Glycogen metabolism is impaired in the brain of male type 2 diabetic Goto-Kakizaki rats.

Diabetes impacts the central nervous system predisposing to cognitive decline. While glucose is the main source of energy fueling the adult brain, brain glycogen is necessary for adequate neuronal function, synaptic plasticity and memory. In this study, we tested the hypothesis that brain glycogen metabolism is impaired in type 2 diabetes (T2D). 13 C magnetic resonance spectroscopy (MRS) during [1-13 C]glucose i.v. infusion was employed to detect 13 C incorporation into whole-brain glycogen in male Goto-Kakizaki (GK) rats, a lean model of T2D, and control Wistar rats. Labeling from [1-13 C]glucose into brain glycogen occurred at a rate of 0.25 ± 0.12 and 0.48 ± 0.22 µmol/g/h in GK and Wistar rats, respectively (p = 0.028), despite similar brain glycogen concentrations. In addition, the appearance of [1-13 C]glucose in the brain was used to evaluate glucose transport and consumption. T2D caused a 31% reduction (p = 0.031) of the apparent maximum transport rate (Tmax ) and a tendency for reduced cerebral metabolic rate of glucose (CMRglc ; -29%, p = 0.062), indicating impaired glucose utilization in T2D. After MRS in vivo, gas chromatography-mass spectrometry was employed to measure regional 13 C fractional enrichment of glucose and glycogen in the cortex, hippocampus, striatum, and hypothalamus. The diabetes-induced reduction in glycogen labeling was most prominent in the hippocampus and hypothalamus, which are crucial for memory and energy homeostasis, respectively. These findings were further supported by changes in the phosphorylation rate of glycogen synthase, as analyzed by Western blotting. Altogether, the present results indicate that T2D is associated with impaired brain glycogen metabolism.

Non-invasive diagnosis and metabolic consequences of congenital portosystemic shunts in C57BL/6 J mice.

This study demonstrates the suitability of magnetic resonance imaging (MRI) and magnetic resonance angiography (MRA) for the imaging of congenital portosystemic shunts (PSS) in mice, a vascular abnormality in which mesenteric blood bypasses the liver and is instead drained directly to the systemic circulation. The non-invasive diagnosis performed in tandem with other experimental assessments permits further characterization of liver, whole-body and brain metabolic defects associated with PSS. Magnetic resonance measurements were performed in a 26-cm, horizontal-bore, 14.1-T magnet. MRA was obtained with a three-dimensional gradient echo sequence (GRE; in-plane resolution, 234 × 250 × 234 µm3 ) using a birdcage coil. Two-dimensional GRE MRI with high spatial resolution (in-plane resolution, 100 × 130 µm2 ; slices, 30 × 0.3 mm) was performed using a surface coil. Brain- (dorsal hippocampus) and liver-localized 1 H magnetic resonance spectroscopy (MRS) was also performed with the surface coil. Whole-body metabolic status was evaluated with an oral glucose tolerance test (OGTT). Both MRA and anatomical MRI allowed the identification of hepatic vessels and the diagnosis of PSS in mice. The incidence of PSS was about 10%. Hepatic lipid content was higher in PSS than in control mice (5.1 ± 2.8% versus 1.8 ± 0.6%, p = 0.02). PSS mice had higher brain glutamine concentration than controls (7.3 ± 1.0 µmol/g versus 2.7 ± 0.6 µmol/g, p < 0.0001) and, conversely, lower myo-inositol (4.2 ± 0.6 µmol/g versus 6.0 ± 0.4 µmol/g, p < 0.0001), taurine (9.7 ± 1.2 µmol/g versus 11.0 ± 0.4 µmol/g, p < 0.01) and total choline (0.9 ± 0.1 µmol/g versus 1.2 ± 0.1 µmol/g, p < 0.001) concentrations. Fasting blood glucose and plasma insulin were lower in PSS than in control mice (4.7 ± 0.5mM versus 8.8 ± 0.6mM, p < 0.0001; and 0.04 ± 0.03 µg/L versus 0.3 ± 0.2 µg/L, p = 0.02, respectively). Glucose clearance during OGTT was delayed and less efficient in PSS mice than in controls. Thus, given the non-negligible incidence of PSS in inbred mice, the undiagnosed presence of PSS will, importantly, have an impact on experimental outcomes, notably in studies addressing brain, liver or whole-body metabolism.

Increased hepatic fatty acid polyunsaturation precedes ectopic lipid deposition in the liver in adaptation to high-fat diets in mice.

