

¹ Department of Medicine, State University of New York at Stony Brook, Stony Brook, New York
² Department of Pediatrics, Harbor-UCLA Medical Center, Torrance, California
³ Department of Human Genetics, University of California, Los Angeles, California
⁴ Department of Preventative Medicine, State University of New York at Stony Brook, Stony Brook, New York
⁵ Department of Medicine, University of California, Los Angeles, California
⁶ Molecular Biology Institute, University of California, Los Angeles, California
⁷ VA Greater Los Angeles Healthcare System, Los Angeles, California
⁸ Department of Pharmacological Sciences, State University of New York at Stony Brook, Stony Brook, New York
⁹ Department of Physiology and Biophysics, State University of New York at Stony Brook, Stony Brook, New York

Address correspondence and reprint requests to Irwin J. Kurland, MD, PHD, State University of New York at Stony Brook, HSC T-15 Room 060, Stony Brook, NY 11794-8154. E-mail: irwin.kurland{at}stonybrook.edu

Abbreviations: FNS, fraction of newly synthesized palmitate molecules; HGP, hepatic glucose production; PDH, pyruvate dehydrogenase; PDK4, pyruvate dehydrogenase kinase isoform 4; PEPCK, phosphoenolpyruvate carboxykinase; RQ, respiratory quotient; TCA, tricarboxylic acid

	ABSTRACT

TOP ABSTRACT RESEARCH DESIGN AND METHODS RESULTS DISCUSSION REFERENCES

 
Fatty liver is a common feature of both obesity and lipodystrophy, reflecting compromised adipose tissue function. The lipin-deficient fatty liver dystrophy (fld) mouse is an exception, as there is lipodystrophy without a fatty liver. Using a combination of indirect calorimetry and stable-isotope flux phenotyping, we determined that fld mice exhibit abnormal fuel utilization throughout the diurnal cycle, with increased glucose oxidation near the end of the fasting period and increased fatty acid oxidation during the feeding period. The mechanisms underlying these alterations include a twofold increase compared with wild-type mice in tissue glycogen storage during the fed state, a 40% reduction in hepatic glucose production in the fasted state, and a 27-fold increase in de novo fatty acid synthesis in liver during the fed state. Thus, the inability to store energy in adipose tissue in the fld mouse leads to a compensatory increase in glycogen storage for use during the fasting period and reliance upon hepatic fatty acid synthesis to provide fuel for peripheral tissues during the fed state. The increase in hepatic fatty acid synthesis and peripheral utilization provides a potential mechanism to ameliorate fatty liver in the fld that would otherwise occur as a consequence of adipose tissue dysfunction.

Glucose and free fatty acids are two major energy substrates in vertebrate species. A switch between glucose/fatty acid utilization and storage has been evolutionarily adapted in these species to meet energy demands during the diurnal cycle of fasting and feeding. Upon prolonged fasting, energy is derived predominantly from fatty acid ß-oxidation, and glucose homeostasis is maintained by hepatic gluconeogenesis. After feeding, when fuel supply exceeds the body’s energy demands, excess glucose is stored in liver and muscle as glycogen and is also converted to fatty acids for storage in adipose tissue as triglyceride. Clearly, selective utilization of glucose versus fatty acid substrates upon transition from fasting to feeding is an important process to both meet energy demands and maintain glucose and lipid homeostasis.

In the past few years, accumulated evidence has revealed a key role for adipose tissue in the regulation of metabolic homeostasis. Adipose tissue is not merely a depot for fuel storage (1,2). Conditions characterized by either excess body fat (obesity) or underdeveloped adipose tissue (lipodystrophy) are associated with insulin resistance and diabetes in humans and animal models (3–5). Despite the fact that obesity and lipodystrophy represent opposite extremes of adiposity, some of the same mechanisms may contribute to metabolic dysregulation in these two extremes. For example, impaired fatty acid trapping in adipocytes in both obesity and lipodystrophy promotes ectopic lipid accumulation in tissues such as liver and muscle, with subsequent impairment of insulin signaling and action (6–9).

