Insulin Signaling Research Articles

Exploring The Differences : A Comparative Analysis of Pharmaceutical Quality of Different Brands of Metformin Hydrochloride Tablets

Type 2 diabetic patients can regulate their increased blood sugar levels using metformin, an antidiabetic medicine approved by the FDA. It decreases the quantity of glucose produced by the liver, lessens the glucose absorption from the intestines, and raises insulin sensitivity. For best effects, metformin should be used in conjunction with dietary modifications and physical activity. The first-line oral hypoglycemic medication for type 2 DM(Diabetes Mellitus) is thought to be metformin. The recommended medication for obese people is MET. AMPK- Adenosine monophosphate-activated protein kinase, a liver enzyme crucial to insulin signaling, is triggered by metformin. Metformin’s inhibitory effect on hepatic cell glucose synthesis necessitates AMPK activation. This study aims to compare the pharmaceutical quality of three different brands of metformin hydrochloride tablets. All the three brands were evaluated for uniformity of weight, friability, hardness, dissolution, disintegration and assay was performed. All the brands tested were within the specification, thus all the tablets passed the pharmaceutical quality test, however, variations exist between brands, which could be due to differences in excipients, pressure during compression, manufacturing procedure etc.

Brain-targeted Tet-1 peptide-PLGA nanoparticles for berberine delivery against STZ-induced Alzheimer’s disease in a rat model: Alleviation of hippocampal synaptic dysfunction, Tau pathology, and amyloidogenesis

Alzheimer’s disease (AD) is an age-related neurodegenerative disorder that causes severe dementia and memory loss. Surface functionalized poly(lactic-co-glycolic acid) nanoparticles have been reported for better transport through the blood–brain barrier for AD therapy. This study investigated the improved therapeutic potential of berberine-loaded poly(lactic-co-glycolic acid)/Tet-1 peptide nanoparticles (BBR/PLGA-Tet NPs) in a rat model of sporadic AD. BBR was loaded into the PLGA-Tet conjugate. BBR/PLGA-Tet NPs were physicochemically and morphologically characterized. AD was achieved by bilateral intracerebroventricular (ICV) injection of streptozotocin (STZ). Cognitively impaired rats were divided into STZ, STZ + BBR, STZ + BBR/PLGA-Tet NPs, and STZ + PLGA-Tet NPs groups. Cognitive improvement was assessed using the Morris Water Maze. Brain acetylcholinesterase and monoamine oxidase activities, amyloid β42 (Aβ42), and brain glycemic markers were estimated. Further, hippocampal neuroplasticity (BDNF, pCREB, and pERK/ERK), Tau pathogenesis (pGSK3β/GSK3β, Cdk5, and pTau), inflammatory, and apoptotic markers were evaluated. Finally, histopathological changes were monitored. ICV-STZ injection produces AD-like pathologies evidenced by Aβ42 deposition, Tau hyperphosphorylation, impaired insulin signaling and neuroplasticity, and neuroinflammation. BBR and BBR/PLGA-Tet NPs attenuated STZ-induced hippocampal damage, enhanced cognitive performance, and reduced Aβ42, Tau phosphorylation, and proinflammatory responses. BBR/PLGA-Tet NPs restored neuroplasticity, cholinergic, and monoaminergic function, which are critical for cognition and brain function. BBR/PLGA-Tet NPs may have superior therapeutic potential in alleviating sporadic AD than free BBR due to their bioavailability, absorption, and brain uptake.

Down-regulation of O-GlcNAcylation alleviates insulin signaling pathway impairment following arsenic exposure via suppressing the AMPK/mTOR-autophagy pathway

Impairment of the insulin signaling pathway is a key contributor to insulin resistance under arsenic exposure. Specifically, O-GlcNAcylation, an important post-translational modification, plays a crucial role in insulin resistance. Nevertheless, the concrete effect and mechanism of O-GlcNAcylation in arsenic-induced impairment of the insulin signaling pathway remain elusive. Herein, C57BL/6 mice were continuously fed arsenic-containing food, with a total arsenic concentration of 30 mg/kg. We observed that the IRS/Akt/GSK-3β insulin signaling pathway was impaired, and autophagy was activated in mouse livers and HepG2 cells exposed to arsenic. Additionally, O-GlcNAcylation expression in mouse livers and HepG2 cells was elevated, and the key O-GlcNAcylation homeostasis enzyme, O-GlcNAc transferase (OGT), was upregulated. In vitro, non-targeted metabolomic analysis showed that metabolic disorder was induced, and inhibition of O-GlcNAcylation restored the metabolic profile of HepG2 cells exposed to arsenic. In addition, we found that the compromised insulin signaling pathway was dependent on AMPK activation. Inhibition of AMPK mitigated autophagy activation and impairment of insulin signaling pathway under arsenic exposure. Furthermore, down-regulation of O-GlcNAcylation inhibited AMPK activation, thereby suppressing autophagy activation, and improving the impaired insulin signaling pathway. Collectively, our findings indicate that arsenic can impair the insulin signaling pathway by regulating O-GlcNAcylation homeostasis. Importantly, O-GlcNAcylation inhibition alleviated the impaired insulin signaling pathway by suppressing the AMPK/mTOR-autophagy pathway. This indicates that regulating O-GlcNAcylation may be a potential intervention for the impaired insulin signaling pathway induced by arsenic.

