Intergenerational inheritance induced by a high-fat diet causes hyperphagia and reduced hypothalamic sensitivity to insulin and leptin in the second-generation of rats.

OBJECTIVE: The aim was to investigate the intergenerational inheritance induced by a high-fat diet on sensitivity to insulin and leptin in the hypothalamic control of satiety in second-generation offspring, which were fed a control diet. METHODS: Progenitor rats were fed a high-fat or a control diet for 59 d until weaning. The first-generation and second-generation offspring were fed the control diet until 90 d of age. Body mass and adiposity index of the progenitors fed the high-fat diet and the second-generation offspring from progenitors fed the high-fat diet were evaluated as were the gene expression of DNA methyltransferase 3a, angiotensin-converting enzyme type 2, angiotensin II type 2 receptor, insulin and leptin signaling pathway (insulin receptor, leptin receptor, insulin receptor substrate 2, protein kinase B, signal transducer and transcriptional activator 3, pro-opiomelanocortin, and neuropeptide Agouti-related protein), superoxide dismutase activity, and the concentration of carbonyl protein and satiety-regulating neuropeptides, pro-opiomelanocortin and neuropeptide Agouti-related protein, in the hypothalamus. RESULTS: The progenitor group fed a high-fat diet showed increased insulin resistance and reduced insulin-secreting beta-cell function and reduced food intake, without changes in caloric intake. The second-generation offspring from progenitors fed a high-fat diet, compared with second-generation offspring from progenitors fed a control diet group, had decreased insulin-secreting beta-cell function and increased food and caloric intake, insulin resistance, body mass, and adiposity index. Furthermore, second-generation offspring from progenitors fed a high-fat diet had increased DNA methyltransferase 3a, neuropeptide Agouti-related protein, angiotensin II type 1 receptor, and nicotinamide adenine dinucleotide phosphate oxidase p47phox gene expression, superoxide dismutase activity, and neuropeptide Agouti-related protein concentration in the hypothalamus. In addition, there were reduced in gene expression of the insulin receptor, leptin receptor, insulin receptor substrate 2, pro-opiomelanocortin, angiotensin II type 2 receptor, angiotensin-converting enzyme type 2, and angiotensin-(1-7) receptor and pro-opiomelanocortin concentration in the second-generation offspring from progenitors fed the high-fat diet. CONCLUSIONS: Overall, progenitors fed a high-fat diet induced changes in the hypothalamic control of satiety of the second-generation offspring from progenitors fed the high-fat diet through intergenerational inheritance. These changes led to hyperphagia, alterations in the hypothalamic pathways of insulin, and leptin and adiposity index increase, favoring the occurrence of different cardiometabolic disorders in the second-generation offspring from progenitors fed the high-fat diet fed only with the control diet.

Computational Screening and Experimental Validation of Inhibitor Targeting the Complex Formation of Grb14 and Insulin Receptor.

The development of drugs targeting gene products associated with insulin resistance holds the potential to enhance our understanding of type 2 diabetes mellitus (T2DM). The virtual screening, based on a three-dimensional (3D) protein structure, is a potential technique to accelerate the development of molecular target drugs. Among the targets implicated in insulin resistance, the genetic characterization and protein function of Grb14 have been clarified without contradiction. The Grb14 gene displays significant variations in T2DM, and its gene product is known to inhibit the function of the insulin receptor (IR) by directly binding to the tyrosine kinase domain. In the present study, a virtual screening, based on a 3D structure of the IR tyrosine kinase domain (IRß) in complex with part of Grb14, was conducted to find compounds that can disrupt the complex formation between Grb14 and IRß. First, ten compounds were selected from 154,118 compounds via hierarchical in silico structure-based drug screening, composed of grid docking-based and genetic algorithm-based programs. The experimental validations suggested that the one compound can affect the blood glucose level. The molecular dynamics simulations and co-immunoprecipitation analysis showed that the compound did not completely suppress the protein-protein interaction between Grb14 and IR, though competitively bound to IR with the tyrosine kinase pseudosubstrate region in Grb14.

Elimination of Vitamin D Signaling Causes Increased Mortality in a Model of Overactivation of the Insulin Receptor: Role of Lipid Metabolism.