The lipodystrophic fatty liver dystrophy (fld) mouse represents a unique model in which to further elucidate the role of adipose tissue in metabolic homeostasis. Lipodystrophy in the fld mouse results from mutation in the Lpin1 gene, which encodes lipin (10). Lipin is abundantly expressed in adipose tissue and skeletal muscle and at lower levels in other tissues that have a role in lipid and glucose homeostasis, including liver and pancreatic ß-cells (10,11). Lipodystrophy in the fld mouse can be attributed to a combination of impaired lipid storage in adipose tissue and altered energy metabolism in muscle. Lipin is required for adipocyte differentiation and triacylglycerol accumulation, such that fld mice have no normal adipocytes and an 80% reduction in adipose tissue mass despite normal food intake (12,13). Lipodystrophy due to lipin deficiency has unique metabolic features. In muscle, lipin deficiency leads to elevated energy expenditure and enhanced expression of fatty acid oxidation genes in muscle (14). The roles of lipin in fat storage in adipocytes and energy expenditure in muscle have been confirmed in tissue-specific lipin transgenic mouse models. Thus, mice with enhanced lipin expression in adipose tissue are predisposed to obesity and compared with wild-type mice exhibit accelerated weight gain and a doubling of fat mass on a high-fat diet (14). Mice with enhanced lipin expression specifically in muscle exhibit even more pronounced obesity, which is evident on a chow diet and is attributable to reduced energy expenditure (14). Thus, the levels of lipin in adipose tissue and muscle, and possibly other tissues such as liver, are an important determinant of metabolic homeostasis.

Here, we further investigate how lipin deficiency leads to altered metabolism in the fld mouse using stable-isotope–based metabolic flux phenotyping in conjunction with gas chromatography/mass spectrometry technology. Stable isotope flux phenotyping uses administration of stable-isotope–labeled substrates to determine the metabolic fate of the labeled molecules in vivo and is a powerful tool for the identification of metabolic alterations occurring as a result of genetic perturbation (15–17). We determined that lipin deficiency results in altered metabolic flux between liver, muscle, and adipose tissue and impaired switching between fatty acid and glucose energy substrates. These studies suggest a role for lipin in the coordination of peripheral glucose and fatty acid storage and utilization throughout the diurnal cycle.

	RESEARCH DESIGN AND METHODS

TOP ABSTRACT RESEARCH DESIGN AND METHODS RESULTS DISCUSSION REFERENCES

 
BALB/cByJ-+/fld mice were obtained from The Jackson Laboratory (Bar Harbor, ME) and bred to produce lipin-deficient fld (fld/fld) and wild-type (+/+ and +/fld) mice for studies. Both wild-type and fld mice ranged from 7 to 9 months of age, with average body weight of 28.3 + 1.7 g for wild-type and 21.6 + 0.8 g for fld mice. Animals were fed standard laboratory chow diet (4.5% fat and 50% carbohydrate by weight) (Lab Diet; Purina 5001, St. Louis, MO) and maintained under 12-h light/dark conditions (6:00 A.M./6:00 P.M.). Ten mice of each type were used for fasting and refeeding experiments of each stable isotope tracer study. Of the 10 mice, 5 (3 male and 2 female) were used for fasting and 5 (3 male and 2 female) for refeeding studies. The high-fat/high-carbohydrate diet contained 35% fat and 33% carbohydrate (by weight) (Diet F3282; Bioserve, Frenchtown, NJ). All animal studies were performed under approved institutional protocols and according to guidelines established in the Guide for the Care and Use of Laboratory Animals.

Indirect calorimetry.
Measurements of respiratory quotient (RQ) were performed using an Oxymax indirect calorimetry system (Columbus Instruments, Columbus, OH). Mice were individually housed in the chamber for 48 h with lights on from 0600 to 1800 in an ambient temperature of 22–24°C. Food was available ad libitum during the dark cycle (feeding phase 1800–0600) and removed during the light cycle (fasting phase 0600–1800). RQ measurements were made under Oxymax system settings as follows: air flow, 0.6 l/min; sample flow, 0.5l/min; settling time, 6 min; and measuring time, 3 min. Six mice of each genotype were measured.

