Taurine Supplementation for 48-Months Improved Glucose Tolerance and Changed ATP-Related Enzymes in Avians

Taurine supplementation was administered for 48 months to avians in an effort to assess its influence, if any, on the organism’s bioenergetics (i.e., the study of the transformation of energy in living organisms) and glucose homeostasis by measuring their respective blood panels and responsivity to a Glucose Tolerance Test (GTT). Taurine is the second most abundant CNS neurotransmitter [1] and is essential for regulating mitochondrial determinants of ATP and antioxidant functions [2], providing neuroprotection [3, 4], and free radical scavenging of advanced glycation end products. The avian pancreas is comprised α- and β-cells that produce glucagon and insulin in response to metabolic demands that may be comparable with mammals. In mammals, the γ-amino butyric acid-A receptor (GABA_AR)-expressing β-cells produce insulin, and taurine protects against free radicals/oxidative stress [5, 6].

Avians have unique naturally occurring high plasma glucose concentrations (P_Glu; 180–350 mg/dL) that would otherwise create chronic hyperglycemia in mammals [7]. Moreover, avians have been reported to have higher taurine plasma levels than mammals and produce more insulin than glucagon through a specie-specific well-regulated hyperglycemia [7], serving functional purposes that are not fully understood. Further, avian resting P_Glu ranges between 200 and 450 mg/dL yet Diabetes Mellitus (DM) is rarely observed (i.e., needs to consistently exceed 50–600 mg/dL) [7‒9]. Notably, the avian pancreas is distinctive from mammals as it is comprised a greater α-: β-cell ratio and an 8–10 times higher glucagon concentration than what is observed in mammals [10]. Additionally, avian homeostasis requires 2–3 times more glucagon than insulin [7, 10], yet it remains unclear whether taurine would affect glucose tolerance in the avian metabolic system (AMS) similarly or differentially from reports established on mammals. The AMS evolved independently of other vertebrates [7, 11] punctuated by genetic deletions [12] (e.g., lacking the advanced glycation end products-receptor gene) [13], contributing to its resistance to diabetes at levels that would otherwise be medically alarming in mammals. Moreover, the AMS in avians also presents with ATP muscle production that differs significantly from mammals since the AMS is continuously in an “on-state,” readily supplying antioxidant-rich metabolism that is necessary for on-demand needs for extreme migratory flight behaviors. Interestingly, the AMS has been proposed for translational metabolic research as a pathology-free model of type 2 DM [8, 9, 13]. However, this area of research with the avian model system warrants further investigation to confirm if such a translational metabolic model is appropriate, limited, or whether it may serve other purposes not fully understood.

Consistent with the prior reports suggesting to use the AMS for translational metabolic research [8, 9, 13], the present preliminary study evaluated whether taurine supplementation in avians would influence their insulin functions similarly to mammals when presented with a GTT. In addition to the GTT, the avian’s health in response to taurine treatment in an adult mixed-sex set of pigeons was determined by a blood panel consisting of Low-Density Lipoprotein (LDL), Very Low-Density Lipoprotein (VLDL), High-Density Lipoprotein (HDL), triglycerides, and cholesterol. Further, the effect of taurine on ATP-related metabolic enzymes was assessed by measuring Glutamate dehydrognase (GLDH), Lacate dehydrogenase (LDH), and Creatine kinase (CK).

Eighteen aged white Carneau pigeons (Columba livia) were individually housed and randomly divided into a Taurine supplemented (mean [M] = 13.11 years, standard deviation [SD] = 4.43), and a Control (M = 11.88 years, SD = 3.41) group. The pigeons were of mixed sex as in adulthood; visually they are difficult if not impossible to sex [14]. The pigeons were maintained at 80–90% free-feeding weight throughout the experiment. Baseline blood panels and Mean Plasma Glucose (M P_Glu) levels were randomly drawn during the last 2 years of the 48 months comprising the preliminary study. The College of Staten Island (CSI) IACUC approved all procedures. Prior to the present preliminary study, the pigeons were randomly selected from a group of long-term working pigeons that supported undergraduate students to learn the principles of shaping and chaining behaviors as well as schedules of reinforcement through the Psychology Department Behavioral Learning and Memory Experimental Laboratory course consistent with the 3 R’s in animal research (i.e., replace, refine, reduce) [15‒17].

