Respiratory Physiology & Neurobiology 205 (2015) 92–98
Contents lists available at ScienceDirect
Respiratory Physiology & Neurobiology journal homepage: www.elsevier.com/locate/resphysiol
T3 supplementation affects ventilatory timing & glucose levels in type 2 diabetes mellitus model Stephen S. Bollinger a , Nathen Y. Weltman a , A. Martin Gerdes b , Evelyn H. Schlenker a,∗ a b
Division of Basic Biomedical Sciences, Sanford School of Medicine, University of South Dakota, Vermillion, SD, USA Department of Biomedical Sciences, New York Institute of Technology-College of Osteopathic Medicine, Old Westbury, NY, USA
a r t i c l e
i n f o
Article history: Accepted 29 October 2014 Available online 5 November 2014 Keywords: Type 2 diabetes mellitus Triiodothyronine Ventilation Blood glucose
a b s t r a c t Type II diabetes mellitus (T2DM) can affect ventilation, metabolism, and fasting blood glucose levels. Hypothyroidism may be a comorbidity of T2DM. In this study T2DM was induced in 20 female Sprague Dawley rats using Streptozotocin (STZ) and Nicotinamide (N). One of experimental STZ/N groups (N = 10 per group) was treated with a low dose of triiodothyronine (T3 ). Blood glucose levels, metabolism and ventilation (in air and in response to hypoxia) were measured in the 3 groups. STZ/N-treated rats increased fasting blood glucose compared to control rats eight days and 2 months post-STZ/N injections indicating stable induction of T2DM state. Treatments had no effects on ventilation, metabolism or body weight. After one month of T3 supplementation, there were no physiological indications of hyperthyroidism, but T3 supplementation altered ventilatory timing and decreased blood glucose levels compared to STZ/N rats. These results suggest that low levels of T3 supplementation could offer modest effects on blood glucose and ventilatory timing in this T2M model. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Type 2 diabetes mellitus (T2DM) is a prevalent endocrine disorder that affects approximately 9.3% of the general population and 26.9% of individuals aged 65 and older in the United States (Prevention, 2011, 2014). Although T2DM primarily manifests in the middle-aged and elderly, it is becoming more common in children and adolescents (Adebayo and Willis, 2014). Complications associated with T2DM are frequent and increase morbidity and mortality significantly (Gregg et al., 2014). Hypothyroidism is the second most prevalent pathological endocrine deficiency behind diabetes mellitus in the United States (Bope and Kellerman, 2012). The incidence of overt hypothyroidism is approximately 2–5% in the general population. Hypothyroidism is a frequent comorbidity in T2DM. For example, in a study of 386 Brazilian patients with Type 1 Diabetes Mellitus (T1DM) or T2DM, 13.1% of T2DM patients (aged 60.7 ± 10.6) also exhibited thyroid dysfunction (TD) (Palma et al., 2013). In a study of 1092 T2DM patients (aged 65.53 ± 11.77) attending an outpatient clinic in Greece, Papazafiropoulou et al. (2010) found that 12.3% of patients displayed TD. Radaideh et al. (2004) found rates of 12.5% for TD in a group of 908 T2DM patients in Jordan. The prevalence of TD
∗ Corresponding author. Tel.: +1 605 677 5160; fax: +1 605 677 6381. E-mail address: [email protected] (E.H. Schlenker). http://dx.doi.org/10.1016/j.resp.2014.10.020 1569-9048/© 2014 Elsevier B.V. All rights reserved.
observed in the abovementioned studies falls within the median range of previous studies (2–17%, Feely and Isles, 1979; Gray et al., 1980; Perros et al., 1995; Smithson, 1998) (Feely and Isles, 1979; Gray et al., 1980; Perros et al., 1995; Smithson, 1998). A study by Celani et al. (1994) of 159 female and 131 male T2DM patients (aged 60.6 ± 11.9 years), who had been hospitalized due to poor diabetic control or had recent onset diabetes, found thyroid disease rates of 31.4%. Although T2DM is known to affect the respiratory system, this area has not been well studied. For example, in 16 patients (aged 63 ± 8 years) with T2DM, Paredi et al. (1999) found a negative correlation between pulmonary function and glycemic control. In a study of 25 patients (aged 56.9 ± 7.9 years) with T2DM, Guvener et al. (2003) found that diabetics had a decreased alveolar gas exchange capacity when compared to healthy controls. The ratio of carbon monoxide diffusion (DLCO ) and alveolar ventilation (VA) was significantly lower in T2DM patients. This decline in pulmonary gas exchange also significantly correlated with increased age and duration of T2DM. The adverse effects of hypothyroidism on the respiratory system and ventilatory control have been recently reviewed (Schlenker, 2012). These include decreased ventilatory responses to hypercapnia, diminished maximal breathing capacity, and decreased DLCO (Wilson and Bedell, 1960; Zwillich et al., 1975). These findings were more frequently observed in female patients and patients with high thyroid stimulating hormone levels (Ladenson et al.,
S.S. Bollinger et al. / Respiratory Physiology & Neurobiology 205 (2015) 92–98
1988). In each of these studies, ventilatory responses to hypoxia and hypercapnia were restored to normal levels after T3 or T4 supplementation. According to Dempsey et al. (2011), factors that may affect regulation of ventilation in myxedema include depressed central nervous system activity, weakened respiratory muscles, change in some lung volume subdivisions, abnormal contractile properties of the upper airway musculature, abnormalities in pulmonary gas exchange, and obesity; which can lead to alveolar hypoventilation and CO2 retention. Interestingly, the role of hypothyroidism in T2DM on ventilation is not well documented. Thus, the purpose of the present study is to utilize combining Streptozotocin (STZ) and Nicotinamide (N) to induce T2DM in a rat model (STZ/N). The addition of N has been shown to preserve beta-cell levels in the pancreas to approximately 40% of normal when compared with STZ administration alone (Masiello et al., 1998). These treatments increase blood glucose and glycosylated hemoglobin levels (Kumar et al., 2012). Ventilation and metabolism has not been investigated in the STZ/N model of T2DM. Since hypothyroidism commonly occurs in the setting of T2DM, we investigated the relationship between thyroid supplementation on ventilation and metabolism in STZ/N treated animals. An intricate relationship exists between thyroid function and glycemic control. Therefore, the comorbid hypothyroid condition in T2DM may contribute to hyperglycemia in T2DM. Consequently, we expected that T3 supplementation would help regulate blood glucose of STZ/N rats. A recent paper used this approach to study effects of the STZ/N model and T3 supplementation on cardiovascular function and coronary vasculature (Weltman et al., 2014). 2. Methods 2.1. Animals and treatments All experiments were approved by The University of South Dakota Institutional Animal Care and Use Committee. Age matched, female Sprague Dawley rats (Harlan, Indianapolis, IN, USA) were used in these experiments. All animals were kept on a 12-h light/dark cycle and food and water were provided ad libitum. Rats were randomized into untreated control (n = 10) or STZ/N treatment (n = 20). Rats in the STZ/N groups received intraperitoneal injections of the N (200 mg/kg) dissolved in sterile saline 15 min prior to an intraperitoneal injection of STZ (65 mg/kg) dissolved in sterile citrate dextrose solution (pH 4.5) using established methods (Masiello et al., 1998). One month after STZ/N treatment, SD rats treated with STZ/N were further divided into T3 treated (STZ/N/T3 ; n = 10) and non-T3 treated (STZ/N; n = 10) groups. T3 was administered non-invasively from a stock solution (40 g/ml T3 , 50% ethanol, 48.5% glycerol) dissolved in drinking water (final concentration in water: 0.03 g/ml T3 ). Baseline fasting blood glucose levels were measured prior to STZ/N, eight days post-STZ/N injections, and after one month of T3 supplementation by tail vein sampling using an Ascensia Contour BG meter (Bayer, Pittsburgh, PA). Body weight and water intake were monitored in all animals. Experimental procedures commenced one month after the T3 supplementation was initiated. 2.2. Physiological measurements During the first two weeks after induction of T2DM, the rats were acclimated to the animal facility. The rats were handled individually in the lab environment for about 5 min. After acclimatizing, the rats were placed in a cylindrical Plexiglas chamber (22 cm long and 15.5 cm in diameter). The rats were then allowed time
93
to acclimate to the chamber until ventilatory patterns stabilized. Ventilation was then evaluated for each rat during exposure to air. To measure ventilatory parameters we used, a cylindrical Plexiglas chamber (22 cm long and 15.5 cm in diameter) contained ports to measure the gas flow rate through the chamber (Gilmont rotameter), O2 and CO2 levels (Vacuumed O2 and CO2 analyzer2), and chamber temperature (Digitec digital thermometer). A Statham pressure transducer measured pressure changes associated with breathing to determine tidal volume (VT ), inspiratory (TI ) and expiratory (TE ) times, breathing frequency (F), and minute ventilation (V˙ E ), the product of breathing frequency and tidal volume when the chamber was closed. Calibration of the chamber was accomplished by injecting 0.4 ml of air into the closed chamber. A small leak stabilized the pressure recordings, but acted like flow box (Szewczak and Powell, 2003). Thus, we can only consider “relative” VT and V˙ E values that were normalized by body weight (1000/body weight). Oxygen and CO2 levels of gas entering and leaving the chamber as well as flow rate were used to measure oxygen consumption (V˙ O2 ) and CO2 production (V˙ CO2 ). Values were corrected to STPD values. After the rat was removed from the chamber, its rectal temperature was measured using a thermometer and thermocouple (Model BAT-12 Sensortec) and body weight before returning the rat to her home cage. After one month of treatment with T3 , physiological parameters were measured for all rats. First, the rats were allowed 5 min to acclimate to the laboratory environment. Each rat was then placed into the plethysmographic chamber and allowed to acclimate until she exhibited uniform breathing patterns. The metabolism and ventilation for rats was first measured during exposure to air. After recording these measurements, the rats were then exposed to hypoxic air containing 10% O2 in nitrogen for 5 min and ventilatory measurements were taken the last minute while the rats were still exposed to hypoxic air inside the chamber. After these measurements were conducted, the chamber was flushed out with air for 10 min prior to post-hypoxia respiratory measurements. Subsequently, rectal body temperature and body weight were measured before returning the rats to their cages. Following the ventilatory and metabolic measurements, fasting blood glucose obtained from tail vein blood was again measured using an Ascensia Contour BG meter (Bayer, Pittsburgh, PA). 2.3. Statistical analysis Ventilatory parameters, body temperature, body weight, metabolic values and baseline fasting blood glucose values are presented as means and standard deviation (SD). Inferential statistics included one-way analysis of variance and post hoc Holms tests for measured and calculated physiological parameters. Since fasting blood glucose levels were not normally distributed for the STZ/N and STZ/N/T3 groups after one month of treatment, this variable was analyzed using a Kruskal–Wallis test and post-hoc MannWhitney tests. Correlation between inspiratory times and blood glucose levels was used to determine associations between TI and blood glucose levels. Significance was accepted at P < 0.05. Data were analyzed using GraphPad version 6.0. 3. Results Prior to STZ/N administration, body weights and resting glucose levels were similar among the three groups (Weltman et al., 2014). Mean body weights were 249 ± 10.5 g, 246 ± 24.2 g, and 245 ± 13.2 g, for the control, STZ/N, and STZ/N/T3 , respectively. Mean fasting blood glucose levels were 101.0 ± 16.9 mg/dl, 103.7 ± 11.6 mg/dl, and 97.0 ± 12.9 mg/dl for control, STZ/N, and STZ/N/T3 , respectively. Free T3 , total T3 , and total T4 showed
94
S.S. Bollinger et al. / Respiratory Physiology & Neurobiology 205 (2015) 92–98
Table 1 Body temperature (BT), body weight (BW), oxygen consumption (V˙ O2 ), CO2 production (V˙ CO2 ) and blood glucose levels in the three groups.
