2.5.1. Streptozotocin (STZ)-Induced Diabetes Model
The effects of naringenin were examined in a few studies using streptozotocin (STZ)-induced animal models. T2DM was induced in vivo in neonatal Wistar rats by an intraperitoneal (i.p) injection of STZ (100 mg/kg) in the study by Li, et al., (2006) followed 21 days later by intestinal brush border membrane vesicle (BBMV) and renal cortical BBMV in vitro tissue isolation (
Table 5) [
65]. Naringenin treatment dose-dependently reduced intestinal sleeve and kidney glucose uptake. Additionally, naringenin dose-dependently decreased Na+-dependent glucose uptake activities in the diabetic rat kidney BBMVs [
65]. New Zealand White rabbits were also used in the above study [
65], mimicking the results in the neonatal Wistar rats. Naringenin treatment dose-dependently reduced glucose uptake in rabbit intestinal BBMVs [
65]. These studies provide evidence of an effect of naringenin to inhibit intestinal glucose absorption as well as renal glucose reabsorption, contributing to an attenuation of diabetic hyperglycemia (
Table 5).
Naringenin treatment (50 mg/kg b.w./day) for 5 days of STZ and nicotinamide-induced diabetic Wistar rats resulted in reduced blood glucose, total cholesterol and triglyceride levels [
66]. Additionally, serum high-density lipoprotein (HDL) levels were increased by naringenin treatment (
Table 5) [
66].
Naringin (30 mg/kg b.w.) and vitamin C (25 mg/kg b.w.) co-administered for 21 days in Wistar rats resulted in the attenuation of the hyperglycemia and oxidative stress induced by the single i.p. injection of STZ (45 mg/kg b.w.) (
Table 5) [
67]. Naringin treatment significantly reduced serum glucose levels and increased serum insulin levels, attenuating the diabetic phenotype. Additionally, treatment with naringin increased liver and kidney hexokinase activity and reduced liver and kidney glucose-6-phosphatase (G6Pase) and fructose-1,6-bisphosphatase (F16BPase) activities. Hexose, hexosamine, fucose and sialic acid levels were decreased in the plasma, kidney and liver of diabetic rats with naringin treatment [
67]. Therefore, the combined treatment of naringin and vitamin C provided antihyperglycemic and antioxidant effects attenuating the diabetic phenotype.
Administration of naringenin (1% and 2% of diet) for 10 weeks in STZ-induced diabetic mice resulted in significantly reduced blood glucose and urea levels, while serum insulin levels were increased (
Table 5) [
68]. Renal TNF-a, interleukin (IL)-1B, IL-6 and MCP-1 mRNA and protein levels were reduced. Furthermore, kidney type IV collagen, fibronectin and transforming growth factor-β1, mRNA and protein levels were reduced. This was accompanied with the suppression of kidney NF-κB p65 mRNA and protein levels [
68].
In the study by Sharma, et al., (2011), Wistar albino male rats fed a high-fat diet (55% fat, 2% cholesterol) and administered a single i.p. injection of STZ (40 mg/kg b.w.) to induce diabetes, were treated with naringin (25, 50 and 100 mg/kg b.w./day) for 28 days (
Table 5) [
69]. Naringin decreased serum glucose and insulin levels in a dose-dependent manner. Overall lipid profile was improved with naringin treatment with serum triglyceride, triacylglycerol, LDL cholesterol and non-esterified fatty acid levels reduced, and HDL-cholesterol levels increased [
69]. Additionally, treatment with naringin prevented pancreatic islet cell destruction and preserved β-cell insulin granule content. Liver and kidney PPARγ protein levels, and liver HSP-72 and HSP-27 protein levels were increased with naringin treatment. Pro-inflammatory cytokine NF-κB p 65 protein levels in the pancreas, liver and kidney and serum TNF-α, IL-6 and CBP levels were significantly reduced with naringin. Superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities were increased and thiobarbituric acid reactive substances (TBARS) levels were reduced in the kidney, pancreas and liver with naringin treatment [
69].