Metabolic flux analysis during fasted/fed conditions.
Metabolic flux analysis was performed using stable-isotope–labeled glucose or lactate as described previously (17,18). Briefly, fasting of animals was initiated at 4:00 P.M. At 7:00 P.M., animals were anesthetized under 5% isoflurane, and a preactivated Alza miniosmotic pump (model 2001D; Alza, Palo Alto, CA) was quickly inserted subcutaneously into each mouse. The entire process of anesthesia and minipump implantation was usually <3 min/mouse. The mini pump contained either 50 mg [U-¹³C₃]lactate or 50 mg [U-¹³C₆]glucose, each dissolved in 220 µl water. All stable isotope tracers used in this study were 99.9% enriched and purchased from Isotec (Miamisburg, OH). The quantity of tracer is sufficient to last through a 24-h infusion at a factory-calibrated pump rate of 8 µl/h. Blood and tissue (liver and skeletal muscle) samples from animals in fasting conditions were collected between 10:00 and 11:00 A.M. the next morning. Alternatively, animals undergoing refeeding were given mouse chow at 10:00 A.M., and blood and tissue samples were collected at 3:00 to 4:00 P.M. Under these experimental conditions, isotopic steady states are achieved at 15 h after fasting (12 h after infusion) and at 4–5 h after refeeding (17). Thus, data reported here were obtained under both metabolic and isotopic steady states. Animals were killed by an overdose of isoflurane anesthesia, and tissue samples were rapidly dissected free, snap frozen in liquid nitrogen, and stored at –80°C until further processing.

Blood plasma was isolated by centrifugation at 4°C. Plasma glucose and lactate concentrations were determined by a Cobas Mira analyzer (Roche Molecular Biochemicals) using reagents provided by Raichem (San Diego, CA). Glucose UV Reagent (catalog no. 80017) was used for glucose determinations, and Stat-Pack Rapid Lactate Test (catalog no. 869218) was used for lactate. Glucose residues derived from liver and muscle glycogen were prepared and determined as previously described (19). Gas chromatography and mass spectrometry conditions for measurement of glucose and lactate derivatives were as previously described (17). For glucose and lactate isotopomer determination, the ion clusters monitored were from 327 to 336 m/z.

Hepatic fatty acids were extracted after saponification of liver homogenates in 30% KOH (potassium hydroxide) and 100% ethanol using petroleum ether. Fatty acids were converted to their methylated derivatives using 0.5 N methanolic HCl (20). A glass capillary column BPX70 (SGE, Austin, TX) measuring 30 m x 250 µm (inner diameter) was used to separate fatty-acid derivatives. The gas chromatography conditions were carrier gas (helium) flow rate 1 ml/min, injector temperature 250°C, and oven temperature programmed from 120 to 220°C at 5°C/min. Under these conditions, palmitate derivative gives mass spectra 270 m/z at retention time 9.09 min and stearate 298 m/z at 11.86 min.

Mass isotopomer distribution was determined using methods that correct for the contribution of derivatizing agent and natural ¹³C abundance to mass isotopomer distribution of the compound of interest (19,21). This distribution was used to determine isotope incorporation and dilution by mass isotopomer distribution analysis according to Hellerstein et al. (22). Hepatic glucose and lactate production rates were determined using the following equation: production rate (mg · kg^–1 · min^–1) = infusion rate x (1/E_Tracer – 1), where infusion rate is the stable-isotope infusion rate, and E_Tracer is the plasma enrichment of stable isotope.

Hepatic glucose production (HGP) from the lactate was determined using the following equations as previously described (17):

(1)

(2)

(3)

In Eq. 3, M represents enrichment of plasma glucose molecules containing 1 to 6 (M1–M6) 13C carbons derived from 13C lactate, and m represents plasma enrichment of lactate molecules containing 1 to 3 (m1–m3) 13C carbons. Apparently, the fractional contribution of lactate to gluconeogenesis, and HGP from lactate, is influenced by multiple factors, such as, for example, dilution of plasma 13C lactate (decrease in m values) after feeding by metabolism of glucose and amino acids in the gut, resulting in increased fractional contribution of lactate to gluconeogenesis. However, plasma 13C glucose could also be diluted (decrease in M values) after feeding by absorption of dietary glucose in the gut, which would decrease fractional contribution of lactate to gluconeogenesis. Thus, multiple tracer dilutions are taken into account in the calculations. De novo fatty-acid synthesis was estimated by measuring incorporation of 13C (derived from infused [U-13C3]lactate) into the palmitate and stearate in the liver. De novo synthesis of palmitate was further quantitatively determined by calculating palmitate synthesis rate, as a fractional rate of lipogenesis, as described previously (23–25). In this study, we used [U-13C3]lactate as isotopic tracer, instead of deuterium oxide, as in the previous study (23–25). In liver, a portion of [U-13C3]lactate is converted to [U-13C2]acetyl-CoA, which is a m2 isotopomer of acetyl-CoA and used for fatty acid synthesis. Incorporation of 1–8 molecules of m2 [U-13C2]acetyl-CoA into palmitate will yield 1 molecule of m2, m4, m6, ... , and m16 palmitate isotopomers, respectively. Based on binomial probability of isotopomer distribution, the enrichment of palmitate isotopomers from de novo fatty acid synthesis could be determined using following equation:

