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Biomedical subjects

G Toffolo

Publications and source records attributed to G Toffolo.

At least 37 records · Page 2Linked to original sources
PubMed1996

Intracellular lactate- and pyruvate-interconversion rates are increased in muscle tissue of non-insulin-dependent diabetic individuals.

The contribution of muscle tissues of non-insulin-dependent diabetes mellitus (NIDDM) patients to blood lactate appearance remains undefined. To gain insight on intracellular pyruvate/lactate metabolism, the postabsorptive forearm metabolism of glucose, lactate, FFA, and ketone bodies (KB) was assessed in seven obese non-insulin-dependent diabetic patients (BMI = 28.0 +/- 0.5 kg/m2) and seven control individuals (BMI = 24.8 +/- 0.5 kg/m2) by using arteriovenous balance across forearm tissues along with continuous infusion of [3-13C1]-lactate and indirect calorimetry. Fasting plasma concentrations of glucose (10.0 +/- 0.3 vs. 4.7 +/- 0.2 mmol/liter), insulin (68 +/- 5 vs. 43 +/- 6 pmol/liter), FFA (0.57 +/- 0.02 vs. 0.51 +/- 0.02 mmol/liter), and blood levels of lactate (1.05 +/- 0.04 vs. 0.60 +/- 0.06 mmol/liter), and KB (0.48 +/- 0.04 vs. 0.29 +/- 0.02 mmol/liter) were higher in NIDDM patients (P < 0.01). Forearm glucose uptake was similar in the two groups (10.3 +/- 1.4 vs. 9.6 +/ 1.1 micromol/min/liter of forearm tissue), while KB uptake was twice as much in NIDDM patients as compared to control subjects. Lactate balance was only slightly increased in NIDDM patients (5.6 +/- 1.4 vs. 3.3 +/- 1.0 micromol/min/liter; P = NS). A two-compartment model of lactate and pyruvate kinetics in the forearm tissue was used to dissect out the rates of lactate to pyruvate and pyruvate to lactate interconversions. In spite of minor differences in the lactate balance, a fourfold increase in both lactate- (44.8 +/- 9.0 vs. 12.6 +/- 4.6 micromol/min/liter) and pyruvate-(50.4 +/- 9.8 vs. 16.0 +/- 5.0 micromol/min/liter) interconversion rates (both P < 0.01) were found. Whole body lactate turnover, assessed by using the classic isotope dilution principle, was higher in NIDDM individuals (46 +/- 9 vs. 21 +/- 3 micromol/min/kg; P < 0.01). Insights into the physiological meaning of this parameter were obtained by using a whole body noncompartmental model of lactate/pyruvate kinetics which provides a lower and upper bound for total lactate and pyruvate turnover (NIDDM = 46 +/- 9 vs. 108 +/- 31; controls = 21 +/- 3 - 50 +/-13 micromol/min/kg). In conclusion, in the postabsorptive state, despite a trivial lactate release by muscle, lactate- and pyruvate-interconversion rates are greatly enhanced in NIDDM patients, possibly due to concomitant impairment in the oxidative pathway of glucose metabolism. This finding strongly suggest a major disturbance in intracellular lactate/pyruvate metabolism in NIDDM.

Adult↗
PubMed1996

Resistance exercise and growth hormone administration in older men: effects on insulin sensitivity and secretion during a stable-label intravenous glucose tolerance test.

