Högskolan i Skövde

Gliflozins, such as dapagliflozin, belong to a class of drugs that inhibit the sodium-glucose cotransporter 2. Gliflozins have been found to raise glucagon levels, a hormone secreted from pancreatic islet a-cells, which can trigger ketosis. However, the precise mechanisms through which gliflozins increase glucagon secretion remain poorly understood. In addition, gliflozins induce osmotic diuresis, resulting in increased urine volume and plasma osmolality. In this study, we investigated the hypothesis that a compensatory increase in arginine-vasopressin (AVP) mediates dapagliflozin-induced increases in glucagon in vivo. We show that dapagliflozin does not increase glucagon secretion in the perfused mouse pancreas, neither at clinical nor at supra-clinical doses. In contrast, AVP potently increases glucagon secretion. In vivo, dapagliflozin increased plasma glucagon, osmolality, and AVP. An oral load with hypertonic saline amplified dapagliflozin-induced glucagon secretion. Notably, a similar increase in glucagon could also be elicited by dehydration, evoked by 24-h water restriction. Conversely, blockade of vasopressin 1b receptor signaling, with either pharmacological antagonism or knockout of the receptor, resulted in reduced dapagliflozin-induced glucagon secretion in response to both dapagliflozin and dehydration. Finally, blocking vasopressin 1b receptor signaling in a mouse model of type 1 diabetes diminished the glucagon-promoting and ketogenic effects of dapagliflozin. Collectively, our data suggest that AVP is an important regulator of glucagon release during both drug-induced and physiological dehydration.

NEW & NOTEWORTHY Gliflozin-induced ketogenic effects partly result from increased glucagon levels. This study shows that dapagliflozin-triggered glucagon secretion is not directly mediated by the pancreas but rather linked to arginine-vasopressin (AVP). Dehydration, common in diabetic ketoacidosis, elevates AVP, potentially explaining the increased ketoacidosis risk in gliflozin-treated patients. Thus, our results highlight AVP as a potential therapeutic target to mitigate the risk of ketoacidosis associated with gliflozin treatments in patients with diabetes.

Background: The adipocyte hormone adiponectin improves insulin sensitivity and there is an inverse correlation between adiponectin levels and type-2 diabetes risk. Previous research shows that adiponectin remodels the adipose tissue into a more efficient metabolic sink. For instance, mice that overexpress adiponectin show increased capacity for hyperplastic adipose tissue expansion as evident from smaller and metabolically more active white adipocytes. In contrast, the brown adipose tissue (BAT) of these mice looks “whiter” possibly indicating reduced metabolic activity. Here, we aimed to further establish the effect of adiponectin on adipose tissue expansion and adipocyte mitochondrial function as well as to unravel mechanistic aspects in this area. Methods: Brown and white adipose tissues from adiponectin overexpressing (APN tg) mice and littermate wildtype controls, housed at room and cold temperature, were studied by histological, gene/protein expression and flow cytometry analyses. Metabolic and mitochondrial functions were studied by radiotracers and Seahorse-based technology. In addition, mitochondrial function was assessed in cultured adiponectin deficient adipocytes from APN knockout and heterozygote mice. Results: APN tg BAT displayed increased proliferation prenatally leading to enlarged BAT. Postnatally, APN tg BAT turned whiter than control BAT, confirming previous reports. Furthermore, elevated adiponectin augmented the sympathetic innervation/activation within adipose tissue. APN tg BAT displayed reduced metabolic activity and reduced mitochondrial oxygen consumption rate (OCR). In contrast, APN tg inguinal white adipose tissue (IWAT) displayed enhanced metabolic activity. These metabolic differences between genotypes were apparent also in cultured adipocytes differentiated from BAT and IWAT stroma vascular fraction, and the OCR was reduced in both brown and white APN heterozygote adipocytes. In both APN tg BAT and IWAT, the mesenchymal stem cell-related genes were upregulated along with an increased abundance of Lineage−Sca1+CD34− “beige-like” adipocyte precursor cells. In vitro, the adiponectin receptor agonist Adiporon increased the expression of the proliferation marker Pcna and decreased the expression of Cd34 in Sca1+ mesenchymal stem cells. Conclusions: We propose that the seemingly opposite effect of adiponectin on BAT and IWAT is mediated by a common mechanism; while reduced adiponectin levels are linked to lower adipocyte OCR, elevated adiponectin levels stimulate expansion of adipocyte precursor cells that produce adipocytes with intrinsically higher metabolic rate than classical white but lower metabolic rate than classical brown adipocytes. Moreover, adiponectin can modify the adipocytes' metabolic activity directly and by enhancing the sympathetic innervation within a fat depot. 