Vitamin D (VD) deficiency has been associated with cancer and diabetes. Insulin signaling through the insulin receptor (IR) stimulates cellular responses by activating the PI3K/AKT pathway. PTEN is a tumor suppressor and a negative regulator of the pathway. Its absence enhances insulin signaling leading to hypoglycemia, a dangerous complication found after insulin overdose. We analyzed the effect of VD signaling in a model of overactivation of the IR. We generated inducible double KO (DKO) mice for the VD receptor (VDR) and PTEN. DKO mice showed severe hypoglycemia, lower total cholesterol and increased mortality. No macroscopic tumors were detected. Analysis of the glucose metabolism did not show clear differences that would explain the increased mortality. Glucose supplementation, either systemically or directly into the brain, did not enhance DKO survival. Lipidic liver metabolism was altered as there was a delay in the activation of genes related to ß-oxidation and a decrease in lipogenesis in DKO mice. High-fat diet administration in DKO significantly improved its life span. Lack of vitamin D signaling increases mortality in a model of overactivation of the IR by impairing lipid metabolism. Clinically, these results reveal the importance of adequate Vitamin D levels in T1D patients.

Hypothalamic miR-1983 Targets Insulin Receptor ß and the Insulin-mediated miR-1983 Increase Is Blocked by Metformin.

MicroRNAs (miRNAs) expressed in the hypothalamus are capable of regulating energy balance and peripheral metabolism by inhibiting translation of target messenger RNAs (mRNAs). Hypothalamic insulin resistance is known to precede that in the periphery, thus a critical unanswered question is whether central insulin resistance creates a specific hypothalamic miRNA signature that can be identified and targeted. Here we show that miR-1983, a unique miRNA, is upregulated in vitro in 2 insulin-resistant immortalized hypothalamic neuronal neuropeptide Y-expressing models, and in vivo in hyperinsulinemic mice, with a concomitant decrease of insulin receptor ß subunit protein, a target of miR-1983. Importantly, we demonstrate that miR-1983 is detectable in human blood serum and that its levels significantly correlate with blood insulin and the homeostatic model assessment of insulin resistance. Levels of miR-1983 are normalized with metformin exposure in mouse hypothalamic neuronal cell culture. Our findings provide evidence for miR-1983 as a unique biomarker of cellular insulin resistance, and a potential therapeutic target for prevention of human metabolic disease.

Activation of Insulin Signaling by Botanical Products.

Type 2 diabetes (T2D) is a worldwide health problem, ranked as one of the leading causes for severe morbidity and premature mortality in modern society. Management of blood glucose is of major importance in order to limit the severe outcomes of the disease. However, despite the impressive success in the development of new antidiabetic drugs, almost no progress has been achieved with regard to the development of novel insulin-sensitizing agents. As insulin resistance is the most eminent factor in the patho-etiology of T2D, it is not surprising that an alarming number of patients still fail to meet glycemic goals. Owing to its wealth of chemical structures, the plant kingdom is considered as an inventory of compounds exerting various bioactivities, which might be used as a basis for the development of novel medications for various pathologies. Antidiabetic activity is found in over 400 plant species, and is attributable to varying mechanisms of action. Nevertheless, relatively limited evidence exists regarding phytochemicals directly activating insulin signaling, which is the focus of this review. Here, we will list plants and phytochemicals that have been found to improve insulin sensitivity by activation of the insulin signaling cascade, and will describe the active constituents and their mechanism of action.

Carnitine promotes recovery from oxidative stress and extends lifespan in C. elegans.

Carnitine is required for transporting fatty acids into the mitochondria for ß-oxidation. Carnitine has been used as an energy supplement but the roles in improving health and delaying aging remain unclear. Here we show in C. elegans that L-carnitine improves recovery from oxidative stress and extends lifespan. L-carnitine promotes recovery from oxidative stress induced by paraquat or juglone and improves mobility and survival in response to H2O2 and human amyloid (Aß) toxicity. L-carnitine also alleviates the oxidative stress during aging, resulting in moderate but significant lifespan extension, which was dependent on SKN-1 and DAF-16. Long-lived worms with germline loss (glp-1) or reduced insulin receptor activity (daf-2) recover from aging-associated oxidative stress faster than wild-type controls and their long lifespans were not further increased by L-carnitine. A new gene, T08B1.1, aligned to a known carnitine transporter OCTN1 in humans, is required for L-carnitine uptake in C. elegans. T08B1.1 expression is elevated in daf-2 and glp-1 mutants and its knockdown prevents L-carnitine from improving oxidative stress recovery and prolonging lifespan. Together, our study suggests an important role of L-carnitine in oxidative stress recovery that might be important for healthy aging in humans.