Table 2 Ventilatory Parameters of Control, STZ/N and STZ/N-T3 treated rats during exposure to baseline air, hypoxia and air following hypoxia (post-hypoxia).
Variables
Control
STZ/N
STZ/N/T3
Variable
Control
STZ/N
STZ/N/T3
BT in ◦ C BW in grams V˙ O2 (ml/min/1000 × BW) V˙ CO2 (ml/min/1000 × BW) Blood glucose (mg/dl)
38.1 (0.2) 263.8 (4.3) 16.0 (1.2) 11.2 (1) 117.5 (15.5)
38.3 (0.1) 258.5 (11.1) 17.2 (1.8) 12.3 (1.4) 399 (75.9)*
38.3 (0.1) 254.9 (5.8) 16.7 (1.1) 11.9 (0.7) 262.9 (68.9)*†
V˙ E air F VT V˙ E hypoxia F VT V˙ E post hypoxia F VT
139.1 (5.5) 87 (3) 1.6 (0.06) 237.2 (10.8) 123 (4) 1.93 (0.08) 124.8 (4.5) 84 (3) 1.49 (0.05)
147.8 (7.9) 87 (2) 1.7 (0.07) 265.3 (13.4) 119 (4) 2.24 (0.12) 122.1 (5.2) 80 (2) 1.53 (0.07)
141.2 (10.6) 89 (6) 1.6 (0.07) 250.5 (18.6) 120 (5) 2.08 (0.12) 124.3 (5.7) 87 (3) 1.43 (0.03)
Values are means and (SD) for each variable. There were 10 animals per group. The asterisk denotes differences compared to control rats and dagger difference between STZ/N and the STZ/N/T3 .
that all rats used in the study were initially euthyroid (Weltman et al., 2014). Fasting blood glucose measurements eight days after STZ/N injection were 116.7 ± 19.6 mg/dl in the control group and, 253.8 ± 162.4 mg/dl in the STZ/N group indicating successful elevation of fasting blood glucose by the STZ/N treatment. After one month of T3 treatment, there were no differences in body temperature, body weight, oxygen consumption, or CO2 production among groups (Table 1). In contrast, there were significant differences in blood glucose levels (P < 0.01) with the STZ/N group having higher levels than the control or STZ/N/T3 groups. However, the latter group’s values were significantly greater than that of the control group. After 30 days of T3 supplementation, ventilatory measurements were determined in the 3 groups of rats exposed to air, hypoxia, and post-hypoxia. Representative waveforms from one animal in each of the 3 groups are shown in Fig. 1. During exposure to baseline air, there were no differences in F, relative VT , or relative V˙ E among the three groups (Table 2). However, TI was significantly larger in the STZ/N/T3 group compared to
Values are means and SD (in parentheses) of 10 animals per group. Ventilation (V˙ E ) is in ml × (1000/BW)/min. frequency (F) is in breaths per minute and tidal volume (VT ) is in ml × (1000/BW).
control and STZ/N (P = 0.023) and showed greater variability (Fig. 2). Although TE was not significantly different among the three groups, the variability in the STZ/N/T3 group was greater than that of the other groups (Fig. 2). This also contributed to a significant increase in TI /Ttot in STZ/N/T3 treated group relative to the control (Fig. 2), but a decrease of VT /TI in the STZ/N/T3 treated group relative to that in the two other groups (Fig. 2). During exposure to hypoxia, ventilation increased in all groups to a similar extent due to an increase in relative tidal volume and frequency (Table 2). There were no differences in TI or TE values among the three groups (data not shown). Following exposure to hypoxia, there was no difference in minute ventilation among the three groups while exposed to room air (Table 2). However, TI was again significantly higher (P = 0.0008) in the STZ/N/T3 group (Fig. 3). TE was lower in the STZ/N/T3 group compared to the STZ/N group (P = 0.015), but not different from
Fig. 1. Representative waveforms in one rat from each of these groups: control, STZ/N and STZ/N/T3 . Each rat was exposed to air, hypoxia and then air following hypoxia (post hypoxia).
S.S. Bollinger et al. / Respiratory Physiology & Neurobiology 205 (2015) 92–98
*
T I a ir
T E a ir
0 .8
95
0 .3
#
T I (s e c )
0 .4
0 .2
0 .1
0 .2
3 Z T S
3 /T /N Z
tr n o
S
C
Z T
T
o
l
3
0
/T /N
Z T S
S
C
o
n
tr
o
l
/N
0 .0
#
5
/N
0 .1
10
Z
0 .2
*
T
0 .3
V T /T I a ir
15
S
0 .4
V T /T I ( ( m l* 1 0 0 0 /B W ) /s e c )
S
*
T I/T T o t a ir
0 .5
/T /N
Z T
n o
T
c
Z
S
T
/N
tr
/T
o
l
3
/N Z
l o tr n o c
/N
0 .0
0 .0
S
T E (s e c )
0 .6
Fig. 2. Inspiratory time (TI) expiratory time (TE), time spent during inspiration relative to the total time of a breath (TI/Tot) and inspiratory flow rate (VT /TI ) of Control, STZ/N and STZ/N/T3 during exposure to air prior to hypoxia. There are 10 rats per group. Data are presented individually with group means and SD indicated by the horizontal lines. The asterisk denotes significant differences between the STZ/N/T3 group and the control group and the # denotes significant differences between the STZ/N/T3 group and the STZ/N group.
the control group. Subsequently, TI /tot was greater in the STZ/N/T3 group compared to the two other groups (P < 0.001, Fig. 3) and VT /TI was lower in the STZ/N/T3 group (P < 0.05). Thus, T3 treatment appears to effect ventilatory timing, but not on other ventilatory parameters during air exposures. To determine if TI and blood glucose were related and impacted by T3 treatment, correlation analysis was conducted separately in the control, STZ/N and the STZ/N/T3 treated groups. There was no significant correlation between blood glucose levels and TI for the control or STZ/N groups. However, there was a significant association between blood glucose levels and TI in the STZ/N/T3 group (r = 0.864, P = 0.0013). Thus, T3 supplementation affects the relationship between TI and glucose levels.
