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Abstract

Metabolic activity of brown adipose tissue (BAT) is activated by β₃-adrenoceptor agonists and norepinephrine transporter (NET) blockers and is measurable using [¹⁸F]fluorodeoxyglucose ([¹⁸F]FDG) positron emission tomography/computed tomography (PET/CT) in rats. Using the streptozotocin (STZ)-treated rat model of type 1 diabetes mellitus (T1DM), we investigated BAT activity in this rat model under fasting and nonfasting conditions using [¹⁸F]FDG PET/CT. Drugs that enhance BAT activity may have a potential for therapeutic development in lowering blood sugar in insulin-resistant diabetes. Rats were rendered diabetic by administration of STZand confirmed by glucose measures. [¹⁸F]FDG was injected in the rats (fasted or nonfasted) pretreated with either saline or β₃-adrenoceptor agonist CL316,243 or the NET blocker atomoxetine for PET/CT scans. [¹⁸F]FDG metabolic activity was computed as standard uptake values (SUVs) in interscapular brown adipose tissue (IBAT) and compared across the different drug treatment conditions. Blood glucose levels > 500 mg/dL were established for the STZ-treated diabetic rats. Under fasting conditions, average uptake of [¹⁸F]FDG in the IBAT of STZ-treated diabetic rats was approximately 70% lower compared to that of normal rats. Both CL316,243 and atomoxetine activated IBAT in normal rats had an SUV > 5, whereas activation in STZ-treated rats was significantly lower. The agonist CL316,243 activated IBAT up to threefold compared to saline in the fasted STZ-treated rat. In the nonfasted rat, the IBAT activation was up by twofold by CL316243. Atomoxetine had a greater effect on lowering blood sugar levels compared to CL316,243 in the nonfasted rats. A significant reduction in metabolic activity was observed in the STZ-treated diabetic rodent model. Increased IBAT activity in the STZ-treated diabetic rat under nonfasted conditions using the β₃-adrenoceptor agonist CL316,243 suggests a potential role of BAT in modulating blood sugar levels. Further studies are needed to evaluate the therapeutic role of β3-adrenoceptor agonists in insulin-resistant T1DM.

BROWN ADIPOSE TISSUE (BAT) has attracted increasing attention as a potential therapeutic organ for diabetes and obesity.¹ Activation of BAT is being explored as a potential therapeutic mechanism for obesity because BAT regulates the breakdown of lipids and glucose.² We recently showed that the Zucker obese (fa/fa) rat had a significantly lowered BAT activity compared to the Zucker lean (Fa/Fa) rat.³ This observation is consistent with the low BAT activity found in obese humans.⁴ Thus, the obese Zucker rat model may be a good model to explore therapeutic approaches for certain aspects of obesity in humans.⁵

The high blood sugar levels in type 1 diabetes mellitus (T1DM) occurs due to the loss of insulin-secreting islet cells in the pancreas. Patients managed using exogenous insulin can often encounter insulin resistance and an inability to control and manage blood sugar levels leading to several complications.⁶ Because of the thermogenic properties of BAT, there may be value to examine the role of BAT activity in managing blood sugar in T1DM. Recently, efforts have been made to transplant BAT in a T1DM mice model (treated with streptozotocin [STZ]) to gain glycemic control without insulin.⁷

Treatment with STZ decreases insulin-producing cells in pancreatic islets, resulting in elevated blood glucose levels.^8,9 This loss in pancreatic islets cells mimics the islet cell loss in humans; thus, the STZ-treated rodents are considered a good model of human T1DM.¹⁰ Metabolism in interscapular brown adipose tissue (IBAT) has been shown to be significantly reduced in STZ-treated T1DM rodents.^11,12

We and others have shown significant activation of IBAT metabolic activity using CL316,243, a β₃-adrenoceptor selective agonist, which enhances the uptake of [¹⁸F] fluorodeoxyglucose ([¹⁸F]FDG) in BAT of normal rats using positron emission tomography (PET), ex vivo autoradiographic and histologic methods,¹³ and mice,^14,15 and by atomoxetine, a norepinephrine transporter (NET) blocker.¹⁶ Thus, β₃-adrenoceptor agonists enhance [¹⁸F]FDG uptake of IBAT in vivo, signifying higher thermogenesis and a possible therapeutic role of these agonists for reducing diabetes and obesity. Since a lower IBAT activity in STZ-treated rodents has previously been reported,¹¹ the objective in this preliminary report was to evaluate the effects of CL316,243 and atomoxetine on IBAT activity in the STZ-treated Sprague Dawley rat model of T1DM.

