Brain access of incretins and incretin receptor agonists to their central targets relevant for appetite suppression and weight loss.

New incretin-based pharmacotherapies provide efficient and safe therapeutic options to curb appetite and produce weight loss in patients with obesity. Delivered systemically, these molecules produce pleiotropic metabolic benefits, but the target sites mediating their weight-suppressive action are located within the brain. Recent research has increased our understanding of the neural circuits and behavioral mechanisms involved in the anorectic and metabolic consequences of glucagon-like peptide 1 (GLP-1)-based weight loss strategies, yet little is known about how these drugs access their functional targets in the brain to produce sustained weight loss. The majority of brain cells expressing incretin receptors are located behind the blood-brain barrier, shielded from the circulation and fluctuations in the availability of peripheral signals, which is a major challenge for the development of CNS-targeted therapeutic peptides. GLP-1 receptor (GLP-1R) agonists with increased half-life and enhanced therapeutic benefit do not cross the blood-brain barrier, yet they manage to access discrete brain sites relevant to the regulation of energy homeostasis. In this review, we give a brief overview of the different routes for peptide hormones to access the brain. We then examine the evidence informing the routes employed by incretins and incretin receptor agonists to access brain targets relevant for their appetite and weight-suppressive actions. We highlight existing controversies and suggest future directions to further establish the functionally relevant access routes for GLP-1-based weight loss compounds, which might guide the development and selection of the future generation of incretin receptor polypharmacologies.

Kappa-Opioid Receptor Blockade Ameliorates Obesity Caused by Estrogen Withdrawal via Promotion of Energy Expenditure through mTOR Pathway.

Weight gain is a hallmark of decreased estradiol (E2) levels because of menopause or following surgical ovariectomy (OVX) at younger ages. Of note, this weight gain tends to be around the abdomen, which is frequently associated with impaired metabolic homeostasis and greater cardiovascular risk in both rodents and humans. However, the molecular underpinnings and the neuronal basis for these effects remain to be elucidated. The aim of this study is to elucidate whether the kappa-opioid receptor (k-OR) system is involved in mediating body weight changes associated with E2 withdrawal. Here, we document that body weight gain induced by OVX occurs, at least partially, in a k-OR dependent manner, by modulation of energy expenditure independently of food intake as assessed in Oprk1-/-global KO mice. These effects were also observed following central pharmacological blockade of the k-OR system using the k-OR-selective antagonist PF-04455242 in wild type mice, in which we also observed a decrease in OVX-induced weight gain associated with increased UCP1 positive immunostaining in brown adipose tissue (BAT) and browning of white adipose tissue (WAT). Remarkably, the hypothalamic mTOR pathway plays an important role in regulating weight gain and adiposity in OVX mice. These findings will help to define new therapies to manage metabolic disorders associated with low/null E2 levels based on the modulation of central k-OR signaling.

A GLP-1:CCK fusion peptide harnesses the synergistic effects on metabolism of CCK-1 and GLP-1 receptor agonism in mice.

Combination approaches for the treatment of metabolic diseases such as obesity and diabetes are becoming increasingly relevant. Co-administration of a glucagon-like peptide-1 receptor (GLP-1R) agonist with a cholecystokinin receptor-1 (CCKR1) agonist exert synergistic effects on weight loss in obese rodents. Here, we report on the effects of a novel fusion peptide (C2816) comprised of a stabilized GLP-1R agonist, AC3174, and a CCKR1-selective agonist, AC170222. C2816 was constructed such that AC3174 was linked to the N-terminus of AC170222, thus preserving the C-terminal amide of the CCK moiety. In functional in vitro assays C2816 retained full agonism at GLP-1R and CCKR1 at lower potency compared to parent molecules, whereas a previously reported fusion peptide in the opposite orientation, (pGlu-Gln)-CCK-8/exendin-4, exhibited no activity at either receptor. Acutely, in vivo, C2816 increased cFos in key central nuclei relevant to feeding behavior, and reduced food intake in wildtype (WT), but less so in GLP-1R-deficient (GLP-1RKO), mice. In sub-chronic studies in diet-induced obese (DIO) mice, C2816 exerted superior reduction in body weight compared to co-administration of AC3174 and AC170222 albeit at a higher molar dose. These data suggest that the synergistic pharmacological effects of GLP-1 and CCK pathways can be harnessed in a single therapeutic peptide.

