Galantamine improves glycemic control and diabetic nephropathy in Leprdb/db mice

Galantamine, a centrally acting acetylcholinesterase inhibitor, has been shown to attenuate inflammation and insulin resistance in patients with metabolic syndrome. We investigated the effects of galantamine on glycemic control and development of diabetic nephropathy (DN) in Leprdb/db mice. Galantamine significantly reduced food intake, body weight, blood glucose and HbA1c levels. Insulin resistance (HOMA-IR, QUICKI), HOMA-β and elevations in plasma inflammatory cytokine levels (TNF-α, IL-6 and HMGB-1) were all attenuated by galantamine. Galantamine also ameliorated diabetes-induced kidney injury as evidenced by improvements in renal function (BUN, creatinine, albuminuria), histologic injury and apoptosis. Improved glycemic control and nephropathy were associated with increased circulating GLP-1, decreased renal P-38 MAPK and caspase-1 activation and reduced SGLT-2 expression. These findings provide insights into the mechanisms by which galantamine improves glycemic control and attenuates DN in the Leprdb/db mouse model.

Galantamine improves DN in db/db mice.The db/db mouse develops obesity by 4 weeks followed by hyperglycemia with glucosuria and albuminuria by 8 weeks of age.In db/db mice, DN is characterized by albuminuria, podocyte loss, and mesangial matrix expansion and increase in glomerular size 12,13,27 .Elevations in plasma BUN and creatinine are also seen.Histologic evidence of DN develops from 8 to 16 weeks including glomerular hypertrophy and mesangial matrix expansion.Increases in the extracellular matrix proteins type IV collagen and fibronectin, nodular glomerulosclerosis, and tubulointerstitial injury (tubular atrophy, dilatation, apoptosis) are seen by 4 months 12,13 .Homozygous db/db mice aged 16-17 weeks were used for the study of DN.Blood samples were collected at weeks 1 and 8 from mice given galantamine or saline for 8 weeks (4 mg/ kg, i.p., once daily), while urinary samples were collected weekly for 8 weeks.After 8 weeks of treatment plasma and kidneys were analyzed for renal function, histology, apoptosis, SGLT-2 expression, activation of P38 MAPK signaling and caspase-1 activation.
Vehicle-treated db/db mice demonstrate a significant increase in plasma BUN (Fig. 5A) by week 8 (**P < 0.01, week 1 vs. week 8).In contrast, galantamine-treated db/db mice demonstrate reductions in plasma BUN (Fig. 4A) and creatinine (Fig. 4B) (*P < 0.05, week 1 vs. week 8).Compared with vehicle-treated controls, the galantaminetreated db/db mice demonstrate improvements in plasma BUN and creatinine at 8 weeks (P < 0.05, vehicle vs. galantamine).Consistent with this finding, albuminuria in the galantamine-treated db/db mice is markedly improved (Fig. 4C).Urinary creatinine levels were similar between the groups (Fig. 4D), whereas the calculated albumin to creatinine ratio (ACR, Fig. 4E) was increased in the saline treated db/db mice compare to the galantamine-treated group (*P < 0.05, vehicle vs. galantamine).Collectively, these results provide evidence galantamine attenuates the development of DN in the db/db mouse model.
Next, we examined the effects of galantamine on renal histology.Kidneys were harvested from db/+ mice and from db/db mice treated with saline or galantamine for 8 weeks.H&E (Fig. 5A) or PAS (Fig. 5B) stained sections were assessed by a renal pathologist blinded to the experimental group.Characteristic histologic changes of DN (glomerular diameter, mesangial matrix expansion, nodular glomerulosclerosis, acute pyelonephritis and acute tubular necrosis) were quantified using the scoring system in Table 1.Compared with db/+ mice, the db/db mice showed signs of significant chronic glomerular injury: moderate mesangial matrix expansion with nodular formation and glomerular hypertrophy (glomerular diameter of 95 μm in db/db mice vs. 75 μm in db/+ mice).In addition, db/db mice showed more active tubular injury with inflammation (clusters of neutrophils and lymphocytes in the renal pelvis and cortical tubulointerstitium shown in supplemental file: s1.file).Administration of galantamine for 8 weeks reduced the histologic changes (glomerular hypertrophy, mesangial matrix expansion, tubular injury) and renal impairment scores in db/db mice, an effect that was significant in comparison to the saline control (Fig. 5C, *P < 0.05).These findings further confirm our hypothesis that galantamine improves histologic evidence of DN in db/db mice.
Apoptosis of tubular epithelial cells is a major feature of DN in the db/db mouse model which is triggered by the release of cytochrome c from damaged mitochondria.Galantamine-treated db/db mice demonstrated decreased levels of cytochrome c in the kidney (Fig. 6A) compared with vehicle treated mice (*P < 0.05, galantamine vs. vehicle).The apoptotic environment was assessed by measuring renal levels of pro-apoptotic proteins Figure 1.Effects of galantamine on food intake, body weight and glycemic control.All mice (7-8 weeks old) received galantamine (4 mg/kg, i.p., once daily) or vehicle for 4 weeks.Food intake and body weight were monitored daily.Food intake in db/+ mice in (A) and db/db mice in (B) were calculated daily.The changes in body weight (%) in db/+ mice in (C) and db/db mice in (D) were analyzed.The data is represented as mean ± SE (n = 5/group), *P < 0.05.OGTT was performed at week 4 in db/db mice in (E) and db/+ mice in (F) mice, as quantified by AUC analysis in (G) HbA1C in (D) was assayed using A1CNow + .The data is represented as mean ± SE (n = 5-12/group), *P < 0.05.
(BAX and cleaved caspase-3) and the anti-apoptotic protein Bcl2.Kidneys from db/db mice demonstrate a proapoptotic environment characterized by increased levels of Bax (Fig. 6B) and cleaved caspase-3 (Fig. 6C) with reductions in Bcl2 (Fig. 6B).All of these changes were significantly attenuated by galantamine.Consistent with this observation, the relative abundance of TUNEL-positive cells in kidneys from db/db mice were reduced by galantamine-treatment (Fig. 6D), with quantification of apoptotic cells shown in Fig. 6E (**P < 0.01, vehicle vs. galantamine).

