Paula G.F. Quaresma

Diet-induced obesity induces endoplasmic reticulum stress and insulin resistance in the amygdala of rats

Insulin acts in the hypothalamus, decreasing food intake (FI) by the IR/PI3K/Akt pathway. This pathway is impaired in obese animals and endoplasmic reticulum (ER) stress and low‐grade inflammation are possible mechanisms involved in this impairment. Here, we highlighted the amygdala as an important brain region for FI regulation in response to insulin. This regulation was dependent on PI3K/AKT pathway similar to the hypothalamus. Insulin was able to decrease neuropeptide Y (NPY) and increase oxytocin mRNA levels in the amygdala via PI3K, which may contribute to hypophagia. Additionally, obese rats did not reduce FI in response to insulin and AKT phosphorylation was decreased in the amygdala, suggesting insulin resistance. Insulin resistance was associated with ER stress and low‐grade inflammation in this brain region. The inhibition of ER stress with PBA reverses insulin action/signaling, decreases NPY and increases oxytocin mRNA levels in the amygdala from obese rats, suggesting that ER stress is probably one of the mechanisms that induce insulin resistance in the amygdala.

Topiramate Treatment Improves Hypothalamic Insulin and Leptin Signaling and Action and Reduces Obesity in Mice

Topiramate (TPM) treatment has been shown to reduce adiposity in humans and rodents. The reduction in adiposity is related to decreased food intake and increased energy expenditure. However, the molecular mechanisms through which TPM induces weight loss are contradictory and remain to be clarified. Whether TPM treatment alters hypothalamic insulin, or leptin signaling and action, is not well established. Thus, we investigate herein whether short-term TPM treatment alters energy balance by affecting insulin and leptin signaling, action, or neuropeptide expression in the hypothalamus of mice fed with a high-fat diet. As expected, short-term treatment with TPM diminished adiposity in obese mice mainly due to reduced food intake. TPM increased anorexigenic signaling by enhancing the leptin-induced leptin receptor/Janus kinase 2/signal transducer and activator of transcription 3 pathway and the insulin-induced insulin receptor substrate/Akt/forkhead box O1 pathway in parallel to reduced phosphatase protein expression in the hypothalamus of obese mice. These effects were independent of body weight. TPM also raised anorexigenic neuropeptides such as POMC, TRH, and CRH mRNA levels in obese mice. In addition, TPM increased the activation of the hypothalamic MAPK/ERK pathway induced by leptin, accompanied by an increase in peroxisome proliferator-activated receptor-coactivator α and uncoupling protein 1 protein levels in brown adipose tissue. Furthermore, TPM increased AMP-activated protein kinase and acetyl-coenzyme A carboxylase phosphorylation in peripheral tissues, which may help improve energy metabolism in these tissues. Together, these results provide novel insights into the molecular mechanisms through which TPM treatment reduces adiposity.

Tub Has a Key Role in Insulin and Leptin Signaling and Action In Vivo in Hypothalamic Nuclei

Mutation of tub gene in mice induces obesity, suggesting that tub could be an important regulator of energy balance. In the current study, we investigated whether insulin, leptin, and obesity can modulate Tub in vivo in hypothalamic nuclei, and we investigated possible consequences on energy balance, neuropeptide expression, and hepatic glucose metabolism. Food intake, metabolic characteristics, signaling proteins, and neuropeptide expression were measured in response to fasting and refeeding, intracerebroventricular insulin and leptin, and Tub antisense oligonucleotide (ASO). Tub tyrosine phosphorylation (Tub-p-tyr) is modulated by nutritional status. Tub is a substrate of insulin receptor tyrosine kinase (IRTK) and leptin receptor (LEPR)–Janus kinase 2 (JAK2) in hypothalamic nuclei. After leptin or insulin stimulation, Tub translocates to the nucleus. Inhibition of Tub expression in hypothalamus by ASO increased food intake, fasting blood glucose, and hepatic glucose output, decreased O2 consumption, and blunted the effect of insulin or leptin on proopiomelanocortin, thyroid-releasing hormone, melanin-concentrating hormone, and orexin expression. In hypothalamus of mice administered a high-fat diet, there is a reduction in leptin and insulin-induced Tub-p-tyr and nuclear translocation, which is reversed by reducing protein tyrosine phosphatase 1B expression. These results indicate that Tub has a key role in the control of insulin and leptin effects on food intake, and the modulation of Tub may contribute to insulin and leptin resistance in DIO mice.

