Hibernating mammals have the ability to decrease their metabolic rate and survive up to 6 months without food in an inactive state where body temperatures approach 0°C. In hibernating 13-lined ground squirrels (Spermophilus tridecemlineatus), oxygen consumption holds at 1/30 to 1/50 of the aroused condition and heart rates are as low as 3–10 beats/min, compared with 200–300 beats/min when the animal is active. This seasonal adaptation requires a metabolic shift away from the oxidation of carbohydrates and towards the combustion of stored fatty acids as the primary source of energy. A key element in this fuel switch is the differential expression of the gene encoding pyruvate dehydrogenase kinase isoenzyme 4. Pyruvate dehydrogenase kinase isoenzyme 4 inhibits pyruvate dehydrogenase and thus minimizes carbohydrate oxidation by preventing the flow of glycolytic products into the tricarboxylic acid cycle. Hibernators also exploit the low-temperature activity of PTL (pancreatic triacylglycerol lipase) in both heart and white adipose tissue. Lipolytic activity at body temperatures associated with hibernation was examined using recombinant ground squirrel and human PTL expressed in yeast. Enzymes from both humans and ground squirrel displayed high activity at temperatures as low as 0°C and showed Q10=1.2–1.5 over the temperature range 37–7°C. These studies indicate that low-temperature lipolysis is a general property of PTL and does not require protein modifications unique to mammalian cells and/or the hibernating state.

Introduction

Hibernation is a natural adaptation that allows certain mammals to survive physiological extremes which would normally lead to death in humans. Hibernating mammals have the ability to decrease their metabolic rate and survive up to 6 months without food in an inactive state where body temperatures approach 0°C. Energy reserves in the form of fat sustain vital functions during long bouts of torpor at near freezing temperatures. During deep hibernation, oxygen consumption holds at 1/30 to 1/50 of the aroused condition and heart rate can be as low as 3–10 beats/min, compared with 200–300 beats/min when the animal is awake and active. This amazing transformation of the whole-animal physiology is completely reversible and serves as an adaptation to conserve energy reserves during extended periods of harsh climate and little or no food.

Remarkably, hibernation occurs in seven different orders of mammals [1], including a species of primate (fat-tailed dwarf lemur [2]). This biological adaptation is the product of tens of millions of years of evolution and results from the selective expression of the genes common to all mammals (reviewed in [3]). In the present study, we have focused on the regulatory aspects of the hibernating state by identifying genes that are differentially expressed in the heart during hibernation in the 13-lined ground squirrel, Spermophilus tridecemlineatus.

We used a PCR-based gene expression screen to isolate cDNAs of the genes showing increased levels of expression during hibernation in the ground squirrel heart. We chose the heart since it is a contractile organ that must continue to work despite suboptimal conditions of temperature and oxygen consumption. We found that genes encoding PTL (pancreatic triacylglycerol lipase) and PDK4 (pyruvate dehydrogenase kinase isoenzyme 4) are up-regulated in the heart when hibernation begins and that steady-state levels of both mRNAs remain high, whereas metabolism and body temperature are greatly decreased [4]. This functional genomics approach has allowed us to construct and test model biochemical pathways specific to the heart of the hibernating animal (Figure 1).

Model showing the metabolic involvement of PDK4 and PTL in the heart of a hibernating 13-lined ground squirrel

Figure 1
Model showing the metabolic involvement of PDK4 and PTL in the heart of a hibernating 13-lined ground squirrel

Names of metabolic pathways are shown in italics. Arrows with a single arrowhead indicate a single reaction. Continuous arrows with two or more arrowheads indicate multistep pathways. DHAP, dihydroxyacetone phosphate; ffa, non-esterified fatty acid; G-3-P, L-glycerol 3-phosphate; PDH, pyruvate dehydrogenase; TCA, tricarboxylic acid; TG, triacylglycerol. Modified from Andrews, M.T., Squire, T.L., Bowen, C.M. and Rollins, M.B. (1998) Low-temperature carbon utilization is regulated by novel gene activity in the heart of a hibernating mammal. Proc. Natl. Acad. Sci. U.S.A. 95, 8392–8397. © 1998 National Academy of Sciences, U.S.A.

Figure 1
Model showing the metabolic involvement of PDK4 and PTL in the heart of a hibernating 13-lined ground squirrel

Names of metabolic pathways are shown in italics. Arrows with a single arrowhead indicate a single reaction. Continuous arrows with two or more arrowheads indicate multistep pathways. DHAP, dihydroxyacetone phosphate; ffa, non-esterified fatty acid; G-3-P, L-glycerol 3-phosphate; PDH, pyruvate dehydrogenase; TCA, tricarboxylic acid; TG, triacylglycerol. Modified from Andrews, M.T., Squire, T.L., Bowen, C.M. and Rollins, M.B. (1998) Low-temperature carbon utilization is regulated by novel gene activity in the heart of a hibernating mammal. Proc. Natl. Acad. Sci. U.S.A. 95, 8392–8397. © 1998 National Academy of Sciences, U.S.A.