OBJECTIVE: We monitored hepatic lipid content (HLC) and fatty acid (FA) composition in the context of enhanced lipid handling induced by a metabolic high-fat diet (HFD) challenge and fasting. MATERIALS AND METHODS: Mice received a control diet (10% of kilocalories from fat, N = 14) or an HFD (45% or 60% of kilocalories from fat, N = 10 and N = 16, respectively) for 26 weeks. A subset of five mice receiving an HFD (60% of kilocalories from fat) were switched to the control diet for the final 7 weeks. At nine time points, magnetic resonance spectroscopy was performed in vivo at 14.1 T, interleaved with glucose tolerance tests. RESULTS: Glucose intolerance promptly developed with the HFD, followed by a progressive increase of fasting insulin level, simultaneously with that of HLC. These metabolic defects were normalized by dietary reversal. HFD feeding immediately increased polyunsaturation of hepatic FA, before lipid accumulation. Fasting-induced changes in hepatic lipids (increased HLC and FA polyunsaturation, decreased FA monounsaturation) in control-diet-fed mice were not completely reproduced in HFD-fed mice, not even after dietary reversal. CONCLUSION: A similar adaptation of hepatic lipids to both fasting and an HFD suggests common mechanisms of lipid trafficking from adipose tissue to the liver. Altered hepatic lipid handling with fasting indicates imperfect metabolic recovery from HFD exposure.

Sexual dimorphism in hepatic lipids is associated with the evolution of metabolic status in mice.

Ectopic lipid accumulation in the liver is implicated in metabolic disease in an age- and sex-dependent manner. The role of hepatic lipids has been well established within the scope of metabolic insults in mice, but has been insufficiently characterized under standard housing conditions, where age-related metabolic alterations are known to occur. We studied a total of 10 male and 10 female mice longitudinally. At 3, 7 and 11 months of age, non-invasive 1 H-magnetic resonance spectroscopy (1 H-MRS) was used to monitor hepatic lipid content (HLC) and fatty acid composition in vivo, and glucose homeostasis was assessed with glucose and insulin challenges. At the end of the study, hepatic lipids were comprehensively characterized by nuclear magnetic resonance (NMR) and liquid chromatography-mass spectrometric analyses of liver tissue samples. In males, HLC increased from 1.4 ± 0.1% at 3 months to 2.9 ± 0.3% at 7 months (p < 0.01) and 2.7 ± 0.3% at 11 months (p < 0.05), in correlation with fasting insulin levels (p < 0.01, r = 0.51) and parameters from the insulin tolerance test (ITT; p < 0.001, r = -0.69 versus area under the curve; p < 0.01, r = -0.57 versus blood glucose drop at 1 h post-ITT; p < 0.01, r = 0.55 versus blood glucose at 3 h post-ITT). The metabolic performance of females remained the same throughout the study, and HLC was higher than that of males at 3 months (2.7 ± 0.2%, p < 0.01), but comparable at 7 months (2.2 ± 0.2%) and 11 months (2.2 ± 0.1%). Strong sexual dimorphism in bioactive lipid species, including diacylglycerols (higher in males, p < 0.0001), phosphatidylinositols (higher in females, p < 0.001) and omega-3 polyunsaturated fatty acids (higher in females, p < 0.01), was found to be in good correlation with metabolic scores at 11 months. Therefore, in mice housed under standard conditions, sex-specific composition of bioactive lipids is associated with metabolic protection in females, whose metabolic performance was independent of hepatic cytosolic lipid content.

Technical and experimental features of Magnetic Resonance Spectroscopy of brain glycogen metabolism.

In the brain, glycogen is a source of glucose not only in emergency situations but also during normal brain activity. Altered brain glycogen metabolism is associated with energetic dysregulation in pathological conditions, such as diabetes or epilepsy. Both in humans and animals, brain glycogen levels have been assessed non-invasively by Carbon-13 Magnetic Resonance Spectroscopy (13C-MRS) in vivo. With this approach, glycogen synthesis and degradation may be followed in real time, thereby providing valuable insights into brain glycogen dynamics. However, compared to the liver and muscle, where glycogen is abundant, the sensitivity for detection of brain glycogen by 13C-MRS is inherently low. In this review we focus on strategies used to optimize the sensitivity for 13C-MRS detection of glycogen. Namely, we explore several technical perspectives, such as magnetic field strength, field homogeneity, coil design, decoupling, and localization methods. Furthermore, we also address basic principles underlying the use of 13C-labeled precursors to enhance the detectable glycogen signal, emphasizing specific experimental aspects relevant for obtaining kinetic information on brain glycogen.

Characterization of hepatic fatty acids in mice with reduced liver fat by ultra-short echo time (1)H-MRS at 14.1 T in vivo.

