The effect of neuronal mIndy on the development of the metabolic syndrome

PhD student: Anica Kurzbach
Supervisor at TUD: Andreas Birkenfeld
Supervisor at KCL: Cynthia Andoniadou
Start date: 01.04.2015  Date of defense: 09.03.2020  Dr. rer. medic.

The mammalian plasma membrane di-and tricarboxylate transporter mINDY (mammalian I’m not dead, yet), initially shown to regulate life-span in the fruit fly Drosophila melanogaster, preferentially transports citrate. Early studies in mice showed that systemic deletion of mIndy protects mice not only from diet- and aging-induced obesity and insulin resistance but also from lipid accumulations in the liver as observed in the context of caloric restriction. The citrate transporter is highly expressed in the liver. Strikingly, liver-specific mIndy knockout rodent models did not fully recapitulate the phenotype of whole-body knockout mice. Hence, the effects of mIndy are not mediated exclusively by its action in the liver.

The gene is also expressed in the brain – primarily in the neurons. As the brain is a major regulator of whole-body energy homeostasis, the aim of this study was to investigate the role of neuronal mIndy using a neuron-specific mIndy knockout (NINKO) mouse model generated with the Cre/loxP gene targeting system.

NINKO mice and controls were metabolically characterized using nuclear magnetic resonance to determine body composition and by indirect calorimetry to investigate energy expenditure, respiratory exchange ratio and food intake. Investigations were performed in mice fed normal chow diet as well as high fed diet. Interestingly, NINKO mice were shown to be protected against diet-induced obesity because of increased energy expenditure. Moreover, they showed lower respiratory exchange ratio, pointing at a switch to  lipid oxidation. Notably, food intake remained unchanged.

Next, to assess insulin sensitivity, the gold standard hyperinsulinemic-euglycemic (HE-) clamp was performed in HFD-challenged mice. NINKO mice revealed improved overall insulin sensitivity due to improved hepatic insulin sensitivity in spite of unaltered organ-specific glucose uptake. One explanation for this observation could be the decreased AMPK phosphorylation in the hypothalamus, which was previously shown to suppress hepatic glucose output.

Multiple reasons might underlie elevated energy expenditure, including higher activity of the brown adipose tissue. Thus, I performed cold exposure studies in HFD-fed NINKO mice. My investigations revealed no differences in cold tolerance in these mice compared to controls. However, AMPK phosphorylation and expression of genes encoding proteins involved in mitochondrial respiration were higher in the brown adipose tissue of these mice, hinting at elevated ATP production instead of releasing energy as heat. The latter represents a possible explanation for the higher energy expenditure in NINKO mice. Moreover, as mitochondrial respiration in brown adipocytes is mainly driven by fatty acid oxidation, my observations could explain the lower respiratory exchange ratio in NINKO mice.

In summary, reduced mIndy expression in neurons reduces hypothalamic AMPK phosphorylation, likely leading to improved hepatic insulin sensitivity. Furthermore, reduced neuronal mIndy expression also increases AMPK phosphorylation and mitochondrial respiration in brown adipose tissue, indicating greater energy expenditure, rising whole-body energy expenditure and lipid oxidation, resulting in lower body weight.

Altogether, the present work shows for the first time the important function of the citrate transporter mINDY in neuronal tissues. In combination with previous work, my observations support the role of mIndy in glucose and energy metabolism and make it an interesting target to treat metabolic diseases. Yet, the results of the present study indicate that for a pharmacological inhibitor of mINDY to be effective, it must penetrate the blood brain barrier. Further studies are expected to elucidate the potential of mINDY as a clinically relevant and druggable target.

Publications:

The anorexigenic peptide neurotensin relates to insulin sensitivity in obese patients after BPD or RYGB metabolic surgery. C. von Loeffelholz, L.C. Gissey, T. Schumann, C. Henke, A. Kurzbach, J. Struck, A. Bergmann, M. Hanefeld, U. Schatz, S.R. Bornstein, G. Casella, G. Mingrone, A.L. Birkenfeld. Int J Obes (Lond). 2018;42:2057-2061. 

The longevity gene INDY (I'm Not Dead Yet) in metabolic control: Potential as pharmacological target.  D.M. Willmes, A. Kurzbach, C. Henke, T. Schumann, G. Zahn, A. Heifetz, J. Jordan, S.L. Helfand, A.L. Birkenfeld. Pharmacol Ther. 2018;185:1-11.

Are Lifestyle Therapies Effective for NAFLD Treatment?  N.N. El-Agroudy*, A. Kurzbach*, R.N. Rodionov, J. O'Sullivan, M. Roden, A.L. Birkenfeld, D.H. Pesta. Trends Endocrinol Metab. 2019;30:701-709.

Disruption of the sodium-dependent citrate transporter SLC13A5 in mice causes alterations in brain citrate levels and neuronal network excitability in the hippocampus.  C. Henke, K. Tollner, R.M. van Dijk, N. Miljanovic, T. Cordes, F. Twele, S. Broer, V. Ziesak, M. Rohde, S.M. Hauck, C. Vogel, L. Welzel, T. Schumann, D.M. Willmes, A. Kurzbach, N.N. El-Agroudy, S.R. Bornstein, S.A. Schneider, J. Jordan, H. Potschka, C.M. Metallo, R. Kohling, A.L. Birkenfeld, W. Loscher. Neurobiol Dis. 2020;143:105018.

VEGF-Trap is a potent modulator of vasoregenerative responses and protects dopaminergic amacrine network integrity in degenerative ischemic neovascular retinopathy.  J.E. Rojo Arias, M. Economopoulou, D.A. Juarez Lopez, A. Kurzbach, K.H. Au Yeung, V. Englmaier, M. Merdausl, M. Schaarschmidt, M. Ader, H. Morawietz, R.H.W. Funk, J. Jaszai. J Neurochem. 2020;153:390-412. 

Deletion of the diabetes candidate gene Slc16a13 in mice attenuates diet-induced ectopic lipid accumulation and insulin resistance.  T. Schumann, J. König, C. von Loeffelholz, D.F. Vatner, D. Zhang, R.J. Perry, M. Bernier, J. Chami, C. Henke, A. Kurzbach, N.N. El-Agroudy, D.M. Willmes, D. Pesta, R. de Cabo, J.F. O Sullivan, E. Simon, G.I. Shulman, B.S. Hamilton, A.L. Birkenfeld. Commun Biol. 2021;4(1):826. 

The longevity gene mIndy (I'm Not Dead, Yet) affects blood pressure through sympathoadrenal mechanisms.  D.M. Willmes, M. Daniels, A. Kurzbach, S. Lieske, N. Bechmann, T. Schumann, C. Henke, N.N. El-Agroudy, A.C. Da Costa Goncalves, M. Peitzsch, A. Hofmann, W. Kanczkowski, K. Kräker, D.N. Müller, H. Morawietz, A. Deussen, M. Wagner, A. El-Armouche, S.L. Helfand, S.R. Bornstein, R. de Cabo, M. Bernier, G. Eisenhofer, J. Tank, J. Jordan, A.L. Birkenfeld. JCI Insight. 2021;6(2):136083.