Water or Taurine solution was provided ad libitum for 48 months. Taurine (Sigma/Aldrich: St. Louis, MO; T0625) was dissolved in dH₂O and supplemented weekly at 500 mg/L (i.e., 0.05% solution w/v [3.99 mM]). Pigeons, regardless of treatment, weighed approximately 500 g (M = 545.42 g, SD = 45.18) at 100% of their ad libitum weight and drank an estimated 40–80 mL/day individually. This was consistent with other reports [18, 19] and yielded an estimated taurine dosage of 20–40 mg/day p.o.

Dextrose (Sigma/Aldrich: St. Louis, MO; 50-99-7) was dissolved in dH₂O at 14.99 g/L (i.e., 15% solution w/v [83.21 mm]; total volume of 5 mL). The pigeon’s blood samples were drawn by a veterinarian from the basilic vein of the wing and immediately analyzed with a standard glucometer (Free Style, Abbott^®). Briefly, blood samples taken from the same basilic vein were drawn by the veterinarian at the following Time Points (i.e., which were defined as the time from initial P_Glu levels and at equidistant time periods thereafter to measure the physiological changes in P_Glu levels) as a pharmacokinetic assessment of glucose tolerance: baseline at 0 min prior to dextrose administration and at 15, 30, and 60 min thereafter.

Another blood sample from the basilic vein of the opposite wing was collected in heparin-coated tubes, immediately stored at −80°C to prevent degradation, and then sent out for external analysis to the University of Miami Avian and Wildlife Laboratory (UMAWL; Miami, FL, USA) for blood panel analyses. The UMAWL has an extensive avian physiological, metabolic, and pathological reference database used to compare experimental samples.

A repeated measures ANOVA was conducted using IBM SPSS v24 (Armonk, NY, USA) to assess the biochemical and hematological data as between-group differences and the effects of Taurine Treatment and Time Point of the GTT, and the interactions. The degrees of freedom are reported as within- and between-subjects across the respective Time Points. A two-tailed t test was used to assess the between-group differences for blood panel measures with a significance criteria set at α = 0.05. Data are reported as the Mean ± SEM.

The baseline M P_Glu levels for pigeons were invariant to fasting and age (N = 60). Pigeons that were fed within 1 h of the GTT had an M P_Glu = 208.69, SD = 20.43 mg/dL (N = 36), while the pigeons that were fed after 24 h food deprivation had an M P_Glu = 204.04, SD = 20.35 mg/dL (N = 24). A group of young pigeons (N = 6; M = 5.33 years, SD = 0.81; data not shown) exhibited identical baseline blood panels compared to the Control group as a viability/health internal control measure/reference. Thus, the aged Control pigeons presented with the same glucose metabolism as the young pigeons. Figure 1 shows that the M P_Glu levels were not significantly different at baseline but were significantly higher than baseline at each of the other three Time Points F_{(3, 42)} = 21.445, p < 0.001***. The effect of Taurine was significant at the 15-, 30-, and 60-min Time Points F_(1,14) = 4.436, p < 0.05^#, when compared to Control pigeons. Notably, the interaction between Taurine × Time Points approached significance F_(3,42) = 2.693, p = 0.058. Since the pigeon’s baseline M P_Glu levels were not influenced by taurine treatment, it is possible that the pharmacodynamics and pharmacokinetics of taurine supplementation in the pigeons may have delayed effects which may explain the influences observed for the absorption of glucose at the 15-min time point and consistently thereafter.

Table 1 shows the blood panels from both groups were within the AMS reference ranges, except for CK enzyme. This suggested that the majority of the enzymes from the blood panels drawn were indicative of normal health in these pigeons regardless of age and treatment. Taurine affected the ATP-related enzyme measures resulting in higher GLDH, t₍₆₎ = 5.278, p < 0.01^## (i.e., nearly a 4x increase), LDH, t₍₈₎ = 3.704, p < 0.01^## (i.e., a bit more than a ×2 increase), and CK levels, t₍₈₎ = 2.887, p < 0.05^# (i.e., a bit more than a ×2 increase), when compared to Control pigeons. This preliminary data on aged mixed-sex pigeons treated with taurine orally for 48 months suggests that, with all other blood panel enzymes falling within normal ranges, the GLDH may be elevated due to taurine potentially having a unique specie-specific influence in avian mitochondrial activity. Further, these differences in avians may perhaps be less likely to be related to liver damage or leakage, whereas in the case of mammals (i.e., humans and rodents), these elevated enzymes would clinically manifest in liver or muscle injury since the age-matched controls did not show similar elevated GLDH profiles. Moreover, the LDH was elevated by taurine and could further perhaps serve to facilitate the two-fold increase in CK production as one possible mechanism to further augment the bioavailability of ATP reserves for readying on-demand flight in avians. The blood panels indicated no significant differences in LDL, HDL, VLDL, triglycerides, and cholesterol suggesting no cardiovascular issues observed in the aged mixed-sex pigeons under taurine treatment, which were all within the normal UMAWL AMS reference ranges. Thus, taurine treatment did not cause cardiac pathophysiological changes nor liver or muscle injury detected by the veterinarian upon physical examination nor through the blood panel analytes. A possible suggestion pertaining to the effects of taurine in avians is that it may serve to influence energy-dependent metabolic enzyme levels (i.e., GLDH, LDH, and CK) for an altered pathway for on-demand energy production (i.e., holding potential energy reserves) that may be unique to avians.

