Discussion
We found that bolus administration of tofogliflozin to the ICV increases food intake for 5 days, whereas intraperitoneal injection does not. Thus, we consider the effects of ICV infusion attributable to direct actions within the CNS rather than due to leakage into the bloodstream. All three SGLT2 inhibitors induced hyperphagia when administered to the CNS. In addition, dapagliflozin showed a dose-dependent effect on food intake. The effect of ICV tofogliflozin was the strongest in the LV, while less or no effects in the 3V or 4V, respectively. ICV infusion of SGLT2 inhibitors activated AMPK in the LH, and upregulated c-fos in the ARC and LH. The hyperphagic effect of tofogliflozin was blocked by systemic administration of liraglutide.
In several previous reports, oral administration of SGLT2 inhibitors was credited with increasing food intake in rats.25–29 In rats with diet-induced obesity, chronic dapagliflozin administration caused compensatory food intake and more than three times the weight loss in rats with paired food consumption.29 In a human study, a dissociation between expected and actual weight loss of approximately 8 kg was observed.4 In another study, steady state energy intake in the canagliflozin-treated subjects was calculated to have increased by ~350 kcal/day.30 To our knowledge, no research has focused on whether this hyperphagic effect is exerted via the CNS. Our observations in the present study raise the possibility that the clinically observed hyperphagic effects associated with SGLT2 inhibitors are, at least partially, exerted via the CNS. If true, this would imply that reduced transfer of SGLT2 inhibitors to the CNS would result in more effective weight loss.
In the present study, tofogliflozin induced a greater increase in food intake than did 2-DG or phlorizin. Several studies have reported that ICV phlorizin increases food intake.14–17 One showed that 0.26 μmol ICV phlorizin induced a stronger hyperphagic effect than 0.13 μmol, while the effects of 0.26 μmol and 0.52 μmol were similar.17 Based on this report, we compared 0.52 μmol of phlorizin with the same molar amount of tofogliflozin and found that the latter had a stronger effect on food intake. Two other previous studies demonstrated that approximately 20 μmol ICV 2-DG induced a significant hyperphagic effect.15 31 We thus employed 20 μmol 2-DG, a dose considered sufficient to increase food intake. This amount of 2-DG tended to induce a hyperphagic effect in the present study, but its effect was smaller than that of tofogliflozin. Therefore, tofogliflozin was more effective in increasing food intake than phlorizin and 2-DG. These observations suggest that higher SGLT2/SLC5A2 selectivity contributes to enhanced food intake, supporting the presence of SGLT2/SLC5A2 in the rat CNS. The function of SGLT2/SLC5A2 itself might be associated with food intake.
The hyperphagic effect seems to be common to SGLT2 inhibitors, because all three SGLT2 inhibitors we used significantly increased food intake. There was, however, a difference in the degree of hyperphagia among groups given the same molar amount of each drug. SGLT2/SLC5A2 selectivity differs among these drugs,32 possibly contributing to this difference. SGLT1/SLC5A1 and SGLT3/SLC5A4 are present in the rat hypothalamus as glucose sensors.33 In humans, tofogliflozin is known to be less selective for SGLT3 versus SGLT2 than the other two SGLT2 inhibitors.32 Taken together with reports showing that SGLT3/SLC5A4 functions as a glucose sensor in the CNS,34 additional effects on SGLT3/SLC5A4 may explain the stronger effects of tofogliflozin. Mice and rats have two isoforms of SGLT3/SLC5A4, unlike humans who have only one isoform.35 It is thus difficult to extrapolate findings from rats to humans.35 In addition, selectivity of SGLT2 inhibitors for SGLT2/SLC5A2 compared with SGLT1/SLC5A1 is reportedly lower in rats than in humans.36 Therefore, if the hyperphagic effect of SGLT2 inhibitors is SGLT2/SLC5A2-specific, it might be stronger in humans. Another possible reason is that differences in the excretion of SGLT2 inhibitors from the CNS may contribute to changes in hyperphagia. However, to the best of our knowledge, no studies have reported the rate or mechanism of SGLT2 inhibitors excretion in the CNS. Future experiments using ICV-radiolabelled SGLT2 inhibitors may clarify why ICV tofogliflozin had the strongest effect on food intake.