Polycystic ovary syndrome (PCOS) is associated with a low-grade inflammation, but it is unknown how hyperandrogenism, the hallmark of PCOS, affects the immune system. Using a PCOS-like mouse model, it is demonstrated that hyperandrogenism affects immune cell populations in reproductive, metabolic, and immunological tissues differently in a site-specific manner. Co-treatment with an androgen receptor antagonist prevents most of these alterations, demonstrating that these effects are mediated through androgen receptor activation. Dihydrotestosterone (DHT)-exposed mice displayed a drastically reduced eosinophil population in the uterus and visceral adipose tissue (VAT). A higher frequency of natural killer (NK) cells and elevated levels of IFN-γ and TNF-α are seen in uteri of androgen-exposed mice, while NK cells in VAT and spleen displayed a higher expression level of CD69, a marker of activation or tissue residency. Distinct alterations of macrophages in the uterus, ovaries, and VAT are also found in DHT-exposed mice and can potentially be linked to PCOS-like traits of the model. Indeed, androgen-exposed mice are insulin-resistant, albeit unaltered fat mass. Collectively, it is demonstrated that hyperandrogenism causes tissue-specific alterations of immune cells in reproductive organs and VAT, which can have considerable implications on tissue function and contribute to the reduced fertility and metabolic comorbidities associated with PCOS.

Women with polycystic ovary syndrome (PCOS) exhibit sustained elevation incirculating androgens during pregnancy, an independent risk factor linked topregnancy complications and adverse outcomes in offspring. Yet, furtherstudies are required to understand the effects of elevated androgens on celltype-specific placental dysfunction and fetal development. Therefore, aPCOS-like mouse model induced by continuous androgen exposure isexamined. The PCOS-mice exhibited impaired placental and embryonicdevelopment, resulting in mid-gestation lethality. Co-treatment with theandrogen receptor blocker, flutamide, prevents these phenotypes includinggerm cell specification . Comprehensive profiling of the placenta bywhole-genome bisulfite and RNA sequencing shows a reduced proportion oftrophoblast precursors, possibly due to the downregulation of Cdx2expression. Reduced expression of Gcm1, Synb, and Prl3b1 is associated withreduced syncytiotrophoblasts and sinusoidal trophoblast giant cells, impairsplacental labyrinth formation. Importantly, human trophoblast organoidsexposed to androgens exhibit analogous changes, showing impairedtrophoblast differentiation as a key feature in PCOS-related pregnancycomplications. These findings provide new insights into the potential cellulartargets for future treatments.

