High-fat High-sucrose Diet Mice Research Articles

Metformin does not prevent the alteration of reticulum-mitochondria Ca2+ coupling and the progression towards early diabetic cardiomyopathy with HFpEF in a diet-induced mouse model of T2D

Diabetic cardiomyopathy, as a major complication of type 2 diabetes (T2D), shows a rising prevalence nowadays. Reticulum-mitochondria Ca2+ uncoupling was recently reported as an early but reversible trigger of cardiac dysfunction using a diet-induced murine model of DCM with heart failure and preserved ejection fraction (HFpEF). Metformin is the first-line treatment prescribed to treat T2D patients, with known protective effects against myocardial infarction. We here aimed to determine if metformin could hinder the alteration of the reticulum-mitochondria Ca2+ coupling and the cardiac dysfunction induced by a high-fat high-sucrose diet (HFHSD) in mice. Mice were either fed a HFHSD or a standard diet for 16 weeks. During the last 6 weeks of diet, HFHSD mice received a daily oral gavage with either metformin or vehicle. Reticulum-mitochondria Ca2+ coupling was assessed: 1) structrally by proximity ligation assay between IP3R and VDAC; and 2) functionally by Ca2+ imaging of the mitochondrial Ca2+ sensor, 4mtD3cpv, expressed by intramyocardial adenoviral injection. Echocardiography was perfomed on lightly anesthetized animals to assess systolic function and hypertrophy. Metformin rescued the glucose tolerance and insulinemia in HFHSD mice, but partially for the cardiac insulin resistance. However, in the HFHSD cardiomyocyte, metformin did not preclude the reduction of the proximity between IP3R and VDAC, and of the histamine-driven reticulum-mitochondria Ca2+ transfer through the IP3R-VDAC complex. Echocardiography also revealed no prevention of HFHSD-induced cardiac hypertrophy and strain rate reduction. Our data report that 6 weeks of metformin treatment did not preserve the reticulum-mitochondria Ca2+ coupling in the diabetic heart and did not prevent the progression towards early DCM with HFpEF.

Investigation into the Role of the Skeletal Muscle BBSome in Insulin Sensitivity

The BBSome is a protein complex composed of eight Bardet‐Biedl syndrome (BBS) proteins including BBS1. Humans and mice lacking a functional BBSome display impaired insulin sensitivity, hyperglycemia, and type 2 diabetes, highlighting the importance of this protein complex in the control of insulin action and glucose homeostasis. However, the contribution of the BBSome in insulin sensitive tissues such as the skeletal muscle to the control of insulin sensitivity is not clear. We hypothesized that BBSome deficiency in the skeletal muscle will lead to insulin resistance and diabetes. To test this, we generated a novel conditional knockout mouse model lacking the Bbs1 gene specifically in the skeletal muscle. This was achieved by crossing mice expressing floxed alleles of the Bbs1 gene (Bbs1fl/fl) with mice expressing Cre‐recombinase in the skeletal muscle (MCKCre). These mice were further bred with tdTomato reporter mice to validate Cre‐recombinase using red tdTomato protein fluorescence. Body weight, glucose handling (measured using glucose tolerance test, GTT) and insulin sensitivity (measured using insulin tolerance test, ITT) of Bbs1fl/fl/MCKCre mice were compared to littermate controls. Furthermore, we used Western blot to test the ability of insulin to activate Akt, a key signaling pathway associated with the insulin receptor. We found that both male and female Bbs1fl/fl/MCKCre mice have no significant alteration in body weight relative to wild type controls (31.4±1.5 vs 31.1±1.0 g in males and 24.1±2.2 vs 23.0±1.7 g in females at 16 weeks of age). There was also no significant change in glucose handling (GTT AUC: 30608±2106 vs 30741±3480 in males and 28836±4466 vs 26530±3334 AUC in females) and insulin sensitivity (ITT AUC: 11017±1285 vs 9935±1080 in males and 8223±792 vs 5803±978 AUC in females) between Bbs1fl/fl/MCKCre mice and controls. This was further confirmed by the lack of difference in insulin‐induced Akt activation in skeletal muscle, liver, and adipose tissue. These findings suggest that BBS1 protein in skeletal muscle is not required for insulin sensitivity. Next, we assessed whether challenging the mice with high fat/high sucrose diet (HFHSD), starting at weaning, could reveal a role for the skeletal muscle BBSome in insulin sensitivity. The weight gain evoked by HFHSD was comparable between Bbs1fl/fl/MCKCre mice and controls (32.3±3.1 vs 33.3±1.0 g in males at 10 weeks of age). Once they reach 16 weeks of age, the HFHSD mice will be subject to glucose tolerance and insulin sensitivity tests to determine if obesity exaggerates hyperglycemia and insulin resistance in Bbs1fl/fl/MCKCre mice.