Analyzing structural differences between insulin receptor (IR) and IGF1R for designing small molecule allosteric inhibitors of IGF1R as novel anti-cancer agents.

IR and insulin-like growth factor-1 receptor (IGF-1R) share high degree of sequence and structural similarity that hinders the development of anticancer drugs targeting IGF1R, which is dysregulated in many cancers. Although IR and IGF1R mediate their activities through similar signalling pathways, yet they show different physiological effects. The exact molecular mechanism(s) how IR and IGF1R exert their distinct functions remain largely unknown. Here, we performed in silico analysis and generated GFP-fusion proteins of wild type IR and its K1079R mutant to analyze their subcellular localization, cytoplasmic and nuclear activities in comparison to IGF1R and its K1055R mutant. We showed that, like K1055R mutation in IGF1R, K1079R mutation does not impede the subcellular localization and nuclear activities of IR. Although K1079R mutation significantly decreases the kinase activity of IR but not as much as K1055R mutation, which was seen to drastically reduce the kinase activity of IGF1R. Moreover, K1079 residue in IR is seen to be sitting in a pocket which is different than the allosteric inhibitor binding pocket present in its homologue (IGF1R). This is for the first time such a study has been conducted to identify structural differences between these receptors that could be exploited for designing small molecule allosteric inhibitor(s) of IGF1R as novel anti-cancer drugs.

Jaceosidin Ameliorates Insulin Resistance and Kidney Dysfunction by Enhancing Insulin Receptor Signaling and the Antioxidant Defense System in Type 2 Diabetic Mice.

Emerging evidence has shown that flavonoids extracted from Artemisia have beneficial effects on metabolic disorders. However, whether and how jaceosidin ameliorates insulin resistance and diabetic nephropathy in type 2 diabetes mellitus is largely unknown. For 8 weeks, db/db diabetic mice were fed with or without jaceosidin. Oral jaceosidin supplementation reduced fasting blood glucose levels and insulin resistance through the upregulation of insulin receptor downstream pathways in the liver and skeletal muscles. While jaceosidin did not noticeably alter kidney filtration function, this dietary intervention contributed to attenuating the accumulation of advanced glycation end products in diabetic kidneys. The levels of VEGF-a (vascular endothelial growth factor-a) proteins in the diabetic kidneys were markedly diminished by jaceosidin treatments, which increased the expression and activity of Cu (copper) and Zn-SOD (zinc-superoxide dismutase). Therefore, it is suggested that jaceosidin supplementation elicits antidiabetic effects and treats diabetic nephropathy by augmenting insulin signaling, suppressing fibrosis, and enhancing antioxidant activity.

Intra-Pancreatic Insulin Nourishes Cancer Cells: Do Insulin-Receptor Antagonists such as PGG and EGCG Play a Role?

Harboring insulin-producing cells, the pancreas has more interstitial insulin than any other organ. In vitro, insulin activates both insulin receptor (IR) and insulin-like growth factor-1 receptor (IGF1R) to stimulate pancreatic cancer cells. Whether intra-pancreatic insulin nourishes pancreatic cancer cells in vivo remains uncertain. In the present studies, we transplanted human pancreatic cancer cells orthotopically in euglycemic athymic mice whose intra-pancreatic insulin was intact or was decreased following pretreatment with streptozotocin (STZ). In the next eight weeks, the tumor carriers were treated with one of the IR/IGF1R antagonists penta-O-galloyl-[Formula: see text]-D-glucose (PGG) and epigallocatechin gallate (EGCG) or treated with vehicle. When pancreatic tumors were examined, their fraction occupied with living cells was decreased following STZ pretreatment and/or IR/IGF1R antagonism. Using Western blot, we examined tumor grafts for IR/IGF1R expression and activity. We also determined proteins that were downstream to IR/IGF1R and responsible for signal transduction, glycolysis, angiogenesis, and apoptosis. We demonstrated that STZ-induced decrease in intra-pancreatic insulin reduced IR/IGF1R expression and activity, decreased the proteins that promoted cell survival, and increased the proteins that promoted apoptosis. These suggest that intra-pancreatic insulin supported local cancer cells. When tumor carriers were treated with PGG or EGCG, the results were similar to those seen following STZ pretreatment. Thus, the biggest changes in examined proteins were usually seen when STZ pretreatment and PGG/EGCG treatment concurred. This suggests that intra-pancreatic insulin normally combated pharmacologic effects of PGG and EGCG. In conclusion, intra-pancreatic insulin nourishes pancreatic cancer cells and helps the cells resist IR/IGF1R antagonism.

