TIGAR deficiency enhances skeletal muscle thermogenesis by increasing neuromuscular junction cholinergic signaling
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Curated by eLife
Evaluation Summary:
In this study, Tang and colleagues report that the deletion of the fructose-2,6-phosphatase TIGAR leads resistance to cold-induce hypothermia. Using different complementary approaches, they found that this phenotype originates from alteration in cholinergic neurons. In particular, they found that deleting TIGAR in ChAT-expressing neurons recapitulates the phenotype of the global knock-out. Overall, this is a well-performed study that provides evidence for a role of TIGAR in regulating the neuromuscular junction.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)
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Abstract
Cholinergic and sympathetic counter-regulatory networks control numerous physiological functions, including learning/memory/cognition, stress responsiveness, blood pressure, heart rate, and energy balance. As neurons primarily utilize glucose as their primary metabolic energy source, we generated mice with increased glycolysis in cholinergic neurons by specific deletion of the fructose-2,6-phosphatase protein TIGAR. Steady-state and stable isotope flux analyses demonstrated increased rates of glycolysis, acetyl-CoA production, acetylcholine levels, and density of neuromuscular synaptic junction clusters with enhanced acetylcholine release. The increase in cholinergic signaling reduced blood pressure and heart rate with a remarkable resistance to cold-induced hypothermia. These data directly demonstrate that increased cholinergic signaling through the modulation of glycolysis has several metabolic benefits particularly to increase energy expenditure and heat production upon cold exposure.
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Author Response:
Reviewer #1 (Public Review):
My main concern relates to the title, which does not appear to be supported by the data. One can't conclude that the reported effects are strictly due to altered glycolysis in cholinergic neurons without directly assessing glucose metabolism in these neurons. Moreover,TIGAR functions by blocking glycolysis and directing the pathway into the pentose phosphate shunt. Therefore, the resulting effect of deleting TIGAR in a neuronal population might be multiple.
The authors show convincingly that deleting TIGAR from ChAT-expressing neurons, but not adipose or muscle cells, protects mice from cold-induced hypothermia. It is however unclear whether this leads to alteration in energy expenditure per se. This it important considering the first argument of the discussion highlighting how …
Author Response:
Reviewer #1 (Public Review):
My main concern relates to the title, which does not appear to be supported by the data. One can't conclude that the reported effects are strictly due to altered glycolysis in cholinergic neurons without directly assessing glucose metabolism in these neurons. Moreover,TIGAR functions by blocking glycolysis and directing the pathway into the pentose phosphate shunt. Therefore, the resulting effect of deleting TIGAR in a neuronal population might be multiple.
The authors show convincingly that deleting TIGAR from ChAT-expressing neurons, but not adipose or muscle cells, protects mice from cold-induced hypothermia. It is however unclear whether this leads to alteration in energy expenditure per se. This it important considering the first argument of the discussion highlighting how approaches to increase energy expenditure through the development/activation of brown/beige adipose tissue thermogenesis have failed. Moreover, it is unclear if TIGAR also affects heat dissipation considering the impact of its deletion from ChAT-expressing neurons on blood pressure and heart rate, two parameters that will likely influence the tail vasoactivity. Evaluating energy expenditure and heat loss appears to be necessary to support the conclusion that the resistance to hypothermia is exclusively dependent on shivering thermogenesis.
One key aspect that may deserve discussion is a potential contribution of the sympathetic nervous system to the observed phenotype. The focus of the manuscript is on acetylcholine but one can't disqualify that sympathetic compensations may happen following the deletion of TIGAR in ChAT-expressing neurons.
There are many data that are not shown but that would worth be included (lines 99, 113, 119, 159, 168, 181, 221,
- We have changed the title to better reflect the specific findings in this study.
- We now present in the Discussion section the potential roles of other mechanisms besides cholinergic signaling (sympathetic, vascular, behavioral) that could also contribute to temperature regulation in this model system.