Age-dependent impact of streptozotocin on metabolic endpoints and Alzheimer's disease pathologies in 3xTg-AD mice

Alzheimer's disease (AD) is a multifactorial neurodegenerative disease with a complex origin, thought to involve a combination of genetic, biological and environmental factors. Insulin dysfunction has emerged as a potential factor contributing to AD pathogenesis, particularly in individuals with diabetes, and among those with insulin deficiency or undergoing insulin therapy. The intraperitoneal administration of streptozotocin (STZ) is widely used in rodent models to explore the impact of insulin deficiency on AD pathology, although prior research predominantly focused on young animals, with no comparative analysis across different age groups. Our study aimed to fill this gap by analyzing the impact of insulin dysfunction in 7 and 23 months 3xTg-AD mice, that exhibit both amyloid and tau pathologies. Our objective was to elucidate the age-specific consequences of insulin deficiency on AD pathology.STZ administration led to insulin deficiency in the younger mice, resulting in an increase in cortical amyloid-β (Aβ) and tau aggregation, while tau phosphorylation was not significantly affected. Conversely, older mice displayed an unexpected resilience to the peripheral metabolic impact of STZ, while exhibiting an increase in both tau phosphorylation and aggregation without significantly affecting amyloid pathology. These changes were paralleled with alterations in signaling pathways involving tau kinases and phosphatases. Several markers of blood-brain barrier (BBB) integrity declined with age in 3xTg-AD mice, which might have facilitated a direct neurotoxic effect of STZ in older mice.Overall, our research confirms the influence of insulin signaling dysfunction on AD pathology, but also advises careful interpretation of data related to STZ-induced effects in older animals.

Obesity-induced tissue alterations resist weight loss: A mechanistic review.

Interventions aimed at weight control often have limited effectiveness in combating obesity. This review explores how obesity-induced dysfunction in white (WAT) and brown adipose tissue (BAT), skeletal muscle, and the brain blunt weight loss, leading to retention of stored fat. In obesity, increased adrenergic stimulation and inflammation downregulate β-adrenoreceptors and impair catecholaminergic signalling in adipocytes. This disrupts adrenergic-mediated lipolysis, diminishing lipid oxidation in both white and brown adipocytes, lowering thermogenesis and blunting fat loss. Emerging evidence suggests that WAT fibrosis is associated with worse weight loss outcomes; indeed, limiting collagen and laminin-α4 deposition mitigates WAT accumulation, enhances browning, and protects against high-fat-diet-induced obesity. Obesity compromises mitochondrial oxidative capacity and lipid oxidation in skeletal muscle, impairing its ability to switch between glucose and lipid metabolism in response to varying nutrient levels and exercise. This dysfunctional phenotype in muscle is exacerbated in the presence of obesity-associated sarcopenia. Additionally, obesity suppresses sarcolipin-induced sarcoplasmic reticulum calcium ATPase (SERCA) activation, resulting in reduced oxidative capacity, diminished energy expenditure, and increased adiposity. In the hypothalamus, obesity and overnutrition impair insulin and leptin signalling. This blunts central satiety signals, favouring a shift in energy balance toward energy conservation and body fat retention. Moreover, both obese animals and humans demonstrate impaired dopaminergic signalling and diminished responses to nutrient intake in the striatum, which tend to persist after weight loss. This may result in enduring inclinations toward overeating and a sedentary lifestyle. Collectively, the tissue adaptations described pose significant challenges to effectively achieving and sustaining weight loss in obesity.

Comparative Study on Anti-Diabetic Activity of Momordica charantia and Psidium guajava

Diabetes is a metabolic disease characterised by decreased insulin signalling and persistent hyperglycaemia. The most frequent progression of diabetes is known as diabetes mellitus which is defined as persistent hyperglycaemia brought on by either peripheral insulin resistance or impaired pancreatic β cell production of insulin. This condition is caused by the disruptions in the digestion of carbohydrates, fats and proteins which are caused by defects in the production, release and regulation of insulin. The breakdown of carbohydrates is significantly aided by the intestinal digestive enzymes α-glucosidase and α-amylase. One type of antidiabetic treatment is to lower the blood glucose levels after a meal by blocking the enzymes α-glucosidase and α-amylase. This could be a key tactic in blood glucose control. When compared to other commercial pharmaceuticals used to treat diabetes, herbal treatments are thought to be more in harmony with the human body and to have less harmful side effects. This herbal remedy is reasonably priced as well. The aim of the current study is to compare the anti- diabetic activity of the methanolic extracts of Momordica charantia and Psidium guajava. The methods used in the determination of invitro antidiabetic activity are alpha amylase and alpha glucosidase inhibitory assays. In the current study, when compared the individual inhibition activity of Momordica charantia and Psidium guajava on the intestinal enzymes (alpha amylase and alpha glucosidase), Momordica charantia has the highest inhibition that is 79.83% at 0.8 mL concentration while the Psidium guajava has shown only 70.58% inhibition at 0.8 mL concentration. Which suggests that the presence of bio active compounds may be responsible for the plants many medicinal uses, including the treatment of diabetes.