Polycystic ovary syndrome (PCOS) is a common endocrine disorder in women that is associated with an increased risk of anxiety and depression and with a lower health-related quality of life (HRQoL). PCOS is closely associated with obesity, which per se can lead to symptoms of anxiety and depression and lower HRQoL. The first-line treatment for PCOS is weight loss through lifestyle intervention, which has been shown to improve all symptoms of the syndrome. The aim of this study was to investigate symptoms of anxiety and depression and HRQoL in women with severe obesity (BMI ≥ 35) with and without PCOS, and to evaluate the effect of a one-year structured weight loss intervention. A total of 246 women with severe obesity (PCOS n = 63, non-PCOS n = 183) were included. The comprehensive psychopathological rating scale self-rating scale for affective symptoms (CPRS-S-A) and the short form-36 (SF-36) were used to assess symptoms of anxiety and depression and HRQoL. In total 72 women of the 246 women with severe obesity completed a one-year weight loss programme and were followed up and compared with baseline data. In women with severe obesity, there were no differences in symptoms of anxiety and depression and HRQoL between women with and without PCOS at baseline. Clinically relevant anxiety symptoms were present in 71.3% (PCOS) and 65.6% (non-PCOS), and depression symptoms were present in 56.4% (PCOS) and 52.2% (non-PCOS). Significant weight loss improved physical HRQoL in all women, but reduced symptoms of anxiety and depression only in women without PCOS. There were no differences when comparing the changes between the groups. Women with severe obesity are severely affected by symptoms of anxiety and depression, independent of PCOS. Weight loss improved symptoms of anxiety and depression in women without PCOS, but there were no differences between groups in change from baseline to follow-up.Trial registration number: Clinical trial.gov: NCT01319162, March 18, 2011. Date of registration and enrolment of the first subject September 2011.

Polycystic ovary syndrome (PCOS) is associated with symptoms of moderate to severe anxiety and depression. Hyperandrogenism is a key feature together with lower levels of the adipocyte hormone adiponectin. Androgen exposure leads to anxiety-like behavior in female offspring while adiponectin is reported to be anxiolytic. Here we test the hypothesis that elevated adiponectin levels protect against the development of androgen-induced anxiety-like behavior. Pregnant mice overexpressing adiponectin (APNtg) and wildtypes were injected with vehicle or dihydrotestosterone to induce prenatal androgenization (PNA) in the offspring. Metabolic profiling and behavioral tests were performed in 4-month-old female offspring. PNA offspring spent more time in the closed arms of the elevated plus maze, indicating anxiety-like behavior. Intriguingly, neither maternal nor offspring adiponectin overexpression prevented an anxiety-like behavior in PNA-exposed offspring. However, adiponectin overexpression in dams had metabolic imprinting effects, shown as lower fat mass and glucose levels in their offspring. While serum adiponectin levels were elevated in APNtg mice, cerebrospinal fluid levels were similar between genotypes. Adiponectin overexpression improved metabolic functions but did not elicit anxiolytic effects in PNA-exposed offspring. These observations might be attributed to increased circulating but unchanged cerebrospinal fluid adiponectin levels in APNtg mice. Thus, increased adiponectin levels in the brain are likely needed to stimulate anxiolytic effects. 

Background: Polycystic ovary syndrome’s (PCOS) main feature is hyperandrogenism, which is linked to a higher risk of metabolic disorders in women. Gene expression analyses in adipose tissue and skeletal muscle reveal dysregulated metabolic pathways in women with PCOS, but these differences do not necessarily lead tochanges in protein levels and biological function. Methods: To advance our understanding of the molecular alterations in PCOS, we performed global proteomic and phosphorylation site analysis using tandem mass spectrometry. Adipose tissue and skeletal muscle were collected at baseline from 10 women with and without PCOS, and in women with PCOS after 5 weeks of treatment with electrical stimulation. Results: Perilipin-1, a protein that typically coats the surface of lipid droplets in adipocytes, was increased whereas proteins involved in muscle contraction and type I muscle fiber function were downregulated in PCOS muscle. Proteins in the thick and thin filaments had many altered phosphorylation sites, indicating differences in protein activity and function. The upregulated proteins in muscle post treatment were enriched in pathways involved in extracellular matrix organization and wound healing, which may reflect a protective adaptation to repeated contractions and tissue damage due to needling. A similar, albeit less pronounced, upregulation in extracellular matrix organization pathways was also seen in adipose tissue. Conclusions: Our results suggest that hyperandrogenic women with PCOS have higher levels of extramyocellular lipids and fewer oxidative insulin-sensitive type I muscle fibers. These could be key factors leading insulin resistance in PCOS muscle while electric stimulation-induced tissue remodeling may be protective.

