The Mitochondrial Permeability Transition Pore Regulator Cyclophilin D Exhibits Tissue-Specific Control of Metabolic Homeostasis

<div><p>The mitochondrial permeability transition pore (mPTP) is a key regulator of mitochondrial function that has been implicated in the pathogenesis of metabolic disease. Cyclophilin D (CypD) is a critical regulator that directly binds to mPTP constituents to facilitate the pore opening. We previously found that global CypD knockout mice (KO) are protected from diet-induced glucose intolerance; however, the tissue-specific function of CypD and mPTP, particularly in the control of glucose homeostasis, has not been ascertained. To this end, we performed calcium retention capacity (CRC) assay to compare the importance of CypD in the liver versus skeletal muscle. We found that liver mitochondria are more dependent on CypD for mPTP opening than skeletal muscle mitochondria. To ascertain the tissue-specific role of CypD in metabolic homeostasis, we generated liver-specific and muscle-specific CypD knockout mice (LKO and MKO, respectively) and fed them either a chow diet or 45% high-fat diet (HFD) for 14 weeks. MKO mice displayed similar body weight gain and glucose intolerance compared with wild type littermates (WT), whereas LKO mice developed greater visceral obesity, glucose intolerance and pyruvate intolerance compared with WT mice. These findings demonstrate that loss of muscle CypD is not sufficient to alter whole body glucose metabolism, while the loss of liver CypD exacerbates obesity and whole-body metabolic dysfunction in mice fed HFD.</p></div

CypD is important for metabolic control in the liver under high-fat diet conditions.

<p>(A) Body weight at basal levels and following 11 wk of HFD; (B) Epididymal fat, (C) liver and (D) plantaris muscle tissue weight normalized to tibia length in HFD fed mice; (E) Blood glucose following i.p. injection of glucose and (F) area under the glucose curve under normal chow conditions; (G) Blood glucose following i.p. injection of glucose and (H) area under the glucose curve following 11 wk of HFD; (I) Blood glucose following i.p. injection of pyruvate and (J) area under the curve following 14 wk of HFD; (K) Representative Oil red O staining of liver sections from WT and LKO mice following 14 wk of HFD and (L) average lipid droplet size. ***p < 0.001 basal vs. HFD, * and **p < 0.05 WT vs. LKO; GTT and PTT n = 9–11; Oil Red O quantification n = 4.</p

CypD is more important for calcium overload induced mitochondrial permeability transition in the liver compared with skeletal muscle.

<p>Representative calcium retention capacity assay of a single experiment on isolated mitochondria from skeletal muscle (A) and liver (B) of WT and CypD KO mice fed HFD for 10 weeks. (C) The average number of calcium additions required before irreversible mPTP opening was observed as indicated by fluorescence plateau and quantified for skeletal muscle and liver mitochondria. For gastrocnemius WT = 9, KO = 5; for liver WT = 7 and KO = 4. *p < 0.05.</p

Deletion of CypD does not alter other mPTP protein abundance.

<p>(A) Western blot for CypD protein in liver, plantaris muscle, heart and epididymal fat tissue. MKO mice have muscle-specific deletion of CypD in skeletal muscle and heart (A), while LKO mice have liver-specific deletion (B). Littermate floxed mice are used as WT showing normal CypD expression. Representative western blot and quantification, respectively, in skeletal muscle (C and E) and liver (D and F) for VDAC, ATP Synthase and ANT. Quantification is normalized to the loading control, β-actin. *p < 0.05; n = 4–6.</p

Individual islet respirometry reveals functional diversity within the islet population of mice and human donors

