The Potential Role of a Novel Metformin-Methylglyoxal Imidazolinone Metabolite (IMZ) in Alleviating Complications from T2DM

Reactive dicarbonyls, such as methylglyoxal (MGO), are elevated in the plasma of patients with type-two diabetes mellitus (T2DM). These endogenous electrophiles covalently modify proteins, which may contribute to diabetic complications. The T2DM first-line therapy, metformin (MF), significantly reduces adverse diabetic endpoints and mortality more effectively than other anti-hyperglycemic agents, the mechanism(s) of which remain unclear. We previously identified and characterized the product of the MF and MGO reaction as a novel imidazolinone metabolite (IMZ) in plasma and urine from patients with T2DM. The formation of IMZ, via scavenging of MGO by MF, represents a possible alternative mechanism of MF drug efficacy, in addition to its antigluconeogenesis properties. Imidazoline receptors (I1R-I3R) are novel targets for drug development in disorders associated with T2DM because they are involved in insulin secretion/sensitization and glucose homeostasis. Thus, we examined the ability of IMZ to modulate insulin-mediated cell signaling pathways in PC12 (express high levels of I1R and lack the α2-adrenergic receptor) and insulin resistant HEPG2 (a well-established model for assessing human liver metabolism) cells. In addition, the effects of IMZ on insulin secretion in MIN6 (best reflect normal pancreatic β-cell physiological conditions) cells were explored. Combination treatment of insulin and IMZ at physiologically relevant concentrations (1 pM & 1 nM) increased AKT and ERK1/2 phosphorylation above levels seen with insulin treatment alone in PC12 cells. This potentiation was not observed in the presence of the I1R antagonists efaroxan and idazoxan. IMZ also restored insulin sensitivity in insulin resistant HEPG2 cells via the AKT signaling pathway. Moreover, insulin secretion was increased in MIN6 cells exposed to various concentrations of IMZ (1 pM-1 uM) and this augmentation was inhibited by the I3R antagonist KU14R and siRNA mediated knockdown of nischarin (mouse homolog of imidazoline receptor antisera- selected protein). In addition, we explored the antidiabetic effects and possible intracellular signaling mechanisms engaged by IMZ in the well-established genetically obese db/db diabetic mouse model. Our results revealed that seven-day treatment of IMZ significantly alleviated insulin resistance, islet hypertrophy, hyperlipidemia, hepatic steatosis, adipocyte hypertrophy, and renal histopathological changes in db/db mice. In addition, IMZ promoted hepatic SIRT1 expression and the downregulation of SREBP1, ChREBP, ACC, and FAS at the protein and mRNA level in db/db mice. Finally, we investigated the long-term protective effects and potential mechanisms of activity exerted by IMZ in db/db mice alongside a clinically relevant dose of MF. The results from these studies demonstrated that IMZ significantly alleviated hyperglycemia, insulin resistance, islet hypertrophy, hyperlipidemia, hepatic steatosis, adipocyte hypertrophy, and renal histopathological changes similarly to MF in db/db mice treated with IMZ for 60 days. In addition, IMZ promoted hepatic downregulation of the lipogenesis and fibrosis markers ACACA, FASN, FN1, COL1A1, and TGFβ1 at the protein and mRNA level in db/db mice comparably to MF. The work in this dissertation therefore describes for the first time the protective effects of a MF metabolite in vitro and in vivo. Taken together, these findings support the hypothesis that the formation of IMZ may contribute to the antidiabetic properties of MF, with the potential to provide the structural basis of an effective agent in the management of T2DM. Although the precise underlying mechanism(s) of IMZ’s pharmacological action remain to be fully elucidated, the present work opens an intriguing window on the mechanism of the antidiabetic properties of MF

Mechanistic basis of altered morphine disposition in nonalcoholic steatohepatitis

