Design, Synthesis, and Biological Evaluation of 1,4-Dihydropyridine-Indole as a Potential Antidiabetic Agent via GLUT4 Translocation Stimulation.

In the quest for the discovery of antidiabetic compounds, a series of 27 1,4-dihydropyridine-indole derivatives were synthesized using a diversity approach. These compounds were systematically evaluated for their antidiabetic activity, starting with an in vitro assessment for GLUT4 translocation stimulation in L6-GLUT4myc myotubes, followed by in vivo antihyperglycemic activity evaluation in a streptozotocin (STZ)-induced diabetic rat model. Among the synthesized compounds, 12, 14, 15, 16, 19, 27, and 35 demonstrated significant potential to stimulate GLUT4 translocation in skeletal muscle cells. Compound 19 exhibited the highest potency and was selected for in vivo evaluation. A notable reduction of 21.6% (p < 0.01) in blood glucose levels was observed after 5 h of treatment with compound 19 in STZ-induced diabetic rats. Furthermore, pharmacokinetic studies affirmed that compound 19 was favorable to oral exposure with suitable pharmacological parameters. Overall, compound 19 emerged as a promising lead compound for further structural modification and optimization.

NOD1 activation in 3T3-L1 adipocytes confers lipid accumulation in HepG2 cells.

AIMS: Activation of specific innate immune receptors has been characterized to modulate nutrient metabolism in individual metabolic tissue directly or indirectly via secretory molecules. Activation of the nucleotide-binding oligomerization domain-containing protein 1 (NOD1) in adipocytes has been reported to induce lipolysis linked with insulin resistance and inflammatory response. These cues are positioned to modulate metabolic action in distal organs through paracrine/endocrine signaling. Here, we assessed the role of NOD1-mediated lipolysis and inflammatory response in adipocytes to affect lipid metabolism in hepatocytes. MAIN METHODS: Human hepatoma cells (HepG2) were exposed to conditioned medium obtained from 3 T3-L1 adipocytes pretreated with NOD1 ligand (iE-DAP) and the effects on lipid accumulation, inflammation and insulin response were assessed. Activation of mechanisms leading to hepatic lipid accumulation was investigated by gene expression analysis. KEY FINDINGS: The conditioned medium from NOD1-activated 3 T3-L1 adipocytes (CM-DAP) induced lipid accumulation in HepG2 cells, driven by both lipolysis and inflammatory responses. The CM-DAP-induced lipid accumulation was independent to de novo lipogenesis and resulted from the enhanced transport of fatty acids inside and consequent increase in rate of triglycerides synthesis in hepatocytes. Moreover, CM-DAP-induced lipid accumulation instigated the expression of the markers of fatty acid oxidation and VLDL assembly for the export of triglycerides from hepatocyte. Furthermore, CM-DAP-induced lipid accumulation was associated with induction of inflammatory response and impairment of insulin signaling in HepG2 cells. SIGNIFICANCE: Beyond showing liver-specific mechanisms to adipocytes-derived factors, our findings support the involvement of adipose tissue as a mediator in NOD1-mediated biological responses to modulate hepatic metabolism.