White adipose tissue browning, defined by accelerated mitochondrial metabolism and biogenesis, is considered a promising mean to treat or prevent obesity-associated metabolic disturbances. We hypothesize that redox stress acutely leads to increased production of reactive oxygen species (ROS), which activate electrophile sensor nuclear factor erythroid 2-Related Factor 2 (NRF2) that over time results in an adaptive adipose tissue browning process. To test this, we have exploited adipocyte-specific NRF2 knockout mice and cultured adipocytes and analyzed time- and dose-dependent effect of NAC and lactate treatment on antioxidant expression and browning-like processes. We found that short-term antioxidant treatment with N-acetylcysteine (NAC) induced reductive stress as evident from increased intracellular NADH levels, increased ROS-production, reduced oxygen consumption rate (OCR), and increased NRF2 levels in white adipocytes. In contrast, and in line with our hypothesis, longer-term NAC treatment led to a NRF2-dependent browning response. Lactate treatment elicited similar effects as NAC, and mechanistically, these NRF2-dependent adipocyte browning responses in vitro were mediated by increased heme oxygenase-1 (HMOX1) activity. Moreover, this NRF2-HMOX1 axis was also important for β3-adrenergic receptor activation-induced adipose tissue browning in vivo. In conclusion, our findings show that administration of exogenous antioxidants can affect biological function not solely through ROS neutralization, but also through reductive stress. We also demonstrate that NRF2 is essential for white adipose tissue browning processes. 

The transgenerational maternal effects of polycystic ovary syndrome (PCOS) in female progeny are being revealed. As there is evidence that a male equivalent of PCOS may exists, we ask whether sons born to mothers with PCOS (PCOS-sons) transmit reproductive and metabolic phenotypes to their male progeny. Here, in a register-based cohort and a clinical case-control study, we find that PCOS-sons are more often obese and dyslipidemic. Our prenatal androgenized PCOS-like mouse model with or without diet-induced obesity confirmed that reproductive and metabolic dysfunctions in first-generation (F1) male offspring are passed down to F3. Sequencing of F1–F3 sperm reveals distinct differentially expressed (DE) small non-coding RNAs (sncRNAs) across generations in each lineage. Notably, common targets between transgenerational DEsncRNAs in mouse sperm and in PCOS-sons serum indicate similar effects of maternal hyperandrogenism, strengthening the translational relevance and highlighting a previously underappreciated risk of transmission of reproductive and metabolic dysfunction via the male germline. 

Adiponectin administration to pregnant mice decreases nutrient transport and fetal growth. An adiponectin deficiency, on the other hand, as seen in obese women during pregnancy, alters fetal growth; however, the mechanism is unclear. To determine the role of adiponectin on placenta function and fetal growth, we used adiponectin knockout, adiponectin heterozygote that displays reduced adiponectin levels, and wild-type mice on a control diet or high fat/high sucrose (HF/HS) diet. Triglycerides (TGs) in the serum, liver, and placenta were measured using colorimetric assays. Gene expression was measured using quantitative RT-PCR. Adiponectin levels did not affect fetal weight, but it reduced adiponectin levels, increased fetal serum and placenta TG content. Wildtype dams on a HF/HS diet protected the fetuses from fatty acid overload as judged by increased liver TGs in dams and normal serum and liver TG levels in fetuses, while low adiponectin was associated with increased fetal liver TGs. Low maternal adiponectin increased the expression of genes involved in fatty acid transport; Lpl and Cd36 in the placenta. Adiponectin deficiency does not affect fetal growth but induces placental dysfunction and increases fetal TG load, which is enhanced with obesity. This could lead to imprinting effects on the fetus and the development of metabolic dysfunction in the offspring.