4. Discussion To our knowledge, the present findings represent the first study to examine the effects of T3 supplementation on ventilatory and metabolic parameters in T2DM using the STZ/N model. We evaluated parameters of metabolism, ventilation and blood glucose levels in a female rat model for T2DM 60 days after STZ/N injections and after 30 days of T3 hormone supplementation. No significant changes were found between the STZ/N and control groups for ventilatory parameters during exposure to air or hypoxia, V˙ O2 , or V˙ CO2 . This suggests that the short-term induction of T2DM with STZ and pretreatment with N did not adversely affect metabolic or ventilatory function in this study. Interestingly, supplementation of STZ/N-treated rats with T3 altered inspiratory time during exposure to air, but not during exposure to hypoxia. Moreover,
T3 supplementation decreased blood glucose values relative to treatment with STZ/N alone. Finally there was a significant association between blood glucose levels and TI only in the STZ/N/T3 group. 4.1. Ventilatory and metabolic results in diabetic models The ventilatory and metabolic results in the STZ/N model reported in this study contrast with previous studies of diabetic rodents (Ramadan et al., 2006; Saiki et al., 2005). We have previously explored parameters of altered control of ventilation in a STZ rat model of diabetes mellitus. In this four-week study of male SD rats treated with STZ, we observed a progressive reduction of VT , V˙ E , and V˙ CO2 compared to the control group and a STZ diabetic group treated with insulin (STZ/I) (Hein et al., 1994). Hypercapnic (3, 6, 9%) and hypoxic (10%) ventilatory responses were also significantly less in the STZ group than the control group and the STZ/I group. Ventilation in air and ventilatory responses following exposures to hypercapnia and hypoxia were similar for the control and STZ/I group indicating that the insulin therapy ameliorated the effects of the STZ on ventilation. Saiki et al. (2005) studied metabolism and respiratory responses to hypoxia in induced diabetic male Wistar rats. In their study, the researchers took respiratory measurements five weeks after STZ injection. During exposure to air and hypoxia, the STZ group did not show significant differences in V˙ E from the control group; however the V˙ CO2 levels in the STZ group were significantly increased in comparison to the control group. Subsequent shortterm administration of insulin increased V˙ E during exposure to hypoxia in comparison to the control group due to an increase
96
S.S. Bollinger et al. / Respiratory Physiology & Neurobiology 205 (2015) 92–98
T I p o s t h y p o x ia
T E p o s t h y p o x ia
0 .8
0 .6
0 .4
0 .2
0 .1
0 .2
0 .0
3 /T /N Z
tr n o
S
C
Z T S
T
o
l
3
0
/T /N
Z T S
n
tr
o
l
/N
0 .0
/N
0 .1
5
Z
0 .2
*
10
T
0 .3
V T /T i p o s t h y p o x ia 15
S
#
V T /T i ( m l* 1 0 0 0 /B W /s e c )
*
0 .4
o
/T /N Z T S
T S
C
T I/ T t o t p o s t h y p o x i a 0 .5
C
3
/N Z S
o C
Z
T
n
/T /N
T S
to
3
/N Z
l o tr n o
l
0 .0
T i/T to t
*
0 .3
T I (s e c )
T E (s e c )
#
Fig. 3. Inspiratory time (TI ) expiratory time (TE ), time spent during inspiration relative to the total time of a breath (TI /Tot ) and inspiratory flow rate (VT /TI ) of Control, STZ/N and STZ/N/T3 rats during exposure to air following exposure to hypoxia. There are 10 rats per group. Data are presented individually with group means and SD indicated by the horizontal lines. The asterisk denotes significant differences between the STZ/N/T3 group and the control group and the # denotes significant differences between the STZ/N/T3 group and the STZ/N group.
in VT . The authors speculated that, independent of blood glucose level, the intrinsic hormonal effects of insulin might be important for the respiratory control mechanisms. Yamazaki et al. (2002) evaluated hypercapnic and hypoxic ventilatory responses in long-term STZ (60 mg/kg in saline) induced diabetic male Sprague-Dawley rats. The study consisted of twenty-six rats split into control, STZ, and STZ/I groups. Basic ventilatory parameters such as respiratory rate, VT , and V˙ E were not significantly altered for the STZ group in comparison to the control group, but ventilatory responses to hypercapnia and hypoxia were reduced significantly starting sixteen weeks after STZ injections and lasted until the end of the twenty-eight week study. Respiratory rate, VT , and V˙ E decreased with age in all experimental groups. Ventilation for all groups increased linearly with increasing concentrations of CO2 in air, but the average slope value of the regression line was lower for the STZ group in compared to that of the control group. As the study progressed and the rats aged, the average slope value of this regression line increased with aging for the control group but remained at a lower level for the STZ group. Hypoxic ventilation measurements increased linearly with decreasing concentrations of O2 in air. Moreover, the average slope value of the regression line for hypoxic ventilation increased with aging for the control group but remained lower for the STZ group. These reduced responses were also seen for the STZ/I group, indicating that daily insulin treatment was not sufficient to mediate these ventilatory changes. This contradicts results seen in the shortterm studies by Hein et al. (1994) and Saiki et al. (2005) where insulin supplementation was sufficient to counteract reduced
ventilatory responses. The researchers therefore concluded that chronic diabetic condition might impair the central and peripheral chemosensitivity, but not the bulbar respiratory network responsible for the regulation of the basic respiratory rhythm. Finally, a study by Polotsky et al. (2001) explored the effects of T1DM induction on control of ventilation for two strains of mice. In this study, researchers used two models of T1DM: (1) STZ-induced diabetes (200 mg/kg) in fifty-nine C57BL/6J male mice on a regular diet or with induced obesity from a high fat diet; and (2) spontaneous T1DM in ten NOD-Ltj female mice. In both of these models, T1DM resulted in depression of hypercapnic ventilatory responses that was associated with duration of hyperglycemia and increased glycosylation of the diaphragm. For the STZ group, V˙ E and hypercapnic ventilatory responses significantly declined seven days after STZ injection; however they found no significant differences between the STZ and control group for V˙ O2 , V˙ CO2 , or the respiratory exchange ratio. In the present study, the relationship between TI (and other ventilatory variables) and blood glucose levels was not significant, suggesting that in the STZ/N model 8 weeks of hyperglycemia alone did not affect control of ventilation. 4.2. Thyroid hormone effects on blood glucose and TI Work by Stratton et al. (2000) does suggest that modest reductions in hyperglycemia has the potential to prevent death from complications related to diabetes. Two months after STZ/N injections and after one-month of T3 supplementation, fasting blood glucose levels for the STZ/N and STZ/N/T3 groups continued
S.S. Bollinger et al. / Respiratory Physiology & Neurobiology 205 (2015) 92–98
to be significantly higher than control, but the latter group’s values were lower than those of the STZ/N group. This suggests that T3 supplementation may either ameliorate hyperglycemia or slow the progression of hyperglycemia. Exact mechanisms for this interaction are currently unknown. This observation correlates with suggestions made by Fernandez-Real et al. (2006) that thyroid function is intrinsically related to variables responsible for insulin resistance. The interaction of thyroid hormone supplementation on fasting blood glucose levels could therefore also serve to prevent the manifestation in vascular complications in T2DM due to chronic and severe hyperglycemia. In this study, we treated rats with T3 instead of T4 , as it is more readily available to be utilized by tissues throughout the body. According to Manjunath (2012), T4 is a precursor to T3 and deiodinases in tissues such as the brain and pituitary gland can convert T4 to T3 . Importantly, T3 is bound to thyroid hormone receptors with 10–15 times greater affinity than T4 leading to increases in hormonal potency. By using the more readily available form of thyroid hormone (T3 ), we increased the likelihood that T3 supplementation would stimulate thyroid receptors in tissues having low thyroid hormone levels. A novel finding in the present study was that T3 supplementation increased TI both before and following hypoxia. The significant correlation between TI and blood glucose levels only found in the STZ/N/T3 group suggests that T3 may have affected this relationship. Thyroid hormone treatment has been reported to augment ventilation as seen in the dystrophic hamsters who display a euthyroid sick syndrome (low T3 , normal T4 levels) as well as in SHHF rats (Schlenker and Burbach, 1995; Schlenker et al., 2003). Moreover, Weltman et al. (2014) recently also showed that T3 supplementation improved cardiac function, the number of cardiac arterioles and reversed the fetal gene expression in the heart in STZ/N female rats. Thus, the present study indicates that this level of T3 supplementation may have subtle effects on blood glucose levels as well as on ventilatory timing. 4.3. Limitations of the study The present study has several limitations. The first limitation was that our plethysmograph acted like a flow box. Thus, our values for tidal volume and minute ventilation can only be considered relative, not absolute and are considerably lower than those previously reported in rats (Fournier et al., 2012). In addition, the duration of the present study was relatively short. The consequences of thyroid hormone supplementation in the STZ/N model and effects of T3 supplementation for a longer time period needs to be determined. However, in the Weltman et al. (2014) paper, serum T3 levels were measured in animals who had been treated for a longer period with STZ/N and STZ/N/T3 and they reported that T3 levels were not different among the three groups, whereas TSH levels were markedly increased in the STZ/N group, compared to the control and STZ/N/T3 groups (Weltman et al., 2014). Importantly T3 levels in cardiac tissues in the study by Weltman et al. (2014) were decreased in the STZ/N group relative to control and the STZ/N/T3 groups. Thus, in the present study measurement of T3 serum levels may not have shown differences among the groups. Another limitation of this study is that we only used female Sprague-Dawley rats. Further limitations of this study involve the extent and mechanism by which thyroid hormone supplementation can affect blood glucose levels and inspiratory timing. Future studies should seek to identify specific impacts of thyroid hormone levels on underlying mechanisms affecting blood glucose levels both in short-term and long-term settings. In regard to the possible translation applicability of T3 supplementation, patients would need to receive oral low doses of T3 rather than the drinking water administration used in the present study.