Moringa oleifera impedes protein glycation and exerts reno-protective effects in streptozotocin-induced diabetic rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Moringa oleifera is a valued plant with wide distribution in tropical and subtropical regions of the world. It is traditionally used for the treatment of fever, infections, rheumatism, cancer, improving cardiac, renal and hepatic functions, and regulating blood glucose level. The plant has been scientifically reported for the anti-inflammatory, antioxidant, renoprotective, and anti-diabetic properties. Diabetic patients are prone to develop end-stage renal diseases due to incidence of diabetes-induced renal dysfunctions. Given that, increased production and accumulation of advanced glycation end-products (AGEs) play a conspicuous role in the development of diabetes-linked renal dysfunctions, nature-based interventions with AGEs inhibitory activity can prevent renal dysfunctions leading to renoprotection. AIM OF THE STUDY: The study aimed to demonstrate the preventive effects of the ethanolic extract of the leaves of Moringa oleifera (EEMO) on protein glycation and its further assessment for the renoprotective effect in diabetic rats. MATERIALS AND METHODS: Antiglycation activity of EEMO was assessed in vitro using bovine serum albumin. For reno-protective activity assessment, streptozotocin (STZ)-induced diabetic rats were orally treated with EEMO (100 mg/kg) or standard antiglycation agent aminoguanidine (100 mg/kg) for consecutive 8 weeks. The effects on glucose homeostasis, renal functions, and renal morphology were assessed by clinical biochemistry, molecular and histological examination. RESULTS: Presence of EEMO efficiently prevented glucose-, fructose- or methylglyoxal-mediated glycation of protein. Under in vivo set-up, compared to diabetic control rats, EEMO treatment effectively improved the glucose tolerance and body weight, and reduced the serum levels of triglycerides and total cholesterol. Additionally, EEMO administration significantly ameliorated renal dysfunctions in diabetic rats characterized by improved levels of creatinine, urea nitrogen, and uric acid in serum, and total protein level in urine, accompanied by improved kidney morphology. The diabetes-associated pro-inflammatory response characterized by upregulated expression of the inducible nitric oxide synthase (iNos), activation of nuclear factor kappa B (NF-κB) and the raised levels of inflammatory factors, interleukin-1 beta (IL-1ß) and interleukin-6 (IL-6) in renal tissue was significantly attenuated in EEMO-treated rats. Moreover, EEMO treatment diminished renal reactive oxygen species (ROS) levels in diabetic animals. CONCLUSIONS: Our study demonstrated that EEMO prevented AGEs formation and ameliorated renal dysfunctions in diabetic rats by blocking inflammatory/oxidative pathways. Our observations justify M. oleifera as a potential source of therapeutic interventions for diabetic nephropathy management.

Convergence of Fructose-Induced NLRP3 Activation with Oxidative Stress and ER Stress Leading to Hepatic Steatosis.

High fructose flux enhances hepatocellular triglyceride accumulation (hepatic steatosis), which is a prime trigger in the emergence of hepatic ailments. Nevertheless, the pathophysiology underlying the process is not completely understood. Emerging evidences have revealed the inputs from multiple cues including inflammation, oxidative stress, and endoplasmic reticulum (ER) stress in the development of hepatic steatosis. Here, we substantiated the role of NLRP3 inflammasome and its convergence with oxidative and ER stress leading to hepatic steatosis under high fructose diet feeding. Male SD rats were fed on 60% high fructose diet (HFrD) for 10 weeks and treated with antioxidant quercetin or NLRP3 inflammasome inhibitor glyburide during the last 6 weeks, followed by metabolic characterization and analysis of hepatic parameters. HFrD-induced hepatic steatosis was associated with the activation of NLRP3 inflammasome, pro-inflammatory response, oxidative, and ER stress in liver. Treatment with quercetin abrogated HFrD-induced oxidative stress, along with attenuation of NLRP3 activation in the liver. On the other hand, inhibition of NLRP3 signaling by glyburide suppressed HFrD-induced oxidative and ER stress. Both glyburide or quercetin treatment significantly attenuated hepatic steatosis, associated with mitigated expression of the lipogenic markers in liver. Our findings verified the association of NLRP3 inflammasome with oxidative and ER stress in fructose-induced lipogenic response and indicate that in addition to be a target of oxidative/ER stress, NLRP3 can act as a trigger for oxidative/ER stress to activate a vicious cycle where these cues act in a complex manner to propagate inflammatory response, leading to hepatic steatosis.

Fructose-mediated NLRP3 activation induces inflammation and lipogenesis in adipose tissue.

Adipose tissue plays a crucial role in energy intake and regulation of metabolic homeostasis. Fructose consumption implicates in development and progression of metabolic dysfunctions. Fructose is a lipogenic sugar known to induce inflammatory response. However, the role of specific inflammatory signal such as nucleotide-binding and oligomerization domain-like receptor, leucine-rich repeat and pyrin domain containing protein 3 (NLRP3) in fructose-induced inflammatory response and its relevance to lipogenesis in adipose tissue are elusive. We assessed NLRP3 activation and its significance in inflammatory response and lipogenesis in epididymal adipose tissue of 60% fructose diet (HFrD)-fed rats. The long-term consumption of HFrD led to impairment of glucose metabolism, development of visceral adiposity, insulin resistance, and elevation of serum triglycerides level, accompanied by activation of NLRP3 in adipose tissue. NLRP3 inflammasome activation in adipose tissue was associated with up-regulated expression of Nlrp3, Asc, and Caspase-1, and raised caspase-1 activity, which resulted in increased expression of IL-1ß and IL-18 and secretion of IL-1ß. Moreover, lipid accumulation and expression of transcription factors exacerbating accumulation of lipids were augmented in adipose tissue of HFrD-fed rats. Treatment with glyburide, quercetin or allopurinol corrected HFrD-induced dyslipidemia or hyperuricemia, and blocked NLRP3 activation, leading to mitigated inflammatory signaling and lipid accumulation in adipose tissue, improved glucose tolerance and insulin sensitivity in HFrD-fed rats. These data suggest the role of NLRP3 inflammasome to establish linkage among inflammation, lipid accumulation and insulin resistance in adipose tissue, and targeting NLRP3 inflammasome may be a plausible approach for prevention and management for fructose-induced metabolic impairments.