TXNIP in Agrp neurons regulates adiposity, energy expenditure, and central leptin sensitivity.

Thioredoxin interacting protein (TXNIP) has recently been described as a key regulator of energy metabolism through pleiotropic actions that include nutrient sensing in the mediobasal hypothalamus (MBH). However, the role of TXNIP in neurochemically specific hypothalamic subpopulations and the circuits downstream from MBH TXNIP engaged to regulate energy homeostasis remain unexplored. To evaluate the metabolic role of TXNIP activity specifically within arcuate Agrp neurons, we generated Agrp-specific TXNIP gain-of-function and loss-of-function mouse models using Agrp-Ires-cre mice, TXNIP (flox/flox) mice, and a lentivector expressing the human TXNIP isoform conditionally in the presence of Cre recombinase. Overexpression of TXNIP in Agrp neurons predisposed to diet-induced obesity and adipose tissue storage by decreasing energy expenditure and spontaneous locomotion, without affecting food intake. Conversely, Agrp neuronal TXNIP deletion protected against diet-induced obesity and adipose tissue storage by increasing energy expenditure and spontaneous locomotion, also without affecting food intake. TXNIP overexpression in Agrp neurons did not primarily affect glycemic control, whereas deletion of TXNIP in Agrp neurons improved fasting glucose levels and glucose tolerance independently of its effects on body weight and adiposity. Bidirectional manipulation of TXNIP expression induced reciprocal changes in central leptin sensitivity and the neural regulation of lipolysis. Together, these results identify a critical role for TXNIP in Agrp neurons in mediating diet-induced obesity through the regulation of energy expenditure and adipose tissue metabolism, independently of food intake. They also reveal a previously unidentified role for Agrp neurons in the brain-adipose axis.

Median eminence myelin continuously turns over in adult mice.

OBJECTIVE: Oligodendrocyte progenitor cell differentiation is regulated by nutritional signals in the adult median eminence (ME), but the consequences on local myelination are unknown. The aim of this study was to characterize myelin plasticity in the ME of adult mice in health or in response to chronic nutritional challenge and determine its relevance to the regulation of energy balance. METHODS: We assessed new oligodendrocyte (OL) and myelin generation and stability in the ME of healthy adult male mice using bromodeoxyuridine labelling and genetic fate mapping tools. We evaluated the contribution of microglia to ME myelin plasticity in PLX5622-treated C57BL/6J mice and in Pdgfra-Cre/ERT2;R26R-eYFP;Myrffl/fl mice, where adult oligodendrogenesis is blunted. Next, we investigated how high-fat feeding or caloric restriction impact ME OL lineage progression and myelination. Finally, we characterized the functional relevance of adult oligodendrogenesis on energy balance regulation. RESULTS: We show that myelinating OLs are continuously and rapidly generated in the adult ME. Paradoxically, OL number and myelin amounts remain remarkably stable in the adult ME. In fact, the high rate of new OL and myelin generation in the ME is offset by continuous turnover of both. We show that microglia are required for continuous OL and myelin production, and that ME myelin plasticity regulates the recruitment of local immune cells. Finally, we provide evidence that ME myelination is regulated by the body's energetic status and demonstrate that ME OL and myelin plasticity are required for the regulation of energy balance and hypothalamic leptin sensitivity. CONCLUSIONS: This study identifies a new mechanism modulating leptin sensitivity and the central control of energy balance and uncovers a previously unappreciated form of structural plasticity in the ME.

Nutrient-sensing hypothalamic TXNIP links nutrient excess to energy imbalance in mice.

Nutrient excess in obesity and diabetes is emerging as a common putative cause for multiple deleterious effects across diverse cell types, responsible for a variety of metabolic dysfunctions. The hypothalamus is acknowledged as an important regulator of whole-body energy homeostasis, through both detection of nutrient availability and coordination of effectors that determine nutrient intake and utilization, thus preventing cellular and whole-body nutrient excess. However, the mechanisms underlying hypothalamic nutrient detection and its impact on peripheral nutrient utilization remain poorly understood. Recent data suggest a role for thioredoxin-interacting protein (TXNIP) as a molecular nutrient sensor important in the regulation of energy metabolism, but the role of hypothalamic TXNIP in the regulation of energy balance has not been evaluated. Here we show in mice that TXNIP is expressed in nutrient-sensing neurons of the mediobasal hypothalamus, responds to hormonal and nutrient signals, and regulates adipose tissue metabolism, fuel partitioning, and glucose homeostasis. Hypothalamic expression of TXNIP is induced by acute nutrient excess and in mouse models of obesity and diabetes, and downregulation of mediobasal hypothalamic TXNIP expression prevents diet-induced obesity and insulin resistance. Thus, mediobasal hypothalamic TXNIP plays a critical role in nutrient sensing and the regulation of fuel utilization.

