Abstract Disrupted light-dark cycles and mistimed feeding are recognized metabolic stressors. In this study, we investigated the independent and combined effects of a 12:12 light:dark cycle, advanced by 6 h every 6 days (Chronic Disruption; CD), and time-restricted feeding on the metabolic health of male C57BL/6J mice. Ad libitum mice under the CD cycle gained significantly more body weight and body fat compared to animals under a control light cycle (LD), despite similar caloric intake and wheel running activity. Using specialized equipment to dissociate light cues from nutrient access, we demonstrate that light-cycle disruption induces metabolic dysfunction specifically through fragmented and misaligned eating patterns. Crucially, restricting food access to a 12-h window synchronized with the shifting dark phase abrogated the weight gain and adiposity observed in the ad libitum group. Conversely, shifting the feeding window under a stable light cycle significantly reduced food intake and weight gain, revealing that feeding regularity is a primary determinant of energy balance independent of light-cycle stability. Overall, while the light-dark cycle is the dominant zeitgeber for activity, the temporal consolidation of food intake is the primary driver of metabolic alignment, offering a potential intervention for managing metabolic risk in shift workers.

The immune response exhibits strong circadian rhythmicity, with enhanced bacterial clearance often synchronized with an organism's active phase. Despite providing the bulk of cellular antibacterial defense, the neutrophil clockwork is poorly understood. Here, we used larval zebrafish to explore the role of clock genes in neutrophils during infection. Per2 was required in neutrophils for reactive oxygen species (ROS) production and bacterial killing by enhancing infection-responsive expression of high-mobility group box 1a (hmgb1a). The Cry binding domain of Per2 was required for regulation of neutrophil bactericidal activity, and neutrophils lacking Cry1a had elevated bactericidal activity and infection-responsive hmgb1a expression. A conserved cis-regulatory element with BMAL1 and nuclear factor κB binding motifs gated infection-responsive hmgb1a expression to the light phase. Mutagenesis of the BMAL1 motif in neutrophils blunted the priming effect of light on bactericidal activity and hmgb1a expression. These findings identify a light-responsive cell-intrinsic timer that controls time-of-day variations in antibacterial activity.

beta-Catenin is a bifunctional molecule that is an effector of the wingless-related integration site (Wnt) signaling to control gene expression and contributes to the regulation of cytoskeleton and neurotransmitter vesicle trafficking. In its former role, beta-catenin binds transcription factor 7-like 2 (TCF7L2), which shows strong genetic associations with the pathogenesis of obesity and type-2 diabetes. Here, we sought to determine whether beta-catenin plays a role in the neuroendocrine regulation of body weight and glucose homeostasis. Bilateral injections of adeno-associated virus type-2 (AAV2)-mCherry-Cre were placed into the arcuate nucleus of adult male and female beta-catenin(flox) mice, to specifically delete beta-catenin expression in the mediobasal hypothalamus (MBH-beta-cat KO). Metabolic parameters were then monitored under conditions of low-fat (LFD) and high-fat diet (HFD). On LFD, MBH-beta-cat KO mice showed minimal metabolic disturbances, but on HFD, despite having only a small difference in weekly caloric intake, the MBH-beta-cat KO mice were significantly heavier than the control mice in both sexes (p < 0.05). This deficit seemed to be due to a failure to show an adaptive increase in energy expenditure seen in controls, which served to offset the increased calories by HFD. Both male and female MBH-beta-cat KO mice were highly glucose intolerant when on HFD and displayed a significant reduction in both leptin and insulin sensitivity compared with controls. This study highlights a critical role for beta-catenin in the hypothalamic circuits regulating body weight and glucose homeostasis and reveals potential mechanisms by which genetic variation in this pathway could impact on development of metabolic disease.

