NAD(P)H fluorescence, mitochondrial membrane potential and respiration rate were measured and manipulated in isolated liver cells from fed and starved rats in order to characterize control of mitochondrial respiration and phosphorylation. Increased mitochondrial NADH supply stimulated respiration and this accounted for most of the stimulation of respiration by vasopressin and extracellular ATP. From the response of respiration to NADH it was estimated that the control coefficient over respiration of the processes that supply mitochondrial NADH was about 0.15-0.3 in cells from fed rats.Inhibition of the ATP synthase with oligomycin increased the mitochondrial membrane potential and decreased respiration in cells from fed rats, while the uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone had the opposite effect. There was a unique relationship between respiration and membrane potential irrespective of the ATP content of the cells indicating that phosphorylation potential controls respiration solely via phosphorylation (rather than by controlling NADH supply).From the response of respiration to the mitochondrial membrane potential (dyM) it was estimated that the control coefficients over respiration rate in cells from fed rats were: 0.29 by the processes that generate dyM, 0.49 by the process of ATP synthesis, transport and consumption, and 0.22 by the processes that cycle protons across the inner mitochondrial membrane other than via ATP synthesis (e. g. the passive proton leak). Control coefficients over the rate of mitochondrial ATP synthesis were 0.23, 0.84 and -0.07, respectively, by the same processes. The control distribution in cells from starved rats was similar.The short-term control of cellular respiration and oxidative phosphorylation has not been well characterized in any cell type (for review see [l]). However, many mechanisms of control have been demonstrated with isolated enzymes and isolated mitochondria, and this has led to several hypotheses as to how respiration may be controlled in cells. The simplest hypothesis, arising from the work of Chance and Williams on isolated mitochondria [2, 31, is that mitochondrial respiration in cells is controlled almost exclusively by ATP usage acting via phosphorylation potential on the rate of oxidative phosphorylation and thus via proton-motive force on respiration rate. In accordance with this hypothesis several different means by which phosphorylation potential may control phosphorylation have been proposed, including thermodynamic control (assuming that phosphorylation is close to equilibrium) [4] or kinetic control of the adenine-nucleotide carrier by ADP or the ADP/ATP ratio [5].A second group of hypotheses suggests that mitochondrial respiration and phosphorylation are also controlled by NADH supply acting via mitochondrial NADH on the respiratory chain (see for example . Several different agents have been proposed to increase respiration by increasing mitochondrial NADH supply including (a) respiratory substrates such as fatty acids, lactate ...

The conidiation rhythm in the fungus Neurospora crassa is a model system for investigating the genetics of circadian clocks. Null mutants at the frq (frequency) locus ( frq 9 and frq 10 ) make no functional frq gene products and are arrhythmic under standard conditions. The white-collar strains (wc-1 and wc-2) are insensitive to most effects of light, and are also arrhythmic. All three genes are proposed to be central components of the circadian oscillator. We have been investigating two mutants, cel (chain-elongation) and chol-1 (choline-requirer), which are defective in lipid synthesis and affect the period and temperature compensation of the rhythm. We have constructed the double mutant strains chol-1 frq 9 , chol-1 frq 10 , chol-1 wc-1, chol-1 wc-2, cel frq 9 , cel frq 10 , and cel wc-2. We find that these double mutant strains are robustly rhythmic when assayed under lipid-deficient conditions, indicating that free-running rhythmicity does not require the frq, wc-1, or wc-2 gene products. The rhythms in the double mutant strains are similar to the cel and chol-1 parents, except that they are less sensitive to light. This suggests that the frq, wc-1, and wc-2 gene products may be components of a pathway that normally supplies input to a core oscillator to transduce light signals and sustain rhythmicity. This pathway can be bypassed when lipid metabolism is altered. T he filamentous fungus Neurospora crassa is an excellent model system for investigating the mechanism of circadian rhythmicity (1, 2) because of the wealth of genetic and biochemical techniques available. Its rhythm of asexual spore-formation (conidiation) produces easily assayed bands of conidiospores in cultures growing on solid agar medium. A number of mutations are available that affect circadian rhythmicity, and molecular analyses of some of these genes have contributed to models for circadian oscillators that are currently thought to be applicable to many other organisms. This paper reports the interactions between clock-affecting mutations that are said to be ''arrhythmic'' with no functional circadian clock and mutations affecting lipid synthesis that also have effects on rhythmicity. Our results raise questions about the proposed roles of the well-known clock genes and point toward an important role for lipid metabolism in the mechanism of rhythmicity.The current model for the oscillator mechanism in Neurospora is a transcription͞translation feedback loop involving the rhythmic expression of the frq (frequency) gene and its protein product FRQ (2). Both short-period and long-period alleles are known at this locus. Mutations in the FRQ protein affect temperature compensation of the circadian rhythm (3) and sensitivity to light-induced phase resetting (4, 5). Both the RNA and protein products of the frq locus are rhythmically accumulated (6, 7). The level of frq mRNA is affected by light pulses and steps (8) and by temperature steps (9). The FRQ protein is found in the nucleus, and nuclear localization is necessary for its function (10). T...

This paper analyzes published and unpublished data on phase resetting of the circadian oscillator in the fungus Neurospora crassa and demonstrates a correlation between period and resetting behavior in several mutants with altered periods: As the period increases, the apparent sensitivity to resetting by light and by cycloheximide decreases. Sensitivity to resetting by temperature pulses may also decrease. We suggest that these mutations affect the amplitude of the oscillator and that a change in amplitude is responsible for the observed changes in both period and resetting by several stimuli. As a secondary hypothesis, we propose that temperature compensation of period in Neurospora can be explained by changes in amplitude: As temperature increases, the compensation mechanism may increase the amplitude of the oscillator to maintain a constant period. A number of testable predictions arising from these two hypotheses are discussed. To demonstrate these hypotheses, a mathematical model of a time-delay oscillator is presented in which both period and amplitude can be increased by a change in a single parameter. The model exhibits the predicted resetting behavior: With a standard perturbation, a smaller amplitude produces type 0 resetting and a larger amplitude produces type 1 resetting. Correlations between period, amplitude, and resetting can also be demonstrated in other types of oscillators. Examples of correlated changes in period and resetting behavior in Drosophila and hamsters raise the possibility that amplitude changes are a general phenomenon in circadian oscillators.

The molecular mechanism of circadian rhythmicity is usually modeled by a transcription/translation feedback oscillator in which clock proteins negatively feed back on their own transcription to produce rhythmic levels of clock protein mRNAs, which in turn cause the production of rhythmic levels of clock proteins. This mechanism has been applied to all model organisms for which molecular data are available. This review summarizes the increasing number of anomalous observations that do not fit the standard molecular mechanism for the model organisms Acetabularia, Synechococcus, Drosophila, Neurospora, and mouse. The anomalies fall into 2 classes: observations of rhythmicity in the organism when transcription of clock genes is held constant, and rhythmicity in the organism when clock gene function is missing in knockout mutants. It is concluded that the weight of anomalies is now so large that the standard transcription/translation mechanism is no longer an adequate model for circadian oscillators. Rhythmic transcription may have other functions in the circadian system, such as participating in input and output pathways and providing robustness to the oscillations. It may be most useful to think in terms of a circadian system that uses a noncircadian oscillator consisting of metabolic feedback loops, which acquires its circadian properties from additional regulatory molecules such as the products of canonical clock genes.