Materials and Methods

All animal studies were approved by the Institutional Animal Health Care and Use Committee of University of California-Irvine. Healthy male Sprague Dawley rats (8 weeks old, 250 g; Charles River Laboratories, Wilmington, MA) were used for the study. For chemical destruction of pancreatic beta cells, rats (n = 4) were administered STZ at a dose of 55 mg/kg.⁹ Rats were classified as diabetic when nonfasting serum blood glucose levels rose above 350 mg/dL for 3 consecutive days. All rats (normal n = 6; diabetic [treated with STZ] n = 4) were either nonfasted or fasted for 24 hours before [¹⁸F]FDG administration for a PET/computed tomography (CT) study. Either CL316,243¹³ (Tocris Bioscience, Ellisville, MO), atomoxetine¹⁶ (Sigma-Aldrich, St. Louis, MO), or normal saline was administered 30 minutes before [¹⁸F]FDG administration to the diabetic rats. Saline-treated rats were studied first, followed by atomoxetine-treated rats and, finally, CL316,243- treated rats. There was at least a 48-hour time interval between each set of experiments under the different conditions. The animals were not randomized due to the small size of the study. After [¹⁸F]FDG (15-22 MBq) administration intravenously, the rats were awake for 60 minutes. All saline- and drug-treated rats were placed in a supine position in a rat holder, anesthetized using isoflurane, and then maintained under anesthesia for upper body PET and CT.

The Inveon PET and CT scanners (Siemens Medical Solutions, Knoxville, TN) were used in the docked mode for combined PET/CT experiments described previously.¹³ Rats were anesthetized with 4% isoflurane for induction and 2.5% maintenance during the scan. The rat holder was placed on the PET/CT bed, and all animals had a CT scan after the PET scan for attenuation correction and anatomic delineation of PET images. For quantitative PET analysis, regions of interest were drawn on respective PET images using ASI Pro and IRW (Siemens Medical Solutions, Knoxville, TN). The magnitude of BAT [¹⁸F]FDG activation was expressed as standard uptake value (SUV) calculated from [¹⁸F]FDG activity in each volume of interest (KBq/cm³), injected dose (MBq), and body weight of each animal (kg) (KBq/cm³/(MBq/kg)). Statistical differences between groups were determined using the Student t-test. A p value of < .05 was considered statistically significant.

Results

As reported previously,⁹ a single high dose of STZ was enough to cause hyperglycemia in rats and blood glucose was > 500 mg/dL compared to 125 to 150 mg/dL in age-matched nondiabetic rats. The diabetic rats maintained nonfasting blood glucose levels > 500 mg/dL during the course of this study. Fasted normal rats pretreated with CL316,243 exhibited high [¹⁸F]FDG uptake in IBAT with an average SUV of 5.81. IBAT activation by atomoxetine in the fasted rats was also high, with an average value of 5.10 compared to the modest activation (SUV of 0.93) in saline-treated normal rats (Figure 1).

Figure 1.

Uptake of [¹⁸F]FDG in fasted rats: (A) PET image of a normal fasted rat pretreated with saline, (B) PET image of a fasted STZ rat pretreated with saline, and (C) coregistered PET/CT image of a fasted STZ rat pretreated with saline showing uptake of [¹⁸F]FDG in the IBAT area (arrows). (D) PET image of a normal fasted rat pretreated with atomoxetine (ATX), (E) PET image of a fasted STZ rat pretreated with atomoxetine, and (F) coregistered PET-CT image of fasted STZ rats pretreated with atomoxetine showing uptake of [¹⁸F]FDG in the IBAT area. (G) PET image of a normal fasted rat pretreated with CL316,243 (CL) (H) PET image of a fasted STZ rat pretreated with CL316,243 and (I) coregistered PET/CT image of a fasted STZ rat pretreated with CL316,243 showing uptake of [¹⁸F]FDG in the IBAT area. (J) Graph showing a comparison in uptake of [¹⁸F]FDG under different treatment conditions in diabetics (n = 4) versus normal rats (n = 6) in a fasted state (p < .05).