- We have now included some of the data that was originally indicated as data not shown but have eliminated some of these data from the text as they are superfluous and do not provide important information for any of the conclusions drawn.
Reviewer #3 (Public Review):
Strengths: The study is nicely written and presented. The investigation of whole-body TIGAR knockout (TKO) clearly demonstrates resistance to cold exposure, and the authors logically follow potential sources through the obvious tissue candidates.
Both skeletal muscle and adipose specific TIGAR knockouts were generated, neither of which recapitulated the effect of the TKO. Other obvious candidates, such as UCP1 content in adipose and basal oxidative capacity and contractility of skeletal muscle were ruled out using ex vivo techniques.
Nevertheless, pharmacological interventions indicated that muscle contraction was necessary for protection from cold exposure and that the loss of TIGAR overcame competitive antagonism of the nicotinic acetylcholine receptor. These data were supportive of a role for skeletal muscle contraction, particularly at the level of cholinergic signaling.
A cholinergic neuron specific TIGAR knockout was produced. Loss of TIGAR was molecularly confirmed, and this mouse recapitulated the whole-body knockout's resistance to cold exposure.
Tracer studies are largely compelling and confirm that loss of TIGAR increases substrate dependence on glucose oxidation in a cell model.
Weaknesses: The TKO mice were not characterized for body weight, body composition or energy expenditure, leaving some room for alternative or additive mechanisms.
Although the tracer data demonstrate that loss of TIGAR causes the cell model to increase reliance on glycolysis compared to other unlabeled substrates, the data do not necessarily demonstrate an increase in the absolute rate of glycolysis or total acetyl-CoA production as intimated in the discussion. It is also unclear why media glutamate is examined for tracer incorporation rather than tissue glutamate.
There are some minor weaknesses related to the description of the methods. For example, the 18O studies need clarification. It will be unclear to most readers how this method works.
- We now include body composition, food intake, activity and energy expenditure data in new Figures S1D-H.
- Following the stable isotope label from 1,2- 13C glucose into glutamate was used in these tracer analyses to non-invasively assess the differences in carbon flux between pyruvate carboxylase and pyruvate dehydrogenase, allowing us to use the cells for assessment of acetyl CoA and acetyl carnitine in the same experiment. This media tracer data indicates an increase in PDH flux (m1) in TKO cells compared to that in control cells, which, along with the corresponding cellular data for acetyl-CoA and acetyl-carnitines levels, all elevated in the TKO SH-SY5Y cells that are also consistent with an increase rate of glycolysis (new Figure S7C and D).
- We have further clarified the methods for the use of 18O labeled water.
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Evaluation Summary:
In this study, Tang and colleagues report that the deletion of the fructose-2,6-phosphatase TIGAR leads resistance to cold-induce hypothermia. Using different complementary approaches, they found that this phenotype originates from alteration in cholinergic neurons. In particular, they found that deleting TIGAR in ChAT-expressing neurons recapitulates the phenotype of the global knock-out. Overall, this is a well-performed study that provides evidence for a role of TIGAR in regulating the neuromuscular junction.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
My main concern relates to the title, which does not appear to be supported by the data. One can't conclude that the reported effects are strictly due to altered glycolysis in cholinergic neurons without directly assessing glucose metabolism in these neurons. Moreover,TIGAR functions by blocking glycolysis and directing the pathway into the pentose phosphate shunt. Therefore, the resulting effect of deleting TIGAR in a neuronal population might be multiple.
The authors show convincingly that deleting TIGAR from ChAT-expressing neurons, but not adipose or muscle cells, protects mice from cold-induced hypothermia. It is however unclear whether this leads to alteration in energy expenditure per se. This it important considering the first argument of the discussion highlighting how approaches to increase energy …
Reviewer #1 (Public Review):
My main concern relates to the title, which does not appear to be supported by the data. One can't conclude that the reported effects are strictly due to altered glycolysis in cholinergic neurons without directly assessing glucose metabolism in these neurons. Moreover,TIGAR functions by blocking glycolysis and directing the pathway into the pentose phosphate shunt. Therefore, the resulting effect of deleting TIGAR in a neuronal population might be multiple.
