T2 e Studio al seguito

[buddha]

Cosa ne pensate?

3,5-Diiodo-L-thyronine powerfully reduces adiposity in rats by increasing the burning of fats

SPECIFIC AIMS

Thyroid hormones (THs), thyroxine (T4) and 3,3',5-triiodo-L-thyronine (T3) are well known to stimulate metabolism while simultaneously lowering metabolic efficiency. This effect has long been the focus of research into the potential use of THs as drugs to stimulate weight loss. However, the concomitant induction of a thyrotoxic state and of several side effects (i.e., increase in heart rate, increases in thyroid and heart mass, and decrease in skeletal muscle mass and in serum TSH levels) has greatly limited their use as weight-lowering agents. Recent evidence suggests that 3,5-diiodo-L-thyronine (T2), a naturally occurring iodothyronine, stimulates metabolic rate via mechanisms involving the mitochondrial apparatus. In addition, T2 can induce metabolic inefficiency, possibly by stimulating energy loss via mechanisms involving mitochondrial proton leakage/redox slippage. Such inefficiency in energy transduction should result in reduced energy storage. In view of these metabolic effects of T2 and the very low affinity of T2 for nuclear T3 receptors, we thought it conceivable that in rats fed a high-fat diet (HFD), long-term treatment with T2 might result in a reduced adiposity and less body weight gain without inducing a clinical syndrome related to the thyrotoxic state.

PRINCIPAL FINDINGS

1. T2 decreases body weight gain and adiposity without inducing a thyrotoxic state
In rats fed an HFD and treated long-term (30 days) with T2 (DT2 rats) serum levels of TSH, FT3, and FT4 showed no substantial differences either from rats fed an HFD (D rats) or the normal animals fed a standard diet (N). As result of a TRH test, no significant difference in the response of serum TSH to TRH injection was observed among the samples from the three groups, thus indicating an intact hypothalamo-pituitary-thyroid (HPT) axis after T2 treatment. The weights of thyroid glands and heart did not change significantly from the normal euthyroid values, whereas visceral white adipose tissue and liver weights were significantly decreased in DT2 rats. Moreover, no change in heart rate was observed after T2 administration. Over the entire period of treatment, D rats consumed ∼5% more food than N rats, while DT2 animals consumed ∼6% more than the D rats. At the end of the treatment period, the D rats were overweight, weighing ∼13% more than N rats. The DT2 rats, on the other hand, were 13% lighter than the D animals, with the result that their body weight was not different from that of N animals. The DT2 rats accumulated much less fat in their adipose tissue than the D animals, the visceral fat pad tissue weighting 19.3 ± 2.97 g per rat in D rats against 10.2 ± 2.4 g in DT2 ones (P<0.05). In N rats, the corresponding weight was 9.4 ± 1.5 g. The muscle mass did not vary after T2 administration to both N and D rats. Determinations made at the end of the treatment period showed that energy expenditure over a 24 h period was markedly greater in DT2 rats (+29%) than in D rats. Respiratory quotient decreased in D rats (vs. N rats) and further in DT2 animals (vs. D), thus indicating a shift in metabolism toward an increase in the utilization of lipids as substrate.

The livers of D rats were lighter in color than the DT2 livers, suggesting that they contained more fat (Fig. 1A ). Stained sections showed that D livers contained abundant fat droplets, whereas these were not apparent at all in DT2 livers (Fig. 1B , C).



View larger version (109K):
In this window
In a new window

Figure 1. Histological analyses of livers obtained from D and DT2 rats. A) Livers of D and DT2 rats. The livers of D rats (B) exhibited steatosis, with the presence of abundant fat droplets, while DT2 livers showed a complete absence of droplets (C).
2. T2 increases hepatic fatty acid oxidation and decreases serum levels of cholesterol and triglycerides
The fatty acid oxidation rate (reported as nmol O/min mg protein) in the liver of N rats was 61 ± 4. The rate was 30% higher in D rats, and it was further increased (+42% vs. D rats) in DT2 rats (79±5 and 112±7, respectively; P<0.05 in each case). In the muscle, the fatty acid oxidation rate was not significantly influenced by T2, thus implicating the liver as a major player in the effects seen here with T2.

When compared with those in the D group, a decrease in the serum levels of cholesterol (18%) and triglycerides (52%) was observed in DT2 rats, the levels reaching values not significantly different from those seen in N rats.

3. T2 activates CPT system and AMPK phosphorylation in liver
To ascertain whether the stimulation of fatty acid oxidation in mitochondria from both D and DT2 rats might be dependent on the carnitine palmitoyl-transferase (CPT) system, CPT activity as well as acetyl-coenzymeA-carboxylase (ACC) activity and AMP-activated protein kinase (AMPK)-phosphorylation levels (P-AMPK, which is known to inhibit ACC activity under several physiological conditions) were measured. Although the CPT1 mRNA expression levels did not vary among the groups, the CPT system activity was significantly greater (by 38%) in D rats than in N rats, and significantly greater (by 52%) in DT2 rats than in D rats (Fig. 2A ).