97
4.4. Overall conclusions In this study we utilized the STZ/N model to successfully induce a T2DM state in female Sprague-Dawley rats. No effects were noted on ventilation, metabolism or body weight. After one month of T3 supplementation there were no physiological indications that this low dose of T3 induced hyperthyroidism. T3 supplementation altered ventilatory timing and decreased blood glucose levels compared to STZ/N treated rats. These results suggest that low levels of T3 supplementation could offer modest effects on blood glucose levels and ventilatory timing in this T2M model. Further research is necessary to establish the effects of the STZ/N model on metabolism, ventilation, and blood glucose in both short-term and long-term models, as well as how thyroid hormone supplementation can affect these manifestations by acting on areas of the brain involved in regulation of breathing. Acknowledgements This project was supported by Grant numbers RO1HL09316001A1 and RO1HL103671 (AMG) from the National Heart, Lung, and Blood Institute. This research was also supported by an American Diabetes Association (ADA) Clinical Scientist Training Award 7-10CST-01 (NYW & AMG) and Center for Undergraduate Grant (SSB). References Adebayo, O., Willis, G.C., 2014. The changing face of diabetes in America. Emerg. Med. Clin. North Am. 32, 319–327. Bope, E., Kellerman, R., 2012. Conn’s Current Therapy 2012. Saunders Elsevier 2012, Philadelphia, PA, USA. Celani, M.F., Bonati, M.E., Stucci, N., 1994. Prevalence of abnormal thyrotropin concentrations measured by a sensitive assay in patients with type 2 diabetes mellitus. Diabetes Res. (Edinburgh, Scotland) 27, 15–25. Dempsey, J.A., Olson, E.B., Skatrud, J.B., 2011. Hormones and Neurochemicals in the Regulation of Breathing, Comprehensive Physiology. John Wiley & Sons, Inc. Feely, J., Isles, T.E., 1979. Screening for thyroid dysfunction in diabetics. Br. Med. J. 1, 1678. Fernandez-Real, J.M., Lopez-Bermejo, A., Castro, A., Casamitjana, R., Ricart, W., 2006. Thyroid function is intrinsically linked to insulin sensitivity and endotheliumdependent vasodilation in healthy euthyroid subjects. J. Clin. Endocrinol. Metab. 91, 3337–3343. Fournier, S., Kinkead, R., Joseph, V., 2012. Influence of housing conditions from weaning to adulthood on the ventilatory, thermoregulatory, and endocrine responses to hypoxia of adult female rats. J. Appl. Physiol. 112, 1474–1481. Gray, R.S., Borsey, D.Q., Seth, J., Herd, R., Brown, N.S., Clarke, B.F., 1980. Prevalence of subclinical thyroid failure in insulin-dependent diabetes. J. Clin. Endocrin. Metab. 50, 1034–1037. Gregg, E.W., Williams, D.E., Geiss, L., 2014. Changes in diabetes-related complications in the United States. N. Engl. J. Med. 371, 286–287. Guvener, N., Tutuncu, N.B., Akcay, S., Eyuboglu, F., Gokcel, A., 2003. Alveolar gas exchange in patients with type 2 diabetes mellitus. Endocr. J. 50, 663–667. Hein, M.S., Schlenker, E.H., Patel, K.P., 1994. Altered control of ventilation in streptozotocin-induced diabetic rats. Proc. Soc. Exp. Biol. Med. 207, 213–219. Kumar, S., Sharma, S., Vasudeva, N., Ranga, V., 2012. In vivo anti-hyperglycemic and antioxidant potentials of ethanolic extract from Tecomella undulata. Diabetol. Metab. Syndr. 4, 33. Ladenson, P.W., Goldenheim, P.D., Ridgway, E.C., 1988. Prediction and reversal of blunted ventilatory responsiveness in patients with hypothyroidism. Am. J. Med. 84, 877–883. Manjunath, S., 2012. Clinical and Metabolic Study of Subclinical Thyroid Disorders in Type 2 Diabetes, General Medicine. Rajiv Gandhi University of Health Sciences, Bangalore, India, pp. 141. Masiello, P., Broca, C., Gross, R., Roye, M., Manteghetti, M., Hillaire-Buys, D., Novelli, M., Ribes, G., 1998. Experimental NIDDM: development of a new model in adult rats administered streptozotocin and nicotinamide. Diabetes 47, 224–229. Palma, C.C.S.S.V., Pavesi, M., Nogueira, V., Clemente, E.L., Vasconcellos, M.d.F.B.M.P., Pereira, L., Pacheco, F., Braga, T., Bello, L., Soares, J., dos Santos, S.C., Campos, V.P.L.C., Gomes, M., 2013. Prevalence of thyroid dysfunction in patients with diabetes mellitus. Diabetol. Metab. Syndr. 5, 58. Papazafiropoulou, A., Sotiropoulos, A., Kokolaki, A., Kardara, M., Stamataki, P., Pappas, S., 2010. Prevalence of thyroid dysfunction among Greek type 2 diabetic patients attending an outpatient clinic. J. Clin. Med. Res. 2, 75–78. Paredi, P., Biernacki, W., Invernizzi, G., Kharitonov, S.A., Barnes, P.J., 1999. Exhaled carbon monoxide levels elevated in diabetes and correlated with glucose
98
S.S. Bollinger et al. / Respiratory Physiology & Neurobiology 205 (2015) 92–98
concentration in blood: a new test for monitoring the disease? Chest 116, 1007–1011. Perros, P., McCrimmon, R.J., Shaw, G., Frier, B.M., 1995. Frequency of thyroid dysfunction in diabetic patients: value of annual screening. Diabet. Med. 12, 622–627. Polotsky, V.Y., Wilson, J.A., Haines, A.S., Scharf, M.T., Soutiere, S.E., Tankersley, C.G., Smith, P.L., Schwartz, A.R., O’Donnell, C.P., 2001. The impact of insulindependent diabetes on ventilatory control in the mouse. Am. J. Respir. Crit. Care Med. 163, 624–632. Prevention, C.f.D.C.a, 2011. National Diabetes Fact Sheet: National Estimates and General Information on Diabetes and Prediabetes in the United States. U.S. Department of Health and Human Services, Centers for Disease Control and Prevention, Atlanta, GA. Prevention, C.f.D.C.a., 2014. National Diabetes Statistics Report: Estimates of Diabetes and Its Burden in the United States. US Department of Health and Human Services, Atlanta, GA. Radaideh, A.R., Nusier, M.K., Amari, F.L., Bateiha, A.E., El-Khateeb, M.S., Naser, A.S., Ajlouni, K.M., 2004. Thyroid dysfunction in patients with type 2 diabetes mellitus in Jordan. Saudi Med. J. 25, 1046–1050. Ramadan, W., Petitjean, M., Loos, N., Geloen, A., Vardon, G., Delanaud, S., Gros, F., Dewasmes, G., 2006. Effect of high-fat diet and metformin treatment on ventilation and sleep apnea in non-obese rats. Respir. Physiol. Neurobiol. 150, 52–65. Saiki, C., Seki, N., Furuya, H., Matsumoto, S., 2005. The acute effects of insulin on the cardiorespiratory responses to hypoxia in streptozotocin-induced diabetic rats. Acta Physiol. Scand. 183, 107–115. Schlenker, E.H., 2012. Effects of hypothyroidism on the respiratory system and control of breathing: Human studies and animal models. Respir. Physiol. Neurobiol. 181, 123–131.
Schlenker, E., Burbach, J., 1995. Thyroxine affects ventilation, lung morphometry, and necrosis of diaphragm in dystrophic hamsters. Am. J. Physiol. 268, R779–R785. Schlenker, E.H., Tamura, T., Gerdes, A.M., 2003. Gender-specific effects of thyroid hormones on cardiopulmonary function in SHHF rats. J. Appl. Physiol. 95, 2292–2298. Smithson, M.J., 1998. Screening for thyroid dysfunction in a community population of diabetic patients. Diabetic med 15, 148–150 (a journal of the British Diabetic Association). Stratton, I.M., Adler, A.I., Neil, H.A., Matthews, D.R., Manley, S.E., Cull, C.A., Hadden, D., Turner, R.C., Holman, R.R., 2000. Association of glycaemia with macrovascular and microvascular complications of type 2 diabetes (UKPDS 35): prospective observational study. BMJ 321, 405–412 (Clinical research ed.). Szewczak, J.M., Powell, F.L., 2003. Open-flow plethysmography with pressure-decay compensation. Respir. Physiol. Neurobiol. 134, 57–67. Weltman, N.Y., Ojamaa, K., Schlenker, E.H., Chen, Y.F., Zucchi, R., Saba, A., Colligiani, D., Rajagopalan, V., Pol, C.J., Gerdes, A.M., 2014. Low-dose t3 replacement restores depressed cardiac t3 levels, preserves coronary microvasculature and attenuates cardiac dysfunction in experimental diabetes mellitus. Mol. Med. 20, 302–312, http://dx.doi.org/10.2119/molmed.2013.00040. Wilson, W.R., Bedell, G.N., 1960. The pulmonary abnormalities in myxedema. J. Clin. Invest. 39, 42–55. Yamazaki, H., Okazaki, M., Takeda, R., Haji, A., 2002. Hypercapnic and hypoxic ventilatory responses in long-term streptozotocin-diabetic rats during conscious and pentobarbital-induced anesthetic states. Life Sci. 72, 79–89. Zwillich, C.W., Pierson, D.J., Hofeldt, F.D., Lufkin, E.G., Weil, J.V., 1975. Ventilatory control in myxedema and hypothyroidism. N. Engl. J. Med. 292, 662–665.
