Moderate Caloric Restriction, But Not Physiological Hyperleptinemia Per Se, Enhances Mitochondrial Oxidative Capacity in Rat Liver and Skele
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内分泌学杂志 2005年第4期
Clinica Medica (R.B., M.Z., A.B., G.B., L.V.-S., G.G.), Department of Clinical, Morphological and Technological Sciences, and Centro Servizi Polivalenti di Ateneo (CSPA)-Animal Facility (M.S.), University of Trieste, Trieste 34100, Italy
Address all correspondence and requests for reprints to: Rocco Barazzoni, M.D., Ph.D., Clinica Medica, University of Trieste, Ospedale Cattinara, Strada di Fiume 447, 34100 Trieste, Italy. E-mail: barazzon@units.it.
Abstract
The study aimed at determining, in lean tissues from nonobese rats, whether physiological hyperleptinemia with leptin-induced reduced caloric intake and/or calorie restriction (CR) per se: 1) enhance mitochondrial-energy metabolism gene transcript levels and oxidative capacity; and 2) reduce triglyceride content. Liver and skeletal muscles were collected from 6-month-old Fischer 344 rats after 1-wk leptin sc infusion (0.4 mg/kg · d: leptin + 3-fold leptinemia vs. ad libitum-fed control) or moderate CR (–26% of those fed ad libitum) in pair-fed animals (CR). After 1 wk: 1) leptin and CR comparably enhanced transcriptional expression of mixed muscle mitochondrial genes (P < 0.05 vs. control); 2) CR independently increased (P < 0.05 vs. leptin-control) hepatic mitochondrial-lipooxidative gene expression and oxidative capacity; 3) hepatic but not muscle mitochondrial effects of CR were associated (P < 0.01) with increased activated insulin signaling at AKT level (P < 0.05 vs. leptin-control); 4) liver and muscle triglyceride content were comparable in all groups. In additional experiments, assessing time course of posttranscriptional CR effects, 3-wk superimposable CR (P < 0.05): 1) increased both liver and muscle mitochondrial oxidative capacity; and 2) selectively reduced muscle triglyceride content. Thus, in nonobese adult rat: 1) moderate CR induces early increments of mitochondrial-lipooxidative gene expression and time-dependent increments of oxidative capacity in liver and mixed muscle; 2) sustained moderate CR alters tissue lipid distribution reducing muscle but not liver triglycerides; 3) mitochondrial-lipid metabolism changes are tissue-specifically associated with hepatic AKT activation; 4) short-term physiological hyperleptinemia has no independent stimulatory effects on muscle and liver mitochondrial-lipooxidative gene expression. Increased lean tissue oxidative capacity could favor substrate oxidation over storage during reduced nutrient availability.
Introduction
MITOCHONDRIA ARE THE key site of tissue substrate utilization, and reduced mitochondrial gene expression and function is rapidly emerging as an important contributor to metabolic alterations in insulin-resistant states (1, 2, 3). Changes in fat disposal at the mitochondrial level could play a pivotal adaptive role during changes in nutrient availability and could alter lean tissue lipid content (4, 5), in turn an important determinant of insulin action (6).
Leptin is an anorexigenic hormone secreted by adipose tissue able to reduce spontaneous food intake in rodents (7). Acute or supraphysiological leptin increments are reported to enhance energy expenditure and tissue substrate utilization (7, 8, 9, 10, 11), contributing to body weight loss. The role of leptin in the in vivo regulation of intermediate metabolism in lean tissues is, however, controversial (12), partly due to discrepancies among the above reports (7, 8, 9, 10, 11) and the known association of increased plasma leptin with obesity (7) and obesity-related impaired lean tissue lipid disposal (13, 14). In particular, the potential effects of sustained physiological increments in circulating leptin on mitochondrial-lipid metabolism and their tissue distribution in vivo in adult individuals remain incompletely understood (12). Importantly, metabolic adaptation to calorie restriction (CR) per se involves increased body ability to use available substrates (15, 16, 17, 18) in the presence of reduced circulating leptin (7). Such effects of CR are at least partially independent from extent of weight loss and duration of reduced nutrient intake (19, 20), and they extend to insulin-stimulated conditions in insulin-sensitive tissues, representing an integral component of metabolic effects of dietary treatment of insulin-resistant states (17, 18). Absolute and relative impacts of leptin and CR per se on mitochondrial-lipid metabolism, as well as their molecular mechanisms and tissue distribution, remain incompletely defined.
The current study was therefore aimed at determining whether physiological hyperleptinemia and/or moderate CR per se enhance mitochondrial-lipid metabolism transcriptional gene expression, mitochondrial oxidative capacity as reflected by mitochondrial enzyme activities, and their impact on triglyceride content in metabolically relevant (21) muscle and liver tissues in a lean adult rat model. In addition to mixed type I-type II fiber gastrocnemius, mitochondrial gene expression and function were investigated in type I soleus muscle. Patterns of transcriptional expression of representative mitochondrial-lipid metabolism genes included flux-generating respiratory chain enzyme cytochrome c oxidase (CO) (subunits I and III) (22), uncoupling proteins (UCPs) (23, 24), the rate-limiting enzyme of fatty acid oxidation carnitine palmitoyl transferase (CPT)-I as well as lipogenic enzymes acetyl-coenzyme A carboxylase (ACC) and fatty acid synthase (FAS). CO and citrate synthase (CS) were selected as markers of tissue oxidative capacity for their flux-generating role in the respiratory chain and tricarboxylic acid cycle, respectively (22). Transcriptional expression of fasting-associated regulators of lipid oxidation peroxisome proliferator-activated receptor (PPAR)- and PPAR-coactivator (PGC)-1 (25, 26) was measured to determine their role in potential metabolic changes during sustained moderate CR. Changes in insulin signaling at the AKT level were determined to assess their potential association with changes in mitochondrial-lipid metabolism during CR (1, 2, 3, 27, 28, 29, 30).
Materials and Methods
Animals and experimental protocol
Twenty-four 6-month-old Fischer 344 male rats were purchased from Harlan Italy (San Pietro al Natisone, Italy). All animals were kept in individual cages in a controlled environment (t = 22 C; 12-h light, 12-h dark cycle) in the Animal Facility of the University of Trieste and fed a standard commercial chow diet (Harlan 2018, Harlan; 3.4 Kcal/g). The experimental protocol was approved by the local Committee for Animal Studies and Animal Care, and experimental procedures were carried out in keeping with institutional guidelines. Results of ghrelin measurements showing the physiological interplay between leptin and ghrelin regulation in vivo were previously published (31). After at least 2 wk from arrival, rats were randomly assigned to undergo either recombinant murine leptin (Research Diagnostics Inc., Flanders, NJ) (n = 8, rate of infusion 0.4 mg/d) or vehicle (5 mM NaCitrate, pH 7.4) infusion with pair-feeding (n = 8) via osmotic minipumps (2ML1, Alza, Mountain View, CA) for 7 d. A third group of rats received vehicle infusion but was allowed free access to food (n = 8). Food intake was measured each day in all rats, and food was presented to pair-fed animals in the afternoon about 2 h before beginning of the dark cycle. After 7 d of treatment, rats were killed in the morning 2–3 h into the light cycle. Food was withdrawn from animals to be killed 90 min before injection of an ip overdose of sodium pentobarbital (70 mg/kg body weight). Gastrocnemius and soleus muscles and liver tissue were then quickly removed in this order and immediately frozen in liquid nitrogen and stored at –80 C until analysis. Blood was drawn through cardiac puncture and plasma was also stored at –80 C.