To assess the effects of 16 weeks of heavy resistance exercise training (RE) on insulin sensitivity and secretion in healthy older men aged 64 to 75 years (N = 15), stable-label ([6,6,2H2]glucose) intravenous glucose tolerance tests (IVGTTs) were performed before and 7 days after the last bout of exercise. Glucose disappearance rate (Rd) and an index of insulin sensitivity (Si*) were derived using the minimal model of labeled glucose disappearance, and insulin secretion parameters were derived from C-peptide and glucose concentrations measured during the IVGTT, using a minimal model of C-peptide secretion and kinetics. Each subject trained at an intensity of 70% to 95% maximum strength 4 d/wk for 16 weeks on Nautilus (DeLand, FL) weight-training equipment. In conjunction with exercise, six men received daily injections of recombinant human growth hormone ([rhGH] 12.5 to 24 microg/kg/d) and the other nine received placebo injections. GH/placebo injections were administered in a double-blind randomized fashion. The RE program was supervised and progressive in nature, consisting of both upper-and lower-body exercises, and significantly increased muscle strength (P < .05) with no additional benefit from rhGH except for a tendency toward a greater increase in fat-free mass (FFM) in the RE + GH group (P = .06). Peak glucose Rd increased following RE (P < 01), and there was a trend for an improved Si* (ie, from 6.79 +/- 1.14 to 8.42 +/- 0.89 x 10(4) per min/[microU/mL], P = .06). Peak glucose Rd and Si* were unchanged in the RE + GH group following treatment. First- and second-phase insulin secretion were not affected by RE or RE + GH. Glucose tolerance, quantified as the glucose disappearance constant (Kg) between 10 and 32 minutes of the IVGTT, was unchanged by exercise or hormone treatment. These findings support those of a recent study that used the hyperinsulinemic-euglycemic clamp technique (Miller et al, J Appl Physiol 77:1122-1127, 1994), and suggest that when healthy older men engage in RE, whole-body glucose Rd and Si* are improved, and these beneficial effects are not only due to the acute effects of the last bout of exercise. Additionally, in six subjects who received GH, glucose Rd and Si* were not significantly improved following the RE program. Although this may suggest that GH can diminish improvements in glucose Rd and Si* that result from RE, further study is needed to confirm this observation.

Aged↗
PubMed1996

Epinephrine exerts opposite effects on peripheral glucose disposal and glucose-stimulated insulin secretion. A stable label intravenous glucose tolerance test minimal model study.

Epinephrine (EPI) plays a pivotal role in regulating glucose metabolism both in splanchnic and peripheral tissues. Nevertheless, previous studies did not clarify the mechanisms by which EPI affect both glucose disposal processes in peripheral tissues and beta-cell secretion. The aim of this study was to investigate, in six normal volunteers, the effects of elevated EPI concentration on peripheral glucose disposal and insulin secretion by using the stable labeled (either [6,6-2H2] or [2-2H1]glucose) intravenous glucose tolerance test (IVGTT) in conjunction with the minimal models of labeled glucose disappearance and C-peptide secretion. Elevated plasma EPI concentration significantly decreased glucose effectiveness (SG*) by 29% (0.0059 +/- 0.0013 vs. 0.0083 +/- 0.0011 min-1, P < 0.05), and even more, 61%, insulin sensitivity (SI*); (22 +/- 6 x 10(4) vs. 54 +/- 20 x 10(4) min-1.pmol.l-1; P < 0.01). These findings are not due to an isotopic effect induced by an enhanced glycogen breakdown, because the [2-2H1]glucose tracer, which is not incorporated into glycogen, gave results similar to those of [6,6-2H2]glucose tracer. No differences were observed in first phase cell sensitivity, phi 1, in the EPI study (199 +/- 91 vs. 245 +/- 144 10(9), NS), but there was a significant increase in the second-phase cell sensitivity to glucose phi 2, (15.2 +/- 1.7 vs. 17.7 +/- 4.4 10(9).min-1, P < 0.05). In conclusion, EPI selectively impairs peripheral glucose metabolism because of its unique ability to simultaneously and independently decrease glucose effectiveness and insulin sensitivity. Furthermore, EPI enhances phi 2, the ratio between the C-peptide amount secreted during the second phase and the area under the curve of the glucose signal, indicating that the observed increase of C-peptide concentration is due not only to the augmented glucose signal but also to a specific EPI-mediated enhancement of beta-cell responsivity to glucose.

Adult↗
PubMed1995

Kinetic analysis of thyroid hormone action on glucose metabolism in man.