A fundamental switch in fuel selection

PDK4 prevents the flow of glycolytic intermediates into the tricarboxylic acid cycle and, therefore, spares glucose and limits carbohydrate oxidation. PTL hydrolyses triacylglycerols to liberate non-esterified fatty acids to be used as fuel. Involvement of PDK4 and PTL in the intermediary metabolism of the ground squirrel heart contributes to the observed RQ (respiratory quotient) of 0.7 seen during hibernation. RQ is a unitless value representing the moles of CO2 respired per mol of O2 consumed. RQ=1.0 indicates combustion of carbohydrates; however, RQ=0.7 indicates that fat is the major substrate for energy production.

Novel expression of PTL

Until recently, PTL was thought to be expressed exclusively in the pancreatic acinar cells and secreted into the small intestine as a means to digest dietary fat. However, we found that PTL is differentially expressed in the heart of 13-lined ground squirrels, where it provides low-temperature lipolysis during hibernation [4]. We have since discovered that PTL is expressed in other ground squirrel tissues, including the main fat storage depot, WAT (white adipose tissue) [5,6]. The unusual expression of PTL may be facilitated by a retroviral insertion in the promoter region of the 13-lined ground squirrel PTL gene [7]. Documentation of endogenous retroviral elements providing alternative promoters or enhancers for neighbouring genes has been provided for the pleiotrophin gene [8], the endothelin B receptor and the apolipoprotein C–I genes [9], and for the Mid1 gene in humans [10]. We propose that insertion of the retrovirus into the promoter region of this ground squirrel gene enabled novel expression of PTL mRNA in a broad range of tissues during hibernation [5,7]. Production of PTL, therefore, confers a selective advantage to the organism in the form of low-temperature lipolysis during hibernation [4,7] and, as a result, has maintained the retroviral sequence in the 13-lined ground squirrel lineage.

Human enzymic activity at 0°C

An obvious explanation for the expression of PTL in heart and WAT is that PTL retains high activity at low temperatures. To determine whether this cold lipolysis is unique to ground squirrel PTL or may be the result of a hibernation-specific modification of the lipase, we expressed cDNAs for both human and 13-lined ground squirrel PTL in the yeast Pichia pastoris [11]. Expression of the two recombinant PTLs produces identical-length 449-amino-acid proteins lacking the 16-amino-acid N-terminal signal peptide. Since both lipases were expressed in the same yeast background, we would not anticipate post-translational modifications that are unique to a specific mammalian tissue and/or state of animal activity.

Activity of the two recombinant lipases was assayed using triacylglycerol substrates, tributyrin and triolein [11]. Figure 2 shows that both human and ground squirrel PTL perform remarkably well at low temperatures. At 0°C, the enzyme from ground squirrel still maintains 48 and 33% of the maximal activity (seen at 37°C) with tributyrin and triolein respectively. Human PTL showed 42% maximal activity at 0°C using tributyrin and an amazing 55% maximal activity using triolein. We conclude that low-temperature lipolysis is a property of both human and 13-lined ground squirrel PTL and that PTL does not require modifications specific to mammalian cells to function in the cold. This result shows that the low-temperature catalysis seen with a protein in hibernators is also found in humans [6].

Lipolytic activity of human and 13-lined ground squirrel recombinant PTLs

Figure 2
Lipolytic activity of human and 13-lined ground squirrel recombinant PTLs

cDNAs encoding human and ground squirrel PTLs were expressed in P. pastoris as described by Yang and Lowe [11]. Each purified recombinant PTL was assayed using the pH-stat method with two triacylglycerol substrates, 4-carbon tributyrin (TB) and 18-carbon triolein (TO). Assays were performed at 0, 7, 17, 27 and 37°C. Results are averages of two independent trials.

Figure 2
Lipolytic activity of human and 13-lined ground squirrel recombinant PTLs

cDNAs encoding human and ground squirrel PTLs were expressed in P. pastoris as described by Yang and Lowe [11]. Each purified recombinant PTL was assayed using the pH-stat method with two triacylglycerol substrates, 4-carbon tributyrin (TB) and 18-carbon triolein (TO). Assays were performed at 0, 7, 17, 27 and 37°C. Results are averages of two independent trials.