Effect of Metformin on T2D-Induced MAM Ca2+ Uncoupling and Contractile Dysfunction in an Early Mouse Model of Diabetic HFpEF.

Diabetic cardiomyopathy (DCM) is a leading complication in type 2 diabetes patients. Recently, we have shown that the reticulum-mitochondria Ca2+ uncoupling is an early and reversible trigger of the cardiac dysfunction in a diet-induced mouse model of DCM. Metformin is a first-line antidiabetic drug with recognized cardioprotective effect in myocardial infarction. Whether metformin could prevent the progression of DCM remains not well understood. We therefore investigated the effect of a chronic 6-week metformin treatment on the reticulum-mitochondria Ca2+ coupling and the cardiac function in our high-fat high-sucrose diet (HFHSD) mouse model of DCM. Although metformin rescued the glycemic regulation in the HFHSD mice, it did not preserve the reticulum-mitochondria Ca2+ coupling either structurally or functionally. Metformin also did not prevent the progression towards cardiac dysfunction, i.e., cardiac hypertrophy and strain dysfunction. In summary, despite its cardioprotective role, metformin is not sufficient to delay the progression to early DCM.

The Role of Brown Adipose Tissue in the Development and Treatment of Nonalcoholic Steatohepatitis: An Exploratory Gene Expression Study in Mice.

Brown adipose tissue (BAT) might be a beneficial mediator in the development and treatment of nonalcoholic steatohepatitis (NASH). We aim to evaluate the gene expression of BAT activity-related genes during the development and the dietary and surgical treatment of NASH. BAT was collected from male C57BL/6J mice that received a high fat-high sucrose diet (HF-HSD) or a normal chow diet (NCD) for 4 and 20 weeks (n=8-9 per dietary group and timepoint) and from mice that underwent dietary intervention (return to NCD) (n=8), roux-en-y gastric bypass (RYGB) (n=6), or sham procedure (n=6) after 12 weeks HF-HSD. Expression of BAT genes involved in lipid metabolism (Cd36 and Cpt1b; p<0.05) and energy expenditure (Ucp1 and Ucp3; p<0.05) were significantly increased after 4 weeks HF-HSD compared with NCD, whereas in the occurrence of NASH after 20 weeks HF-HSD no difference was observed. We observed no differences in gene expression regarding lipid metabolism or energy expenditure at 8 weeks after dietary intervention (no NASH) compared with HF-HSD mice (NASH), nor in mice that underwent RYGB compared with SHAM. However, dietary intervention and RYGB both decreased the BAT gene expression of inflammatory cytokines (Il1b, Tnf-α and MCP-1; p<0.05). Gene expression of the batokine neuregulin 4 was significantly decreased after 20 weeks HF-HSD (p<0.05) compared with NCD, but was restored by dietary intervention and RYGB (p<0.05). In conclusion, BAT is hallmarked by dynamic alterations in the gene expression profile during the development of NASH and can be modulated by dietary intervention and bariatric surgery.

Reduced reticulum\u2013mitochondria Ca2+ transfer is an early and reversible trigger of mitochondrial dysfunctions in diabetic cardiomyopathy

Type 2 diabetic cardiomyopathy features Ca2+ signaling abnormalities, notably an altered mitochondrial Ca2+ handling. We here aimed to study if it might be due to a dysregulation of either the whole Ca2+ homeostasis, the reticulum–mitochondrial Ca2+ coupling, and/or the mitochondrial Ca2+ entry through the uniporter. Following a 16-week high-fat high-sucrose diet (HFHSD), mice developed cardiac insulin resistance, fibrosis, hypertrophy, lipid accumulation, and diastolic dysfunction when compared to standard diet. Ultrastructural and proteomic analyses of cardiac reticulum–mitochondria interface revealed tighter interactions not compatible with Ca2+ transport in HFHSD cardiomyocytes. Intramyocardial adenoviral injections of Ca2+ sensors were performed to measure Ca2+ fluxes in freshly isolated adult cardiomyocytes and to analyze the direct effects of in vivo type 2 diabetes on cardiomyocyte function. HFHSD resulted in a decreased IP3R–VDAC interaction and a reduced IP3-stimulated Ca2+ transfer to mitochondria, with no changes in reticular Ca2+ level, cytosolic Ca2+ transients, and mitochondrial Ca2+ uniporter function. Disruption of organelle Ca2+ exchange was associated with decreased mitochondrial bioenergetics and reduced cell contraction, which was rescued by an adenovirus-mediated expression of a reticulum-mitochondria linker. An 8-week diet reversal was able to restore cardiac insulin signaling, Ca2+ transfer, and cardiac function in HFHSD mice. Therefore, our study demonstrates that the reticulum–mitochondria Ca2+ miscoupling may play an early and reversible role in the development of diabetic cardiomyopathy by disrupting primarily the mitochondrial bioenergetics. A diet reversal, by counteracting the MAM-induced mitochondrial Ca2+ dysfunction, might contribute to restore normal cardiac function and prevent the exacerbation of diabetic cardiomyopathy.

