Conditional Deletion of ß-Catenin in the Mediobasal Hypothalamus Impairs Adaptive Energy Expenditure in Response to High-Fat Diet and Exacerbates Diet-Induced Obesity.

ß-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, ß-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 ß-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 ß-catenin flox mice, to specifically delete ß-catenin expression in the mediobasal hypothalamus (MBH-ß-cat KO). Metabolic parameters were then monitored under conditions of low-fat (LFD) and high-fat diet (HFD). On LFD, MBH-ß-cat KO mice showed minimal metabolic disturbances, but on HFD, despite having only a small difference in weekly caloric intake, the MBH-ß-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-ß-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 ß-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.

Dietary wheat gluten induces astro- and microgliosis in the hypothalamus of male mice.

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.

Obesity pharmacotherapy: incretin action in the central nervous system.

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.

Leptin regulates glucose homeostasis via the canonical Wnt pathway in the zebrafish.

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.

Central signalling cross-talk between insulin and leptin in glucose and energy homeostasis.

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/ß-catenin pathway. Leptin activates WNT/ß-catenin signalling, leading to inhibition of glycogen synthase kinase-3ß, 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.

Hyperleptinemia as a contributing factor for the impairment of glucose intolerance in obesity.

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.

A New Zealand green-lipped mussel oil-enriched high-fat diet exhibits beneficial effects on body weight and metabolism in mice.

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.

Docosahexaenoic acid prevents palmitate-induced insulin-dependent impairments of neuronal health.

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.

Hypothalamic leptin sensitivity and health benefits of time-restricted feeding are dependent on the time of day in male mice.

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.

Chronic Light Cycle Disruption Alters Central Insulin and Leptin Signaling as well as Metabolic Markers in Male Mice.

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.

"Insulin-like" effects of palmitate compromise insulin signalling in hypothalamic neurons.

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.

Timing Matters: Circadian Effects on Energy Homeostasis and Alzheimer's Disease.

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 3ß (GSK3ß) signalling, and central inflammation are discussed, and therapeutic interventions targeting those mechanisms are mentioned briefly.

SerpinA3N is a novel hypothalamic gene upregulated by a high-fat diet and leptin in mice.

BACKGROUND: 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. RESULTS: Mouse global array data identified serpinA3N as a novel gene highly upregulated by both a HFD and leptin challenge. In situ hybridisation showed serpinA3N expression upregulation by HFD and leptin in all major hypothalamic nuclei in agreement with transcriptomic gene expression data. Immunohistochemistry and studies in the hypothalamic clonal neuronal cell line, mHypoE-N42 (N42), confirmed that alpha 1-antichymotrypsin (α1AC), the protein encoded by serpinA3, is localised to neurons and revealed that it is secreted into the media. SerpinA3N expression in N42 neurons is upregulated by palmitic acid and by leptin, together with IL-6 and TNFα, and all three genes are downregulated by the anti-inflammatory monounsaturated fat, oleic acid. Additionally, palmitate upregulation of serpinA3 in N42 neurons is blocked by the NFκB inhibitor, BAY11, and the upregulation of serpinA3N expression in the hypothalamus by HFD is blunted in IL-1 receptor 1 knockout (IL-1R1 -/- ) mice. CONCLUSIONS: These data demonstrate that serpinA3 expression is implicated in nutritionally mediated hypothalamic inflammation.

Feeding and GLP-1 receptor activation stabilize ß-catenin in specific hypothalamic nuclei in male rats.

ß-catenin is a multifunctional protein that can act in the canonical Wnt/ß-catenin pathway to regulate gene expression but can also bind 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 ß-catenin in regulating trafficking of insulin vesicles in ß-cells and showing that changes in nutrient levels rapidly alter levels of ß-catenin in these cells. Given the importance of neuroendocrine hormone secretion in the regulation of whole body glucose homeostasis, the objective of this study was to investigate whether ß-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 collected at specific timepoints for analysis of ß-catenin expression by immunohistochemistry and Western blotting. Changes in gene expression were assessed by RT-qPCR. Using immunohistochemistry, feeding acutely increased detectable cytoplasmic levels of ß-catenin ('stabilized ß-catenin') in neurons in specific regions of the hypothalamus involved in metabolic regulation, including the arcuate, dorsomedial and paraventricular nuclei of the hypothalamus. Feeding-induced elevations in ß-catenin in these nuclei were associated with increased transcription of several genes that are known to be responsive to Wnt/ß-catenin signalling. The effect of feeding was mimicked by administration of the GLP-1 agonist exendin-4, and was characterized by cAMP-dependent phosphorylation of ß-catenin at serine residues 552 and 675. The data suggest that ß-catenin/TCF signalling is involved in metabolic sensing in the hypothalamus. This article is protected by copyright. All rights reserved.