Circulating GLP-1 levels and renal SGLT-2 expression.
To explore potential mechanisms by which galantamine improves glycemic control and DN we measured circulating glucagon-like peptide-1 (GLP-1) levels, renal Na + /glucose cotransporter-2 (SGLT-2) protein, mRNA expression and urinary glucose levels.GLP-1 is released by activation of the α7nAChRs on intestinal L cells and reduces postprandial elevations in blood  glucose by multiple mechanisms 28 .SGLT-2 is a sodium-dependent glucose transporter found in renal proximal tubule (PTC) cells which reabsorbs up to 90% of filtered glucose in the kidney.Changes in the abundance or activity of renal SGLT-2 can significantly impact glycemic control by altering urinary glucose absorption.
Galantamine administration significantly increased plasma levels of GLP-1 (Fig. 7A; *P < 0.05).In addition, galantamine-treated db/db mice also demonstrated decreased levels of SGLT-2 protein in renal tubules by     www.nature.com/scientificreports/cleaved caspase-1 were decreased in kidney from db/db mice treated with galantamine (*P < 0.05, vehicle vs. galantamine).These results provide evidence that the p38 and caspase-1 signaling pathways are downregulated by galantamine in kidney from db/db mice.

Discussion
The current study examines the effects of galantamine, a centrally acting acetylcholinesterase inhibitor approved for treating Alzheimer's disease on glycemic control and DN in the db/db mouse model.The db/db mouse has a mutant leptin receptor which results in impaired satiety, hyperphagia and obesity which is commonly used to study T2DM and its complications.Our results provide evidence galantamine acts to improve glycemic control by reducing food intake, promoting weight loss, reducing systemic inflammation, improving insulin sensitivity, stimulating GLP-1 secretion, and decreasing renal SGLT-2 expression.While improvements in glycemic control undoubtedly help to prevent renal injury, galantamine also appears to attenuate renal apoptosis and inflammation, likely via its effects on the p38 MAPK and NLRP3 inflammasome/Caspase-1 pathways.Perhaps more interesting is the support our study provides for an important role of the CAP in the regulation of obesity, diabetes, and DN.The effects of galantamine on food intake and weight loss in our study are supported by the results of others which show reductions of food intake and body weight in obese high-fat diet-fed mice 30 , the streptozotocininduced (STZ) rat model of diabetes 31 , and in galantamine-treated patients with dementia 32 .Of interest, treatment with specific α7nAChR agonists like GTS-21 do not seem to impact food intake or body weight 33 .However, like galantamine, vagal nerve stimulation (VNS) has also been shown to suppress appetite, increase circulating GLP-1 and decrease inflammation [34][35][36] .The improvements in glycemic control and insulin resistance in our study are supported by similar findings in high-fat diet mice 30 and the STZ rat model 31 .Galantamine was also shown to reduce inflammatory cytokine levels, insulin resistance and blood glucose levels in a double-blind clinical trial in patients with metabolic syndrome (ClinicalTrials.govIdentifier: NCT02283242) 37 .These changes were associated with alterations in autonomic tone suggesting involvement of the vagus nerve.Our results that galantamine treatment reduced inflammatory cytokines (TNF-α, IL-6, HMGB-1), improved insulin resistance and glycemic control in the db/db mice are consistent with those of the clinical trial of galantamine in patients with metabolic syndrome.Of interest, previous work from our laboratory showed that treatment of db/db mice with α7nAChR agonists (GTS-21 or PNU-282987) increased GLP-1 secretion and improved oral glucose tolerance by a mechanism requiring an intact GLP-1 receptor 21 .This suggests that some, but perhaps not all galantamine's effects (e.g., decreased food and weight loss) on glycemic control and inflammation are mediated by the α7nAChR and GLP-1 receptor at the tissue level.
Galantamine likely improves glycemic control via multiple mechanisms including reduced food intake, body weight, increased circulating GLP-1 and decreased renal SGLT-2 expression.The relative importance of changes in food intake and body weight are difficult to ascertain since pair-feeding controls were not part of our study design.Endogenous GLP-1 regulates glycemia via its actions on the gastrointestinal tract, the vagus nerve, as well as pancreatic β cells (↑insulin synthesis, and secretion, ↓ apoptosis) and α cell (↓glucagon secretion).For a comprehensive review on GLP-1 regulation of glycemic control, see the article by Drucker 38 .
One of the more novel findings of our study are the reductions in renal SGLT-2 expression and glucosuria as a potential mechanism for improved glycemia in galantamine-treated db/db mice.Located on the luminal surface of the proximal renal tubule, SGLT-2 is a sodium-glucose cotransporter that is normally responsible for the reabsorption ~ 90% of the glucose filtered by the glomerulus 39 .SGLT-2 expression appears to be increased in the diabetic kidney and contributes to the development of hyperglycemia in T2DM.Consistent with this finding, SGLT-2 inhibitors (e.g., canagliflozin, empagliflozin) are highly effective in treating hyperglycemia in T2DM by enhancing glycosuria.Therefore, the reduction in renal SGLT-2 expression and increased glucosuria observed in galantamine-treated db/db mice likely contributed to the improvements on glycemic control we observed in our study.