Objective: Islets from the same pancreas show remarkable variability in glucose sensitivity. While mitochondrial respiration is essential for glucose-stimulated insulin secretion, little is known regarding heterogeneity in mitochondrial function at the individual islet level. This is due in part to a lack of high-throughput and non-invasive methods for detecting single islet function. Methods: We have developed a novel non-invasive, high-throughput methodology capable of assessing mitochondrial respiration in large-sized individual islets using the XF96 analyzer (Agilent Technologies). Results: By increasing measurement sensitivity, we have reduced the minimal size of mouse and human islets needed to assess mitochondrial respiration to single large islets of >35,000 μm2 area (∼210 μm diameter). In addition, we have measured heterogeneous glucose-stimulated mitochondrial respiration among individual human and mouse islets from the same pancreas, allowing population analyses of islet mitochondrial function for the first time. Conclusions: We have developed a novel methodology capable of analyzing mitochondrial function in large-sized individual islets. By highlighting islet functional heterogeneity, we hope this methodology can significantly advance islet research. Keywords: Islets, Mitochondria, Respirometry, Glucos

Mitochondrial Proton Leak Regulated by Cyclophilin D Elevates Insulin Secretion in Islets at Nonstimulatory Glucose Levels

Fasting hyperinsulinemia precedes the development of type 2 diabetes. However, it is unclear whether fasting insulin hypersecretion is a primary driver of insulin resistance or a consequence of the progressive increase in fasting glycemia induced by insulin resistance in the prediabetic state. Herein, we have discovered a mechanism that specifically regulates non-glucose-stimulated insulin secretion (NGSIS) in pancreatic islets that is activated by nonesterified free fatty acids, the major fuel used by β-cells during fasting. We show that the mitochondrial permeability transition pore regulator cyclophilin D (CypD) promotes NGSIS, but not glucose-stimulated insulin secretion, by increasing mitochondrial proton leak. Islets from prediabetic obese mice show significantly higher CypD-dependent proton leak and NGSIS compared with lean mice. Proton leak-mediated NGSIS is conserved in human islets and is stimulated by exposure to nonesterified free fatty acids at concentrations observed in obese subjects. Mechanistically, proton leak activates islet NGSIS independently of mitochondrial ATP synthesis but ultimately requires closure of the KATP channel. In summary, we have described a novel nonesterified free fatty acid-stimulated pathway that selectively drives pancreatic islet NGSIS, which may be therapeutically exploited as an alternative way to halt fasting hyperinsulinemia and the progression of type 2 diabetes

Quadrivalent vaccine against human papillomavirus to prevent high-grade cervical lesions

BACKGROUND: Human papillomavirus types 16 (HPV-16) and 18 (HPV-18) cause approximately 70% of cervical cancers worldwide. A phase 3 trial was conducted to evaluate a quadrivalent vaccine against HPV types 6, 11, 16, and 18 (HPV-6/11/16/18) for the prevention of high-grade cervical lesions associated with HPV-16 and HPV-18. METHODS: In this randomized, double-blind trial, we assigned 12,167 women between the ages of 15 and 26 years to receive three doses of either HPV-6/11/16/18 vaccine or placebo, administered at day 1, month 2, and month 6. The primary analysis was performed for a per-protocol susceptible population that included 5305 women in the vaccine group and 5260 in the placebo group who had no virologic evidence of infection with HPV-16 or HPV-18 through 1 month after the third dose (month 7). The primary composite end point was cervical intraepithelial neoplasia grade 2 or 3, adenocarcinoma in situ, or cervical cancer related to HPV-16 or HPV-18. RESULTS: Subjects were followed for an average of 3 years after receiving the first dose of vaccine or placebo. Vaccine efficacy for the prevention of the primary composite end point was 98% (95.89% confidence interval [CI], 86 to 100) in the per-protocol susceptible population and 44% (95% CI, 26 to 58) in an intention-to-treat population of all women who had undergone randomization (those with or without previous infection). The estimated vaccine efficacy against all high-grade cervical lesions, regardless of causal HPV type, in this intention-to-treat population was 17% (95% CI, 1 to 31). CONCLUSIONS: In young women who had not been previously infected with HPV-16 or HPV-18, those in the vaccine group had a significantly lower occurrence of high-grade cervical intraepithelial neoplasia related to HPV-16 or HPV-18 than did those in the placebo group