Diabetes was induced in albino Wistar rats by intraperitoneal injections of STZ (50 mg/kg b.w.) and nicotinamide (110 mg/kg b.w.) in a study by Annadurai, et al., (2012), followed by oral administration of naringenin (50 mg/kg b.w./day) for 21 days [
70]. Treatment with naringenin significantly decreased fasting serum glucose and glycosylated hemoglobin HbA1c levels, while insulin levels were increased. Additionally, naringenin significantly increased the activities of pancreatic antioxidant enzymes (SOD, catalase, GSH-Px and glutathione-S-transferase) and non-enzymatic antioxidants (glutathione (GSH), vitamin C and vitamin E) plasma levels (
Table 5) [
70].
Administration of naringin (50 mg/kg b.w.) for 4 weeks in male albino high-fat fed/STZ-induced diabetic rats reversed the diabetic phenotype [
71]. Naringin treatment significantly decreased serum glucose and HbA1c levels, while insulin levels were increased. Serum and hepatic lipid peroxide and nitric oxide levels were reduced in diabetic rats treated with naringin. Serum GSH levels were significantly increased with naringin treatment. Pro-inflammatory cytokines, TNF-α and IL-6 serum levels were significantly increased in diabetic animals and naringin treatment normalized (reduced) to levels similar to control (
Table 5) [
71].
Administration of naringenin (25 mg/kg p.o.) in high-fat diet fed/STZ-induced diabetic rats reduced the maltose- and sucrose-loaded glycemic response by 49.72% and 49.96%, respectively (
Table 5) [
72]. Additionally, naringenin significantly inhibited the activity of intestinal α-glucosidase resulting in decreased carbohydrate absorption and reduced postprandial blood glucose levels [
72].
Treatment of STZ-induced diabetic rats with naringenin (50 and 100 mg/kg) significantly decreased serum glucose levels, and increased serum SOD antioxidant activity [
73]. In addition, treatment with naringenin protected against diabetic hyperalgesia and tactile allodynia (
Table 5) [
73].
Administration of naringenin (50 mg/kg b.w.) for 30 days in male Wistar STZ-induced diabetic rats decreased blood glucose levels and decreased hepatocyte ROS and lipid peroxidation [
74]. These anti-hyperglycemic effects were accompanied by an increase in hepatocyte mitochondrial membrane potential and decreased hepatocyte mRNA and protein levels of apoptosis regulators Bax and Bcl-2 [
74]. Overall, treatment with naringenin provided hepatoprotection from STZ-induced diabetes.
Naringenin treatment (25 and 50 mg/kg b.w./day) for 5 weeks in STZ-induced diabetic rats reduced serum glucose levels and increased insulin levels [
75]. This was accompanied with a dose dependent decrease in serum pro-inflammatory cytokine levels TNF-α, IL-1β, and IL-6 with naringenin treatment. Sciatic nerve insulin growth factor (IGF) and nerve growth factor (NGF) levels were significantly increased in naringenin treated-diabetic rats compared to control diabetic rats. Additionally, SOD and catalase activities were significantly increased, while TBARs and GSH levels were decreased following naringenin treatment. This resulted in an attenuation of the diabetic phenotype with decreased sciatic nerve axonal degenerative histological changes (
Table 5) [
75].
Intragastric administration of naringenin (50 or 100 mg/kg/day) for 6 weeks in STZ-induced diabetic rats significantly decreased blood glucose levels [
76]. In addition, lipid, malonaldehyde and ICAM-1 serum levels were decreased with naringenin treatment and overall, a significant alleviation of the diabetic phenotype was seen (
Table 5) [
76].
Treatment of STZ and high-fat/high-sucrose fed-induced diabetic rats with naringenin (50 mg/kg b.w./day) for 6 weeks resulted in decreased serum glucose and urinary protein levels, indicating better kidney function (
Table 5) [
77]. This was accompanied with increased creatinine clearance and reduced glomerular area, reflecting improved renal filtration function. Naringenin reduced collagen (Col4) and fibronectin mRNA and protein levels indicating less deposition of extracellular matrix proteins in the kidneys. Furthermore, kidney TGF-β1, TGFBR1, smad2 and smad7 protein levels were decreased following naringenin treatment, while let-7a levels were increased. These results indicate naringenin acts to alleviate nephropathy in STZ-induced diabetic rats [
77].
