Jakob Appel Østergaard

Assessment of early diabetic renal changes with hyperpolarized [1‐13C]pyruvate

This experimental study explores a novel magnetic resonance imaging/spectroscopic (MRI/MRS) method that measures changes in renal metabolism in a diabetic rat model. This hyperpolarized metabolic MRI/MRS method allows monitoring of metabolic processes in seconds by >10 000‐fold enhancement of the MR signal. The method has shown that the conversion of pyruvate to bicarbonate, i.e. pyruvate dehydrogenase (PDH) activity, is significantly altered in the myocardium already at the onset of diabetes, and the predominant Warburg effect is a valuable cancer maker via the lactate dehydrogenase (LDH) activity. We hypothesize that a similar change in PDH and LDH could be found in the early diabetic kidney.

High altitude may alter oxygen availability and renal metabolism in diabetics as measured by hyperpolarized [1-13C]pyruvate magnetic resonance imaging

The kidneys account for about 10% of the whole body oxygen consumption, whereas only 0.5% of the total body mass. It is known that intrarenal hypoxia is present in several diseases associated with development of kidney disease, including diabetes, and when renal blood flow is unaffected. The importance of deranged oxygen metabolism is further supported by deterioration of kidney function in patients with diabetes living at high altitude. Thus, we argue that reduced oxygen availability alters renal energy metabolism. Here, we introduce a novel magnetic resonance imaging (MRI) approach to monitor metabolic changes associated with diabetes and oxygen availability. Streptozotocin diabetic and control rats were given reduced, normal, or increased inspired oxygen in order to alter tissue oxygenation. The effects on kidney oxygen metabolism were studied using hyperpolarized [1-(13)C]pyruvate MRI. Reduced inspired oxygen did not alter renal metabolism in the control group. Reduced oxygen availability in the diabetic kidney altered energy metabolism by increasing lactate and alanine formation by 23% and 34%, respectively, whereas the bicarbonate flux was unchanged. Thus, the increased prevalence and severity of nephropathy in patients with diabetes at high altitudes may originate from the increased sensitivity toward inspired oxygen. This increased lactate production shifts the metabolic routs toward hypoxic pathways.

Insufficient insulin administration to diabetic rats increases substrate utilization and maintains lactate production in the kidney

Good glycemic control is crucial to prevent the onset and progression of late diabetic complications, but insulin treatment often fails to achieve normalization of glycemic control to the level seen in healthy controls. In fact, recent experimental studies indicate that insufficient treatment with insulin, resulting in poor glycemic control, has an additional effect on progression of late diabetic complications, than poor glycemic control on its own. We therefore compared renal metabolic alterations during conditions of poor glycemic control with and without suboptimal insulin administration, which did not restore glycemic control, to streptozotocin (STZ)‐diabetic rats using noninvasive hyperpolarized 13C‐pyruvate magnetic resonance imaging (MRI) and blood oxygenation level–dependent (BOLD) 1H‐MRI to determine renal metabolic flux and oxygen availability, respectively. Suboptimal insulin administration increased pyruvate utilization and metabolic flux via both anaerobic and aerobic pathways in diabetic rats even though insulin did not affect kidney oxygen availability, HbA1c, or oxidative stress. These results imply direct effects of insulin in the regulation of cellular substrate utilization and metabolic fluxes during conditions of poor glycemic control. The study demonstrates that poor glycemic control in combination with suboptimal insulin administration accelerates metabolic alterations by increasing both anaerobic and aerobic metabolism resulting in increased utilization of energy substrates. The results demonstrate the importance of tight glycemic control in insulinopenic diabetes, and that insulin, when administered insufficiently, adds an additional burden on top of poor glycemic control.

Identification of Individuals With Undiagnosed Diabetes and Pre-Diabetes in a Danish Cohort Attending Dental Treatment.

