Recombinant resistin. The entire open reading frame (ORF) of resis- tin were cloned into pFM1, which contains an internal ribosome entry site followed by the ORF of GFP as previously described (19). Stably transfected HEK 293-T cells were sorted by FACS for high levels of GFP expression and, therefore, high resistin expression. Five sorts of enrichment established a line of cells capable of produc- ing milligram quantities of recombinant protein per liter of media. Serum-free media was used to collect the secreted protein from con- fluent cells for 2 days. The media was then harvested, spun down to remove cells and the pH adjusted to pH 6.0 with 20 mM Bis-Tris pH 6.0. The media was then filtered through a 0.22-µm filter and loaded directly onto an Econo-S ion exchange column (Bio-Rad Laborato- ries Inc., Hercules, California, USA). The protein was then eluted with a step salt gradient from 200 mM to 300 mM NaCl, followed by a 300 mM to 1 M NaCl continuous gradient. Two major peaks were apparent, with resistin eluting in the second peak between approxi- mately 350 and 500 mM NaCl. This eluate was then concentrated by centrifugation in a centricon YM-3 (Millipore, Bedford, Massachu- setts, USA) and loaded onto a Superdex 200 size filtration column (Amersham-Pharmacia, Piscataway, New Jersey, USA). The major peak contained at least 99% pure recombinant protein.
action on glucose uptake in adipose tissue and, thus, preventing further adipocyte differentiation (37). Recent investigations of human resistin in relation to obesity have shown higher serum resistin levels in obese subjects compared with lean subjects (39,28), which positively correlated with the changes in BMI and visceral fat area (28,40,41). The implication that resistin is important in human adipose tissue has been corroborated by studies showing increased protein expression with obesity (28), as well as protein secretion from isolated adipocytes (42). These recent observations are concomitant with initial studies that showed increased serum resistin levels and gene expression levels in abdominal depots in states of increased adiposity. A further study has shown a significant reduction in circulating resistin levels following moderate weight loss and post-gastric bypass (43). Contrary with the studies suggesting a role for resistin in obesity, (44) have reported resistin was undetectable in serum of obese mice, with the same study indicating reductions of resistin mRNA and protein expression in obesity. Others have reported no association of resistin expression with increased adiposity, despite observing elevated circulating levels (45, 36,38). However, it has been suggested recently that resistin mRNA expression does not necessarily correlate with protein expression (36).Possible explanations for such diverse observations include differences in post-transcriptional and post-translational modifications, consequently affecting secretory rates of resistin. Increased serum levels may enhance transcript degradation rates via negative feedback mechanisms, or the initiation and recruitment of inhibitors of translation. The secreted form of resistin is considered to have paracrine properties, and this may imply the majority of regulation occurs at the protein level. Similarly, Rajala and co-workers (36). Further recent human studies have shown no correlation of serum or plasma levels of resistin with any markers of adiposity (46). Heilbronn et al., reported no relationship between resistin serum levels and percentage body fat, visceral adiposity and BMI. However, the authors (47) suggested that the lack of correlation of serum resistin and increased adiposity was partly due to the confounding variable of age, as non-obese subjects were significantly younger than obese subjects (47).
the rates of glucose infusion required to maintain euglycemia were markedly lower in OF rats compared with control rats fed a stan- dard chow (SC) diet (7.9 ± 0.7 versus 15.4 ± 0.2 mg/kg/min; P < 0.01) when SCR was administered (Figure 3B). On the other hand, when Scd1 ASO was given, the rate of glucose infusion required to main- tain euglycemia in OF rats was markedly increased up to the lev- els observed in control SC rats (15.2 ± 0.4 mg/kg/min; Figure 3B). Scd1 ASO did not significantly alter the levels of glucoregulatory hormones (Supplemental Table 1) and the rate of glucose uptake (Figure 3B). Conversely, this intervention markedly suppressed liver glucose production (Figure 3, B and D), and this effect entirely accounted for its effect on glucose metabolism. Glucose produc- tion represents the net contribution of gluconeogenesis and gly- cogenolysis (Figure 3C). However, a portion of glucose entering the liver via phosphorylation of glucose is also a substrate for dephosphorylation via glucose-6-phosphatase (Glc-6-Pase, encod- ed in rodents by the G6pc gene), creating a futile (glucose) cycle (Figure 3C). To delineate the mechanisms by which Scd1 inhibition modulates liver glucose homeostasis, we estimated the in vivo flux through Glc-6-Pase (Glc-6-Pase flux) and the relative contribution of glucose cycling, gluconeogenesis, and glycogenolysis to glucose output (Figure 3, D and E). Treatment with Scd1 ASO markedly and similarly decreased the rates of glucose cycling and Glc-6-Pase flux (Figure 3D) in parallel to its effects on glucose production (Figure 3D). The decrease in glucose production was accounted for by a marked inhibition of gluconeogenesis as well as glycogenolysis (Figure 3E and Supplemental Table 2).
