Nitric oxide signaling regulates mitochondrial number and function

Obesity affects many millions of children and adults worldwide and poses a major health problem. Weight gain results when energy intake exceeds energy expenditure. Energy can be dissipated in the form of work or heat. In order to combat obesity and associated disease, including diabetes, heart attack, and stroke, understanding how our body regulates energy balance will be of fundamental importance. Recent findings have revealed that the brain regulates energy expenditure. Environmental cues, such as cold, exercise, and food-intake signal the sympathetic nervous system to trigger the release of the hormone, noradrenaline, which in turn innervates brown adipose tissue (BAT) by binding to the β-adrenergic receptor (reviewed in Lowell and Spiegelman¹). BAT is the major site of adaptive thermogenesis, which protects the body from cold and controls the response to changes in diet. β-adrenergic receptor stimulation then leads to mitochondrial biogenesis (mitochondrial proliferation and activation). The mitochondrion can be viewed as a cellular furnace where fatty acids and glucose are oxidized, and energy is stored as ATP or wasted as heat, thus regulating cellular energy balance. The exact pathways that lead to mitochondrial proliferation in response to β-adrenergic receptor signaling in BAT are not entirely understood. Clues have emerged indicating that transcriptional control is implicated in this process.^2,3,4,5,6 Now Nisoli et al.⁷ have made the intriguing discovery that the gas nitric oxide (NO) links β-adrenergic receptor signaling with mitochondrial biogenesis by increasing the activity of a master transcriptional regulator of the mitochondrial biogenesis program. Here we will highlight the observations made by Nisoli and colleagues and speculate on their potential implications for the study of energy metabolism, aging, and cell death.

NO regulates mitochondrial biogenesis. BAT plays an important role in controlling body temperature and energy expenditure. Differentiation of brown adipocytes is accompanied by multiplication and functional activation of mitochondria.⁸ This event coincides with the activation of UCP-1, an uncoupling protein of the mitochondrial inner membrane that discharges the proton gradient, thus dissociating electron transport from ATP synthesis in the respiratory chain and generating heat in response to cold or energy intake.^1,9 The proton leak induced by UCP-1 activity not only generates heat, but also reduces free radical production, thereby regulating redox balance. Nisoli et al.⁷ report, in an article recently published in Science, that NO regulates mitochondrial biogenesis in brown adipocytes and various cell lines. NO is known to have pleiotropic physiological effects depending on the target tissue and cell type (reviewed in Bredt and Snyder¹⁰). For instance, in blood vessels, NO functions as a vasodilator, while in the nervous system, NO acts as a neurotransmitter. However, if produced in excess and in the appropriate redox state, NO can be neurotoxic. NO also regulates the binding and release of oxygen from hemoglobin, thereby regulating oxygen delivery to tissues.¹¹ NO is produced by three isoforms of the nitric oxide synthase (NOS) enzyme. How then does NO trigger an increase in mitochondrial biogenesis? Nisoli et al.⁷ have found that β-adrenergic receptor activation leads to Ca²⁺- and cAMP-mediated activation of endothelial NOS (eNOS) (Figure 1). In turn eNOS generates NO, which induces activation of the master regulator of mitochondrial biosynthesis, PGC-1α (peroxisome proliferator-activated receptor γ coactivator-1α). PGC-1α has been shown to be upregulated after cold exposure and changes in diet.^1,2,3,4,5,6 Moreover, PGC-1α transgenic mice exhibit increased numbers of mitochondria in heart and skeletal muscle.⁵ PGC-1α is a key transcriptional cofactor of PPARγ, a nuclear hormone receptor, which increases the expression of nuclear respiratory factors (NRF) and the mitochondrial transcription factor A (mtTFA). This cascade of events increases the expression of nuclear and mitochondrial genes encoding components of the respiratory chain complexes and regulators of mtDNA replication. Furthermore, antisense inhibition of PGC-1α revealed that NO-induced mitochondrial biogenesis is dependent on PGC-1α. Importantly, the effect of NO on adipose tissue is dependent on cyclic guanosine 3′, 5′-monophosphate (cGMP), thus making it less likely that NO mediates mitochondrial biogenesis by directly affecting mitochondria. It is of note that NO has previously been shown to directly block mitochondrial respiration by competing for the oxygen binding site of cytochrome c oxidase.^12,13,14

