Kristina Wallenius

Here, we developed and validated a high throughput proteomics workflow that enables combined profiling of global proteomes and post-translational modifications (PTMs) without conventional enrichment or fractionation. Using mass spectrometry-based methods, we identified PTM landscapes, particularly N-glycopeptides and non-enzymatic glycated peptides, from the same sample prepared for global proteomics. Applying this methodology to investigate longitudinal serum in women at 6 and 10 years post-gestational diabetes mellitus (GDM), we identified PTM signatures, including N-glycosylation and non-enzymatic glycation, associated with development of type 2 diabetes (T2D) and prediabetes, which were confirmed in an independent T2D cohort.

Aim: To characterise glycaemic outcomes 4–6 years postpartum according to gestational diabetes mellitus (GDM) diagnostic thresholds and evaluate whether continuous glucose monitoring (CGM) identifies differences not captured by conventional clinical measures. Methods: Women recruited before and after introduction of WHO GDM criteria were followed at 4 and 6 years postpartum. At each follow‐up, participants underwent oral glucose tolerance testing (OGTT), HbA1c measurement, body composition assessment and 14‐day CGM. Based on pregnancy OGTT results and the diagnostic criteria in use at the time of testing, women were classified as meeting EASD 1991 criteria (GDM _OLD ; higher glucose thresholds), meeting WHO 2013 criteria but not EASD 1991 without diagnosis and treatment (GDM _WHO −) or meeting WHO 2013 criteria with diagnosis and treatment (GDM _WHO +). Results: Women classified as GDM _OLD had higher fasting glucose at both follow‐ups compared with women meeting only WHO 2013 criteria, and showed greater hyperglycaemic exposure and glycaemic variability on CGM. Time above range, coefficient of variation and mean amplitude of glycaemic excursions were consistently higher in GDM _OLD group, and differences remained significant after adjustment for pregnancy glucose levels. Within the WHO‐defined groups, conventional clinical measures did not differ between women with and without a GDM diagnosis, whereas CGM detected modest differences in mean glucose and time above range over time. Approximately 50% of women meeting only WHO 2013 criteria had prediabetes. CGM metrics were strongly associated with OGTT‐derived glucose and HbA1c but only weakly with anthropometric and blood lipid measures. BMI and body fat percentage did not differ between groups. Conclusions: Long‐term glycaemic risk aligns with diagnostic thresholds used during pregnancy. Women diagnosed by older, higher thresholds exhibit a persistently less favourable glycaemic profile. Women meeting the lower WHO 2013 glucose thresholds represent a large group with a high prevalence of prediabetes, regardless of diagnosis during pregnancy. CGM detects modest differences in free‐living glycaemic patterns not apparent from conventional clinical measures.

We characterized the longitudinal serum protein signatures of women 6 and 10 years after having gestational diabetes mellitus (GDM) to identify factors associated with the development of type 2 diabetes mellitus (T2D) and prediabetes in this at-risk post-GDM population, aiming to discover potential biomarkers for early diagnosis and prevention of T2D. Our study identified 75 T2D-associated serum proteins and 23 prediabetes-associated proteins, some of which were validated in an independent T2D cohort. Machine learning (ML) performed on the longitudinal proteomics highlighted protein signatures associated with progression to post-GDM diabetes. We also proposed prognostic biomarker candidates that were differentially regulated in healthy participants at 6 years postpartum who later progressed to having T2D. Our longitudinal study revealed T2D risk factors for post-GDM populations who are relatively young and healthy, providing insights for clinical decisions and early lifestyle interventions.

Excessive fructose intake is a risk factor for the development of obesity and its complications. Targeting ketohexokinase (KHK), the first enzyme of fructose metabolism, has been investigated for the management of metabolic dysfunction–associated steatotic liver disease (MASLD). We compared the effects of systemic, small molecule inhibitor of KHK enzymatic activity with hepatocyte-specific, N-acetylgalactosamine siRNA–mediated knockdown of KHK in mice on an HFD. We measured KHK enzymatic activity, extensively quantified glycogen accumulation, performed RNA-Seq analysis, and enumerated hepatic metabolites using mass spectrometry. Both KHK siRNA and KHK inhibitor led to an improvement in liver steatosis; however, via substantially different mechanisms, KHK knockdown decreased the de novo lipogenesis pathway, whereas the inhibitor increased the fatty acid oxidation pathway. Moreover, KHK knockdown completely prevented hepatic fructolysis and improved glucose tolerance. Conversely, the KHK inhibitor only partially reduced fructolysis, but it also targeted triokinase, mediating the third step of fructolysis. This led to the accumulation of fructose-1 phosphate, resulting in glycogen accumulation, hepatomegaly, and impaired glucose tolerance. Overexpression of wild-type, but not kinase-dead, KHK in cultured hepatocytes increased hepatocyte injury and glycogen accumulation after treatment with fructose. The differences between KHK inhibition and knockdown are, in part, explained by the kinase-dependent and -independent effects of KHK on hepatic metabolism.