(4)

where p is the enrichment of endogenous, unlabeled acetyl-CoA, and q is the enrichment of [U-¹³C₂]acetyl-CoA. Thus, the enrichment of palmitate isotopomers is p⁸ for m0 palmitate, 8p⁷q for m2 palmitate, 28p⁶q² for m4 palmitate,... , and q⁸ for m16 palmitate. From the ratio of two consecutive palmitate isotopomers, the value of p and q could be determined. For instance, the ratio of

(5)

which gives

(6)

Since p + q = 1, P = 1 – q = 1 – (1 – p) = p/[p + (1 – p)] = [p/(1 – p)]/[1 + p/(1 – p)], which combines with Eq. 6 and gives Eq. 7:

(7)

From actual measurement of hepatic m2 and m4 palmitate values, the value p can be calculated using Eq. 4.
Theoretically, when 100% of acetyl-CoA is used for de novo fatty acid synthesis, the theoretical M2 palmitate enrichment is expected to be M2 palmitate = 8p⁷q = 8p⁷ 1 – p, where the value p is obtained from Eq. 4. The de novo synthesis rate of palmitate in the liver is then determined from the ratio of (measured m2 palmitate enrichment)/(theoretic M2 palmitate enrichment) and expressed as a fraction of newly synthesized palmitate molecules (FNS) (23–25).

RNA quantitation.
Total RNA was isolated from liver or skeletal muscle with Trizol (Invitrogen, Carlsbad, CA) and used as template for cDNA synthesis using oligo dT primers. Real-time PCR was performed with the iCycler iQ real-time detection system (BioRad, Hercules, CA) using SYBR Green PCR QuantiTect reagent (Qiagen, Valencia, CA). Each assay was performed in triplicate using 25–50 ng cDNA. In addition, a standard curve of four serial dilution points of control cDNA (ranging from 100 ng to 100 pg), as well as a no-template control, were included in each assay. The relative concentration of genes of interest was determined by plotting the threshold cycle (C_t) versus the log of the serial dilution points and normalizing to expression levels of an endogenous control gene, TBP (TATA box binding protein). Primers used for real-time PCR were designed to span introns and were analyzed by conventional PCR and agarose gel electrophoresis to verify specificity before use in real-time assays. Primer sequences are given in Table 1.

	RESULTS

TOP ABSTRACT RESEARCH DESIGN AND METHODS RESULTS DISCUSSION REFERENCES

 
Abnormal metabolic fuel utilization throughout the diurnal cycle in the fld mouse.
Triglyceride in adipose tissue and glycogen in liver and muscle are the two major energy storage sources in vertebrate species in the fasted state. Although glycogen is utilized during initial fasting periods, glycogen reserves are depleted after prolonged fasting, and fatty acids derived largely from adipose tissue then become the major energy substrate. The reduced capacity for fat storage and lipolysis in the fld mouse raised the possibility that compensatory adaptations in energy substrate use may occur in these animals. This hypothesis is supported by the previous observation that energy expenditure is increased in fld mice and that the average RQ is altered as assessed by indirect calorimeter (14). The RQ reflects the type of metabolic fuels utilized and is calculated as the ratio of carbon dioxide produced to oxygen consumed, where oxidation of exclusively carbohydrate would produce a theoretical RQ of 1.00 and exclusive utilization of fatty acids an RQ of 0.70 (26). To further investigate how lipin deficiency alters energy substrate utilization, we measured RQ throughout the diurnal cycle. We found striking abnormalities in the fld mouse, particularly in the transition between fed and fasted states.