Central Regulation of Glucose Homeostasis.

The ability of the brain to directly control glucose levels in the blood independently of its effects on food intake and body weight has been known ever since 1854 when Claude Bernard, a French physiologist, discovered that lesioning the floor of the fourth ventricle in rabbits led to a rise of sugar in the blood. Despite this outstanding discovery at that time, it took more than 140 years before progress started to be made in identifying the underlying mechanisms of brain-mediated control of glucose homeostasis. Technological advances including the generation of brain insulin receptor null mice revealed that insulin action specifically in the central nervous system is required for the regulation of glucose metabolism, particularly in the modulation of hepatic glucose production. Furthermore, it was established that the hormone leptin, known for its role in regulating food intake and body weight, actually exerts its most potent effects on glucose metabolism, and that this function of leptin is mediated centrally. Under certain circumstances, high levels of leptin can replicate the actions of insulin, thus challenging the idea that life without insulin is impossible. Disruptions of central insulin signaling and glucose metabolism not only lead to impairments in whole body glucose homeostasis, they also have other serious consequences, including the development of Alzheimer's disease which is sometimes referred to as type 3 diabetes reflecting its common etiology with type 2 diabetes. © 2017 American Physiological Society. Compr Physiol 7:471-764, 2017.

Photoperiodic and Diurnal Regulation of WNT Signaling in the Arcuate Nucleus of the Female Djungarian Hamster, Phodopus sungorus.

The WNT pathway was shown to play an important role in the adult central nervous system. We previously identified the WNT pathway as a novel integration site of the adipokine leptin in mediating its neuroendocrine control of metabolism in obese mice. Here we investigated the implication of WNT signaling in seasonal body weight regulation exhibited by the Djungarian hamster (Phodopus sungorus), a seasonal mammal that exhibits profound annual changes in leptin sensitivity. We furthermore investigated whether crucial components of the WNT pathway are regulated in a diurnal manner. Gene expression of key components of the WNT pathway in the hypothalamus of hamsters acclimated to either long day (LD) or short day (SD) photoperiod was analyzed by in situ hybridization. We detected elevated expression of the genes WNT-4, Axin-2, Cyclin-D1, and SFRP-2, in the hypothalamic arcuate nucleus, a key energy balance integration site, during LD compared with SD as well as a diurnal regulation of Axin-2, Cyclin-D1, and DKK-3. Investigating the effect of photoperiod as well as leptin on the activation (phosphorylation) of the WNT coreceptor LRP-6-(Ser1490) by immunohistochemistry, we found elevated activity in the arcuate nucleus during LD relative to SD as well as after leptin treatment (2 mg/kg body weight). These findings indicate that differential WNT signaling may be associated with seasonal body weight regulation and is partially regulated in a diurnal manner in the adult brain. Furthermore, they suggest that this pathway plays a key role in the neuroendocrine regulation of body weight and integration of the leptin signal.

Central inhibition of IKKß/NF-κB signaling attenuates high-fat diet-induced obesity and glucose intolerance.

Metabolic inflammation in the central nervous system might be causative for the development of overnutrition-induced metabolic syndrome and related disorders, such as obesity, leptin and insulin resistance, and type 2 diabetes. Here we investigated whether nutritive and genetic inhibition of the central IκB kinase ß (IKKß)/nuclear factor-κB (NF-κB) pathway in diet-induced obese (DIO) and leptin-deficient mice improves these metabolic impairments. A known prominent inhibitor of IKKß/NF-κB signaling is the dietary flavonoid butein. We initially determined that oral, intraperitoneal, and intracerebroventricular administration of this flavonoid improved glucose tolerance and hypothalamic insulin signaling. The dose-dependent glucose-lowering capacity was profound regardless of whether obesity was caused by leptin deficiency or high-fat diet (HFD). To confirm the apparent central role of IKKß/NF-κB signaling in the control of glucose and energy homeostasis, we genetically inhibited this pathway in neurons of the arcuate nucleus, one key center for control of energy homeostasis, via specific adeno-associated virus serotype 2-mediated overexpression of IκBα, which inhibits NF-κB nuclear translocation. This treatment attenuated HFD-induced body weight gain, body fat mass accumulation, increased energy expenditure, and reduced arcuate suppressor of cytokine signaling 3 expression, indicative for enhanced leptin signaling. These results reinforce a specific role of central proinflammatory IKKß/NF-κB signaling in the development and potential treatment of DIO-induced comorbidities.

Acyl ghrelin acts in the brain to control liver function and peripheral glucose homeostasis in male mice.