Our results also show a beneficial effect of galantamine on renal function, albuminuria, histologic injury, and apoptosis in the db/db mouse model.Some of these findings are potentially attributable to the ability of galantamine to activate the α7nAChR since GTS-21 has also been shown to attenuate renal injury, histology, inflammation, and apoptosis in the db/db mouse model.However, galantamine's effects on renal SGLT-2 represent another potential mechanism for its protective effects on DN in our study.SGLT-2 inhibitors have been shown to attenuate the development of DN in patients with T2DM.Several mechanisms have been identified for the beneficial effects of SGLT-2 inhibitors on DN including: (1) the restoration of tubuloglomerular feedback with reversal of afferent arteriole vasodilation and glomerular hyperperfusion; (2) a reduction in hyperglycemiamediated reactive oxygen species (ROS); and (3) reduced renal tubular injury and inflammation 40,41 .Particularly interesting is the ability of SGLT-2 inhibitors to attenuate podocytopathy, elevate glomerular nephrin expression 42 and alter glomerular distribution and size 15 in db/db mice.Renal SGLT-2 expression and activity are regulated by filtered glucose levels, oxidative stress-induced inflammation, angiotensin II and its effects on epidermal growth factor activation 43 .Therefore, it is tempting to speculate that galantamine-mediated effects on glycemic control and inflammation are involved in the changes in renal SGLT-2 levels we observed in our study.Additional studies will be required to further delineate the relative importance of these SGLT-2 mediated mechanisms in the beneficial effects of galantamine on DN.
In the current study, down-regulation of renal apoptosis by galantamine was characterized by a reduction in the relative abundance of pro-apoptotic proteins (cytochrome c, BAX, and caspase-3) and increase in the anti-apoptotic protein (Bcl-2).There is evidence that SGLT-2 inhibitors provide an antioxidant mechanism of renoprotection [44][45][46] .However, our laboratory has also shown that α7nAChR activation by GTS-21 reduces apoptosis via regulating cytochrome c, BAX, Bcl-2, and caspase-3 in db/db mice 22 26 .The finding GTS-21 attenuates p38 MAPK activity and renal apoptosis in db/db mice is consistent with the effects of galantamine on p38 MAPK activity in the current study 22 .Our results also provide evidence galantamine attenuates renal caspase-1 activation by the NLRP3 inflammasome in the db/db model of DN.These findings are consistent with Shahzad et al. who demonstrate a protective effect of caspase inhibition and caspase-1 deficiency on DN in their study 47 .They also find that podocyte-specific caspase-1 genetic deficiency improves DN.Collective these studies support the idea that therapies like galantamine which enhance glycemic control, reduce renal inflammation, apoptosis, P38 MAPK and caspase-1 activation work through similar signaling pathways to prevent the progression of DN 48 .Nonetheless, there are several limitations to our study.First, while the murine model of T2DM is useful experimentally, mutations in the leptin receptor are rare in humans.In addition, there are differences in insulin secretion and glucose metabolism between humans and mice, therefore our findings may not fully translate to the human condition.We also acknowledge that HOMA-IR and -B were designed and validated to study insulin sensitivity and β cell function in humans, despite this limitation they are commonly used to compare insulin resistance and β cell function in murine models of T2DM.Although we show galantamine ameliorates both glycemia and DN in the db/db model, the relative importance of vagal activation vs. α7nAChR activation and decreased renal SGLT-2 expression requires additional study.Finally, although we demonstrated galantamine improves both glycemia and DN in the db/db model, the pleiotropic effects of galantamine and our experimental design prohibit us from explicitly delineating the exact mechanisms for improvements in T2DM and DN.
In conclusion, our study provides evidence that galantamine acts to improve glycemic control in the db/db mouse by reducing food intake, enhancing weight loss, decreasing systemic inflammation, improving insulin sensitivity, stimulating GLP-1 secretion and decreasing renal SGLT-2 expression.Galantamine also attenuates the development of DN by improving glycemic control, reducing renal apoptosis and inflammation, in part through its effects on the 38 MAPK and NLRP3 inflammasome/Caspase-1 pathways.Collectively our data provide support for a potentially important role of the CAP in the regulation of obesity, diabetes and DN.