Targeting Metabolic Syndrome Pathways: Carrot microRNAs As Potential Modulators.

Metabolic syndrome is a condition characterized by metabolic alterations that culminate in chronic noncommunicable diseases of high morbidity and mortality, such as cardiovascular diseases, type 2 diabetes, nonalcoholic fatty liver disease, and colon cancer. Developing new therapeutic strategies with a multifactorial approach is important since current therapies focus on only one or two components of the metabolic syndrome. In this sense, plant-based gene regulation represents an innovative strategy to prevent or modulate human metabolic pathologies, including metabolic syndrome. Here, using a computational and systems biology approach, it was found that carrot microRNAs can modulate key BMPs/SMAD signaling members, C/EBPs, and KLFs involved in several aspects associated with metabolic syndrome, including the hsa04350:TGF-beta signaling pathway, hsa04931:insulin resistance, hsa04152:AMPK signaling pathway, hsa04933:AGE-RAGE signaling pathway in diabetic complications, hsa04010:MAPK signaling pathway, hsa04350:TGF-beta signaling pathway, hsa01522:endocrine resistance, and hsa04910:insulin signaling pathway. These data demonstrated the potential applications of carrot microRNAs as effective food-based therapeutics for obesity and associated metabolic diseases.

Interaction of serotonin/GLP-1 circuitry in a dual preclinical model for psychiatric disorders and metabolic dysfunction

Isolation of rodents throughout adolescence is known to induce many behavioral abnormalities which resemble neuropsychiatric disorders. Separately, this paradigm has also been shown to induce long-term metabolic changes consistent with a pre-diabetic state. Here, we investigate changes in central serotonin (5-HT) and glucagon-like peptide 1 (GLP-1) neurobiology that dually accompany behavioral and metabolic outcomes following social isolation stress throughout adolescence. We find that adolescent-isolation mice exhibit elevated blood glucose levels, impaired peripheral insulin signaling, altered pancreatic function, and fattier body composition without changes in bodyweight. These mice further exhibited disruptions in sleep and enhanced nociception. Using bulk and spatial transcriptomic techniques, we observe broad changes in neural 5-HT, GLP-1, and appetitive circuits. We find 5-HT neurons of adolescent-isolation mice to be more excitable, transcribe fewer copies of Glp1r (mRNA; GLP-1 receptor), and demonstrate resistance to the inhibitory effects of the GLP-1R agonist semaglutide on action potential thresholds. Surprisingly, we find that administration of semaglutide, commonly prescribed to treat metabolic syndrome, induced deficits in social interaction in group-housed mice and rescued social deficits in isolated mice. Overall, we find that central 5-HT circuitry may simultaneously influence mental well-being and metabolic health in this model, via interactions with GLP-1 and proopiomelanocortin circuitry.

Mitochondrial ALDH2 improves ß-cell survival and function against doxorubicin-induced apoptosis by targeting CK2 signaling.

The aim of this study was to better understand how the chemotherapy drug doxorubicin contributes to the development of β-cell dysfunction and to explore its relationship with mitochondrial aldehyde dehydrogenase-2 (ALDH2). In order to investigate this hypothesis, doxorubicin was administered to INS-1 cells, a rat insulinoma cell line, either with or without several target protein activators and inhibitors. ALDH2 activity was detected with a commercial kit and protein levels were determined with western blot. Mitochondrial ROS, membrane potential, and lipid ROS were determined by commercial fluorescent probes. The cell viability was measured by CCK-assay. Exposure of INS-1 cells to doxorubicin decreased active insulin signaling resulting in elevated ALDH2 degradation, compared with control cells by the induction of acid sphingomyelinase mediated ceramide induction. Further, ceramide induction potentiated doxorubicin induced mitochondrial dysfunction. Treatment with the ALDH2 agonist, ALDA1, blocked doxorubicin-induced acid sphingomyelinase activation which significantly blocked ceramide induction and mitochondrial dysfunction mediated cell death. Treatment with the ALDH2 agonist, ALDA1, stimulated casein kinase-2 (CK2) mediated insulin signaling activation. CK2 silencing neutralized the function of ALDH2 in the doxorubicin treated INS-1 cells. Mitochondrial ALDH2 activation could inhibit the progression of doxorubicin induced pancreatic β-cell dysfunction by inhibiting the acid sphingomyelinase induction of ceramide, by regulating the activation of CK2 signaling. Our research lays the foundation of ALDH2 activation as a therapeutic target for the precise treatment of chemotherapy drug induced β-cell dysfunction.