Morphine is metabolized in humans to morphine-3-glucuronide (M3G) and the pharmacologically active morphine-6-glucuronide (M6G). The hepatobiliary disposition of both metabolites relies upon multidrug resistance-associated proteins Mrp3 and Mrp2, located on the sinusoidal and canalicular membrane, respectively. Nonalcoholic steatohepatitis (NASH), the severe stage of nonalcoholic fatty liver disease, alters xenobiotic metabolizing enzyme and transporter function. The purpose of this study was to determine whether NASH contributes to the large interindividual variability and postoperative adverse events associated with morphine therapy. Male Sprague-Dawley rats were fed a control diet or a methionine- and choline-deficient diet to induce NASH. Radiolabeled morphine (2.5 mg/kg, 30 µCi/kg) was administered intravenously, and plasma and bile (0-150 or 0-240 minutes), liver and kidney, and cumulative urine were analyzed for morphine and M3G. The antinociceptive response to M6G (5 mg/kg) was assessed (0-12 hours) after direct intraperitoneal administration since rats do not produce M6G. NASH caused a net decrease in morphine concentrations in the bile and plasma and a net increase in the M3G/morphine plasma area under the concentration-time curve ratio, consistent with upregulation of UDP-glucuronosyltransferase Ugt2b1. Despite increased systemic exposure to M3G, NASH resulted in decreased biliary excretion and hepatic accumulation of M3G. This shift toward systemic retention is consistent with the mislocalization of canalicular Mrp2 and increased expression of sinusoidal Mrp3 in NASH and may correlate to increased antinociception by M6G. Increased metabolism and altered transporter regulation in NASH provide a mechanistic basis for interindividual variability in morphine disposition that may lead to opioid-related toxicity

Mechanistic Basis of Altered Morphine Disposition in Nonalcoholic Steatohepatitis

Morphine is metabolized in humans to morphine-3-glucuronide (M3G) and the pharmacologically active morphine-6-glucuronide (M6G). The hepatobiliary disposition of both metabolites relies upon multidrug resistance-associated proteins Mrp3 and Mrp2, located on the sinusoidal and canalicular membrane, respectively. Nonalcoholic steatohepatitis (NASH), the severe stage of non-alcoholic fatty liver disease, alters xenobiotic metabolizing enzyme and transporter function. The purpose of this study was to determine whether NASH contributes to the large interindivid-ual variability and postoperative adverse events associated with morphine therapy. Male Sprague-Dawley rats were fed a control diet or a methionine- and choline-deficient diet to induce NASH. Radiolabeled morphine (2.5 mg/kg, 30 mCi/kg) was administered intravenously, and plasma and bile (0–150 or 0–240minutes), live

Metformin Scavenges Methylglyoxal To Form a Novel Imidazolinone Metabolite in Humans

Methylglyoxal (MG) is a highly reactive dicarbonyl compound involved in the formation of advanced glycation endproducts (AGE). Levels of MG are elevated in patients with type-2 diabetes mellitus (T2DM), and AGE have been implicated in the progression of diabetic complications. The antihyperglycemic drug metformin (MF) has been suggested to be a scavenger of MG. The present work examined and characterized unequivocally the resulting scavenged product from the metformin–MG reaction. The primary product was characterized by ¹H, ¹³C, 2D-HSQC, and HMBC NMR and tandem mass spectrometry. X-ray diffraction analysis determined the structure of the metformin and MG-derived imidazolinone compound as (<i>E</i>)-1,1-dimethyl-2-(5-methyl-4-oxo-4,5-dihydro-1<i>H</i>-imidazol-2-yl)­guanidine (IMZ). A LC-MS/MS multiple reaction monitoring method was developed to detect and quantify the presence of IMZ in metformin-treated T2DM patients. Urine from >90 MF-treated T2DM patients was analyzed, with increased levels of MF directly correlating with elevations in IMZ. Urinary MF was detected in the range of 0.17 μM to 23.0 mM, and simultaneous measurement of IMZ concentrations were in the range of 18.8 nM to 4.3 μM. Since plasma concentrations of MG range from 40 nM to 4.5 μM, the level of IMZ production may be of therapeutic significance. Thus, in addition to lowering hepatic gluconeogenesis, metformin also scavenges the highly reactive MG <i>in vivo</i>, thereby reducing potentially detrimental MG protein adducts, with subsequent reductions in diabetic complications