Diabetes Metab J.  2023 Nov;47(6):784-795. 10.4093/dmj.2022.0261.

Altered Metabolic Phenotypes and Hypothalamic Neuronal Activity Triggered by Sodium-Glucose Cotransporter 2 Inhibition

• Lee HG
• Jung IH
• Park BS
• Yang HR
• Kim KK
• Tu TH
• Yeh JY
• Lee S
• Yang S
• Lee BJ
• Kim JG
• Nam-Goong IS

• KMID: 2548155
• DOI: http://doi.org/10.4093/dmj.2022.0261

Abstract

Background
Sodium-glucose cotransporter 2 (SGLT-2) inhibitors are currently used to treat patients with diabetes. Previous studies have demonstrated that treatment with SGLT-2 inhibitors is accompanied by altered metabolic phenotypes. However, it has not been investigated whether the hypothalamic circuit participates in the development of the compensatory metabolic phenotypes triggered by the treatment with SGLT-2 inhibitors.
Methods
Mice were fed a standard diet or high-fat diet and treated with dapagliflozin, an SGLT-2 inhibitor. Food intake and energy expenditure were observed using indirect calorimetry system. The activity of hypothalamic neurons in response to dapagliflozin treatment was evaluated by immunohistochemistry with c-Fos antibody. Quantitative real-time polymerase chain reaction was performed to determine gene expression patterns in the hypothalamus of dapagliflozin-treated mice.
Results
Dapagliflozin-treated mice displayed enhanced food intake and reduced energy expenditure. Altered neuronal activities were observed in multiple hypothalamic nuclei in association with appetite regulation. Additionally, we found elevated immunosignals of agouti-related peptide neurons in the paraventricular nucleus of the hypothalamus.
Conclusion
This study suggests the functional involvement of the hypothalamus in the development of the compensatory metabolic phenotypes induced by SGLT-2 inhibitor treatment.

Keyword

Appetite; Dapagliflozin; Energy metabolism; Hypothalamus; Obesity

Figure

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  Fig. 1. (A) Experiment 1: To examine the effects of dapagliflozin (sodium-glucose cotransporter 2 inhibitor, Sigma-Aldrich) on metabolic phenotypes, mice were randomly divided into two groups and fed a standard diet (SD, 10% calories from fat, Research Diets Inc.). For treatment, SD-fed mice were administered with vehicle (0.9% saline, oral injection) or dapagliflozin (1 mg/kg/day, oral injection) for 5 weeks. (B) Experiment 2: To test the effects of dapagliflozin on metabolic phenotypes during overnutrition, mice were fed a high-fat diet (HFD, 60% of calories from fat, Research Diets Inc.) for 13 weeks. Eight weeks after the HFD treatment, mice were treated with dapagliflozin (1 mg/kg/day, oral injection), while HFD treatment was continued.

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  Fig. 2. Effect of dapagliflozin on variations in food intake and body weight. Body weight and food intake were evaluated in vehicle- or dapagliflozin-treated mice under standard diet (SD) or high-fat diet (HFD) feeding conditions using an indirect calorimetry system. No significant alterations in body weight were found in dapagliflozin-treated mice under (A) SD or (B) HFD feeding conditions. (C) Cumulative food intake over 48 hours was enhanced in dapagliflozin-treated mice under SD feeding conditions. (D) Meal frequency during the dark period and (E) meal size during the light period were higher in dapagliflozin-treated mice than those in vehicle-treated mice under SD feeding conditions. (F) Cumulative food intake was increased in dapagliflozin-treated mice compared with that in vehicle-treated mice under HFD feeding conditions. (G) No significant difference in meal frequency and (H) increased meal size were observed in dapagliflozin-treated mice under HFD feeding conditions. Data are presented as the mean±standard error of the mean (n=6 mice for SD group, n=7 mice for HFD group). aP<0.05.