Potential Effect of Chloroquine in Modulating Hyperglycemia and PTEN Receptor Linked Dyslipidemia on Alloxan-induced Diabetic Mice

Background: Diabetes mellitus is a life-threatening disease associated with worsening of glycemic control, progressive metabolic dysfunctions and disruption of the Phosphatase and Tensin Homolog (PTEN) receptor. Purpose: To evaluate the effect of chloroquine on blood glucose level and lipid profile of alloxan-induced diabetic mice and to observe its interaction with PTEN, a negative regulator of insulin signaling pathway. Methodology: Thirty mice were used for the experiment. A group (n = 5) was kept as healthy control while the others were pre-treated with 140 mg/kg of alloxan monohydrate in distilled water. The alloxan-induced diabetic mice were randomly divided into four groups: diabetic model control group, 60 mg/kg chloroquine, 120 mg/kg chloroquine, and 10 mg/kg glibenclamide treatment groups respectively. Treatment was done once daily for seven days and the blood glucose level was investigated acutely and then daily throughout the experimental period. A molecular docking study was also conducted to evaluate the interaction of chloroquine with PTEN using PYRX software, while an automated COBAS C 311 machine was used to analyze the lipid profile after the treatment period. Results: Oral administration of chloroquine gradually and significantly lowered the raised blood glucose level in a time- and dose-dependent manner. A repeated study with 120 mg/kg of chloroquine revealed a decline in blood sugar from 221.5 ± 3.6 on day 1 to 85.5 ± 2.4 on day 7 in comparison to glibenclamide, whose sugar level was reduced to 75.5 ± 3.7 at the end of day 7. The docking study revealed a non-competitive inhibition with an inhibition score of -6.1 in comparison to metformin, which had a score of -4.4, and glibenclamide, which had a score of -9.0. This interaction resulted in a conformational change of the receptor, hence, enhancing glucose uptake and reducing the raised hyperglycemia. Treatment with chloroquine was also observed to reduce the total cholesterol and triglyceride levels from 162 in the model group to 144 and 141 to 116 mmol/L, respectively. The levels of low-density lipoprotein cholesterol (LDL-C) and very low-density lipoprotein (VLDL) following treatment with chloroquine were not statistically different from the non-diabetic healthy mice. These values were very similar to those obtained with glibenclamide-treated diabetic mice. Conclusion: From the results, chloroquine possesses potent antidiabetic properties and can improve dyslipidemia imposed by hyperglycemia probably owing to its inhibition of PTEN, a negative regulator of insulin resistance.

Regulation of placental amino acid transport in health and disease.

Abnormal fetal growth, i.e., intrauterine growth restriction (IUGR) or fetal growth restriction (FGR) and fetal overgrowth, is associated with increased perinatal morbidity and mortality and is strongly linked to the development of metabolic and cardiovascular disease in childhood and later in life. Emerging evidence suggests that changes in placental amino acid transport may contribute to abnormal fetal growth. This review is focused on amino acid transport in the human placenta, however, relevant animal models will be discussed to add mechanistic insights. At least 25 distinct amino acid transporters with different characteristics and substrate preferences have been identified in the human placenta. Of these, System A, transporting neutral nonessential amino acids, and System L, mediating the transport of essential amino acids, have been studied in some detail. Importantly, decreased placental Systems A and L transporter activity is strongly associated with IUGR and increased placental activity of these two amino acid transporters has been linked to fetal overgrowth in human pregnancy. An array of factors in the maternal circulation, including insulin, IGF-1, and adiponectin, and placental signaling pathways such as mTOR, have been identified as key regulators of placental Systems A and L. Studies using trophoblast-specific gene targeting in mice have provided compelling evidence that changes in placental Systems A and L are mechanistically linked to altered fetal growth. It is possible that targeting specific placental amino acid transporters or their upstream regulators represents a novel intervention to alleviate the short- and long-term consequences of abnormal fetal growth in the future.

Berberine attenuates obesity-induced insulin resistance by inhibiting miR-27a secretion.