Activation of IBAT in the fasted diabetic rats was remarkably reduced compared to that in the normal rats. In the saline-treated diabetic rat, a reduction of > 70% of IBAT activity was observed (SUV of 0.25 for diabetic rat versus 0.93 for normal rat). With CL316,243 pretreatment, diabetic rats exhibit only 13% of ¹⁸F-FDG IBAT activity compared to normal rats (SUV of 0.75 for diabetic rat versus 5.81 for normal rat). Atomoxetine-pretreated diabetic rats exhibited greater than 90% reduction of IBAT activity compared to normal rats (SUV of 0.19 for diabetic rat versus 5.10 for normal rat; see Figure 1). Atomoxetine pretreatment in the diabetic rats decreased blood glucose dramatically but failed to activate IBAT more than saline-pretreated rats (Table 1). On the other hand, CL316,243 showed a threefold increase in IBAT activity compared to saline-treated diabetic rats (SUV of 0.75 for CL316,243 versus 0.25 for saline; see Table 1). Interestingly, the blood sugar levels with CL316,243 pretreatment increased after the PET scan.

Table 1.

Blood Glucose and [¹⁸F]FDG Uptake in IBAT of Streptozotocin-Treated Rats^*

Drug Treatment	Fasted			Nonfasted

	Blood Glucose	SUV	[¹⁸F]FDG, % Injected Dose/cc in IBAT	Blood Glucose	SUV	[¹⁸F]FDG, % Injected Dose/cc in IBAT
Saline	108 ± 48^†	0.25 ± 0.03	0.14 ± 0.04	600 ± 1^†	0.17 ± 0.05	0.09 ± 0.03
	152 ± 74^‡			537 ± 71^‡
Atomoxetine	105 ± 18^†	0.19 ± 0.06	0.10 ± 0.03	557 ± 69^†	0.19 ± 0.05	0.09 ± 0.02
	22 ± 8^‡			418 ± 129^‡
CL316,243	129 ± 62^†	0.75 ± 0.48	0.34 ± 0.15	600^{† §}	0.30§	0.15§
	202 ± 101^‡			539^{‡ §}

[¹⁸F]FDG = [¹⁸F]fluorodeoxyglucose; IBAT = interscapular brown adipose tissue; SUV = standard uptake value.

Diabetic Sprague Dawley rats treated with streptozotocin (n = 4).

†

Blood sugar measured before administration of saline or drug.

‡

Blood sugar measured after the PET scan, approximately 2 hours after administration of saline or drug (atomoxetine or CL316,243).

Standard deviation not calculated due to fewer animals.

Nonfasted normal rats pretreated with CL316,243 had the highest [¹⁸F]FDG uptake in IBAT, with an average SUV of 9.70 as reported previously.¹³ Atomoxetine pretreatment in the nonfasted rat had a significantly blunted effect on IBAT activation, with an average SUV of 1.26 and comparable to SUV levels in the saline-pretreated rats.¹⁶ IBAT activity in the nonfasted diabetic rats was dramatically reduced and had SUVs of 0.17, 0.19, and 0.30 in rats pretreated with saline, atomoxetine, and CL316,243, respectively (see Table 1). Thus, there was no difference between saline and atomoxetine treatment, whereas CL316,243 had an approximately twofold greater activation compared to saline (SUV of 0.30 for CL316,243 versus 0.17 for saline; see Table 1 and Figure 2).

Figure 2.

Uptake of [¹⁸F]FDG in nonfasted rats: (A) PET image of a normal rat pretreated with CL316,243 (CL), (B) PET image of an STZ rat pretreated with CL316,243, and (C) coregistered PET/CT image of an STZ rat pretreated with CL316,243 showing uptake of [¹⁸F]FDG in the IBAT area (arrows). (D) Graph showing a comparison in uptake of [¹⁸F]FDG under different treatment conditions (saline, atomoxetine, and CL316,243) in diabetics (n = 4) versus normal (n = 6) in a nonfasted state (p < .05).

In the nonfasted diabetic rats, atomoxetine had a greater effect on lowering blood glucose levels (557 mg/dL before and 418 mg/dL after atomoxetine), whereas the effect of CL316,243 was small (600 mg/dL before and 539 mg/dL after CL316,243). Similar lowering of blood glucose levels by atomoxetine was also observed in the fasted rats. The large difference in blood glucose levels between the fasted and nonfasted states (see Table 1) seemed to have little effect on the [¹⁸F]FDG uptake in the saline group and the atomoxetine group. In the case of the CL316,243 group (see Table 1), the higher blood glucose levels in the nonfasted states may have had a small diluting effect on the IBAT [¹⁸F]FDG uptake, thus lowering the [¹⁸F]FDG uptake.