Administration of naringenin (5–10 mg/kg b.w.) for 10 weeks in STZ-induced diabetic rats significantly decreased serum glucose levels, total cholesterol, triglyceride, LDL, VLDL, creatine, albumin and urea levels (
Table 5) [
78]. Kidney tissue malondialdehyde levels were also reduced following naringenin treatment. Treatment with naringenin increased SOD, catalase and GSH enzyme activities in the diabetic kidney. Naringenin treatment improved kidney tissue histology through reduced apoptotic activity. Additionally, a significant decrease in renal tissue IL-1 expression occurred with naringenin treatment [
78].
Oral administration of naringenin (50 mg/kg b.w./day) for 5 weeks in STZ (65 mg/kg b.w.)-induced diabetic rats significantly reduced serum TBARs levels and increased GSH levels [
79]. Additionally, naringenin increased the levels of neuroprotective factors (BDNF), tropomyosin related kinase B (TrkB) and synaptophysin in the diabetic retina. Anti-apoptotic Bcl-2 protein expression was improved following naringenin treatment, while pro-apoptotic Bax and caspase-3 protein expression was reduced. This was accompanied with the overall improvement of the diabetic phenotype, with decreased fasting blood glucose levels and increased insulin levels with naringenin treatment (
Table 5) [
79].
Oral administration of naringenin (100 mg/kg b.w./day) for 4 weeks in nicotineamide (120 mg/kg b.w.)/STZ (50 mg/kg b.w.)-induced diabetic rats alleviated the diabetic phenotype via insulinotropic effects and insulin improving action [
80]. Naringenin administration (100 mg/kg b. w./day for 4 weeks) restored the lowered insulin and C-peptide serum levels and liver glycogen content (
Table 5). The serum lipid profile was improved, HDL were increased while, total cholesterol, triglyceride, LDL, and FFAs levels were reduced and comparable to levels seen in healthy non-diabetic animals. The activities of liver G6Pase and glycogen phosphorylase were decreased with naringenin treatment. GLUT4, insulin receptor β subunit and adiponectin mRNA levels were enhanced in adipose tissue following naringenin treatment [
80].
Administration of naringenin (100 mg/kg/day) for 15 days in STZ-induced diabetic rats significantly reduced blood glucose levels [
81]. Naringenin treatment restored body weight and lipid serum levels in diabetic animals to levels similar to those found in the control non-diabetic group. Naringenin treatment normalized oxidative stress biomarkers in the liver and pancreas and increased PPARγ and GLUT4 mRNA and protein levels in adipose tissue (
Table 5) [
81].
Overall, these studies (
Table 5) indicate that naringenin administration in STZ-induced diabetic animals resulted in restoration of blood glucose and lipid levels, increased GLUT4 translocation in adipose tissue, and reduced diabetic nephropathy. In addition, naringenin administration resulted in anti-inflammatory and antioxidant properties.
2.5.4. Diet-Induced Diabetes Model
Apart from the studies using STZ- and alloxan-induced diabetic animal models, several studies exist using diet-induced diabetic animal models (
Table 8). Supplementation of naringin (0.05%) for 8 weeks in male rabbits fed a high-cholesterol (0.5% dietary intake) diet resulted in decreased plasma LDL and cholesterol levels and increased HDL levels [
86]. In addition, hepatic 3-hydroxy-3-methylglutaryl CoA reductase activity was increased, and acyl-CoA acyltransferase activity was reduced with naringin supplementation. Naringin supplementation significantly increased total fecal sterol content indicating increased intestinal cholesterol absorption [
86].