Diet reversal rescues mouse diabetic cardiomyopathy by counteracting the decreased reticulum-mitochondria interactions and the ensuing mitochondrial Ca2+ uptake impairment

Type 2 diabetic cardiomyopathy has been linked to Ca2+ signaling alterations, notably a decreased mitochondrial Ca2+ uptake. Recent discovery of Ca2+ microdomains between cardiac mitochondria and reticulum launched a new investigation avenue for cardiometabolic diseases. Hereby, we aimed to investigate if the mitochondrial Ca2+ mishandling is due to a dysregulation of the reticulum-mitochondria interactions or of the mitochondrial Ca2+ uniporter in the diabetic mouse heart. Mice fed for 16 weeks with a high fat high sucrose diet (HFHSD) displayed a cardiac insulin resistance combined to cardiac fibrosis, hypertrophy and contractile dysfunction (decreased strain rate) versus standard diet-fed mice (SD). Fractionation of HFHSD mice hearts revealed a decreased protein content of their mitochondria associated-reticulum membranes, together with a lower expression of tethering proteins quantified by mass spectrometry. On functional terms, reduced IP3R-VDAC proximity and co-immunoprecipitation were associated with a reduced IP3-stimulated Ca2+ transfer to mitochondria in isolated HFHSD cardiomyocytes, yet no changes in mitochondrial calcium uniporter protein expression and function were detected. Moreover, HFHSD cardiomyocytes displayed no significant changes in the reticular Ca2+ release level and the amplitude of cytosolic Ca2+ transients. Eight weeks of diet reversal restored the cardiac insulin signaling, MAM content, Ca2+ transfer and cardiac function in HFHSD mice. Therefore, our data indicate that disruption of the cardiac reticulum-mitochondria interactions is an early and reversible event impairing mitochondrial Ca2+ handling and contributing to the cardiac contractile dysfunction in the diabetic mouse heart, which could be counteracted by diet reversal.

Insulin‐like Growth Factor 1 Improves Cardiac Function after Acute Myocardial Infarction in a Prediabetic Mouse Model

BackgroundType 2 diabetes mellitus (T2DM) is a major comorbidity worsening the outcome after acute myocardial infarction (MI). Reduced cardiac glucose uptake may be the underlying mechanism compromising cardiac function even under prediabetic conditions. Insulin‐like growth factor 1 (IGF1) is an anabolic hormone similar to insulin in molecular structure and signaling. IGF1 controls proliferation, differentiation and metabolism of cells. In previous studies we have shown that short‐term IGF1 treatment after MI improved cardiac function and reduced scar size in mouse hearts. Here we investigate whether IGF1 can maintain cardiac function also after MI under conditions of prediabetes.MethodsMice were fed standard chow or high fat/high sucrose diet (HFHSD) for 10 weeks to induce obesity and prediabetes. Phenotyping was performed by measurements of blood glucose, insulin levels, glucose (GTT) and insulin tolerance tests (ITT). Mice were subjected to 45 min left anterior descending coronary artery occlusion and 1‐week reperfusion. IGF1 or vehicle were given over 3 days using osmotic mini pumps. Heart function was analyzed echocardiographically before and one week after MI including strain analysis. In addition, a transcriptome analysis was carried out for both the area at risk and the remote area by next‐generation sequencing.ResultsHFHSD led to increased fasting blood glucose levels with up to 200 mg/dl and glucose intolerance. In line with this results, plasma insulin levels were elevated in HFHSD mice with 1.3 ng/L in comparison to 0.4 ng/l in standard chow mice. However, exogenous insulin was able to lower blood glucose in the ITT, indicating prediabetes but no T2DM. Functional analysis by EC indicated a slight improvement of ejection fraction (EF) in HFHSD mice with 71.1% in comparison to 64.0% in the control group. However, cardiac function after MI under prediabetic conditions (EF= 35.3%) worsened to the same extent as in the control group (EF= 34.4%). Treatment of IGF1 after MI preserved cardiac function not only in the control group but also in the prediabetes group, with higher EF (control: 45.5%; IGF1: 48.2%), higher stroke volume and lower end‐systolic volume. Strain analysis revealed that this improvement is rather by an improvement of the remote area than by the area at risk.ConclusionDespite a similar signal transduction mediated by IGF1 and insulin, IGF1 was able to preserve cardiac function after MI in a prediabetic mouse model. Thus, an IGF1‐based therapeutic intervention could be a promising approach to improve the worsened cardiac outcome after myocardial infarction even under prediabetic conditions and may also be protective under T2DM.Support or Funding InformationThis work was funded by the German Research Foundation (CRC 1116 “Master Switches in Cardiac Ischemia”, TP A06).