Alterations in the hepatic lipid content (HLC) and fatty acid composition are associated with disruptions in whole body metabolism, both in humans and in rodent models, and can be non-invasively assessed by (1)H-MRS in vivo. We used (1)H-MRS to characterize the hepatic fatty-acyl chains of healthy mice and to follow changes caused by streptozotocin (STZ) injection. Using STEAM at 14.1 T with an ultra-short TE of 2.8 ms, confounding effects from T2 relaxation and J-coupling were avoided, allowing for accurate estimations of the contribution of unsaturated (UFA), saturated (SFA), mono-unsaturated (MUFA) and poly-unsaturated (PUFA) fatty-acyl chains, number of double bonds, PU bonds and mean chain length. Compared with in vivo (1) H-MRS, high resolution NMR performed in vitro in hepatic lipid extracts reported longer fatty-acyl chains (18 versus 15 carbons) with a lower contribution from UFA (61 ± 1% versus 80 ± 5%) but a higher number of PU bonds per UFA (1.39 ± 0.03 versus 0.58 ± 0.08), driven by the presence of membrane species in the extracts. STZ injection caused a decrease of HLC (from 1.7 ± 0.3% to 0.7 ± 0.1%), an increase in the contribution of SFA (from 21 ± 2% to 45 ± 6%) and a reduction of the mean length (from 15 to 13 carbons) of cytosolic fatty-acyl chains. In addition, SFAs were also likely to have increased in membrane lipids of STZ-induced diabetic mice, along with a decrease of the mean chain length. These studies show the applicability of (1)H-MRS in vivo to monitor changes in the composition of the hepatic fatty-acyl chains in mice even when they exhibit reduced HLC, pointing to the value of this methodology to evaluate lipid-lowering interventions in the scope of metabolic disorders.

Advances in the use of 3D colorectal cancer models for novel drug discovery.

INTRODUCTION: Colorectal cancer (CRC) is one of the most common and deadly tumors worldwide. CRC in vitro and in vivo models that recapitulate key features of human disease are essential to the development of novel and effective therapeutics. However, two-dimensional (2D) in vitro culture systems are considered too simple and do not represent the complex nature of the human tumor. However, three-dimensional (3D) models have emerged in recent years as more advanced and complex cell culture systems, able to closely resemble key features of human cancer tissues. AREAS COVERED: The authors' review the currently established in vitro cell culture models and describe the advances in the development of 3D scaffold-free models to study CRC. The authors also discuss intestinal spheroids and organoids. As well as in vitro models for drug screening and metastatic CRC (mCRC). EXPERT OPINION: The ideal CRC in vitro model is not yet established. Spheroid-based 3D models represent one of the most used approaches to recapitulate the tumor environment, overcoming some limitations of 2D models. Mouse and patient-derived organoids are more advanced models that can mimic more closely the characteristics and properties of CRC, with the possibility of including cells derived from patients with metastatic CRC.

Neurochemical Modifications in the Hippocampus, Cortex and Hypothalamus of Mice Exposed to Long-Term High-Fat Diet.

Metabolic syndrome and diabetes impact brain function and metabolism. While it is well established that rodents exposed to diets rich in saturated fat develop brain dysfunction, contrasting results abound in the literature, likely as result of exposure to different high-fat diet (HFD) compositions and for varied periods of time. In the present study, we investigated alterations of hippocampal-dependent spatial memory by measuring Y-maze spontaneous alternation, metabolic profiles of the hippocampus, cortex and hypothalamus by 1H magnetic resonance spectroscopy (MRS), and levels of proteins specific to synaptic and glial compartments in mice exposed for 6 months to different amounts of fat (10, 45, or 60% of total energy intake). Increasing the dietary amount of fat from 10 to 45% or 60% resulted in obesity accompanied by increased leptin, fasting blood glucose and insulin, and reduced glucose tolerance. In comparison to controls (10%-fat), only mice fed the 60%-fat diet showed increased fed glycemia, as well as plasma corticosterone that has a major impact on brain function. HFD-induced metabolic profile modifications measured by 1H MRS were observed across the three brain areas in mice exposed to 60%- but not 45%-fat diet, while both HFD groups displayed impaired hippocampal-dependent memory. HFD also affected systems involved in neuro- or gliotransmission in the hippocampus. Namely, relative to controls, 60%-fat-fed mice showed reduced SNAP-25, PSD-95 and syntaxin-4 immunoreactivity, while 45%-fat-fed mice showed reduced gephyrin and syntaxin-4 immunoreactivity. For both HFD levels, reductions of the vesicular glutamate transporter vGlut1 and levels of the vesicular GABA transporter were observed in the hippocampus and hypothalamus, relative to controls. Immunoreactivity against GFAP and/or Iba-1 in the hypothalamus was higher in mice exposed to HFD than controls, suggesting occurrence of gliosis. We conclude that different levels of dietary fat result in distinct neurochemical alterations in the brain.

Oral administration of peptides and proteins: nanoparticles and cyclodextrins as biocompatible delivery systems.

This review discusses drawbacks to peptide and protein oral formulations related to these drugs' chemical and physical instability. Means used to overcome such limitations are mentioned and discussed in parallel with manufacturing considerations, metabolism, absorption mechanisms and the efflux systems that peptides and proteins experience as they travel through the gastrointestinal tract. Special focus is given to the use of delivery systems based on nanoparticles and cyclodextrins. Advantages of these systems relate to the protection from degradation, enhancement of absorption, targeting and controlling the release of the drug. Biodistribution and safety issues are discussed once material from the delivery system is expected to be absorbed by the body and thus interact with biological components. Operating parameters regarding nanoparticle manufacture and composition are also overviewed since nanoparticle physicochemical characteristics influence the ability to successfully entrap the intended drug as well as interaction with body.