The authors show convincingly that deleting TIGAR from ChAT-expressing neurons, but not adipose or muscle cells, protects mice from cold-induced hypothermia. It is however unclear whether this leads to alteration in energy expenditure per se. This it important considering the first argument of the discussion highlighting how approaches to increase energy expenditure through the development/activation of brown/beige adipose tissue thermogenesis have failed. Moreover, it is unclear if TIGAR also affects heat dissipation considering the impact of its deletion from ChAT-expressing neurons on blood pressure and heart rate, two parameters that will likely influence the tail vasoactivity. Evaluating energy expenditure and heat loss appears to be necessary to support the conclusion that the resistance to hypothermia is exclusively dependent on shivering thermogenesis.
One key aspect that may deserve discussion is a potential contribution of the sympathetic nervous system to the observed phenotype. The focus of the manuscript is on acetylcholine but one can't disqualify that sympathetic compensations may happen following the deletion of TIGAR in ChAT-expressing neurons.
There are many data that are not shown but that would worth be included (lines 99, 113, 119, 159, 168, 181, 221,
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Reviewer #2 (Public Review):
This is an excellent study that introduces new players for the regulation of energy expenditure with sophisticated approaches to neuronal and peripheral metabolism. The experiments are well executed with appropriate controls and careful interpretation. The paper is clearly written and relays the conceptual advance that TIGAR controls acetyl choline levels to fuel changes in thermogenesis.
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Reviewer #3 (Public Review):
Strengths:
The study is nicely written and presented. The investigation of whole-body TIGAR knockout (TKO) clearly demonstrates resistance to cold exposure, and the authors logically follow potential sources through the obvious tissue candidates.Both skeletal muscle and adipose specific TIGAR knockouts were generated, neither of which recapitulated the effect of the TKO. Other obvious candidates, such as UCP1 content in adipose and basal oxidative capacity and contractility of skeletal muscle were ruled out using ex vivo techniques.
Nevertheless, pharmacological interventions indicated that muscle contraction was necessary for protection from cold exposure and that the loss of TIGAR overcame competitive antagonism of the nicotinic acetylcholine receptor. These data were supportive of a role for skeletal …
Reviewer #3 (Public Review):
Strengths:
The study is nicely written and presented. The investigation of whole-body TIGAR knockout (TKO) clearly demonstrates resistance to cold exposure, and the authors logically follow potential sources through the obvious tissue candidates.Both skeletal muscle and adipose specific TIGAR knockouts were generated, neither of which recapitulated the effect of the TKO. Other obvious candidates, such as UCP1 content in adipose and basal oxidative capacity and contractility of skeletal muscle were ruled out using ex vivo techniques.
Nevertheless, pharmacological interventions indicated that muscle contraction was necessary for protection from cold exposure and that the loss of TIGAR overcame competitive antagonism of the nicotinic acetylcholine receptor. These data were supportive of a role for skeletal muscle contraction, particularly at the level of cholinergic signaling.
A cholinergic neuron specific TIGAR knockout was produced. Loss of TIGAR was molecularly confirmed, and this mouse recapitulated the whole-body knockout's resistance to cold exposure.
Tracer studies are largely compelling and confirm that loss of TIGAR increases substrate dependence on glucose oxidation in a cell model.
Weaknesses:
The TKO mice were not characterized for body weight, body composition or energy expenditure, leaving some room for alternative or additive mechanisms.Although the tracer data demonstrate that loss of TIGAR causes the cell model to increase reliance on glycolysis compared to other unlabeled substrates, the data do not necessarily demonstrate an increase in the absolute rate of glycolysis or total acetyl-CoA production as intimated in the discussion. It is also unclear why media glutamate is examined for tracer incorporation rather than tissue glutamate.
There are some minor weaknesses related to the description of the methods. For example, the 18O studies need clarification. It will be unclear to most readers how this method works.
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