Thyroid hormone action on insulin's effect on glucose kinetics was investigated with the use of a physiological three compartment model. In six healthy volunteers before and after 14 days of thyroxine treatment (300 micrograms/day), a bolus of [3-H3]glucose was injected and the time course of plasma radioactivity was followed closely for 150 min. Then a hyperinsulinemic (1 mU.min-1.kg-1) and euglycemic clamp was started, and euglycemia was maintained for another 250 min. A second bolus of the tracer was then given at 240 min, and the plasma radioactivity was followed for 160 min. Insulin stimulated basal plasma glucose clearance fourfold (p < 0.001) and completely suppressed basal hepatic glucose production (p < 0.001). Concomitantly, the total distribution volume of glucose was increased by 19% (p < 0.05); this change was accompanied by about 50% expansion of the slowly exchanging glucose pool (putatively representing the insulin-dependent compartment). Thyroxine treatment increased plasma triiodothyronine by about 20% (0.1 > p > 0.05) but did not affect basal glucose turnover, insulin-stimulated plasma glucose clearance or the insulin-induced suppression of endogenous glucose output. However, thyroxine treatment blunted the insulin-induced increases in total distribution volume and the slowly exchanging pool of glucose (p = NS vs the basal state). We conclude that minor changes in plasma triiodothyronine (such as occur during overfeeding) do not interfere with the ability of insulin to stimulate the rate of disappearance of glucose or suppress endogenous glucose release; however, our data suggest that they induce finer changes in glucose kinetics, possibly reflecting acceleration or intracellular glucose degradation.

Adult↗
PubMed1995

Estimation of beta-cell sensitivity from intravenous glucose tolerance test C-peptide data. Knowledge of the kinetics avoids errors in modeling the secretion.

Parametric models of insulin secretion are used to measure indexes of beta-cell function from plasma C-peptide concentration during an intravenous glucose tolerance test (IVGTT). Since the models have been usually assessed against plasma C-peptide data, both secretory and kinetic parameters need to be simultaneously estimated. However, undesired compensations between the two sets of parameters may arise. In this study, in order to evaluate IVGTT insulin secretion models, we have analyzed IVGTT data from seven normal subjects for whom individual C-peptide kinetics were known from a separate experiment. Three different beta-cell models have been examined: the minimal model M1 (Diabetes 37:223-231, 1988); a variation of a published model, M2 (Math Biosci 27:319-332, 1975); and a new one, M3. A two-compartment model was used to describe C-peptide kinetics. The results suggest the inadequacy of M1 since kinetic parameter estimates were consistently biased versus the known individual values, and systematic errors were present in the prediction of C-peptide data when kinetic parameters were fixed to the known individual values. M2 performs better than M1 since it reproduces C-peptide data satisfactorily when the individually known description of the kinetics is adopted. M3 retains the second-phase description of M2 but improves the description of first-phase release. M3 is thus proposed to reconstruct the insulin secretion time course and to estimate parameters of first- and second-phase sensitivity to glucose. We also show the robustness of M3, i.e., standard values of C-peptide kinetic parameters can be used when individual values are not available without a loss of accuracy in the estimated secretion parameters. Finally, the shortcomings of using a simplified single-compartment description of C-peptide kinetics are discussed.

Adult↗
PubMed1994

The effects of hormonal replacement therapy on insulin sensitivity in surgically postmenopausal cynomolgus monkeys (Macaca fascicularis).

OBJECTIVE: Our purpose was to evaluate the effect of hormone replacement therapy on insulin resistance in postmenopausal cynomolgus monkeys (Macaca fascicularis). STUDY DESIGN: We studied 37 surgically postmenopausal cynomolgus monkeys that were fed a moderately atherogenic diet for 12 weeks with either no treatment (control), conjugated equine estrogens, medroxyprogesterone acetate, combination conjugated equine estrogens and medroxyprogesterone acetate, or tamoxifen. Insulin sensitivity and glucose effectiveness were determined by the frequent-sampling intravenous tolerance test by means of the minimal model analysis. RESULTS: There were no differences in body weight, total plasma cholesterol, or body fat distribution between control and conjugated equine estrogens, medroxyprogesterone acetate, or combination treatment groups. However, compared with control animals (insulin sensitivity = 5.9 +2- 1.2 x 10(-4) min-1 microU-1 ml) or conjugated equine estrogens treatment (6.3 +/- 1.1 x 10(-4) min-1 microU-1 ml) insulin sensitivity was significantly decreased in animals treated with medroxyprogesterone acetate (2.9 +/- 0.4 x 10(-4) min-1 microU-1 ml, p < 0.001) or conjugated equine estrogens and medroxyprogesterone acetate (2.8 +/- 0.6 x 10(-4) min-1 microU-1 ml, p < 0.001). Although insulin sensitivity was shown to be decreased in the tamoxifen-treated animals (insulin sensitivity = 4.6 +/- 0.6 x 10(-4) min-1 microU -1 ml), the difference was not statistically significant compared with the control or conjugated equine estrogens-treated animals. No significant differences were seen for glucose effectiveness comparing control animals (glucose effectiveness = 0.043 +/- 0.006 min-1) to animals treated with medroxyprogesterone acetate (glucose effectiveness = 0.046 +/- 0.009 min-1), conjugated equine estrogens and medroxyprogesterone acetate (0.048 +/- 0.008 min-1) or tamoxifen (0.039 +/- 0.006 min-1). CONCLUSION: These results suggest that progestins alone or in combination with estrogens can induce insulin resistance in postmenopausal monkeys while having no effect on plasma lipid concentrations or glucose effectiveness.