Additional experiments were designed to assess potential time course of posttranscriptional effects of CR on mitochondrial enzyme activities as well as on triglyceride content in lean tissues (see Results and Discussion). Rats of same age, strain, and body weight (see Results) were divided into two groups receiving either ad libitum control diet (Harlan 2018, Harlan; 3.4 Kcal/g) or CR superimposable to that in the pair-fed group for 3 wk. Identical experimental procedures were used for liver, mixed gastrocnemius muscle, and blood collection.
RNA and DNA analysis
Total RNA was isolated from 40–60 mg of tissues by the guanidinium method (Tri Reagent, MRC, Inc., Cincinnati, OH). Transcript levels of pivotal regulators of lipid metabolism were measured by real-time PCR (7900 Sequence Detection System, Applied Biosystems, Foster City, CA) as previously described (32). Total RNA (1 μg) was reverse-transcribed (RNA Reverse Transcription KIT, Applied Biosystems) and amplified using primers (300 nM) and probes (50 nM) selected using the Primer Express Software (Applied Biosystems) (Table 1). Target and housekeeping gene were run separately and their final quantitation was achieved using a relative standard curve. Results for each gene were divided by the corresponding 28S rRNA abundance and expressed as percent of average control value. CO subunit I and III and UCP mRNA levels were measured by Northern blotting of total RNA as previously described (33, 34).
TABLE 1. Forward (FP) and reverse (RP) primer and probe sequences for real-time PCR gene quantitation for ACC, fatty acid synthase (FAS), CPT-I, PPAR- and 28S rRNA
Total DNA was extracted from liver and skeletal muscles using the Wizard Genomic DNA Isolation kit (Promega Corp., Madison, WI). Mitochondrial (mt) DNA copy number was measured by Southern blotting using cDNA probes for mtDNA-encoded CO I and nuclear-encoded 28S rRNA genes as previously described (33). CO bands in each tissue were normalized to the corresponding 28S rRNA band and individual results were expressed as a percentage of the average value for control ad libitum-fed animals.
Western blot
For measurement of activated (phosphorylated-P) AKT, total tissue proteins were extracted from liver and muscles and quantitated as described (32, 34). Forty micrograms of total protein (six to seven animals per group) were separated on 12% acrylamide gels and transferred to polyvinylidene difluoride membranes (Bio-Rad Laboratories, Hercules, CA) that were blocked and hybridized overnight at 4 C to rabbit antibodies for Ser473-P-AKT (Cell Signaling, Beverly, MA). The secondary antibody was peroxidase-conjugated goat antirabbit IgG (Jackson ImmunoResearch, West Grove, PA) used at a 1:10,000 dilution for 1 h at room temperature. Membranes were then exposed to films for 4–8' (Kodak Biomax MR, Kodak, Rochester, NY), and resulting images were quantitated by densitometry. Similar to mRNA measurement, individual results from all experiments were expressed as a percentage of the average value for vehicle-treated animals.
CO and CS enzyme activity, tissue triglycerides, and plasma biochemical profile
CO as well as CS enzyme activity was measured spectrophotometrically from tissue homogenates in liver and muscles (32, 33). Triglyceride content was measured from 35–40 mg of liver and gastrocnemius muscle (32). Samples were homogenized in 2:1 chloroform: methanol solution in a 20:1 volume: weight ratio and kept at 4 C overnight with gentle shaking. Phase separation was performed using H2SO4 (1 mmol/liter), lipid phase was dried under nitrogen and dissolved in 100 μl ethanol. Triglyceride content was then measured using a commercially available kit (TG, Roche Diagnostics Corp., Indianapolis, IN). Plasma leptin, insulin, and ghrelin concentrations were measured by RIA using commercially available kits (Linco, St. Louis, MO). Blood glucose was measured with an AccuCheck glucose monitor (Roche Diagnostic Corp.).
Statistical analysis
Results in the three groups were compared using one-way ANOVA. Student’s t test for unpaired data was then used to compare results between two groups. P values of less than 0.05 were considered statistically significant. In follow-up experiments, Student’s t test for unpaired data was used to compare results in the two experimental groups.
Results
Food intake, body weight, and metabolic profile
Compared with ad libitum-fed rats leptin reduced overall spontaneous food intake by 26% and superimposable changes were induced in pair-fed animals. As expected, leptin-treated and pair-fed rats lost moderate but statistically significant amounts of body weight compared with control ad libitum-fed animals, resulting in approximately 4% net loss at the end of the study period (Table 2). Plasma leptin concentration increased less than 3-fold in the leptin-treated group, whereas it was reduced in the pair-fed group compared with control animals (both P < 0.05 vs. control). Plasma ghrelin was increased (P < 0.05 vs. control and leptin) in pair-fed but not in leptin-treated rats. Plasma insulin concentration was similarly reduced in leptin and pair-fed groups (P < 0.05 vs. control), whereas glucose concentration was comparable in all animals. Circulating free fatty acids were reduced in calorie-restricted (CR) (P < 0.05 vs. control and leptin) but not in leptin-treated animals.
TABLE 2. Body weight changes, food intake, plasma leptin, insulin, ghrelin, glucose, and free fatty acid concentrations in the three experimental groups
Moderate short-term CR but not physiological hyperleptinemia favor expression of mitochondrial-lipooxidative over lipogenic genes in liver
Tissue patterns of mitochondrial-lipid metabolism gene expression.
In liver leptin treatment reduced expression of lipogenic genes ACC and FAS (P < 0.05 vs. control, Fig. 1) with no changes in mitochondrial-lipooxidative genes. In addition, leptin treatment was associated with muscle group-specific effects. In mixed type I-type II fiber gastrocnemius muscle leptin resulted in higher CO I, CO III, and UCP3 but not UCP2 transcript levels (P < 0.05 vs. control; Fig. 2). CPT-I and ACC transcript levels were not altered after leptin treatment (Fig. 2). No effect of leptin on mitochondrial-UCP gene expression were observed in highly oxidative type I soleus muscle (Table 3).
FIG. 1. Moderate short-term CR but not physiological hyperleptinemia favor expression of mitochondrial-lipooxidative over lipogenic genes in liver. A, Effects of CR and leptin on liver CO subunit I and III and UCP-2 mRNA levels (Northern blotting). B, Effects of CR and leptin on liver CPT-I, ACC, FAS mRNA levels (real-time PCR). Bars represent average ± SE from eight animals per group. Under each bar (A) are shown representative bands from two animals of each group. Top bands show signals from each target gene and bottom bands show signals from the 28S rRNA probe. *, Statistically different results (P < 0.05 or less) by ANOVA and Student’s t test for unpaired data compared with control; $, statistically different results (P < 0.05 or less) by ANOVA and Student’s t test for unpaired data compared with control and leptin.
FIG. 2. Moderate short-term CR and moderate hyperleptinemia comparably enhance expression of mitochondrial genes in gastrocnemius muscle. Effects of CR and leptin on mRNA levels of gastrocnemius CO subunit I and III (A), UCP 2 and 3 (B), and CPT-I and ACC. Bars represent average ± SE from eight animals per group. Under each bar (A and B) are shown representative bands from two animals of each group. Top bands show signals from each target gene and bottom bands show signals from the 28S rRNA probe. *, Statistically different results (P < 0.05 or less) by ANOVA and Student’s t test for unpaired data compared with control.