In the differentially tuned AMS, chronic taurine supplementation for 48 months in mixed-sex pigeons was found to enhance P_Glu levels through a GTT similar to what has been observed in mammals [5]. This could be due to avians having a specie-specific enhanced pancreatic function and health. The present study did not sacrifice the avians nor were biopsies relevant (i.e., as these were working pigeons for an experimental psychology curriculum), and it remains to be elucidated on whether the exogenous taurine supplementation increased the serum or tissue taurine concentrations endogenously. Further, how the taurine synthesis in the liver of avians (i.e., given the differences in structure when compared to mammals) may uniquely regulate methionine and cysteine remains to be elucidated. The latter point is important as long-term taurine supplementation in mammals (i.e., the 48 months conducted in the present study) may suppress endogenous taurine synthesis and further alter the role in conditions of reduced protein synthesis, increased protein catabolism, and/or excretion such as in the cases of diabetes or nephropathy. Again, the latter examples are based upon clinical findings in mammals, but the differences that require more study in avians may prove fruitful for preclinical researchers to fully understand and appreciate its advantages and limitations as a model for diabetes as one example for preclinical research. Moreover, taurine serves as an agonist to the GABA_AR, thereby protecting the pancreatic β-cells. Glucose homeostasis can be disrupted by high-fat/high-fructose diets that produce insulin resistance and type 2 DM (i.e., metabolic syndrome) that can be recovered by taurine supplementation [18]. Further, taurine may remediate insulin resistance [18] by altering endocrine activity and subsequently remodeling pancreatic β-cells [5, 6, 19, 20]. The relationship between taurine and insulin production may be an evolutionarily conserved functional trait across species for regulating bioenergetics via glucose metabolism [7, 21, 22]. However, the findings from the present study pose more questions than answers, for example, despite these conserved functional traits, do avians have unique specie-specific metabolic functional traits, and to what end can they be considered a valid model system for mammalian pathological states.

An evolutionarily unconserved trait in avian muscle metabolism is unparalleled by an insistent energy system that is always in the “on-state” required for adaptive flight maneuvers. Notably, GLDH critically regulates amino acid metabolism and is present in high concentrations within liver, heart, muscle, and kidney mitochondria where it catalyzes the (i.e., reversible) oxidative deamination of l-glutamate to α-ketoglutarate [1, 7, 8, 11, 13], thereby providing energy via the citric acid cycle-dependent ATP. The GLDH activity is particularly important within pancreatic β-cells, which secretes insulin in an ATP:ADP ratio-dependent manner. As glutamate is metabolized into α-ketoglutarate, this ratio rises consequentially; and as a result, more insulin is secreted. Relatedly, LDH provides a sustainable metabolic pathway for pyruvate production feedback directly into the citric acid cycle, ultimately producing ATP. Therefore, LDH becomes overactive during periods of extreme muscle activity in mammals and its bioavailability is activity-dependent. Interestingly, this preliminary study showed that taurine further increased avian LDH levels absent of significant changes in muscle enzyme activity [23] and any other physical pathological states that could be observed as gross motoric and/or behavioral traits upon veterinary examination throughout the study. Further, there was an inverse relationship between the M P_Glu levels and GLDH, LDH, and CK, respectively. Finally, as CK catalyzes the reversible transfer of high-energy phosphate from ATP to creatine, it facilitates phosphocreatine energy storage. In muscle cells, this extra energy buffer regulates ATP homeostasis as well as in the mitochondrial complex I in the electron transport chain. It is well established that CK values decrease after exercise in mammals, with high levels often associated with muscle injury [23].