Discussion

Our findings of the effects of β₃-adrenoceptor agonists in the obese Zucker rats suggested a downregulation of the β₃- adrenoceptor in BAT, particularly IBAT.³ This appears to be consistent with reports that β₃-adrenoceptor levels may be reduced in the obese Zucker rat model. In an effort to understand the role of β₃-adrenoceptor activation in BAT in type 1 diabetes, we investigated the STZ-treated diabetic rat model. Our efforts here were to understand if enhanced BAT metabolic activity may affect blood sugar levels. This may have the potential for improved management of type 1 and type 2 diabetes mellitus.

Compared to our previous observations of [¹⁸F]FDG uptake in IBAT in nondiabetic Sprague Dawley rats,¹³ [¹⁸F]FDG uptake in the IBAT of STZ-treated Sprague Dawley rats was significantly reduced. IBAT metabolic activity in the diabetic animal was reduced more than 70% (see Figure 1, A and B). Metabolism in IBAT has been shown to be significantly affected in STZ-treated rodents with type 1 diabetes,^11,12 and our findings of lower [¹⁸F]FDG measures support these findings in the diabetic animal. This lowered metabolic activity in the diabetic rat may be due to anomalies in the adrenergic pathway in BAT or to mitochondrial changes.¹⁰ In the presence of the agonist CL316,243, activation of β₃-adrenoceptors in the IBAT was significant in the diabetic rat and threefold greater than saline treatment (see Figure 1, G and H, and Table 1). This is in contrast to the lower IBAT activation by CL316,243 in the Zucker fat rat, which is likely due to the lower β₃-adrenoceptor levels in the Zucker fat rat model.³ When the NET blocker atomoxetine was used, activation of IBAT was similar in the saline- and atomoxetine-treated conditions (see Figure 1, B and E), suggesting weak effects of norepinephrine or perhaps little effect of atomoxetine on the norepinephrine levels in IBAT in the diabetic rat. It has been reported that the norepinephrine turnover in the STZ-treated rats is lowered.¹⁷ Thus, in contrast to nondiabetic rats, the effects of atomoxetine in the diabetic rats were weak.

The extent to which BAT may be activated under nonfasted conditions was evaluated to understand the potential value of BAT in altering blood glucose levels (Figure 2). In saline-treated animals, IBAT activation was significantly reduced in the diabetic rat (see Figure 2D). In the presence of the agonist CL316,243, activation of β₃-adrenoceptors in the IBAT was significant in the nondiabetic animals, whereas it was twofold higher in the diabetic animals compared to the saline-treated animals (see Table 1). Atomoxetine had little effect on IBAT metabolic activity in the diabetic rat, but the blood glucose level was lowered by approximately 25% (see Table 1).

Summary

Our preliminary findings of a significant reduction of IBAT activity in the STZ-induced T1DM model is similar to our recently reported results from the leptin receptor–deficient fa/fa Zucker rat model.³ Comparing the two diabetic models, it appears that the reduction in IBAT activity in the Zucker rat may be driven by impaired β₃-adrenoceptor signaling,¹⁸ whereas for the reduction in the STZ-treated rats, the impairment may be driven by mitochondrial dysfunction.¹⁰ Our results also suggest that IBAT is activated by stimulation of β₃-adrenoceptor in this T1DM rat model and is able to enhance metabolic activity. However, attempts to alter norepinephrine levels using atomoxetine had little effect, possibly due to impaired norepinephrine turnover.¹⁷ A recent study using atomoxetine in obese women had a small effect on short-term weight loss.¹⁹ The sensitivity to detect this enhanced BAT metabolic activity via the β₃-adrenoceptor by Cl316,243 activation using the [¹⁸F]FDG PET/CT method may therefore be useful in the development and evaluation of novel treatment strategies.²⁰ Recent studies in rats and humans have confirmed activation of IBAT using a clinically used β₃-adrenoceptor agonist in normal subjects.^21,22 To further validate the significance of the IBAT STZ model and treatment paradigms presented here, a larger study is warranted.

Footnotes

Acknowledgments

We would like to thank Kimberly Schade and Christopher Liang for technical support and helpful discussions.

Financial disclosure of authors: This research was supported by National Institutes of Health grants RC1 DK087352 (to J.M.) and R21 DK092917 (to J.M.).

Financial disclosure of reviewers: None reported.

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Initial Assessment of β 3 -Adrenoceptor-Activated Brown Adipose Tissue in Streptozotocin-Induced Type 1 Diabetes Rodent Model Using [ 18 F]Fluorodeoxyglucose Positron Emission Tomography/Computed Tomography

Abstract

Materials and Methods

Results

Discussion

Summary

Footnotes

Acknowledgments

References