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Proceedings of the Society for Experimental Biology and Medicine 224:172-177 (2000)
© 2000 Society for Experimental Biology and Medicine

Original Article

Tissue-Specific Expression of the Uncoupling Protein Family in Streptozotocin-Induced Diabetic Rats

Shuji Hidaka, Hironobu Yoshimatsu, Tetsuya Kakuma, Hiroshi Sakino, Seiya Kondou, Reiko Hanada, Kyoko Oka2,, Yasushi Teshima, Mamoru Kurokawa3, and Toshiie Sakata1,


Department of Internal Medicine I, School of Medicine, Oita Medical University, Oita, 879–5593, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The vulnerability of streptozotocin (STZ)-induced diabetic rats to cold stress has been established. One of the elements controlling body temperature is thermogenesis, in which uncoupling protein (UCP) is known to play an important role. We have examined UCP2 and UCP3 expressions in brown adipose tissue (BAT), white adipose tissue (WAT), and skeletal muscle (MSL) during the acute and chronic phases of STZ-induced diabetes in rats. The long-term effect and the effect of insulin treatment thereafter were also unexplored previously and are examined in this study. In the acute phase of diabetes (2.5 days after STZ injection), UCP2 gene expression in BAT, WAT, and MSL, and UCP3 expression in the muscle were significantly increased. In the chronic phase of diabetes (21 days after STZ injection), UCP2 and UCP3 expression in the MSL were restored to the control levels without insulin supplementation. UCP2 in BAT and WAT remained high in the chronic phase, whereas UCP3 expression in BAT and WAT, which did not change in the acute phase, was significantly decreased. Insulin supplementation restored UCP2 expression in BAT and WAT, but over-corrected UCP3 in WAT above the control and did not affect UCP3 expression in BAT. Insulin supplementation depressed UCP3 expression in the MSL below control. These results indicate that the effects of STZ-induced diabetes on UCPs gene expression are tissue-specific as well as dependent on the duration of diabetes.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
One of the agents believed to regulate nonshivering heat production is uncoupling protein 1 (UCP1), a protein located in the inner mitochondrial membrane in brown adipose tissue (BAT). UCP1 dissipates the electrochemical gradient generated by respiratory activity (1-3). Mitochondrial respiration uncoupled from oxidative phosphorylation thus causes energy to disperse as heat instead of being used in ATP synthesis (1, 2).

UCP2, cloned recently as a second member of the UCP family, is ubiquitously expressed in human and rodent tissues (4, 5). Another member of the UCP family, UCP3, is preferentially expressed in skeletal muscle (MSL) and BAT (6-8). Proton leakage occurs in mitochondria of cell types other than the brown adipocyte (9, 10). UCP2 and UCP3 are candidates to explain nonshivering heat production in such cells.

Since streptozotocin (STZ)-induced diabetic rats are sensitive to cold temperatures, and UCP plays a role in the production of heat, the role of insulin in thermoregulation has been investigated (11, 12). STZ-induced insulin deficiency has been shown to produce a marked reduction of UCP1 expression in BAT (13, 14). High-fat diet, which produces hyperinsulinemia, was found to upregulate UCP1 expression in BAT (11).

High-fat diet was also found to upregulate the gene expression of UCP2 in white adipose tissue (WAT) (6) and UCP3 in the MSL (10). Fasting, which causes hypoglycemia, hypoinsulinemia, and elevated free fatty acids (FFAs), upregulated the gene expression of both UCP2 (15) and UCP3 (16) in the MSL. The present study was designed to investigate the effects of STZ-induced diabetes and subsequent insulin replacement on UCPs gene expression in BAT, WAT, and MSL.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals.
Male Wistar King A (WKA) rats, 13–16 weeks of age, were used in these studies. All rats were housed in a room illuminated daily from 0700 to 1900 hr (a 12:12-hr light:dark cycle) with temperature at 21° ± 1°C and humidity at 55% ± 5%. All animals were allowed free access to standard pelleted rat diet (CE-2, CLEA Japan Ltd., Tokyo, Japan) and tap water.