TABLE 3. Transcript levels of CO subunit I, subunit III, UCP2, UCP3, and COX activity in soleus muscle in the three experimental groups
At variance with leptin treatment, pair feeding independently favored the expression of hepatic mitochondrial-lipooxidative over lipogenic genes (CO III, UCP2, CPT-I: P < 0.05 vs. control and leptin—Fig. 1). Pair-feeding and leptin treatment were associated with superimposable patterns of transcriptional changes in skeletal muscles. In particular, moderate CR with adaptive hypoleptinemia lead to similar increments in mixed muscle mitochondrial and UCP transcript levels (P < 0.05 vs. control; Fig. 2).
Mitochondrial DNA copy number was measured in gastrocnemius and liver tissues to assess whether changes in mitochondrial-encoded transcripts were associated with increased template availability. Mitochondrial DNA copy number was comparable in the three experimental groups in both tissues, although a trend for reduced mtDNA was observed in gastrocnemius muscle from CR animals (control vs. leptin vs. CR: liver, 100 ± 6 vs. 96 ± 14 vs. 95 ± 16; gastrocnemius, 100 ± 18 vs. 97 ± 21 vs. 68 ± 10, arbitrary units; all P > 0.10).
Tissue mitochondrial enzyme activities and triglyceride levels (Table 4).
After 1 wk of moderate CR, mitochondrial CO activity, an index of tissue oxidative capacity (22), was higher in CR compared with leptin-treated and ad libitum-fed rats in liver (P < 0.05 CR vs. control and leptin, P = 0.05 leptin vs. control). At variance with the liver, skeletal muscle CO activities in CR rats were comparable with those in control and leptin-treated animals. Tissue triglyceride content was comparable in
TABLE 4. PPAR- and PGC-1 mRNA (arbitrary units), CO enzyme activity (micromoles per minute per gram of tissue) and triglyceride content (milligrams per gram wet weight) in liver and gastrocnemius muscle in the three experimental groups
Tissue PPAR, PGC-1 expression, and AKT phosphorylation.
Changes in mitochondrial-lipid metabolism gene expression after both leptin treatment and CR per se were not associated with changes in PPAR and PGC-1 transcriptional expression in any tissue (Table 4). Hepatic but not muscle mitochondrial changes were associated with increased P-AKT in CR compared with both leptin and control groups (Fig. 3). A positive correlation was observed between hepatic P-AKT and CO enzyme activity (Liver: r = 0.83; P < 0.0001; data not shown). Liver CO enzyme activity was in turn negatively correlated with plasma free fatty acid concentration (r = –0.68; P = 0.001, Fig. 4). A similar negative correlation was observed between liver CPT-I transcript levels and circulating free fatty acids (r = –0.57; P = 0.005; data not shown).
FIG. 3. Effects of moderate short-term CR and moderate hyperleptinemia on liver and gastrocnemius muscle activated P-AKT. Bars represent average ± SE from six to seven animals per group. Under each bar are shown representative bands from two animals of each group. *, Statistically different results (P < 0.05 or less) by Student’s t test for unpaired data compared with control.
FIG. 4. Liver CO enzyme activity is negatively correlated with plasma free fatty acid (FFA) concentration (r = –0.68; P < 0.001).
Three-week moderate CR enhances lean tissue mitochondrial enzyme activity and reduces muscle triglyceride content with tissue-specific effects on AKT phosphorylation
Mitochondrial protein synthesis is a key posttranscriptional step of mitochondrial gene expression, and its basal rate is substantially higher in liver than in skeletal muscle (35). This observation led us to test the hypothesis that changes in posttranscriptional steps of CO expression would require longer time and occur after more sustained CR in muscle than in the liver. In additional experiments, Fischer 344 rats of identical age and initial weight (control vs. CR: 374 ± 4 vs. 371 ± 4 g) underwent superimposable CR (n: 10–12 each; control vs. CR: 94 ± 4 vs. 72 ± 2.5 g/wk, –27% food intake) for 3 wk. Plasma metabolic profile was also comparable to that observed in 1-wk CR animals (control vs. CR: plasma leptin: 9.6 ± 2.1 vs. 6.6 ± 1.4 ng/ml; plasma insulin: 6.8 ± 1.2 vs. 3.2 ± 0.4 ng/ml, P < 0.05; plasma ghrelin: 2019 ± 169 vs. 3489 ± 339 pg/ml, P < 0.05; blood glucose: 7.9 ± 0.2 vs. 8.3 ± 0.2 mmol/liter; plasma free fatty acids 0.306 ± 0.020 vs. 0.232 ± 0.023 mmol, P < 0.05). Similar to the 1-wk protocol hepatic CO activity and P-AKT were increased after 3-wk CR (Fig. 5). In addition, 3-wk CR also increased gastrocnemius muscle mitochondrial oxidative capacity in the absence of changes in P-AKT. Activity of CS, a key enzyme of the Krebs cycle and also a marker of mitochondrial oxidative capacity, was also increased in both tissues in CR animals. Increased mitochondrial enzyme activities were associated with reduced triglyceride content in gastrocnemius muscle but not in liver indicating tissue-specificity of lipid deposition and altered tissue lipid distribution during reduced nutrient availability (Fig. 5).
FIG. 5. Effects of 3-wk CR on CO and CS enzyme activities and triglyceride content (TG) in liver (A and B) and gastrocnemius muscle (C and D); effects of 3-wk CR on P-AKT content (E) in both tissues. Bars represent average ± SE from eight to nine animals per group. *, Statistically different results (P < 0.05 or less) by Student’s t test for unpaired data compared with control.
Discussion
The current study shows that: 1) moderate CR induces early increments of mitochondrial-lipooxidative gene expression and time-dependent increments of lean tissue oxidative capacity in liver and mixed skeletal muscle of nonobese rats; 2) increased lean tissue oxidative capacity is associated with reduced tissue triglyceride content in muscle but not in liver tissue; 3) mitochondrial-lipid metabolism changes are associated with increased insulin signaling activation at the AKT level in liver but not skeletal muscle; and 4) short-term physiological hyperleptinemia has no independent stimulatory effects on muscle and liver mitochondrial-lipooxidative gene expression.
Moderate CR favors expression of mitochondrial-lipooxidative genes
The current data identify increased expression of oxidative genes in both liver and mixed skeletal muscle as an early response to moderate reduction of nutrient availability in nonobese rats. Mitochondrial oxidative capacity as reflected by the activity of the flux-generating enzymes CO (respiratory chain) and CS (tricarboxylic acid cycle) was also time-dependently increased by moderate CR in both tissues. Mitochondrial transcript levels and oxidative capacity are positively associated with insulin-stimulated substrate disposal and insulin sensitivity in lean tissues in vivo (3, 36, 37, 38, 39). Increased insulin sensitivity in CR animals in the current study is supported by reduced circulating levels of insulin and free fatty acids as well as by tissue-specific increment of activated insulin-signaling molecules. The current results therefore indicate the involvement of early mitochondrial changes in increased insulin action commonly observed in CR states and dietary treatment of insulin-resistant conditions (16, 17, 18, 19, 20).