Berberine (BBR) is an alkaloid found in plants. It has neuroprotective, anti-inflammatory and lipid-lowering activity. However, the efficacy of treatment with BBR and the mechanisms through which it acts need further study. This study investigated the therapeutic effects and the mechanism of action of BBR on obesity-induced insulin resistance in peripheral tissues. High-fat-fed C57BL/6J mice and low-fat-fed C57BL/6J mice with miR-27a overexpression were given BBR intervention (100 mg/kg, po), and the oral glucose tolerance test (OGTT) and insulin tolerance test (ITT) were performed. Palmitic acid-stimulated hypertrophic adipocyte models were treated with BBR (10 μM). Related indicators and protein expression levels were examined. The AUCs of the OGTT and the ITT in the BBR intervention group were reduced significantly (p < 0.01) (p < 0.05), and the serum biochemical parameters, including FBG, TC, TG and LDL-C were significantly reduced after BBR intervention. In the invitro experiments, the triglyceride level and volume of lipid droplets decreased significantly after BBR intervention (p < 0.01) (p < 0.05). Likewise, BBR ameliorates skeletal muscle and pancreas insulin signalling pathways invivo and invitro. The results showed that BBR significantly ameliorated insulin resistance, reduced body weight and percent body fat and improved serum biochemical parameters in mice. Likewise, BBR reduced triglyceride level and lipid droplet volume in hypertrophic adipocytes, BBR improved obesity effectively. Meanwhile, BBR ameliorated the histomorphology of the pancreas, and skeletal muscle and pancreas insulin related signalling pathways of islets in invitro and invivo experiments. The results further demonstrated that BBR inhibited miR-27a levels in serum from obese mice and supernatant of hypertrophic adipocytes. miR-27a overexpression in low-fat fed mice indicated that miR-27a caused insulin resistance, and BBR intervention significantly improved the miR-27a induced insulin resistance status. This study demonstrates the important role of BBR in obesity-induced peripheral insulin resistance and suggest that the mechanism of its effect may be inhibition of miR-27a secretion.

Crithmum maritimum Extract Restores Lipid Homeostasis and Metabolic Profile of Liver Cancer Cells to a Normal Phenotype

Hepatocellular carcinoma (HCC) is an alarming epidemiological clinical problem worldwide. Pharmacological approaches currently available do not provide adequate responses due to poor effectiveness, high toxicity, and serious side effects. Our previous studies have shown that the wild edible plant Crithmum maritimum L. inhibits the growth of liver cancer cells and promotes liver cell differentiation by reducing lactic acid fermentation (Warburg effect). Here, we aimed to further characterise the effects of C. maritimum on lipid metabolism and markers of cellular metabolic health, such as AMP-activated protein kinase (AMPK), Sirtuin 1 (SIRT1), and Sirtuin 3 (SIRT3), as well as the insulin signalling pathway. To better mimic the biological spectrum of HCC, we employed four HCC cell lines with different degrees of tumorigenicity and lactic acid fermentation/Warburg phenotype. Lipid accumulation was assessed by Oil Red O (ORO) staining, while gene expression was measured by real-time quantitative PCR (RT-qPCR). The activation of AMPK and insulin signalling pathways was determined by Western blotting. Results indicate that C. maritimum prevents lipid accumulation, downregulates lipid and cholesterol biosynthesis, and modulates markers of metabolic health, such as AMPK, SIRT1 and SIRT3. This modulation is different amongst HCC cell lines, revealing an important functional versatility of C. maritimum. Taken together, our findings corroborate the importance of C. maritimum as a valuable nutraceutical, reinforcing its role for the improvement of metabolic health.

237 Disrupted insulin signaling due to an acute immune response in swine

Abstract Insulin is an anabolic hormone involved in glucose uptake and synthesis of fats, proteins, and glycogen. Altered insulin signaling can occur due to metabolic state, pathogen exposure, and autoimmune disorders. Lipopolysaccharide (LPS), an endotoxin secreted produced by gram-negative bacteria, can induce a severe, systemic immune response. In pigs, chronic LPS exposure has induced insulin resistance. However, the effects of acute exposure to LPS on insulin signaling and resistance have not been elucidated. Crossbred nursery pigs [n = 58, initial body weight (BW) = 12.1 kg ± 0.23 kg] were randomly assigned into two experimental subsets, where 10 animals were designated for repeated blood sampling and 48 animals underwent timed euthanasia for tissue collection. For repeated blood sampling, jugular catheters were placed under anesthesia and the animals were given 48 h to recover before being randomly assigned to treatments. These treatments were an intraperitoneal injection of either 15µg/kg BW LPS or an equal volume of 0.9% Saline (n = 5 pigs/treatment). Blood was collected for 48 h followed by euthanasia. The other 48 pigs were randomly assigned to the same treatments, and then randomly assigned to euthanasia timepoints of 0, 3, 6, 12, 24 or 48 h post injection for tissue collection. Among the catheterized pigs, 2-h post LPS challenge there was a decrease in serum insulin concentration (P &amp;lt; 0.05) and mild hyperglycemia (P &amp;lt; 0.05) post challenge. At 12- and 24-h post LPS challenge there was a decrease in insulin (P &amp;lt; 0.05) and a trend hyperglycemia was also apparent at 24 h (P = 0.063). In liver tissue, insulin related genes exhibited alterations post LPS injection. Phosphoinositide 3-kinase (pi3k) and insulin receptor substrate 1 (irs1) expression were decreased at 3 and 6 h (p&amp;lt; 0.05) and protein kinase b (akt) expression were decreased at 3 h (P &amp;lt; 0.05) post challenge. The expression of insulin inhibitor growth factor receptor bound protein 10 (grb10) was decreased at 6-, 12-, and 24-h after challenge (P &amp;lt; 0.05). Fructose-1-6-bisphosphatase (f16bp) expression was reduced at 3-, 6-, and 12-h (P &amp;lt; 0.05) in the liver while phosphoenolpyruvate carboxykinase (pepck) expression was decreased at 3- and 6-h (P &amp;lt; 0.01) post challenge. Other metabolic genes were decreased at 3-, 6-, and 12-h post LPS challenge, including fatty acid synthase (fas; P &amp;lt; 0.05), carbohydrate response element binding protein (chrebp; P &amp;lt; 0.05), and acetyl CoA carboxylase (acc; P &amp;lt; 0.05). Protein abundance of Akt 3 h after LPS injection was decreased in the liver (P &amp;lt; 0.05). In skeletal muscle 6 h post LPS injection, insulin receptor (insr), insulin-like growth factor receptor (igfr) and fas were all increased (P &amp;lt; 0.05) These results indicate disrupted insulin signaling at various levels after an acute immune response.