Agriophyllum oligosaccharides ameliorate hepatic injury in type 2 diabetic db/db mice targeting INS-R/IRS-2/PI3K/AKT/PPAR-γ/Glut4 signal pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Agriophyllum squarrosum (L.) Moq. is a traditional Mongol medicine generally used to treat diabetes. OBJECTIVE: To investigate the protective effects and potential mechanisms of Agriophyllum oligosaccharides (AOS) on liver injury in type 2 diabetic db/db mice. MATERIALS AND METHODS: The db/db mice were divided into the model group (Model), metformin group (MET), high-dose AOS group (HAOS), and low-dose AOS group (LAOS). Nondiabetic littermate control db/m mice were used as the normal control group (Control). Mice in AOS groups were treated with AOS (380 or 750 mg/kg) daily, for 8 weeks. At 8 weeks, blood samples were collected to detect lipid and enzyme parameters concerning hepatic function, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), total protein (TP), albumin (ALB), globulin (GLB), triglyceride (TG), total cholesterol (TC), and high-density lipoprotein cholesterol (HDL-C). Random blood glucose (RBG) test, oral glucose tolerance test (OGTT), and oral maltose tolerance test (OMTT) were also conducted. Microscopy was used to observe morphological changes in the liver of AOS-treated groups. Real-time PCR was used to detect the mRNA expression, including insulin receptor substrate 2 (IRS-2), phosphatidylinositol 3 kinase (PI3K), protein kinase B (AKT), peroxisome proliferator-activated receptor (PPAR)-γ, insulin receptor (INS-R), and Glut4. Furthermore, western blotting was performed to identify proteins, including phosphorylation of IRS-2 (p-IRS-2), PI3K, p-AKT, PPAR-γ, INS-R, and Glut4. Hepatic protein expression of p-IRS-2, PI3K, p-AKT, PPAR-γ, INS-R, and Glut4 was observed using immunohistochemical staining. RESULTS: AOS treatment significantly decreased RBG, OGTT, and OMTT in mice, as well as serum ALT and AST activities. AOS groups demonstrated significantly higher expressions of p-IRS-2, PI3K, PPAR-γ, p-AKT, INS-R, and Glut4 protein and IRS-2, PI3K, AKT, PPAR-γ, INS-R, and Glut4 mRNA in the liver tissue of db/db mice; the degeneration and necrosis of hepatocytes and formation of collagen fibres markedly reduced, improving the structural disorder in the liver. CONCLUSION: The results suggest that AOS could protect the liver in type 2 diabetes, in part by activating insulin in the INS-R/IRS2/PI3K/AKT/Glut4/PPAR-γ signal pathway, facilitating hepatocyte proliferation, and further reducing the blood glucose levels.

Dynamic changes of blood glucose, serum biochemical parameters and gene expression in response to exogenous insulin in Arbor Acres broilers and Silky fowls.