Preparation of Acute-Phase STZ-DM Rats.
A solution of STZ (Sigma, St. Louis, MO) was freshly prepared on Day 1 of the experiment by dissolving it to a final concentration of 0.1 M (pH 4.5) in sterile sodium citrate buffer. Based on matched body weight and age, 10 male WKA rats were assigned to one of two groups, the STZ-induced diabetic (STZ-DM) group (n = 5) or the citrate buffer control (CIT-CON) group (n = 5). Rats were made diabetic by injecting STZ (60 mg/kg) into the tail vein at 1850–1900 hr under light ether anesthesia. The CIT-CON group was injected with citrate buffer solution containing no STZ. The rats in the STZ-DM group were considered diabetic when their blood glucose concentrations, measured from an intravenous catheter (see below), were 350 mg/dl in the fed state 2.5 days after the STZ injection.

Preparation of STZ-DM Rats at the Chronic Phase and Insulin Supplementation.
Twelve male WKA rats were assigned to one of three groups, the STZ-DM rats with insulin treatment (STZ-INS; n = 4), the STZ-DM rats with saline treatment (STZ-SAL; n = 4), or the CIT-CON rats with saline treatment (CIT-SAL; n = 4). After STZ treatment proceeded as described above, the STZ-INS group was treated with subcutaneous injections of 3 U insulin/day (Lente insulin, Shimizu Pharmaceutical Co., Ltd., Shizuoka, Japan) at 1850–1900 hr for 6 days, starting on Day 14 and continuing to Day 20 following the STZ injection. The STZ-SAL and the CIT-SAL groups were similarly injected with an equal volume of saline.

Blood Sampling and Tissue Extirpation.
Blood samples were collected from each rat through a chronically indwelling silicone catheter implanted in the right external jugular vein with its end at a point just outside of the atrium (17). Surgical catheterizations were done under ether anesthesia 7 days prior to the STZ injections. Just prior to removal of the tissue of each rat, blood samples were taken at 0700–0710 hr. Serum samples were frozen immediately at –20°C until humoral factor levels were measured. All tissue samples, surgically removed at 0710–0720 hr under ether anesthesia, were frozen immediately in liquid nitrogen. All tissue samples were stored at –80°C until RNA extraction.

Assays of Blood- and Adipocyte-Born Humoral Factors.
Serum insulin and leptin levels were quantitated using an insulin radioimmunoassay (RIA) kit (Rat insulin [125I] assay system; Amersham, Buckinghamshire, England; %CVs: 5.1%) and a rat leptin RIA kit (Linco, Inc., St. Louis, MO; %CVs: 4.1%), respectively. Serum glucose, triglyceride, and FFAs concentrations were measured with commercially available kits (Merckauto Glucose, Kanto Chemical Co., Tokyo, Japan; L Type Wako Triglyceride, Wako Pure Chemical Ltd., Osaka, Japan; NEFA-SS'Eiken', Eiken Chemical Co., Ltd., Tokyo, Japan).

RNA Extraction and Northern Blot Analysis.
Total RNA was extracted using the standard acid guanidinium phenol-chloroform method (18). Northern blot analysis was performed as described previously (19). cDNA probes for the rat UCP2 (20), UCP3 (20), and ob were prepared by reverse transcription-polymerase chain reaction using the following primers: ob-sense, 5'-TGT GGC TTT GGT CCT ATC TG-3'; and ob-antisense, 5'-TGC TCA AAG CCA CCA CCT CT-3'. The identity of the appropriately sized product was confirmed by mapping with multiple restriction endonucleases and sequencing. The hybridization signals were analyzed with a BIO-image analyzer BAS 2000 (Fuji Photo Film Co., Tokyo, Japan). The density of 28S ribosomal RNA stained with ethidium bromide and detected with ultraviolet transillumination was used to monitor the amount of total RNA in each sample (21).

