Within mitochondria, the ability to produce energy relies upon the architectural hallmarks of double membranes and cristae invaginations. Herein, we describe novel features of mitochondrial cristae structure, which correspond to the energetic state of the organelle. In concordance with high-energy demand, mitochondria of Drosophila indirect flight muscle exhibited extensive intra-mitochondrial membrane switches between densely packed lamellar cristae that resulted in a spiral-like cristae network and allowed for bidirectional matrix confluency. This highly interconnected architecture is expected to allow rapid equilibration of membrane potential and biomolecules across integrated regions. In addition, mutant flies with mtDNA replication defect and an accelerated aging phenotype accumulated mitochondria that contained subsections of swirling membrane alongside normal cristae. The swirling membrane had impaired energy production capacity as measured by protein composition and function. Furthermore, mitochondrial fusion and fission dynamics were affected in the prematurely aged flies. Interestingly, the normal cristae that remained in the mitochondria with swirling membranes maintained acceptable function that camouflaged them from quality control elimination. Overall, structural features of mitochondrial cristae were described in three-dimension from serial section electron tomographic analysis which reflect energetic state and mtDNA-mediated aging.

Obese men have lower circulating testosterone than men with an optimal body mass index. Elevated fatty acids (FAs) caused by obesity have been reported to suppress the steroidogenesis of Leydig cells. Recent studies have demonstrated that autophagy regulates steroidogenesis in endocrine cells; however, few studies have investigated the molecular mechanisms of FA-impaired steroidogenesis. To study FA regulation in the steroidogenesis of Leydig cells, MA-10 cells were treated with an FA mixture and co-treated with 8-Br-cAMP to stimulate the steroidogenesis capacity. We showed that FAs led to cellular lipid accumulation and decreased steroidogenesis of MA-10 cells, and FA-suppressed steroidogenesis was largely recovered by P5 treatment but not by 22R-OHC treatment, suggesting the primary defect was the deficiency of CYP11A1. To examine the involvement of autophagy in the steroidogenesis of Leydig cells, we treated MA-10 cells with autophagy regulators, including rapamycin, bafilomycin, and chloroquine. Inhibition of late-stage autophagy including FA-upregulated Rubicon suppressed the steroidogenesis of MA-10 cells. More interestingly, Rubicon played a novel regulatory role in the steroidogenesis of MA-10 cells, independent of inhibitors of late-stage autophagy. Collectively, this study provides novel targets to investigate the interaction between FAs and steroidogenesis in steroidogenic cells.

During the prediabetic development, the changes in β-cell function and tissue-specific insulin resistance have been described. However, there are conflicting views in insulin secretory capacity between early clinical observation and recent proposed mathematical model. On the basis of digestive and metabolic similarities with humans, swine have great potential as an animal model to investigate the progressive mechanisms of prediabetes. The aim of this study was to investigate the insulin secretory response and tissue-specific insulin resistance in a dietary-induced prediabetic porcine model. Adult male Taiwan Lee-Sung miniature pigs were randomized into two groups: (1) low-fat diet and (2) high-fat plus high-fructose diet (HFHF; 20.9% crude fat and 17.8% fructose). During the 12-month dietary intervention, body weights and blood glucose levels were measured monthly. Intravenous glucose tolerance test was used for measuring glucose tolerance and insulin secretory capacity. At the end of the experiment, liver and soleus muscle specimens were collected for ex vivo insulin sensitivity testing. The results showed that the HFHF group had obesity, hyperinsulinemia, and dyslipidemia, but normal fasting glucose levels. The HFHF pigs exhibited enhanced first- and second-phase insulin secretion and high 2-h postload glucose levels in intravenous glucose tolerance test. Furthermore, the skeletal muscle specimens from the HFHF group were desensitized to insulin stimulation as shown by the lack of AKT Ser473 phosphorylation; however, the liver specimens remained a normal response. In conclusion, the HFHF diet-fed pigs developed isolated impaired glucose tolerance corresponding to prediabetes with an intense insulin secretory response and skeletal muscle insulin resistance.

Aims: Studies have observed changes in autophagic flux in the adipose tissue of type 2 diabetes patients with obesity. However, the role of autophagy in obesity-induced insulin resistance is unclear. We propose to confirm the effect of a high-fat diet (HFD) on autophagy and insulin signaling transduction from adipose tissue to clarify whether altered autophagy-mediated HFD induces insulin resistance, and to elucidate the possible mechanisms in autophagy-regulated adipose insulin sensitivity. Methods: Eight-week-old male C57BL/6 mice were fed with HFD to confirm the effect of HFD on autophagy and insulin signaling transduction from adipose tissue. Differentiated 3T3-L1 adipocytes were treated with 1.2 mM fatty acids (FAs) and 50 nM Bafilomycin A1 to determine the autophagic flux. 2.5 mg/kg body weight dose of Chloroquine (CQ) in PBS was locally injected into mouse epididymal adipose (10 and 24 h) and 40 µM of CQ to 3T3-L1 adipocytes for 24 h to evaluate the role of autophagy in insulin signaling transduction. Results: The HFD treatment resulted in a significant increase in SQSTM1/p62, Rubicon expression, and C/EBP homologous protein (CHOP) expression, yet the insulin capability to induce Akt (Ser473) and GSK3β (Ser9) phosphorylation were reduced. PHLPP1 and PTEN remain unchanged after CQ injection. In differentiated 3T3-L1 adipocytes treated with CQ, although the amount of phospho-Akt stimulated by insulin in the CQ-treated group was significantly lower, CHOP expressions and cleaved caspase-3 were increased and bafilomycin A1 induced less accumulation of LC3-II protein. Conclusion: Long-term high-fat diet promotes insulin resistance, late-stage autophagy inhibition, ER stress, and apoptosis in adipose tissue. Autophagy suppression may not affect insulin signaling transduction via phosphatase expression but indirectly causes insulin resistance through ER stress or apoptosis.