Silky chicken is a breed of chickens with black skin and slow growth rate used in Chinese traditional medicine, whereas Arbor Acres broiler is a well-known commercial breed in the poultry industry, it is featured by a large size, rapid-growth rate, high feed-conversion rate and strong adaptability. The difference in their rate of growth may be primarily related to different mechanism for glucose metabolism. Here we compared the insulin sensitivity of the two breeds; we investigated the temporal changes (at 0 min, 120 min and 240 min) of serum insulin and other biochemical parameters and determined the spatio-temporal changes of gene mRNA abundance in response to exogenous insulin (80 µg/kg body weight). The results indicated that: (1) Silky chickens showed stronger blood glucose recovery than broilers in the insulin resistance test. (2) The serum urea level in Silky chickens was twice of broilers; exogenous insulin significantly up-regulated serum uric acid level in Silky fowls in a time-dependent manner and increased serum cholesterol content at 120 min. (3) Two breeds showed distinctly different temporal changed in serum insulin in response to exogenous insulin stimulation. The fasting serum insulin concentration of broilers was three-fold of Silky chickens at the basal state; it decreased significantly after insulin injection and the levels at 120 min and 240 min of broilers were only 23% (P < 0.01) and 14% (P < 0.01) of the basal state, respectively. Whereas the serum insulin content in Silky chickens showed stronger recovery, and the 240 min level was close to the 0 min level. (4) GLUT2, GLUT12, neuropeptide Y and insulin receptor (IR) were predominantly expressed in the liver, pectoralis major, olfactory bulb and pancreas, respectively, where these genes presented stronger insulin sensitivity. In addition, the IR mRNA level was strongly positively with the GLUT12 level. In conclusion, our findings suggested that Silky chickens have a stronger ability to regulate glucose homeostasis than broilers, owing to their higher IR levels in the basal state, stronger serum insulin homeostasis and candidate genes functioning primarily in their predominantly expressed tissue in response to exogenous insulin.

Trilactic glyceride regulates lipid metabolism and improves gut function in piglets.

Glycerol-lactate esters are energy supplements for exercise, but effects of trilactic glyceride (TLG) on intestinal function and hepatic metabolism are unknown. We found that dietary supplementation with 0.5% TLG to weanling piglets decreased plasma concentrations of low-density lipoprotein and gamma-glutamyl transferase but increased those of D-xylose and high-density lipoprotein. TLG supplementation enhanced mRNA levels for fatty acid synthase (FASN) and SLC27A2 in white adipose tissue; insulin receptor in duodenum; aquaporin-8 in ileum, jejunum and colon; aquaporin-10 in duodenum and ileum; nuclear factor like-2 in jejunum and colon; glutathione S-transferase and phosphoenolpyruvate carboxykinase-1 in intestines; and abundances of claudin-1 and occludin proteins. TLG supplementation decreased mRNA levels for: hepatic hormone-sensitive lipase E, lipoprotein lipase, FASN, insulin-like growth factor-binding protein-3, and SLC27A2; and intestinal lipoprotein lipase, FASN and NADPH oxidase. Furthermore, TLG supplementation enhanced abundances of genus Bifidobacterium, while reducing abundances of family Enterobacteriaceae in ileum, colon and cecum; jejunal caspase-3 protein and diarrhea rate. In conclusion, dietary supplementation with TLG modulated lipid metabolism and alleviated diarrhea by improving intestinal function and regulating intestinal microflora in piglets.

Adrenalectomy impairs insulin-induced hypophagia and related hypothalamic changes.

Adrenalectomy (ADX) induces hypophagia and glucocorticoids counter-regulate the peripheral metabolic effects of insulin. This study evaluated the effects of ADX on ICV (lateral ventricle) injection of insulin-induced changes on food intake, mRNA expression of hypothalamic neuropeptides (insulin receptor (InsR), proopiomelanocortin, cocaine and amphetamine-regulated transcript (Cart), agouti-related protein, neuropeptide Y (Npy) in the arcuate nucleus of the hypothalamus (ARC), corticotrophin-releasing factor in the paraventricular nucleus of the hypothalamus) and hypothalamic protein content of insulin signaling-related molecules (insulin receptor substrate (IRS) 1, protein kinase B (AKT), extracellular-signal-regulated kinase (ERK1/2), c-Jun N-terminal kinase (JNK), protein tyrosine phosphatase-1B (PTP1B) and T cell protein tyrosine phosphatase (TCPTP)) Compared with sham animals, ADX increased the hypothalamic content of pJNK/JNK, PTP1B and TCPTP, as well as decreased mRNA expression of InsR, and corticosterone (B) treatment reversed these effects. Insulin central injection enhanced hypothalamic content of pAKT/AKT and Cart mRNA expression, decreased Npy mRNA expression and food intake only in sham rats, without effects in ADX and ADX + B rats. Insulin did not alter the hypothalamic phosphorylation of IRS1 and ERK1/2 in the three experimental groups. These data demonstrate that ADX reduces the expression of InsR and increases insulin counter-regulators in the hypothalamus, as well as ADX abolishes hypophagia, activation of hypothalamic AKT pathway and changes in Cart and Npy mRNA expression in the ARC induced by insulin. Thus, the higher levels of insulin counter-regulatory proteins and lower expression of InsR in the hypothalamus are likely to underlie impaired insulin-induced hypophagia and responses in the hypothalamus after ADX.