Recently, it has been shown that resistin mRNA and protein are both present in mouse hypothalamus (33, 34) and that resistin activates a certain subset of hypothalamic neurons in vitro (35). With work from our laboratory as well as by others highlighting the importance of the brain-liver circuit in controlling hepatic glucose homeostasis in response to hypothalamically initiated hormonal (i.e., insulin and leptin) (36–38) and nutritional (i.e., FFA and glucose) signals (39–41), resistin also seemed a likely can- didate to act via hypothalamic pathways. Since the effects are at least in part mediated via interactions with receptors within the CNS, it is postulated herein that resistin regulates glucose fluxes and signaling in the liver both directly via hepatic effects and indi- rectly through a central (hypothalamic) site of action (Figure 1A). In this study, we investigated whether the brain also plays a role in mediating the diabetogenic effects of physiological hyperresis- tinemia and identify potential mechanisms by which resistin mod- ulates hepatic glucose fluxes. To determine whether an increase in resistin made available to the CNS would modulate peripheral insulin action, we made use of the hyperinsulinemic-euglycemic clamp combined with icv and mediobasal hypothalamus (MBH) infusions of recombinant resistin. Furthermore, MBH adminis- tration of a specific anti-mouse resistin antibody (Rs Ab) was uti- lized to assess what contribution central resistin action made to the effect of circulating resistin on whole-body glucose homeosta- sis. Lastly, we aimed to further investigate the complex relation- ship between inflammation and insulinresistance in mediating resistin’s effects on glucose fluxes. The adipose-derived hormone resistin rapidly stimulates glucose production (GP) and induces hepaticinsulinresistance in rodents (17–20).
Previous studies using stearoyl-CoA desaturase–1–deficient (SCD1-defi- cient) mice have shown that this enzyme plays an important role in many diseases of altered cellular metabolism including obesity, insulinresistance, and dyslipidemia. Although SCD1 activity is highest in lipogenic tissues such as the liver and adipose tissue, it is also present at lower levels in most tissues. To better understand the role of SCD1 in liver metabolism it is nec- essary to explore SCD1 deficiency in a more focused, tissue-specific man- ner. This commentary focuses on 2 recent studies published in the JCI that address this question using antisense oligonucleotide inhibition of SCD1. First, Jiang et al. have previously reported that long-term inhibition of SCD1 prevents the development of high-fat diet–induced obesity and hepatic ste- atosis. Second, Gutiérrez-Juárez et al. show in this issue that short-term inhi- bition of hepatic SCD1 is sufficient to prevent diet-inducedhepaticinsulinresistance, signifying an important role of hepatic SCD1 in liver insulin sen- sitivity (see related article beginning on page 1686).
HFD feeding for twelve weeks significantly increased the body weight and fasting plasma glucose concentrations as compared with animals fed with control diet (Supplementary Fig. S1A). After 12 weeks of HFD feeding, the fasting plasma insulin concentrations were significantly increased and were approximately 1.7 times higher than before. As shown in Fig. 1A, during the period of spexin treatment, the body weight curve of the HFD + spexin group and the HFD group was gradually separated, while the difference was not significant between the spexin+control groups and the control group. After 8 weeks treatment of spexin, the body weight of the rats at 26 weeks of age in the HFD + spexin group was significantly lower than those in the HFD group (P < 0.05) (Fig. 1B). Exogenous spexin intervention had no significant effect on blood glucose and the area under the curve of glucose (P > 0.05, Fig. 1C, D). Spexin reduced the insulin concentrations in the HFD-fed rats, while no obvious changes were observed in the normal diet-fed group (Fig. 1E). However, Spexin treatment reduced the serum FFA levels (both P < 0.01) but not the serum TG level, when compared with the levels observed in the HFD group (Fig. 1F, G). Exogenous spexin also reduced the HOMA-IR value in the HFD-fed rats but not normal diet-fed rats (Fig. 1H).
