Transcriptionbased Mechanisms Of Metabolic Reprogramming In Muscle In Response To Overnutrition

Understanding of metabolic reprogramming and fuel selection in skeletal muscle under different physiological conditions has deepened as a result of new knowledge about transcription factors that serve as broad metabolic regulators. For example, the family of peroxisome proliferator-activated receptors (PPARs) are powerful global regulators of metabolism according to nutritional status (59-61). The three major PPAR subtypes, PPARa, 8, and 7 have distinct tissue distributions that reflect their discrete but overlapping functions. PPARa is expressed most abundantly in skeletal muscle, the heart, and the liver, where it plays a key role in regulating pathways of ^-oxidation (61). Although PPAR7, the target of the insulin-sensitizing thiazolidinediones, is expressed primarily in adipose tissue (62), recent studies have demonstrated that muscle-specific deletion of PPAR7 in mice resulted in whole-body insulin resistance, suggesting the low levels of this receptor in muscle are physiologically important (63). PPARS, the most ubiquitous and least characterized of these receptors, has been shown to regulate both fatty acid oxidation and cholesterol efflux, apparently sharing many duties with PPARa (60,64). Recent findings also suggest that PPARS participates in the adaptive metabolic and histologic (fiber-type switching) response of skeletal muscle to endurance exercise (65).

Pharmacological activation of either PPARa or PPARS results in the robust induction of genes that influence lipid metabolism, including several associated with lipid trafficking, interorgan lipid transport and cholesterol efflux, fatty acid oxidation, glucose sparing and uncoupling proteins (UCPs) (60,64). Interestingly, a similar set of genes is upregulated by diverse circumstances that raise circulating free fatty acids, including obesity, diabetes, overnight starvation, high-fat feeding, and acute exercise (60,64,66). Studies in PPARa-null mice indicate that this nuclear receptor is essential for regulating both constitutive and inducible expression of genes involved in fatty acid oxidation in the liver and heart (61). However, skeletal muscles from PPARa-null mice are remarkably unperturbed with regard to lipid metabolism, and retain their ability to upregulate several known PPAR-target genes in response to starvation and exercise, perhaps owing to functional redundancy between PPARa and PPARS (60,64).

The nutritionally responsive PPAR receptors are themselves regulated by interactions with a variety of co-activators and corepressors. Promiment among these in terms of regulation of skeletal muscle physiology are the PPAR7 Coactivator-1 (PGC-1) proteins, PGC-1a andPGC-1(3. PGCla was originally identified as a PPAR7 interacting protein responsible for regulating mitochondrial replication in brown fat (67). Subsequent studies identified a second isoform (PGC1^) and determined that both proteins are widely expressed and function as promiscuous coactivators of a number of nuclear hormone receptors, as well as other kinds of transcription factors (68). In addition to its interactions with PPARs to regulate lipid metabolism, PGC1a stimulates mitochondrial biogenesis via coactivation of the nuclear respiratory factor (69) and regulates genes involved in oxidative phosphorylation through interactions with estrogen-related receptor a (70) in muscle. PGC1a also coactivates myocyte enhancer factor-2 (69), a muscle-specific transcription factor involved in fiber-type programming. PGC1a is more abundant in red/oxidative muscle and is induced by exercise, whereas its expression is decreased both by inactivity and chronic high-fat feeding (71,72). In contrast, PGC1^ mRNA levels are unaltered by these manipulations.

UPREGULATION OF FATTY ACID OXIDATION AS A MECHANISM FOR GENERATING LIPID SPECIES THAT IMPAIR INSULIN ACTION—A UNIFYING HYPOTHESIS?