Throughout a 12-h fast, the RQ in wild-type mice drops as expected from 0.9 to 0.7, reflecting a shift from reliance largely upon carbohydrate during feeding to a reliance almost exclusively on fatty acids by the end of the fasting period (Fig. 1A). During the same period, the RQ in fld mice also diminishes, but the response is blunted. They maintain significantly higher RQ values than wild-type mice during the last 5 h of the fast, indicating failure to rely exclusively on fatty acid fuels. The most pronounced difference between wild-type and fld mice occurs following the transition from the fasted to fed state. Immediately upon introduction of chow diet (4.5% fat and 50% carbohydrate by weight), for wild-type mice there is a sharp transition from fatty acid utilization to a preference for carbohydrate substrates (RQ 0.90) (Fig. 1A). The RQ remains relatively high until the beginning of the fasting period, which induces a gradual decline to an RQ of 0.70 after 8 h of fasting. In contrast, fld mice do not undergo a pronounced shift to carbohydrate utilization upon feeding, reaching a maximum RQ of only 0.83 after 5 h of feeding (Fig. 1A). The fld mice therefore fail to undergo the normal switch to predominantly carbohydrate metabolism at any point during the feeding period. Paradoxically, the RQ in fld mice increases to its highest level (0.85–0.89) as soon as fasting begins (illustrated in both the initial and second fasting periods shown in Fig. 1A). The switch to carbohydrates at the beginning of the fasted period, and substantial utilization of fatty acids during the fed state, are the reverse of what occurs in normal mammals and indicate a fundamental alteration in metabolism in the lipin-deficient fld mouse.

View larger version (34K): [in this window] [in a new window]   FIG. 1. Indirect calorimetric measurements of RQ during successive 12-h light/dark periods. Each mouse was measured individually in the Oxymax chamber, and results averaged for six female mice of each genotype and diet group. Food was available ad libitum during the dark cycle (feeding phase 1800–0600) and removed during the light cycle (fasting phase 0600–1800) (see RESEARCH DESIGN AND METHODS). A: RQ for chow-fed wild-type vs. fld mice. B: RQ for chow-fed wild-type vs. high-fat/high-carbohydrate–fed wild-type mice. C: Chow-fed fld vs. high-fat/high-carbohydrate–fed fld mice. D: High-fat/high-carbohydrate–fed wild-type vs. fld mice. Statistical differences between the wild-type and fld mice were determined by Student’s t test (n = 5): *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. Data are presented as means ± SEM. HC, high carbohydrate; HF, high fat; WT, wild type.

Increased tissue glycogen storage in the fld mouse.
The reduced capacity for fat storage and elevated RQ observed in fld mice after prolonged fasting raised the possibility that glycogen may have increased importance as an energy source in these animals during fasting. Consistent with this possibility, measurement of tissue glycogen content showed that fld mice had about twice as much glycogen in both liver and skeletal muscle as wild-type mice when refed for 5 h with chow diet after an 18-h overnight fast (Fig. 2). This could not be attributed to differences in food intake, as wild-type and fld mice consumed the same amount of food during the 5-h refeeding period (wild-type 2.01 ± 0.40 vs. fld 2.22 ± 0.39 g). The liver glycogen content in both wild-type and fld mice after 18-h fasting were dramatically reduced to 4.5 mg/g tissue weight and showed no significant difference between the wild-type and fld mice (data not shown). Together with the RQ data, these results suggest that fld mice store excess glycogen during the fed state for use after prolonged fasting, when fatty acids released by adipose tissue lipolysis would normally be used but are unavailable in the fld mouse due to lack of adipose tissue.

View larger version (11K): [in this window] [in a new window]   FIG. 2. Tissue glycogen storage after 5-h refeeding. Before refeeding, wild-type and fld mice maintained on regular chow diet were fasted for 18 h. Liver and skeletal muscle glycogen contents were extract from these animals after 5 h refeeding (n = 4 for each genotype). Glycogen content was determined by measuring the amount of glucose residues derived from glycogen. Statistical differences were determined by Student’s t test. Data are presented as means ± SEM. WT, wild type.

View larger version (8K): [in this window] [in a new window]   FIG. 3. HGP rate in wild-type and fld mice after an 18-h fast. HGP in the 18-h fasting state was estimated using constant infusion of [U-13C6]glucose, which was delivered by Alza osmotic minipump. HGP (mg · kg–1 · min–1) = [U-13C6]glucose infusion rate x (1/M6 plasma glucose enrichment – 1). Statistical differences between the wild-type and fld mice (n = 5 for each genotype) was determined by Student’s t test. Data are presented as means ± SEM. WT, wild type.