This preliminary study showed that 48 months of taurine supplementation in a mixed-sex and aged set of pigeons can increase glucose resistance as a possible new insight for the AMS. Over a 50-year history, the pigeons in the CSI aviary, of which the subjects from the present study were sampled, have had consistently elevated CK levels compared to free-flight avians [24]. These pigeons were sampled by from a group of untreated avians used within a Behavioral Learning and Memory Experimental Laboratory course used to teach undergraduate students the principles of shaping and chaining behaviors as well as schedules of reinforcement; all of which had no influence on the results of the present study. Notably, since sex was not examined fully to assess whether it could affect glucose metabolism and muscle enzyme levels future study will have to address this matter more carefully. Further, the present study used a 48-month taurine supplementation model as a means to simulate long-term positive health effects through the pharmacological treatment of the aged avians consistent with its use as a neuroprotective agent in mammals. Future work may also consider short-term or drug-treatment reversal designs to understand how and whether exogenous taurine supplementation in avians might suppress endogenous taurine production in response to a range of treatment regimens. The taurine treatment also altered ATP-related enzymatic activity through elevating CK levels, consistent with the “on-state” of avian muscle tissues. These preliminary findings warrant the need for further investigation on taurine relationships and/or influences with avian bioenergetic metabolism and enhanced energy storage mechanisms for on-demand flight. Interestingly, the observed elevated muscle enzyme serum levels in response to taurine treatment were absent of any clinical or physical pathology as observed by the veterinarian. However, if the same muscle enzyme serum levels observed in the avians from this preliminary study were found in mammals, they would be biomarkers of hepatic and muscular injury. These differences in the glucose metabolism and muscle enzyme levels in response to taurine in avians suggest that the AMS may need to be reevaluated for its utility as a preclinical model for medical diseases. In recent years, a new body of research is being reported on a broad range of taurine therapeutics and bioenergetics in regulating: oligodendrocytes in neuropathophysiological conditions [25], bioenergetics of the mitochondria in the brain [26], neuroprotection in aged rats [27], how the brain responds to stress and posttraumatic stress disorder-like symptoms in rats [28], recovering fronto-executive functions and anxiety-like behaviors in lead poisoned rats [29‒35], mitigating against brain excitability and epilepsy [36], to bioenergetic adaptations in skeletal muscle function [37], its ability to influence motor learning following oral supplementation [38], its sex-dependent influences on rat’s active avoidance learning processes [39], the way rats respond to resistance training [40] and even in humans in runner adaptations to running trails [41] and improving power during severe and intense exercise [42]. This rich emerging literature will certainly guide the field in next approaches to improving our understanding of the tradeoffs between taurine bioenergetics and therapeutics across different model systems [43, 44].

Once the advantages and disadvantages, as well as limitations, are clearly understood, then perhaps a new understanding of taurine supplementation in avians at the amino acid/mitochondrial metabolic levels can be further comprehended. Given what is known and what was observed in this preliminary study, as one possible explanation, aged avians of mixed sex may prove useful as a unique animal model for investigating how avians manage to remain healthy and possess high GLDH, LDH, and CK serum levels to have “on-demand” energy stores with an uncanny efficiency of delivery to its skeletal and smooth muscles. Further, once the bioenergetics of the AMS are fully understood, then perhaps the AMS may prove useful when investigating taurine influences on these physiological biomarkers in a wide-range of complex metabolic disorders such as metabolic syndrome, type 2 DM, senescence [23, 24], and other syndromes comorbid with oxidative stress [3]. Unfortunately, more work is required in this area before such an avian AMS model can be fully deployed at the preclinical pharmacological levels to advance it as a new tool for metabolic research. This is just the beginning of what might be a rather intriguing journey that the avian model might lead us into.

We thank the CSI vivarium staff for their excellent care of the pigeons, the many undergraduate research assistants, and Dr. Mark Valitutto. We would like to dedicate this manuscript to E.F.M. as he passed away prior to the time of this submission.

The College of Staten Island (CSI) IACUC approved all procedures for protocol #11-020. Outside of the present study, these pigeons were working pigeons that supported student learning through the Psychology Department Experimental Psychology courses consistent with the 3 R’s in animal research (i.e., replace, refine, reduce).

The authors declare no conflicts of interest.

The work was supported in part by PSC-CUNY grant (PSCREG-41-1064) awarded to E.F.M.

N.G. and E.F.M. conceived on the study and analyzed the data with L.S.N. S.K. performed all the blood draws associated with the GTT. All authors contributed to the manuscript. It is with great sadness that E.F.M. passed away prior to this work being published. May he rest in peace and his words that he lived by as he valued mindfulness continue with us “Invite joy, to practice joy” be remembered by all who were lucky enough to have known and been graced by him.

All data generated or analyzed during this study are included in this article. Further inquiries can be directed to the corresponding author.