We attempted to identify brain regions responsible for the hyperphagic effect induced by SGLT2 inhibitors. ICV tofogliflozin infusion into the 3V caused a milder increase in food intake than that into the LV. ICV tofogliflozin into the 4V produced no significant hyperphagic effect. Conversely, body weight tended to be reduced by 4V-ICV administration. These results are in contrast to those of previous studies using phlorizin and 5-thioglucose.16 17 37 ICV phlorizin in both the LV and 4V reportedly induced a similar hyperphagic effect.17 In another report, ICV infusion of 5-thioglucose into both the LV and the 4V increased food intake, and this effect was abolished only in the LV after obstruction of the cerebral aqueduct.37 According to one report, SGLT2/SLC5A2 was detected in the hypothalamus, amygdala, periaqueductal gray, and the nucleus of the solitary tract by immunohistochemical analysis.12 Another report showed that SGLT2/SLC5A2 is present in ependymal cells and choroid plexus epithelial cells.11 Thus, there is still no consensus regarding which CNS locations express SGLT2/SLC5A2. GLUTs are the primary transporter of glucose uptake in the CNS, and the contribution of SGLTs is low.38 Although differing in selectivity, tofogliflozin and phlorizin caused completely different effects when administered 4V-ICV, despite being SGLT inhibitors. These results suggest that the tofogliflozin target exists mainly in the forebrain, suggesting that SGLT2/SLC5A2, as a target of tofogliflozin, is present in the forebrain. This assumption is consistent with the sites previously suggested to harbor SGLT2/SLC5A2.12 Another possibility is that spinal fluid flows from the LV to the lower ventricles, and the SGLT2 inhibitor thereby reaches more SGLT2-expressing ependymal cells expressed around the cerebral ventricle, which may explain why the effect is stronger in the upper ventricles.
In the present study, ICV administration of tofogliflozin increased c-fos expression and tended to suppress AgRP and NPY, without significant changes in POMC in the ARC. c-fos belongs to the Fos family,39 and various stimuli are known to selectively induce its expression.40 41 Oral administration of SGLT2 inhibitors has been reported to enhance c-fos expression in the PVH, ARC, and LH, consistent with our observations.12 AMPK is known to be the main sensor of cellular energy status, and coordinates metabolism at the whole-body level.42 In the hypothalamus, regulation of AMPK phosphorylation is a mechanism for detecting nutritional changes.33 43 Early researchers referred to the LH and VMH as feeding and satiety centers, respectively.44 More recently, the LH has been suggested to be a core node within a distributed feeding network that integrates different feeding drives and motivates behaviour.45 The present study showed that ICV infusion of tofogliflozin activated AMPK in the LH, consistent with a previous report that showed that systemic hypoglycemia could induce AMPK phosphorylation in the LH.46 These observations suggest that SGLT2/SLC5A2 in the CNS directly or indirectly regulates feeding via AMPK phosphorylation in the LH. It was reported that the ARC projects to the LH,47 which may play an important role in feeding.48 Taken together, it is possible that tofogliflozin acts on the LH to enhance feeding, which in turn may induce compensatory suppression of AgRP and NPY expression. Systemic administration of exendin-4, a GLP-1 receptor agonist, has been reported to decrease AMPK phosphorylation in the LH.49 In the present study, systemic injection of liraglutide blocked ICV infused tofogliflozin-mediated hyperphagia. One possible mechanism involves inhibition by GLP-1 of ICV administered SGLT2 inhibitor driven AMPK phosphorylation in the LH. Our findings may further clarify the role of the LH in food intake. Further studies using ICV-administrated AMPK inhibitors (ie, SBI-0206965) or allosteric activators (ie, A-769662, MK-8722, or PF-06409577) with tofogliflozin are necessary to further demonstrate that SGLT2 inhibitors enhance feeding via AMPK phosphorylation in the LH. The combination of SGLT2 inhibitors and GLP-1 receptor agonists reportedly results in greater weight loss than the use of a single agent in humans.50 Our results may provide a basic research perspective impetus for investigating the usefulness of combining SGLT2 inhibitors and GLP-1 receptor agonists.
The present study has several limitations. We did not prove that SGLT2/SLC5A2 regulates food intake. To determine the function of SGLT2/SLC5A2 in the brain, it is necessary to conduct experiments using knockout models. However, administering an SGLT2 inhibitor into the LV dramatically enhanced food intake, suggesting that the SGLT2 inhibitor acts directly in the CNS to induce hyperphagia. Another limitation is the dose of SGLT2 inhibitor administered to the CNS in this experiment, which would be higher than that of clinical use. To our knowledge, it is not known how much of the orally administered SGLT2 inhibitors could be transferred to the CNS. Thus, in addition to the acute and chronic effects on the CNS, as shown in this study, chronic energy loss via the excretion of urinary glucose could also induce compensatory food intake.
In summary, our study suggests that direct ICV administration of an SGLT2 inhibitor into the CNS increases food intake via phosphorylation of AMPK in the LH. While the physiological function of SGLT2 in the CNS has not been confirmed, our results support the theory that CNS SGLT2/SLC5A2 plays a regulatory role in food intake. This result may explain the increased food intake observed with the use of SGLT2 inhibitors in clinical practice. If we can develop SGLT2 inhibitors with lower or absent BBB penetration, diabetic treatment with reduced compensatory food intake might become possible.