We now return to the issue of how the seemingly discrepant hypotheses of obesity-related muscle insulin resistance (a condition of up-regulated or down-regulated fatty acid oxidation?) can be reconciled. One emergent idea is that lipid-induced upregulation of the enzymatic machinery for ^-oxidation of fatty acids is not coordinated with downstream metabolic pathways such as the tricarboxylic acid (TCA) cycle and electron transport chain (71,73). This idea came to light via the observation that isolated mitochondria from rats fed on a high-fat diet had the same rate of [14C] palmitate oxidation to CO2 as mitochondria isolated from muscles of standard chow-fed control rats, but with a larger accumulation of radiolabeled intermediates in an acid-soluble pool (71) (Fig. 1A, B). This suggests that insulin resistant muscles from fat-fed rats have a higher rate of "incomplete" fatty acid oxidation. Consistent with this idea is the previously discussed study in which hepatic expression of malonyl-CoA decarboxylase (MCD) caused near-complete reversal of severe muscle insulin resistance in rats fed a high-fat diet (22). In this study, metabolic profiling of 36 acyl-carnitine species by tandem mass spectrometry revealed a unique decrease in the concentration of one lipid-derived metabolite, ^-OH-butyrylcarnitine (C4-OH), in muscle of MCD-overexpressing animals (22) (Fig. 2A). Moreover, muscle concentrations of this metabolite correlated positively with serum levels of nonesterified fatty acids (Fig. 2B) but not circulating ketones, suggesting that its production occurs locally within the muscle as a consequence of increased lipid delivery. Further studies revealed that exposure of L6 myotubes to elevated concentrations of fatty acids not only induces enzymes of

Fig. 1. Fatty acid oxidation in rat muscle mitochondria. Mitochondria were isolated from whole gastrocnemius muscles harvested in the ad lib fed or 24 h starved state from rats fed on a either a standard chow (SC) or high fat (HF) diet for 12 wk. Mitochondria were incubated in the presence of 150 ^M [1-14C]palmitate and radiolabel incorporation in CO2 (A) was determined as a measure of complete oxidation, whereas label incorporation into acid soluble metabolites (ASM) (B) was measured to assess incomplete fatty acid oxidation. Complete and incomplete oxidation rates were normalized to total mitochondrial protein. Data are from Koves et al. (71).

Fig. 1. Fatty acid oxidation in rat muscle mitochondria. Mitochondria were isolated from whole gastrocnemius muscles harvested in the ad lib fed or 24 h starved state from rats fed on a either a standard chow (SC) or high fat (HF) diet for 12 wk. Mitochondria were incubated in the presence of 150 ^M [1-14C]palmitate and radiolabel incorporation in CO2 (A) was determined as a measure of complete oxidation, whereas label incorporation into acid soluble metabolites (ASM) (B) was measured to assess incomplete fatty acid oxidation. Complete and incomplete oxidation rates were normalized to total mitochondrial protein. Data are from Koves et al. (71).

fatty acid oxidation, such as CPT-1, but also increases the expression of the ketogenic enzyme, mitochondrial HMG CoA synthase (Fig. 2C), while having no effect on expression of key enzymes of the TCA cycle or the electron transport chain (22). Thus, this work suggests that de novo ketogenesis (typically thought of as a hepatic program) is induced in skeletal muscle to provide an outlet for accumulating acetyl CoA, made necessary by increased P-oxidative flux occurring without a coordinated adjustment in TCA cycle activity. The profile of other acylcarnitine species obtained by tandem MS also support the notion of incomplete P-oxidation in animal models of insulin resistance. Such profiles demonstrate that multiple fatty acylcarnitine metabolites, including long-chain acylcarnitines such as palmityl- and oleyl-carnitine, were abnormally high in obese compared to lean rats (22,71). Moreover, rats fed a standard chow diet exhibited decreased levels of acylcarnitines in muscle during the transition from the fasted to the fed states, whereas in comparison, rats on the high-fat diet exhibited little or no change (Fig. 3A). Finally, a 3-wk exercise intervention in mice fed on a chronic high-fat diet lowered muscle acylcarnitine levels (Fig. 3B), in association with increased TCA cycle activity and restoration of glucose tolerance (71).