BACKGROUND It is estimated that 3.6% and 13.6% of the Danish population have undiagnosed type 2 diabetes mellitus and prediabetes, respectively. Periodontitis is an established complication associated with diabetes (DM). Identification of individuals with DM and prediabetes is important to reduce DM-related complications, including periodontitis. The objective of this study is to identify individuals with undiagnosed DM or prediabetes among patients attending a dental setting for diagnosis and treatment. METHODS A total of 291 adults with no history of DM was included in the study (periodontitis patients: n = 245; non-periodontitis control individuals: n = 46). Participants answered questionnaires concerning general health, including family history of DM. Body mass index, waist circumference, fat percentage, and glycated hemoglobin (HbA1c) level were recorded chairside. Periodontal examination was performed and radiographic bone level measured. All individuals were informed about their HbA1c test result and were referred to their physician if HbA1c level was above guideline levels for DM or prediabetes according to the American Diabetes Association. RESULTS Nine (3.1%) and 79 (27.1%) individuals were identified with HbA1c levels corresponding to guideline levels for DM and prediabetes, respectively. Higher proportions of patients with undiagnosed DM and prediabetes were observed in the periodontitis group (32.7%) than in the control group (17.4%) (P = 0.054). Identification of DM and prediabetes based on a diagnosis of periodontitis yielded a sensitivity of 0.91 and a specificity of 0.19. CONCLUSIONS This study confirms that individuals with undiagnosed DM and prediabetes can be identified in the dental office by chairside recordings of HbA1c levels. Routine measurement of HbA1c levels in dental offices, eventually restricted to those at risk, may help identification of individuals with DM and prediabetes at early stages of disease, which may prevent future complications.

Antioxidant treatment attenuates lactate production in diabetic nephropathy

The early progression of diabetic nephropathy is notoriously difficult to detect and quantify before the occurrence of substantial histological damage. Recently, hyperpolarized [1-13C]pyruvate has demonstrated increased lactate production in the kidney early after the onset of diabetes, implying increased lactate dehydrogenase activity as a consequence of increased nicotinamide adenine dinucleotide substrate availability due to upregulation of the polyol pathway, i.e., pseudohypoxia. In this study, we investigated the role of oxidative stress in mediating these metabolic alterations using state-of-the-art hyperpolarized magnetic resonance (MR) imaging. Ten-week-old female Wistar rats were randomly divided into three groups: healthy controls, untreated diabetic (streptozotocin treatment to induce insulinopenic diabetes), and diabetic, receiving chronic antioxidant treatment with TEMPOL (4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl) via the drinking water. Examinations were performed 2, 3, and 4 wk after the induction of diabetes by using a 3T Clinical MR system equipped with a dual tuned 13C/1H-volume rat coil. The rats received intravenous hyperpolarized [1-13C]pyruvate and were imaged using a slice-selective 13C-IDEAL spiral sequence. Untreated diabetic rats showed increased renal lactate production compared with that shown by the controls. However, chronic TEMPOL treatment significantly attenuated diabetes-induced lactate production. No significant effects of diabetes or TEMPOL were observed on [13C]alanine levels, indicating an intact glucose-alanine cycle, or [13C]bicarbonate, indicating normal flux through the Krebs cycle. In conclusion, this study demonstrates that diabetes-induced pseudohypoxia, as indicated by an increased lactate-to-pyruvate ratio, is significantly attenuated by antioxidant treatment. This demonstrates a pivotal role of oxidative stress in renal metabolic alterations occurring in early diabetes.

Diabetes-induced changes in mannan-binding lectin levels and complement activation in a mouse model of type 1 diabetes.

Circulating mannan‐binding lectin (MBL) levels are elevated in type 1 diabetes. Further, high MBL levels are associated with the development of diabetic nephropathy. In animals, a direct effect of MBL on diabetic kidney changes is observed. We hypothesized that MBL levels and detrimental complement activation increase as a consequence of diabetes. We measured plasma MBL before and 7 weeks after inducing diabetes by streptozotocin. Mice have two MBLs, MBL‐A and MBL‐C. Diabetes induction led to an increase in MBL‐C concentration, whereas no change during the study was found in the control group. The increase in MBL‐C was associated with the increasing plasma glucose levels. In accordance with the observed changes in circulating MBL levels, liver expression of Mbl2mRNA (encoding MBL‐C) was increased in diabetes. Mbl1expression (encoding MBL‐A) did not differ between diabetic and control animals. The estimated half‐life of recombinant human MBL was significantly prolonged in mice with diabetes compared with control mice. Complement activation in plasma and glomeruli did not differ between groups. We demonstrate for the first time that MBL levels increase after induction of diabetes and in parallel with increasing plasma glucose. Our findings support the previous clinical observations of increased MBL in type 1 diabetes. This change may be explained by alternations in both MBL production and turnover.

Mannan-binding lectin in diabetic kidney disease: the impact of mouse genetics in a type 1 diabetes model.