Insulin resistance corresponds with a progressive increase in NOD1 in high fat diet-fed mice.

PURPOSE: Innate immune components participate in obesity-induced inflammation, which can contribute to endocrine dysfunction during metabolic diseases. However, the chronological activation of specific immune proteins such as Nucleotide-binding oligomerization domain-containing protein 1 (NOD1) and relevance to cellular crosstalk during the progression of obesity-associated insulin resistance (IR) is not known. METHODS: The NOD1 signaling in various insulin-sensitive metabolic tissues during the progression of diet-insulin resistance was assessed in C57BL/6J mice fed with 60% high-fat diet (HFD) for 4, 8, 12, and 16 weeks. Intestinal permeability was measured using FITC-dextran. NOD1 activating potential was analyzed using HEK-Blue mNOD1 cells. RESULTS: HFD-fed mice showed progressive induction of glucose intolerance and impairment of insulin signaling in key metabolic tissues. We found a time-dependent increase in intestinal permeability coupled with transport and accumulation of NOD1 activating ligand in the serum of HFD-fed mice. We also observed a progressive accumulation of γ-D-glutamyl-meso-diaminopimelic acid (DAP), a microbial peptidoglycan ligand known to activate NOD1, in serum samples of the HFD-fed mice. There was also a progressive increase in transcripts levels of NOD1 in bone marrow-derived macrophages during HFD-feeding. In addition, skeletal muscle, adipose and liver, the key insulin sensitive metabolic tissues also had a time-dependent increase in transcripts of NOD1 and Rip2 and a corresponding activation of pro-inflammatory responses in these tissues. CONCLUSION: These data highlight the correlation of inflammation and insulin resistance to NOD1 activation in the bone marrow derived macrophages and insulin responsive metabolic tissues during high fat diet feeding in mice.

NOD1 activation induces oxidative stress via NOX1/4 in adipocytes.

Activation of innate immune components promotes cell autonomous inflammation in adipocytes. Oxidative stress links pattern recognition receptor-mediated detection of inflammatory ligands and the immune response. Reactive oxygen species (ROS) may mediate the effect of nucleotide-binding oligomerization domain protein-1 (NOD1) activation on inflammation in adipocytes. Here, we define the potential role of NADPH oxidase (NOX)-derived ROS in NOD1-mediated inflammatory response in adipocytes. Differentiated 3T3-L1 adipocytes were treated with NOD1 activating ligand D-gamma-Glu-meso-diaminopimelic acid (iE-DAP) to evaluate the oxidative stress and contribution of NOX as source of intracellular ROS. NOD1 activation potently induced ROS generation in 3T3-L1 adipocytes. Of the NOX family members, expression of NOX1 and NOX4 was increased upon NOD1 activation, in a PKCδ-dependent manner. siRNA-mediated down-regulation of NOX1 or NOX4 inhibited NOD1-mediated ROS production and increased the expression of antioxidant defense enzyme catalase and superoxide dismutase (SOD). siRNA-mediated lowering of NOX1 or NOX4 also suppressed NOD1-mediated activation of JNK1/2 and NF-κB, and consequent activation of inflammatory response in 3T3-L1 adipocytes. In summary, our findings demonstrate that NOD1 activation provokes oxidative stress in adipocytes via NOX1/4 and that oxidative stress, at least in part, contributes to induction of inflammatory response. Defining the source of ROS after immune response engagement may lead to new therapeutic strategies for adipose tissue inflammation.