Statistical Analysis.
Data were expressed as the mean ± SEM. Statistical significances between two groups were assessed by unpaired Student's t test and between three groups by one-way analysis of variance (ANOVA) followed by Scheffe's multiple comparison test. Probability of 0.05 was defined as the threshold for statistical significance.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The STZ-DM group showed weight loss (CIT-CON versus STZ-DM, 312 ± 5 vs 279 ± 6 g, P < 0.01), hyperglycemia (126 ± 2 vs 462 ± 22 mg/dl, P < 0.0001), and hypoinsulinemia (11.7 ± 0.9 vs 2.4 ± 0.1 ng/ml, P < 0.0001) 2.5 days after STZ treatment. In the STZ-DM group, serum FFAs levels were increased by more than three-fold (0.32 ± 0.01 vs 1.07 ± 0.07 mEq/l, P < 0.0001) accompanied by an increase in serum triglyceride levels by more than four-fold (86 ± 8 vs 360 ± 65 mg/dl, P < 0.01) and decreases both in serum leptin levels by 64% (2.8 ± 0.2 vs 1.0 ± 0.1 ng/ml, P < 0.0001) and in ob mRNA by 90% (P < 0.0001) (Fig. 1B)Go. BAT UCP1 expression in the STZ-DM group decreased by 95% (P < 0.0001) in the acute diabetic phase of this group. In contrast, UCP2 expression in the STZ-DM group increased by 1.7-fold in BAT (P < 0.01), by 2.0-fold in WAT (P < 0.01), and by 2.9-fold in MSL (P < 0.001), compared with that in the CIT-CON group (Fig. 1A-C)Go. UCP3 expression in MSL dramatically increased by 7.3-fold (P < 0.0001) in the STZ-DM group (Fig. 1C)Go, whereas the slight increases in BAT and WAT were not statistically significant (Fig. 1A-B)Go. As we reported recently (20), heart UCP3 expression is also dramatically increased in acute phase STZ-DM rats. These results are similar to the foregoing findings except BAT and WAT UCP2 (22). These discrepancies might be due to the fact that these studies were done in different species of rats, at different duration after administration of STZ, and using different doses of STZ.



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  		Figure 1.   Regulation of uncoupling proteins (UCPs) and ob mRNA expression in brown adipose tissue (BAT), white adipose tissue (WAT), and skeletal muscle (MSL) of STZ-induced diabetic (STZ-DM) rats. Total RNA (20 µg/lane) was analyzed with each lane containing RNA from one rat. Representative Northern blots and corresponding ethidium bromide-stained 28S ribosomal RNA (rRNA) electrophoretically separated on an agarose gel are shown in the left panel. Quantitation of the percentage changes in UCPs and ob mRNA levels in the STZ-DM rats from those in the citrate-treated control (CIT-CON) rats. (A) BAT. (B) WAT. (C) MSL. Values, mean ± SEM (n = 5 per group). a = P < 0.01; b = P < 0.001; c = P < 0.0001 vs CIT-CON rats.

 
To gain further insight into the mechanism by which the UCPs are specially regulated in STZ-DM rats, we examined the relatively long-term effects of prolonged diabetic state and the effects of insulin supplementation on UCPs gene expression. These long-term results and the effect of insulin treatment thereafter have not been reported previously. Changes in serum metabolites at the chronic diabetic phase were the same as those at the acute diabetic phase. Insulin supplementation returned serum FFAs concentration to that in the controls (CIT-SAL versus STZ-SAL versus STZ-INS, 0.39 ± 0.02 vs 0.74 ± 0.02 vs 0.34 ± 0.02 mEq/l, respectively; no significant (NS) difference between CIT-SAL and STZ-INS), leptin concentration (2.4 ± 0.2 vs 0.7 ± 0.4 vs 2.0 ± 0.2 ng/ml; NS between CIT-SAL and STZ-INS) and insulin concentration (7.7 ± 1.1 vs 2.1 ± 0.2 vs 8.5 ± 1.4 ng/ml; NS between CIT-SAL and STZ-INS). The insulin supplementation also restored body weight (353 ± 4 vs 259 ± 5 vs 307 ± 7 g), serum concentrations of glucose (95 ± 2 vs 524 ± 8 vs 295 ± 59 mg/dl), and triglyceride (59 ± 4 vs 254 ± 21 vs 140 ± 9 mg/dl), but each was incompletely restored relative to that in the CIT-SAL group (CIT-SAL versus STZ-INS: body weight, P < 0.001; glucose, P < 0.001; triglyceride, P < 0.05). These data have been described in detail elsewhere (20). BAT UCP1 and UCP2 mRNA, and WAT UCP2 and ob mRNA maintained each down- or upregulated expression in the later phase of diabetes (CIT-SAL versus STZ-SAL: BAT UCPs, P < 0.0001; WAT UCP2, P < 0.05; WAT ob, P < 0.001) (Fig. 2)Go. MSL UCP2 and UCP3 expression, each of which showed a drastic increase in the acute phase, returned to the CIT-SAL level in the chronic diabetic phase without insulin supplementation (Fig. 2)Go, although serum FFAs levels remained high. UCP3 expression in BAT and WAT, which did not change in the early phase, decreased in the chronic diabetic phase (P < 0.05 for each) (Fig. 2)Go. Insulin treatment restored expression of BAT and WAT UCP2, and over-corrected WAT UCP3 more than that in the controls (P < 0.01 vs CIT-SAL, P < 0.0001 vs STZ-SAL), but downregulated MSL UCP3 below the control levels (P < 0.001 vs CIT-SAL, P < 0.0001 vs STZ-SAL). BAT UCP1 and MSL UCP2 were affected by insulin supplementation, but changes were not statistically significant (Fig. 2)Go. BAT UCP3 expression was not affected by insulin supplementation.