Administration of high-fat fed Ldlr
−/− mice with naringenin (1–3% wt/wt) for 4 weeks corrected the excess VLDL production, reduced hepatic steatosis and attenuated dyslipidemia [
87]. Importantly, these improvements were seen with naringenin administration without an effect on caloric intake or fat absorption [
87]. Naringenin treatment significantly increased hepatic fatty acid oxidation, liver Pgc1α, Cpt1α and Aco mRNA levels and mitochondrial DNA content. In addition, naringenin administration reduced hepatic cholesterol and cholesterol ester synthesis, prevented muscle and liver lipogenesis and muscle triglyceride accumulation and reduced plasma glucose and insulin levels (
Table 8) [
87].
Naringenin treatment (3% wt/wt) of high-fat fed Ldlr
−/− mice for 6 months resulted in decreased fasting plasma triglyceride and cholesterol levels [
38]. In addition, aortic plaque deposits and atherosclerosis in the aortic arch and abdomen was reduced with naringenin treatment. Liver triglyceride and cholesteryl ester mass was reduced by 80% indicating reduced hepatic steatosis with naringenin treatment (
Table 8) [
38].
Administration of naringenin (50 mg/kg b.w.) for 45 days in Wistar rats fed a high-fructose diet (60 g/100 g) decreased plasma glucose, insulin, triglyceride and free fatty acid levels [
43]. Treatment with naringenin restored liver hexokinase, pyruvate kinase, G6Pase and F16BPase activities to levels comparable to levels seen in healthy non-diabetic animals. In addition, administration of naringenin improved insulin sensitivity and enhanced liver protein tyrosine kinase (PTK), while reduced protein tyrosine phosphatase (PTP) activity (
Table 8) [
43].
Treatment of high-fat/high-sucrose fed rats with naringenin (0.003%, 0.006% and 0.012% of diet) for 6 weeks reduced the total plasma and liver triglyceride and cholesterol levels (
Table 8) [
88]. In addition, naringenin treatment decreased adiposity and triglyceride content in adipose tissue. Liver PPARα, CPT-1 and UCP2 protein levels were increased with naringenin [
88].
C57BL/6 mice fed a high-fat diet (37.1% fat, 42.2% carbohydrate and 20.5% protein) for 20 weeks resulted in the development of obesity, dyslipidemia, liver dysfunction and insulin resistance [
89]. Importantly, treatment with naringin (0.2 g/kg diet) for 20 weeks attenuated these changes (
Table 8). Naringin treatment increased hepatic fatty acid oxidation and increased AMPK phosphorylation/activation [
89]. Furthermore, treatment of hepatocytes, isolated from C57BL/6 mice fed a high-fat diet, with naringin resulted in increased p-AMPKα and p-IRS1 protein levels (
Table 8) [
89].
Treatment of high-fat fed C57BL/6J mice with naringenin (0.5%–3% of dietary intake) for 4 months significantly reduced blood glucose levels, and increased insulin levels [
54]. In addition, naringenin treatment decreased TNF-α, MCP-1 and TLR2 expression in adipose tissue (
Table 8) [
54].
In a study by Alam, et al. (2013) administration of naringin (100 mg/kg/day) for 16 weeks in high-fat/high-cholesterol fed Wistar rats resulted in improved glucose tolerance, with decreased serum glucose, insulin, cholesterol, triglyceride and non-esterified fatty acid (NEFA) concentrations (
Table 8) [
90]. Liver mitochondrial function was also improved with naringenin treatment, resulting in reduced inflammatory cell infiltration, collagen deposition, plasma aspartate transaminase (AST) and alanine transaminase (ALT) activity and increased mitochondrial respiration state 3 rates [
90].
Administration of naringenin (3% wt/wt) for 12 weeks in high-fat/high-cholesterol (HFHC) fed Ldlr
−/− mice increased hepatic fatty acid oxidation and attenuation of fatty acid synthesis (
Table 8) [
91]. Plasma glucose, insulin, total cholesterol, triglyceride, VLDL and LDLD levels were significantly reduced with naringenin treatment. Additionally, naringenin attenuated ApoB100 section by 80% in the HFHC-induced mice. Hepatic
Srebf1c and
Acox1 mRNA levels were reduced, while
Fgf21, Pgc1a, and
Cpt1a mRNA levels were increased with naringenin treatment. In addition, hepatic pro-inflammatory cytokine (
Tnfa, Il1b, Ccl2, and
Ccl3) mRNA levels were reduced [
91].
Treatment with naringenin (50 and 100 mg/kg/day) for 14 days had no effect on blood glucose levels of diabetic C57BL/6J mice fed a high-fat diet in a study by Yoshida, et al., (2014) [
92]. However, naringenin significantly repressed MCP-1 levels in adipose tissue and suppressed overall macrophage infiltration [
92]. These data indicate a potential of naringenin to prevent macrophage infiltration into adipose tissues and, therefore, prevent the inflammatory responses contributing to insulin resistance and diabetes (
Table 8).
Naringenin treatment (25 mg/kg p.o.) for 2 h in albino Wistar rats fed a high-fat diet significantly reduced the maltose- and sucrose-loaded glycemic response by 50.64% and 51.02%, respectively [
72]. Moreover, naringenin significantly inhibited α-glucosidase enzymatic activity leading to delayed intestinal carbohydrate absorption and reduced blood glucose levels (
Table 8) [
72].
Naringenin treatment (3% wt/wt) for 16 weeks of high fat diet-induced diabetic C57BL6/J and FGF21
−/− mice significantly reduced dyslipidemia and improved glucose tolerance [
93]. Naringenin treatment in both HFD-C57BL6/J and HFD-FGF21
−/− mice significantly reduced plasma glucose and insulin levels (
Table 8). Furthermore, treatment with naringenin reduced HFD-induced visceral and subcutaneous adipose tissue accumulation. Plasma leptin and TNFα levels were decreased with naringenin treatment to levels comparable to healthy non-diabetic animals. In addition, hepatic and white adipose
Pgc1a and
Cpt1a mRNA levels were significantly reduced, while
Pnpla2 and
Lipe mRNA levels were increased with naringenin treatment [
93].
Naringenin treatment (50 mg/kg b.w.) for 90 days in high-cholesterol (10 g cholesterol/kg and 1 g cholic acid/ kg) fed male Wistar rats significantly prevented renal failure (
Table 8) [
94]. Plasma and urine urea levels were significantly reduced, while creatinine clearance rates were increased with naringenin treatment. Histological parameters, white blood cells and platelets were increased with the high-cholesterol diet, and significantly reduced with naringenin treatment. Additionally, naringenin significantly decreased the lipid profile and kidney pro-oxidant inflammation marker (NTPDases, CD73, iNOS, TNF-α, IL-6 and NF-κB) mRNA levels [
94]. These data indicate that naringenin treatment improves renal function and may protect against diabetic nephropathy.
In a study by Krishnamoorthy et al., (2017), oral administration of naringenin (50 mg/kg b.w./day) for 6 weeks in high fructose-fed diabetic rats resulted in a significant increase in GLUT4 translocation in skeletal muscle [
48]. This was accompanied by increased in skeletal muscle AMPK phosphorylation and increased SIRT1 and PGC-1α protein levels (
Table 8) [
48].
Naringenin treatment (3% wt/wt) for 12 weeks in high-fat/high-cholesterol (24% fat and 0.2% cholesterol caloric intake) fed Ldlr
−/− mice significantly increased energy expenditure and hepatic fatty acid oxidation [
95]. Naringenin treatment significantly decreased body weight, epididymal fat accumulation, and visceral and subcutaneous fat volume. Adipose tissue inflammation was significantly reduced with decreased mRNA levels of pro-inflammatory genes,
Tnfa,
Ccl2 and
Ccl3. Naringenin also significantly reduced fasting serum insulin and glucose levels comparable to healthy non-diabetic animals. Skeletal muscle and hepatic triglyceride accumulation was reduced, while hepatic fatty oxidation gene (
Pgc1a and
Cpt1a) expression was increased with naringenin treatment (
Table 8) [
95].
Overall, these studies indicate that naringenin and naringin treatment of diet-induced diabetic animals resulted in restoration of serum glucose and lipid levels, increased liver and skeletal muscle fatty acid oxidation and increased skeletal muscle GLUT4 translocation (
Table 8).