Contents lists available at ScienceDirect
Respiratory Physiology & Neurobiology journal homepage: www.elsevier.com/locate/resphysiol
T3 supplementation affects ventilatory timing & glucose levels in type 2 diabetes mellitus model Stephen S. Bollinger a , Nathen Y. Weltman a , A. Martin Gerdes b , Evelyn H. Schlenker a,∗ a b
Division of Basic Biomedical Sciences, Sanford School of Medicine, University of South Dakota, Vermillion, SD, USA Department of Biomedical Sciences, New York Institute of Technology-College of Osteopathic Medicine, Old Westbury, NY, USA
a r t i c l e
i n f o
Article history: Accepted 29 October 2014 Available online 5 November 2014 Keywords: Type 2 diabetes mellitus Triiodothyronine Ventilation Blood glucose
a b s t r a c t Type II diabetes mellitus (T2DM) can affect ventilation, metabolism, and fasting blood glucose levels. Hypothyroidism may be a comorbidity of T2DM. In this study T2DM was induced in 20 female Sprague Dawley rats using Streptozotocin (STZ) and Nicotinamide (N). One of experimental STZ/N groups (N = 10 per group) was treated with a low dose of triiodothyronine (T3 ). Blood glucose levels, metabolism and ventilation (in air and in response to hypoxia) were measured in the 3 groups. STZ/N-treated rats increased fasting blood glucose compared to control rats eight days and 2 months post-STZ/N injections indicating stable induction of T2DM state. Treatments had no effects on ventilation, metabolism or body weight. After one month of T3 supplementation, there were no physiological indications of hyperthyroidism, but T3 supplementation altered ventilatory timing and decreased blood glucose levels compared to STZ/N rats. These results suggest that low levels of T3 supplementation could offer modest effects on blood glucose and ventilatory timing in this T2M model. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Type 2 diabetes mellitus (T2DM) is a prevalent endocrine disorder that affects approximately 9.3% of the general population and 26.9% of individuals aged 65 and older in the United States (Prevention, 2011, 2014). Although T2DM primarily manifests in the middle-aged and elderly, it is becoming more common in children and adolescents (Adebayo and Willis, 2014). Complications associated with T2DM are frequent and increase morbidity and mortality significantly (Gregg et al., 2014). Hypothyroidism is the second most prevalent pathological endocrine deficiency behind diabetes mellitus in the United States (Bope and Kellerman, 2012). The incidence of overt hypothyroidism is approximately 2–5% in the general population. Hypothyroidism is a frequent comorbidity in T2DM. For example, in a study of 386 Brazilian patients with Type 1 Diabetes Mellitus (T1DM) or T2DM, 13.1% of T2DM patients (aged 60.7 ± 10.6) also exhibited thyroid dysfunction (TD) (Palma et al., 2013). In a study of 1092 T2DM patients (aged 65.53 ± 11.77) attending an outpatient clinic in Greece, Papazafiropoulou et al. (2010) found that 12.3% of patients displayed TD. Radaideh et al. (2004) found rates of 12.5% for TD in a group of 908 T2DM patients in Jordan. The prevalence of TD
∗ Corresponding author. Tel.: +1 605 677 5160; fax: +1 605 677 6381. E-mail address: [email protected] (E.H. Schlenker). http://dx.doi.org/10.1016/j.resp.2014.10.020 1569-9048/© 2014 Elsevier B.V. All rights reserved.
observed in the abovementioned studies falls within the median range of previous studies (2–17%, Feely and Isles, 1979; Gray et al., 1980; Perros et al., 1995; Smithson, 1998) (Feely and Isles, 1979; Gray et al., 1980; Perros et al., 1995; Smithson, 1998). A study by Celani et al. (1994) of 159 female and 131 male T2DM patients (aged 60.6 ± 11.9 years), who had been hospitalized due to poor diabetic control or had recent onset diabetes, found thyroid disease rates of 31.4%. Although T2DM is known to affect the respiratory system, this area has not been well studied. For example, in 16 patients (aged 63 ± 8 years) with T2DM, Paredi et al. (1999) found a negative correlation between pulmonary function and glycemic control. In a study of 25 patients (aged 56.9 ± 7.9 years) with T2DM, Guvener et al. (2003) found that diabetics had a decreased alveolar gas exchange capacity when compared to healthy controls. The ratio of carbon monoxide diffusion (DLCO ) and alveolar ventilation (VA) was significantly lower in T2DM patients. This decline in pulmonary gas exchange also significantly correlated with increased age and duration of T2DM. The adverse effects of hypothyroidism on the respiratory system and ventilatory control have been recently reviewed (Schlenker, 2012). These include decreased ventilatory responses to hypercapnia, diminished maximal breathing capacity, and decreased DLCO (Wilson and Bedell, 1960; Zwillich et al., 1975). These findings were more frequently observed in female patients and patients with high thyroid stimulating hormone levels (Ladenson et al.,
S.S. Bollinger et al. / Respiratory Physiology & Neurobiology 205 (2015) 92–98
1988). In each of these studies, ventilatory responses to hypoxia and hypercapnia were restored to normal levels after T3 or T4 supplementation. According to Dempsey et al. (2011), factors that may affect regulation of ventilation in myxedema include depressed central nervous system activity, weakened respiratory muscles, change in some lung volume subdivisions, abnormal contractile properties of the upper airway musculature, abnormalities in pulmonary gas exchange, and obesity; which can lead to alveolar hypoventilation and CO2 retention. Interestingly, the role of hypothyroidism in T2DM on ventilation is not well documented. Thus, the purpose of the present study is to utilize combining Streptozotocin (STZ) and Nicotinamide (N) to induce T2DM in a rat model (STZ/N). The addition of N has been shown to preserve beta-cell levels in the pancreas to approximately 40% of normal when compared with STZ administration alone (Masiello et al., 1998). These treatments increase blood glucose and glycosylated hemoglobin levels (Kumar et al., 2012). Ventilation and metabolism has not been investigated in the STZ/N model of T2DM. Since hypothyroidism commonly occurs in the setting of T2DM, we investigated the relationship between thyroid supplementation on ventilation and metabolism in STZ/N treated animals. An intricate relationship exists between thyroid function and glycemic control. Therefore, the comorbid hypothyroid condition in T2DM may contribute to hyperglycemia in T2DM. Consequently, we expected that T3 supplementation would help regulate blood glucose of STZ/N rats. A recent paper used this approach to study effects of the STZ/N model and T3 supplementation on cardiovascular function and coronary vasculature (Weltman et al., 2014). 2. Methods 2.1. Animals and treatments All experiments were approved by The University of South Dakota Institutional Animal Care and Use Committee. Age matched, female Sprague Dawley rats (Harlan, Indianapolis, IN, USA) were used in these experiments. All animals were kept on a 12-h light/dark cycle and food and water were provided ad libitum. Rats were randomized into untreated control (n = 10) or STZ/N treatment (n = 20). Rats in the STZ/N groups received intraperitoneal injections of the N (200 mg/kg) dissolved in sterile saline 15 min prior to an intraperitoneal injection of STZ (65 mg/kg) dissolved in sterile citrate dextrose solution (pH 4.5) using established methods (Masiello et al., 1998). One month after STZ/N treatment, SD rats treated with STZ/N were further divided into T3 treated (STZ/N/T3 ; n = 10) and non-T3 treated (STZ/N; n = 10) groups. T3 was administered non-invasively from a stock solution (40 g/ml T3 , 50% ethanol, 48.5% glycerol) dissolved in drinking water (final concentration in water: 0.03 g/ml T3 ). Baseline fasting blood glucose levels were measured prior to STZ/N, eight days post-STZ/N injections, and after one month of T3 supplementation by tail vein sampling using an Ascensia Contour BG meter (Bayer, Pittsburgh, PA). Body weight and water intake were monitored in all animals. Experimental procedures commenced one month after the T3 supplementation was initiated. 2.2. Physiological measurements During the first two weeks after induction of T2DM, the rats were acclimated to the animal facility. The rats were handled individually in the lab environment for about 5 min. After acclimatizing, the rats were placed in a cylindrical Plexiglas chamber (22 cm long and 15.5 cm in diameter). The rats were then allowed time
93
to acclimate to the chamber until ventilatory patterns stabilized. Ventilation was then evaluated for each rat during exposure to air. To measure ventilatory parameters we used, a cylindrical Plexiglas chamber (22 cm long and 15.5 cm in diameter) contained ports to measure the gas flow rate through the chamber (Gilmont rotameter), O2 and CO2 levels (Vacuumed O2 and CO2 analyzer2), and chamber temperature (Digitec digital thermometer). A Statham pressure transducer measured pressure changes associated with breathing to determine tidal volume (VT ), inspiratory (TI ) and expiratory (TE ) times, breathing frequency (F), and minute ventilation (V˙ E ), the product of breathing frequency and tidal volume when the chamber was closed. Calibration of the chamber was accomplished by injecting 0.4 ml of air into the closed chamber. A small leak stabilized the pressure recordings, but acted like flow box (Szewczak and Powell, 2003). Thus, we can only consider “relative” VT and V˙ E values that were normalized by body weight (1000/body weight). Oxygen and CO2 levels of gas entering and leaving the chamber as well as flow rate were used to measure oxygen consumption (V˙ O2 ) and CO2 production (V˙ CO2 ). Values were corrected to STPD values. After the rat was removed from the chamber, its rectal temperature was measured using a thermometer and thermocouple (Model BAT-12 Sensortec) and body weight before returning the rat to her home cage. After one month of treatment with T3 , physiological parameters were measured for all rats. First, the rats were allowed 5 min to acclimate to the laboratory environment. Each rat was then placed into the plethysmographic chamber and allowed to acclimate until she exhibited uniform breathing patterns. The metabolism and ventilation for rats was first measured during exposure to air. After recording these measurements, the rats were then exposed to hypoxic air containing 10% O2 in nitrogen for 5 min and ventilatory measurements were taken the last minute while the rats were still exposed to hypoxic air inside the chamber. After these measurements were conducted, the chamber was flushed out with air for 10 min prior to post-hypoxia respiratory measurements. Subsequently, rectal body temperature and body weight were measured before returning the rats to their cages. Following the ventilatory and metabolic measurements, fasting blood glucose obtained from tail vein blood was again measured using an Ascensia Contour BG meter (Bayer, Pittsburgh, PA). 2.3. Statistical analysis Ventilatory parameters, body temperature, body weight, metabolic values and baseline fasting blood glucose values are presented as means and standard deviation (SD). Inferential statistics included one-way analysis of variance and post hoc Holms tests for measured and calculated physiological parameters. Since fasting blood glucose levels were not normally distributed for the STZ/N and STZ/N/T3 groups after one month of treatment, this variable was analyzed using a Kruskal–Wallis test and post-hoc MannWhitney tests. Correlation between inspiratory times and blood glucose levels was used to determine associations between TI and blood glucose levels. Significance was accepted at P < 0.05. Data were analyzed using GraphPad version 6.0. 3. Results Prior to STZ/N administration, body weights and resting glucose levels were similar among the three groups (Weltman et al., 2014). Mean body weights were 249 ± 10.5 g, 246 ± 24.2 g, and 245 ± 13.2 g, for the control, STZ/N, and STZ/N/T3 , respectively. Mean fasting blood glucose levels were 101.0 ± 16.9 mg/dl, 103.7 ± 11.6 mg/dl, and 97.0 ± 12.9 mg/dl for control, STZ/N, and STZ/N/T3 , respectively. Free T3 , total T3 , and total T4 showed
94
S.S. Bollinger et al. / Respiratory Physiology & Neurobiology 205 (2015) 92–98
Table 1 Body temperature (BT), body weight (BW), oxygen consumption (V˙ O2 ), CO2 production (V˙ CO2 ) and blood glucose levels in the three groups.