The potential impact of mitochondrial changes on tissue lipid content is a relevant issue because lean tissue lipid deposition is associated with insulin resistance (6). The current observations support a link between changes in oxidative enzyme activities and skeletal muscle lipid content in nonobese rats, in agreement with previous reports in obese or insulin-resistant models (40). A more complex relationship between lipid content and mitochondrial function is in turn suggested in the liver. Relative excess deposition of liver triglycerides in CR animals is consistent with fatty liver reported during chronic or marked undernutrition (13). Contributors to excess liver fat in the presence of increased mitochondrial oxidative capacity under the current experimental conditions could include altered hepatic lipid uptake and/or secretion, as suggested by previous reports (13). Although its potential adaptive role remains to be elucidated, increased hepatic fat deposition could sustain glucose and lipoprotein production while contributing to reduce lipid content and insulin resistance in nonliver tissues during CR. Whereas mixed muscle phosphorylated AKT was unchanged by CR under basal conditions, consistent with previous reports (28), a substantial tissue-specific increase in hepatic AKT activation occurred in CR animals in the current study, suggesting a link between in vivo activation of insulin signaling and enhanced mitochondrial oxidative capacity in the liver. Liver AKT overexpression (30) or activation through phosphatase and tension homolog knockout (29) lead to parallel increments of hepatic lipid content and systemic insulin sensitivity in ad libitum-fed rodent models. Also in indirect agreement with the current data, liver fat deposition after AKT overexpression in ad libitum-fed rats was independent of enhanced tissue lipogenic gene expression (30). The current findings, therefore, suggest liver AKT activation as a potential contributor to maintained hepatic lipid content (13), as well as increased systemic insulin action (16, 17, 18) in the presence of reduced nutrient availability. A role of hepatic responses in systemic adaptive changes in lipid metabolism and disposal is also supported by the association between hepatic mitochondrial oxidative capacity and circulating free fatty acids.
PPAR- and PGC-1 are reported to be associated with adaptive lipid metabolism changes during fasting (25, 26). Lack of changes in their transcript levels does not support their potential role in metabolic adaptation to moderate CR, although indirectly indicating that no major fasting effects occurred under the current experimental conditions. CR is associated with increased circulating ghrelin that was recently demonstrated to have a profound tissue-specific impact on liver and muscle mitochondrial function and lipid metabolism in ad libitum-fed rats (32). Stimulation by 4-d exogenous ghrelin treatment of mixed (but not soleus) muscle mitochondrial enzyme activities and concomitant triglyceride depletion is in excellent agreement with the current data and with a role of hyperghrelinemia in muscle metabolic changes during CR. Ghrelin also favored liver triglyceride deposition without suppressing tissue mitochondrial oxidative capacity in further agreement with the current data (32). At variance with the current results, enhanced hepatic lipogenic gene expression was, however, observed in the quoted study (32), suggesting that additional factors, possibly including reduced insulin concentration, contributed to the differential gene expression profile observed in CR animals.
In excellent indirect agreement with the current results, muscle transcript levels of nuclear-encoded mitochondrial genes and/or liver and muscle oxidative capacities were reduced after short-term overfeeding (41, 42). Differential effects of CR in different muscle groups are consistent with larger increments in UCP and lipooxidative gene expression in gastrocnemius than soleus muscle after acute starvation in rat (43, 44). Thus, short-term adaptation to reduced nutrient availability involves increased energy gene expression in mixed but not in highly oxidative muscle, and this differential response is likely to be partly due to higher basal oxidative capacity and reserve in slow-twitch muscle fibers (45).
CR states are associated with variable weight loss and reduction of whole-body energy expenditure to limit weight changes (46, 47). Tissue-specific increments of mitochondrial gene expression and oxidative capacity may potentially limit reduction of total energy expenditure and directly contribute to negative energy balance and weight loss (46, 47). It is possible that more prolonged CR associated with weight stabilization results in differential changes in basal mitochondrial oxidative capacity as suggested by previous long-term studies in rat skeletal muscle (48).
Physiological hyperleptinemia has no independent stimulatory effects on patterns of lean tissue mitochondrial-lipid metabolism gene expression
Despite a number of relevant studies on metabolic effects of leptin (7, 12), the impact of sustained physiological increments of circulating leptin on energy metabolism gene expression and lipid content in lean tissues remain largely undetermined. This issue is important because chronic moderate hyperleptinemia occurs in common human obesity, in turn associated with reduced oxidative lipid disposal and excess lipid deposition in lean tissues (13, 14). Also importantly, most studies of in vivo leptin effects have used growing, incompletely mature rodents that are potentially more responsive to leptin effects than their adult counterpart because aging affects leptin sensitivity (7, 12). The moderate leptin dose in the current physiologically relevant setting did reduce spontaneous food intake and cause weight loss. The current findings, however, indicate that moderate increments of circulating leptin (in a range comparable to variations observed in circadian rhythms and after moderate increments of body weight and insulin resistance) do not independently stimulate lean tissue mitochondrial gene expression-oxidative capacity and have no net effect on lipid content in major lean tissues in vivo when compared with the effects of pair-feeding.
The data are intriguing in suggesting that physiological leptin increments negatively affect liver mitochondrial gene expression and oxidative capacity, preventing increased mitochondrial oxidative capacity induced by pair feeding and resulting in no net changes in tissue triglyceride content. Importantly, the current findings are supported by reports of high liver transcripts for nuclear-encoded CO subunits and UCP2 as well as increased proton leak in leptin-deficient ob/ob mice (49, 50, 51, 52). The data further suggest that a negative impact of physiological leptin increments on in vivo AKT phosphorylation contributed to negative leptin effect on liver mitochondria. Previous studies reported no changes of AKT phosphorylation during leptin treatment in cultured hepatocytes (53, 54). In indirect agreement with the current findings, however, leptin also activated upstream steps of the insulin-signaling cascade, thus indeed preventing a potential increase of AKT activating phosphorylation in the quoted studies (53, 54). Reduced liver triglyceride content although notably not increased lipooxidative gene expression (55) was reported in vivo during sustained supraphysiological circulating leptin increments induced by gene therapy in rodents (55, 56, 57). Different leptin dose as well as route of leptin administration could have contributed to differential results. In addition, animals in these studies were not mature (usual age of 2 months) and could have therefore responded differently to leptin treatment (57). Importantly, no pair-fed control animals were studied in some of these reports (55, 57), making it difficult to assess the potential relative contribution of leptin and CR to the quoted findings.
Lack of independent in vivo leptin effects on skeletal muscle mitochondrial oxidative capacity and phosphorylated AKT is in indirect agreement with similar increments in skeletal muscle glucose disposal reported in leptin-treated and pair-fed lean rats during moderate hyperleptinemia (58) and intracerebroventricular leptin infusion (59). In skeletal muscle, leptin administration acutely stimulates fat oxidation in vitro (60) and in vivo (11). During sustained leptin treatment in lean female rat, increased fatty acid oxidation was, however, reported in soleus muscle after contraction ex vivo but not under basal conditions (61) in keeping with the current observations. The recent report of lack of leptin effects on energy gene expression in cultured myocytes provides additional support for the current in vivo findings (62).
In conclusion, moderate CR induces early tissue-specific increments of mitochondrial-lipooxidative gene expression in lean tissues of nonobese adult rat and causes time-dependent increments of tissue oxidative capacity. Increased lean tissue oxidative capacity leads to triglyceride depletion in muscle but not liver, thus resulting in altered lean tissue lipid distribution. Mitochondrial changes are not associated with increased expression of PPAR- and PGC-1, but they are paralleled by increased AKT activation in liver. Physiological hyperleptinemia has no independent stimulatory effects on muscle and liver mitochondrial-lipooxidative gene expression. These results identify increased expression of lean tissue mitochondrial-lipooxidative genes and altered tissue lipid distribution as early responses that could favor substrate oxidation over storage during reduced nutrient availability and contribute to metabolic effects of dietary treatment in insulin-resistant states.