Behavioural and neurochemical mechanisms underpinning the feeding-suppressive effect of GLP-1/CCK combinatorial therapy.

OBJECTIVES: Combinatorial therapies are under intense investigation to develop more efficient anti-obesity drugs; however, little is known about how they act in the brain to produce enhanced anorexia and weight loss. The goal of this study was to identify the brain sites and neuronal populations engaged during the co-administration of GLP-1R and CCK1R agonists, an efficient combination therapy in obese rodents. METHODS: We measured acute and long-term feeding and body weight responses and neuronal activation patterns throughout the neuraxis and in specific neuronal subsets in response to GLP-1R and CCK1R agonists administered alone or in combination in lean and high-fat diet fed mice. We used PhosphoTRAP to obtain unbiased molecular markers for neuronal populations selectively activated by the combination of the two agonists. RESULTS: The initial anorectic response to GLP-1R and CCK1R co-agonism was mediated by a reduction in meal size, but over a few hours, a reduction in meal number accounted for the sustained feeding suppressive effects. The nucleus of the solitary tract (NTS) is one of the few brain sites where GLP-1R and CCK1R signalling interact to produce enhanced neuronal activation. None of the previously categorised NTS neuronal subpopulations relevant to feeding behaviour were implicated in this increased activation. However, we identified NTS/AP Calcrl+ neurons as treatment targets. CONCLUSIONS: Collectively, these studies indicated that circuit-level integration of GLP-1R and CCK1R co-agonism in discrete brain nuclei including the NTS produces enhanced rapid and sustained appetite suppression and weight loss.

Nutritional regulation of oligodendrocyte differentiation regulates perineuronal net remodeling in the median eminence.

The mediobasal hypothalamus (MBH; arcuate nucleus of the hypothalamus [ARH] and median eminence [ME]) is a key nutrient sensing site for the production of the complex homeostatic feedback responses required for the maintenance of energy balance. Here, we show that refeeding after an overnight fast rapidly triggers proliferation and differentiation of oligodendrocyte progenitors, leading to the production of new oligodendrocytes in the ME specifically. During this nutritional paradigm, ME perineuronal nets (PNNs), emerging regulators of ARH metabolic functions, are rapidly remodeled, and this process requires myelin regulatory factor (Myrf) in oligodendrocyte progenitors. In genetically obese ob/ob mice, nutritional regulations of ME oligodendrocyte differentiation and PNN remodeling are blunted, and enzymatic digestion of local PNN increases food intake and weight gain. We conclude that MBH PNNs are required for the maintenance of energy balance in lean mice and are remodeled in the adult ME by the nutritional control of oligodendrocyte differentiation.

Mediobasal hypothalamic leucine sensing regulates food intake through activation of a hypothalamus-brainstem circuit.

In response to nutrient stimuli, the mediobasal hypothalamus (MBH) drives multiple neuroendocrine and behavioral mechanisms to regulate energy balance. While central leucine reduces food intake and body weight, the specific neuroanatomical sites of leucine sensing, downstream neural substrates, and neurochemical effectors involved in this regulation remain largely unknown. Here we demonstrate that MBH leucine engages a neural energy regulatory circuit by stimulating POMC (proopiomelanocortin) neurons of the MBH, oxytocin neurons of the paraventricular hypothalamus, and neurons within the brainstem nucleus of the solitary tract to acutely suppress food intake by reducing meal size. We identify central p70 S6 kinase and Erk1/2 pathways as intracellular effectors required for this response. Activation of endogenous leucine intracellular metabolism produced longer-term reductions in meal number. Our data identify a novel, specific hypothalamus-brainstem circuit that links amino acid availability and nutrient sensing to the control of food intake.

Nutrient sensing in the nucleus of the solitary tract mediates non-aversive suppression of feeding via inhibition of AgRP neurons.

