Paradoxical Increase in TAG and DAG Content Parallel the Insulin Sensitizing Effect of Unilateral DGAT1 Overexpression in Rat Skeletal Muscle

BACKGROUND: The involvement of muscle triacylglycerol (TAG) storage in the onset of insulin resistance is questioned and the attention has shifted towards inhibition of insulin signalling by the lipid intermediate diacylglycerol (DAG). The enzyme 1,2-acylCoA:diacylglyceroltransferase-1 (DGAT1) esterifies a fatty acyl-CoA on DAG to form TAG. Therefore, the aim of the present study was to investigate if unilateral overexpression of DGAT1 in adult rat Tibialis anterior (TA) muscle will increase conversion of the lipid intermediate DAG into TAG, thereby improving muscle insulin sensitivity. METHODOLOGY/PRINCIPAL FINDINGS: The DGAT1 gene construct was injected in the left TA muscle of male rats on chow or high-fat (45% kcal) diet for three weeks, followed by application of one 800 V/cm and four 80 V/cm pulses, using the contralateral leg as sham-electroporated control. Seven days after electroporation, muscle specific insulin sensitivity was assessed with a hyperinsulinemic euglycemic clamp using 2-deoxy-[3H]glucose. Here, we provide evidence that unilateral overexpression of DGAT1 in TA muscle of male rats is associated with an increased rather than decreased DAG content. Strikingly, this increase in DAG content was accompanied by improved muscle insulin sensitivity. Interestingly, markers of muscle lipolysis and mitochondrial function were also increased in DGAT1 overexpressing muscle. CONCLUSIONS/SIGNIFICANCE: We conclude that unilateral DGAT1 overexpression can rescue insulin sensitivity, possibly by increasing DAG and TAG turnover in skeletal muscle. In case of a proper balance between the supply and oxidation of fatty acids in skeletal muscle, the lipid intermediate DAG may not exert harmful effects on insulin signalling

The hypothalamo-pituitary-adrenal axis in multiple sclerosis

During multiple sclerosis (MS), an inflammatory demyelinating disease of the central nervous system (CNS), activation of the hypothalamo-pituitary-adrenal (HPA) axis is considered to modulate the immune system in such a way that the probability of recovery from a relapse is increased. In a series of postmortem studies we observed a significant activation of corticotropin releasing hormone (CRH) neurons and increased cortisol in the cerebrospinal fluid (CSF) of MS patients, indicating activation of the HPA axis in this disease. On the other hand, sepsis, while elevating cortisol in control subjects, did not associate with a further increase of cortisol in MS patients. Thus, the activated HPA-system in MS does not respond to an acute inflammatory stimulus. In order to investigate the role of chronic inflammation in the CNS in the activation of the HPA axis in MS, MS lesions in the hypothalamus were quantified and interleukin (IL)-6 levels in the CSF were determined. There was no difference in IL-6 levels between MS and control patients. A positive correlation was found between cortisol and IL-6 in control subjects with sepsis, but not in MS patients with sepsis or MS and control groups without sepsis. Thus, IL-6 in the CSF of MS patients is not the cause of the activation of the HPA system in MS. We found a remarkably high incidence (95% of the patients) of MS lesions in the hypothalamus, of which the majority (60%) were active. The more active lesions were present in the hypothalamus, the shorter the disease duration to the moment of death, indicative of a worse disease course. Preliminary data show suppression of the activation of CRH neurons by active hypothalamic MS lesions. We propose that this suppression of CRH neurons by active hypothalamic MS lesions causes the concomitant unfavorable disease course via an inadequate cortisol response during relapses of M

Direct deep UV lithography to micropattern PMMA for stem cell culture

Microengineering is increasingly being used for controlling the microenvironment of stem cells. Here, a novel method for fabricating structures with subcellular dimensions in commonly available thermoplastic poly(methyl methacrylate) (PMMA) is shown. Microstructures are produced in PMMA substrates using Deep Ultraviolet lithography, and the effect of different developers is described. Microgrooves fabricated in PMMA are used for the neuronal differentiation of mouse embryonic stem cells (mESCs) directly on the polymer. The fabrication of 3D, curvilinear patterned surfaces is also highlighted. A 3D multilayered microfluidic chip is fabricated using this method, which includes a porous polycarbonate (PC) membrane as cell culture substrate. Besides directly manufacturing PMMA-based microfluidic devices, an application of the novel approach is shown where a reusable PMMA master is created for replicating microstructures with polydimethylsiloxane (PDMS). As an application example, microchannels fabricated in PDMS are used to selectively expose mESCs to soluble factors in a localized manner. The described microfabrication process offers a remarkably simple method to fabricate for example multifunctional topographical or microfluidic culture substrates outside cleanrooms, thereby using inexpensive and widely accessible equipment. The versatility of the underlying process could find various applications also in optical systems and surface modification of biomedical implants

Diminished aromatase immunoreactivity in the hypothalamus, but not in the basal forebrain nuclei in Alzheimer's disease