Manganese-mediated MRI signals correlate with functional β-cell mass during diabetes progression.

Diabetes diagnostic therapy and research would strongly benefit from noninvasive accurate imaging of the functional β-cells in the pancreas. Here, we developed an analysis of functional β-cell mass (BCM) by measuring manganese (Mn(2+)) uptake kinetics into glucose-stimulated β-cells by T1-weighted in vivo Mn(2+)-mediated MRI (MnMRI) in C57Bl/6J mice. Weekly MRI analysis during the diabetes progression in mice fed a high-fat/high-sucrose diet (HFD) showed increased Mn(2+)-signals in the pancreas of the HFD-fed mice during the compensation phase, when glucose tolerance and glucose-stimulated insulin secretion (GSIS) were improved and BCM was increased compared with normal diet-fed mice. The increased signal was only transient; from the 4th week on, MRI signals decreased significantly in the HFD group, and the reduced MRI signal in HFD mice persisted over the whole 12-week experimental period, which again correlated with both impaired glucose tolerance and GSIS, although BCM remained unchanged. Rapid and significantly decreased MRI signals were confirmed in diabetic mice after streptozotocin (STZ) injection. No long-term effects of Mn(2+) on glucose tolerance were observed. Our optimized MnMRI protocol fulfills the requirements of noninvasive MRI analysis and detects already small changes in the functional BCM.

Imeglimin normalizes glucose tolerance and insulin sensitivity and improves mitochondrial function in liver of a high-fat, high-sucrose diet mice model.

Imeglimin is the first in a new class of oral glucose-lowering agents currently in phase 2b development. Although imeglimin improves insulin sensitivity in humans, the molecular mechanisms are unknown. This study used a model of 16-week high-fat, high-sucrose diet (HFHSD) mice to characterize its antidiabetic effects. Six-week imeglimin treatment significantly decreased glycemia, restored normal glucose tolerance, and improved insulin sensitivity without modifying organs, body weights, and food intake. This was associated with an increase in insulin-stimulated protein kinase B phosphorylation in the liver and muscle. In liver mitochondria, imeglimin redirects substrate flows in favor of complex II, as illustrated by increased respiration with succinate and by the restoration of respiration with glutamate/malate back to control levels. In addition, imeglimin inhibits complex I and restores complex III activities, suggesting an increase in fatty acid oxidation, which is supported by an increase in hepatic 3-hydroxyacetyl-CoA dehydrogenase activity and acylcarnitine profile and the reduction of liver steatosis. Imeglimin also reduces reactive oxygen species production and increases mitochondrial DNA. Finally, imeglimin effects on mitochondrial phospholipid composition could participate in the benefit of imeglimin on mitochondrial function. In conclusion, imeglimin normalizes glucose tolerance and insulin sensitivity by preserving mitochondrial function from oxidative stress and favoring lipid oxidation in liver of HFHSD mice.

Voluntary exercise improves insulin sensitivity and adipose tissue inflammation in diet-induced obese mice

Exercise promotes weight loss and improves insulin sensitivity. However, the molecular mechanisms mediating its beneficial effects are not fully understood. Obesity correlates with increased production of inflammatory cytokines, which in turn, contributes to systemic insulin resistance. To test the hypothesis that exercise mitigates this inflammatory response, thereby improving insulin sensitivity, we developed a model of voluntary exercise in mice made obese by feeding of a high fat/high sucrose diet (HFD). Over four wk, mice fed chow gained 2.3 +/- 0.3 g, while HFD mice gained 6.8 +/- 0.5 g. After 4 wk, mice were subdivided into four groups: chow-no exercise, chow-exercise, HFD-no exercise, HFD-exercise and monitored for an additional 6 wk. Chow-no exercise and HFD-no exercise mice gained an additional 1.2 +/- 0.3 g and 3.3 +/- 0.5 g respectively. Exercising mice had higher food consumption, but did not gain additional weight. As expected, GTT and ITT showed impaired glucose tolerance and insulin resistance in HFD-no exercise mice. However, glucose tolerance improved significantly and insulin sensitivity was completely normalized in HFD-exercise animals. Furthermore, expression of TNF-alpha, MCP-1, PAI-1 and IKKbeta was increased in adipose tissue from HFD mice compared with chow mice, whereas exercise reversed the increased expression of these inflammatory cytokines. In contrast, expression of these cytokines in liver was unchanged among the four groups. These results suggest that exercise partially reduces adiposity, reverses insulin resistance and decreases adipose tissue inflammation in diet-induced obese mice, despite continued consumption of HFD.