Figure 1

Nitric oxide/cGMP-mediated control of mitochondrial biogenesis and energy balance. Changes in cold, exercise, or food intake are detected by the brain. Sympathetic neurons innervate brown adipocytes by releasing noradrenaline (NA) and activating the β-adrenergic receptor (βAR), which in turn results in a rise in intracellular Ca²⁺ and cAMP. These messengers activate endothelial nitric oxide synthase (eNOS) and thus NO synthesis. High levels of NO can directly block mitochondrial respiration. However, lower concentrations of NO activate guanylate cyclase (GC) to increase cGMP, which in turn activates PGC-1α. In combination with the nuclear hormone receptor PPARγ PGC-1α promotes the expression of nuclear respiration factor (NRF), uncoupling protein 1 (UCP-1), mitochondrial transcription factor A (mtTFA), and components of the respiratory chain complex. mtTFA then translocates to mitochondria where it regulates the expression of mitochondrial genes and mitochondrial DNA replication. Uncoupling electron transport from ATP production by UCP-1 triggers a decline ROS in production, ATP levels, and ΔΨ_m, with a concomitant increase in heat, mitochondrial proliferation, and respiration.

Significantly, Nisoli et al.⁷ now show that eNOS knockout mice exhibit a reduced number of BAT mitochondria, decreased UCP-1 and PPAR-γ expression, defective energy expenditure, increased body weight, insulin resistance, and hypertension.^15,16,17 This is a phenotype characteristic of a metabolic syndrome and often occurs during aging.

Extending lifespan: the insulin connection. Is there a link between the NO-mediated pathway of mitochondrial proliferation proposed by Nisoli and colleagues and the regulation of aging? In this regard, a number of observations might be of relevance. NO-mediated mitochondrial biogenesis promotes leanness due to adaptive thermogenesis and a reduction in reactive oxygen species; both of these effects have previously been proposed to increase longevity (reviewed in Spiegelman and Flier¹⁸ and Guarente and Kenyon¹⁹). In contrast, as mentioned above, eNOS knockout mice are insulin-resistant and manifest increased body weight.¹⁶ Thus, it is interesting to speculate that insulin signaling and NO-mediated mitochondrial biogenesis may regulate both body weight and lifespan. Along these lines, levels of UCP proteins and mitochrondrial density are decreased during aging, while free radicals increase (reviewed in Florez-Duquet and McDonald²⁰).

Paradoxically however, insulin receptor signaling has been proposed to accelerate aging (reviewed in Guarente and Kenyon¹⁹). Genetic studies on Caenorabditis elegans revealed that animals lacking DAF-2, a molecule with structural similarity to human insulin growth factor 1 (IGF-1) receptor, doubled their lifespan.^21,22 In addition, Holzenberger et al.²³ recently reported that mice heterozygous for IGF-1 receptor deficiency exhibit increased life expectancy. Insulin-related genes also have critical functions in growth and glucose metabolism. Loss of insulin signaling results in dwarfism, diabetes, and obesity. Recently, however, Blüher et al ²⁴ reported that mice carrying a fat cell-specific insulin receptor knockout (FIRKO) display enhanced longevity and reduced body weight, despite normal food intake. Caloric restriction has been proposed to counteract the aging process by decreasing metabolism and free radical formation, but FIRKO mice manifest increased lifespan and are resistant to obesity, despite normal energy intake. Moreover, FIRKO mice display an increased rate of metabolism. These findings imply that increased energy expenditure and metabolism may be more effective in the delay of aging process than a decreased food intake.

Bi-functionality of NO in cell survival and cell death. NO has the ability to either inhibit or promote cell death.²⁵ How can the same molecule have apparently opposite functions? The molecular basis for this is currently unclear. Several mechanisms have been proposed to account for the protective effects of NO. One such mechanism is the S-nitrosylation (or transfer of NO to a regulatory cysteine thiol) of proteins involved in cell death, such as effector caspases, resulting in the inactivation of the caspase cascade.^26,27 In addition, S-nitrosylation of NMDA receptors and other proteins have been proposed to reduce cell death (reviewed in Lipton²⁸). The observations of Nisoli et al ⁷ suggest yet another mechanism whereby NO may promote survival, i.e. by UCP-1-mediated uncoupling of mitochondria and reduction in free radical production.