In 1963, Philip Randle put forth the notion that FAs may themselves induce insulinresistance (53). Indeed, increasing levels of circulating FAs highly correlate with insulinresistance (54). Further, clinical trials testing agents that reduce FAs have shown increased insulin sensitivity outcomes (55–58). One of the many actions of insulin is to inhibit adipose tissue lipolysis and hence FA release into the circulation. GH has also been proposed to be a major regulator of adipose tissue lipolysis (15). Indeed, acromegalic patients have enhanced lipolysis and increased adipose expression of genes that regulate lipolysis (59). Two independent trials reported that administration of the antilipolytic drug acipimox increases insulin sensitivity in GH-treated patients (19, 20). Collectively, these data support a predominant role for lipolysis in GH-mediated insulinresistance. Our work here supports these findings. We showed that GH-mediated lipolysis correlates with hepaticinsulinresistance and that GH promoted adipose tissue lipolysis through JAK2, but indirectly via perturbation of insulin action. Therefore, the predominant diabetogenic mecha- nism of GH is likely due to increased adipose tissue lipolysis, promoting hepaticinsulinresistance.
Acute or chronic exercise has been showed to induce numerous metabolic and hemodynamic factors that can contribute to the improvements in glucose homeostasis in individuals with insulinresistance [15-18]. These adaptive responses include enhanced insulin action on the skeletal muscle glucose transport system, reduced hormonal stimulation of hepatic glucose production, improved blood flow to skeletal muscle, and norma- lization of an abnormal blood lipid profile. In fact, the beneficial effects of an acute bout of exercise and of chronic exercise training on insulin action in insulin- resistant states are well established. Our group and other groups have demonstrated that the accomplishment of exercise (chronic and acute) improves insulinresistance; therefore, exercise protocols are performed when the diet-induced obesity (reversal treatment manner) is al- ways installed in mice and rats [19-24]. In addition, Marinho and colleagues  have shown the involvement of APPL1 in the improvement of insulinresistance in the liver of exercise training mice. Few are knowledgeable about the efficacy of exercise training on insulin signaling/ insulinresistance when applied simultaneously to the onset
a model of insulinresistance (IR), induced by prolonged high fat diet with high content of saturated fats was used to investigate the effect of N-stearoylethanolamine (NSe) on the composition of free fatty acids (FFA), plasma lipoprotein spectrum and content of proinflammatory cytokine TNFα in rats. The results of this work showed a rise in the content of monounsaturated fatty acids (18:1 n-9) and a reduction in the level of polyunsaturated fatty acids (20:4 n-6) in plasma of rats with experimental IR. These findings are accompa- nied by the increased TNFα production and significant changes in plasma lipoprotein profile of rats with the fat overload. particularly, a decreased high-density lipoprotein (hDl) cholesterol level and increased low- density (LDL) and very low-density lipoprotein (VLDL) cholesterol level were detected. The NSE administra - tion to obese rats with IR restored the content of mono- and polyunsaturated FFa, increased hDl cholesterol content and reduced lDl cholesterol level. In addition, the IR rats treated with NSe showed normalization in the serum TNFα level. Our results showed the restoration of plasma lipid profile under NSE administration in rats with obesity-induced IR. considering the fact that plasma lipid composition displays the lipid metabolism in general, the NSE actions may play a significant role in the prevention of IR-associated complications.