Methods
Animal models.Our studies with mice were approved by the Institutional Animal Care and Use Committee of SUNY Upstate Medical University (IACUC # 423).These studies were performed in accordance with the National Institutes of Health and ARRIVE guidelines for the use of laboratory animals.Heterozygous Lepr db/+ (db/+) mice (strain: BKS.Cg-Dock7 m +/+ Lepr db /J, Cat.No. 000642) were purchased from The Jackson Laboratory (Bar Harbor, ME).Homozygous Lepr db/db (db/db) mice were obtained by breeding db/+ mice.Meanwhile, the obtained db/+ littermates as non-diabetes control were used to evaluate the effect of galantamine in diabetic and non-diabetic mice since db/+ mice do not exhibit the diabetic phenotype.Breeding of mice was performed at the Animal Core Facility at SUNY Upstate Medical University.Mice were housed in a temperature-controlled room at 22 °C.Mice aged 7-8 (for glycemic control study) 12 and 16-17 (for DN study) 12,13 weeks were randomly assigned to different treatment groups and matched for age and sex.Mice were housed in separate cages to measure daily food intake and body weight.

Oral glucose tolerance test (OGTT).
All mice were fasted overnight prior to administration of glucose (2 g/kg body weight) by oral gavage.Tail blood samples were obtained before (time 0) and at 30, 60, 90 and 120 min after the glucose challenge for determination of blood glucose concentrations using an AimStrip Plus Blood Glucose Meter Kit (Cat.No. 37321, Germaine ® Laboratories, Inc, San Antonio, TX).The area under the curve (AUC) for OGTTs was calculated using the trapezoidal rule. 26and the quantitative insulin sensitivity check index (QUICKI).HOMA is a method for assessing beta-cell function (HOMA-β) and insulin resistance (HOMA-IR) from fasting glucose and insulin 49 .HOMA-β has been reported to correlate with β-cell function assessed by oral glucose tolerance test or hyperglycemic clamp test 50,51 .QUICKI is a useful index of insulin sensitivity and has linear correlation with glucose clamp determinations of insulin sensitivity 52 .The formulas used to calculate the three parameters are: HOMA-β = 360 × fasting insulin (μU/mL)/(fasting glucose (mg/ dL) − 63).HOMA-IR = fasting glucose (mg/dL) × fasting insulin (μU/mL)/405.QUICKI = 1/[log (fasting insulin (μU/mL) + log (fasting glucose (mg/dL)].