Baicalein improves glucose metabolism in insulin resistant HepG2 cells.

Insulin resistance (IR) is the primary pathogenesis of Type 2 diabetes mellitus (T2DM). Scutellaria baicalensis Georgi is a traditional Chinese herbal medicine, often used in the clinical treatment of T2DM. Baicalein which is considered to have anti-IR effects is one of its active ingredients. IR-induced HepG2 cells were used to investigate the effect of baicalein on glucose metabolism and insulin-signaling pathway, using metformin as a positive control. We found that the use of both baicalein and metformin increased the glucose consumption of IR cells, as well as increasing the pyruvate kinase (PK) and glucokinase (GCK) activity. Also increased was the expression levels of insulin receptor (InsR), insulin receptor substrate-1 (IRS-1), phosphoinositide 3-kinase (PI3K), protein kinase B (AKT) pathway and glucose transporter 2 (GLUT2). Reduced expression levels were found in that of glucose 6 phosphatase (G6Pase) and phosphoenolpyruvate carboxykinase (PEPCK) mRNA. The results confirmed that baicalein (10-6 and 10-5 mol/L) promotes glucose uptake and glycolysis, inhibits gluconeogenesis of hepatocytes to improve glucose metabolism, and may be as a result from regulation of InsR/IRS-1/PI3K/AKT pathway. Additionally, baicalein has large concentration range on inhibiting IR, and at lower concentrations has strong anti-IR hepatocyte activity.

Hypothalamic insulin receptor expression and DNA promoter methylation are sex-specifically altered in adult offspring of high-fat diet (HFD)-overfed mother rats.

Maternal overnutrition around reproduction has been shown to increase the offspring's risk for "diabesity," mediated by altered hypothalamic neuropeptide expression. In this report, a possible contribution of altered hypothalamic sensing capacity for the peripheral satiety signals glucose, insulin and leptin will be addressed, taking into account potential sex differences. Specifically, we evaluated the effects a maternal high-fat diet (HFD) overfeeding has in rats pre- and during pregnancy and lactation on the hypothalamic gene expression patterns of insulin and leptin receptors (InsR, ObRb) and glucose transporter 3 (Glut3) as well as DNA methylation in the offspring at adult age (day 200 of life). Maternal HFD consumption resulted in a metabolic syndrome phenotype, i.e., obesity, hyperleptinemia, hyperinsulinemia, impaired glucose tolerance and increased homeostatic model assessment of insulin resistance. Interestingly, in turn, insulin resistance was more pronounced in male offspring, accompanied by decreased hypothalamic InsR-mRNA. This was linked with hypermethylation of an activating transcription factor binding site within the hypothalamic InsR promoter. The degree of methylation correlated inversely with respective InsR expression, while InsR expression itself was inversely related to phenotypic "diabesity." Expression of ObRb and Glut3 mRNA was not significantly changed. In conclusion, sex-specific alterations of hypothalamic InsR expression and DNA promoter methylation in adult offspring of HFD-overfed dams may lead to hypothalamic insulin resistance and "diabesity," with males predisposed to this epigenetic malprogramming.

Aspalathin-Enriched Green Rooibos Extract Reduces Hepatic Insulin Resistance by Modulating PI3K/AKT and AMPK Pathways.