a kind of decompensated state due to the combined effects of physiological IR and chron- ic IR in late pregnancy that cannot be overcome by the compensatory secreted insulin of islet beta cells . This kind of chronic IR may already exist before pregnancy. And after preg- nancy, under the effects of placental hormone secretion, IR is further strengthened while the amount of insulin secretion is limited, so the increase of insulin is not enough to offset the pregnancy-induced IR and blood glucose is increased from the middle-late stage of preg- nancy thus resulting in GDM . This study showed that even the blood glucose was well controlled in GDM group, insulin and HOMA-IR were also significantly increased compared to the control group. It was promoted that periph- eral insulin target organ of GDM patients had severer IR than the normal pregnancy women. And it was also suggested that IR was an impor- tant cause for GDM. The GDM pregnant women of 37~40 pregnant week still showed a higher level of HOMA-IR and FINS than those in healthy control group after the satisfactory control of blood glucose by controlling diet, exercise and insulin therapy, indicating the damage of islet β-cells which provided insulin and modulated the glucose metabolism in pregnant women was not permanent. These cells maintained the normal glucose metabolism by increasing the secretion of insulin against insulin resis- tance. Insulinresistance existed not only in midtrimester but in the full-term pregnancy when blood glucose was satisfactorily and per- sistently controlled and further affected the pregnancy outcome of pregnant women with GDM.
Insulinresistance (IR) is a major public health problem that can lead to many dangerous medical disorders and early mortality. This study aimed to explore the effectiveness of resveratrol (RSV) to counteract the neuro-complications accompanying high fat, high fructose (HFHF) diet experimentally induced-IR in rats. IR was induced by the ingestion of HFHF diet for 70 days, 80 juvenile rats were used, and the treatments were given orally for the diet latest 10 days. Rats’ general behavior was assessed by open field test (OFT) and forced swimming test (FST). On biochemical level; neuro- complications were assessed by measuring brain levels of monoamines and their metabolites as well as the levels of 8-hydroxyguanosine (8-OHDG), tumor necrosis factor-α (TNF-α), malondialdehyde (MDA), reduced and oxidized glutathione (GSH and GSSG) and nitric oxide (NO x ). Oral RSV (20 and 40 mg/kg p.o) increased the activity of
Both insulinresistance and inflammation are complex disease states that involve a number of highly controlled signalling pathways [18, 19, 43, 53, 56, 96, 100, 164, 199, 291-295]. There has been much research in investigating the involvement of numerous cells and proteins on altering these pathways in the development of obesity-induced inflammation [112, 113, 156, 247, 295-299]; however despite this a number of specific mechanisms such as when and how inflammation is induced in diet-induced obesity, and a detailed time-course of how its induction impacts on vascular and muscular insulin-mediated responses, is still not fully understood [26, 117, 148, 198, 210]. What is clear from previous research is that the induction of inflammatory signalling, especially within diet-induced obesity, may be reliant on a number of other signals and responses to occur before inflammation can be initiated. Due to the strong association between obesity and inflammation, the adipose tissue has been highlighted as an important site in which the regulation of inflammatory expression is conducted [19, 27, 164, 165, 300]. The view that adipose tissue is solely a site for energy storage is now outdated. Adipose tissue is now recognised as a complex and active organ capable of multiple functions and that, in addition to its primary role in lipid storage, also contributes towards endocrine function [163, 165]. The adipose tissue is known to secrete a number of hormones including leptin and adiponectin which are important for metabolic and energy homeostasis [163-165]. In addition to this the adipose tissue also secretes or induces a number of inflammatory factors including cytokines and macrophage markers. However the exact mechanisms and pathways by which this occurs are still not fully understood.
Animals and diets. Mice received ad libitum access to food and water and were generated and housed in the Yale Animal Resources Center at 23°C under a 12-hour light/12-hour dark cycles (0700–1900h). Male mice were studied at 14 to 19 weeks of age. The following diets were used: regular chow diet (Harlan Teklad TD2018: 18% fat, 58% carbo- hydrate, and 24% protein) and a HFD (Research Diets D12492: 60% fat, 20% carbohydrate, and 20% protein). For all animal studies, sam- ple sizes were preselected to yield 90% power (at α = 0.05) to detect 20% differences in metabolic parameters, with an expected SD of 10%. Mice were randomly allocated to experimental groups, and weight matching was ensured before beginning experimental protocols. The investigators were not always blinded to genotype during the studies.