In contrast, excessive amounts of NO can combine with superoxide anions (O₂⁻) to form toxic peroxynitrite (ONOO⁻).²⁹ While NO reversibly inhibits mitochondrial respiration,^12,13,14 ONOO⁻ irreversibly blocks many components of the mitochondrial respiratory chain. This process can trigger cell death by apoptosis (if ATP levels are sufficient) or necrosis (if prolonged ATP deficits arise).^30,31,32 Additionally, NO-mediated S-nitrosylation can activate metalloproteinases, thus degrading the extracellualr matrix and promoting cell death by anoikis.³³ Lastly, NO may trigger DNA damage and poly (ADP)-ribose synthetase (PARP)-mediated neuronal cell death.³⁴

Mitochondrial proliferation during cell death. In contrast to NO-induced biogenesis of mitochondria in BAT, there is mounting evidence indicating that mitochondria also proliferate during apoptotic cell death initiated by a wide variety of stimuli in a number of different cell types.³⁵ Nevertheless, the role of mitochondrial proliferation during cell death remains contentious. In fact, mitochondrial proliferation may constitute an early stress response to adapt to new energy demands and to repair injuries, thus promoting cell survival. Notably, mitochondrial uncouplers, such as carbonylcyanide p-(trifluoromethyoxy) phenyl-hydrazone (FCCP), induce mitocho-ndrial proliferation, but do not induce cell death. It is currently unclear whether UCP-1 and related factors play a role in the mitochondrial proliferation events observed during apoptosis. Along these lines, recent studies suggest that the brain mitochondrial uncoupling protein-2 (UCP-2) exhibits protective effects against brain injuries.³⁶

On the other hand, it has been suggested that mitochondrial proliferation, also referred to as mitochondrial fission, might be an intricate part of the cell death program. Support for this notion is provided by the recent findings of Youle et al.³⁵ They report that overexpression of a dominant-negative form of Drp-1, a highly conserved dynamin-related GTPase that is a component of the mitochondrial division/fission machinery, blocked apoptotic mitochondrial proliferation, release of cytochrome c from mitochondria, and cell death.³⁵ In addition, Drp-1 and the proapoptotic Bax protein colocalized to scission sites on mitochondria, suggesting that the mitochondrial fission machinery cooperates with the cell death machinery via Bcl-2 family members.³⁷ Interestingly, NO can upregulate Bax and trigger its translocation to mitochondria.³⁸ Furthermore, NO-induced p38 MAP kinase activation has been shown to induce Bax translocation to mitochondria.³⁹ Mitochondrial uncouplers promote Bax translocation to mitochondria, suggesting that a decrease in mitochondrial membrane potential (Δψ_m) may suffice to facilitate Bax membrane insertion.⁴⁰

Thus, it is interesting to speculate that the dual function of NO, in either promoting or preventing cell death, may have as part of its mechanistic basis the regulation of mitochondrial biogenesis. We propose that small amounts of NO may stimulate mitochondrial proliferation, thereby conveying a survival signal. Conversely, excessive amounts of NO combined with free radicals may, in addition to generating mitochondria, recruit the cell death machinery and thus commit cells to die.

Conclusions/future directions. Mitochondrial biogenesis requires the choreographed expression of diverse transcriptional activators, such as PGC-1, PPARγ, NRF-1, and mtTFA. Nisoli et al.⁷ have shown that NO acts as a key messenger to activate the mitochondrial biogenesis program (Figure 1). These findings imply that NO regulates energy balance, may prevent obesity, and may play a role in determining lifespan. Thus, these results may provide new therapeutic opportunities to combat obesity and associated diseases. In future experiments it will be interesting to test whether NO also regulates mitochondrial proliferation in other cell types and tissues. The question of lifespan in eNOS knockout mice, eNOS transgenic mice, or PGC-1α transgenic mice should also be explored. Lastly, it will be important to elucidate the precise mechanism by which NO/cGMP activates PGC-1α to trigger mitochondrial biogenesis.