Gene expression in the liver of the rat induced in synthetic torpor
Authors: Hitrec Timna , Tinganelli Walter , Romani Fabrizio , Simoniello Palma , Squarcio Fabio , Piscitiello Emiliana , Marchesano Valentina , Luppi Marco , Occhinegro Alessandra , Sioli Maximiliano , Helm Alexander , Compagnone Gaetano , Morganti Alessio G , Amici Roberto , Negrini Matteo , Zoccoli Antonio , Durante Marco , Cerri Matteo 
Published: April 2020

Torpor/hibernation is a survival strategy used by several mammals in periods of unfavorable environmental conditions, such as food shortage and low ambient temperature. It is characterized by an active reduction in metabolic rate followed by a decrease in body temperature. The decrease of metabolism and therefore in oxygen demand could be beneficial in many clinical conditions, such as, but not only, transplant and stroke. Recently, rats (non‐hibernating mammals) were successfully induced in a torpor‐mimicking state called synthetic torpor (Cerri, Annu Rev Physiol., 2017) by central inhibition of Raphe Pallidus (RPa), a key brainstem area involved in thermoregulation (Cerri et al., J Neurosci., 2013). Even though this model is very promising, it is still not known how this condition of induced etherotermy acts on a cellular level on a non‐hibernating mammal.

Eight male Sprague‐Dawley rats, adapted to a standard Light‐Dark cycle of 12h‐12h (Light: 07 AM–07 PM), were used. Under general anesthesia (Diazepam, 5mg/kg, i.m., Ketamine, 100 mg/kg, i.p.), animals were implanted with a hypothalamic thermistor and a microcannula within the RPa. After recovery from surgery, starting at 7 AM, rats were divided in two groups: i) animals induced in a state of synthetic torpor by the repeated microinjections of the GABA‐A agonist muscimol (1mM, 120nl), within the RPa for 9 hours (Synthetic torpor, n=4); ii) homeothermic animals, injected with artificial cerebrospinal fluid (aCSF) within the RPa (Control, n =4), for 9 hours. At 4 PM, rats were euthanized and the liver was dissected and processed for gene expression analysis by RNA‐Seq.

Analysis of the Differential Expression (DE) shows that in synthetic torpor a total of 1107 mRNA sequences were significantly up‐regulated, among which genes involved in the metabolic switch to beta‐oxidation of free fatty acids (fatty acid binding proteins (FABP), Peroxisome proliferator‐activated receptor (PPAR) family, Pyruvate dehydrogenase lipoamide kinase isozyme 4 (PDK4), Carnitine Palmitoyltransferase 1α (CPT1α)) and 1337 significantly down‐regulated.

In a preliminary analysis, we report that several upregulated genes in synthetic torpor are known to be upregulated in hibernation as well. These preliminary findings suggest that the specific adaptation observed in natural torpor may be replicated also in non hibernators.

Significantly upregulated mRNA expression of genes involved in fatty acid metabolism, in rats induced in synthetic torpor (Synthetic torpor, n=4) vs normothermic ones (Control, n=4) expressed in values of log2FC (FC=fold change). On the right side, their corresponding known function, and a list of hibernating species where a significant upregulation of the genes has been observed in literature.

mRNA

log2FC

Function

Upregulated in

Fatty‐acid binding proteins (FABP)

Facilitation of β‐oxidation in mitochondria

Myotis lucifugus (Eddy& Storey, 2004)
Ictidomys tridecemlineatus (Epperson et al., 2010)

 FABP5

1.05

 FABP9

1.65

 FABP12

1.42

Peroxisome proliferator‐activated receptor (PPAR) family

Safe deposition and consumption of fatty acid, avoiding the associated inflammatory risks (Nunn et al. 2007). Linked to the integration of nutritional information and circadian rhythms (Kohsaka & Bass, 2007; Green et al. 2008).

Spermophilus tridecemlineatus (Eddy et al., 2004)
Myotis lucifugus (Eddy& Storey, 2004)

 PPARGC1a

3.40

 PPARGC1b

1.80

Pyruvate dehydrogenase lipoamide kinase isozyme 4

Shift away from the oxidation of carbohydrates and toward the combustion of stored fatty acids as the primary source of energy during torpor (Buck et al., 2002)

Ictidomys Tridecemlineatus (Buck et al., 2002)

 PDK4

4.03

Carnitine Palmitoyltransferase 1α

Fatty acid catabolism

Urocitellus parryii (Williams et al., 2011)

 CPT1α

1.08