Nut-based products are a good source of high-quality plant protein in addition to mono- and polyunsaturated fatty acids, and may aid low-glycaemic dietary strategies important for the prevention of type 2 diabetes (T2D). In particular, they may be advantageous in populations susceptible to dysglycaemia, such as Asian Chinese. The present study aimed to compare effects of a higher-protein nut bar (HP-NB, also higher in total fibre and unsaturated fats, comprising mixed almonds and peanuts) vs. an isoenergetic higher-carbohydrate cereal bar (HC-CB) within the diet of 101 Chinese adults with overweight and normo- or hyperglycaemia. Ectopic pancreas and liver fat were characterised using magnetic resonance imaging and spectroscopy (MRI/S) as a secondary outcome. Participants were randomized to receive HP-NB or HC-CB daily as a 1 MJ light meal or snack replacement, in addition to healthy eating advice. Anthropometry and clinical indicators of T2D risk were assessed fasted and during an oral glucose tolerance test (OGTT), pre- and post-intervention. No significant difference was observed between diet groups for body weight, body mass index, waist or hip circumference, blood pressure, glucoregulatory markers, lipid profile or inflammatory markers over 12 weeks (all, p > 0.05). No difference was observed between glycaemic subgroups or those with normal versus high ectopic organ fat. Although HP-NB can attenuate postprandial glycaemia following a meal, no effects were observed for either fasting or glucose-mediated outcomes following longer-term inclusion in the habitual diet of Chinese adults with overweight, including at-risk subgroups.

Butein, a rare chalcone found in the toxic plant Toxicodendron vernicifluum, has been shown to regulate glucose homeostasis via inhibition of the nuclear factor kappa-B kinase subunit beta (IKKβ)/nuclear factor kappa B (NF-κB) pathway in the brain. Here, we investigated whether the nonpoisonous plant Dahlia pinnata could be a source of butein as a potential treatment for type 2 diabetes (T2D). In mice fed a high-fat diet (HFD) to induce glucose intolerance, an oral D. pinnata petal extract improved glucose tolerance at doses of 3.3 mg/kg body weight and 10 mg/kg body weight. Surprisingly, this effect was not mediated by butein alone but by butein combined with the closely related flavonoids, sulfuretin and/or isoliquiritigenin. Mechanistically, the extract improved systemic insulin tolerance. Inhibition of phosphatidylinositol 3-kinase to block insulin signaling in the brain abrogated the glucoregulatory effect of the orally administered extract. The extract reinstated central insulin signaling and normalized astrogliosis in the hypothalamus of HFD-fed mice. Using NF-κB reporter zebrafish to determine IKKβ/NF-κB activity, a potent anti-inflammatory action of the extract was found. A randomized controlled crossover clinical trial on participants with prediabetes or T2D confirmed the safety and efficacy of the extract in humans. In conclusion, we identified an extract from the flower petals of D. pinnata as a novel treatment option for T2D, potentially targeting the central regulation of glucose homeostasis as a root cause of the disease.

Gluten, which is found in cereals such as wheat, rye and barley, makes up a major dietary component in most western nations, and has been shown to promote body mass gain and peripheral inflammation in mice. In the current study, we investigated the impact of gluten on central inflammation that is typically associated with diet-induced obesity. While we found no effect of gluten when added to a low-fat diet (LFD), male mice fed high fat diet (HFD) enriched with gluten increased body mass and adiposity compared with mice fed HFD without gluten. We furthermore found that gluten, when added to the LFD, increases circulating C-reactive protein levels. Gluten regardless of whether it was added to LFD or HFD led to a profound increase in the number of microglia and astrocytes in the arcuate nucleus of the hypothalamus, as detected by immunohistochemistry for ionised calcium binding adaptor molecule 1 (Iba-1) and glial fibrillary acidic protein (GFAP), respectively. In mice fed LFD, gluten mimicked the immunogenic effects of HFD exposure and when added to HFD led to a further increase in the number of immunoreactive cells. Taken together, our results confirm a moderate obesogenic effect of gluten when fed to mice exposed to HFD and for the first-time report gluten-induced astro- and microgliosis suggesting the development of hypothalamic injury in rodents.