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  		Figure 2.   Regulation of UCPs and ob mRNA expression in the BAT, WAT, and MSL of STZ-DM rats with saline treatment (STZ-SAL) and STZ-DM rats with insulin treatment (STZ-INS). Total RNA (20 µg/lane) was analyzed with each lane containing RNA from one rat. Representative Northern blots and corresponding ethidium bromide-stained 28S rRNA electrophoretically separated on an agarose gel are shown in the left panel. Quantitation of the percentage changes in UCPs and ob mRNA levels in the STZ-SAL and STZ-INS rats from those in the CIT-CON rats with saline treatment (CIT-SAL) rats. (A) BAT. (B) WAT. (C) MSL. Values, mean ± SEM (n = 4 per group). a = P < 0.05; b = P < 0.01; c = P < 0.001; d = P < 0.0001 vs CIT-SAL group. e = P < 0.05; f = P < 0.0001 vs STZ-SAL group.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The increases in UCP2 mRNA in the whole tissues examined and UCP3 mRNA in MSL of STZ-DM rats were unexpected as energy expenditure and thermogenesis were decreased in STZ-DM rats (23, 24). We deduced that the downregulation of BAT UCP1, which was found in the previous (14) and present studies, in effect reduced energy expenditure to accommodate the energy deprivation of diabetic state. Fasting, another model for low energy availability, has also been reported to decrease BAT UCP1, to increase UCP2 (15) and UCP3 (16) mRNA expression in various tissues. One possible explanation for such UCP upregulation is that UCP2 and UCP3 may play a protective role to prevent an excessive decrease in body temperature (16). Another possible explanation is that UCP2 and UCP3 may not play a definite role in the maintenance of body temperature because upregulation of MSL UCP2 and UCP3 during fasting is induced equally well even under thermoneutral conditions (25, 26).

A question can be raised as to whether insulin deficiency per se or the resulting humoral and metabolic abnormalities in STZ-DM may affect UCPs expression in the present study. Lack of insulin per se is not likely to upregulate the UCPs except for BAT UCP1 directly. First, insulin treatment for 3 days, which induced a hyperinsulinemic hypoglycemic state, did not alter MSL UCP2 and UCP3 expression (data not shown). Second, insulin alone did not acutely regulate UCP2 or UCP3 gene expression under conditions of euglycemic hyperinsulinemic clamp (27). Leptin, as another candidate, has been shown to upregulate UCP2 (28) and UCP3 (16) gene expression. However, the decreases in both·ob mRNA expression in WAT and serum leptin concentration were obvious in the present STZ-DM rats. In other words, leptin cannot be a mediator of UCP2 or UCP3 gene expression under conditions of decreased leptin level in STZ-DM rats.