Analogues of the hepatokine fibroblast growth factor 21 (FGF21) are in clinical development for type 2 diabetes and nonalcoholic steatohepatitis (NASH) treatment. Although their glucose-lowering and insulin-sensitizing effects have been largely unraveled, the mechanisms by which they alleviate liver injury have only been scarcely addressed. Here, we aimed to unveil the mechanisms underlying the protective effects of FGF21 on NASH using APOE*3-Leiden.CETP mice, a well-established model for human-like metabolic diseases. Liver-specific FGF21 overexpression was achieved in mice, followed by administration of a high-fat high-cholesterol diet for 23 weeks. FGF21 prevented hepatic lipotoxicity, accompanied by activation of thermogenic tissues and attenuation of adipose tissue inflammation, improvement of hyperglycemia and hypertriglyceridemia, and upregulation of hepatic programs involved in fatty acid oxidation and cholesterol removal. Furthermore, FGF21 inhibited hepatic inflammation, as evidenced by reduced Kupffer cell (KC) activation, diminished monocyte infiltration, and lowered accumulation of monocyte-derived macrophages. Moreover, FGF21 decreased lipid- and scar-associated macrophages, which correlated with less hepatic fibrosis as demonstrated by reduced collagen accumulation. Collectively, hepatic FGF21 overexpression limits hepatic lipotoxicity, inflammation, and fibrogenesis. Mechanistically, FGF21 blocks hepatic lipid influx and accumulation through combined endocrine and autocrine signaling, respectively, which prevents KC activation and lowers the presence of lipid- and scar-associated macrophages to inhibit fibrogenesis.

Circadian rhythms play a critical role in regulating metabolism, including daily cycles of feeding/fasting. Glucokinase (GCK) is central for whole-body glucose homeostasis and oscillates according to a circadian clock. GCK activators (GKAs) effectively reduce hyperglycemia, but their use is also associated with hypoglycemia, hyperlipidemia, and hepatic steatosis. Given the circadian rhythmicity and natural postprandial activation of GCK, we hypothesized that GKA treatment would benefit from being timed specifically during feeding periods. Acute treatment of obese Zucker rats with the GKA AZD1656 robustly increased flux into all major metabolic pathways of glucose disposal, enhancing glucose elimination. Four weeks of continuous AZD1656 treatment of obese Zucker rats improved glycemic control; however, hepatic steatosis and inflammation manifested. In contrast, timing AZD1656 to feeding periods robustly reduced hepatic steatosis and inflammation in addition to improving glycemia, whereas treatment timed to fasting periods caused overall detrimental metabolic effects. Mechanistically, timing AZD1656 to feeding periods diverted newly synthesized lipid toward direct VLDL secretion rather than intrahepatic storage. In line with increased hepatic insulin signaling, timing AZD1656 to feeding resulted in robust activation of AKT, mTOR, and SREBP-1C after glucose loading, pathways known to regulate VLDL secretion and hepatic de novo lipogenesis. In conclusion, intermittent AZD1656 treatment timed to feeding periods promotes glucose disposal when needed the most, restores metabolic flexibility and hepatic insulin sensitivity, and thereby avoids hepatic steatosis. Thus, chronotherapeutic approaches may benefit the development of GKAs and other drugs acting on metabolic targets.

Analogues of the hepatokine FGF21 are in clinical development for type 2 diabetes and nonalcoholic steatohepatitis (NASH) treatment. Although their glucose-lowering and insulin-sensitizing effects have been largely unraveled, the mechanisms by which they alleviate liver injury have only been scarcely addressed. Here, we aimed to unveil the mechanisms underlying the protective effects of FGF21 on NASH using APOE*3-Leiden.CETP mice, a well-established model for human-like metabolic diseases. Liver-specific FGF21 overexpression was achieved in mice, followed by administration of a high-fat high-cholesterol diet for 23 weeks. FGF21 prevented hepatic lipotoxicity, accompanied by activation of thermogenic tissues and attenuation of adipose tissue inflammation, improvement of hyperglycemia and hypertriglyceridemia, and upregulation of hepatic programs involved in fatty acid oxidation and cholesterol removal. Furthermore, FGF21 inhibited hepatic inflammation, as evidenced by reduced Kupffer cell (KC) activation, diminished monocyte infiltration and lowered accumulation of monocyte-derived macrophages. Moreover, FGF21 decreased lipid- and scar-associated macrophages, which correlated with less hepatic fibrosis as demonstrated by reduced collagen accumulation. Collectively, hepatic FGF21 overexpression limits hepatic lipotoxicity, inflammation and fibrogenesis. Mechanistically, FGF21 blocks hepatic lipid influx and accumulation through combined endocrine and autocrine signaling, respectively, which prevents KC activation and lowers the presence of lipid- and scar-associated macrophages to inhibit fibrogenesis.