The prevalence of obesity is rising, creating an urgent need for efficacious therapies. Recent clinical trials show that tirzepatide, a dual agonist of receptors for the incretin hormones glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP), yields more weight loss than selective GLP-1 receptor (GLP-1R) agonists. Incretin receptors in the central nervous system (CNS) may contribute to these effects. Yet exactly how each receptor regulates body weight from within the CNS is not clearly understood. It remains especially unclear how GIP receptor (GIPR) signalling contributes to the effects of tirzepatide because both stimulation and inhibition of CNS GIPRs yield weight loss in preclinical models. We summarise current knowledge on CNS incretin receptor pharmacology to provide insight into the potential mechanisms of action of dual GIPR/GLP-1R agonists, with tirzepatide as the exemplar. In addition, we discuss recent developments in incretin-based dual- and tri-agonism for inducing weight loss in obese individuals.

Insulin and leptin are essential hormones for the regulation of glucose- and energy homeostasis. The hypothalamus is regarded as the neuronal centre for metabolic control and the signalling machinery of both hormones is abundantly expressed in this brain region. While the brain does not play a major role in glucose disposal central insulin signalling is essential for maintenance of whole-body glucose- and energy homeostasis and interaction with the hormone leptin, secreted by adipocytes, is critical in this process. Emerging evidence suggests that maintenance of metabolic health depends on the balance between circulating leptin levels and leptin sensitivity. In this chapter we will present recent discoveries in this field with a focus on crosstalk of both hormones and its importance for neuroendocrine control of glucose- and energy homeostasis. We will furthermore highlight how the insulin sensitising hormones glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide 1 (GLP-1) may interact with insulin and leptin in the brain. Understanding the interplay of these hormone and their intracellular signalling machinery may provide a promising avenue for novel weight loss strategies.

Leptin is best known for its role in adipostasis, but it also regulates blood glucose levels. The molecular mechanism by which leptin controls glucose homeostasis remains largely unknown. Here, we use a zebrafish model to show that Wnt signaling mediates the glucoregulatory effects of leptin. Under normal feeding conditions, leptin regulates glucose homeostasis but not adipostasis in zebrafish. In times of nutrient excess, however, we found that leptin also regulates body weight and size. Using a Wnt signaling reporter fish, we show that leptin activates the canonical Wnt pathway in vivo. Utilizing two paradigms for hyperglycemia, it is revealed that leptin regulates glucose homeostasis via the Wnt pathway, as pharmacological inhibition of this pathway impairs the glucoregulatory actions of leptin. Our results may shed new light on the evolution of the physiological function of leptin.

Obesity has emerged as a major risk factor for insulin resistance leading to the development of type 2 diabetes (T2D). The condition is characterized by high circulating levels of the adipose‐derived hormone leptin and a state of chronic low‐grade inflammation. Pro‐inflammatory signaling in the hypothalamus is associated with a decrease of central leptin‐ and insulin action leading to impaired systemic glucose tolerance. Intriguingly, leptin not only regulates body weight and glucose homeostasis but also acts as a pro‐inflammatory cytokine. Here we demonstrate that increasing leptin levels (62,5 µg/kg/d, PEGylated leptin) in mice fed a high‐fat diet (HFD) exacerbated body weight gain and aggravated hypothalamic micro‐ as well as astrogliosis. In contrast, administration of a predetermined dose of a long‐acting leptin antagonist (100 µg/kg/d, PESLAN) chosen to block excessive leptin signaling during diet‐induced obesity (DIO) showed the opposite effect and significantly improved glucose tolerance as well as decreased the total number of microglia and astrocytes in the hypothalamus of mice fed HFD. These results suggest that high levels of leptin, such as in obesity, worsen HFD‐induced micro‐and astrogliosis, whereas the partial reduction of hyperleptinemia in DIO mice may have beneficial metabolic effects and improves hypothalamic gliosis.