Acknowledgments
We are very grateful to Ms. A. De Santis, A. Semolic, and M. Sturma for excellent technical assistance.
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Address all correspondence and requests for reprints to: Rocco Barazzoni, M.D., Ph.D., Clinica Medica, University of Trieste, Ospedale Cattinara, Strada di Fiume 447, 34100 Trieste, Italy. E-mail: barazzon@units.it.
Abstract
The study aimed at determining, in lean tissues from nonobese rats, whether physiological hyperleptinemia with leptin-induced reduced caloric intake and/or calorie restriction (CR) per se: 1) enhance mitochondrial-energy metabolism gene transcript levels and oxidative capacity; and 2) reduce triglyceride content. Liver and skeletal muscles were collected from 6-month-old Fischer 344 rats after 1-wk leptin sc infusion (0.4 mg/kg · d: leptin + 3-fold leptinemia vs. ad libitum-fed control) or moderate CR (–26% of those fed ad libitum) in pair-fed animals (CR). After 1 wk: 1) leptin and CR comparably enhanced transcriptional expression of mixed muscle mitochondrial genes (P < 0.05 vs. control); 2) CR independently increased (P < 0.05 vs. leptin-control) hepatic mitochondrial-lipooxidative gene expression and oxidative capacity; 3) hepatic but not muscle mitochondrial effects of CR were associated (P < 0.01) with increased activated insulin signaling at AKT level (P < 0.05 vs. leptin-control); 4) liver and muscle triglyceride content were comparable in all groups. In additional experiments, assessing time course of posttranscriptional CR effects, 3-wk superimposable CR (P < 0.05): 1) increased both liver and muscle mitochondrial oxidative capacity; and 2) selectively reduced muscle triglyceride content. Thus, in nonobese adult rat: 1) moderate CR induces early increments of mitochondrial-lipooxidative gene expression and time-dependent increments of oxidative capacity in liver and mixed muscle; 2) sustained moderate CR alters tissue lipid distribution reducing muscle but not liver triglycerides; 3) mitochondrial-lipid metabolism changes are tissue-specifically associated with hepatic AKT activation; 4) short-term physiological hyperleptinemia has no independent stimulatory effects on muscle and liver mitochondrial-lipooxidative gene expression. Increased lean tissue oxidative capacity could favor substrate oxidation over storage during reduced nutrient availability.
Introduction
MITOCHONDRIA ARE THE key site of tissue substrate utilization, and reduced mitochondrial gene expression and function is rapidly emerging as an important contributor to metabolic alterations in insulin-resistant states (1, 2, 3). Changes in fat disposal at the mitochondrial level could play a pivotal adaptive role during changes in nutrient availability and could alter lean tissue lipid content (4, 5), in turn an important determinant of insulin action (6).
Leptin is an anorexigenic hormone secreted by adipose tissue able to reduce spontaneous food intake in rodents (7). Acute or supraphysiological leptin increments are reported to enhance energy expenditure and tissue substrate utilization (7, 8, 9, 10, 11), contributing to body weight loss. The role of leptin in the in vivo regulation of intermediate metabolism in lean tissues is, however, controversial (12), partly due to discrepancies among the above reports (7, 8, 9, 10, 11) and the known association of increased plasma leptin with obesity (7) and obesity-related impaired lean tissue lipid disposal (13, 14). In particular, the potential effects of sustained physiological increments in circulating leptin on mitochondrial-lipid metabolism and their tissue distribution in vivo in adult individuals remain incompletely understood (12). Importantly, metabolic adaptation to calorie restriction (CR) per se involves increased body ability to use available substrates (15, 16, 17, 18) in the presence of reduced circulating leptin (7). Such effects of CR are at least partially independent from extent of weight loss and duration of reduced nutrient intake (19, 20), and they extend to insulin-stimulated conditions in insulin-sensitive tissues, representing an integral component of metabolic effects of dietary treatment of insulin-resistant states (17, 18). Absolute and relative impacts of leptin and CR per se on mitochondrial-lipid metabolism, as well as their molecular mechanisms and tissue distribution, remain incompletely defined.
The current study was therefore aimed at determining whether physiological hyperleptinemia and/or moderate CR per se enhance mitochondrial-lipid metabolism transcriptional gene expression, mitochondrial oxidative capacity as reflected by mitochondrial enzyme activities, and their impact on triglyceride content in metabolically relevant (21) muscle and liver tissues in a lean adult rat model. In addition to mixed type I-type II fiber gastrocnemius, mitochondrial gene expression and function were investigated in type I soleus muscle. Patterns of transcriptional expression of representative mitochondrial-lipid metabolism genes included flux-generating respiratory chain enzyme cytochrome c oxidase (CO) (subunits I and III) (22), uncoupling proteins (UCPs) (23, 24), the rate-limiting enzyme of fatty acid oxidation carnitine palmitoyl transferase (CPT)-I as well as lipogenic enzymes acetyl-coenzyme A carboxylase (ACC) and fatty acid synthase (FAS). CO and citrate synthase (CS) were selected as markers of tissue oxidative capacity for their flux-generating role in the respiratory chain and tricarboxylic acid cycle, respectively (22). Transcriptional expression of fasting-associated regulators of lipid oxidation peroxisome proliferator-activated receptor (PPAR)- and PPAR-coactivator (PGC)-1 (25, 26) was measured to determine their role in potential metabolic changes during sustained moderate CR. Changes in insulin signaling at the AKT level were determined to assess their potential association with changes in mitochondrial-lipid metabolism during CR (1, 2, 3, 27, 28, 29, 30).
Materials and Methods
Animals and experimental protocol
Twenty-four 6-month-old Fischer 344 male rats were purchased from Harlan Italy (San Pietro al Natisone, Italy). All animals were kept in individual cages in a controlled environment (t = 22 C; 12-h light, 12-h dark cycle) in the Animal Facility of the University of Trieste and fed a standard commercial chow diet (Harlan 2018, Harlan; 3.4 Kcal/g). The experimental protocol was approved by the local Committee for Animal Studies and Animal Care, and experimental procedures were carried out in keeping with institutional guidelines. Results of ghrelin measurements showing the physiological interplay between leptin and ghrelin regulation in vivo were previously published (31). After at least 2 wk from arrival, rats were randomly assigned to undergo either recombinant murine leptin (Research Diagnostics Inc., Flanders, NJ) (n = 8, rate of infusion 0.4 mg/d) or vehicle (5 mM NaCitrate, pH 7.4) infusion with pair-feeding (n = 8) via osmotic minipumps (2ML1, Alza, Mountain View, CA) for 7 d. A third group of rats received vehicle infusion but was allowed free access to food (n = 8). Food intake was measured each day in all rats, and food was presented to pair-fed animals in the afternoon about 2 h before beginning of the dark cycle. After 7 d of treatment, rats were killed in the morning 2–3 h into the light cycle. Food was withdrawn from animals to be killed 90 min before injection of an ip overdose of sodium pentobarbital (70 mg/kg body weight). Gastrocnemius and soleus muscles and liver tissue were then quickly removed in this order and immediately frozen in liquid nitrogen and stored at –80 C until analysis. Blood was drawn through cardiac puncture and plasma was also stored at –80 C.