Table 2 Ventilatory Parameters of Control, STZ/N and STZ/N-T3 treated rats during exposure to baseline air, hypoxia and air following hypoxia (post-hypoxia).
Variables
Control
STZ/N
STZ/N/T3
Variable
Control
STZ/N
STZ/N/T3
BT in ◦ C BW in grams V˙ O2 (ml/min/1000 × BW) V˙ CO2 (ml/min/1000 × BW) Blood glucose (mg/dl)
38.1 (0.2) 263.8 (4.3) 16.0 (1.2) 11.2 (1) 117.5 (15.5)
38.3 (0.1) 258.5 (11.1) 17.2 (1.8) 12.3 (1.4) 399 (75.9)*
38.3 (0.1) 254.9 (5.8) 16.7 (1.1) 11.9 (0.7) 262.9 (68.9)*†
V˙ E air F VT V˙ E hypoxia F VT V˙ E post hypoxia F VT
139.1 (5.5) 87 (3) 1.6 (0.06) 237.2 (10.8) 123 (4) 1.93 (0.08) 124.8 (4.5) 84 (3) 1.49 (0.05)
147.8 (7.9) 87 (2) 1.7 (0.07) 265.3 (13.4) 119 (4) 2.24 (0.12) 122.1 (5.2) 80 (2) 1.53 (0.07)
141.2 (10.6) 89 (6) 1.6 (0.07) 250.5 (18.6) 120 (5) 2.08 (0.12) 124.3 (5.7) 87 (3) 1.43 (0.03)
Values are means and (SD) for each variable. There were 10 animals per group. The asterisk denotes differences compared to control rats and dagger difference between STZ/N and the STZ/N/T3 .
that all rats used in the study were initially euthyroid (Weltman et al., 2014). Fasting blood glucose measurements eight days after STZ/N injection were 116.7 ± 19.6 mg/dl in the control group and, 253.8 ± 162.4 mg/dl in the STZ/N group indicating successful elevation of fasting blood glucose by the STZ/N treatment. After one month of T3 treatment, there were no differences in body temperature, body weight, oxygen consumption, or CO2 production among groups (Table 1). In contrast, there were significant differences in blood glucose levels (P < 0.01) with the STZ/N group having higher levels than the control or STZ/N/T3 groups. However, the latter group’s values were significantly greater than that of the control group. After 30 days of T3 supplementation, ventilatory measurements were determined in the 3 groups of rats exposed to air, hypoxia, and post-hypoxia. Representative waveforms from one animal in each of the 3 groups are shown in Fig. 1. During exposure to baseline air, there were no differences in F, relative VT , or relative V˙ E among the three groups (Table 2). However, TI was significantly larger in the STZ/N/T3 group compared to
Values are means and SD (in parentheses) of 10 animals per group. Ventilation (V˙ E ) is in ml × (1000/BW)/min. frequency (F) is in breaths per minute and tidal volume (VT ) is in ml × (1000/BW).
control and STZ/N (P = 0.023) and showed greater variability (Fig. 2). Although TE was not significantly different among the three groups, the variability in the STZ/N/T3 group was greater than that of the other groups (Fig. 2). This also contributed to a significant increase in TI /Ttot in STZ/N/T3 treated group relative to the control (Fig. 2), but a decrease of VT /TI in the STZ/N/T3 treated group relative to that in the two other groups (Fig. 2). During exposure to hypoxia, ventilation increased in all groups to a similar extent due to an increase in relative tidal volume and frequency (Table 2). There were no differences in TI or TE values among the three groups (data not shown). Following exposure to hypoxia, there was no difference in minute ventilation among the three groups while exposed to room air (Table 2). However, TI was again significantly higher (P = 0.0008) in the STZ/N/T3 group (Fig. 3). TE was lower in the STZ/N/T3 group compared to the STZ/N group (P = 0.015), but not different from
Fig. 1. Representative waveforms in one rat from each of these groups: control, STZ/N and STZ/N/T3 . Each rat was exposed to air, hypoxia and then air following hypoxia (post hypoxia).
S.S. Bollinger et al. / Respiratory Physiology & Neurobiology 205 (2015) 92–98
*
T I a ir
T E a ir
0 .8
95
0 .3
#
T I (s e c )
0 .4
0 .2
0 .1
0 .2
3 Z T S
3 /T /N Z
tr n o
S
C
Z T
T
o
l
3
0
/T /N
Z T S
S
C
o
n
tr
o
l
/N
0 .0
#
5
/N
0 .1
10
Z
0 .2
*
T
0 .3
V T /T I a ir
15
S
0 .4
V T /T I ( ( m l* 1 0 0 0 /B W ) /s e c )
S
*
T I/T T o t a ir
0 .5
/T /N
Z T
n o
T
c
Z
S
T
/N
tr
/T
o
l
3
/N Z
l o tr n o c
/N
0 .0
0 .0
S
T E (s e c )
0 .6
Fig. 2. Inspiratory time (TI) expiratory time (TE), time spent during inspiration relative to the total time of a breath (TI/Tot) and inspiratory flow rate (VT /TI ) of Control, STZ/N and STZ/N/T3 during exposure to air prior to hypoxia. There are 10 rats per group. Data are presented individually with group means and SD indicated by the horizontal lines. The asterisk denotes significant differences between the STZ/N/T3 group and the control group and the # denotes significant differences between the STZ/N/T3 group and the STZ/N group.
the control group. Subsequently, TI /tot was greater in the STZ/N/T3 group compared to the two other groups (P < 0.001, Fig. 3) and VT /TI was lower in the STZ/N/T3 group (P < 0.05). Thus, T3 treatment appears to effect ventilatory timing, but not on other ventilatory parameters during air exposures. To determine if TI and blood glucose were related and impacted by T3 treatment, correlation analysis was conducted separately in the control, STZ/N and the STZ/N/T3 treated groups. There was no significant correlation between blood glucose levels and TI for the control or STZ/N groups. However, there was a significant association between blood glucose levels and TI in the STZ/N/T3 group (r = 0.864, P = 0.0013). Thus, T3 supplementation affects the relationship between TI and glucose levels.
