Diabetes Metab J.  2021 Nov;45(6):921-932. 10.4093/dmj.2020.0187.

Ipragliflozin, an SGLT2 Inhibitor, Ameliorates High-Fat Diet-Induced Metabolic Changes by Upregulating Energy Expenditure through Activation of the AMPK/ SIRT1 Pathway

• Lee JY
• Lee M
• Lee JY
• Bae J
• Shin E
• Lee Yh
• Lee BW
• Kang ES
• Cha BS

• KMID: 2522731
• DOI: http://doi.org/10.4093/dmj.2020.0187

Abstract

Background
Sodium-glucose co-transporter 2 (SGLT2) inhibitors are a new class of antidiabetic drugs that exhibit multiple extraglycemic effects. However, there are conflicting results regarding the effects of SGLT2 inhibition on energy expenditure and thermogenesis. Therefore, we investigated the effect of ipragliflozin (a selective SGLT2 inhibitor) on energy metabolism.
Methods
Six-week-old male 129S6/Sv mice with a high propensity for adipose tissue browning were randomly assigned to three groups: normal chow control, 60% high-fat diet (HFD)-fed control, and 60% HFD-fed ipragliflozin-treated groups. The administration of diet and medication was continued for 16 weeks.
Results
The HFD-fed mice became obese and developed hepatic steatosis and adipose tissue hypertrophy, but their random glucose levels were within the normal ranges; these features are similar to the metabolic features of a prediabetic condition. Ipragliflozin treatment markedly attenuated HFD-induced hepatic steatosis and reduced the size of hypertrophied adipocytes to that of smaller adipocytes. In the ipragliflozin treatment group, uncoupling protein 1 (Ucp1) and other thermogenesis-related genes were significantly upregulated in the visceral and subcutaneous adipose tissue, and fatty acid oxidation was increased in the brown adipose tissue. These effects were associated with a significant reduction in the insulin-to-glucagon ratio and the activation of the AMP-activated protein kinase (AMPK)/sirtuin 1 (SIRT1) pathway in the liver and adipose tissue.
Conclusion
SGLT2 inhibition by ipragliflozin showed beneficial metabolic effects in 129S6/Sv mice with HFD-induced obesity that mimics prediabetic conditions. Our data suggest that SGLT2 inhibitors, through their upregulation of energy expenditure, may have therapeutic potential in prediabetic obesity.

Keyword

Adipose tissue; Obesity; Sodium-glucose transporter 2 inhibitors; Thermogenesis

Figure

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  Fig. 1. Biochemical characterization of the mice. Male 129S6/Sv mice were fed with a normal chow (NC) or high-fat diet (HFD) and were treated with vehicle or ipragliflozin (Ipra) for 16 weeks. (A) Changes in body weight. (B) Food intake (kcal per day). (C) Changes in the random blood glucose levels. (D) Serum glucose levels after an oral glucose tolerance test and area under the curve. (E) Serum glucose levels after an insulin tolerance test and area under the curve. Data are presented as the mean±standard error. NC control (unfilled circle), HFD control (filled triangle), and Ipra-treated HFD mice (filled square). aP<0.05 vs. the NC group, bP<0.01 vs. the NC group, cP<0.05 vs. the HFD group.

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  Fig. 2. Effects of ipragliflozin (Ipra) on pancreatic islets. (A) Serum insulin concentration, (B) serum glucagon concentration, (C) ratio of serum insulin-to-glucagon levels, and (D) serum β-hydroxybutyrate concentration. (E) Immunohistochemical analysis of insulin using pancreatic tissue sections. Scale bar, 500 μm (upper) and 100 μm (lower). (F) Immunohistochemical analysis of glucagon using pancreatic tissue sections. Magnification, ×200. (G) Relative glucagon-positive area (%) in a pancreatic section. Data are presented as the mean±standard error. NC, normal chow; HFD, high-fat diet; NS, not statistically significant. aP<0.05, bP<0.01.