View larger version (12K): [in this window] [in a new window]   FIG. 4. Lactate production rate (A), plasma lactate concentration (B), and HGP from lactate (C) in wild-type and fld mice in the 18-h fasted/5-h refed states. Animals were maintained on regular chow diet. Lactate production rate and HGP from lactate were estimated using constant infusion of [U-13C3]lactate, delivered by Alza osmotic minipump. Lactate production rate (mg · kg–1 · min–1) = [U-13C3]- lactate infusion rate x (1/m3 plasma lactate enrichment – 1). HGP from lactate was determined by the product of lactate production rate and the fraction of lactate that is converted to plasma glucose. Two-way ANOVA indicated that differences in lactate production rate (A) and plasma lactate concentration (B) were statistically different (P < 0.01) for both genotype and feeding status. Differences in HGP from lactate (C) were statistically different (P < 0.001) for genotype only. Data are presented as mean ± SEM. n = 5 mice for each genotype at each feeding status. WT, wild type.

Increased hepatic fatty acid synthesis with lipin deficiency.
To determine the fate of lactate in the fld mice, we followed the conversion of infused [U-¹³C₃]lactate to hepatic fatty acids after 5 h of refeeding. Through the action of lactate dehydrogenase and pyruvate dehydrogenase (PDH), [U-¹³C₃]lactate can be converted to [U-¹³C₂]acetyl-CoA, an m2 isotopomer that serves as a direct substrate for fatty acid synthesis. Incorporation of one molecule of m2 acetyl-CoA into fatty acid will yield an m2 fatty acid isotopomer, such as m2 palmitate or m2 stearate. As shown in Fig. 5, enrichment of m2 palmitate and m2 stearate extracted from the liver of refed fld mice was 29-fold and 5-fold higher than in wild-type mice, indicating that a greater proportion of the infused [U-¹³C₃]lactate was directed to fatty acid synthesis in the fld mouse. To eliminate the possibility that the difference in hepatic m2 fatty acid enrichment between wild-type and fld mice was due to the increased enrichment of plasma [U-¹³C₃]lactate concentration (Table 2), we determined the de novo synthesis rate of palmitate, which is a fractional rate of lipogenesis, in the liver using a modification of methods described by Hellerstein and coworkers (23–25).

View larger version (15K): [in this window] [in a new window]   FIG. 5. Hepatic fatty acid synthesis in the livers of wild-type and fld mice after 5-h refeeding. Animals were maintained on regular chow diet. Fatty acids were extracted from livers of mice (n = 4 for each genotype) that had undergone 18-h fasting and 5-h refeeding, with constant infusion of [U-13C3]lactate for 23 h. Incorporation of the stable-isotope–labeled lactate into hepatic fatty acids was used to estimate the relative rate of hepatic fatty acid synthesis. Increased rate of de novo fatty acid synthesis would result in higher enrichment of 13C-labeled fatty acids in the liver. Data are presented as means ± SEM. Statistical differences were determined by Student’s t test. WT, wild type.

Lipin regulates metabolic steps in hepatic fatty acid synthesis and peripheral glucose utilization.
To determine whether the changes revealed by metabolic flux analysis are reflected by altered gene expression in pathways controlling net fatty acid synthesis and substrate utilization, we examined mRNA levels of enzymes that are involved in gluconeogenesis, tricarboxylic acid (TCA) cycle, and fatty-acid/triglyceride synthesis using real-time RT-PCR. These enzymes are phosphoenolpyruvate carboxykinase (PEPCK), pyruvate carboxylase, pyruvate kinase, pyruvate dehydrogenase kinase isoform 4 (PDK4), ATP:citrate lyase, acetyl-CoA carboxylase, and fatty acid synthase.

PEPCK and pyruvate carboxylase are two key enzymes that commit pyruvate/lactate to hepatic gluconeogenesis. The mRNA levels of these two enzymes in the fld mice were significantly lower than in the wild-type mice in both fasted and refed states (Fig. 6A). In response to refeeding, PEPCK gene expression in both wild-type and fld mice were downregulated by a similar degree, indicating that 5-h refeeding had a similar suppressive effect on hepatic gluconeogenesis regardless of genotype. However, liver pyruvate carboxylase mRNA levels in the fld mice remained unchanged in response to refeeding, in contrast to a significant downregulation of pyruvate carboxylase mRNA in the wild-type mice (Fig. 6A). This gene expression profile of PEPCK and pyruvate carboxylase supports our earlier observation of decreased hepatic gluconeogenesis from lactate/pyruvate. Since pyruvate carboxylase plays a important role in replenishment of TCA cycle intermediates (by the conversion of pyruvate to oxaloacetate) to facilitate fatty acid synthesis (28,29), the suppressed PEPCK and unchanged pyruvate carboxylase gene expressions in the refed fld mice would favor the redirection of metabolic flux toward fatty acid synthesis.