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  Fig. 3. Treatment with dapagliflozin leads to a decrease in energy expenditure in high-fat diet (HFD)-fed mice. The patterns of energy expenditure were measured in vehicle- or dapagliflozin-treated mice using an indirect calorimetry system. Under standard diet (SD) feeding conditions, dapagliflozin-treated mice displayed no alteration in (A) oxygen consumption (VO2), (B) carbon dioxide emission (VCO2), (C) respiratory exchange ratio (RER), or (D) energy expenditure compared with control mice. Under HFD feeding conditions, dapagliflozin-treated mice showed reduced (E) VO2, (F) VCO2, and (H) energy expenditure with no alteration in (G) RER compared with control mice. Data are presented as the mean±standard error of the mean (n=6 mice for SD group, n=8 mice for HFD group). aP<0.05, bP<0.01.

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  Fig. 4. Treatment with dapagliflozin alters the neuronal activities in the hypothalamic nuclei of mice. (A) Representative images showing immunosignals of c-Fos-positive cells in hypothalamic nuclei of vehicle- or dapagliflozin-treated mice under standard diet (SD) feeding conditions. (B) Dapagliflozin treatment resulted in an increasing number of c-Fos-positive cells in the hypothalamic arcuate nucleus (Arc) and lateral hypothalamus (Lh) and a decreasing number of c-Fos-positive cells in the hypothalamic ventromedial hypothalamus (Vmh) and paraventricular nucleus (Pvn). (C) Representative images showing immunosignals of c-Fos-positive cells in the hypothalamic nuclei of vehicle- or dapagliflozin-treated mice under high-fat diet (HFD) feeding conditions. (D) Dapagliflozin treatment resulted in an increase in c-Fos-positive cells in the hypothalamic Arc and a decrease in c-Fos-positive cells in the hypothalamic Lh in HFD-fed mice. Data are presented as the mean±standard error of the mean (n=5 mice for SD-vehicle-treated group, n=4 mice for SD-dapagliflozin-treated group, n=6 mice for HFD-vehicle-treated group; HFD-dapagliflozin-treated group). Scale bar=100 µm. Dmh, dorsomedial hypothalamus. aP<0.05, bP<0.01.

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  Fig. 5. Dapagliflozin treatment enhances immunosignals of agouti-related peptide (Agrp) in the paraventricular nucleus (Pvn) of high-fat diet (HFD)-fed mice. (A) Representative images showing immunosignals of Agrp fibers in the hypothalamic Pvn of standard diet (SD)-fed mice. Treatment with dapagliflozin did not alter (B) fiber density and (C) particle numbers of Agrp-positive immunosignals in the Pvn of SD-fed mice. (D) Representative image showing immunosignals of Agrp fibers in the hypothalamic Pvn of HFD-fed mice. An increase in the (E) fiber density and (F) particle numbers of Agrp-positive immunosignals was observed in the Pvn of dapagliflozin-treated mice under HFD feeding conditions. Data are presented as the mean±standard error of the mean (n=5 mice for SD-vehicle-treated group, n=4 mice for SD-dapagliflozin-treated group, n=6 mice for HFD-vehicle-treated group; HFD-dapagliflozin-treated group). Scale bar=100 µm. aP<0.05, bP<0.01.

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  Fig. 6. The expression patterns of hypothalamic mRNA in response to dapagliflozin treatment. The levels of mRNA involved in appetite regulation were analyzed in the hypothalamus of vehicle- or dapagliflozin-treated mice under standard diet (SD) or high-fat diet (HFD) feeding conditions. (A) The agouti-related peptide (Agrp) mRNA level shows an elevation in the hypothalamus of dapagliflozin-treated mice compared with that in control mice under SD feeding conditions. The mRNA levels of (B) pro-opiomelanocortin (Pomc) and (C) neuropeptide Y (Npy) did not respond to dapagliflozin treatment under SD or HFD feeding conditions. Dapagliflozin treatment did not affect the mRNA expression of (D) suppressor of cytokine signaling 3 (Socs3) and (E) forkhead box protein O1 (Foxo1) in the hypothalamus of SD- or HFD-fed mice. Data are presented as the mean±standard error of the mean (n=4 mice for SD-vehicle-treated group; HFD-vehicle-treated group; n=5 mice for SD-dapagliflozin-treated group; HFD-dapagliflozin-treated group). aP<0.05.

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