A nitroalkene derivative of salicylate alleviates diet-induced obesity by activating creatine metabolism and non-shivering thermogenesis

Abstract

Obesity-related type II diabetes (diabesity) has increased global morbidity and mortality dramatically. Previously, the ancient drug salicylate demonstrated promise for the treatment of type II diabetes, but its clinical use was precluded due to high dose requirements. In this study, we present a nitroalkene derivative of salicylate, 5-(2-nitroethenyl)salicylic acid (SANA), a molecule with unprecedented beneficial effects in diet-induced obesity (DIO). SANA reduces DIO, liver steatosis and insulin resistance at doses up to 40 times lower than salicylate. Mechanistically, SANA stimulated mitochondrial respiration and increased creatine-dependent energy expenditure in adipose tissue. Indeed, depletion of creatine resulted in the loss of SANA action. Moreover, we found that SANA binds to creatine kinases CKMT1/2, and downregulation CKMT1 interferes with the effect of SANA in vivo. Together, these data demonstrate that SANA is a first-in-class activator of creatine-dependent energy expenditure and thermogenesis in adipose tissue and emerges as a candidate for the treatment of diabesity.

Keywords: Adipose tissue; creatine; energy expenditure; obesity; salicylate derivative; thermogenesis.

Figure 1.. SANA protects against diet-induced obesity.

A) SANA synthesis route (left) and characterization of its electrophilic properties (right). SANA (10 μM) was incubated with β-Mercaptoethanol (BME, 100 μM) or reduced glutathione (GSH, 100 μM). Spectra of the reaction were obtained in the 200–600 nm range every 60 s. B) Representative picture of mice fed with high-fat diet (HFD) or HFD+SANA (or salicylate, SAL) at 400 mg/kg/day (PO) C) Weight gain of the mice shown in B). D-E) Weight gain expressed as percent of initial weight in mice in normal diet (ND) or fed with HFD alone or supplemented with different doses of SANA (in D) or SAL (in E). Compounds were administered PO at 200, 300 and 400 mg/kg/day. F) Percent of weight gain in mice fed with ND alone and ND+SANA (or SAL) at 400 mg/kg/day after 4 weeks of treatment or HFD+SANA (or SAL) at 400 mg/kg/day after 8 weeks. G) Cumulative food intake in mice treated as described in B.H) Representative picture of mice treated as described in B. Arrows point to perigonadal fat depots. I) Total fat mass was measured by EchoMRI and J) Quantitation of different fat depots of mice treated as described in B). Brown adipose tissue (iscap_BAT), inguinal subcutaneous (i_WAT), subscapular subcutaneous (sScap_WAT) and perigonadal (p_WAT) white adipose. K) Total lean mass was measured by EchoMRI in the same conditions described in B).

Figure 2.. SANA protects against glucose intolerance and liver steatosis in response to DIO.

A-C) Liver macroscopic appearance (A), H&E liver staining (B) and liver weight (C) in mice fed with ND, HFD, HFD+SANA or SAL at 400 mg/kg/day (PO). D) Liver transaminases in plasma/serum and E-G) Fasting glucose, glucose tolerance test (GTT) and GTT area under the curve (AUC) in mice treated as described in C). H-J) Quantitation of insulin (H), NEFA (nonesterified fatty acids) (I) and Leptin (J), in plasma/serum from mice treated as described in C). KO) SANA was delivered orally in solution (by gavage) at 10 mg/kg/day and compared with the dose of 400 mg/kg/day PO. K) Weight gain. L) Cumulative food intake, M) Fasting glucose levels at week 8. N) GTT at week 8. O) Insulin plasma levels at week 8.

Figure 3.. Treatment of obese mice with SANA promotes weight loss and amelioration of glucose intolerance and liver damage.

Obese mice were treated with SANA at 200 mg/kg/day PO. A) Weight gain and percent of initial weight. B) Representative picture and C) Cumulative food intake. D) Fasting glucose levels measured at week 8. E) GTT at week 8. F) Free-fatty acids levels in plasma at week 8. G) Liver macroscopic appearance and H&E liver staining and H) Liver transaminases in plasma/serum from mice at week 8. I) Percent of initial weight and representative picture of obese mice treated with SANA or SAL (200 mg/kg/day PO), in combination or not with metformin (MET 300 mg/kg/day, gavage). J) Cumulative food intake and K) evolution of fasting glucose.

Figure 4.. Proteomic analysis of whole tissue and isolated mitochondria from iWAT to SANA.