Additional experiments were designed to assess potential time course of posttranscriptional effects of CR on mitochondrial enzyme activities as well as on triglyceride content in lean tissues (see Results and Discussion). Rats of same age, strain, and body weight (see Results) were divided into two groups receiving either ad libitum control diet (Harlan 2018, Harlan; 3.4 Kcal/g) or CR superimposable to that in the pair-fed group for 3 wk. Identical experimental procedures were used for liver, mixed gastrocnemius muscle, and blood collection.
RNA and DNA analysis
Total RNA was isolated from 40–60 mg of tissues by the guanidinium method (Tri Reagent, MRC, Inc., Cincinnati, OH). Transcript levels of pivotal regulators of lipid metabolism were measured by real-time PCR (7900 Sequence Detection System, Applied Biosystems, Foster City, CA) as previously described (32). Total RNA (1 μg) was reverse-transcribed (RNA Reverse Transcription KIT, Applied Biosystems) and amplified using primers (300 nM) and probes (50 nM) selected using the Primer Express Software (Applied Biosystems) (Table 1). Target and housekeeping gene were run separately and their final quantitation was achieved using a relative standard curve. Results for each gene were divided by the corresponding 28S rRNA abundance and expressed as percent of average control value. CO subunit I and III and UCP mRNA levels were measured by Northern blotting of total RNA as previously described (33, 34).
TABLE 1. Forward (FP) and reverse (RP) primer and probe sequences for real-time PCR gene quantitation for ACC, fatty acid synthase (FAS), CPT-I, PPAR- and 28S rRNA
Total DNA was extracted from liver and skeletal muscles using the Wizard Genomic DNA Isolation kit (Promega Corp., Madison, WI). Mitochondrial (mt) DNA copy number was measured by Southern blotting using cDNA probes for mtDNA-encoded CO I and nuclear-encoded 28S rRNA genes as previously described (33). CO bands in each tissue were normalized to the corresponding 28S rRNA band and individual results were expressed as a percentage of the average value for control ad libitum-fed animals.
Western blot
For measurement of activated (phosphorylated-P) AKT, total tissue proteins were extracted from liver and muscles and quantitated as described (32, 34). Forty micrograms of total protein (six to seven animals per group) were separated on 12% acrylamide gels and transferred to polyvinylidene difluoride membranes (Bio-Rad Laboratories, Hercules, CA) that were blocked and hybridized overnight at 4 C to rabbit antibodies for Ser473-P-AKT (Cell Signaling, Beverly, MA). The secondary antibody was peroxidase-conjugated goat antirabbit IgG (Jackson ImmunoResearch, West Grove, PA) used at a 1:10,000 dilution for 1 h at room temperature. Membranes were then exposed to films for 4–8' (Kodak Biomax MR, Kodak, Rochester, NY), and resulting images were quantitated by densitometry. Similar to mRNA measurement, individual results from all experiments were expressed as a percentage of the average value for vehicle-treated animals.
CO and CS enzyme activity, tissue triglycerides, and plasma biochemical profile
CO as well as CS enzyme activity was measured spectrophotometrically from tissue homogenates in liver and muscles (32, 33). Triglyceride content was measured from 35–40 mg of liver and gastrocnemius muscle (32). Samples were homogenized in 2:1 chloroform: methanol solution in a 20:1 volume: weight ratio and kept at 4 C overnight with gentle shaking. Phase separation was performed using H2SO4 (1 mmol/liter), lipid phase was dried under nitrogen and dissolved in 100 μl ethanol. Triglyceride content was then measured using a commercially available kit (TG, Roche Diagnostics Corp., Indianapolis, IN). Plasma leptin, insulin, and ghrelin concentrations were measured by RIA using commercially available kits (Linco, St. Louis, MO). Blood glucose was measured with an AccuCheck glucose monitor (Roche Diagnostic Corp.).
Statistical analysis
Results in the three groups were compared using one-way ANOVA. Student’s t test for unpaired data was then used to compare results between two groups. P values of less than 0.05 were considered statistically significant. In follow-up experiments, Student’s t test for unpaired data was used to compare results in the two experimental groups.
Results
Food intake, body weight, and metabolic profile
Compared with ad libitum-fed rats leptin reduced overall spontaneous food intake by 26% and superimposable changes were induced in pair-fed animals. As expected, leptin-treated and pair-fed rats lost moderate but statistically significant amounts of body weight compared with control ad libitum-fed animals, resulting in approximately 4% net loss at the end of the study period (Table 2). Plasma leptin concentration increased less than 3-fold in the leptin-treated group, whereas it was reduced in the pair-fed group compared with control animals (both P < 0.05 vs. control). Plasma ghrelin was increased (P < 0.05 vs. control and leptin) in pair-fed but not in leptin-treated rats. Plasma insulin concentration was similarly reduced in leptin and pair-fed groups (P < 0.05 vs. control), whereas glucose concentration was comparable in all animals. Circulating free fatty acids were reduced in calorie-restricted (CR) (P < 0.05 vs. control and leptin) but not in leptin-treated animals.
TABLE 2. Body weight changes, food intake, plasma leptin, insulin, ghrelin, glucose, and free fatty acid concentrations in the three experimental groups
Moderate short-term CR but not physiological hyperleptinemia favor expression of mitochondrial-lipooxidative over lipogenic genes in liver
Tissue patterns of mitochondrial-lipid metabolism gene expression.
In liver leptin treatment reduced expression of lipogenic genes ACC and FAS (P < 0.05 vs. control, Fig. 1) with no changes in mitochondrial-lipooxidative genes. In addition, leptin treatment was associated with muscle group-specific effects. In mixed type I-type II fiber gastrocnemius muscle leptin resulted in higher CO I, CO III, and UCP3 but not UCP2 transcript levels (P < 0.05 vs. control; Fig. 2). CPT-I and ACC transcript levels were not altered after leptin treatment (Fig. 2). No effect of leptin on mitochondrial-UCP gene expression were observed in highly oxidative type I soleus muscle (Table 3).
FIG. 1. Moderate short-term CR but not physiological hyperleptinemia favor expression of mitochondrial-lipooxidative over lipogenic genes in liver. A, Effects of CR and leptin on liver CO subunit I and III and UCP-2 mRNA levels (Northern blotting). B, Effects of CR and leptin on liver CPT-I, ACC, FAS mRNA levels (real-time PCR). Bars represent average ± SE from eight animals per group. Under each bar (A) are shown representative bands from two animals of each group. Top bands show signals from each target gene and bottom bands show signals from the 28S rRNA probe. *, Statistically different results (P < 0.05 or less) by ANOVA and Student’s t test for unpaired data compared with control; $, statistically different results (P < 0.05 or less) by ANOVA and Student’s t test for unpaired data compared with control and leptin.
FIG. 2. Moderate short-term CR and moderate hyperleptinemia comparably enhance expression of mitochondrial genes in gastrocnemius muscle. Effects of CR and leptin on mRNA levels of gastrocnemius CO subunit I and III (A), UCP 2 and 3 (B), and CPT-I and ACC. Bars represent average ± SE from eight animals per group. Under each bar (A and B) are shown representative bands from two animals of each group. Top bands show signals from each target gene and bottom bands show signals from the 28S rRNA probe. *, Statistically different results (P < 0.05 or less) by ANOVA and Student’s t test for unpaired data compared with control.