Background. Mannan-binding lectin (MBL) is involved in the development of diabetic nephropathy. MBL is a part of the innate immune system where it can activate the complement system. Serum MBL level predicts later renal impairment in diabetes patients. Direct involvement of MBL in the development of diabetic kidney disease is observed in one animal strain. However, this involvement may differ among the animal strains. We thus examined the impact of the genetic background on the role of MBL in diabetic nephropathy. Materials/Methods. C57BL/6JBomTac and 129S6/SvEvTac mice were compared. In both strains, experimental type 1 diabetes was induced in wild-type (WT) and MBL-knockout (MBL-KO) mice by streptozotocin. Nondiabetic WT and MBL-KO mice were used as controls. We tested if MBL modified the diabetes-induced kidney changes by two-way ANOVA allowing for interaction. Results. MBL aggravated diabetes-induced kidney growth and glomerulus enlargement in C57BL/6JBomTac mice. MBL did not modify diabetes effects on glomerular basement membrane thickness or mesangial volume in any strain. Diabetes-induced changes in renal gene transcription of growth factors and matrix components were unaffected by MBL. Conclusions. Strain-specific MBL effects were found on downstream diabetic kidney changes. This emphasizes the importance of genetic background in this model of diabetic complications.

Investigation of metabolic changes in STZ‐induced diabetic rats with hyperpolarized [1‐13C]acetate

In the metabolism of acetate several enzymes are involved, which play an important role in free fatty acid oxidation. Fatty acid metabolism is altered in diabetes patients and therefore acetate might serve as a marker for pathological changes in the fuel selection of cells, as these changes occur in diabetes patients. Acetylcarnitine is a metabolic product of acetate, which enables its transport into the mitochondria for energy production. This study investigates whether the ratio of acetylcarnitine to acetate, measured by noninvasive hyperpolarized [1‐13C]acetate magnetic resonance spectroscopy, could serve as a marker for myocardial, hepatic, and renal metabolic changes in rats with Streptozotocin (STZ)‐induced diabetes in vivo. We demonstrate that the conversion of acetate to acetylcarnitine could be detected and quantified in all three organs of interest. More interestingly, we found that the hyperpolarized acetylcarnitine to acetate ratio was independent of blood glucose levels and prolonged hyperglycemia following diabetes induction in a type‐1 diabetes model.

Increased autoreactivity of the complement-activating molecule mannan-binding lectin in a type 1 diabetes model

Background. Diabetic kidney disease is the leading cause of end-stage renal failure despite intensive treatment of modifiable risk factors. Identification of new drug targets is therefore of paramount importance. The complement system is emerging as a potential new target. The lectin pathway of the complement system, initiated by the carbohydrate-recognition molecule mannan-binding lectin (MBL), is linked to poor kidney prognosis in diabetes. We hypothesized that MBL activates complement upon binding within the diabetic glomerulus. Methods. We investigated this by comparing complement deposition and activation in kidneys from streptozotocin-induced diabetic mice and healthy control mice. Results. After 20 weeks of diabetes, glomerular deposition of MBL was significantly increased. Diabetic animals had 2.0-fold higher (95% CI 1.6–2.5) immunofluorescence intensity from anti-MBL antibodies compared with controls (P < 0.001). Diabetes and control groups did not differ in glomerular immunofluorescence intensity obtained by antibodies against complement factors C4, C3, and C9. However, the circulating complement activation product C3a was increased in diabetes as compared to control mice (P = 0.04). Conclusion. 20 weeks of diabetes increased MBL autoreactivity in the kidney and circulating C3a concentration. Together with previous findings, these results indicate direct effects of MBL within the kidney in diabetes.

Effects of TNF‐α blocking on experimental periodontitis and type 2 diabetes in obese diabetic Zucker rats

OBJECTIVE Tumour necrosis factor α (TNF-α) is considered a key signalling modulator in the pathogenesis of both periodontitis (PD) and type 2 diabetes mellitus (DM2). This study aims at elucidating the effect of TNF-α blocking on the interplay between PD and DM2. METHODS Obese diabetic Zucker rats and their lean littermates were divided into five treatment groups with or without periodontitis. Anti-TNF-α treatment was provided with Etanercept injections. Diabetic state was evaluated by oral glucose tolerance test, the homeostatic model assessment, free fatty acids and blood glucose. Systemic inflammation was assessed by measurement of interleukin (IL)-1β, IL-6 and TNF-α in plasma. Kidney complications were evaluated by real-time rtPCR, creatinine clearance rate, urinary albumin excretion and increase in weight. PD was evaluated by registration of alveolar bone level. RESULTS After 4 weeks the diabetic state was modified by Etanercept treatment with lower insulin levels and lower homeostatic model assessment. Furthermore, while kidney complications were reduced by Etanercept treatment, PD had no effect. PD was influenced by diabetic state, but the impact was attenuated by Etanercept treatment. CONCLUSION In this study anti-TNF-α treatment improved glucose tolerance and compensated for the increased periodontal disease in obese diabetic Zucker. PD did not influence diabetic parameters assessed including complications of the rats kidneys.