Galantamine drug delivery protocol and animal care.
1.The effect of galantamine on glycemic control, food intake, body weight and inflammation in db/db and db/+ mice.Mice were divided into 4 groups at 7-8 weeks of age: (1) db/+ Vehicle, (2) db/+ Galantamine, db/db Vehicle, and (4) db/db Galantamine.Mice were then transferred to individual housing.These groups of mice were injected intraperitoneally (IP) once a day (Q.D.) for 4 weeks with the vehicle or Galantamine (4 mg/kg).Body weight and food intake were monitored daily and were calculated.OGTT was performed at week 4 of galantamine treatment.Blood was also collected at this time for the measurements of insulin, glucose, inflammatory mediator (HMGB-1) and HB Ac1. 2. The effect of galantamine on DN in db/db mice.Mice were divided into 2 groups at 12-13 weeks of age: (1) db/db Vehicle, and (2) db/db Galantamine.These groups of mice were injected intraperitoneally (IP) once a day (Q.D.) for 8 weeks with the vehicle or Galantamine (4 mg/kg).Body weight and food intake were

Figure 2 .
Figure 2. Effects of galantamine on insulin resistance and β-cell function.All mice received galantamine (4 mg/ kg, i.p., once daily) or vehicle for 4 weeks.Glucose levels were checked in blood using glucometer.The plasma was collected for measuring insulin.HOMA-IR in (A), HOMA-β in (B) and QUICHI in (C) were calculated from the levels of glucose and insulin.The data is represented as mean ± SE (n = 3-15/group), *P < 0.05.

Figure 4 .
Figure 4. Effects of galantamine on renal function and injury.All mice (12-13 weeks old) received galantamine (4 mg/kg, i.p., once daily) or vehicle for 8 weeks.Blood samples were collected at weeks 1 and 8 for measuring plasma BUN in (A) and creatinine in (B).Urinary samples were collected weekly for 8 weeks for measuring urinary albumin in (C), creatinine in (D).ACR in (E) was calculate using urinary albumin and creatinine.The data is represented as mean ± SE (n = 3-5/group), *P < 0.05, **P < 0.01.

Figure 5 .
Figure 5. Histological assessment of DN.All mice (12-13 weeks old) were treated with galantamine (4 mg/kg, i.p., once daily) or vehicle for 8 weeks.Kidney sections were stained with H&E and PAS to assess the glomeruli and mesangial matrix, and detect acute pyelonephritis and ATN.Representative histological sections of the kidney are shown with H&E stain in (A) and PAS stain in (B).Magnification 400 ×.Histological changes in DN were characterized by glomerular diameter, acute pyelonephritis, ATN, percentage of glomeruli with nodular formulation and glomeruli mesangial expansion (green arrow).Quantification of renal injury was performed as described in "Methods" is shown in (C).The data is represented as mean ± SE (n = 4-6/group), *P < 0.05.

Figure 6 .Figure 7 .
Figure 6.Galantamine attenuates mitochondrial dysfunction and renal apoptosis.The db/db mice (12-13 weeks old) were treated with galantamine (4 mg/kg, i.p., once daily) or vehicle for 8 weeks.Western blot was used to measure apoptotic proteins (cytochrome c in (A), BAX and Bcl2 in (B), and caspase-3 in (C).Densitometry data for the individual proteins was normalized to β-actin and GAPDH and is presented as relative densitometry units (RDU).Histological sections were stained using TUNEL assay to detect apoptotic cells in (D).Magnification 400 ×.Immunofluorescence stain shows TUNEL-positive cells (dark brown, black arrow), nuclei (blue).Quantification of TUNEL-positive cells was performed and represented in (E).The data is represented as mean ± SE (n = 4/group), *P < 0.05.

Figure 8 .
Figure 8. Activation of the caspase-1 and p38 MAPK pathways in db/db mice.Mice (12-13 weeks old) were treated with galantamine (4 mg/kg, i.p., once daily) or vehicle for 8 weeks.Renal lysate was prepared, then caspase-1 and p38 protein were assayed by Western blot.Densitometry data for phosphorylated and total and cleaved caspase-1 in (A), total and phosphorylated p38 in (B) were normalized to β-actin and presented as RDU.The data is represented as mean ± SE (n = 4/group), NS no significant difference, *P < 0.05.
. Our current results showing reduction of renal p38 MAPK activation by galantamine are consistent with Lim et al. who reported that knockout of the p38 MAPK signaling pathway in db/db mice protects against renal dysfunction, albuminuria, renal hypertrophy, loss of podocytes, activation of mesangial cells, and glomerular fibrosis a