TABLE 3. Transcript levels of CO subunit I, subunit III, UCP2, UCP3, and COX activity in soleus muscle in the three experimental groups
At variance with leptin treatment, pair feeding independently favored the expression of hepatic mitochondrial-lipooxidative over lipogenic genes (CO III, UCP2, CPT-I: P < 0.05 vs. control and leptin—Fig. 1). Pair-feeding and leptin treatment were associated with superimposable patterns of transcriptional changes in skeletal muscles. In particular, moderate CR with adaptive hypoleptinemia lead to similar increments in mixed muscle mitochondrial and UCP transcript levels (P < 0.05 vs. control; Fig. 2).
Mitochondrial DNA copy number was measured in gastrocnemius and liver tissues to assess whether changes in mitochondrial-encoded transcripts were associated with increased template availability. Mitochondrial DNA copy number was comparable in the three experimental groups in both tissues, although a trend for reduced mtDNA was observed in gastrocnemius muscle from CR animals (control vs. leptin vs. CR: liver, 100 ± 6 vs. 96 ± 14 vs. 95 ± 16; gastrocnemius, 100 ± 18 vs. 97 ± 21 vs. 68 ± 10, arbitrary units; all P > 0.10).
Tissue mitochondrial enzyme activities and triglyceride levels (Table 4).
After 1 wk of moderate CR, mitochondrial CO activity, an index of tissue oxidative capacity (22), was higher in CR compared with leptin-treated and ad libitum-fed rats in liver (P < 0.05 CR vs. control and leptin, P = 0.05 leptin vs. control). At variance with the liver, skeletal muscle CO activities in CR rats were comparable with those in control and leptin-treated animals. Tissue triglyceride content was comparable in
TABLE 4. PPAR- and PGC-1 mRNA (arbitrary units), CO enzyme activity (micromoles per minute per gram of tissue) and triglyceride content (milligrams per gram wet weight) in liver and gastrocnemius muscle in the three experimental groups
Tissue PPAR, PGC-1 expression, and AKT phosphorylation.
Changes in mitochondrial-lipid metabolism gene expression after both leptin treatment and CR per se were not associated with changes in PPAR and PGC-1 transcriptional expression in any tissue (Table 4). Hepatic but not muscle mitochondrial changes were associated with increased P-AKT in CR compared with both leptin and control groups (Fig. 3). A positive correlation was observed between hepatic P-AKT and CO enzyme activity (Liver: r = 0.83; P < 0.0001; data not shown). Liver CO enzyme activity was in turn negatively correlated with plasma free fatty acid concentration (r = –0.68; P = 0.001, Fig. 4). A similar negative correlation was observed between liver CPT-I transcript levels and circulating free fatty acids (r = –0.57; P = 0.005; data not shown).
FIG. 3. Effects of moderate short-term CR and moderate hyperleptinemia on liver and gastrocnemius muscle activated P-AKT. Bars represent average ± SE from six to seven animals per group. Under each bar are shown representative bands from two animals of each group. *, Statistically different results (P < 0.05 or less) by Student’s t test for unpaired data compared with control.
FIG. 4. Liver CO enzyme activity is negatively correlated with plasma free fatty acid (FFA) concentration (r = –0.68; P < 0.001).
Three-week moderate CR enhances lean tissue mitochondrial enzyme activity and reduces muscle triglyceride content with tissue-specific effects on AKT phosphorylation
Mitochondrial protein synthesis is a key posttranscriptional step of mitochondrial gene expression, and its basal rate is substantially higher in liver than in skeletal muscle (35). This observation led us to test the hypothesis that changes in posttranscriptional steps of CO expression would require longer time and occur after more sustained CR in muscle than in the liver. In additional experiments, Fischer 344 rats of identical age and initial weight (control vs. CR: 374 ± 4 vs. 371 ± 4 g) underwent superimposable CR (n: 10–12 each; control vs. CR: 94 ± 4 vs. 72 ± 2.5 g/wk, –27% food intake) for 3 wk. Plasma metabolic profile was also comparable to that observed in 1-wk CR animals (control vs. CR: plasma leptin: 9.6 ± 2.1 vs. 6.6 ± 1.4 ng/ml; plasma insulin: 6.8 ± 1.2 vs. 3.2 ± 0.4 ng/ml, P < 0.05; plasma ghrelin: 2019 ± 169 vs. 3489 ± 339 pg/ml, P < 0.05; blood glucose: 7.9 ± 0.2 vs. 8.3 ± 0.2 mmol/liter; plasma free fatty acids 0.306 ± 0.020 vs. 0.232 ± 0.023 mmol, P < 0.05). Similar to the 1-wk protocol hepatic CO activity and P-AKT were increased after 3-wk CR (Fig. 5). In addition, 3-wk CR also increased gastrocnemius muscle mitochondrial oxidative capacity in the absence of changes in P-AKT. Activity of CS, a key enzyme of the Krebs cycle and also a marker of mitochondrial oxidative capacity, was also increased in both tissues in CR animals. Increased mitochondrial enzyme activities were associated with reduced triglyceride content in gastrocnemius muscle but not in liver indicating tissue-specificity of lipid deposition and altered tissue lipid distribution during reduced nutrient availability (Fig. 5).
FIG. 5. Effects of 3-wk CR on CO and CS enzyme activities and triglyceride content (TG) in liver (A and B) and gastrocnemius muscle (C and D); effects of 3-wk CR on P-AKT content (E) in both tissues. Bars represent average ± SE from eight to nine animals per group. *, Statistically different results (P < 0.05 or less) by Student’s t test for unpaired data compared with control.
Discussion
The current study shows that: 1) moderate CR induces early increments of mitochondrial-lipooxidative gene expression and time-dependent increments of lean tissue oxidative capacity in liver and mixed skeletal muscle of nonobese rats; 2) increased lean tissue oxidative capacity is associated with reduced tissue triglyceride content in muscle but not in liver tissue; 3) mitochondrial-lipid metabolism changes are associated with increased insulin signaling activation at the AKT level in liver but not skeletal muscle; and 4) short-term physiological hyperleptinemia has no independent stimulatory effects on muscle and liver mitochondrial-lipooxidative gene expression.
Moderate CR favors expression of mitochondrial-lipooxidative genes
The current data identify increased expression of oxidative genes in both liver and mixed skeletal muscle as an early response to moderate reduction of nutrient availability in nonobese rats. Mitochondrial oxidative capacity as reflected by the activity of the flux-generating enzymes CO (respiratory chain) and CS (tricarboxylic acid cycle) was also time-dependently increased by moderate CR in both tissues. Mitochondrial transcript levels and oxidative capacity are positively associated with insulin-stimulated substrate disposal and insulin sensitivity in lean tissues in vivo (3, 36, 37, 38, 39). Increased insulin sensitivity in CR animals in the current study is supported by reduced circulating levels of insulin and free fatty acids as well as by tissue-specific increment of activated insulin-signaling molecules. The current results therefore indicate the involvement of early mitochondrial changes in increased insulin action commonly observed in CR states and dietary treatment of insulin-resistant conditions (16, 17, 18, 19, 20).