The role of exosome derived miRNAs in inter-cell crosstalk among insulin-related organs in type 2 diabetes mellitus.

Exosomes are small extracellular vesicles secreted by almost all cell types, and carry diverse cargo including RNA, and other substances. Recent studies have focused exosomal microRNAs (miRNAs) on various human diseases, including type 2 diabetes mellitus (T2DM) and metabolic syndrome (METS) which accompany the occurrence of insulin resistance. The regulation of insulin signaling has connected with some miRNA expression which play a significant regulatory character in insulin targeted cells or organs, such as fat, muscle, and liver. The miRNAs carried by exosomes, through the circulation in the body fluids, mediate all kinds of physiological and pathological process involved in the human body. Studies have found that exosome derived miRNAs are abnormally expressed and cross-talked with insulin targeted cells or organs to affect insulin pathways. Further investigations of the mechanisms of exosomal miRNAs in T2DM will be valuable for the diagnostic biomarkers and therapeutic targets of T2DM. This review will summarize the molecular mechanism of action of the miRNAs carried by exosomes which are secreted from insulin signaling related cells, and elucidate the pathogenesis of insulin resistance to provide a new strategy for the potential diagnostic biomarkers and therapeutic targets for the type 2 diabetes.

The microbiome: a link between obesity and breast cancer risk

Globally, breast cancer is the leading cause of cancer incidence and mortality among all female cancers. Hereditary factors only account for 5-10% of breast cancers, highlighting the importance of non-hereditary factors, such as obesity. The increasing prevalence of obesity underscores the need to understand its contribution to breast cancer risk. Multiple mechanisms may mediate pro-carcinogenic effects of obesity, including altered adipokine levels, local and systemic inflammation, disruption of insulin and insulin-like growth factor signaling, increased estrogen levels, and alterations of the microbiome. In this review, we focus on the link between gut microbiome alterations and breast cancer risk in the context of obesity. First, we discuss how obesity influences the gut microbiome. Next, we describe the effect of such microbiome alterations on breast carcinogenesis, highlighting underlying molecular mechanisms. Finally, we review preclinical data on the interactions between host and bacteria, current challenges to study the obesity-microbiome connection, and future perspectives in this field.

Abstract PO3-02-03: Ketogenic diet and letrozole to inhibit PI3K signaling in ER+ breast cancer

Abstract Obesity is associated with an increased risk of breast cancer recurrence, similar in magnitude to the reduction in risk seen with the use of adjuvant chemotherapy. However, whether and how obesity causes that increased risk of recurrence remains unknown. Plausible mechanisms include obesity-mediated increases in estrogen signaling, inflammatory conditions resulting from obesity, or effects on insulin or other growth factor signaling pathways resulting from the insulin-resistant state that often accompanies obesity. While phosphatidyl inositol 3-kinase (PI3K) signaling is the normal, biological effector of insulin signaling, abnormal activation of PI3K is of particular concern in breast cancer, where it is associated with resistance to endocrine therapies and HER2-targeted therapies. We hypothesize that at least part of the adverse outcome associated with obesity results from aberrant activation of PI3K signaling in tumors. This may confer resistance to established therapies, or directly stimulate tumor growth (particularly in tumors with existing PI3K pathway mutations), or both. We tested a dietary intervention designed to alter insulin resistance pathophysiology coupled with endocrine therapy in the well-established neoadjuvant treatment paradigm in breast cancer. This will allow us to study the effects of the dietary intervention directly on tumor biology. The primary objective of this neoadjuvant study was to assess feasibility and tolerability of 2 weeks of a very low-carbohydrate ketogenic diet in combination with letrozole for patients with early stage operable ER+ disease. Up to 36 patients will be enrolled for this pilot and feasibility study (24 in the treatment group and 12 controls). Baseline metabolic parameters will be measured and the treatment group will begin a dietitian-supervised 2-week diet to induce a ketogenic state, along with letrozole 2.5 mg daily. The control group will receive only letrozole. At the end of 2 weeks, patients will proceed with surgical treatment of their breast cancer. We will report the primary outcome measure of feasibility, assessed by determining 1) the proportion of patients in the diet group achieving ketosis, 2) adherence to the diet and 3) participant-reported measures of stress, fatigue, and emotional health. For both groups, cell proliferation will be measured in a tumor biopsy obtained from the surgical specimen and compared with the pre-treatment diagnostic biopsy. Correlative studies will evaluate tumor markers of insulin/PI3K signaling before and after the intervention and between diet and control. Citation Format: Brent Rexer, Vandana Abramson, Sara Nunnery, Sonya Reid, Laura Kennedy, Melinda Sanders, Jessica Bennett, Paula Gonzalez-Ericsson, Violeta Sanchez. Ketogenic diet and letrozole to inhibit PI3K signaling in ER+ breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-02-03.