4. Discussion To our knowledge, the present findings represent the first study to examine the effects of T3 supplementation on ventilatory and metabolic parameters in T2DM using the STZ/N model. We evaluated parameters of metabolism, ventilation and blood glucose levels in a female rat model for T2DM 60 days after STZ/N injections and after 30 days of T3 hormone supplementation. No significant changes were found between the STZ/N and control groups for ventilatory parameters during exposure to air or hypoxia, V˙ O2 , or V˙ CO2 . This suggests that the short-term induction of T2DM with STZ and pretreatment with N did not adversely affect metabolic or ventilatory function in this study. Interestingly, supplementation of STZ/N-treated rats with T3 altered inspiratory time during exposure to air, but not during exposure to hypoxia. Moreover,
T3 supplementation decreased blood glucose values relative to treatment with STZ/N alone. Finally there was a significant association between blood glucose levels and TI only in the STZ/N/T3 group. 4.1. Ventilatory and metabolic results in diabetic models The ventilatory and metabolic results in the STZ/N model reported in this study contrast with previous studies of diabetic rodents (Ramadan et al., 2006; Saiki et al., 2005). We have previously explored parameters of altered control of ventilation in a STZ rat model of diabetes mellitus. In this four-week study of male SD rats treated with STZ, we observed a progressive reduction of VT , V˙ E , and V˙ CO2 compared to the control group and a STZ diabetic group treated with insulin (STZ/I) (Hein et al., 1994). Hypercapnic (3, 6, 9%) and hypoxic (10%) ventilatory responses were also significantly less in the STZ group than the control group and the STZ/I group. Ventilation in air and ventilatory responses following exposures to hypercapnia and hypoxia were similar for the control and STZ/I group indicating that the insulin therapy ameliorated the effects of the STZ on ventilation. Saiki et al. (2005) studied metabolism and respiratory responses to hypoxia in induced diabetic male Wistar rats. In their study, the researchers took respiratory measurements five weeks after STZ injection. During exposure to air and hypoxia, the STZ group did not show significant differences in V˙ E from the control group; however the V˙ CO2 levels in the STZ group were significantly increased in comparison to the control group. Subsequent shortterm administration of insulin increased V˙ E during exposure to hypoxia in comparison to the control group due to an increase
96
S.S. Bollinger et al. / Respiratory Physiology & Neurobiology 205 (2015) 92–98
T I p o s t h y p o x ia
T E p o s t h y p o x ia
0 .8
0 .6
0 .4
0 .2
0 .1
0 .2
0 .0
3 /T /N Z
tr n o
S
C
Z T S
T
o
l
3
0
/T /N
Z T S
n
tr
o
l
/N
0 .0
/N
0 .1
5
Z
0 .2
*
10
T
0 .3
V T /T i p o s t h y p o x ia 15
S
#
V T /T i ( m l* 1 0 0 0 /B W /s e c )
*
0 .4
o
/T /N Z T S
T S
C
T I/ T t o t p o s t h y p o x i a 0 .5
C
3
/N Z S
o C
Z
T
n
/T /N
T S
to
3
/N Z
l o tr n o
l
0 .0
T i/T to t
*
0 .3
T I (s e c )
T E (s e c )
#
Fig. 3. Inspiratory time (TI ) expiratory time (TE ), time spent during inspiration relative to the total time of a breath (TI /Tot ) and inspiratory flow rate (VT /TI ) of Control, STZ/N and STZ/N/T3 rats during exposure to air following exposure to hypoxia. There are 10 rats per group. Data are presented individually with group means and SD indicated by the horizontal lines. The asterisk denotes significant differences between the STZ/N/T3 group and the control group and the # denotes significant differences between the STZ/N/T3 group and the STZ/N group.
in VT . The authors speculated that, independent of blood glucose level, the intrinsic hormonal effects of insulin might be important for the respiratory control mechanisms. Yamazaki et al. (2002) evaluated hypercapnic and hypoxic ventilatory responses in long-term STZ (60 mg/kg in saline) induced diabetic male Sprague-Dawley rats. The study consisted of twenty-six rats split into control, STZ, and STZ/I groups. Basic ventilatory parameters such as respiratory rate, VT , and V˙ E were not significantly altered for the STZ group in comparison to the control group, but ventilatory responses to hypercapnia and hypoxia were reduced significantly starting sixteen weeks after STZ injections and lasted until the end of the twenty-eight week study. Respiratory rate, VT , and V˙ E decreased with age in all experimental groups. Ventilation for all groups increased linearly with increasing concentrations of CO2 in air, but the average slope value of the regression line was lower for the STZ group in compared to that of the control group. As the study progressed and the rats aged, the average slope value of this regression line increased with aging for the control group but remained at a lower level for the STZ group. Hypoxic ventilation measurements increased linearly with decreasing concentrations of O2 in air. Moreover, the average slope value of the regression line for hypoxic ventilation increased with aging for the control group but remained lower for the STZ group. These reduced responses were also seen for the STZ/I group, indicating that daily insulin treatment was not sufficient to mediate these ventilatory changes. This contradicts results seen in the shortterm studies by Hein et al. (1994) and Saiki et al. (2005) where insulin supplementation was sufficient to counteract reduced
ventilatory responses. The researchers therefore concluded that chronic diabetic condition might impair the central and peripheral chemosensitivity, but not the bulbar respiratory network responsible for the regulation of the basic respiratory rhythm. Finally, a study by Polotsky et al. (2001) explored the effects of T1DM induction on control of ventilation for two strains of mice. In this study, researchers used two models of T1DM: (1) STZ-induced diabetes (200 mg/kg) in fifty-nine C57BL/6J male mice on a regular diet or with induced obesity from a high fat diet; and (2) spontaneous T1DM in ten NOD-Ltj female mice. In both of these models, T1DM resulted in depression of hypercapnic ventilatory responses that was associated with duration of hyperglycemia and increased glycosylation of the diaphragm. For the STZ group, V˙ E and hypercapnic ventilatory responses significantly declined seven days after STZ injection; however they found no significant differences between the STZ and control group for V˙ O2 , V˙ CO2 , or the respiratory exchange ratio. In the present study, the relationship between TI (and other ventilatory variables) and blood glucose levels was not significant, suggesting that in the STZ/N model 8 weeks of hyperglycemia alone did not affect control of ventilation. 4.2. Thyroid hormone effects on blood glucose and TI Work by Stratton et al. (2000) does suggest that modest reductions in hyperglycemia has the potential to prevent death from complications related to diabetes. Two months after STZ/N injections and after one-month of T3 supplementation, fasting blood glucose levels for the STZ/N and STZ/N/T3 groups continued
S.S. Bollinger et al. / Respiratory Physiology & Neurobiology 205 (2015) 92–98
to be significantly higher than control, but the latter group’s values were lower than those of the STZ/N group. This suggests that T3 supplementation may either ameliorate hyperglycemia or slow the progression of hyperglycemia. Exact mechanisms for this interaction are currently unknown. This observation correlates with suggestions made by Fernandez-Real et al. (2006) that thyroid function is intrinsically related to variables responsible for insulin resistance. The interaction of thyroid hormone supplementation on fasting blood glucose levels could therefore also serve to prevent the manifestation in vascular complications in T2DM due to chronic and severe hyperglycemia. In this study, we treated rats with T3 instead of T4 , as it is more readily available to be utilized by tissues throughout the body. According to Manjunath (2012), T4 is a precursor to T3 and deiodinases in tissues such as the brain and pituitary gland can convert T4 to T3 . Importantly, T3 is bound to thyroid hormone receptors with 10–15 times greater affinity than T4 leading to increases in hormonal potency. By using the more readily available form of thyroid hormone (T3 ), we increased the likelihood that T3 supplementation would stimulate thyroid receptors in tissues having low thyroid hormone levels. A novel finding in the present study was that T3 supplementation increased TI both before and following hypoxia. The significant correlation between TI and blood glucose levels only found in the STZ/N/T3 group suggests that T3 may have affected this relationship. Thyroid hormone treatment has been reported to augment ventilation as seen in the dystrophic hamsters who display a euthyroid sick syndrome (low T3 , normal T4 levels) as well as in SHHF rats (Schlenker and Burbach, 1995; Schlenker et al., 2003). Moreover, Weltman et al. (2014) recently also showed that T3 supplementation improved cardiac function, the number of cardiac arterioles and reversed the fetal gene expression in the heart in STZ/N female rats. Thus, the present study indicates that this level of T3 supplementation may have subtle effects on blood glucose levels as well as on ventilatory timing. 4.3. Limitations of the study The present study has several limitations. The first limitation was that our plethysmograph acted like a flow box. Thus, our values for tidal volume and minute ventilation can only be considered relative, not absolute and are considerably lower than those previously reported in rats (Fournier et al., 2012). In addition, the duration of the present study was relatively short. The consequences of thyroid hormone supplementation in the STZ/N model and effects of T3 supplementation for a longer time period needs to be determined. However, in the Weltman et al. (2014) paper, serum T3 levels were measured in animals who had been treated for a longer period with STZ/N and STZ/N/T3 and they reported that T3 levels were not different among the three groups, whereas TSH levels were markedly increased in the STZ/N group, compared to the control and STZ/N/T3 groups (Weltman et al., 2014). Importantly T3 levels in cardiac tissues in the study by Weltman et al. (2014) were decreased in the STZ/N group relative to control and the STZ/N/T3 groups. Thus, in the present study measurement of T3 serum levels may not have shown differences among the groups. Another limitation of this study is that we only used female Sprague-Dawley rats. Further limitations of this study involve the extent and mechanism by which thyroid hormone supplementation can affect blood glucose levels and inspiratory timing. Future studies should seek to identify specific impacts of thyroid hormone levels on underlying mechanisms affecting blood glucose levels both in short-term and long-term settings. In regard to the possible translation applicability of T3 supplementation, patients would need to receive oral low doses of T3 rather than the drinking water administration used in the present study.