Animals↗
PubMed1993

Estimation of protein fractional synthetic rate from tracer data.

The fractional synthetic rate (FSR) is a key parameter characterizing protein turnover that is estimated from tracer kinetic data. Formulas to estimate this parameter usually assume a precursor-product model. Assuming this model is correct, we discuss these formulas to estimate the FSR in the steady and non-steady state both for the radioactive and stable isotope tracer. Then we deal with the non-steady-state case where the FSR becomes time varying and derive formulas for its estimation. A non-steady-state case study on the flooding-dose technique for measuring protein turnover is presented.

Homeostasis↗
PubMed1993

Estimating the fractional synthetic rate of plasma apolipoproteins and lipids from stable isotope data.

The use of isotopic tracer studies to quantitate parameters characterizing apolipoprotein metabolism is enjoying a resurgence. This is due in large part to the availability of a number of stable isotopes and methods to measure them accurately in small quantities. Most experimental protocols in which stable isotopes are used call for endogenous labeling of the apolipoprotein of interest by an infusion of a labeled amino acid. Unlike the radioactively labeled amino acid counterpart in which turnover studies have traditionally been carried out for 72 hours to 14 days, the duration of the stable isotope experiment is normally less than 24 hours. This has contributed to some problems related to estimating the kinetic parameters because simplistic formulas whose underlying assumptions are not applicable to the lipoprotein system under study are often invoked. This is particularly true for the fractional synthetic rate (FSR). The purpose of this review is to address some of these problems. We derive the formula commonly used to estimate the FSR. In so doing, the underlying assumptions are carefully delineated. We then discuss several ways in which the formula is applied. Finally, we discuss the implications of these assumptions when the formula is applied to specific lipoprotein systems.

Apolipoproteins↗
PubMed1992

Tracer-to-tracee ratio for analysis of stable isotope tracer data: link with radioactive kinetic formalism.

A kinetic formalism for the analysis of stable isotope transient tracer data is developed by establishing the link with the formalism available for radioactive tracer data. The crucial variable is the tracer-to-tracee ratio. By expressing the measurements in terms of this ratio, the conventional kinetic formalism used for radioactive data can be applied to estimate noncompartmental parameters using stable isotope tracer data. The tracer-to-tracee ratio also plays an important role in compartmental modeling. By considering the tracer masses in the compartments as state variables the system-experiment model can be written in a format analogous to that usually adopted for the radioactive tracer. Finally, it is shown that the tracer-to-tracee ratio also plays a role in a test of the endogenous steady-state assumption.

Animals↗
PubMed1992

V-A and A-V modes in whole body and regional kinetics: domain of validity from a physiological model.

In turnover studies, both at whole body and regional level, sources of tracer and tracee are in general nonidentical thus resulting in nonuniformity of specific activity (SA). Guidelines are available in literature to deal with the heterogeneous SA problem, and either the V-A or A-V modes, based on the arterial and mixed venous blood SA, respectively, have been recommended for different substrates. In particular, the A-V mode is considered the method of choice for studying lactate, amino acids, free fatty acid, etc. Guidelines are based on specific models chosen to describe kinetic and circulatory events of the substance under study but are often conflicting. A unitary physiological framework to understand assumptions of various models is also lacking. In this paper, we first review these models to assess their domain of validity. In particular, we point out major drawbacks that relate to the tissue compartment being treated as a lumped well-mixed pool with a single SA value. We then attempt to handle the nonuniform tissue SA by employing a more physiological model. The tissue system is thought to be made up of elementary units connected in parallel and categorized according to their functional ability to affect incoming SA. Potential changes of SA within individual units are examined. Thus each tissue unit may provide a different contribution to the overall change in SA, as measured in mixed venous blood. A spatial profile of SA is also identified both along the direction of blood flow and transversely toward the inner cellular space. This distributed model allows assessment of the domain of validity of V-A and A-V modes. We show that, in general, the V-A mode underestimates the production rate both at whole body and regional level, whereas the A-V mode can either under- or overestimate it.