Co-ordinate expression of PDK4 in different tissues

During hibernation, PDK4 mRNA was seen at low levels in the adrenals, brainstem, cerebrum, kidney, liver, pancreas and testes, and at much higher levels in heart, skeletal muscles and WAT [12]. Our investigation of PDK4 expression in 13-lined ground squirrels was conducted throughout the hibernation season to determine whether there was evidence for a common regulation among the three tissues showing the highest mRNA levels. Total RNA from heart, skeletal muscles from the thigh (quadriceps femoris) and abdominal WAT were prepared from individual animals at various states of activity from August to March. Expression of the PDK4 gene was similar in all the three tissues throughout this time period [12]. PDK4 mRNA levels were relatively unchanged until the animal entered hibernation in October. Hibernating animals in October show a slight non-significant increase in expression in all three tissues relative to August levels. Later in the hibernation season (December–March), there was a further increase in mRNA levels, with the largest increase relative to August values seen during interbout arousals. After hibernation ceased in the spring, PDK4 message decreased back to near August levels [12].

Induction of hibernation-associated gene activity by circulating effector molecules

Identification of the specific genes associated with the hibernating state allows researchers to identify and test the endogenous and exogenous factors that control differential gene expression during hibernation. Factors important in hibernation-related gene activity may be environmental such as lower ambient temperatures and shorter daylight hours and/or internal factors such as circannual rhythms and changes in metabolites and hormone levels.

Co-ordinate activation of the gene encoding PDK4 in different tissues throughout the body points to circulating effector molecules that regulate its expression during hibernation [12]. Figure 3 presents a model showing changes in serum insulin and fatty acid levels that regulate PDK4 expression and, ultimately, the switch from carbohydrate- to fat-based catabolism during hibernation. Insulin has been shown to repress PDK4 gene activity, whereas specific fatty acids act as ligands for peroxisome proliferator-activated receptor α, which activates the PDK4 gene in a ligand-dependent manner [13].

Model showing regulation of the switch from carbohydrate to fatty acids as the primary source of fuel during hibernation

Figure 3
Model showing regulation of the switch from carbohydrate to fatty acids as the primary source of fuel during hibernation

Effects of serum levels of insulin secreted from the pancreas and non-esterified fatty acids secreted from WAT on PDK4 gene expression, carbohydrate oxidation and fatty acid oxidation in heart and skeletal muscles in active animals that are fattening (Active; September–October) and in animals that are hibernating (Hibernation; December–January). Lines with arrowheads indicate up-regulation or activation and lines with blunt ends indicate down-regulation or inhibition. ——, the active or predominant mode of regulation; - - -, minor pathways. Short arrows pointing up or down indicate an increase or decrease, respectively, in concentration or activity. HSL, hormone-sensitive lipase; PPARα, peroxisome proliferator activated receptor α; PPARαa, PPARαi, active and inactive PPARα respectively; TG, triacylglycerol. Modified from Carey, H.V., Andrews, M.T. and Martin, S.L. (2003) Mammalian hibernation: cellular and molecular responses to depressed metabolism and low temperature. Physiol. Rev. 83, 1153–1181, used with permission.

Figure 3
Model showing regulation of the switch from carbohydrate to fatty acids as the primary source of fuel during hibernation

Effects of serum levels of insulin secreted from the pancreas and non-esterified fatty acids secreted from WAT on PDK4 gene expression, carbohydrate oxidation and fatty acid oxidation in heart and skeletal muscles in active animals that are fattening (Active; September–October) and in animals that are hibernating (Hibernation; December–January). Lines with arrowheads indicate up-regulation or activation and lines with blunt ends indicate down-regulation or inhibition. ——, the active or predominant mode of regulation; - - -, minor pathways. Short arrows pointing up or down indicate an increase or decrease, respectively, in concentration or activity. HSL, hormone-sensitive lipase; PPARα, peroxisome proliferator activated receptor α; PPARαa, PPARαi, active and inactive PPARα respectively; TG, triacylglycerol. Modified from Carey, H.V., Andrews, M.T. and Martin, S.L. (2003) Mammalian hibernation: cellular and molecular responses to depressed metabolism and low temperature. Physiol. Rev. 83, 1153–1181, used with permission.

Thirteen-lined ground squirrel BAC library approved by genome institute

In March 2004, the National Human Genome Research Institute announced that they would construct a BAC (bacterial artificial chromosome) library of the 13-lined ground squirrel genome. This decision was made after considerable input from the hibernation research community and weighing the advantages of various squirrel species. The resulting BAC library may be a prelude to a 13-lined ground squirrel genome project and will allow researchers to isolate genes that are important for surviving the physiological extremes of hibernation.

Energy: Generation and Information: A Focus Topic at BioScience2004, held at SECC Glasgow, U.K., 18–22 July 2004. Edited by J. Arthur (Rowett Research Institute, Aberdeen, U.K.), P. Newsholme (University College Dublin, Ireland), M. Murphy (MRC-Dunn Human Nutrition Unit, Cambridge, U.K.) and R. Reece (Manchester, U.K.).

Abbreviations

     
  • PDK4

    pyruvate dehydrogenase kinase isoenzyme 4

  •  
  • PTL

    pancreatic triacylglycerol lipase

  •  
  • WAT

    white adipose tissue

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