Abstract RF02-02: BBO-10203, a first-in-class, orally bioavailable, selective covalent small molecule that inhibits RAS-driven PI3Kalpha activity without affecting glucose metabolism

Abstract PI3Kα is the most mutated kinase and the second most mutated oncogene in human cancer. Activation of PI3Ka can be achieved by receptor tyrosine kinases such as insulin receptor and insulin-like growth factor receptor 1 and/or by directly interacting with RAS family members. Previous elegant preclinical studies have established that RAS-driven PI3Ka activation is important in tumor cells but may not be involved in cell types controlling glucose metabolism. Alpelisib, a small molecule inhibitor of the kinase activity of PI3Ka, has been approved for the treatment of ER+ PIK3CA mutant breast cancer in combination with fulvestrant after endocrine therapy in advanced or metastatic breast cancer based on an improvement in PFS versus fulvestrant alone. Inhibition of PI3Ka activity by alpelisib in normal tissues resulted in a severe (G3/4) hyperglycemia rate of 37% with frequent dose interruptions and discontinuations. Additionally, preclinical studies have demonstrated that the dysregulation of glucose homeostasis resulting from PI3Ka kinase inhibition leads to hyperinsulinemia that increases pathway flux, rendering kinase inhibitors less effective. Here, we report on a novel covalent small molecule designed to inhibit RAS-mediated activation of the AKT pathway via PI3Ka without the resultant hyperglycemia associated with direct inhibition of PI3Ka kinase activity. BBO-10203 disrupts the physical interaction between RAS and PI3Ka in tumor cells resulting in potent signaling pathway inhibition. This agent selectively binds to PI3Ka and disrupts its interaction with K-,H-, and N-RAS with low single digit nanomolar potency (~5 nM). Breaking the interaction between these two oncogenes inhibits basal pAKT cellular levels (BT-474/KYSE-410 IC50: ~5 nM) in HER2 amplified (HER2amp) and wild-type or mutant PI3Ka cell lines. Even though BBO-10203 does not inhibit the kinase activity of PI3Ka, its effects on cancer cell signaling inhibition and transcriptional regulation highly resemble those of alpelisib. BBO-10203 displays excellent drug-like properties and oral bioavailability. Single dose treatment of KYSE-410 (HER2amp/KRASG12C) tumor bearing mice with increasing doses (1-100 mg/kg, PO) of BBO-10203 results in dose and time dependent inhibition of pAKT in vivo. Maximal inhibition (~80%) is achieved at 30 mg/kg and lasts for 24 hours. Repeated dose treatment of tumor bearing mice with BBO-10203 is well tolerated and results in significant efficacy in PIK3CA mutant as well as HER2amp human xenograft models. In the KYSE-410 xenograft model, BBO-10203 daily oral dosing of 30 mg/kg results in significant tumor regressions. Importantly, treatment with BBO-10203 does not affect insulin signaling in differentiated adipocytes in vitro, nor does it impact glucose metabolism in vivo at 3-times the maximal efficacious dose level in xenograft studies. In conclusion, we have identified a novel approach to inhibit the PI3Ka signaling pathway by blocking its interaction with, and activation by RAS. This approach can achieve strong pAKT inhibition in tumor cells without changes in glucose metabolism. Clinical investigation of BBO-10203 for the treatment of both ER+/PIK3CA mutant and HER2amp breast cancer is warranted. Citation Format: Pedro Beltran, Simanshu Dhirendra, Rui Xu, Ming Chen, Daniel Czyzyk, Sofia Donovan, Siyu Feng, Cindy Feng, Lijuan Fu, Felice Lightstone, Ken Lin, Anna Maciag, Dwight Nissley, Erin Riegler, Kerstin Sinkevicius, Andrew Stephen, James Stice, David Turner, Bin Wang, Keshi Wang, Yue Yang, Cathy Zhang, Frank McCormick, Eli Wallace. BBO-10203, a first-in-class, orally bioavailable, selective covalent small molecule that inhibits RAS-driven PI3Kalpha activity without affecting glucose metabolism [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr RF02-02.