Questions have been raised as to whether dietary carbohydrate intake is directly related to the development of type 2 diabetes. Of particular importance, fructose-inducedinsulinresistance has been previously shown in animals. However, the implications of such findings for humans are un- clear as these models typically use very high doses of sugars and from sources not commonly con- sumed. Little is known about how the typical consumption of sugar in humans affects risk factors for diabetes. 355 weight-stable (weight change < 3% in previous 30 days) individuals aged 20 - 60 years old drank sugar-sweetened low fat milk every day for 10 weeks as part of their usual diet. Added sugar was provided in the milk as either high fructose corn syrup or sucrose at 8%, 18% or 30% of the calories required to maintain body weight. Insulinresistance was measured using the Homeostasis Model Assessment (HOMA IR) on fasting measures and a standard Oral Glucose To- lerance Test (OGTT) was used to measure insulin and glucose areas under the curve resistance (AUC30 g * AUC30 I) and whole body insulin sensitivity and hepaticinsulinresistance using the Matsuda Composite Insulin Sensitivity Index (ISI). There was a small increase in weight in the en- tire cohort (169.1 ± 30.6 vs 171.6 ± 31.8 lbs, p < 0.001), which was greater in the 30% level than in the 8% or 18% levels (p < 0.05). Glucose, insulin, HOMA, glucose AUC, insulin AUC, Matsuda insulin sensitivity index, and hepaticinsulinresistance did not vary by sugar level (p > 0.05) nor by sugar type (p > 0.05). In the entire cohort insulin sensitivity decreased as evidenced by an increase in HOMA IR (1.8 ± 1.3 vs 2.3 ± 3.4, p < 0.01) and a decrease in the Matsuda ISI (13.1 ± 21.3 vs 11.6 ± 16.1, p < 0.05). Hepaticinsulinresistance was unchanged (2.4 ± 1.7 vs 2.4 ± 1.7 p > 0.05). Neither
At present, there is sufficient evidence that hepatic IR can be induced by long-time high-fat diet (HFD) . Therefore, in this study, in order to provide basic infor- mation for definition and identification of miRNAs asso- ciated with hepatic IR, high-throughput sequencing of small RNAs was performed and analyzed in the livers of normal diet (ND) mice and HFD-inducedhepatic IR mice. Differentially expressed miRNAs were obtained through analysis and comparison. Afterwards, the potential target genes of these differentially expressed miRNAs were pre- dicted, and relevant pathways were analyzed. Meanwhile, some miRNAs were selected and validated by quantitative real-time PCR (q-PCR), and their potential target genes relative to IR or glycolipid metabolism were displayed in our study. Overall, our findings provide a global view of specific miRNA expression profiles in the liver of HFD-inducedhepatic IR mice, which is expected to contribute to future studies of miRNAs and their target genes’ regulatory mechanisms in the pathogenesis of hepatic IR and related diseases.
The consumption of the HFD resulted in hypertriglyc- eridemia, hypercholesterolemia, and increased levels of both LDL-C and HDL-C. Fasting hypertriglyceridemia in IR has largely been attributed to apoB-100 containing TGs rich very low density lipoprotein (VLDL) overpro- duction and secretion by the liver, with a lesser contribu- tion to the impaired VLDL removal . Fructose consumption can promote hepatic lipogenesis because it provides unregulated amounts of lipogenic substrates acetyl-CoA and glycerol-3-phosphate . Fructose can also activate sterol regulatory element binding protein-1c (SREBP-1c) independently of insulin, which then acti- vates genes involved in de-novo lipogenesis . SREBP- 1c over-expression was also reported to inhibit insulin
Our results showed that HZ extract prevented increases in body weight and fat accumulation around the waist in mice fed a 12-week-HFD diet. Food intake efficiency was decreased along, with a reduction in the accumulation of fat that may cause metabolic syndrome. HZ treatment also inhibited the extremely high level of bad cholesterol transportation carriers, LDL-C, in HFD mice, these be- ing considered a sign of health problems. The protective effect on metabolism was examined in terms of concen- trations of glucose, lipids, and other insulinresistance- associated index. Further investigation of the molecular mechanism showed that HZ extract upregulated the genes and proteins associated with fatty acids oxidation, downregulated those related to hepatic de novo lipogen- esis in palmitate-treated human HuS-E/2 hepatocytes. These solid results indicate that HZ has a promising bio- activity in regulating obesity and insulin sensitivity, which may have potential for clinical application in pre- venting from hepatic steatosis and insulinresistance.