IKKε Is Key to Induction of Insulin Resistance in the Hypothalamus, and Its Inhibition Reverses Obesity

IKK epsilon (IKKε) is induced by the activation of nuclear factor-κB (NF-κB). Whole-body IKKε knockout mice on a high-fat diet (HFD) were protected from insulin resistance and showed altered energy balance. We demonstrate that IKKε is expressed in neurons and is upregulated in the hypothalamus of obese mice, contributing to insulin and leptin resistance. Blocking IKKε in the hypothalamus of obese mice with CAYMAN10576 or small interfering RNA decreased NF-κB activation in this tissue, relieving the inflammatory environment. Inhibition of IKKε activity, but not TBK1, reduced IRS-1Ser307 phosphorylation and insulin and leptin resistance by an improvement of the IR/IRS-1/Akt and JAK2/STAT3 pathways in the hypothalamus. These improvements were independent of body weight and food intake. Increased insulin and leptin action/signaling in the hypothalamus may contribute to a decrease in adiposity and hypophagia and an enhancement of energy expenditure accompanied by lower NPY and increased POMC mRNA levels. Improvement of hypothalamic insulin action decreases fasting glycemia, glycemia after pyruvate injection, and PEPCK protein expression in the liver of HFD-fed and db/db mice, suggesting a reduction in hepatic glucose production. We suggest that IKKε may be a key inflammatory mediator in the hypothalamus of obese mice, and its hypothalamic inhibition improves energy and glucose metabolism.

Pioglitazone treatment increases food intake and decreases energy expenditure partially via hypothalamic adiponectin/adipoR1/AMPK pathway

Introduction:Thiazolidinediones (TZDs) enhanced body weight (BW) partially by increased adipogenesis and hyperphagia. Neuronal PPARγ knockout mice on high-fat diet (HFD) are leaner because of enhanced leptin response, although it could be secondary to their leanness. Thus, it still is an open question how TZDs may alter energy balance. Multiple factors regulate food intake (FI) and energy expenditure (EE), including anorexigenic hormones as insulin and leptin. Nonetheless, elevated hypothalamic AMPK activity increases FI and TZDs increase AMPK activity in muscle cells. Thus, the aim of the present study was to investigate whether Pioglitazone (PIO) treatment alters hypothalamic insulin and leptin action/signaling, AMPK phosphorylation, and whether these alterations may be implicated in the regulation of FI and EE.Methods:Swiss mice on HFD (2 months) received PIO (25 mg kg−1 per day-gavage) or vehicle for 14 days. AMPK and AdipoR1 were inhibited via Intracerebroventricular injections using Compound C (CompC) and small interference RNA (siRNA), respectively. Western blot, real-time PCR and CLAMS were done.Results:PIO treatment increased BW, adiposity, FI, NPY mRNA and decreased POMC mRNA expression and EE in HFD mice. Despite higher adiposity, PIO treatment improved insulin sensitivity, glucose tolerance, decreased insulin and increased adiponectin serum levels. This result was associated with, improved insulin and leptin action/signaling, decreased α2AMPKSer491 phosphorylation and elevated Acetyl-CoA carboxylase and AMPKThr172 phosphorylation in hypothalamus. The inhibition of hypothalamic AMPK with CompC was associated with decreased adiposity, FI, NPY mRNA and EE in PIO-treated mice. The reduced expression of hypothalamic AdipoR1 with siRNA concomitantly with PIO treatment reverted PIO induced obesity development, suggesting that adiponectin may be involved in this effect.Conclusions:These results demonstrated that PIO, despite improving insulin/leptin action in hypothalamus, increases FI and decreases EE, partially, by activating hypothalamic adiponectin/AdipoR1/AMPK axis. Suggesting a novel mechanism in the hypothalamus by which TZDs increase BW.