It is reported that an increase in FFAs in fasting rats is a potential mediator of upregulation of MSL UCP3 expression (25, 29-32), which is consistent with the present results that showed an increase in serum FFAs accompanied by upregulation of MSL and cardiac muscle UCP3 (20). When insulin levels are low, MSL and cardiac muscle oxidize fatty acids rather than glucose as its principal fuel source. Furthermore, MSL and cardiac muscle are known to express peroxisome proliferator-activated receptor {alpha} (PPAR {alpha}) (30) and PPAR {gamma}1 (33). It has most recently been reported that the 5' flanking region of the UCP3 gene contains three peroxisome proliferator-response elements (PPREs) (34). Three PPREs in the UCP3 gene may provide the mechanism by which FFAs, ligands for PPAR {alpha} and PPAR {gamma} (35), upregulates MSL and cardiac muscle UCP3 gene expression. On the other hand, Samec et al. (36,37) report that although the regulation of UCPs expression by FFAs is evident in the soleus muscle (slow-twitch fibers, oxidative), regulation of UCPs expression by FFAs was not detected in gastrocnemius and tibial anterior muscles (fast-twitch fibers, glycolytic/oxidative-glycolytic). Our study involved the quadriceps muscle, which is composed of both fast-twich and slow-twitch fibers (29) and is thus a plausible candidate for UCPs upregulation by FFAs. However, as we explain later, there are cases in which serum FFAs levels do not necessarily correlate with changes in UCPs expression, which suggests the existence of other signaling mechanisms. The possibility of alternate signaling mechanisms was also supported by the previous report (36).

The long-term results indicate that the effects of insulin deficiency on UCPs expression appear to be dependent on the duration of the diabetic state. The responses to insulin deficiency and insulin supplementation are tissue dependent. It is of special interest that muscle UCP2 and UCP3 expression was restored to the CIT-SAL levels in the later diabetic phase without insulin treatment, although serum FFAs concentration remained high. In addition, MSL UCP3 mRNA remained below the CIT-SAL level even on the 6th day after insulin supplementation, although serum FFAs concentration was completely recovered to the CIT-SAL level. Thus, a change in serum FFAs level is not necessarily consistent with responses of UCPs. While Zucker fatty (fa/fa) rats showed a low level of UCP3 mRNA expression in MSL (26) despite chronically hyper-free fatty acidemia (20, 38, 39), fasting upregulated expression of MSL UCP2 and UCP3 mRNAs concomitantly with an acute increase in FFA (data not shown). These results indicate that an acute change in FFAs concentrations rather than their sustained elevation must be in part responsible for the elevation of UCP3 mRNA. Improved understanding of these findings may eventually support the concept that upregulation of MSL UCP2 and UCP3 expression may, at least in part, be attributable to acute elevation of serum FFAs concentration.


   Acknowledgments
 
We thank Dr. David S. Knight for help in preparation of the manuscript, and Drs. Seikoh Yasunaga, Seiichi Chiba, Hitoshi Noguchi, and Katsuro Himeno, Department of Internal Medicine I, Oita Medical University for their technical assistance.


   Footnotes
 
This work was supported partly by Grants-in-Aid 07457225 from Japanese Ministry of Education, Science, and Culture of Japan, and by Research Grants from the Ministry of Health and Welfare of Japan, 1996–1997.

1 To whom requests for reprints should be addressed at the Department of Internal Medicine I, School of Medicine, Oita Medical University, 1–1 Idaigaoka, Hasama Oita, 879–5593 Japan. E-mail: sakata{at}oita-med.ac.jp Back

2 Present address: Department of Pediatric Dentistry, Faculty of Dentistry, Kyushu University 61, Fukuoka, 812–8582, Japan. Back

3 Present address: Department of Anatomy, Nagasaki University School of Medicine, Nagasaki, 852–8253, Japan. Back


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 Materials and Methods
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 Discussion
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Received for publication May 27, 1999. Accepted for publication February 24, 2000.




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J. Himms-Hagen and M.-E. Harper
Physiological Role of UCP3 May Be Export of Fatty Acids from Mitochondria When Fatty Acid Oxidation Predominates: An Hypothesis
Experimental Biology and Medicine, February 1, 2001; 226(2): 78 - 84.
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