Recent evidence suggests that peripheral ghrelin regulates glucose metabolism. Here, we designed experiments to examine how central acyl ghrelin infusion affects peripheral glucose metabolism under pair-fed or ad libitum feeding conditions. Mice received intracerebroventricular (icv) infusion of artificial cerebrospinal fluid (aCSF), ghrelin, and allowed to eat ad libitum (icv ghrelin ad lib) or ghrelin and pair-fed to the aCSF group (icv ghrelin pf). Minipumps delivered acyl ghrelin at a dose of 0.25 µg/h at 0.5 µL/h for 7 days. There was no difference in daily blood glucose, insulin, glucagon, triglycerides, or nonesterified fatty acids. Body weight gain and food intake was significantly higher in icv ghrelin ad lib mice. However, both icv ghrelin ad lib and icv ghrelin pf groups exhibited heavier white adipose mass. Icv ghrelin pf mice exhibited better glucose tolerance than aCSF or icv ghrelin ad lib mice during a glucose tolerance test, although both icv ghrelin ad lib and icv ghrelin pf increased insulin release during the glucose tolerance test. Central acyl ghrelin infusion and pair feeding also increased breakdown of liver glycogen and triglyceride, and regulated genes involved in hepatic lipid and glucose metabolism. Icv ghrelin pf mice had an increase in plasma blood glucose during a pyruvate tolerance test relative to icv ghrelin ad lib or aCSF mice. Our results suggest that under conditions of negative energy (icv ghrelin pf), central acyl ghrelin engages a neural circuit that influences hepatic glucose function. Metabolic status affects the ability of central acyl ghrelin to regulate peripheral glucose homeostasis.

The development of diet-induced obesity and glucose intolerance in C57BL/6 mice on a high-fat diet consists of distinct phases.

High-fat (HF) diet-induced obesity and insulin insensitivity are associated with inflammation, particularly in white adipose tissue (WAT). However, insulin insensitivity is apparent within days of HF feeding when gains in adiposity and changes in markers of inflammation are relatively minor. To investigate further the effects of HF diet, C57Bl/6J mice were fed either a low (LF) or HF diet for 3 days to 16 weeks, or fed the HF-diet matched to the caloric intake of the LF diet (PF) for 3 days or 1 week, with the time course of glucose tolerance and inflammatory gene expression measured in liver, muscle and WAT. HF fed mice gained adiposity and liver lipid steadily over 16 weeks, but developed glucose intolerance, assessed by intraperitoneal glucose tolerance tests (IPGTT), in two phases. The first phase, after 3 days, resulted in a 50% increase in area under the curve (AUC) for HF and PF mice, which improved to 30% after 1 week and remained stable until 12 weeks. Between 12 and 16 weeks the difference in AUC increased to 60%, when gene markers of inflammation appeared in WAT and muscle but not in liver. Plasma proteomics were used to reveal an acute phase response at day 3. Data from PF mice reveals that glucose intolerance and the acute phase response are the result of the HF composition of the diet and increased caloric intake respectively. Thus, the initial increase in glucose intolerance due to a HF diet occurs concurrently with an acute phase response but these effects are caused by different properties of the diet. The second increase in glucose intolerance occurs between 12-16 weeks of HF diet and is correlated with WAT and muscle inflammation. Between these times glucose tolerance remains stable and markers of inflammation are undetectable.

Phenotypic characterization of miR-92a-/- mice reveals an important function of miR-92a in skeletal development.

MicroRNAs (miRNAs, miRs) emerged as key regulators of gene expression. Germline hemizygous deletion of the gene that encodes the miR-17∼92 miRNA cluster was associated with microcephaly, short stature and digital abnormalities in humans. Mice deficient for the miR-17∼92 cluster phenocopy several features such as growth and skeletal development defects and exhibit impaired B cell development. However, the individual contribution of miR-17∼92 cluster members to this phenotype is unknown. Here we show that germline deletion of miR-92a in mice is not affecting heart development and does not reduce circulating or bone marrow-derived hematopoietic cells, but induces skeletal defects. MiR-92a-/- mice are born at a reduced Mendelian ratio, but surviving mice are viable and fertile. However, body weight of miR-92a-/- mice was reduced during embryonic and postnatal development and adulthood. A significantly reduced body and skull length was observed in miR-92a-/- mice compared to wild type littermates. µCT analysis revealed that the length of the 5th mesophalanx to 5th metacarpal bone of the forelimbs was significantly reduced, but bones of the hindlimbs were not altered. Bone density was not affected. These findings demonstrate that deletion of miR-92a is sufficient to induce a developmental skeletal defect.