The potential impact of mitochondrial changes on tissue lipid content is a relevant issue because lean tissue lipid deposition is associated with insulin resistance (6). The current observations support a link between changes in oxidative enzyme activities and skeletal muscle lipid content in nonobese rats, in agreement with previous reports in obese or insulin-resistant models (40). A more complex relationship between lipid content and mitochondrial function is in turn suggested in the liver. Relative excess deposition of liver triglycerides in CR animals is consistent with fatty liver reported during chronic or marked undernutrition (13). Contributors to excess liver fat in the presence of increased mitochondrial oxidative capacity under the current experimental conditions could include altered hepatic lipid uptake and/or secretion, as suggested by previous reports (13). Although its potential adaptive role remains to be elucidated, increased hepatic fat deposition could sustain glucose and lipoprotein production while contributing to reduce lipid content and insulin resistance in nonliver tissues during CR. Whereas mixed muscle phosphorylated AKT was unchanged by CR under basal conditions, consistent with previous reports (28), a substantial tissue-specific increase in hepatic AKT activation occurred in CR animals in the current study, suggesting a link between in vivo activation of insulin signaling and enhanced mitochondrial oxidative capacity in the liver. Liver AKT overexpression (30) or activation through phosphatase and tension homolog knockout (29) lead to parallel increments of hepatic lipid content and systemic insulin sensitivity in ad libitum-fed rodent models. Also in indirect agreement with the current data, liver fat deposition after AKT overexpression in ad libitum-fed rats was independent of enhanced tissue lipogenic gene expression (30). The current findings, therefore, suggest liver AKT activation as a potential contributor to maintained hepatic lipid content (13), as well as increased systemic insulin action (16, 17, 18) in the presence of reduced nutrient availability. A role of hepatic responses in systemic adaptive changes in lipid metabolism and disposal is also supported by the association between hepatic mitochondrial oxidative capacity and circulating free fatty acids.
PPAR- and PGC-1 are reported to be associated with adaptive lipid metabolism changes during fasting (25, 26). Lack of changes in their transcript levels does not support their potential role in metabolic adaptation to moderate CR, although indirectly indicating that no major fasting effects occurred under the current experimental conditions. CR is associated with increased circulating ghrelin that was recently demonstrated to have a profound tissue-specific impact on liver and muscle mitochondrial function and lipid metabolism in ad libitum-fed rats (32). Stimulation by 4-d exogenous ghrelin treatment of mixed (but not soleus) muscle mitochondrial enzyme activities and concomitant triglyceride depletion is in excellent agreement with the current data and with a role of hyperghrelinemia in muscle metabolic changes during CR. Ghrelin also favored liver triglyceride deposition without suppressing tissue mitochondrial oxidative capacity in further agreement with the current data (32). At variance with the current results, enhanced hepatic lipogenic gene expression was, however, observed in the quoted study (32), suggesting that additional factors, possibly including reduced insulin concentration, contributed to the differential gene expression profile observed in CR animals.
In excellent indirect agreement with the current results, muscle transcript levels of nuclear-encoded mitochondrial genes and/or liver and muscle oxidative capacities were reduced after short-term overfeeding (41, 42). Differential effects of CR in different muscle groups are consistent with larger increments in UCP and lipooxidative gene expression in gastrocnemius than soleus muscle after acute starvation in rat (43, 44). Thus, short-term adaptation to reduced nutrient availability involves increased energy gene expression in mixed but not in highly oxidative muscle, and this differential response is likely to be partly due to higher basal oxidative capacity and reserve in slow-twitch muscle fibers (45).
CR states are associated with variable weight loss and reduction of whole-body energy expenditure to limit weight changes (46, 47). Tissue-specific increments of mitochondrial gene expression and oxidative capacity may potentially limit reduction of total energy expenditure and directly contribute to negative energy balance and weight loss (46, 47). It is possible that more prolonged CR associated with weight stabilization results in differential changes in basal mitochondrial oxidative capacity as suggested by previous long-term studies in rat skeletal muscle (48).
Physiological hyperleptinemia has no independent stimulatory effects on patterns of lean tissue mitochondrial-lipid metabolism gene expression
Despite a number of relevant studies on metabolic effects of leptin (7, 12), the impact of sustained physiological increments of circulating leptin on energy metabolism gene expression and lipid content in lean tissues remain largely undetermined. This issue is important because chronic moderate hyperleptinemia occurs in common human obesity, in turn associated with reduced oxidative lipid disposal and excess lipid deposition in lean tissues (13, 14). Also importantly, most studies of in vivo leptin effects have used growing, incompletely mature rodents that are potentially more responsive to leptin effects than their adult counterpart because aging affects leptin sensitivity (7, 12). The moderate leptin dose in the current physiologically relevant setting did reduce spontaneous food intake and cause weight loss. The current findings, however, indicate that moderate increments of circulating leptin (in a range comparable to variations observed in circadian rhythms and after moderate increments of body weight and insulin resistance) do not independently stimulate lean tissue mitochondrial gene expression-oxidative capacity and have no net effect on lipid content in major lean tissues in vivo when compared with the effects of pair-feeding.
The data are intriguing in suggesting that physiological leptin increments negatively affect liver mitochondrial gene expression and oxidative capacity, preventing increased mitochondrial oxidative capacity induced by pair feeding and resulting in no net changes in tissue triglyceride content. Importantly, the current findings are supported by reports of high liver transcripts for nuclear-encoded CO subunits and UCP2 as well as increased proton leak in leptin-deficient ob/ob mice (49, 50, 51, 52). The data further suggest that a negative impact of physiological leptin increments on in vivo AKT phosphorylation contributed to negative leptin effect on liver mitochondria. Previous studies reported no changes of AKT phosphorylation during leptin treatment in cultured hepatocytes (53, 54). In indirect agreement with the current findings, however, leptin also activated upstream steps of the insulin-signaling cascade, thus indeed preventing a potential increase of AKT activating phosphorylation in the quoted studies (53, 54). Reduced liver triglyceride content although notably not increased lipooxidative gene expression (55) was reported in vivo during sustained supraphysiological circulating leptin increments induced by gene therapy in rodents (55, 56, 57). Different leptin dose as well as route of leptin administration could have contributed to differential results. In addition, animals in these studies were not mature (usual age of 2 months) and could have therefore responded differently to leptin treatment (57). Importantly, no pair-fed control animals were studied in some of these reports (55, 57), making it difficult to assess the potential relative contribution of leptin and CR to the quoted findings.
Lack of independent in vivo leptin effects on skeletal muscle mitochondrial oxidative capacity and phosphorylated AKT is in indirect agreement with similar increments in skeletal muscle glucose disposal reported in leptin-treated and pair-fed lean rats during moderate hyperleptinemia (58) and intracerebroventricular leptin infusion (59). In skeletal muscle, leptin administration acutely stimulates fat oxidation in vitro (60) and in vivo (11). During sustained leptin treatment in lean female rat, increased fatty acid oxidation was, however, reported in soleus muscle after contraction ex vivo but not under basal conditions (61) in keeping with the current observations. The recent report of lack of leptin effects on energy gene expression in cultured myocytes provides additional support for the current in vivo findings (62).
In conclusion, moderate CR induces early tissue-specific increments of mitochondrial-lipooxidative gene expression in lean tissues of nonobese adult rat and causes time-dependent increments of tissue oxidative capacity. Increased lean tissue oxidative capacity leads to triglyceride depletion in muscle but not liver, thus resulting in altered lean tissue lipid distribution. Mitochondrial changes are not associated with increased expression of PPAR- and PGC-1, but they are paralleled by increased AKT activation in liver. Physiological hyperleptinemia has no independent stimulatory effects on muscle and liver mitochondrial-lipooxidative gene expression. These results identify increased expression of lean tissue mitochondrial-lipooxidative genes and altered tissue lipid distribution as early responses that could favor substrate oxidation over storage during reduced nutrient availability and contribute to metabolic effects of dietary treatment in insulin-resistant states.
Acknowledgments
We are very grateful to Ms. A. De Santis, A. Semolic, and M. Sturma for excellent technical assistance.
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