View larger version (28K):
In this window
In a new window

Figure 2. T2 activates CPT system, AMPK phosphorylation, and mitochondrial proton leak kinetics in rat liver. T2 treatment does not further decrease ACC activity in DT2 rat liver. A) Total CPT activity in N (open bars), D (filled bars), and DT2 (hatched bars) rat livers. Data are mean ± SE for 6 rats in each group. *P< 0.05 compared with N value, °P < 0.05 compared with D value. B) Total ACC activity in N (open bars), D (filled bars), and DT2 (hatched bars) rat livers. Data are mean ± SE for 6 rats in each group. *P< 0.05 compared with N value. C) Phosphorylation and total protein levels for AMPK in liver lysates (30 µg) from N, D, and DT2 rats (a). ACC protein levels in liver lysates (30 µg). Numbers and arrows indicate the two recognized isoforms of ACC (ACC1 and ACC2) (b). D) Proton leak kinetics of liver mitochondria from N (solid square), D (open circle), and DT2 (solid triangle) rats. Data are mean ± SE for 4 rats in each group.
In D rats, the total ACC activity was ∼65% lower than in N rats (Fig. 2B ) despite the phosphorylated form of AMPK being decreased (vs. N, Fig. 2C ). In DT2 animals, the ACC activity was not further decreased (Fig. 2B ) despite the phosphorylated form of AMPK being increased in comparison with that in D rats (Fig. 2C ), thus apparently excluding an involvement of AMPK-ACC-CPT pathway in the observed effects of HFD and T2 on liver fatty acid oxidation when analyzed after 30 days.

The decrease in ACC activity seen in D animals (vs. N) was likely due to a reduced ACC protein content, as shown by the Western blot analysis (Fig. 2C ).

4. T2 activates mitochondrial proton leak in liver
The kinetic response of the proton leak to a change in membrane potential both in liver and skeletal muscle mitochondria from N, D, and DT2 rats was assessed to examine whether T2 might not only stimulate fatty acid oxidation, but also induce a less efficient utilization of lipid substrates through a stimulation of mitochondrial uncoupling. Liver mitochondria from DT2 rats would need to respire more than D and N rats to maintain the same membrane potential, indicating that DT2 liver mitochondria have a higher proton conductance, no differences being observed in proton leak kinetics between D and N rats (Fig. 2D). The same did not occur in skeletal muscle mitochondria as D and DT2 skeletal muscle mitochondria have the same proton conductance. As mitochondrial uncoupling increases the burning of fat, these results are in line with the above-mentioned effects of T2 on adiposity and further support the liver as a principal target of T2.

CONCLUSIONS AND SIGNIFICANCE

Although in the past and to a large extent now, iodothyronines other than T3 have been regarded as inactive, some evidence supports a biological relevance of T2 mainly because of its effects on metabolism. The results reported here provide clear evidence that in rats, T2 is able to increase the burning of fats reducing both adiposity and the serum levels of fatty acids, triglycerides, and cholesterol without inducing thyrotoxicosis and leading the thyroid axis intact.

The liver, which is a major contributor to energy expenditure, appears to be the principal target for T2.

In DT2 rats, hepatic ß oxidation, CPT activity, and P-AMPK content were significantly increased when compared with those in D rats. On the other hand, the ACC activity was not further decreased in DT2 rats despite the higher CPT activity and the higher fatty acid oxidation rate as well as the high level of phosphorylated AMPK (Fig. 2B, C ).

These results suggest that T2 might enhance fatty acid flux into mitochondria by regulating CPT1 activity in a P-AMPK-dependent and ACC-malonyl-CoA-independent way. In line with this notion, it has been recently reported that an AMPK-mediated phosphorylation of a cytoskeletal component leads to a stimulation of CPT1. The high level of the phosphorylated form of AMPK we observed in DT2 rats (Fig. 2C ) is consistent with this possibility.

Together with a stimulation of fatty acid oxidation in the liver, T2 induced a less efficient utilization of lipid substrates through an induction of a thermogenic mechanism such as mitochondrial uncoupling (proton leak) as, it (leading to a decreased ATP synthesis and a greater burning of fat) was greater in DT2 than in D rats, but not different between D and N rats. This suggests that proton leak plays a determining role in the effects exerted by T2 on the efficiency of substrate utilization and, consequently, on adiposity.

Finally, the results reported here (Fig. 3 ), in a scenario in which there is a high level of fatty acid oxidation, reduced fat storage, considerable reductions in serum triglyceride and cholesterol levels, a reduced fat content in the liver (steatosis), and a reduced body weight gain without a reduction in calorie/fat intake, constitute an attractive prospect for humans (especially those in the so-called developed countries that tend increasingly to follow a lifestyle that favors fat accumulation). Overweight, hepatic steatosis, and dyslipidemia are increasing problems in some countries. Diet-induced nonalcohol-induced fatty liver disease (NAFLD) is now widespread in affluent societies in which up to 24% of the general population has been estimated to have NAFLD due to the epidemic increase in the prevalence of obesity in these societies. If the results reported here hold true for humans, then pharmacological administration of T2 might help to counteract the problems above without inducing a thyrotoxic state.