Animals↗
PubMed1990

Pseudoketogenesis in hepatectomized dogs.

Overestimation of ketone body turnover in vivo, measured by tracer kinetics, could occur if specific activity or molar percent enrichment is diluted in extrahepatic tissues by label exchange via reversal of 3-oxoacid-CoA transferase, a process we call pseudoketogenesis. To test this hypothesis, euglycemic hepatectomized dogs were injected with a bolus of acetoacetate (0.8 mmol/kg), 32% enriched in [3,4-13C2]acetoacetate. Concentrations and labeling patterns of blood acetoacetate and R-3-hydroxybutyrate were measured by selected ion-monitoring gas chromatography-mass spectrometry. During the 60 min after bolus injection of [3,4-13C2]acetoacetate, the molar percent enrichment of blood [3,4-13C2]acetoacetate decreased to 73 +/- 3% (n = 5) in controls and to 11.5 +/- 0.8% (n = 3) during infusion of dichloroacetate, an activator of pyruvate dehydrogenase. The enrichment of R-3-hydroxy-[3,4-13C2]butyrate followed closely that of [3,4-13C2]acetoacetate. These dilutions occurred despite a net uptake of ketone bodies. Concomitantly, 10.6 +/- 2.2 (n = 5) and 6.0 +/- 2.9% (n = 3) of [13C]acetoacetate molecules were labeled on all four carbons in control and dichloroacetate-treated dogs, respectively. This uniformly labeled acetoacetate arises from partial equilibration between [3,4-13C2]acetoacetate and [1,2-13C2]acetyl-CoA via the reactions catalyzed by 3-oxoacid-CoA transferase and acetoacetyl-CoA thiolase. Our data demonstrate the reversibility of the 3-oxoacid-CoA transferase in intact extrahepatic tissues and support the concept of pseudoketogenesis. This phenomenon has been quantitated by kinetic analysis of the data.

3-Hydroxybutyric Acid↗
PubMed1990

Myocardial metabolism in insulin-deficient diabetic humans without coronary artery disease.

Eleven insulin-dependent diabetes mellitus (IDDM) patients with angiographically normal coronary arteries and a normal hemodynamic response to an echocardiographic-dipyridamole test and 12 normal controls were studied at rest and after atrial pacing simultaneously sampling arterial and coronary sinus blood. In IDDM patients, despite hyperglycemia [10.0 +/- 2.0 (SE) mmol/l], myocardial glucose uptake was slightly lower than in controls. This process was significantly activated in both groups during atrial pacing. The isotopically calculated net flux of lactate across myocardium, in agreement with the net balance value based on unlabeled lactate-pyruvate arteriovenous differences, showed a net uptake in controls (3.5 +/- 0.6 mumol.min-1.1.73 m-2) and a net release in IDDM (12.4 +/- 2.6; P less than 0.01). Atrial pacing stimulated lactate uptake in both groups. Myocardial uptake of ketone bodies was significantly higher in IDDM (37.0 +/- 6.3 mumol.min-1.1.73 m-2) than in controls (10.1 +/- 3.4 mumol.min-1.1.73 m-2; P less than 0.01). Free fatty acid uptake was also significantly greater in IDDM than in controls (44.1 +/- 7.0 vs. 24.1 +/- 5.1 mumol.min-1.1.73 m-2; P less than 0.01). Alanine and branched amino acids were released by diabetic but not by control hearts at rest. The normalization of blood glucose concentrations restored normal patterns of lactate and ketone body kinetics across diabetic myocardium. In conclusion, 1) at rest, myocardial lactate and amino acid uptake is markedly impaired in IDDM without coronary artery disease, and 2) the metabolic abnormalities of the diabetic myocardium are not a primary phenomenon but rather a consequence of hypoinsulinemia and hyperglycemia because insulin administration, resulting in euglycemia, restored normal patterns of cardiac metabolism.

Adult↗
PubMed1990

Constant specific activity input allows reconstruction of endogenous glucose concentration in non-steady state.