GlycoID Proximity Labeling to Identify O-GlcNAcylated Protein Interactomes in Live Cells.

Cells continuously remodel their intracellular proteins with the monosaccharide O-linked N-acetylglucosamine (O-GlcNAc) to regulate metabolism, signaling, and stress. This protocol describes the use of GlycoID tools to capture O-GlcNAc dynamics in live cells. GlycoID constructs contain an O-GlcNAc binding domain linked to a proximity labeling domain and a subcellular localization sequence. When expressed in mammalian cells, GlycoID tracks changes in O-GlcNAc-modified proteins and their interactomes in response to chemical induction with biotin over time. Pairing the subcellular localization of GlycoID with the chemical induction of activity enables spatiotemporal studies of O-GlcNAc biology during cellular events such as insulin signaling. However, optimizing intracellular labeling experiments requires attention to several variables. Here, we describe two protocols to adapt GlycoID methods to a cell line and biological process of interest. Next, we describe how to conduct a semiquantitative proteomic analysis of O-GlcNAcylated proteins and their interactomes using insulin versus glucagon signaling as a sample application. This articles aims to establish baseline GlycoID protocols for new users and set the stage for widespread use over diverse cellular applications for the functional study of O-GlcNAc glycobiology. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Expression of targeted GlycoID constructs to verify subcellular location and labeling activity in mammalian cells Basic Protocol 2: GlycoID labeling in live HeLa cells for O-GlcNAc proteomic comparisons.

High Fructose Diet (HFrD) Induced Hepatic Insulin Resistance Is Not Present in Liver Androgen Receptor Knockout (LivARKO) Male Mice

Background: Hyperandrogenism (HA), a common symptom of polycystic ovary syndrome (PCOS), involves action of the androgen receptor (AR) and can induce insulin resistance among 35-80% of PCOS women (PMID 35432749: Amisi 2022). The liver plays an essential role in the metabolism of insulin and androgen signaling. HA has been shown to increase insulin resistance in women and female mice. Previous studies have suggested that knocking out liver AR can lead to improvements in metabolic and reproductive functions of PCOS (PMID 34547140: Andrisse 2021). Hypothesis: We hypothesized that liver androgen receptor knockout (LivARKO) mice on a high fructose diet would exhibit increased insulin resistance compared to controls, as measured by expression of phospho-Akt (p-Akt) and phospho-FoxO1 (p-foxo1), proteins involved in insulin signaling. Methods: LivARKO mice were divided into groups based on an assigned diet (Control or High Fructose; Con, Research Diets Inc, RDI D12450J, Protein (20%), Carbs (70%, corn starch-50%, sucrose-maldextrin-20%, fructose-0%), Fat 10% (lard)) and High Fructose (HFrD, RDI D02022704, Protein (20%), Carbs (70%, corn starch-9%, sucrose-maldextrin-1%, fructose-60%), Fat 10% (lard)). The mice were sacrificed and liver tissue samples were extracted for western blotting analysis examining p-Akt S473 and p-foxo1 S256 levels. Results: LivARKO male mice fed the control diet for one month displayed a significant increase in p-AKT expression when stimulated with insulin compared to basal controls. Similarly, LivARKO male mice fed the high fructose diet for one month displayed a significant increase in p-AKT expression when stimulated with insulin compared to basal mice on the same diet. After one month, LivARKO male mice on a control diet stimulated with insulin displayed significantly decreased p-Foxo1 expression compared to non-stimulated basal control. A similar trend was seen between basal and insulin-stimulated groups in the LivARKO-HFrD male mice, though this trend was not significant. Discussion: These findings suggested that the HFrD did not induce hepatic insulin resistance in LivARKO male mice. Here, we observed a similar pattern of increased insulin stimulated p-AKT to what is seen in WT mice on a chow diet (PMCID PMC3296881: Lu 2012); and this suggested that LivARKO did not disrupt insulin signaling in the liver of male mice when fed a control diet, which was similar to what was seen previously (DOI: 10.1002/hep.22252, Lin 2008, insulin increased PI3K activity in chow diet LivARKO male mice). Our data suggested that the HFrD induced hepatic insulin resistance seen in some WT models (such as PMCID PMC3131094: Nagai 2009) is not present in LivARKO male mice.This also suggested that unlike HFD, HFrD did not cause insulin resistance in LivARKO male mice. These results illuminate LivARKO as a potential therapeutic target for insulin resistance. This project was supported by the following grants to Dr. Stanley Andrisse. (1) NIH 1R01DK126892; (2) NIH 1T34GM142610; and (3) NSF 1931045. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.