Energy homeostasis is controlled by an intricate regulatory system centred in the brain. The peripheral adiposity signals insulin and leptin play a crucial role in this system by informing the brain of the energy status of the body and mediating their catabolic effects through signal transduction in hypothalamic areas that control food intake, energy expenditure and glucose metabolism. Disruptions of insulin and leptin signalling can result in diabetes and obesity. The central signalling cross‐talk between insulin and leptin is essential for maintenance of normal healthy energy homeostasis. An important role of leptin in glucoregulation has been revealed. Typically regarded as being controlled by insulin, the control of glucose homeostasis critically depends on functional leptin action. Leptin, on the other hand, is able to lower glucose levels in the absence of insulin, although insulin is necessary for long‐term stabilisation of euglycaemia. Evidence from rodent models and human patients suggests that leptin improves insulin sensitivity in type 1 diabetes. The signalling cross‐talk between insulin and leptin is likely conveyed by the WNT/β‐catenin pathway. Leptin activates WNT/β‐catenin signalling, leading to inhibition of glycogen synthase kinase‐3β, a key inhibitor of insulin action, thereby facilitating improved insulin signal transduction and sensitisation of insulin action. Interestingly, insights into the roles of insulin and leptin in insects and fish indicate that leptin may have initially evolved as a glucoregulatory hormone and that its anorexigenic and body weight regulatory function was acquired throughout evolution. Furthermore, the regulation of both central and peripheral control of energy homeostasis is tightly controlled by the circadian clock, allowing adaptation of homeostatic processes to environmental cues.

To induce diet-induced obesity (DIO) in rodents, diets high in saturated fat and/or carbohydrates are commonly used. In the laboratory, standardised diets evolved over time without paying particular attention to the effect of fat composition on metabolic alterations. In the present study, customised high-fat diets (HFD) enriched with a combination of lard and different concentrations of New Zealand green-lipped mussel (Perna canaliculus) oil or MSC Hoki (Macruronus novaezelandiae, blue grenadier) liver oil, important sources of n-3 PUFA, in comparison with a solely lard-based diet, were fed to lean and DIO male C57BL/6 mice and their effects on metabolic parameters were monitored. Intriguingly, an isoenergetic HFD containing 63 % of total fat in the form of mussel oil and only 28 % in the form of lard attenuated HFD-induced body weight gain after 1 and 4 weeks, respectively. Consistently, changing a lard-enriched HFD to the mussel oil diet reduced body weight markedly even after mice had been exposed to the former diet for 10 months. The weight-reducing effect of the diet was not caused by altered energy intake or expenditure, but was associated with reduced visceral fat mass. Collectively, these data suggest a novel weight-reducing potential of green-lipped mussel oil.

The importance of fatty acids (FAs) for healthy brain development and function has become more evident in the past decades. However, most studies focus on the hypothalamus as an important FA-sensing brain region involved in energy homeostasis. Less work has been done to evaluate the effects of FAs on brain regions such as the hippocampus or cortex, two important centres of learning, memory formation, and cognition. Furthermore, the mechanisms of how FAs modulate the neuronal development and function are incompletely understood. Therefore, this study examined the effects of the saturated FA palmitic acid (PA) and the polyunsaturated FA docosahexaenoic acid (DHA) on primary hippocampal and cortical cultures isolated from P0/P1 Sprague Dawley rat pups. Exposure to PA, but not DHA, resulted in severe morphological changes in primary neurons such as cell body swelling, axonal and dendritic blebbing, and a reduction in synaptic innervation, compromising healthy cell function and excitability. Pharmacological assessment revealed that the PA-mediated alterations were caused by overactivation of neuronal insulin signaling, demonstrated by insulin stimulation and phosphoinositide 3-kinase inhibition. Remarkably, co-exposure to DHA prevented all PA-induced morphological changes. This work provides new insights into how FAs can affect the cytoskeletal rearrangements and neuronal function via modulation of insulin signaling.