In previous studies we have shown in Alzheimer's disease (AD) an enhanced nuclear estrogen receptor (ER) alpha expression in the cholinergic basal forebrain nuclei, i.e. the vertical limb of the diagonal band of Broca (VDB) and the nucleus basalis of Meynert (NBM), and in a number of hypothalamic nuclei, i.e. the supraoptic nucleus (SON), the infundibular nucleus (INF), the medial mamillary nucleus (MMN). We aimed at determining whether the increase in nuclear ERalpha seen in AD patients was related to a rise in local production of estrogens by aromatase (P-450arom), which is a key enzyme that catalyzes the biosynthesis of estrogens from precursor aromatizable androgens. We confirmed for the first time the presence of aromatase mRNA in neurons and glial cells in the human NBM and the tuberomamillary nucleus by RT-QPCR using laser microdissection. Enhanced aromatase immunoreactivity (ir) was indeed observed in the NBM in AD. However, in contrast a decreased aromatase-ir was found in the SON, INF and MMN of AD patients. In addition, P-450arom-ir was clearly diminished in ependymal and choroid plexus cells in AD. While an increase in aromatase-ir was found in the NBM and SON during normal aging, a decrease in staining was observed in the MMN. No sex differences in young control, elderly control or AD patients were present in any of the nuclei studied. In conclusion, brain P-450arom-ir and the relationship of its regulation with plasma sex steroid levels, estrogen and androgen receptors in the human hypothalamus and basal forebrain are region-specific. (C) 2004 Elsevier Inc. All rights reserve

An in vitro model system based on calcium- and phosphate ion-induced hMSC spheroid mineralization

A challenge in regenerative medicine is creating the three-dimensional organic and inorganic in vitro microenvironment of bone, which would allow the study of musculoskeletal disorders and the generation of building blocks for bone regeneration. This study presents a microwell-based platform for creating spheroids of human mesenchymal stromal cells, which are then mineralized using ionic calcium and phosphate supplementation. The resulting mineralized spheroids promote an osteogenic gene expression profile through the influence of the spheroids’ biophysical environment and inorganic signaling and require less calcium or phosphate to achieve mineralization compared to a monolayer culture. We found that mineralized spheroids represent an in vitro model for studying small molecule perturbations and extracellular mediated calcification. Furthermore, we demonstrate that understanding pathway signaling elicited by the spheroid environment allows mimicking these pathways in traditional monolayer culture, enabling similar rapid mineralization events. In sum, this study demonstrates the rapid generation and employment of a mineralized cell model system for regenerative medicine applications

Perilipin 2 improves insulin sensitivity in skeletal muscle despite elevated intramuscular lipid levels

Type 2 diabetes is characterized by excessive lipid storage in skeletal muscle. Excessive intramyocellular lipid storage exceeds intracellular needs and induces lipotoxic events ultimately contributing to the development of insulin resistance. Lipid droplet (LD)-coating proteins may control proper lipid storage in skeletal muscle. Perilipin 2 (PLIN2/ADRP) is one of the most abundantly expressed LD-coating proteins in skeletal muscle. Here we examined the role of PLIN2 in myocellular lipid handling and insulin sensitivity by investigating the effects of in vitro PLIN2 knockdown and in vitro and in vivo overexpression. PLIN2 knockdown decreased LD formation and triacylglycerol storage, marginally increased FA oxidation, and increased incorporation of palmitate into diacylglycerols and phospholipids. PLIN2 overexpression in vitro increased intramyocellular TAG storage paralleled with improved insulin sensitivity. In vivo muscle-specific PLIN2 overexpression resulted in increased LD accumulation and blunted the high-fat diet-induced increase of OXPHOS protein content. Diacylglycerol levels were unchanged, while ceramide levels were increased. Despite the increased intramyocellular lipid accumulation, PLIN2 overexpression improved skeletal muscle insulin sensitivity. We conclude that PLIN2 is essential for lipid storage in skeletal muscle by enhancing the partitioning of excess FAs towards triacylglycerol storage in LDs thereby blunting lipotoxicity-associated insulin resistance

DGAT1 overexpression is associated with an increased mitochondrial function and biogenesis in HFD-fed rats.

<p>(<b>A</b>) Western blot analysis of PGC1α (<b>B</b>) OXPHOS complexes and (<b>C</b>) UCP3 in rat TA muscle. Data are expressed as mean ± SEM (n = 10–12). <b>^#</b> P<0.05 HFD-DGAT1 vs. HFD-control.</p

Three weeks of high-fat feeding is not associated with an increased TA ceramide content compared to rats on CHOW.

<p>DGAT1 overexpression does not result in an increase in TA ceramide content in rats on CHOW or HFD. Data are expressed as mean ± SEM (n = 10–12).</p

DGAT1 overexpression results in bigger lipid droplets in HFD-fed rats.

<p>(<b>A</b>) Electron microscopy revealed an increase in lipid droplet diameter in DGAT1 overexpressing TA muscle of HFD-fed rats. (<b>B</b>) Western blot analysis of OXPAT in rat TA muscle. Data are expressed as mean ± SEM (n = 10–12).</p

Three weeks of high-fat feeding is associated with an increased TA DAG content compared to rats on CHOW.

<p>DGAT1 overexpression leads to an increase in TA DAG content in rats on CHOW and tended to increase DAG content in rats on HFD. Data are expressed as mean ± SEM (n = 6). *P<0.05 CHOW-DGAT1 and HFD-control vs. CHOW-control.</p