These studies also highlighted important roles for PGC1a and PPAR transcription factors in mediating lipid-induced metabolic adaptations (71). Similar to muscle mitochondria from high-fat fed rats, L6 myocytes exposed to increasing fatty acid concentrations exhibited disproportionate increases in the rates of incomplete (assessed by measuring incorporation of the label from [14C] oleate into acid-soluble P-oxidative intermediates) relative to complete (label incorporation into CO2) P-oxidation of fatty acids. Overexpression of PGC1a in lipid-cultured L6 cells caused production of 14CO2 to increase and maintain pace with production of [14C]-labeled acid-soluble P-oxidative intermediates (Fig. 4A). In other words, the ratio of complete to incomplete P-oxidation was dramatically increased by PCG1a expression (Fig. 4B). Consistent with these functional assessments, cDNA microarray analyses showed that fatty acid exposure in the context of low PGC1a activity resulted in the induction of classic PPAR-targeted genes involved in lipid trafficking, glucose sparing and P-oxidation, but with little or no change in other downstream pathways that regulate respiratory capacity. In contrast, high PGC1a expression enabled the coordinated induction of P-oxidative enzymes with equally important downstream targets (e.g., TCA cycle, ETC, and NADH shuttle systems). These findings imply that PGC1a enables tighter coupling between P-oxidation and the TCA cycle.

Taken together, these metabolic studies underscore several important points. First, the accumulation of fatty acylcarnitines in muscle of obese/insulin resistant rats implies increased rather than decreased rates of

Fig. 2. Reversal of insulin resistance corresponds with reduced ^-OH-butyryl-carnitine levels in muscle. A) Tandem mass spectrometry-based analysis of short (SC), medium (MC) and long (LC) chain acyl carnitine species in gastrocnemius muscles. Wistar rats were fed on a high-fat diet for 11 wk before virus treatment and muscles were harvested 5 d after injections of adenoviruses encoding active malonyl-CoA decarboxylase (AdCMV-MCD A5) or an inactive mutated form of the enzyme (AdCMV-MCDmut). B) Linear regression analysis of ^-OH-butyrate (C4-OH) levels in muscle versus serum free fatty acids (FFA). C) Semiquantitative RT-PCR analysis of HMG-CoA synthase 2 (HS2) mRNA, normalized to glucose-6-phosphate dehydrogenase, G6PDH mRNA, in fully differentiated rat L6 myotubes incubated without (L6-control) or with 500 pM oleate (L6-FA) for 24 h. RNA from liver of fasted rats was analyzed as a positive control. Data are from An et al. (22).

Fig. 2. Reversal of insulin resistance corresponds with reduced ^-OH-butyryl-carnitine levels in muscle. A) Tandem mass spectrometry-based analysis of short (SC), medium (MC) and long (LC) chain acyl carnitine species in gastrocnemius muscles. Wistar rats were fed on a high-fat diet for 11 wk before virus treatment and muscles were harvested 5 d after injections of adenoviruses encoding active malonyl-CoA decarboxylase (AdCMV-MCD A5) or an inactive mutated form of the enzyme (AdCMV-MCDmut). B) Linear regression analysis of ^-OH-butyrate (C4-OH) levels in muscle versus serum free fatty acids (FFA). C) Semiquantitative RT-PCR analysis of HMG-CoA synthase 2 (HS2) mRNA, normalized to glucose-6-phosphate dehydrogenase, G6PDH mRNA, in fully differentiated rat L6 myotubes incubated without (L6-control) or with 500 pM oleate (L6-FA) for 24 h. RNA from liver of fasted rats was analyzed as a positive control. Data are from An et al. (22).

Fig. 4. PGCla enhances complete oxidation of fatty acids. Fatty acid oxidation was evaluated in rat L6 myocytes treated with recombinant adenoviruses encoding P-galactosidase (P-gal) or PGCla, compared against a no virus control (NVC) group. Forty eight h after addition of virus, cells were incubated 3 h with 100-500 pM [14C]oleate. A) Complete fatty acid oxidation was determined by measuring 14C-label incorporation into CO2. B) The relationship between incomplete and complete fatty acid oxidation was expressed as a ratio of label incorporated into acid soluble metabolites (ASM) divided by labeling of CO2. Differences among groups were analyzed by ANOVA and Student's f-test, * indicates P < 0.05 comparing PGCla to NVC and P-gal treatments, $ indicates P < 0.05 comparing low and high FA conditions. Data are from Koves et al. (71).