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  Fig. 3. Effects of ipragliflozin (Ipra) on liver panels and hepatic steatosis. (A) Liver weight. (B, C, D) Serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), and triglyceride (TG) levels. (E) Hematoxylin and eosin (H&E) staining and (F) Oil Red O staining of liver sections. Magnification, ×200. (G) Hepatic TG concentration. (H, I) Relative mRNA expression levels of lipogenesis-related and proinflammatory genes. (J) Protein levels of AMP-activated protein kinase (AMPK), pAMPK, and sirtuin 1 (SIRT1) in the liver were determined using Western blot analysis. The graph on the right shows the densitometric analysis of the SIRT1/glyceraldehyde 3-phosphate dehydrogenase (GAPDH) ratio determined from the immunoblots shown on the left. Left, normal chow (NC) control mice. Middle, high-fat diet (HFD) control mice. Right, Ipra-treated HFD mice. Data are presented as the mean±standard error. NS, not statistically significant; Acc, acetyl-CoA carboxylase; Fas, fatty acid synthase; Mcp1, monocyte chemoattractant protein 1. aP<0.05, bP<0.01.

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  Fig. 4. Effects of ipragliflozin (Ipra) on the mass of white adipose tissue and size of adipocytes. (A) Visceral fat weight and (B) subcutaneous fat weight. Data are presented as the mean±standard error. (C) Hematoxylin and eosin (H&E) staining of visceral and subcutaneous fat. Scale bar, 100 μm. (D, E) Histogram of visceral and subcutaneous fat diameters. The median is marked by a vertical line inside the box, the ends of the box are the upper and lower quartiles, and the lower and the upper lines outside the box represent the 5th and 95th percentiles, respectively. Whisker plots represent the outliers. NC, normal chow; HFD, high-fat diet; NS, not statistically significant. aP<0.05, bP<0.01.

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  Fig. 5. Ipragliflozin (Ipra) induced brown fat-like changes in white adipose tissue. (A) Immunohistochemical analysis of the expression of uncoupling protein 1 in the visceral and subcutaneous fat. Scale bar, 200 μm. (B, C) Relative mRNA expression levels of thermogenesis-related genes and sirtuin 1 (SIRT1) in visceral fat and (D, E) subcutaneous fat. (F) Protein levels of AMP-activated protein kinase (AMPK), pAMPK, and SIRT1 in visceral fat were determined using Western blot analysis. The graph on the right shows the densitometric analysis of the SIRT1/glyceraldehyde 3-phosphate dehydrogenase (GAPDH) ratio determined from the immunoblots shown on the left. Left, normal chow (NC) control mice. Middle, high-fat diet (HFD) control mice. Right, Ipra-treated HFD mice. Data are presented as the mean±standard error. Ucp1, uncoupling protein 1; Dio2, type 2 selenodeiodinase; Pgc1α, peroxisome proliferator-activated γ coactivator 1α; Tmem26, transmembrane protein 26. aP<0.05, bP<0.01.e

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  Fig. 6. Effects of ipragliflozin (Ipra) on brown adipose tissue. (A) Representative gross images of interscapular brown adipose tissue. (B) Brown fat weight. (C) Hematoxylin and eosin (H&E) staining of brown fat. Scale bar, 100 μm. (D) Relative mRNA expression levels of thermogenesis-related genes in the brown fat. (E) Relative mitochondrial DNA content analyzed by polymerase chain reaction using primers specific for cytochrome c oxidase subunit II. (F) Relative mRNA expression levels of fatty acid oxidation-related genes. Data are presented as the mean±standard error. NC, normal chow; HFD, high-fat diet; NS, not statistically significant; Ucp1, uncoupling protein 1; Dio2, type 2 selenodeiodinase; Pgc1α, peroxisome proliferator-activated γ coactivator 1α; Acad, acyl-coenzyme A dehydrogenase; Acsl1, acyl-CoA synthetase long-chain family member 1. aP<0.05.

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