The nucleus of the solitary tract (NTS) is emerging as a major site of action for the appetite-suppressive effects of leading pharmacotherapies currently investigated to treat obesity. However, our understanding of how NTS neurons regulate appetite remains incomplete. OBJECTIVES: In this study, we used NTS nutrient sensing as an entry point to characterize stimulus-defined neuronal ensembles engaged by the NTS to produce physiological satiety. METHODS: We combined histological analysis, neuroanatomical assessment using inducible viral tracing tools, and functional tests to characterize hindbrain-forebrain circuits engaged by NTS leucine sensing to suppress hunger. RESULTS: We found that NTS detection of leucine engages NTS prolactin-releasing peptide (PrRP) neurons to inhibit AgRP neurons via a population of leptin receptor-expressing neurons in the dorsomedial hypothalamus. This circuit is necessary for the anorectic response to NTS leucine, the appetite-suppressive effect of high-protein diets, and the long-term control of energy balance. CONCLUSIONS: These results extend the integrative capability of AgRP neurons to include brainstem nutrient sensing inputs.

Calcitonin Receptor Neurons in the Mouse Nucleus Tractus Solitarius Control Energy Balance via the Non-aversive Suppression of Feeding.

To understand hindbrain pathways involved in the control of food intake, we examined roles for calcitonin receptor (CALCR)-containing neurons in the NTS. Ablation of NTS Calcr abrogated the long-term suppression of food intake, but not aversive responses, by CALCR agonists. Similarly, activating CalcrNTS neurons decreased food intake and body weight but (unlike neighboring CckNTS cells) failed to promote aversion, revealing that CalcrNTS neurons mediate a non-aversive suppression of food intake. While both CalcrNTS and CckNTS neurons decreased feeding via projections to the PBN, CckNTS cells activated aversive CGRPPBN cells while CalcrNTS cells activated distinct non-CGRP PBN cells. Hence, CalcrNTS cells suppress feeding via non-aversive, non-CGRP PBN targets. Additionally, silencing CalcrNTS cells blunted food intake suppression by gut peptides and nutrients, increasing food intake and promoting obesity. Hence, CalcrNTS neurons define a hindbrain system that participates in physiological energy balance and suppresses food intake without activating aversive systems.

Mapping neuronal inputs to Kiss1 neurons in the arcuate nucleus of the mouse.

The normal function of the mammalian reproductive axis is strongly influenced by physiological, metabolic and environmental factors. Kisspeptin neuropeptides, encoded by the Kiss1 gene, are potent regulators of the mammalian reproductive axis by stimulating gonadodropin releasing hormone secretion from the hypothalamus. To understand how the reproductive axis is modulated by higher order neuronal inputs we have mapped the afferent circuits into arcuate (ARC) Kiss1 neurons. We used a transgenic mouse that expresses the CRE recombinase in Kiss1 neurons for conditional viral tracing with genetically modified viruses. CRE-mediated activation of these viruses in Kiss1 neurons allows the virus to move transynaptically to label neurons with primary or secondary afferent inputs into the Kiss1 neurons. Several regions of the brain showed synaptic connectivity to arcuate Kiss1 neurons including proopiomelanocortin neurons in the ARC itself, kisspeptin neurons in the anteroventral periventricular nucleus, vasopressin neurons in the supraoptic and suprachiasmatic nuclei, thyrotropin releasing neurons in the paraventricular nucleus and unidentified neurons in other regions including the subfornical organ, amygdala, interpeduncular nucleus, ventral premammilary nucleus, basal nucleus of stria terminalis and the visual, somatosensory and piriform regions of the cortex. These data provide an insight into how the activity of Kiss1 neurons may be regulated by metabolic signals and provide a detailed neuroanatomical map for future functional studies.

Glucose-Dependent Insulinotropic Polypeptide Receptor-Expressing Cells in the Hypothalamus Regulate Food Intake.

Ambiguity regarding the role of glucose-dependent insulinotropic polypeptide (GIP) in obesity arises from conflicting reports asserting that both GIP receptor (GIPR) agonism and antagonism are effective strategies for inhibiting weight gain. To enable identification and manipulation of Gipr-expressing (Gipr) cells, we created Gipr-Cre knockin mice. As GIPR-agonists have recently been reported to suppress food intake, we aimed to identify central mediators of this effect. Gipr cells were identified in the arcuate, dorsomedial, and paraventricular nuclei of the hypothalamus, as confirmed by RNAscope in mouse and human. Single-cell RNA-seq identified clusters of hypothalamic Gipr cells exhibiting transcriptomic signatures for vascular, glial, and neuronal cells, the latter expressing somatostatin but little pro-opiomelanocortin or agouti-related peptide. Activation of Gq-DREADDs in hypothalamic Gipr cells suppressed food intake in vivo, which was not obviously additive with concomitant GLP1R activation. These data identify hypothalamic GIPR as a target for the regulation of energy balance.