A-D) Whole iWAT proteomic analysis from obese mice fed with HFD or HFD+SANA (400 mg/kg/day, PO). A) Heatmap generated showing an individual protein per row and biological replicates of each condition in columns. B) Venn diagram indicating the proteins exclusively detected in each condition (p-value < 0.05). Ckm: Creatine kinase M-type. C) Volcano plot showing proteins found in both conditions with statistically differential abundance (BH q-value < 0.05). Each dot represents a protein detected in at least 4 biological replicates from the total of 6 in both conditions. The darkest dots correspond to statistically differential proteins. D) WebGestalt’s pathway over-representation analysis of proteins overexpressed in HFD+SANA vs HFD. E) Cellular respiration and F) oxygen consumption in white adipocytes isolated from mice treated as described in A-D). G-J) Proteomic analysis of isolated mitochondria from iWAT of mice treated as described in A-D). Gatm: Glycine amidinotransferase, mitochondrial. K) Total creatine and L) phosphocreatine levels in iWAT from mice treated as described in A-D), measured by MS.

Figure 5.. SANA stimulates thermogenesis in the absence of UCP1 activation, an effect that is abolished when creatine metabolism is impaired in vivo.

A) Thermal image of mice fed with HFD or HFD+SANA (400 mg/kg/day, PO). B-D) Analysis of inguinal white adipose tissue B) Representative histological (H&E) image of iWAT. C) Expression of thermogenesis markers by qPCR in iWAT from mice treated as described in A. D) WB analysis of CKMT1, CKMT2, UCP1 in iWAT after SANA treatment as described in A. E) Human differentiated white adipose cells (TERT-hWA) were incubated with SANA (100 μM) for 24 hours. Left, expression of thermogenic markers measured by qPCR. Right, mitochondrial respiration. F-K) Analysis of brown adipose tissue (BAT). F) Expression of thermogenesis markers by qPCR in BAT, including scaffold as a further control; G) total creatine levels in BAT measured by MS and H) creatine kinase activity in BAT isolated mitochondria, from mice treated as described in A. I) State II respiration in isolated mitochondria from BAT measured with pyruvate+malate (PM). J) Inhibited respiration of isolated mitochondria from BAT by GDP K) FFA-dependent respiration of isolated mitochondria from BAT after UCP1 inhibition with GDP. L) Cold challenge from mice treated with SANA (20 mg/kg/day, SC) M) Creatine quantification in iWAT from mice treated as described in L) after 6 hours of cold exposure. N) Effect of the creatine antagonist β-GPA on cold response in mice treated with SANA. O) Electromyogram during cold exposure.

Figure 6.. SANA stimulates energy expenditure and protects against obesity under thermoneutral conditions.

A) Schematic representation of long-term treatment under thermoneutral conditions. Mice were fed with HFD or HFD+SANA (or SAL) at 20 mg/kg/day, SC. B) Weight gain and percent of initial weight. C) Cumulative food intake D) Fasting glycemia at weeks 0 and 8. E) GTT at week 8 F) Schematic representation of acute HFD and SANA treatment under thermoneutral conditions. G) Body weight and H) Representative thermal image of HFD and HFD+SANA treated mice at the end of the acute treatment. I) Surface temperature quantitation from thermal images. J) EE measurements over a 24-hour period at the end of the acute treatment. K) Regression plot of EE shown in J. L) Change in individual EE during HFD treatment at 28°C. M) EE measurement of CL316,243-treated mice injected at the end of the acute treatment. N) Regression plot of EE shown in M). O-P) Thermoneutral-to-cold challenge switch of mice at the end of the acute treatment. O) Representative thermal image after 1 hour incubation at 4°C. P) Surface temperature quantitation from thermal images.

Figure 7.. Identification of SANA-binding proteins followed by in vivo confirmation identified Ckmt1/2 as putative targets of SANA.

A) Workflow scheme describing the screening strategy for the identification of SANA-binding proteins. B) Electrophilic properties of biotinylated SANA (bSANA). bSANA (10 μM) was incubated with β-mercaptoethanol (BME, 100 μM). Spectra of the reaction were obtained in the 200–600 nm range every 60s. C) Venn diagram showing proteins that were identified bound to SANA (control) or bSANA. D) Survival curve of WT and Ckmt1 KO mice treated with SANA (20 mg/kg/day, SC) for 1 week at RT or thermoneutrality. E) Fasting glucose in WT and Ckmt1 KO mice at thermoneutrality after HFD feeding and treatment with SANA (20 mg/kg/day, SC) or vehicle for 1 week.