Rat Gastrocnemius Muscle

Fig. 3. Muscle acylcarnitine profiling in diet-induced insulin resistance and exercise training. A) Gastrocnemius muscles were harvested from rats fed ad libitum (fed) or starved 24 h after 12 wk on either a standard chow (SC) or high fat (HF) diet. B) Gastrocnemius muscles were harvested from mice fed on standard chow (SC) or high fat (HF) diets for 14 wk. During the final 2 wk of the diet half of the mice in each group were kept sedentary (Sed) or exercise trained (Ex) by running wheel. Muscle acylcarnitine profiles were evaluated by tandem mass spectometry and are expressed as a percent of SC-fed controls. Data are from Koves et al. (71).

Fig. 3. Muscle acylcarnitine profiling in diet-induced insulin resistance and exercise training. A) Gastrocnemius muscles were harvested from rats fed ad libitum (fed) or starved 24 h after 12 wk on either a standard chow (SC) or high fat (HF) diet. B) Gastrocnemius muscles were harvested from mice fed on standard chow (SC) or high fat (HF) diets for 14 wk. During the final 2 wk of the diet half of the mice in each group were kept sedentary (Sed) or exercise trained (Ex) by running wheel. Muscle acylcarnitine profiles were evaluated by tandem mass spectometry and are expressed as a percent of SC-fed controls. Data are from Koves et al. (71).

mitochondrial fatty acid uptake and ß-oxidation. Second, experiments in isolated mitochondria from high-fat rats suggest that PPAR-mediated increases in ß-oxidative activity exceeded the capacity of the TCA cycle to fully oxidize the incoming acetyl-CoA. This supports the idea that assessment of complete fat oxidation via measurement of CO2 production provides only a partial view of lipid catabolism. Lastly, the acylcarnitine profiles from fed and fasted rats suggested that mitochondria from obese animals were unable to appropriately adjust mitochondrial fatty acid influx in response to nutritional status, thus supporting the observation of metabolic inflexibility in humans (57).

The foregoing findings now provide a potential reconciliation of current prominent hypotheses of metabolic perturbations leading to muscle insulin resistance (summarized schematically in Fig. 5). The new model holds that fuel oversupply to muscle results in enhanced fatty acid ß-oxidation due both to transcriptional regulation and increased substrate supply. However, in the absence of work (i.e., exercise), the TCA cycle not only remains

Mechanism Metabolism Exercise

Fig. 5. Proposed model of lipid-induced insulin resistance in skeletal muscle. During conditions of overnutrition, starvation and/or inactivity, fatty acid influx and peroxisome proliferator-activated receptor (PPAR)-mediated activation of target genes (in yellow) promotes ß-oxidation without an accompanying increase in tricarboxylic acid (TCA) cycle enzymes. TCA cycle flux and complete fat oxidation is further hampered by a high energy redox state (rising NADH/NAD and acetyl-CoA/free CoA ratios). As a result, metabolic by-products of incomplete fatty acid oxidation (acylcarnitines, ketones and reactive oxygen species (ROS)) accumulate, which in turn gives rise to the accumulation of LC-CoA species and subsequent production of other lipid-derived metabolites, such DAG, ceramide and IMTAG. Together, these mitochondrial and lipid-derived stresses impinge upon insulin signal transduction, thus inhibiting glucose uptake and metabolism (in blue). Exercise combats lipid stress by activating PPAR7 coactivator 1 a (PGCla), which coordinates increased ß-oxidation with the activation of downstream metabolic pathways (in orange), thereby promoting enhanced mitochondrial function and complete fuel oxidation. Tighter coupling of ß-oxidation and TCA cycle activity alleviates mitochondrial stress, lowers intramuscular lipids and restores insulin sensitivity. Abbreviations: ACS; acyl-CoA synthase, ß-Oxd; ß-oxidative enzymes, CD36/FAT; fatty acid transporter, CPT1; carnitine palmitoyltransferase 1, DAG; diacylglycerol, ETC; electron transport chain; Glut4; glucose transporter 4, HS2; mitochondrial HMG-CoA synthase, IMTG; intramuscular triacylglycerol, IR; insulin receptor, LC-CoAs; long-chain fatty acyl-CoAs; PDH; pyruvate dehydrogenase; PDK; pyruvate dehydrogenase kinase, ROS, reactive oxygen species, TF; transcription factor.