Synchronization between biologic clocks and metabolism is crucial for most species. Here, we examined the ability of leptin, important in the control of energy metabolism, to induce leptin signaling at the molecular as well as the behavioral level throughout the 24‐h day in mice fed either a control or a high‐fat diet (HFD). Furthermore, we investigated the effects of time‐restricted feeding (TRF; a limitation of HFD access to 6 h each day) on energy metabolism during different periods throughout the 24‐h day. In control mice, molecular leptin sensitivity was highest at zeitgeber time (ZT)0 (lights on), declining during the light phase, and increasing during the dark phase. Surprisingly, leptin resistance in HFD‐fed mice was only present from the middle of the dark to the middle of the light period. Specifically, when TRF occurred from ZT21 to ZT3 (when leptin resistance in HFD‐fed mice was most profound), it resulted in a disruption of the daily rhythms of locomotor activity and energy expenditure and in increased plasma insulin levels compared with other TRF periods. These data provide evidence that leptin sensitivity is controlled by the circadian rhythm and that TRF periods may be most efficient when aligned with the leptin‐sensitive period.—Boucsein, A., Rizwan, M. Z., Tups, A. Hypothalamic leptin sensitivity and health benefits of time‐restricted feeding are dependent on the time of day in male mice. FASEB J. 33, 12175–12187 (2019). www.fasebj.org

Recent evidence suggests that the circadian timing system plays a role in energy and glucose homeostasis, and disruptions to this system are a risk factor for the development of metabolic disorders. We exposed animals to a constantly shifting lighting environment comprised of a 6-hour advance, occurring every 6 days, to chronically disrupt their circadian timing system. This treatment caused a gradual increase in body weight of 12 +/- 2% after 12 phase shifts, compared with a 6 +/- 1% increase in mice under control lighting conditions. Additionally, after the fifth phase shift, light cycle-disrupted (CD) animals showed a reversal in their diurnal pattern of energy homeostasis and locomotor activity, followed by a subsequent loss of this rhythm. To investigate potential molecular mechanisms mediating these metabolic alterations, we assessed central leptin and insulin sensitivity. We discovered that CD mice had a decrease in central leptin signaling, as indicated by a reduction in the number of phosphorylated signal transducer and activator of transcription 3 immunoreactive cells in the arcuate nucleus of the hypothalamus. Furthermore, CD animals exhibited a marked increase in fasting blood glucose (269.4 +/- 21.1 mg/dL) compared with controls (108.8 +/- 21.3 mg/dL). This dramatic increase in fasting glucose levels was not associated with an increase in insulin levels, suggesting impairments in pancreatic insulin release. Peripheral hyperglycemia was accompanied by central alterations in insulin signaling at the level of phospho Akt and insulin receptor substrate 1, suggesting that light cycle disruption alters central insulin signaling. These results provide mechanistic insights into the association between light cycle disruption and metabolic disease.

Saturated fatty acids are implicated in the development of metabolic diseases, including obesity and type 2 diabetes. There is evidence, however, that polyunsaturated fatty acids can counteract the pathogenic effects of saturated fatty acids. To gain insight into the early molecular mechanisms by which fatty acids influence hypothalamic inflammation and insulin signalling, we performed time-course experiments in a hypothalamic cell line, using different durations of treatment with the saturated fatty acid palmitate, and the omega-3 polyunsaturated fatty acid, docosahexaenoic acid (DHA). Western blot analysis revealed that palmitate elevated the protein levels of phospho(p)AKT in a time-dependent manner. This effect is involved in the pathogenicity of palmitate, as temporary inhibition of the PI3K/AKT pathway by selective PI3K inhibitors prevented the palmitate-induced attenuation of insulin signalling. Similar to palmitate, DHA also increased levels of pAKT, but to a weaker extent. Co-administration of DHA with palmitate decreased pAKT close to the basal level after 8 h, and prevented the palmitate-induced reduction of insulin signalling after 12 h. The monounsaturated fatty acid oleate had a similar effect on the palmitate-induced attenuation of insulin signalling, the polyunsaturated fatty acid linoleate had no effect. Measurement of the inflammatory markers pJNK and pNFκB-p65 revealed tonic elevation of both markers in the presence of palmitate alone. DHA alone transiently induced elevation of pJNK, returning to basal levels by 12 h treatment. Co-administration of DHA with palmitate prevented palmitate-induced inflammation after 12 h, but not at earlier timepoints.

