A sensitive mitochondrial thermometry 2.0 and the availability of thermogenic capacity of brown adipocyte.

The temperature of a living cell is a crucial parameter for cellular events, such as cell division, gene expressions, enzyme activities and metabolism. We previously developed a quantifiable mitochondrial thermometry 1.0 based on rhodamine B methyl ester (RhB-ME) and rhodamine 800 (Rh800), and the theory for mitochondrial thermogenesis. Given that the synthesized RhB-ME is not readily available, thus, a convenient mitochondrial thermometry 2.0 based on tetra-methyl rhodamine methyl ester (TMRM) and Rh800 for the thermogenic study of brown adipocyte was further evolved. The fluorescence of TMRM is more sensitive (∼1.4 times) to temperature than that of RhB-ME, then the TMRM-based mito-thermometry 2.0 was validated and used for the qualitatively dynamic profiles for mitochondrial thermogenic responses and mitochondrial membrane potential in living cells simultaneously. Furthermore, our results demonstrated that the heterogenous thermogenesis evoked by ß3 adrenoceptor agonist only used overall up to ∼46% of the thermogenic capacity evoked by CCCP stimulation. On the other hand, the results demonstrated that the maximum thermogenesis evoked by NE and oligomycin A used up to ∼79% of the thermogenic capacity, which suggested the maximum thermogenic capacity under physiological conditions by inhibiting the proton-ATPase function of the mitochondrial complex V, such as under the cold activation of sympathetic nerve and the co-release of sympathetic transmitters.

Application of a dye-based mitochondrion-thermometry to determine the receptor downstream of prostaglandin E2 involved in the regulation of hepatocyte metabolism.

Temperature distributions inside a living cell reflect the thermodynamics and functions of cellular components. We used a newly-developed method of mitochondrial thermometry based on Rhodamine B methyl ester, which equilibrates as a thermosensitive mixture of nonfluorescent and fluorescent resonance forms. Prostaglandin E2 (PGE2) is released from hepatic non-parenchymal Kupffer cells and acts as an inflammatory factor to impact various functions of hepatocytes. The activity of PGE2 on energy mechanism of hepatocytes has not been fully elucidated and in particular, which PGE2 receptor mediates the functions has been elusive. We identified EP4 as the major receptor of PGE2 via our mitochondrion-thermometry approach and then substantiated this receptor's role in hepatic metabolism. We discovered that PGE2 is able to decrease intracellular temperature of hepatocytes, via increasing some lipogenic genes' expressions, hampering lipolysis and mitochondrial ß-oxidation, reducing intracellular ATP level and elevating cAMP level through EP4 receptor. The redox status of hepatocytes represented by FAD vs FAD + NADH ratio is influenced by PGE2 in an EP4 receptor-dependent manner. Collectively, these data demonstrate that PGE2 regulates metabolism of hepatocytes mainly through EP4 receptor.

Dye-based mito-thermometry and its application in thermogenesis of brown adipocytes.

Mitochondrion is the main intracellular site for thermogenesis and attractive energy expenditure targeting for obesity therapy. Here, we develop a method of mitochondrial thermometry based on Rhodamine B methyl ester, which equilibrates as a thermosensitive mixture of nonfluorescent and fluorescent resonance forms. Using this approach, we are able to demonstrate that the efficacy of norepinephrine-induced thermogenesis is low, and measure the maximum transient rate of temperature increase in brown adipocytes. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s41048-017-0039-6) contains supplementary material, which is available to authorized users.

Sympathetic transmitters control thermogenic efficacy of brown adipocytes by modulating mitochondrial complex V.

Obesity is a worldwide epidemic and results from excessive energy intake or inefficient energy expenditure. It is promising to utilize the thermogenic function of brown adipose tissue for obesity intervention. However, the mechanisms controlling the efficacy of norepinephrine-induced thermogenesis in brown adipocytes remain elusive. Here we demonstrate that norepinephrine (NE) induces low-efficacy thermogenesis, evoking both heterogeneous changes (ΔΨm and ΔpH) and homogenous responses, one of which is that NE stimulation causes large amounts of ATP consumption in brown adipocytes. We reveal that the proton-ATPase activity of mitochondrial complex V is a key factor that antagonizes proton leakage by UCP1 and determines the efficacy of NE-induced thermogenesis in brown adipocytes. Furthermore, to avoid unnecessary and undesired heat production, we reveal that ATP is a necessary sympathetic cotransmitter for the high efficacy and specificity of NE-induced thermogenesis in brown adipocytes as it increases intracellular calcium concentrations and upregulates the ATP synthase activity of complex V. Thus, we demonstrate the modulation mechanism of thermogenic efficacy in brown adipocytes. These findings imply new strategies to partially or fully utilize the thermogenic capacity of brown adipocytes to identify therapeutic targets for the treatment of obesity and diabetes.

Cellular model of neuronal atrophy induced by DYNC1I1 deficiency reveals protective roles of RAS-RAF-MEK signaling.

Neuronal atrophy is a common pathological feature occurred in aging and neurodegenerative diseases. A variety of abnormalities including motor protein malfunction and mitochondrial dysfunction contribute to the loss of neuronal architecture; however, less is known about the intracellular signaling pathways that can protect against or delay this pathogenic process. Here, we show that the DYNC1I1 deficiency, a neuron-specific dynein intermediate chain, causes neuronal atrophy in primary hippocampal neurons. With this cellular model, we are able to find that activation of RAS-RAF-MEK signaling protects against neuronal atrophy induced by DYNC1I1 deficiency, which relies on MEK-dependent autophagy in neuron. Moreover, we further reveal that BRAF also protects against neuronal atrophy induced by mitochondrial impairment. These findings demonstrate protective roles of the RAS-RAF-MEK axis against neuronal atrophy, and imply a new therapeutic target for clinical intervention.

[Secondary structure changes of insulin induced by PEF exposure at different temperatures using Raman spectra and theoretical model analysis].

Recently, biological effects induced by weak electromagnetic fields have been a public concern. Our previous study found temperature and electromagnetic field co-effects on insulin conformation. Therefore, in the present study, Raman spectroscopy was employed to investigate the secondary structure changes of insulin molecule induced by pulsed electric field (PEF) exposure at various temperatures. The content changes in alpha helix of insulin were obtained. Then, protein helix-random coil transition model was used to quantitatively study the experimental results. The theoretical model could figure out the effect of PEF on alpha helix contents of insulin at different temperatures. The protein secondary structure transits from helix to random coil evoked by PEF exposure and change of thermodynamic environment, which could explain the reason for the decline of alpha helix content of insulin caused by PEF exposure together with temperature rising. The results offer experimental basis and theoretical reference for further study of the mechanism of nonthermal effects of weak electromagnetic fields on biological molecule secondary structure.

[Raman spectra of murine skin irradiated by high power millimeter wave].

An experimental study on Raman spectroscopy of normal murine skin and the skin irradiated by high power millimeter wave (HPMM) is presented. It is showed that the Raman spectra of normal skin mainly originate from collagen, and the characteristic peaks are 857, 936 and 1 658 cm(-1). The result showed that after irradiation by HPMM, the relative intensity of the characteristic peaks at 857 and 936 cm(-1) of Raman spectra was decreased. This meant that the collagen was destroyed and even daimaged. It was probably indicated that the skin tissue was damaged and could not be restored. The result also showed that the intensity of the characteristic peak at 1658 cm(-1) of the skin tissue irradiated by millimeter wave with the duration of 20 s decreased. It was considered that the protein in the skin was destroyed. Those results were consistent with macroscopic observation results.