Hypothalamic dopamine signaling regulates brown fat thermogenesis.

Dopamine signaling is a crucial part of the brain reward system and can affect feeding behavior. Dopamine receptors are also expressed in the hypothalamus, which is known to control energy metabolism in peripheral tissues. Here we show that pharmacological or chemogenetic stimulation of dopamine receptor 2 (D2R) expressing cells in the lateral hypothalamic area (LHA) and the zona incerta (ZI) decreases body weight and stimulates brown fat activity in rodents in a feeding-independent manner. LHA/ZI D2R stimulation requires an intact sympathetic nervous system and orexin system to exert its action and involves inhibition of PI3K in the LHA/ZI. We further demonstrate that, as early as 3 months after onset of treatment, patients treated with the D2R agonist cabergoline experience an increase in energy expenditure that persists for one year, leading to total body weight and fat loss through a prolactin-independent mechanism. Our results may provide a mechanistic explanation for how clinically used D2R agonists act in the CNS to regulate energy balance.

A gut-brain axis regulating glucose metabolism mediated by bile acids and competitive fibroblast growth factor actions at the hypothalamus.

OBJECTIVE: Bile acids have been implicated as important regulators of glucose metabolism via activation of FXR and GPBAR1. We have previously shown that FGF19 can modulate glucose handling by suppressing the activity of hypothalamic AGRP/NPY neurons. As bile acids stimulate the release of FGF19/FGF15 into the circulation, we pursued the potential of bile acids to improve glucose tolerance via a gut-brain axis involving FXR and FGF15/FGF19 within enterocytes and FGF receptors on hypothalamic AGRP/NPY neurons. METHODS: A 5-day gavage of taurocholic acid, mirroring our previous protocol of a 5-day FGF19 treatment, was performed. Oral glucose tolerance tests in mice with genetic manipulations of FGF signaling and melanocortin signaling were used to define a gut-brain axis responsive to bile acids. RESULTS: The taurocholic acid gavage led to increased serum concentrations of taurocholic acid as well as increases of FGF15 mRNA in the ileum and improved oral glucose tolerance in obese (ob/ob) mice. In contrast, lithocholic acid, an FXR antagonist but a potent agonist for GPBAR1, did not improve glucose tolerance. The positive response to taurocholic acid is dependent upon an intact melanocortinergic system as obese MC4R-null mice or ob/ob mice without AGRP did not show improvements in glucose tolerance after taurocholate gavage. We also tested the FGF receptor isoform necessary for the bile acid response, using AGRP:Fgfr1-/- and AGRP:Fgfr2-/- mice. While the absence of FGFR1 in AGRP/NPY neurons did not alter glucose tolerance after taurocholate gavage, manipulations of Fgfr2 caused bidirectional changes depending upon the experimental model. We hypothesized the existence of an endogenous hypothalamic FGF, most likely FGF17, that acted as a chronic activator of AGRP/NPY neurons. We developed two short peptides based on FGF8 and FGF17 that should antagonize FGF17 action. Both of these peptides improved glucose homeostasis after a 4-day course of central and peripheral injections. Significantly, daily average blood glucose from continuous glucose monitoring was reduced in all tested animals but glucose concentrations remained in the euglycemia range. CONCLUSIONS: We have defined a gut-brain axis that regulates glucose metabolism mediated by antagonistic fibroblast growth factors. From the intestine, bile acids stimulate FGF15 secretion, leading to activation of the FGF receptors in hypothalamic AGRP/NPY neurons. FGF receptor intracellular signaling subsequently silences AGRP/NPY neurons, leading to improvements of glucose tolerance that are likely mediated by the autonomic nervous system. Finally, short peptides that antagonize homodimeric FGF receptor signaling within the hypothalamus have beneficial effects on glucose homeostasis without inducing hypoglycemia. These peptides could provide a new mode of regulating glucose metabolism.

Rapid sensing of l-leucine by human and murine hypothalamic neurons: Neurochemical and mechanistic insights.