View larger version (15K): [in this window] [in a new window]   FIG. 6. Gene expression profile in liver and skeletal muscle of wild-type and fld mice determined by real-time RT-PCR. Wild-type and fld mice were maintained on regular chow diet. Tissues were taken from these animals either after an 18-h fasting or after an 18-h fasting plus a 5-h refeeding (n = 4 for each genotype at each feeding status). Gene expression results are presented as means ± SEM and expressed as the percentage seen for the expression in wild-type mice tissues in the fasted state. Statistical differences (P < 0.05) were determined by two-way ANOVA, followed by Bonferroni posttests. Statistical differences determined by Bonferroni posttests of multiple comparison were shown between * and #. A: Gene expression of hepatic enzymes involved in gluconeogenesis and TCA cycle entry. All mRNA levels are statistically different for genotype (except for pyruvate kinase), feeding status (except for pyruvate carboxylase), and genotype/feeding interactions (except for PEPCK). B: Gene expression of hepatic fatty acid synthesis enzymes and muscle hexokinase II. All gene expressions were statistically different for genotype (except for ATP:citrate lyase and hexokinase II), feeding status, and genotype/feeding interactions. ACC, acetyl-CoA carboxylase; ACL, ATP:citrate lyase; FAS, fatty acid synthase; HKII, muscle hexokinase II; PC, pyruvate carboxylase; PK, pyruvate kinase; WT, wild type.

In chow-fed fld mice, the increased fatty acid synthesis together with the higher RQ during fasting, as well as the increased glycogen storage and lower RQ during refeeding, suggested an adaptation to conserve glucose for prolonged fasting. This most likely stems from the impaired fat stores and reduced HGP observed in the fld mouse. Evidence supporting glucose conservation in the fld mouse includes our earlier observation of a twofold increase in both liver and skeletal muscle glycogen stores. In addition, skeletal muscle hexokinase II mRNA levels in fld mice were only 20% of those in wild-type mice after an 18-h fast and were upregulated sixfold after refeeding, in contrast to unchanged levels in wild-type mice (Fig. 6B). Skeletal muscle, by virtue of its mass, is a major determinant of energy metabolism. The hexokinase II gene expression profile indicates that glucose uptake by the skeletal muscle in the fasted fld mouse was decreased, perhaps as a compensatory response to spare glucose for utilization by glucose-dependent organs such as brain. The dramatic upregulation of hexokinase II after refeeding in the fld mouse is consistent with the increased skeletal muscle glycogen storage as a means of glucose conservation.

	DISCUSSION

TOP ABSTRACT RESEARCH DESIGN AND METHODS RESULTS DISCUSSION REFERENCES

 
Fatty liver is a common feature in disorders characterized by "too much" (obesity) or "too little" (lipodystrophy) fat, as a result of excess fatty acids or inadequate adipose tissue storage, respectively. Individuals with these disorders are predisposed to insulin resistance and diabetes, which are thought to be partly associated with ectopic deposition of triglyceride and lipotoxicity (32). A major difference between human lipodystrophies and the lipodystrophic fld mouse is the spontaneous resolution of the fatty liver by 2 weeks of age in the mouse (33). In addition, the fld mouse does not develop severe hyperglycemia, as is seen in human lipodystrophies (3,5). These previous observations suggested that partitioning of energy substrates, namely glucose and free fatty acids, in the adult fld mouse may be altered.

The studies presented here demonstrate that lipin deficiency leads to novel changes in mobilizing and utilizing energy substrates in the fld mice during the diurnal metabolic cycle. Indirect calorimetry indicated that fatty acids serve as an important metabolic fuel for fld mice during the fed state, whereas wild-type animals rely primarily on glucose. Using metabolic flux phenotyping analysis, we determined that the fatty acids utilized by fld mice during the fed state are likely available because of a 27-fold increase in de novo hepatic fatty acid synthesis in fld compared with wild-type mice. Previous studies have shown that levels of plasma triglycerides and free fatty acids are comparable between adult wild-type and fld mice under the ad libtum feeding condition (13), and fatty liver and hyperlipidemia were observed only in neonatal fld mice (13,33). Thus, we propose that the reliance of adult fld mice on fatty acids as an energy source may explain, in part, why ectopic fat deposition, and hepatic steatosis, does not occur in adult fld mice. In contrast to the increased utilization of fatty acids during the fed state, fld mice exhibit increased glucose utilization during the fasting period, especially during the final 5 h of a 12-h fast. The increased glycogen storage appears to be possible because of the reduced reliance on glucose oxidation during the fed state and suggests that glycogen serves as a surrogate energy reserve in fld mice in the face of inadequate adipose tissue reserves.