Diet-induced obesity. Male and female mice (6–8 weeks old) were fed either regular mouse chow or a high-fat diet for 14 weeks. The high-fat diet con- tained 33.8% fat (59.9% of calories), 27.1% carbohydrate (21.3% of calories), and 23.9% protein (18.8% of calories) as well as 5.8% fiber, 6.2% moisture, and 3.2% vitamins and minerals (TD.97070; Harlan Teklad). The fatty acid profile was 45% saturated, 24% trans, 24% monounsaturated (cis), and 7% polyunsaturated (cis). The mice had free access to food and water and were kept on a 12-hour light/12-hour dark cycle. Food intake and body weight were measured daily. Mice were sacrificed by cervical dislocation. The liver and combined adipose tissue (inguinal, retroperitoneal, subcutaneous, and epididymal) were removed, weighed, and snap-frozen, and the combined adipose tissue weight expressed as percent of total body weight was defined as adiposity index. Trunk blood was collected for measuring endocrine and biochemical parameters.
The severity of HFD-induced IR was less in obese MIF 2/2 mice, with significantly lower GTT and ITT compared to obese WT mice (Figure 1A–D). Fasting plasma insulin levels increased following the HFD irrespective of genotype, however obese MIF 2/2 mice secreted significantly less insulin in response to glucose compared to obese WT mice (Figure 1E). Baseline GTT and ITT were not different between WT and MIF 2/2 mice. Age- matched chow-fed WT and MIF 2/2 mice GTTs and ITTs were equivalent and significantly lower than obese WT and MIF 2/2 mice (Figure S1A–D). Despite equivalent body weight at baseline and comparable food intake during the intervention, MIF 2/2 mice gained significantly less weight than WT mice in response to HFD (Figure 1F–G). Body mass composition confirmed the lower body weight was due to reduced fat mass (Figure 1H), however we could not determine the body regions of fat distribution. Nevertheless, we measured the weight of various organs and observed both liver and epididymal weights were significantly greater in obese WT compared to MIF 2/2 mice (Table 1). Histological analysis confirmed obese MIF 2/2 mice display a hyperplasic morphology (Figure S2A). Since weight is a key determinant of insulin sensitivity, we sought to distinguish between direct effects of MIF deficiency on insulin sensitivity from secondary effects of reduced weight. Weight-matched (45–47 g) obese MIF 2/2 mice had significantly lower GTT and ITT, compared to equivalently obese WT mice, indicating that improved glucose homeostasis in obese MIF 2/2 mice is indepen- dent of body weight (Figure S2B–E). Fasting plasma triacylglycerol (TAG), NEFA, cholesterol, IL-6 IL-10, IL-12p70 and MCP-1 levels increased equivalently in both genotypes following HFD compared to lean counterparts. Plasma leptin and keratinocyte chemoattractant (KC) levels were lower in HFD MIF 2/2 mice compared WT mice following feeding of a HFD (Table 1).
Prolonged activation of p70 S6 kinase (S6K) by insulin and nutrients leads to inhibition of insulin signaling via negative feedback input to the signaling factor IRS-1. Systemic deletion of S6K protects against diet-induced obesity and enhances insulin sensitivity in mice. Herein, we present evidence suggesting that hypothalamic S6K activation is involved in the pathogenesis of diet-inducedhepaticinsulinresistance. Extending previous findings that insulin suppresses hepatic glucose production (HGP) partly via its effect in the hypothalamus, we report that this effect was blunted by short-term high-fat diet (HFD) feeding, with concomitant suppression of insulin signaling and activation of S6K in the mediobasal hypothalamus (MBH). Constitutive activation of S6K in the MBH mimicked the effect of the HFD in normal chow–fed animals, while suppression of S6K by overexpression of dominant-negative S6K or dominant-negative raptor in the MBH restored the ability of MBH insulin to suppress HGP after HFD feeding. These results suggest that activation of hypothalamic S6K contributes to hepaticinsulinresistance in response to short-term nutrient excess.