OBJECTIVE: Dietary proteins are sensed by hypothalamic neurons and strongly influence multiple aspects of metabolic health, including appetite, weight gain, and adiposity. However, little is known about the mechanisms by which hypothalamic neural circuits controlling behavior and metabolism sense protein availability. The aim of this study is to characterize how neurons from the mediobasal hypothalamus respond to a signal of protein availability: the amino acid l-leucine. METHODS: We used primary cultures of post-weaning murine mediobasal hypothalamic neurons, hypothalamic neurons derived from human induced pluripotent stem cells, and calcium imaging to characterize rapid neuronal responses to physiological changes in extracellular l-Leucine concentration. RESULTS: A neurochemically diverse subset of both mouse and human hypothalamic neurons responded rapidly to l-leucine. Consistent with l-leucine's anorexigenic role, we found that 25% of mouse MBH POMC neurons were activated by l-leucine. 10% of MBH NPY neurons were inhibited by l-leucine, and leucine rapidly reduced AGRP secretion, providing a mechanism for the rapid leucine-induced inhibition of foraging behavior in rodents. Surprisingly, none of the candidate mechanisms previously implicated in hypothalamic leucine sensing (KATP channels, mTORC1 signaling, amino-acid decarboxylation) were involved in the acute activity changes produced by l-leucine. Instead, our data indicate that leucine-induced neuronal activation involves a plasma membrane Ca2+ channel, whereas leucine-induced neuronal inhibition is mediated by inhibition of a store-operated Ca2+ current. CONCLUSIONS: A subset of neurons in the mediobasal hypothalamus rapidly respond to physiological changes in extracellular leucine concentration. Leucine can produce both increases and decreases in neuronal Ca2+ concentrations in a neurochemically-diverse group of neurons, including some POMC and NPY/AGRP neurons. Our data reveal that leucine can signal through novel mechanisms to rapidly affect neuronal activity.

Melanin-Concentrating Hormone acts through hypothalamic kappa opioid system and p70S6K to stimulate acute food intake.

Melanin-Concentrating Hormone (MCH) is one of the most relevant orexigenic factors specifically located in the lateral hypothalamic area (LHA), with its physiological relevance demonstrated in studies using several genetically manipulated mice models. However, the central mechanisms controlling MCH-induced hyperphagia remain largely uncharacterized. Here, we show that central injection of MCH in mice deficient for kappa opoid receptor (k-OR) failed to stimulate feeding. To determine the hypothalamic area responsible for this MCH/k-OR interaction, we performed virogenetic studies and found that downregulation of k-OR by adeno-associated viruses (shOprk1-AAV) in LHA, but not in other hypothalamic nuclei, was sufficient to block MCH-induced food intake. Next, we sought to investigate the molecular signaling pathway within the LHA that mediates acute central MCH stimulation of food intake. We found that MCH activates k-OR and that increased levels of phosphorylated extracellular signal regulated kinase (ERK) are associated with downregulation of phospho-S6 Ribosomal Protein. This effect was prevented when a pharmacological inhibitor of k-OR was co-administered with MCH. Finally, the specific activation of the direct upstream regulator of S6 (p70S6K) in the LHA attenuated MCH-stimulated food consumption. Our results reveal that lateral hypothalamic k-OR system modulates the orexigenic action of MCH via the p70S6K/S6 pathway.

Dietary cysteine alleviates sucrose-induced oxidative stress and insulin resistance.

Diets that promote oxidative stress favor impairment in glucose homeostasis. In this context, increasing the cysteine intake may be beneficial by maintaining glutathione status. We have investigated the effects of dietary cysteine on oxidative stress and glucose homeostasis in rats fed a high-sucrose (HS) diet. Rats were assigned for 6 weeks to a standard diet or to HS diets in which the protein source was either an alpha-lactalbumin-rich whey concentrate (a cysteine-rich protein) or the total milk proteins alone or supplemented with 5.8 or 20 g N-acetylcysteine per kilogram of food. Increasing the cysteine intake prevented HS-induced oxidative stress, as assessed by blood and tissue glutathione and carbonyl levels. At the same time, the HS-induced glucose intolerance, impaired postprandial glycemic control, and decrease in muscle and liver insulin-induced activation of insulin receptor substrate 1 and Akt were prevented by increasing the level of dietary cysteine, a major original finding. Of great interest was the observation that all beneficial effects of cysteine supplementation were duplicated by the consumption of a cysteine-rich protein. These data show that increasing the cysteine intake limits HS-induced impairment of glucose homeostasis and suggest that these effects are mediated by a reduction in oxidative stress.