Adipocyte-derived hormones are established to have important roles in regulating energy metabolism and homeostasis. As with other mouse lipodystrophy models (34,35), it is reasonable to postulate that low levels of adipocyte-secreted hormones may contribute to metabolic alterations in the fld mouse. For example, leptin administration can reverse insulin resistance seen both in human and mouse lipodystrophies (34,36), in part via hypothalamic actions (37,38). Both leptin and adiponectin levels in the fld mouse are substantially reduced (10,12). However, among these lipodystrophic mouse models, only the fld mouse lacks a fatty liver in adulthood. Thus, although impaired adipokine action may contribute, it cannot account for the unique phenotype and metabolic adaptation observed in the fld mouse, suggesting that these are a consequence specifically of lipin deficiency.

Our data reveal that lipin deficiency results in impaired normal diurnal metabolic fuel switching in the fld mice. The key changes in fld compared with wild-type mice were detected at points that direct metabolic flux toward energy storage versus energy expenditure. The integration of metabolic flux studies, calorimetry, and gene expression analysis leads to an integrated view of the altered metabolic profile of the fld mouse, as illustrated in Fig. 7. In the fed state, fld mice cannot store significant amounts of triglyceride in adipose tissue due to impaired adipocyte function. Rather than being stored, fatty acids synthesized in the liver are therefore utilized as energy substrates. As we determined, hepatic fatty acid synthesis is dramatically increased in the fld mouse with refeeding, mainly for peripheral utilization (Fig. 1), sparing glucose molecules for glycogen synthesis. This results in enhanced glucose flux toward glycogen deposition in the liver and muscle. In the fasted state, glycogen stored in the fld liver and muscle becomes a prominent fuel source in the absence of normal adipose tissue stores. The supply of glycerol for gluconeogenesis in fld liver should be reduced, due to decreased lipolysis from limited amounts of adipose tissue. Lactate supply for gluconeogenesis is reduced (Fig. 4) due to increased peripheral glucose oxidation. Glucose uptake by skeletal muscle is also decreased, which could spare glucose for glucose-dependent organs such as brain. Thus, the lipin deficiency results in a reversed diurnal metabolic cycle. This adaptation in fuel selection and energy conservation in the fld mouse may account for the amelioration of fatty liver in the adult animals. It is important to acknowledge that we assessed regulation of metabolic pathway enzymes by examining mRNA expression, which may not be a reliable gauge of resulting enzyme activity due to posttranscriptional effects. However, the stable isotope-based flux phenotyping studies done here provide a "visual" assessment of the fate of labeled molecules as they are metabolized by specific pathways and the rate at which these labeled molecules pass through these pathways.

View larger version (77K): [in this window] [in a new window]   FIG. 7. Changes in metabolic flux adapted by the fld mouse during feeding and fasting. Metabolic flux through pathways in the wild-type mouse is illustrated by broken grey arrows. Only key changes in metabolic flux in the fld mouse are indicated by solid black arrows. The thickness of the black arrows indicates increased (thicker) or decreased (thinner) metabolic flux in the fld mouse, relative to the wild type. FFA, free fatty acids; G-6-P, glucose-6-phosphate; TG, triglyceride; WT, wild type.

	ACKNOWLEDGMENTS

 
This work was supported by National Institutes of Health Grants DK 58132 (to I.J.K.) and HL28481 (to K.R.), a grant from the American Diabetes Association (to I.J.K.), and a grant supporting gas chromatography/mass spectrometry metabolic tool development from the Center for Biotechnology, SUNY Stony Brook and Ingenious Targeting Laboratories (to I.J.K.).

We thank Ping Xu and Sara Bassilian for excellent technical assistance.

	FOOTNOTES

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received for publication February 23, 2006 and accepted in revised form August 4, 2006

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