97
4.4. Overall conclusions In this study we utilized the STZ/N model to successfully induce a T2DM state in female Sprague-Dawley rats. No effects were noted on ventilation, metabolism or body weight. After one month of T3 supplementation there were no physiological indications that this low dose of T3 induced hyperthyroidism. T3 supplementation altered ventilatory timing and decreased blood glucose levels compared to STZ/N treated rats. These results suggest that low levels of T3 supplementation could offer modest effects on blood glucose levels and ventilatory timing in this T2M model. Further research is necessary to establish the effects of the STZ/N model on metabolism, ventilation, and blood glucose in both short-term and long-term models, as well as how thyroid hormone supplementation can affect these manifestations by acting on areas of the brain involved in regulation of breathing. Acknowledgements This project was supported by Grant numbers RO1HL09316001A1 and RO1HL103671 (AMG) from the National Heart, Lung, and Blood Institute. This research was also supported by an American Diabetes Association (ADA) Clinical Scientist Training Award 7-10CST-01 (NYW & AMG) and Center for Undergraduate Grant (SSB). References Adebayo, O., Willis, G.C., 2014. The changing face of diabetes in America. Emerg. Med. Clin. North Am. 32, 319–327. Bope, E., Kellerman, R., 2012. Conn’s Current Therapy 2012. Saunders Elsevier 2012, Philadelphia, PA, USA. Celani, M.F., Bonati, M.E., Stucci, N., 1994. Prevalence of abnormal thyrotropin concentrations measured by a sensitive assay in patients with type 2 diabetes mellitus. Diabetes Res. (Edinburgh, Scotland) 27, 15–25. Dempsey, J.A., Olson, E.B., Skatrud, J.B., 2011. Hormones and Neurochemicals in the Regulation of Breathing, Comprehensive Physiology. John Wiley & Sons, Inc. Feely, J., Isles, T.E., 1979. Screening for thyroid dysfunction in diabetics. Br. Med. J. 1, 1678. Fernandez-Real, J.M., Lopez-Bermejo, A., Castro, A., Casamitjana, R., Ricart, W., 2006. Thyroid function is intrinsically linked to insulin sensitivity and endotheliumdependent vasodilation in healthy euthyroid subjects. J. Clin. Endocrinol. Metab. 91, 3337–3343. Fournier, S., Kinkead, R., Joseph, V., 2012. Influence of housing conditions from weaning to adulthood on the ventilatory, thermoregulatory, and endocrine responses to hypoxia of adult female rats. J. Appl. Physiol. 112, 1474–1481. Gray, R.S., Borsey, D.Q., Seth, J., Herd, R., Brown, N.S., Clarke, B.F., 1980. Prevalence of subclinical thyroid failure in insulin-dependent diabetes. J. Clin. Endocrin. Metab. 50, 1034–1037. Gregg, E.W., Williams, D.E., Geiss, L., 2014. Changes in diabetes-related complications in the United States. N. Engl. J. Med. 371, 286–287. Guvener, N., Tutuncu, N.B., Akcay, S., Eyuboglu, F., Gokcel, A., 2003. Alveolar gas exchange in patients with type 2 diabetes mellitus. Endocr. J. 50, 663–667. Hein, M.S., Schlenker, E.H., Patel, K.P., 1994. Altered control of ventilation in streptozotocin-induced diabetic rats. Proc. Soc. Exp. Biol. Med. 207, 213–219. Kumar, S., Sharma, S., Vasudeva, N., Ranga, V., 2012. In vivo anti-hyperglycemic and antioxidant potentials of ethanolic extract from Tecomella undulata. Diabetol. Metab. Syndr. 4, 33. Ladenson, P.W., Goldenheim, P.D., Ridgway, E.C., 1988. Prediction and reversal of blunted ventilatory responsiveness in patients with hypothyroidism. Am. J. Med. 84, 877–883. Manjunath, S., 2012. Clinical and Metabolic Study of Subclinical Thyroid Disorders in Type 2 Diabetes, General Medicine. Rajiv Gandhi University of Health Sciences, Bangalore, India, pp. 141. Masiello, P., Broca, C., Gross, R., Roye, M., Manteghetti, M., Hillaire-Buys, D., Novelli, M., Ribes, G., 1998. Experimental NIDDM: development of a new model in adult rats administered streptozotocin and nicotinamide. Diabetes 47, 224–229. Palma, C.C.S.S.V., Pavesi, M., Nogueira, V., Clemente, E.L., Vasconcellos, M.d.F.B.M.P., Pereira, L., Pacheco, F., Braga, T., Bello, L., Soares, J., dos Santos, S.C., Campos, V.P.L.C., Gomes, M., 2013. Prevalence of thyroid dysfunction in patients with diabetes mellitus. Diabetol. Metab. Syndr. 5, 58. Papazafiropoulou, A., Sotiropoulos, A., Kokolaki, A., Kardara, M., Stamataki, P., Pappas, S., 2010. Prevalence of thyroid dysfunction among Greek type 2 diabetic patients attending an outpatient clinic. J. Clin. Med. Res. 2, 75–78. Paredi, P., Biernacki, W., Invernizzi, G., Kharitonov, S.A., Barnes, P.J., 1999. Exhaled carbon monoxide levels elevated in diabetes and correlated with glucose
98
S.S. Bollinger et al. / Respiratory Physiology & Neurobiology 205 (2015) 92–98
concentration in blood: a new test for monitoring the disease? Chest 116, 1007–1011. Perros, P., McCrimmon, R.J., Shaw, G., Frier, B.M., 1995. Frequency of thyroid dysfunction in diabetic patients: value of annual screening. Diabet. Med. 12, 622–627. Polotsky, V.Y., Wilson, J.A., Haines, A.S., Scharf, M.T., Soutiere, S.E., Tankersley, C.G., Smith, P.L., Schwartz, A.R., O’Donnell, C.P., 2001. The impact of insulindependent diabetes on ventilatory control in the mouse. Am. J. Respir. Crit. Care Med. 163, 624–632. Prevention, C.f.D.C.a, 2011. National Diabetes Fact Sheet: National Estimates and General Information on Diabetes and Prediabetes in the United States. U.S. Department of Health and Human Services, Centers for Disease Control and Prevention, Atlanta, GA. Prevention, C.f.D.C.a., 2014. National Diabetes Statistics Report: Estimates of Diabetes and Its Burden in the United States. US Department of Health and Human Services, Atlanta, GA. Radaideh, A.R., Nusier, M.K., Amari, F.L., Bateiha, A.E., El-Khateeb, M.S., Naser, A.S., Ajlouni, K.M., 2004. Thyroid dysfunction in patients with type 2 diabetes mellitus in Jordan. Saudi Med. J. 25, 1046–1050. Ramadan, W., Petitjean, M., Loos, N., Geloen, A., Vardon, G., Delanaud, S., Gros, F., Dewasmes, G., 2006. Effect of high-fat diet and metformin treatment on ventilation and sleep apnea in non-obese rats. Respir. Physiol. Neurobiol. 150, 52–65. Saiki, C., Seki, N., Furuya, H., Matsumoto, S., 2005. The acute effects of insulin on the cardiorespiratory responses to hypoxia in streptozotocin-induced diabetic rats. Acta Physiol. Scand. 183, 107–115. Schlenker, E.H., 2012. Effects of hypothyroidism on the respiratory system and control of breathing: Human studies and animal models. Respir. Physiol. Neurobiol. 181, 123–131.
Schlenker, E., Burbach, J., 1995. Thyroxine affects ventilation, lung morphometry, and necrosis of diaphragm in dystrophic hamsters. Am. J. Physiol. 268, R779–R785. Schlenker, E.H., Tamura, T., Gerdes, A.M., 2003. Gender-specific effects of thyroid hormones on cardiopulmonary function in SHHF rats. J. Appl. Physiol. 95, 2292–2298. Smithson, M.J., 1998. Screening for thyroid dysfunction in a community population of diabetic patients. Diabetic med 15, 148–150 (a journal of the British Diabetic Association). Stratton, I.M., Adler, A.I., Neil, H.A., Matthews, D.R., Manley, S.E., Cull, C.A., Hadden, D., Turner, R.C., Holman, R.R., 2000. Association of glycaemia with macrovascular and microvascular complications of type 2 diabetes (UKPDS 35): prospective observational study. BMJ 321, 405–412 (Clinical research ed.). Szewczak, J.M., Powell, F.L., 2003. Open-flow plethysmography with pressure-decay compensation. Respir. Physiol. Neurobiol. 134, 57–67. Weltman, N.Y., Ojamaa, K., Schlenker, E.H., Chen, Y.F., Zucchi, R., Saba, A., Colligiani, D., Rajagopalan, V., Pol, C.J., Gerdes, A.M., 2014. Low-dose t3 replacement restores depressed cardiac t3 levels, preserves coronary microvasculature and attenuates cardiac dysfunction in experimental diabetes mellitus. Mol. Med. 20, 302–312, http://dx.doi.org/10.2119/molmed.2013.00040. Wilson, W.R., Bedell, G.N., 1960. The pulmonary abnormalities in myxedema. J. Clin. Invest. 39, 42–55. Yamazaki, H., Okazaki, M., Takeda, R., Haji, A., 2002. Hypercapnic and hypoxic ventilatory responses in long-term streptozotocin-diabetic rats during conscious and pentobarbital-induced anesthetic states. Life Sci. 72, 79–89. Zwillich, C.W., Pierson, D.J., Hofeldt, F.D., Lufkin, E.G., Weil, J.V., 1975. Ventilatory control in myxedema and hypothyroidism. N. Engl. J. Med. 292, 662–665.