Fig. 5. Proposed model of lipid-induced insulin resistance in skeletal muscle. During conditions of overnutrition, starvation and/or inactivity, fatty acid influx and peroxisome proliferator-activated receptor (PPAR)-mediated activation of target genes (in yellow) promotes ß-oxidation without an accompanying increase in tricarboxylic acid (TCA) cycle enzymes. TCA cycle flux and complete fat oxidation is further hampered by a high energy redox state (rising NADH/NAD and acetyl-CoA/free CoA ratios). As a result, metabolic by-products of incomplete fatty acid oxidation (acylcarnitines, ketones and reactive oxygen species (ROS)) accumulate, which in turn gives rise to the accumulation of LC-CoA species and subsequent production of other lipid-derived metabolites, such DAG, ceramide and IMTAG. Together, these mitochondrial and lipid-derived stresses impinge upon insulin signal transduction, thus inhibiting glucose uptake and metabolism (in blue). Exercise combats lipid stress by activating PPAR7 coactivator 1 a (PGCla), which coordinates increased ß-oxidation with the activation of downstream metabolic pathways (in orange), thereby promoting enhanced mitochondrial function and complete fuel oxidation. Tighter coupling of ß-oxidation and TCA cycle activity alleviates mitochondrial stress, lowers intramuscular lipids and restores insulin sensitivity. Abbreviations: ACS; acyl-CoA synthase, ß-Oxd; ß-oxidative enzymes, CD36/FAT; fatty acid transporter, CPT1; carnitine palmitoyltransferase 1, DAG; diacylglycerol, ETC; electron transport chain; Glut4; glucose transporter 4, HS2; mitochondrial HMG-CoA synthase, IMTG; intramuscular triacylglycerol, IR; insulin receptor, LC-CoAs; long-chain fatty acyl-CoAs; PDH; pyruvate dehydrogenase; PDK; pyruvate dehydrogenase kinase, ROS, reactive oxygen species, TF; transcription factor.

inactivated at a transcriptional level, but moreover, flux through the pathway is inhibited by the high energy redox state that prevails under circumstances of overnutrition. As a result, acetyl CoA accumulates and forces accumulation of other acyl CoA species (as reflected by acylcarnitine profiling). This leads in turn to increased production of other lipid-derived molecules, including TAG, diacylglycerol, ketones, ceramides and reactive oxygen species, as well as other yet unidentified metabolites that could contribute to or reflect mitochondrial stress.

An important question remaining is whether the high rates of fatty acid catabolism in the obese state are insufficient to compensate for increased lipid delivery, thereby allowing excess lipid-derived metabolites to impair insulin signaling, or alternatively, whether persistently high rates of mitochondrial ^-oxidation directly contribute to the development of insulin resistance. These possibilities are not necessarily mutually exclusive. Assuming that insulin resistance originally evolved as a survival mechanism, it is likely that nature has devised several distinct metabolic and molecular roadways leading to the same (dys)functional endpoint. Future studies are certain to reveal new clues as to how these pathways intersect, and perhaps more importantly, how they can be circumvented by behavioral and/or pharmacological therapies.

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Responses

  • yohannes
    What is palmitoyltransferase I and DKA?
    7 years ago
  • harri kunnas
    Which are mechanisms of metabolism?
    7 years ago

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