In vivo studies on the glucose system often require its perturbation by an exogenous input of glucose, whereas glucose turnover is assessed by infusing a glucose tracer. The constant infusion represents the usual format of tracer administration, but it has no clear advantage other than simplicity. Here we propose a different tracer infusion format. It consists of infusing the tracer in parallel with unlabeled glucose so as to maintain a constant specific activity in the infusate. This protocol does not increase experimental complexity and provides new information on the glucose system in non-steady state by allowing reconstruction of the endogenous component of glucose concentration. This reconstruction only requires very general assumptions, such as tracer-tracee indistinguishability and mass conservation; in particular it is independent of the glucose model structure, i.e., number of compartments and their interconnections. A proof of the result is given for a general nonlinear model of the glucose system. The constant specific activity input is also advantageous for non-steady-state calculations, because it reduces the variation in the measured plasma glucose specific activity. The glucose system has served as the prototype, but the protocol is applicable to other blood-borne substances. The radioactive tracer case has been considered, but the same results apply to stable isotope tracers as well; in this case they also become relevant in a somewhat different context, i.e., kinetic studies in steady state.

Animals↗
PubMed1989

Stable-label intravenous glucose tolerance test minimal model.

The minimal model approach to estimating insulin sensitivity (Sl) and glucose effectiveness in promoting its own disposition at basal insulin (SG) is a powerful tool that has been underutilized given its potential applications. In part, this has been due to its inability to separate insulin and glucose effects on peripheral uptake from their effects on hepatic glucose inflow. Prior enhancements, with radiotracer labeling of the dosage, permit this separation but are unsuitable for use in pregnancy and childhood. In this study, we labeled the intravenous glucose tolerance test (IVGTT) dosage with [6,6-2H2]glucose, [2-2H]glucose, or both stable isotopically labeled glucose tracers and modeled glucose kinetics in six postabsorptive, nonobese adults. As previously found with the radiotracer model, the tracer-estimated S*l derived from the stable-label IVGTT was greater than Sl in each case except one, and the tracer-estimated SG* was less than SG in each instance. More importantly, however, the stable-label IVGTT estimated each parameter with an average precision of +/- 5% (range 3-9%) compared to average precisions of +/- 74% (range 7-309%) for SG and +/- 22% (range 3-72%) for Sl. In addition, because of the different metabolic fates of the two deuterated tracers, there were minor differences in basal insulin-derived measures of glucose effectiveness, but these differences were negligible for parameters describing insulin-stimulated processes. In conclusion, the stable-label IVGTT is a simple, highly precise means of assessing insulin sensitivity and glucose effectiveness at basal insulin that can be used to measure these parameters in individuals of all ages, including children and pregnant women.

Adult↗
PubMed1987

In vivo glucose metabolism in the awake rat: tracer and insulin clamp studies.

The goals of this study were twofold: (1) to determine the in vivo dose-response relationship in the conscious, unstressed rat between the plasma insulin concentration and total body glucose uptake, and between plasma insulin and suppression of endogenous glucose production; and (2) to develop a physiologic compartmental model to describe the kinetics of plasma glucose in the rat in the basal state. In order to perform repeat insulin clamp studies in the same rat, chronic catheters were implanted in the aortic arch (via the carotid artery) and in the cardiac atrium (via the jugular vein), exteriorized, and fixed to the back of the skull with a dental cement cap. Insulin was infused at rates of 1.2, 2.4, 4.8, 12, and 24 mU/min.kg, and the plasma glucose was held constant at the basal level by a variable glucose infusion (euglycemic insulin clamp). The resulting steady-state plasma insulin concentrations ranged from 40 to 1,300 microU/mL. The dose-response curve for glucose uptake was sigmoidal in shape: in the basal state, total glucose utilization averaged 6.8 mg/min.kg at an insulin concentration of 9 microU/mL, half-maximal glucose uptake (18.3 mg/kg.min) occurred at a plasma insulin concentration between 70 and 80 microU/mL, and maximal uptake (36.6 mg/kg.min) was seen at an insulin level in excess of 100 microU/mL. Residual endogenous glucose production was evaluated by a prime-continuous infusion of (3-3H)-glucose. The dose-response curve for suppression of endogenous glucose output also was sigmoidal.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