Blocking iNOS and endoplasmic reticulum stress synergistically improves insulin resistance in mice

Objective Recent data show that iNOS has an essential role in ER stress in obesity. However, whether iNOS is sufficient to account for obesity-induced ER stress and Unfolded Protein Response (UPR) has not yet been investigated. In the present study, we used iNOS knockout mice to investigate whether high-fat diet (HFD) can still induce residual ER stress-associated insulin resistance. Methods For this purpose, we used the intraperitoneal glucose tolerance test (GTT), euglycemic-hyperinsulinemic clamp, western blotting and qPCR in liver, muscle, and adipose tissue of iNOS KO and control mice on HFD. Results The results of the present study demonstrated that, in HFD fed mice, iNOS-induced alteration in insulin signaling is an essential mechanism of insulin resistance in muscle, suggesting that iNOS may represent an important target that could be blocked in order to improve insulin sensitivity in this tissue. However, in liver and adipose tissue, the insulin resistance induced by HFD was only partially dependent on iNOS, and, even in the presence of genetic or pharmacological blockade of iNOS, a clear ER stress associated with altered insulin signaling remained evident in these tissues. When this ER stress was blocked pharmacologically, insulin signaling was improved, and a complete recovery of glucose tolerance was achieved. Conclusions Taken together, these results reinforce the tissue-specific regulation of insulin signaling in obesity, with iNOS being sufficient to account for insulin resistance in muscle, but in liver and adipose tissue ER stress and insulin resistance can be induced by both iNOS-dependent and iNOS-independent mechanisms.

Cdc2-like kinase 2 in the hypothalamus is necessary to maintain energy homeostasis

Objective:To investigate whether the Cdc2-like kinase 2 (CLK2) is expressed in hypothalamic neurons and if it is, whether the hypothalamic CLK2 has a role in the regulation of energy balance.Subjects:Swiss mice on chow or high-fat diet (HFD) and db/db mice on chow diet were used to address the role of CLK2 in the hypothalamus.Results:Hypothalamic CLK2Thr343 phosphorylation, which induces CLK2 activity, is regulated in vivo by refeeding, insulin and leptin, in a PI3K (phosphoinositide 3-kinase)-dependent manner. The reduction of CLK2 expression in the hypothalamus, by chronic pharmacological inhibition with TG003 or by chronic knockdown with small interfering RNA was sufficient to abolish the anorexigenic effect of insulin and leptin, to increase body weight, fat mass, food intake and to decrease energy expenditure in mice on chow. In contrast, CLK2Thr343 phosphorylation in the hypothalamus in response to insulin, leptin or refeeding was impaired in mice on HFD or in db/db mice. Chronic CLK2 inhibition in the hypothalamus was associated with a slight increase in the fasting blood glucose levels, reduction in PEPCK (phosphoenolpyruvate carboxykinase) expression in the liver and enhanced glucose production from pyruvate, suggesting a regulation of hepatic glucose production. Further, overexpressing CLK2 in the mediobasal hypothalami of mice on HFD or in db/db mice by adenovirus partially reversed the obese phenotype.Conclusions:Thus, our results suggest that protein CLK2 integrates some important hypothalamic pathways, and may be a promising molecule for new therapeutic approaches for obesity and diabetes.

Statement of Retraction. Tub Has a Key Role in Insulin and Leptin Signaling and Action In Vivo in Hypothalamic Nuclei. Diabetes 2013;62:137-148. DOI: 10.2337/db11-1388.