We previously demonstrated that an aspalathin-enriched green rooibos extract (GRE) reversed palmitate-induced insulin resistance in C2C12 skeletal muscle and 3T3-L1 fat cells by modulating key effectors of insulin signalling such as phosphatidylinositol-4,5-bisphosphate 3-kinase/protein kinase B (PI3K/AKT) and AMP-activated protein kinase (AMPK). However, the effect of GRE on hepatic insulin resistance is unknown. The effects of GRE on lipid-induced hepatic insulin resistance using palmitate-exposed C3A liver cells and obese insulin resistant (OBIR) rats were explored. GRE attenuated the palmitate-induced impairment of glucose and lipid metabolism in treated C3A cells and improved insulin sensitivity in OBIR rats. Mechanistically, GRE treatment significantly increased PI3K/AKT and AMPK phosphorylation while concurrently enhancing glucose transporter 2 expression. These findings were further supported by marked stimulation of genes involved in glucose metabolism, such as insulin receptor (Insr) and insulin receptor substrate 1 and 2 (Irs1 and Irs2), as well as those involved in lipid metabolism, including Forkhead box protein O1 (FOXO1) and carnitine palmitoyl transferase 1 (CPT1) following GRE treatment. GRE showed a strong potential to ameliorate hepatic insulin resistance by improving insulin sensitivity through the regulation of PI3K/AKT, FOXO1 and AMPK-mediated pathways.

Insulin receptor localization in the embryonic avian hypothalamus.

The hypothalamus is a brain region critical for the homeostatic regulation of appetite and energy expenditure. Hypothalamic neuronal activity that is altered during development can produce permanent physiological changes later in life. For example, circulating hormones such as insulin have been shown to influence hypothalamic neuronal projections, leading to altered metabolism in adult rodents. While insulin signaling in the post-hatch chicken has been shown to mirror that of mammals, the developmental role of insulin in the avian embryonic hypothalamus remains largely unexplored. Here we present the earliest known evidence for insulin receptor (InsR) expression in embryonic avian hypothalamic nuclei governing energy homeostasis. RT-PCR analysis reveals InsR mRNA in E8, E10, and E12 neurons while western blot data demonstrate protein expression in E12 avian whole brain and hypothalamic lysates. Immunohistochemical analysis of avian hypothalamic brain slices demonstrates a shift in InsR localization from paraventricular expression in E8 to a more defined concentration of InsR in developmental regions resembling the ventromedial hypothalamus (VMH) and arcuate nucleus (ARC) in E12 time points. In addition, InsR expression appears in a heterogeneous pattern, suggesting receptor localization to subpopulations of avian hypothalamic neurons as early as E8. With increasing evidence suggesting energy homeostasis pathways may be altered via the gestational environment, it is important to understand how insulin signaling may affect embryogenesis. Our research provides evidence for the earliest known embryonic expression of InsR protein in the avian hypothalamus and may suggest a developmental role for insulin signaling in the early patterning of metabolic pathways in the central nervous system.

Beneficial Effects of an Aged Black Garlic Extract in the Metabolic and Vascular Alterations Induced by a High Fat/Sucrose Diet in Male Rats.

Aged black garlic (ABG) is a functional food with antioxidant and anti-inflammatory properties. Recent studies also report its beneficial metabolic effects in a context of obesity or diabetes, although the mechanisms involved are poorly understood. The aim of this work was to analyze the effects of an ABG extract in the vascular and metabolic alterations induced by a high-fat/sucrose diet in rats. For this purpose, male Sprague⁻Dawley rats were fed either a standard chow (controls; n = 12) or a high-fat/sucrose diet (HFD; n = 24) for 16 weeks. From week 8 on, half of the HFD rats were treated with a commercial ABG extract concentrated in S-allyl cysteine and melanoidins (ABG10+®; 250 mg/kg daily by gavage; 5 mL/kg). ABG10+®-treated rats showed lower mean caloric intake, body weight, triglycerides, low density lipoprotein cholesterol (LDL-c), insulin and leptin serum concentrations and higher high density lipoprotein cholesterol (HDL-c) and adiponectin serum concentrations than non-treated rats. In the hypothalamus, ABG10+® treatment induced an increase in the gene expression of proopiomelanocortin (POMC) and a decrease in leptin receptor (ObR) mRNA levels. No significant changes were found in visceral adipose tissue except for an overexpression of ß3-adrenergic receptor (ß3-ADR) in ABG-treated rats. In subcutaneous adipose tissue, ABG10+® treatment decreased adipose weight and downregulated the gene expression of PPAR-γ, LPL, ObR and HSL. In brown adipose tissue, an overexpression of InsR, GLUT-4, UCP-1 and ß3-ADR in ABG10+®-treated rats was found, whereas PPAR-γ mRNA levels were significantly decreased. Regarding vascular function, ABG10+® treatment attenuated the obesity-induced vasoconstriction in response to potassium chloride both in presence/absence of perivascular adipose tissue (PVAT). On the contrary, aorta segments from ABG-treated rats showed and improved relaxation in response to acetylcholine only when PVAT was present, with this fact possible being related to the decreased gene expression of proinflammatory cytokines in this tissue. In conclusion, ABG10+® administration partially improves the metabolic and vascular alterations induced by a high-fat/high-sucrose diet in rats through modifications in the gene expression of proteins and neuropeptides involved in inflammation, fat metabolism and food intake regulation. Further studies are required to assess the bioavailability of ABG between rats and humans.

Insulin action in the brain regulates mitochondrial stress responses and reduces diet-induced weight gain.

OBJECTIVE: Insulin action in the brain controls metabolism and brain function, which is linked to proper mitochondrial function. Conversely, brain insulin resistance associates with mitochondrial stress and metabolic and neurodegenerative diseases. In the present study, we aimed to decipher the impact of hypothalamic insulin action on mitochondrial stress responses, function and metabolism. METHODS: To investigate the crosstalk of insulin action and mitochondrial stress responses (MSR), namely the mitochondrial unfolded protein response (UPRmt) and integrated stress response (ISR), qPCR, western blotting, and mitochondrial activity assays were performed. These methods were used to analyze the hypothalamic cell line CLU183 treated with insulin in the presence or absence of the insulin receptor as well as in mice fed a high fat diet (HFD) for three days and STZ-treated mice without or with insulin therapy. Intranasal insulin treatment was used to investigate the effect of acute brain insulin action on metabolism and mitochondrial stress responses. RESULTS: Acute HFD feeding reduces hypothalamic mitochondrial stress responsive gene expression of Atf4, Chop, Hsp60, Hsp10, ClpP, and Lonp1 in C57BL/6N mice. We show that insulin via ERK activation increases the expression of MSR genes in vitro as well as in the hypothalamus of streptozotocin-treated mice. This regulation propagates mitochondrial function by controlling mitochondrial proteostasis and prevents excessive autophagy under serum deprivation. Finally, short-term intranasal insulin treatment activates MSR gene expression in the hypothalamus of HFD-fed C57BL/6N mice and reduces food intake and body weight development. CONCLUSIONS: We define hypothalamic insulin action as a novel master regulator of MSR, ensuring proper mitochondrial function by controlling mitochondrial proteostasis and regulating metabolism.

Insulin signaling in LepR cells modulates fat and glucose homeostasis independent of leptin.

Hypothalamic neurons detect changes in circulating hormones such as leptin and insulin and put forward outputs to sustain energy and glucose homeostasis. Because leptin and insulin receptors colocalize in ~40-60% of neurons in the hypothalamus, we characterized the metabolic phenotype of mice with selective deletion of the insulin receptor (InsR) in LepR cells. LRΔInsR mice presented no difference in body weight and insulin levels but increased fat mass. In the light phase, LRΔInsR mice exhibited increased food intake, locomotor activity, carbon dioxide production, and respiratory exchange rate. These mice showed reduced fat oxidation and reduced expression of cluster of differentiation 36 and AMP-activated protein kinase-α1 in the liver, increased glucose oxidation in the light phase, and overall reduced basal glucose levels. To verify the impact of InsR deletion in LepR cells in obesity, we generated ob/ ob InsRfl, ob/ ob LRcre, and ob/ ob LRΔInsR mice. The ob/ ob LRΔInsR mice had higher body weight, fat mass, and expression of genes related to fat metabolism in the liver. No difference in food intake despite increased neuropeptide Y and agouti-related peptide expression, and no difference in energy expenditure, fat, or glucose oxidation was found in ob/ ob LRΔInsR compared with LRcre or LRΔInsR controls. Remarkably, basal glucose levels were reduced, and the expression of genes associated with glucose metabolism in the liver was higher. Insulin signaling in LepR cells is required for the proper fat and glucose oxidation. These effects are independent of leptin given that the leptin-deficient ob/ ob LRΔInsR mice also presented reduced glycemia and higher adiposity. The mechanisms underlying these responses remain to be unveiled.