Metabolic syndrome and Alzheimer's disease (AD) are two major health issues in modern society causing an extraordinary financial burden for the global healthcare systems. A tight link between the pathologies of obesity and type 2 diabetes (T2D), and more recently between T2D and AD, has been discovered. Furthermore, in recent years it has become apparent that the circadian clock has an important function in controlling metabolism. This review integrates the role of the circadian clock in the development of these metabolic derangements and vice versa. Common features such as central insulin resistance, altered glycogen synthase kinase 3 beta (GSK3 beta) signalling, and central inflammation are discussed, and therapeutic interventions targeting those mechanisms are mentioned briefly.

β‐catenin is a multifunctional protein that not only acts in the canonical Wnt/β‐catenin pathway to regulate gene expression but also binds to cadherin proteins in adherens junctions, where it plays a key role in regulating cytoskeleton linked with these junctions. Recently, evidence has been presented indicating an essential role for β‐catenin in regulating the trafficking of insulin vesicles in β‐cells and showing that changes in nutrient levels rapidly alter levels of β‐catenin in these cells. Given the importance of neuroendocrine hormone secretion in the regulation of whole body glucose homeostasis, the present study aimed to investigate whether β‐catenin signalling is regulated in the hypothalamus during the normal physiological response to food intake. Rats were subjected to a fasting/re‐feeding paradigm, and then samples were collected at specific timepoints for analysis of β‐catenin expression by immunohistochemistry and western blotting. Changes in gene expression were assessed by a quantitative reverse transcriptase‐polymerase chain reaction. Using immunohistochemistry, feeding acutely increased detectable cytoplasmic levels of β‐catenin (‘stabilised β‐catenin’) in neurones in specific regions of the hypothalamus involved in metabolic regulation, including the arcuate, dorsomedial and paraventricular nuclei of the hypothalamus. Feeding‐induced elevations in β‐catenin in these nuclei were associated with an increased transcription of several genes known to be responsive to Wnt/β‐catenin signalling. The effect of feeding was mimicked by administration of the GLP‐1 agonist exendin‐4 and was also characterised by cAMP‐dependent phosphorylation of β‐catenin at serine residues 552 and 675. These data suggest that β‐catenin/T cell factor signalling is involved in metabolic sensing in the hypothalamus.

Energy homeostasis is regulated by the hypothalamus but fails when animals are fed a high-fat diet (HFD), and leptin insensitivity and obesity develops. To elucidate the possible mechanisms underlying these effects, a microarray-based transcriptomics approach was used to identify novel genes regulated by HFD and leptin in the mouse hypothalamus.

Saturated fatty acids are implicated in the development of metabolic diseases, including obesity and type 2 diabetes. There is evidence, however, that polyunsaturated fatty acids can counteract the pathogenic effects of saturated fatty acids. To gain insight into the early molecular mechanisms by which fatty acids influence hypothalamic inflammation and insulin resistance, we performed time-course experiments in a hypothalamic cell line, using different durations of treatment with the saturated fatty acid palmitate, and the omega-3 polyunsaturated fatty acid, docosahexaenoic acid (DHA). Western blot analysis revealed that palmitate elevated the protein levels of phospho(p)AKT in a time-dependent manner. This effect seems involved in the pathogenicity of palmitate, as temporary inhibition of the PI3K/AKT pathway by selective PI3K inhibitors prevented palmitate-induced insulin resistance. Similarly to palmitate, DHA also increased levels of pAKT, but to a weaker extent. Co-administration of DHA with palmitate decreased pAKT close to the basal level after 8 h, and prevented palmitate-induced insulin resistance after 12 h. Measurement of the inflammatory markers pJNK and pNFκB-p65 revealed tonic elevation of both markers in the presence of palmitate alone. DHA alone transiently induced elevation of pJNK, returning to basal levels by 12 h treatment. Co-administration of DHA with palmitate prevented palmitate-induced inflammation after 12 h, but not at earlier time points.