The above-cited article has been retracted by the American Diabetes Association, the publisher of Diabetes . This article was previously the subject of an expression of concern in the April 2016 issue of the journal (Diabetes 2016;65:1121–1122. DOI: 10.2337/db16-ec04. PMID: 27208021). As noted in the April 2016 expression of concern, the American Diabetes Association asked the corresponding author’s institution, the University of Campinas (Sao Paulo, Brazil), to investigate concerns related to the possible duplication of several images presented in this article. The American Diabetes Association did not receive a response from the university, but the lead author of the article, who is also listed as a guarantor of the study, provided a point-by-point response to address the issues reported in the expression of concern. After reviewing the author’s response, the American Diabetes Association’s Panel on Ethical Scientific Programs (ESP) maintains that the …

Exercise Reduces Hepatic Gluconeogenesis in Obese and Insulin Resistant Animals Through CLK2 Protein (Cdc2-Like Kinase): 1904 Board #56 June 2, 3

High-intensity interval training (HIT) represents a very time-efficient mode of exercise training and induces several health benefits. However, little is known regarding the role of HIT to combat whole body insulin resistance. PURPOSE: To investigate the effect of HIT on whole body insulin resistance in high-fat diet (HFD)-induced obese mice. METHODS: At 5-week postnatal period, a total of 30 male mice (C57BL/6) were randomly assigned to standard chow (SC) (n=10) or HFD (n=20) for 23 weeks. After 15 weeks of dietary treatment, the HFD mice were further assigned to HFD (n=10) or HFD+HIT (HFD+HIT, n=10). The HFD+HIT mice were subjected to HIT on a motor-driven rodent treadmill during the last 8 weeks of the 23-week HFD course. RESULTS: HIT suppressed HFD-induced increases in body weight (HFD 47.3±1.4 vs. HFD+HIT 44.2±1.2g, p=0.023), subcutaneous fat mass (HFD 2.7±0.1 vs. HFD+HIT 2.4±0.1g, p=0.038), serum total cholesterol (HFD 253.5±48.5 vs. HFD+HIT 198.7±7.4mg/, p=0.010), area under the curve of glucose tolerance test (HFD 53070±8585 vs. HFD+ HIT 45401±4312, p=0.031), and area under the curve of insulin tolerance test (HFD 9367±987 vs. HFD+ HIT 6416±619, p=0.016). HIT prevented HFD-induced decreases of total adiponectin in serum (p=0.001) and adipose tissue (p=0.016). Along with improved metabolic risk factors, HIT prevented HFD-induced decreases in proteins of adiponectin receptor 1 (p=0.010), AMP-activated protein kinase (p=0.001), and NAD-dependent deacetylase sirtuin-1 (p=0.010) and HFD-induced decreases in mRNAs of peroxisome proliferator-activated receptor(p=0.027), carnitine palmitoyltransferase I (p=0.023), and acyl CoA oxidase (p=0.030) in skeletal muscle. CONCLUSION: The current findings show that HIT alleviates whole body insulin resistance associated with obesity via the AdipoR1 and AMPK mediated-signaling pathway in skeletal muscle, implying the therapeutic role of HIT to combat whole body insulin resistance. Supported by the National Research Foundation Grant funded by the Korean Government (NRF-2015S1A5B5A02012775).

Expression of Concern. Tub Has a Key Role in Insulin and Leptin Signaling and Action In Vivo in Hypothalamic Nuclei. Diabetes 2013;62:137-148. DOI: 10.2337/db11-1388.

On the basis of the recommendation of the American Diabetes Association’s Panel on Ethical Scientific Programs (ESP), the American Diabetes Association, the publisher of Diabetes, is issuing this expression of concern to alert readers to questions about the reliability of the data in the above-cited article. After readers of the journal contacted Diabetes about potentially duplicated images in the article, the ESP reviewed the following issues: