Phloretamide Prevent Hepatic and Pancreatic Damage in Diabetic Male Rats by Modulating Nrf2 and NF-κB

**Background:** Type 1 diabetes mellitus (T1DM) is frequently complicated by nonalcoholic fatty liver disease (NAFLD), driven by oxidative stress, inflammation, and impaired antioxidant signaling—particularly involving the Nrf2/antioxidant axis. Phloretin, an apple-derived flavonoid, has known anti-diabetic and antioxidant properties mediated through Nrf2 activation. Phloretamide, a polyphenol metabolite of phloretic acid derived from phloretin, had not been previously characterized for its effects on diabetic complications. This study tested whether phloretamide could alleviate NAFLD in streptozotocin (STZ)-induced diabetic rats by modulating oxidative stress and inflammation.

**Methods:** Twelve-week-old male Wistar rats (220–240 g) were divided into 6 groups (n=8 each): (1) non-diabetic control (vehicle), (2) non-diabetic + phloretamide 100 mg/kg, (3) non-diabetic + phloretamide 200 mg/kg, (4) STZ-diabetic (vehicle), (5) STZ + phloretamide 100 mg/kg, (6) STZ + phloretamide 200 mg/kg. Diabetes was induced by a single intraperitoneal injection of STZ (50 mg/kg); rats with fasting glucose >320 mg/dL were included. Treatments were administered orally by gavage for 12 weeks. Measured endpoints included fasting glucose and insulin, HOMA-β, hepatic gluconeogenic enzymes (G-6-Pase, FBP-1), hexokinase, glycogen, serum and hepatic lipids (TG, CHOL, LDL-c, HDL-c, FFAs), oxidative stress markers (MDA, GSH, SOD, CAT, HO-1), inflammatory cytokines (TNF-α, IL-6), NF-κB p65 and Nrf2 levels (mRNA, total, nuclear), Keap-1/Nrf2 ratio, and histology of pancreas and liver.

**Key Results:** STZ-diabetic rats exhibited significantly reduced body weight, fasting hypoinsulinemia, hyperglycemia, elevated hepatic G-6-Pase and FBP-1, depleted hexokinase and glycogen, dyslipidemia (elevated serum and hepatic TG, CHOL, LDL-c, FFAs; reduced HDL-c), increased hepatic MDA, TNF-α, IL-6, NF-κB p65 (total and nuclear), and Keap-1/Nrf2 mRNA ratio, with depleted Nrf2 (total and nuclear) and reduced GSH, SOD, CAT, and HO-1. Pancreatic histology showed shrunken islets with reduced cell number and hemorrhage; livers showed cytoplasmic vacuolation, dilated sinusoids, and immune cell infiltration. Phloretamide at both doses (100 and 200 mg/kg) significantly reversed these changes in a dose-dependent manner in STZ-diabetic rats. Specifically, phloretamide 200 mg/kg increased body weight, raised fasting insulin, reduced fasting glucose, suppressed G-6-Pase and FBP-1, increased hexokinase and glycogen, reduced serum and hepatic TG and CHOL, reduced serum LDL-c and FFAs, increased HDL-c, reduced MDA, TNF-α, IL-6, and NF-κB p65, and increased GSH, SOD, CAT, HO-1, and Nrf2 (mRNA, total, nuclear) while reducing the Keap-1/Nrf2 ratio. However, even at the higher dose, most parameters did not fully return to non-diabetic control levels. In non-diabetic rats, phloretamide also reduced fasting glucose, G-6-Pase, FBP-1, and MDA, and increased hexokinase, glycogen, GSH, SOD, CAT, HO-1, and Nrf2 without affecting insulin, TNF-α, IL-6, or NF-κB.

**Clinical Implications:** This study provides the first in vivo evidence that phloretamide can ameliorate diabetes-associated hepatic steatosis through antioxidant and anti-inflammatory mechanisms involving Nrf2 activation and NF-κB suppression. The dose-dependent improvements in pancreatic beta-cell structure, glucose homeostasis, lipid profiles, and liver histology suggest therapeutic potential for T1DM-associated NAFLD. However, the authors acknowledge key limitations: the observational nature of the data, lack of Nrf2-knockout validation, and absence of pharmacokinetic data on naturally occurring phloretamide concentrations. Further preclinical studies in Nrf2-deficient models and clinical trials are needed before translation to human therapy.